CN101008033A - Gene chip for detecting functional central nerve damage and its production method - Google Patents
Gene chip for detecting functional central nerve damage and its production method Download PDFInfo
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Abstract
The invention relates to gene chip used for detecting functional central nervous damage diagnosis and the preparing method. said gene chip is equipped with oligonucleotide gene probe array, the oligonucleotide biomolecule of said probe array is the special biological gene factor playing improtant role in multiple central nervous system damage process simutaneously, relating to netural cell death and tissue necrosis, inflammation, irritated response, ion dysequilibrium inside cell, signal transmission, abnormal control of cell cycle, abnormal cell construction and nerve degeneration damage morbid physiology and molecular biology active gene, respectively. The gene chip can be used to check the conditioning way, expression level and change rule of biological factor relative to cerebral ischemia damage inside body in the beginning of clinic for high risk group of cerebral apoplexy, and to predict possibility of cerebral apoplexy; and used to conditioning way and change rule of special biological factor relative to nerve damage inside nerve cell after central nervous system damage, and relationship between cell death and disease occurancy, and provides checking criterion for cilinical treatment.
Description
Technical field
The invention belongs to the gene engineering field, relate to a kind of functional central nervous system injury diagnostic detection gene chip and manufacture method thereof, particularly a kind of specific detection and central nervous system injury (comprise cerebral apoplexy or claim brain injury, traumatic brain injury and the Spinal injury that cerebrovascular accident causes) pathologic, physiologic and molecular biology change the oligonucleotide chip of the total gene of main neural wound that is closely related.
Background technology
Central nervous system injury (comprise cerebral apoplexy or claim brain injury, traumatic brain injury and the Spinal injury that cerebrovascular accident causes) is the principal disease that threatens human health.It causes serious disabled anergy and unusual high mortality, and the cost of huge medical rehabilitation.Annual about 430,000 people suffer from cerebral apoplexy (25% patient death) in the U.S., and about 270,000 people suffer from traumatic brain injury (27% patient death), and about 10-12 ten thousand people suffer from Spinal injury (8-10% patient death).Only in the U.S., (the treatment expense that wherein is used for cerebral apoplexy is 63,500,000,000 dollars up to 1,227 hundred million dollars to be used for the treatment expense of central nervous system injury every year, the treatment expense that is used for traumatic brain injury is 48,300,000,000 dollars, and the treatment expense that is used for Spinal injury is 10,900,000,000 dollars).According to according to a preliminary estimate, be used for the medicine of central nervous system injury treatment every year, its potential market exploitation in the U.S. is worth up to 30,000,000,000 dollars.
For a long time, central nervous system injury is considered to a kind of irremediable disease.Because behind the central nervous system injury, its extent of damage also not only is confined to the primary injury zone, its degree of injury is also considerably beyond the degree of primary injury.A kind of process that is called gradual secondary neural tissue injury and nerve cell death often occurs in hindered in back a few hours, a couple of days even several weeks.This Secondary cases nerve injury is the main reason that causes serious dysneuria (comprise paralysis, anergy, numbness, weakness, feel dizzy, obstinate headache, aphasis or language understanding power obstacle, visual disorder, memory loss, disturbance in thinking, equilibrant or the forfeiture of direction distinguishing power, motion and sensory function disappearance and behavior disorder etc.).Yet, the Secondary cases nerve injury is also treated the chance that central nervous system injury provides a time-delay treatment for us, promptly after wound, reach the purpose of interrupting gradual neural tissue injury and nerve cell death by blocking-up Secondary cases nerve injury process in a few hours, thereby alleviate serious sequela such as dysneuria that central nervous system injury causes, paralysis, anergy to greatest extent.
Central nervous system injury, particularly the Secondary cases nerve injury is the pathophysiological change process of a complexity, it can be not only be caused the change that yet can only not cause single biomolecules by the change of single biomolecules, and it relates to the 1-2% (being 200-500 gene) of certified Human genome sum (ten thousand genes of 2-2.5) at least.After the wound because of the large number of biological molecule abnormality change cause that the dysequilibrium of bio-networks in the whole cell is only the true cause that causes the irreversible death of neurocyte.Therefore, the evolution of central nervous system injury should be the unbalance process of biomolecules regulating networks in the cell.These regulating networks are made of thousands of biomolecules (albumen or gene), the big and small cities and towns that these biomolecules are lived as us, interconnect by the bio signal transmission path that is similar to highway and railway, form biomolecules regulating networks in the complete cell.Under normal circumstances, all biomolecules are kept work metastable state (promptly do not increase and do not reduce), thereby to keep the relative equilibrium of whole network, this relative equilibrium be the bring into normal play important foundation of physiological function of human body cell and tissue.After the central nervous system damage, therefore corresponding change takes place for dozens of even hundreds of biomolecules, and (what have increases, the reduction that has), thereby destroyed the relative equilibrium of whole network system, though the resistance mechanisms of human body self also constantly attempts to recover the balance of network, if the unbalance scope of the network that causes of damage is excessive, intensity is strong excessively, surpass the repair ability of self, then network is unbalance will develop towards irreversible direction, finally cause necrocytosis and tissue necrosis.And only can regulate numerous by one that damages in the biomolecules that is changed at the pharmacological agent of single biomolecules, though this adjusting is very effective to corresponding single biomolecules, can change the activity and the working order of this biomolecules, also the function of cell biological molecular regulation network can be therefore partly improved, but the normal equilibrium of whole network can not be recovered effectively.This is that most single medicines can only relief of symptoms and the reason that can not effect a radical cure central nervous system injury.Fearful is, change the activity of single creature molecule and working order and the corresponding signal transmission path function that working order (such as blocking this biomolecules) will inevitably change other associated biomolecules, be not restored in the cellular network balance, normal physiological function exists under the situation of obstacle, the obstacle that can cause other cell function on the contrary, aggravate the unbalance of whole cellular network, Here it is single medicine is not only invalid sometimes on the contrary can the toxigenicity side effect and the possible cause of complication.
Biochip is to grow up twentieth century the mid-90, and melting microtronics, biology, physics, chemistry, computer science is the tip height chiasma type biotechnology of one.Biochip technology has become one of the most far-reaching great biotechnology progress of influence.Biochip not only has great biomedical research and is worth, and has tangible clinical detection application prospect again.Biochip is meant methods such as adopting the synthetic or micro-sampling of photoconduction original position, with the large number of biological macromole such as nucleic acid fragment (oligonucleotide/PNA, cDNA, genomic dna), peptide molecule even tissue slice, cell or the like biological sample solidifies in upholder in an orderly manner (as slide, silicon chip, polyacrylamide gel, solid phase carriers such as nylon membrane) surface, form intensive two-dimentional molecular arrangement, when target molecule in the biological sample to be measured of mark with after probe molecule hybridization on the chip combines, intensity of hybridization signal is carried out fast such as laser confocal scanning or electric charge coupling photography camera by specific instrument, parallel, check and analysis efficiently, thus realize DNA, RNA, polypeptide, the quantity of target molecule and expression level in the high throughput testing of protein and other biological composition and the judgement sample.Because slide/silicon chip commonly used is as solid support, and in the technology of preparing of preparation process analog calculation machine chip, so be referred to as biochip technology.According to the fixed probe difference on the chip, biochip comprises gene chip, protein chip, cell chip, organization chip.As fixed on the fruit chip is peptide or albumen, is used to study the biochip of protein function and transactional analysis, then is called protein chip; As fixed molecule on the fruit chip is oligonucleotide probe or DNA, and it is used to study the biochip of expression of gene situation, then is gene chip.The most outstanding characteristics of biochip are: integrated, parallelization, microminiaturization, automatization and serialization.
The development of biochip technology makes the researchist can promptly observe the expression and the functional status of interior whole genome of cell and protein groups, and biomedical research work has been played huge pushing effect.The utilization biochip obtains gene and protein expression data, by the experimental data finishing analysis of computer and the contrast of theoretical and experimental evidence, can make more accurate mensuration and judgement by pair cell physiology/pathology functional status.Yet traditional high-throughput chip gene expression profile cost consumption is big, and the data of acquisition are various, and many gene expression datas are that the investigator does not need or useless data.Most of biomedical research personnel and clinician more are ready to study the expression variation of some gene/gene families relevant with specified disease and albumen/protein family in research and clinical position, rather than the variation of whole genome and protein groups.Therefore, the small throughput functional classification biochip that is used for specified disease has become the genome times afterwards comprehensively effective analysis tool of system more.
Summary of the invention
The object of the present invention is to provide a kind of functional central nervous system injury diagnostic detection gene chip and manufacture method thereof, this gene chip can be used for specific detection central nervous system injury the molecular biology Changing Pattern and the characteristics of (comprising cerebral apoplexy, traumatic brain injury and Spinal injury).This gene chip can be used for detecting in its body of cerebral apoplexy high risk population preclinical phase and adjusting form, expression level and the Changing Pattern of the closely-related biotic factor of cerebral ischemic injury, with the possibility of prediction cerebral apoplexy generation; And can detect behind the central nervous system injury in the neurocyte with the adjusting form of the closely-related specific biological factor of nerve injury, Changing Pattern and with the mutual relationship of necrocytosis and disease development, detect foundation for clinical treatment provides experiment.
Realize that the object of the invention technical scheme is this functional central nervous system injury diagnostic detection gene chip, immobilized oligonucleotide Oligo gene probe array on the chip, these oligonucleotide biomolecules of oligonucleotide gene probe array all are the specific biological factors that plays a crucial role in multiple central nervous system injury generation evolution simultaneously, relate separately to nerve cell death and tissue necrosis, inflammation and stress reaction, ionic equilibrium imbalance in the cell, the signal conduction, cell cycle regulating is unusual, cellularstructure unusual and neurodegeneration equivalent damage pathologic, physiologic and molecular biology active gene factor.Further these specific biological factors comprise: 1, trauma stress reaction; 2, wound early response; 3, apoptosis and anti-apoptotic; 4, peroxidation stress reaction; 5, redox reaction; 6, inflammatory reaction; 7, cell cycle regulation; 8, neurodegeneration; 9, neural defence protection; 10, albumen is synthetic; 11, ion path and ionic equilibrium are regulated; 12, cell growth circulation; 13, cell transcription is regulated; 14, intracellular transport; 15, cell receptor; 16, cell physiological metabolism; 17, cell adhesion; 18, cell signal transmission; 19, cell tissue structure; 20, the epicyte protein functional gene factor.These gene probes stochastic distribution on chip, every kind of gene all repeats point sample twice at chip, to prevent producing inaccurate detected result because of single point sample error.
Further the oligonucleotide arrays of this gene chip is to use that photomask technology and traditional DNA synthetic chemistry are combined to be made with high-density.Oligonucleotide probe designs according to the oligonucleotide probe principle of design according to the sequence (sequence) of each gene, fills part DNA that estimation may form or the stability of RNA two strands.Oligonucleotide probe (probe) is exactly one section and goal gene or DNA complementary specificity nucleotide sequence, and it can comprise whole gene, also can only be the part of gene; Can be DNA itself, also can be to be transcribed and next RNA by it.Oligonucleotide probe is to have obtained mRNA from corresponding genetic transcription, and the probe that obtains by reverse transcription is called oligonucleotide cDNA probe (Oligo cDNA probe) again again.Oligonucleotide cDNA probe does not contain intron sequences.In addition, also can external artificial synthetic alkali radix few with gene order complementary dna fragmentation.
The specificity of the susceptibility of probe and probe sequence should be gived top priority during design, and primer must have the specificity of sufficient length, so that can combine with template and in position go up to start and synthesize.The sequence of primer and template oligonucleotide 3 ' hold or be close to 3 ' end should be complementary.End product is isometric or lack two ingot dna fragmentations of (this depends primarily on and the position of primer complementary sequence on template) slightly than template with template, in order to obtain the most effective probe as far as possible, the non-marked template strand must be separated from radiolabeled complementary product effectively, otherwise two long complementary strands will be annealed mutually, thus the hybridization of the target sequence of containment and needed testing gene.If two long-chain length are slightly different again.Then separation efficiency cannot be higher, so, should say and design of primers may be become and template template 3 ' 2-3 Nucleotide complementation of end.The designed probe sequence just should be unique, does not promptly have tumor-necrosis factor glycoproteins in template.Combine with goal gene specificity to be measured guaranteeing, can design not phase multiple probe of the same but sequence of a plurality of length, to make final data more reliable for same goal gene.The probe of known array can accurately match with their aim sequence of testing gene, can design hybridization conditions exactly, only not close with sequence non-perfectly matched sequence hybridization is then invalid for the purpose fragment of some unknown nucleotide sequences with aim sequence hybridization to guarantee probe.
OligoArray 2.0 (http://berry.engin.umich.edu/oligoarray2) is that the U.S. newly develops the computer program that is used for the oligonucleotide probe design, this program has 18 to set selection, comprise input and output document title (d, i, o, r and R), the greatest measure of oligonucleotide sequence (n), the length range of oligonucleotide probe (l and L), the temperature of fusion (t and T) of chromosomal GC content (p and P) and oligonucleotide, the threshold value (s) that the aggregated(particle) structure oligonucleotide forms is stablized in restriction, the threshold value (x) of restriction cross hybridization, the parallel hybridization quantity (N) of sequence oligonucleotide probe and testing gene sequence, two are adjoined minor increment between oligonucleotide 5 ' end (g).This program can also be judged some gene order (m) that should not be present in the sequence oligonucleotide probe, and be provided with the ultimate range (D) between receptible oligonucleotide 5 ' end and the list entries 3 ' end.After the necessary parameter of input, OligoArray 2.0 will provide gene recognition mark mark, 5 ' terminal position glycosides probe length, the hybridization free energy (kcal/mol) under the 37.C condition, enthalpy (kcal/mol), entropy (cal/kmol), the temperature of fusion Tm (C) of oligonucleotide probe and the target sequence that is used for the oligonucleotide probe of microarray.
During preparation oligonucleotide cDNA probe, at first need the separation and purification corresponding mRNA, (for example from hematopoietic cell) preparation mRNA from tissue, cell.After having had mRNA to make template, under the effect of reversed transcriptive enzyme, just can synthesize complementary DNA (being cDNA) with it.There is identical base sequence the coding region of cDNA and testing gene, but intron excises in the course of processing.The advantage of oligonucleotide cDNA probe is that it can carry out synthetic with dna synthesizer as required.Usually, no matter whether whether the length of testing gene sequence (dna fragmentation) identical consistent with molecular size, synthetic oligonucleotide cDNA probe length and molecular size should be identical, and its length is generally short than the dna fragmentation of testing gene, and with testing gene DNA complementation.For example, HY Wang etc. just is that 75 kinds of known rat genes and 10 kinds of Arabidopsis controlling genes have designed oligonucleotide chip microarray (the Genome Biol.2003 of probe sequence length identical (being 70-mer); 4 (1): R5).
All oligonucleotide probe length all are designed to 70mer among the present invention.Up-to-date work shows, the oligonucleotide cDNA probe of 70mer is mRNA level in the human body cell sensitively, and every kind of gene only needs single oligonucleotide cDNA probe just can fully monitor its expression level.Every kind of gene on this chip all is two point samples (two point).
The present invention is through the testing result of (thousands of times) in enormous quantities central lesion Gene Expression Profiles chip is carried out the computer generalization data analysis; Screening and bioinformatics integrate to be processed, and nearly thousand examples of finishing and announce according to the whole world (mainly being the U.S.) 43 different experiments chambers are screened by the experimental result of the damage brain that gathers on muroid animal (rat and mouse) the central lesion model and myeloid tissue's wide spectrum genetic chip (having 12000 to 36000 genes is arranged) and the total gene of definite central lesion (both all having found simultaneously two above different experiments chambers and reported this gene to occur behind central lesion that obviously change--expression is increased or reduced). Find and confirmed the total gene of central nervous system injury and the homologous protein thereof of 381 kinds of keys.The total gene of the central nervous system injury of these 381 kinds of keys sees Table 1, these biomolecules all are the specific biological factors that plays a crucial role in multiple central nervous system injury (cerebral apoplexy, traumatic brain injury and Spinal injury) generation evolution simultaneously, they relate separately to nerve cell death and tissue necrosis, inflammation and stress reaction, ionic equilibrium imbalance in the cell, the signal conduction, cell cycle regulating is unusual, cellularstructure unusual and neurodegeneration equivalent damage pathologic, physiologic and molecular biology activity.Utilize microarray technology that these genes are solidified and be arranged on the same chip, draw functional central nervous system injury and detect gene chip.Importance factor is meant this gene in what different experiments chambers is found simultaneously and reports in the table 1.The breadboard quantity of digitized representation (or experimental papers quantity of announcing) .The gene that " unknown function " arranged in the table 1, be because the physiological function of the homologous protein of a lot of genes still can not be affirmed at present, its which kind of effect of concrete performance is also unclear, if it changes behind central nervous system injury, can claim that also it is the central nervous system injury gene.
Through these 381 kinds total genes of damage and homologous protein thereof are carried out the computer integrated processing of biological signaling pathway and signal network, find that they interconnect and constitute the signal delivery network of regulation and control central nervous system injury reaction.This network mainly is made of 4 class signal transmission paths: 1) the dead and damaging dead signal path of neurocyte suicide; 2) inflammation and stress reaction signal path; 3) cause intracellular calcium accumulative signal path; 4) through the neuroprotective signal path of PPAR-alpha.After the central nervous system damage, initial damage signal or harmful neurotransmitter discharge a plurality of acceptors that can activate simultaneously on the primary injury point adjacent cells film, thereby activate this regulation and control central nervous system injury response regulatory signal delivery network.The transmission path of bars more than 100 was involved in the signal transmission of central nervous system injury response regulatory signal delivery network, its transfer mode is " the multi-path flow direction " formula transmission, signal can pass to several or tens of different signal paths through being positioned at the signal path node factor on certain bar path, wherein can produce reaction type or the transmission of staggered form signal between different paths again.The signal path node factor is controlled the flow direction of work damage signal and is compiled.
Utilize biochip technology to study the total gene of these neural wounds and homologous protein, relevant shared signal path and signal transmission regulating networks and the node factor in intravital Changing Pattern of central nervous system injury patient and characteristics.
The present invention is according to the functional classification gene chip of specified disease central nervous system injury design, has special advantages.The biomedical researcher only need pay close attention to own interested and closely-related high specific genes of specified disease and biological signaling pathway, makes research work more simple, rapid and effective.Research that this is new and diagnostic detection instrument certainly will be accelerated the speed and the process of life science.
The functional central nervous system injury diagnostic detection gene chip manufacture method of the present invention comprises that the structure of DNA square formation, original position are synthesized, the point sample of synthetic back probe.
1.DNA the structure of square formation: adopt the method for surface chemistry or the method for combinatorial chemistry to handle carrier-pellet base (sheet glass or film) when preparing oligonucleotide chip earlier, the specific biological factor that will play a crucial role in multiple central nervous system injury generation evolution simultaneously relates separately to nerve cell death and tissue necrosis then, inflammation and stress reaction, ionic equilibrium imbalance in the cell, the signal conduction, cell cycle regulating is unusual, cellularstructure is unusual, the oligonucleotide probe random alignment of neurodegeneration damage pathologic, physiologic and molecular biology active gene is on chip.Glass chip will be selected the DNA-Ready Type II Slide of Clontech for use, and this slide not only can provide nucleic acid binding ability efficiently, can make the crossed contamination between points of probe be reduced to minimum level simultaneously.And film base combines many advantages of slide sheet base and nylon membrane: allow wash-out probe after hybridization as nylon membrane, same chip can be hybridized repeatedly repeatedly; Simultaneously allow the high-density point sample as slide again, each is put all uniformity and separates good; Because the surface of film does not resemble porous the nylon membrane, has significantly reduced non-special absorption, need not after the hybridization to wash repeatedly and just can access clean background, the assurance good signal-to-noise; Film has certain rigid, also can not curl when high-temperature wash, and repeatedly the hybridization of wash-out probe-repeatedly can be out of shape yet, not only easy handling but also be convenient to scan reading and interpretation of result.In order to guarantee the reliable of signal, each gene on the film all is two point (soon every kind of gene in the table 1 repeats a secondary by the principle of random alignment on film).
2. original position is synthetic: original position is synthetic to be to make the method that high-density Oligo chip generally uses at present, and the present invention mainly adopts original position photoetching route of synthesis to make the probe of oligonucleotide (Oligo) gene chip.Original position photoetching synthetic technology utilizes method, photosensitive protective group and the photoetching technique of solid state chemistry to realize that accurately base location and fragment are synthetic.Its know-why is to connect a photosensitive protective group at the monomeric 5 ' C-terminal of synthetic base.The synthetic the first step is to utilize rayed to make the hydroxyl terminal deprotection, and the nucleotide monomer with one 5 ' end protection connects up then, and this process carries out finishing until synthetic repeatedly.Use multiple cloak to produce highdensity array, be exponential growth at synthesis cycle middle probe number with synthesis step still less.The a certain oligonucleotide that contains N Nucleotide, can synthesize 4N by 4N chemical step may structure.For example: ten complete nucleic acid may synthesize 65,536 probes in 8 hours by 32 chemical steps.The probe density of original position photoetching synthetic method preparation is very high, and every square centimeter can have 400,000 different probe prefectures.The original position photoetching is synthetic except the density height, also has the advantage of pinpoint accuracy.Press gene listed in the table 1 and press the oligonucleotide probe of the synthetic required sequence of in-situ synthesis.
3. point sample: synthetic back point sample or crosslinked principle are simple, utilize automatic spot sample device that the Oligo cDNA probe for preparing in advance in the step 2 is put on the carrier-pellet base of special processing by specific high speed point model machine is direct.Wherein the surface treatment of carrier-pellet base is very important.The crosslinked covalent manner that then mainly adopts of oligonucleotide.Need in above-mentioned steps 2 (when oligonucleotide synthesizes), introduce and participate in crosslinked activating group (different amino-acid residues (tRNA)).The point model machine that adopts has the three-dimensional running gear of a cover computer control; A plurality of printing/jet-printing heads; A damping base, porous plate and a plurality of chip of Sheng probe in can putting above.Printing/spray printing pin takes out directly printing or spray printing with probe on chip from porous plate.Connect really that syringe needle contacts with chip when printing, and when spray printing syringe needle and chip maintenance certain distance.The advantage of impact system is the probe density height, common 1 square centimeter printable 2,500 probes.Behind the point sample chip pressed the different of probe and substrate combination, take appropriate means to be cured.Use immediately then or be stored in 4 ℃ standby.
The cardinal principle of Oligo gene chip is to hybridize by the base complementrity pair principle, detects whether homologous segment exists and what of amount.The essential distinction of it and cDNA chip is that Oligo chip fixed probe is specific DNA Oligo fragment (probe), and the cDNA chip then is cDNA.Two important parameters of gene chip are sensitivity and the specificitys that detects.The Tm value is different, and numerous genes is hybridized on same chip because gene length is different for the cDNA chip, and it is same to make that hybridization conditions is difficult to, and makes the resolving power of traditional cDNA chip be restricted.And the Oligo chip sequence is selected through optimizing, and utilizes the Oligo single-stranded probe of synthetic certain-length (about 70-mer) to replace the full-length cDNA point sample, is made into chip.
The Oligo gene chip is upward to press the synthetic fixedly Oligo probe that reaches of array original position at sheet glass or film (Plastic film).It uses the combination of a kind of photomask technology and traditional DNA synthetic chemistry to make the Oligo array with very high density.General Oligo probe length is generally 70-mer, may have the non-specific cross hybridization between the Oligo when detecting the mRNA abundance, and this may cover hybridization signal; Terms of settlement is to adopt the right method of coupling/mismatch (PM/MM) probe, promptly when a special Oligo of design (coupling), design a non-specific Oligo probe simultaneously, this probe only is equipped with a base in interposition and replaces (mismatch), like this can be with the difference between PM and the MM as strength of signal.In addition, for specific Oligo, strength of signal is for the based composition of Oligo relatively more responsive.In order to address this problem, when designing probe, comprise a plurality of Oligo probes for each mRNA to be detected, for example design many to (as design 11-20 to) probe detects a transcript.Different with the cDNA microarray is, in the hybrid experiment with the Oligo chip hybridization be single sample, rather than measure the mixture of sample and check sample in the experiment of cDNA microarray.
The optimization design of Oligo probe is one step of key of making the Oligo chip.The result that can obtain very soon with the Oligo design chips of optimizing, and during with inappropriate Oligo, often draw specious result.Experimental results show that the long Oligo of 70-mer is best suited for chip hybridization,, keep very high gene specific simultaneously because the design of 70-mer Oligo probe can obtain enough high sensitivity.And the hybridization conditions that the Oligo that uses homogeneous length can make gene homogeneous more.Because the single strand dna and the strand cDNA probe that are fixed on the chip do not have competitive inhibition (such as double-stranded DNA) when hybridization, can make the sensitivity of detection higher, background is lower.The problem that should note in the optimization design of Oligo probe is: 1. the estimation DNA that may form or the stability of RNA two strands.No matter be the Oligo of DNA or RNA, the potential possibility that forms duplex structure is all arranged, this structure has a significant impact the effect of Oligo.So, predict that this stability of structure is just very important to design and optimization Oligo; 2. should make when selecting primer in 3 ' the terminal and primer molecule of primer and not produce complementation, note component, melting temperature(Tm) and length and the internal stability of maintenance primer and the uniqueness of primer of primer; 3. according to aminoacid sequence design Oligo the time, the problem that note is would rather be with simple primer (oligonucleotide), the also primer that need not guess, and prevent the mispairing of primer and template.With the uniqueness primer time, with the following total nucleotide concentration of 0.2mmol/L, in case high nucleotide concentration increases the wrong ratio that participates in.When degeneracy Oligo, PCR should carry out (1～3mol/L rather than 0.2mol/L) under than higher primer concentration, because the most of oligomers in reaction mixture are not to be used to cause single-minded reaction, and just produces high background.
The functional central nervous system injury diagnostic detection of the present invention with the application method of gene chip is:
1, sampling: extract mRNA sample blood, tissue or the cell on one's body from patient;
2, sample process: will become cDNA on one's body from the mRNA sample reverse transcription that patient takes also with fluorescein-labelled;
3, hybridization: then the mark mixture is added to functional central nervous system injury diagnostic detection with on the gene chip Oligo cDNA chip, hybridizes, after crossover process is finished, clean microarray with probe;
4, the detection of hybridization collection of illustrative plates and reading: with laser scanner scans and after obtaining fluoroscopic image, image is analyzed, to obtain the fluorescence intensity level of each point on the Oligo chip;
5, interpretation of result: the fluorescence intensity level quantitative response of fluoroscopic image exist in the sample with probe complementary mRNA abundance, just reflected the corresponding expression of gene level of probe.The ratio of the fluorescence intensity of the fluorescence intensity of sample to be detected and normal control sample is represented abundance or the expression level of the mRNA of sample to be detected.When ratio is more than 2, represent that this genetic expression obviously increases; If ratio below (1 ÷ fluorescence intensity value (such as being 0.5,0.45 or 0.25 etc.)), represents that this genetic expression obviously reduces for-2.These variations show that central nervous system injury causes these and the total gene of the closely-related wound of damage to take place obviously to change.If the total gene fluorescence intensity ratio of certain wound be between+2 to+3.99, represent that this gene slightly increases, ratio be between+4 to+5.99 for moderate increases, ratio is for to increase for severe more than+6; Equally, if the total gene fluorescence intensity ratio of certain wound is between-2 to-3.99, represent that this gene slightly reduces, ratio is to reduce for moderate between-4 to-5.99, ratio is for (increasing and reducing of individual gene might not influence the severity of the state of an illness for severe reduces more than-6.The severity of the state of an illness is relevant with the gene dosage that changes, and quantity is many more, and the state of an illness is heavy more).
Functional central nervous system injury diagnostic detection among the present invention is with having the total gene of 381 kinds of central nervous system injurys on the gene chip.The diagnosable central nervous system minor injury that is if the gene below 100 changes; The diagnosable moderate lesion that is if 100-200 gene changes; The diagnosable severe injury that is if 200-300 gene changes; If it is major injury or life-threatening damage that all genes all change diagnosable.The treatment plan that draws thus should be arranged according to the known physiological function and the expression level of change gene, just take anti-inflammatory therapy as inflammatory reaction, the trauma stress reaction just takes to reduce the measure of stress reaction, apoptosis just takes to prevent the treatment measure of necrocytosis, and anti-apoptotic is then taked treatment measure that strengthens anti-apoptotic etc.Just take to reduce its active treatment measure if its expression level increases, just take to strengthen its active treatment measure if its expression level reduces.
The functional central nervous system injury diagnostic detection of the present invention can be used for detecting central nervous system injuries such as cerebral apoplexy, traumatic brain injury and cervical spinal cord damage, chest and pars lumbalis medullae spinalis damage with gene chip.
Oligo chip of the present invention is compared with traditional cDNA chip, and following advantage is arranged:
1. sequence reduces non-specific hybridization through optimizing, and can effectively distinguish the gene that homologous sequence is arranged;
2. minimizing secondary structure;
3. the hybridization temperature homogeneous improves hybridization efficiency;
4. synthetic product concentration homogeneous is avoided causing point sample amount difference because of sample concentration difference;
5. need not amplification, prevent amplification failure influence experiment.
Embodiment
The present invention adopts original position photoetching route of synthesis to make the Oligo gene chip, connects a photosensitive protective group at the monomeric 5 ' C-terminal of synthetic base.The synthetic the first step is to utilize rayed to make the hydroxyl terminal deprotection, and the nucleotide monomer with one 5 ' end protection connects up then, and this process carries out finishing until synthetic repeatedly.Press listed 381 kinds of genes in the table 1, after synthetic 381 kinds utilize at a high speed full-automatic microarray point model machine to press designed microarray matrix point sample to the DNA-Ready Type II Slide slide of U.S. Clontech company production with closely-related specific gene probe of central nervous system injury and negative control physical prospecting pin (DNA Oligo fragment).Slide behind the point sample can preserved about a week under 4 ℃ of conditions.Extract the mRNA sample in patients serum, cerebrospinal fluid or the damaged tissue sample, reverse transcription becomes cDNA and uses fluorescein (Cy5 or Cy3) mark; Then the mark mixture is added on the Oligo gene chip, hybridizes, after crossover process is finished, clean microarray with probe.With laser scanner scans and after obtaining fluoroscopic image, image is analyzed, to obtain the fluorescence intensity level of each point on the Oligo chip.The fluorescence intensity level quantitative response exist in the sample with probe complementary mRNA abundance, just reflected the corresponding expression of gene level of probe.Below be non-limiting qualification example of the present invention:
Embodiment 1: functional central nervous system injury diagnostic detection manufacturing with gene chip
The structure of a.DNA square formation: with in the table 1 listed 381 kinds with the closely-related specific gene of central nervous system injury and negative control thing (bacterium contrast probe or human house-keeping gene (Housekeeping Genes)) by their functional classification random alignment (on good grounds some model machine in the COMPUTER DETECTION program of chip Oligo probe random alignment order and the list of 381 kinds of gene probes producing) on the DNA-ReadyType II Slide slide that U.S. Clontech company produces.This slide method of combinatorial chemistry is handled, and nucleic acid binding ability efficiently not only can be provided, and also farthest prevents crossed contamination between points simultaneously.
B. probe design and original position are synthetic: according to two important parameters of gene chip: the sensitivity of detection and specificity, mainly adopt original position photoetching route of synthesis to make the Oligo gene chip.Original position photoetching synthetic technology utilizes method, photosensitive protective group and the photoetching technique of solid state chemistry to realize that accurately base location and fragment are synthetic.Its know-why is to connect a photosensitive protective group at the monomeric 5 ' C-terminal of synthetic base.The synthetic the first step is to utilize rayed to make the hydroxyl terminal deprotection, and the nucleotide monomer with one 5 ' end protection connects up then, and this process carries out finishing until synthetic repeatedly.Use multiple cloak to produce highdensity array, be exponential growth at synthesis cycle middle probe number with synthesis step still less.Oligo single-stranded probe length after synthetic is 70-mer.The long Oligo of 70-mer is best suited for chip hybridization, because the design of 70-mer Oligo probe can obtain enough high sensitivity, keeps very high gene specific simultaneously.
C. point sample: will synthesize the Oligo probe point sample of getting well to DNA-Ready Type II Slide carrier-pellet base by designed microarray matrix with at a high speed full-automatic microarray point model machine.The point model machine has the three-dimensional running gear of a cover computer control, a plurality of printing/jet-printing heads and a damping base, porous plate and a plurality of chip of Sheng probe in can putting above.Printing/spray printing pin takes out directly printing or spray printing with probe on chip from porous plate.
Embodiment 2: the application of gene chip of functional central nervous system injury diagnostic detection
A.RNA extracts: with the RNA in Trizol reagent, chloroform, Virahol and 75% alcoholic extraction patients serum, cerebrospinal fluid or the damaged tissue, through repeatedly centrifugal, filter, under 4 ℃ of conditions, preserve the RNA that obtains stand-by after the washing.
B. reverse transcription becomes cDNA: with RNA and amine-modified random primer (as 2-hydrogen base VITAMIN B4), Rnase inhibitor and aa-dUTP/dNTPs, DTT and SSII Reverse Transcriptase hybrid reaction after about 2 hours, with EDTA (pH8.0) termination reaction, use NaOH hydrolysis RNA then.Adopt MinElute Filter column produced in USA to extract reverse transcription cDNA, after centrifugal, filtration repeatedly, washing and drying, obtain the cDNA of purification.
C. fluorescent labelling: sample cDNA is carried out fluorescent marker (Cy5 or Cy3) mark with Qia-quick Filter column produced in USA.
D. hybridization: the good cDNA of mark is after poly (dA), Mouse Cot-1 DNA and Yeast tRNA blocking-up are solidified, under 98 ℃ of conditions, make its sex change, be added drop-wise on the Oligo gene chip after mixing with the F-Hyb buffer reagent (50%formamide, 10X SSC and 0.2%SDS) of fresh configuration then.Hybridize in the hybridization case that Oligo gene chip behind the application of sample is 42 ℃ spend the night (16-24 hour).Use then cleaning buffer solution 1 (2X SSC, 0.1%SDS), cleaning buffer solution 2 (0.5X SSC, 0.01%SDS) and cleaning buffer solution 3 (0.06X SSC) clean chip respectively.
E. chip scanning and image data are handled: with being mounted with GenePix Pro 3 Computer Analysis software GenePi4000A scanner scanning chips and obtaining fluoroscopic image.Subsequently image data is carried out quantitative computer analysis and processing.
Use the functional diagnostic detection of the present invention to be with the standard of gene chip diagnosis central nervous system injury:
1. have the gene below 100 kinds (the fluorescence intensity ratio is for more than+2) to take place obviously to increase and/or obviously reduce (the fluorescence intensity ratio is for below-2) in the total gene of 381 kinds of wounds of sum, diagnosable is slight central nervous system injury;
2. have the gene of 101-200 kind that (the fluorescence intensity ratio is for more than+2) and/or obvious reduction the (the fluorescence intensity ratio is for below-2) take place obviously to increase in the total gene of 381 kinds of wounds of sum, diagnosable is the moderate central nervous system injury;
3. have the gene of 201-300 kind that (the fluorescence intensity ratio is for more than+2) and/or obvious reduction the (the fluorescence intensity ratio is for below-2) take place obviously to increase in the total gene of 381 kinds of wounds of sum, diagnosable is the severe central nervous system injury;
4. have the gene of 301-381 kind that (the fluorescence intensity ratio is for more than+2) and/or obvious reduction the (the fluorescence intensity ratio is for below-2) take place obviously to increase in the total gene of 381 kinds of wounds of sum, diagnosable is nervus centralis major injury and life-threatening damage.
Below be the functional central nervous system injury diagnostic detection of the present invention is used for the clinical detection patient with gene chip concrete case:
1. cerebral apoplexy case, traumatic brain injury case, and the case of cervical spinal cord damage (brain injury and the damage of neck marrow can cause breathing the serious hindrance with heart function usually, and then life-threatening):
The gene chip detected result of certain patient's first is, have 99 kinds of genes that (the fluorescence intensity ratio is for more than+2) and/or obvious reduction the (the fluorescence intensity ratio is for below-2) take place obviously to increase in the total gene of 381 kinds of wounds of sum, this patient is diagnosable to be slight central nervous system injury.According to the function of these 99 kinds of genes such as signal transmission, trauma stress reaction, wound early response, anti-apoptotic, redox reaction, inflammatory reaction, growth regulating, neurodegeneration, neural defence protection, ion path and ionic equilibrium adjusting, intracellular transport, apoptosis, cell receptor, cell physiological metabolism, cell growth circulation, cell adhesion, cell tissue structure, epicyte protein, cell transcription regulate, albumen is synthetic, peroxidation stress reaction etc., what genes calculate has change in each functional classification.According to the number gene that changes in each functional classification, determine it mainly is that obvious change has taken place which genoid, should carry out the treatment of which aspect.For example, in 99 genes that this patient has taken place obviously to change, there are 23 genes to belong to the trauma stress response class, 18 genes belong to ion path and ionic equilibrium adjusting class, 16 genes belong to the inflammatory reaction class, 12 genes belong to the apoptosis class, 10 genes belong to signal and transmit class, 8 genes belong to the redox reaction class, 7 genes belong to the cell receptor class, 5 genes belong to peroxidation stress reaction class, show that then the major cause of slight central nervous system injury takes place this patient is too high trauma stress reaction, ion path and ionic equilibrium imbalance, inflammatory reaction strengthens, cell generation apoptosis and signal conductive obstruction etc. should be taked corresponding symptomatic treatment measure.
Another patient's second: the gene chip detected result is, 150 kinds of total genes of wounds take place obviously to increase and/or obviously reduce, and this patient is diagnosable to be the moderate central nervous system injury.According to the functional classification of 150 kinds of genes that changed, what genes measure has change in each functional classification again.According to the number gene that changes in each functional classification, determine it mainly is that obvious change has taken place which function class gene, take corresponding symptomatic treatment measure.
The total gene of the third: 280 kind of wound of patient takes place obviously to increase and/or obviously reduce, and the central nervous system injury degree that shows this patient is a severe injury.According to the functional classification of 280 kinds of genes that changed, what genes measure has change in each functional classification again.According to the number gene that changes in each functional classification, determine it mainly is that obvious change has taken place which function class gene, take corresponding symptomatic treatment measure.
Patient's fourth: the gene chip detected result is that the total gene of 360 kinds of wounds takes place obviously to increase and/or obviously reduce, and this patient is diagnosable to be nervus centralis major injury and life-threatening damage.According to the functional classification of 360 kinds of genes that changed, what genes measure has change in each functional classification again.According to the number gene that changes in each functional classification, determine it mainly is that obvious change has taken place which function class gene, take corresponding symptomatic treatment measure immediately.
Chest and pars lumbalis medullae spinalis the damage case:
The gene chip detected result of certain chest or pars lumbalis medullae spinalis damage patient A is, have 80 kinds of genes that (the fluorescence intensity ratio is for more than+2) and/or obvious reduction the (the fluorescence intensity ratio is for below-2) take place obviously to increase in the total gene of 381 kinds of wounds of sum, this patient is diagnosable to be slight Spinal injury.According to the functional classification of 80 kinds of genes that changed, what genes measure has change in each functional classification.According to the number gene that changes in each functional classification, determine it mainly is that obvious change has taken place which function class gene, take corresponding symptomatic treatment measure.
Certain chest or pars lumbalis medullae spinalis damage patient B gene chip detected result are that the total gene of 180 kinds of wounds takes place obviously to increase and/or obviously reduce, and this patient is diagnosable to be moderate Spinal injury.According to the functional classification of 180 kinds of genes that changed, what genes measure has change in each functional classification.According to the number gene that changes in each functional classification, determine it mainly is that obvious change has taken place which function class gene, take corresponding symptomatic treatment measure.
Certain thoracic spinal cord damage patient C gene chip detected result is that the total gene of 250 kinds of wounds takes place obviously to increase and/or obviously reduce, and this patient is diagnosable to be severe Spinal injury.According to the functional classification of 250 kinds of genes that changed, what genes measure has change in each functional classification.According to the number gene that changes in each functional classification, determine it mainly is that obvious change has taken place which function class gene, take corresponding symptomatic treatment measure.
Certain thoracic spinal cord damage patient D gene chip detected result is that the total gene of 370 kinds of wounds takes place obviously to increase and/or obviously reduce, and then is diagnosed as the spinal cord major injury.According to the functional classification of 370 kinds of genes that changed, what genes measure has change in each functional classification.According to the number gene that changes in each functional classification, determine it mainly is that obvious change has taken place which function class gene, take corresponding symptomatic treatment measure immediately.
Table 1
| Sequence number | Gene is called for short | Gene full name | Function | Importance factor | Gene pool number |
| 1. | c-fos | c-fos | The wound early response factor | 18 | Mm.297978 |
| 2. | Junb | Jun | Cell transcription is regulated | 15 | Mm.275071 |
| 3. | MT1 | Metallothionein 1 | The cell physiological metabolism | 15 | Mm.192991 |
| 4. | Gfap | Glial fibriIlary acidic protein | The cell tissue structure | 14 | Mm.1239 |
| 5. | Hsp70 | Heat shock protein，70kDa | The trauma stress reaction | 13 | Mm.1777 |
| 6. | TNFa | Tumor necrosis factor-alpha | The inflammatory reaction factor | 12 | Mm.3509 |
| 7. | Vim | Vimentin | The cell tissue structure | 12 | Mm.268000 |
| 8. | IL-1b | Interleukin 1，beta | The inflammatory reaction factor | 11 | Mm.222830 |
| 9. | Hsp27 | Heat shock protein，27kDa | The trauma stress reaction | 11 | Mm.21549 |
| 10. | Nfl | Neurofilament，light polypeptide | The cell tissue structure | 11 | Mm.1956 |
| 11. | MT2 | Metallothionein 2 | The cell physiological metabolism | 11 | Mm.147226 |
| 12. | Bdnf | Brain derived neurotrophic factor | Cell growth regulator | 11 | Mm.1442 |
| 13. | Nes | Nestin | The cell tissue structure | 10 | Mm.331129 |
| 14. | Mtap2 | Microtubule-associated protein 2 | The cell tissue structure | 10 | Mm.256966 |
| 15. | TGF | Trahsforming growth factor，beta 1 | Growth regulator ion path and ionic equilibrium | 10 | Mm.248380 |
| 16. | PCNP | Potassium channel protein | Regulate ion path and ionic equilibrium | 10 | Mm.242532 |
| 17. | SCN1 | Sodium channel 1 | Regulate | 10 | Mm.182944 |
| 18. | Egr1 | Early growth response 1 | Cell transcription is regulated | 10 | Mm.181959 |
| 19. | Irf1 | Interferon regulatory factor 1 Calcium/calmodulin dependent | Cell transcription is regulated | 10 | Mm.105218 |
| 20. | CaMK2b | protein kiBase II beta | Cell signal transmits | 9 | Mm.4857 |
| 21. | Selp | P-selectin | The inflammatory reaction factor | 9 | Mm.332590 |
| 22. | HO1 | Heme oxygenase(decycling)1 | The cell physiological metabolism | 9 | Mm.276389 |
| 23. | IL-6 | Interleukin 6 | The inflammatory reaction factor | 9 | Mm.1019 |
| 24. | Cpg21 | MAP kinase phosphatase(cpg21) | Cell signal transmits | 8 | Rn.100548 |
| 25. | Icam1 | Intercellular adhesion molecule 1 | Epicyte protein | 8 | Mm.90364 |
| 26. | Casp3 | Caspase 3 | Apoptosis | 8 | Mm.34405 |
| 27. | PKCa | Protein kinase C，alpha Immediate early gene transcription | Cell signal transmits | 8 | Mm.222178 |
| 28. | NGFI-B | factor NGF1-B | Cell transcription is regulated | 7 | Rn.10000 |
| 29. | GST | Glutathione S-transferase | Neural defence protection | 7 | Mm.37199 |
| 30. | PKCg | Protein KiBase C，gamma | Cell signal transmits | 7 | Mm.368265 |
| 31. | SOCS3 | Suppressor of cytokine signaling 3 | Cell growth circulation | 7 | Mm.3468 |
| 32. | Cox2 | Cyclooxygenase 2 | The trauma stress reaction | 7 | Mm.292547 |
| 33. | Sod2 | Mn superoxide dismutase | Redox reaction | 7 | Mm.290876 |
| 34. | INOS | Incucible nitrjc oxide synthase | The peroxidation stress reaction | 7 | Mm.2894 |
| 35. | SFB | Silencer factor B Growth arrest and DNA damage | Cell transcription is regulated | 7 | Mm.28864 |
| 36. | Gadd45g | inducible 45 gamma | The wound early response factor | 7 | Mm.281298 |
| 37. | SYN2 | Synapsin 2 | Cell signal transmits | 7 | Mm.275845 |
| 38. | c-Jun | c-Jun | Wound early response factor ion path and ionic equilibrium | 7 | Mm.275071 |
| 39. | Kcnk1 | Potassium channel TWIK | Regulate | 7 | Mm.24877 |
| 40. | Hsp60 | Heat shock protein，60 kDa | The trauma stress reaction | 7 | Mm.1777 |
| 41. | Fas | Fas | The inflammatory reaction factor | 7 | Mm.1626 |
| 42. | VEGF | Vascular endothelial growth factor | Cell growth regulator | 7 | Mm.15607 |
| 43. | Npy | Neuropeptide Y | Cell signal transmits | 7 | Mm.154796 |
| 44. | TNFR | Tumor necrosis factor receptor | Cell receptor | 7 | Mm.1258 |
| 45. | BAX | Bcl-2 associated X protein | Apoptosis | 6 | Mm.84073 |
| 46. | Syt11 | Synaptotagmin II Synaptosome-associated protein | The intracellular transport factor | 6 | Mm.5102 |
| 47. | NAP25a | 25A | Epicyte protein | 6 | Mm.45953 |
| 48. | NGF | Nerve growth factor Complement component 1，q | Cell growth regulator | 6 | Mm.378974 |
| 49. | C1QA | subcomponent，alpha polypeptide Calcium-ATPase，isoform 2，plasma | Trauma stress reactive ion path and ionic equilibrium | 6 | Mm.370 |
| 50. | ATP2B2 | membrane Major intrinsic protein of eye lens | Regulate | 6 | Mm.321755 |
| 51. | Mip | fiber 1 | The intracellular transport factor | 6 | Mm.31625 |
| 52. | APOE | Apolipoprotein E | The cell physiological metabolism | 6 | Mm.305152 |
| 53. | Rgs2 | Regulator of G-protein signaling 2 | Cell signal transmits | 6 | Mm.28262 |
| 54. | HMOX | Heme oxygenase | The trauma stress reaction | 6 | Mm.276389 |
| 55. | IGF1 | Insulin-like growth factor 1 | Cell growth regulator | 6 | Mm.268521 |
| 56. | Pafah1b3 | Platelet derived growth factor | Cell growth circulation | 6 | Mm.2675 |
| 57. | FGFR1 | Fibroblast growth factor receptor 1 | Cell signal transmits | 6 | Mm.265716 |
| 58. | PKCb | Protein Kinase C，beta Calcium channel， | Cell signal transmits ion path and ionic equilibrium | 6 | Mm.252776 |
| 59. | Cacnalc | voltage-dependent-L type | Regulate ion path and ionic equilibrium | 6 | Mm.236039 |
| 60. | ATP1A2 | Sodium/potassium-ATPase，alpha 2 | Regulate | 6 | Mm.207432 |
| 61. | IL-1a | Interleukin 1，alpha Signal transducer and activation of | The inflammatory reaction factor | 6 | Mm.15534 |
| 62. | Stat3 | transcription 3 | Cell transcription is regulated | 6 | Mm.1550 |
| 63. | tPA | Tissue plasminogen activator Calcium/calmodulin dependent | The cell physiological metabolism | 6 | Mm.154660 |
| 64. | AMK2A | protein kinase II，alpha Nuclear factor of kappa light | Cell signal transmits | 6 | Mm.131530 |
| 65. | NF-kB | polypeptide gene enhancer in B-cells Synaptosome-associated protein | Cell transcription is regulated | 6 | Mm.102365 |
| 66. | SNAP25b | 25B Activity and neurotransmitter-induced early gene | Epicyte protein | 6 | Hs.167317 |
| 67. | Ania4 | protein 4 | The wound early response factor | 5 | Rn.80575 |
| 68. | CTSL | Cathepsin L Tissue inhibitor of metalloproteinase | Albumen is synthetic | 5 | Mm.930 |
| 69. | TIMP1 | 1 | The cell tissue structure | 5 | Mm.8245 |
| 70. | CREMdel | cAMP responsive element modulator | Cell transcription is regulated | 5 | Mm.5244 |
| taC-G | |||||
| 71. | GAL | Galanin | Cell signal transmits | 5 | Mm.4655 |
| 72. | SNAP25 | Synaptosomal-associated protein 25 | Cell signal transmits | 5 | Mm.45953 |
| 73. | nNOS | Neuronal nitric oxide synthase | The cell physiological metabolism | 5 | Mm.44249 |
| 74. | PDE | Phosphodiesterase | The cell physiological metabolism | 5 | Mm.40678 |
| 75. | HIF1 | Hypoxia-inducible factor 1 | Cell transcription is regulated | 5 | Mm.3879 |
| 76. | mGluR3 | Metabotropic glutamate receptor 3 Homerl，neuronal immediate early | Cell receptor | 5 | Mm.381853 |
| 77. | Homerlc | gene，1 | Cell signal transmits | 5 | Mm.37533 |
| 78. | Ctss | Cathepsin S CCAAT/enhancer binding protein | The cell physiological metabolism | 5 | Mm.3619 |
| 79. | Cebpb | (C/EBP)，beta Eukarvotic translation elongation | The inflammatory reaction factor | 5 | Mm.347407 |
| 80. | EEF1A1 | factor 1，alpha 1 | Unknown Function | 5 | Mm.335315 |
| 81. | SMNF | Survival motor neuron factor | Cell transcription is regulated | 5 | Mm.313687 |
| 82. | p38 | p38 MAP kinase Calcium/calmodulin dependent | Cell signal transmits | 5 | Mm.311337 |
| 83. | AMK1A | protein kinase I，belta 2 | Cell signal transmits | 5 | Mm.289237 |
| 84. | SOD | Cu-Zn superoxide dismutase | Unknown Function | 5 | Mm.276325 |
| 85. | Hsp105 | Heat shock protein，105kDa Activity regulated | The trauma stress reaction | 5 | Mm.270681 |
| 86. | Arc | cytoskeletal-associated protein | The cell tissue structure | 5 | Mm.25405 |
| 87. | nAChR | nACh receptor | Cell receptor ion path and ionic equilibrium | 5 | Mm.247042 |
| 88. | ATP1B2 | Sodium/potassium-ATPase，beta 2 Calcium/calmodulin dependent | Regulate | 5 | Mm.235204 |
| 89. | Camk4 | protein kinase IV | Cell signal transmits | 5 | Mm.222329 |
| 90. | TTR | Transthyretin | The peroxidation stress reaction | 5 | Mm.2108 |
| 91. | TXNRD1 | Thioredoxin reductase | Redox reaction | 5 | Mm.210155 |
| 92. | Slc2a1， Glut1 | Glucose transporter 1 | Ion path and ionic equilibrium are regulated | 5 | Mm.21002 |
| 93. | E2F1 | E2F transcription factor 1 Insulin-like growth factor binding | Cell growth circulation | 5 | Mm.18036 |
| 94. | IGFBP2 | protein 2 | Apoptosis | 5 | Mm.141936 |
| 95. | Rab3 | Ras-related rab3 | Cell signal transmits | 4 | Rn.44409 |
| 96. | Hsp70-2 | Heat shock protein，70kDa 2 | The trauma stress reaction | 4 | Rn.4297 |
| 97. | Lrfn3 | Cell adhesion-like molecule | Epicyte protein | 4 | Rn.11366 |
| 98. | GMFB | Glia maturation factor，beta | Unknown Function | 4 | Rn.10454 |
| 99. | GABTP | GABA transporter protein Transforming growth factor beta | Cell receptor | 4 | Rn.10035 |
| 100. | TGFB | inducible early growth response | The wound early response factor | 4 | Mm.9616 |
| 101. | Lcn2 | Lipocalin 2 | Anti-apoptotic | 4 | Mm.9537 |
| 102. | Ccl2 | chemokine(C-C motif)ligand 2 | The peroxidation stress reaction | 4 | Mm.6272 |
| 103. | Fra-1 | Fos-related antigen 1 | Cell transcription is regulated | 4 | Mm.6215 |
| 104. | DCN | Decorin | The cell tissue structure | 4 | Mm.56769 |
| 105. | HTR1c | 5-HT receptor 1c | Cell receptor | 4 | Mm.4716 |
| Glial cell line-derived neurotrophic | |||||
| 106. | Gdnf | factor | Anti-apoptotic | 4 | Mm.4679 |
| 107. | Egr4 | Early growth response 4 | Cell transcription is regulated | 4 | Mm.44137 |
| 108. | Stk25 | Serine/threonine kinase | Cell signal transmits | 4 | Mm.42126 |
| 109. | MBP | Myelin basic protein | Epicyte protein | 4 | Mm.40461 |
| 110. | PLC | Phospholipase C | Cell signal transmits | 4 | Mm.38009 |
| 111. | COMT | Catechol-O-methyltransferase | The cell physiological metabolism | 4 | Mm.379165 |
| 112. | c-Myc | c-Myc | Cell transcription is regulated | 4 | Mm.347916 |
| 113. | TUBA1 | Tubulin alpha 1 | The cell tissue structure | 4 | Mm.339945 |
| 114. | ABAR | GABA(A)receptor | Cell receptor | 4 | Mm.338713 |
| 115. | Casp8 | Caspase 8 | Apoptosis | 4 | Mm.336851 |
| 116. | CCND2 | Cyclin D2 | Cell growth circulation | 4 | Mm.333406 |
| 117. | GSTYcS MC | Glutathione S-transferase Yc subunit，Mu class | The peroxidation stress reaction | 4 | Mm.304763 |
| 118. | Rps6 | Ribosomal protein S6 | Albumen is synthetic | 4 | Mm.301827 |
| 119. | p53 | p53 | Cell growth circulation ion path and ionic equilibrium | 4 | Mm.293605 |
| 120. | TP1B1 | Sodium/potassium-ATPase，beta 1 | Regulate | 4 | Mm.290083 |
| 121. | Jak1 | Janus kinase 1 | Cell signal transmits | 4 | Mm.289657 |
| 122. | Hbb-b1 | Hemoglobin，beta adult major chain | The intracellular transport factor | 4 | Mm.288567 |
| 123. | SPP1 | Osteopontin | The trauma stress reaction | 4 | Mm.288474 |
| 124. | CALM1 | Calmodulin 1 | Cell signal transmits | 4 | Mm.285993 |
| 125. | Il6r | Interleukin 6 receptor | Cell signal transmits | 4 | Mm.2856 |
| 126. | Plod | Procollagen | Cell adhesion | 4 | Mm.277792 |
| 127. | Mkk2 | MAPK kinase 2 | Cell signal transmits | 4 | Mm.275436 |
| 128. | SV2B | Synaptic vesicle protein 2B Fibroblast growth factor receptor 1， | Cell signal transmits | 4 | Mm.273082 |
| 129. | FGFR1b | beta isoform Eukaryotic translation initiation | Cell signal transmits | 4 | Mm.265716 |
| 130. | EIF4A2 | factor 4，alpha 2 | The cell physiological metabolism | 4 | Mm.260256 |
| 131. | eNos | Endothelial nitric oxide synthase Nuclear factor of kappa light polypeptide gene enhancer in | The trauma stress reaction | 4 | Mm.258415 |
| 132. | Nfkb1 | B-cells，p105 | Cell transcription is regulated | 4 | Mm.256765 |
| 133. | Fosl2 | Fos-related antigen 2 | Cell transcription is regulated | 4 | Mm.24684 |
| 134. | Bcl-XL | Bcl-XL | Apoptosis | 4 | Mm.238213 |
| 135. | APP | Amyloid precursor protein | Neurodegeneration | 4 | Mm.237670 |
| 136. | PON1 | Malondialdehyde | The cell physiological metabolism | 4 | Mm.237657 |
| 137. | CTSD | Cathepsin D | Albumen is synthetic | 4 | Mm.231395 |
| 138. | HTR2 | 5-HT receptor 2 Insulin-like growth factor binding | Cell receptor | 4 | Mm.214351 |
| 139. | IGFBP1 | protein 1 | Cell growth regulator | 4 | Mm.21300 |
| 140. | CTSB | Cathepsin B | Apoptosis | 4 | Mm.2108 |
| 141. | CCNG | Cyclin G | Apoptosis | 4 | Mm.2103 |
| 142. | CLU | Clusterin | Apoptosis | 4 | Mm.200608 |
| 143. | RTN4 | NogoA | Cell growth regulator | 4 | Mm.192580 |
| 144. | Hsp86 | Heat shock protein，86 kDa | The trauma stress reaction | 4 | Mm.1777 |
| 145. | Hsp70-1 | Heat shock protein，70 kDa 1 | The trauma stress reaction | 4 | Mm.1777 |
| 146. | Ania6a | Cyclin ania6a | Cell growth circulation | 4 | Mm.175612 |
| 147. | B2M | Microglobulin，beta 2 | The trauma stress reaction | 4 | Mm.163 |
| 148. | rNFIL-6C | rNFIL-6 C/EBP-related transcription factor | Cell transcription is regulated | 4 | Mm.1583 |
| 149. | SICA3 | Small inducible cytokine A3 solute carrier family 24 (sodium/potassium/calcium | Inflammatory reaction factor ion path and ionic equilibrium | 4 | Mm.1282 |
| 150. | Slc24a2 | exchanger)，member 2 | Regulate | 4 | Mm.127296 |
| 151. | Casp1 | Caspase 1 | Apoptosis | 4 | Mm.1051 |
| 152. | VGF | VGF nerve growth factor inducible | Cell growth regulator | 3 | Rn.9704 |
| 153. | CRBP | Cytosolic retinol binding protein | Cell signal transmits | 3 | Rn.902 |
| 154. | Dio2 | Iodothyronine deiodinase，type II | The cell physiological metabolism | 3 | Rn.88380 |
| 155. | Lgals3 | IgE binding protein | The inflammatory reaction factor | 3 | Rn.764 |
| 156. | GRGK3 | Glutamate receptor GluR-K3 | Cell receptor | 3 | Rn.74049 |
| 157. | CAPN2 | Calpain II 80-kDa subunit | Albumen is synthetic | 3 | Rn.6822 |
| 158. | DCP | Digoxin carrier protein | Unknown Function | 3 | Rn.5641 |
| 159. | MAP1A | Microtubule-associated protein 1A Bcl-2 binding protein with anticell | The cell tissue structure | 3 | Rn.41412 |
| 160. | BAD | death activity | Anti-apoptotic | 3 | Rn.36696 |
| 161. | 14-3-3PES | 14-3-3 protein，eta-subtype Major intrinsic protein of eye lens | Cell signal transmits | 3 | Rn.3324 |
| 162. | Mip2p | fiber 2 precursor | The intracellular transport factor | 3 | Rn.23532 |
| 163. | Erk | MAPK kinase MEKK 1 | The inflammatory reaction factor | 3 | Rn.139371 |
| 164. | MAPKK | MAPK kinase 1 | Cell signal transmits | 3 | Rn.139371 |
| 165. | Nucleop orinp45 | Nucleoporin p45 | Ion path and ionic equilibrium are regulated | 3 | Rn.11099 |
| 166. | AD1AT | AD1 antigen | The peroxidation stress reaction | 3 | Rn.11068 |
| 167. | IGF1R | Insulin-like growth factor 1 receptor | Cell receptor | 3 | Rn.10957 |
| 168. | CX3C | Chemokine CX3C | The inflammatory reaction factor | 3 | Rn.107266 |
| 169. | rBAXa | rBax，alpha | Apoptosis | 3 | Rn.10668 |
| 170. | SNC1 | Synuclein 1 | Cell signal transmits | 3 | Rn.103654 |
| 171. | Ptgi3 | Prostaglandin I 3 kinase | Redox reaction | 3 | Rn.100903 |
| 172. | PSEN1 | Presenilin 1 | Anti-apoptotic | 3 | Mm.998 |
| 173. | APC | Adenomatous polyposis coli protein | Unknown Function | 3 | Mm.7883 |
| 174. | VCAM1 | Vascular cell adhesion molecule 1 | The inflammatory reaction factor | 3 | Mm.76649 |
| 175. | Bcl-2 | Bcl-2 | Apoptosis | 3 | Mm.7660 |
| 176. | RhoA | RhoA | Cell signal transmits | 3 | Mm.757 |
| 177. | SLC32A1 | Vesicular inhibitory amino acid transporter | The intracellular transport factor | 3 | Mm.7444 |
| 178. | Gadd45a | Growth arrest and DNA damage inducible 45 alpha | The wound early response factor | 3 | Mm.72235 |
| 179. | ROCK | Rho-associated coiled kinase | Cell signal transmits | 3 | Mm.6710 |
| 180. | BFGF | Basic fibroblast growth factor | Cell transcription is regulated | 3 | Mm.57094 |
| 181. | GABA-A | Aminobutyric acid-gamma | Ion path and ionic equilibrium are regulated | 3 | Mm.5309 |
| 182. | GABBRgb2 | GABA(B)receptor gb2 | Cell receptor | 3 | Mm.5260 |
| 183. | Sele | E-selectin | Albumen is synthetic | 3 | Mm.5245 |
| 184. | CHST | Chondroitin 4-sulfotransferase | Cell adhesion | 3 | Mm.49071 |
| 185. | MPO | Myeloperoxidase | Redox reaction | 3 | Mm.4668 |
| 186. | Rb1 | Retinoblastoma 1 N-methyl-D-aspartate | Cell growth circulation | 3 | Mm.4480 |
| 187. | GRINA2A | receptor-associated protein(NMDA) 2A N-methyl-D-aspartate receptor-associated protein(NMDA) | Cell receptor | 3 | Mm.41665 |
| 188. | GRINA | 1 | Cell receptor | 3 | Mm.41665 |
| 189. | SOCS2 | Suppressor of cytokine signaling 2 Myelin-associated oligodendrocytic | Anti-apoptotic | 3 | Mm.4132 |
| 190. | Mobp | basic protein | The cell tissue structure | 3 | Mm.40461 |
| 191. | MEKK | MAP kinase phosphatase | Cell signal transmits | 3 | Mm.3994 |
| 192. | Egr2 | Early growth response 2 | Cell transcription is regulated | 3 | Mm.399 |
| 193. | Itm2a | Integral membrane protein 2 | Epicyte protein | 3 | Mm.38868 |
| 194. | ADSL | Adenylosuccinate lyase | The cell physiological metabolism | 3 | Mm.38151 |
| 195. | TUBB1 | Tubulin，beta 1 | The cell tissue structure | 3 | Mm.379811 |
| 196. | Hnt | Neurotrimin | Cell signal transmits | 3 | Mm.379400 |
| 197. | PK | Serine/threonine protein kinase | Albumen is synthetic | 3 | Mm.379270 |
| 198. | Pcna | Proliferating cell nuclear antigen | Cell growth circulation | 3 | Mm.378980 |
| 199. | Gpx2 | Glutathione peroxidase 2 | Neural defence protection | 3 | Mm.371561 |
| 200. | NEFH | Neurofilament，heavy polypeptide Actin-related protein 2/3 complex 41 | The cell tissue structure | 3 | Mm.359679 |
| 201. | p41-Arc | kDa subunit | The cell tissue structure | 3 | Mm.359491 |
| 202. | HLA-C | MHC class I antigen | The inflammatory reaction factor | 3 | Mm.358603 |
| 203. | GLY49a | Glycoprotein 49A Platelet endothelial cell adhesion | The trauma stress reaction | 3 | Mm.358601 |
| 204. | PECAM | molecule Spermidine/spermine N1-acetyl | Cell adhesion | 3 | Mm.343951 |
| 205. | SAT | transferase Potassium channel，voltage gated | The intracellular transport factor | 3 | Mm.340410 |
| 206. | Kcnd2 | Shal-related family，member 2 | The intracellular transport factor | 3 | Mm.320691 |
| 207. | Cd53 | CD53 antigen | Cell signal transmits ion path and ionic equilibrium | 3 | Mm.316861 |
| 208. | KCNA | Potassium channel，voltage-gated | Regulate | 3 | Mm.315769 |
| 209. | COX | Cytochrome C oxidase | Apoptosis | 3 | Mm.3128 |
| 210. | GSTYc1 SAC | Glutathione S-transferase Yc1 subunit，alpha class | The peroxidation stress reaction | 3 | Mm.304763 |
| 211. | Fti1 | Ferritin light chain 1 | The cell physiological metabolism | 3 | Mm.30357 |
| 212. | CAMP | Cyclic AMP | Cell signal transmits | 3 | Mm.297444 |
| 213. | Actb | actin，beta，cytoplasma | The cell tissue structure | 3 | Mm.297 |
| 214. | Mmp2 | Matrix Metalloproteinase 2 | Cell adhesion | 3 | Mm.29564 |
| 215. | TFRC | Transferrin receptor | Unknown Function | 3 | Mm.294740 |
| 216. | IGF2 | Insulin-like growth factor 2 | Cell growth regulator | 3 | Mm.294740 |
| 217. | CCL2 | Small inducible cytokine A2 | The trauma stress reaction | 3 | Mm.290320 |
| 218. | ATP1A1 | Sodium/potassium-ATPase，alpha 1 | Ion path and ionic equilibrium are regulated | 3 | Mm.290083 |
| 219. | PENK | Proenkephalin | Cell signal transmits | 3 | Mm.2899 |
| 220. | Eef2 | Eukaryotic elongation factor 2 | Albumen is synthetic | 3 | Mm.289431 |
| 221. | Pex11a | Peroxisomal biogenesis factor | The cell physiological metabolism | 3 | Mm.286622 |
| 222. | IER3 | Immediate early response 3 | Anti-apoptotic | 3 | Mm.28593 |
| 223. | Ddxl | Nuclear RNA helicase | Cell transcription is regulated | 3 | Mm.28222 |
| 224. | VIP | Vasoactive intestinal peptide Neural precursor cell expressed， | The trauma stress reaction | 3 | Mm.282007 |
| 225. | Nedd4 | developmentally down-regulated gene 4 DnaJ(Hsp40)homolog，subfamily A， | Unknown Function | 3 | Mm.279923 |
| 226. | Dnaja1 | member 1 poly (ADP-ribose) polymerase | The trauma stress reaction | 3 | Mm.27897 |
| 227. | Parp3 | family，member 3 | The intracellular transport factor | 3 | Mm.277779 |
| 228. | JAK2 | Janus kinase 2 | Cell signal transmits | 3 | Mm.275839 |
| 229. | TRKB | Tyrosine kinase(trk)B | Cell signal transmits | 3 | Mm.274346 |
| 230. | NEU1 | Neurodap 1 | Neural defence protection | 3 | Mm.272809 |
| 231. | khc | Kinesin heavey chain | The cell tissue structure | 3 | Mm.271648 |
| 232. | CSPG3 | Neurocan | The cell tissue structure | 3 | Mm.268079 |
| 233. | IL-1r | Interleukin 1 receptor | The inflammatory reaction factor | 3 | Mm.253424 |
| 234. | Mag | Myelin-associated glycoprotein | Cell adhesion | 3 | Mm.241355 |
| 235. | CACNA3 | Calcium channel 3 subunit | Ion path and ionic equilibrium are regulated | 3 | Mm.241121 |
| 236. | Nnat | Neuronatin | Cell growth regulator | 3 | Mm.233903 |
| 237. | IL-4r | Interleukin 4 receptor | The trauma stress reaction | 3 | Mm.233802 |
| 238. | TPR1 | Inositol 1，4，5-triphosphate receptor 1 | Signal transmits ion path and ionic equilibrium | 3 | Mm.227912 |
| 239. | SCN2 | Sodium channel 2 | Regulate | 3 | Mm.220329 |
| 240. | ITGB | Integrin beta subunit | Cell adhesion | 3 | Mm.213873 |
| 241. | MAOA | Monoamine oxidase A | The peroxidation stress reaction | 3 | Mm.21108 |
| 242. | Hmgb1 | High mobility group protein 1 Eukaryotic translation initiation | Cell transcription is regulated | 3 | Mm.207047 |
| 243. | EiF1A | factor 1，alpha | The cell physiological metabolism | 3 | Mm.196220 |
| 244. | Hba-a1 | Hemoglobin alpha，adult chain 1 | The intracellular transport factor | 3 | Mm.196110 |
| 245. | C3 | Complement component C3 | The inflammatory reaction factor | 3 | Mm.19131 |
| 246. | LYZ | Lysozyme | Neural defence protection | 3 | Mm.177539 |
| 247. | Cd48 | CD48 antigen Interferon-related developmental | The inflammatory reaction factor | 3 | Mm.1738 |
| 248. | IFRD1 | regulator 1 | Cell growth circulation | 3 | Mm.168 |
| 249. | Pla2 | Phospholipase A2 | The cell physiological metabolism | 3 | Mm.151951 |
| 250. | Lp1 | Lipoprotein lipase | The cell physiological metabolism | 3 | Mm.1514 |
| 251. | EDN1 | Endothelin 1 | Cell signal transmits | 3 | Mm.14543 |
| 252. | IL-18 | Interleukin 18 | The inflammatory reaction factor | 3 | Mm.1410 |
| 253. | Cox8 | Cytochrome C oxidase subunit VIII | The cell physiological metabolism | 3 | Mm.14022 |
| 254. | Msn | Moesin | The cell tissue structure | 3 | Mm.138876 |
| 255. | MCP2 | Methyl CpG binding protein 2 | Neural defence protection | 3 | Mm.131408 |
| 256. | CA2 | Carbonic anhydrase II | The peroxidation stress reaction | 3 | Mm.1186 |
| 257. | Id3 | Inhibitor of DNA binding 3 | Cell transcription is regulated | 3 | Mm.110 |
| 258. | AIF1 | Allograft inflammatory factor 1 | The trauma stress reaction | 3 | Mm.10747 |
| 259. | Glt | Glutamate | Cell signal transmits | 3 | Mm.10600 |
| 260. | BIK | Bcl-2 interacting killer/like | Neural defence protection | 3 | Hs.475055 |
| 261. | GRGB | Glutamate receptor GluR-B Nerve growth factor-induced protein | Cell receptor | 2 | Rn.91361 |
| 262. | Nr4a1 | B | Cell transcription is regulated | 2 | Rn.9096 |
| 263. | Spp1 | Secreted phosphoprotein 1 | Cell growth regulator | 2 | Rn.8871 |
| 264. | Scd2 | Stearyl-CoA desaturase 2 | The cell physiological metabolism | 2 | Rn.83595 |
| 265. | GRGC | Glutamate receptor GluR-C | Cell receptor | 2 | Rn.74049 |
| 266. | PKCr | Protein kinase C receptor | Cell signal transmits | 2 | Rn.62726 |
| 267. | NLGN2 | Neuroligin 2 | The cell tissue structure | 2 | Rn.55111 |
| 268. | Nrxn1 | Non-processed neurexin I | Cell signal transmits | 2 | Rn.54924 |
| 269. | S100RP | S-100 related protein Macrphage inflammatory protein 1 | The peroxidation stress reaction | 2 | Rn.4083 |
| 270. | MIP1a | alpha Activity and neurotransmitter-induced early gene | The inflammatory reaction factor | 2 | Rn.34673 |
| 271. | Ania3 | 3 | The cell tissue structure | 2 | Rn.34270 |
| 272. | COL1A1 | Collagen，alpha 1-I Basic transcription element binding | The cell tissue structure | 2 | Rn.2953 |
| 273. | BTEB1 | protein 1 | Cell transcription is regulated | 2 | Rn.19481 |
| 274. | GSTYaSAC | Glutathione S-transferase Ya subunit， alpha class | The peroxidation stress reaction | 2 | Rn.144550 |
| 275. | Hmgb2 | High mobility group protein 2 | Cell transcription is regulated | 2 | Rn.141159 |
| 276. | SYN1 | Synapsin 1 | Cell signal transmits | 2 | Rn.129069 |
| 277. | KLK6 | Myelencephalon specific protease | Neurodegeneration | 2 | Rn.10732 |
| 278. | Ngfr1 | Nerve growth factor receptor 1 intercellular adhesion molecule 1 | Cell receptor | 2 | Mm.90787 |
| 279. | ICAM1 | precursor | Epicyte protein | 2 | Mm.90364 |
| 280. | SGCB | Sarcoglycan，beta | The cell tissue structure | 2 | Mm.89310 |
| 281. | TUBA6 | Tubulin，alpha 6 | The cell tissue structure | 2 | Mm.88212 |
| 282. | Bag3 | Bcl-2 associated athanogene | Neural defence protection | 2 | Mm.688 |
| 283. | STX6 | Syntaxin 6 | The intracellular transport factor | 2 | Mm.66264 |
| 284. | Atf4 | Activating transcription factor 4 | Cell transcription is regulated | 2 | Mm.641 |
| 285. | Adr1a | Adrenergic receptor 1A | Cell receptor ion path and ionic equilibrium | 2 | Mm.57064 |
| 286. | SCG2 | Secretogranin II | Regulate | 2 | Mm.5038 |
| 287. | MIP2 | Macrophage inflammatory protein 2 | The inflammatory reaction factor | 2 | Mm.4979 |
| 288. | NCAM | neural cell adhesion molecule | Unknown Function | 2 | Mm.4974 |
| 289. | BFP | Brain finger protein | Cell transcription is regulated | 2 | Mm.49441 |
| 290. | INS | Insulin | Growth regulator | 2 | Mm.46269 |
| 291. | IL-18-1 | Interleukin 18 precursor | The inflammatory reaction factor | 2 | Mm.45579 |
| 292. | Dop | Dopamine Pre-B-cell leukemia transcription | Cell signal transmits | 2 | Mm.44241 |
| 293. | Pbx2 | factor | Cell transcription is regulated | 2 | Mm.43358 |
| 294. | Rgs4 | regulator of G-protein signaling 4 | Cell signal transmits | 2 | Mm.41642 |
| 295. | Fas-L | Fas-L | Apoptosis | 2 | Mm.39760 |
| 296. | Casp2 | Caspase 2 Glyceraldehyde-3-phosphate | Apoptosis | 2 | Mm.3921 |
| 297. | GAPDH | dehydrogenase | The cell physiological metabolism | 2 | Mm.379644 |
| 298. | MNT | Max binding protein | Cell transcription is regulated | 2 | Mm.3759 |
| 299. | TF | Transferrin | Unknown Function | 2 | Mm.37214 |
| 300. | Slc1a2 | Solute carrier family 1，member 2 | The intracellular transport factor | 2 | Mm.371582 |
| 301. | FOSB | Fos B | Cell transcription is regulated | 2 | Mm.362761 |
| 302. | rps4 | Ribosomal protein S4 U2 small nuclear ribonucleoprotein | Albumen is synthetic | 2 | Mm.361382 |
| 303. | U2af1 | auxiliary factor | Cell transcription is regulated | 2 | Mm.360389 |
| 304. | Casp7 | Caspase 7 | Apoptosis | 2 | Mm.35687 |
| 305. | Matn2 | Matrilin 2 | The cell tissue structure | 2 | Mm.3511 |
| 306. | PDHA1 | Pyruvate dehydrogenase | Redox reaction | 2 | Mm.34775 |
| 307. | Hsp84 | Heat shock protein，84kDa | The trauma stress reaction | 2 | Mm.336743 |
| 308. | Ubc | Ubiquitin C | Albumen is synthetic | 2 | Mm.331 |
| 309. | Cd44 | CD44 antigen | Neurodegeneration | 2 | Mm.330428 |
| 310. | GluR3 | Glutamate receptor 3 | Cell receptor | 2 | Mm.327681 |
| 311. | Nr3c2 | Mineralocorticoid receptor | Cell receptor | 2 | Mm.324393 |
| 312. | Hspb1 | Heat shock protein 1，alpha | The trauma stress reaction | 2 | Mm.315997 |
| 313. | NRXN1 | Neurexin I，beta | Unknown Function | 2 | Mm.312068 |
| 314. | AGT | Angiotensinogen | Cell signal transmits | 2 | Mm.301626 |
| 315. | RPS25 | Ribosomal protein S25 | Albumen is synthetic | 2 | Mm.297486 |
| 316. | Kfl9 | Kruppel-like factor 9 | Cell transcription is regulated | 2 | Mm.291595 |
| 317. | Ndu | NADH dehydrogenase | Redox reaction | 2 | Mm.290791 |
| 318. | SPARCL1 | SPARC-like 1 | Ion path and ionic equilibrium are regulated | 2 | Mm.29027 |
| 319. | Zyx | Zyxin vasoactive intestinal peptide receptor | Cell adhesion | 2 | Mm.282303 |
| 320. | VIPr2 | 2 | Cell receptor | 2 | Mm.282007 |
| 321. | Casp6 | Caspase 6 | Apoptosis | 2 | Mm.281379 |
| 322. | CCNC | Cyclin C | Cell growth circulation | 2 | Mm.278584 |
| 323. | Vil2 | Villin2 | The cell tissue structure | 2 | Mm.277812 |
| 324. | PTX3 | Pentaxin related gene | The inflammatory reaction factor | 2 | Mm.276776 |
| 325. | Zipro1 | Zinc finger proliferation 1 | The cell physiological metabolism | 2 | Mm.2760 |
| 326. | PTGS1 | Cyclooxygenase 1 | The cell physiological metabolism | 2 | Mm.275434 |
| 327. | nmr-2 | NMDA receptor | Apoptosis | 2 | Mm.275281 |
| 328. | IFNAR1 | Interferon receptor 1(alpha and beta) Protein tyrosine phosphatase-like | Cell receptor | 2 | Mm.275044 |
| 329. | PTPLA | protein | Albumen is synthetic | 2 | Mm.27286 |
| 330. | No | Nitric oxide | Redox reaction | 2 | Mm.272139 |
| 331. | snap25b | Soluble NSF attachment protein 25B | Cell transcription is regulated | 2 | Mm.271992 |
| 332. | snap25a | Soluble NSF attachment protein 25A | Cell transcription is regulated | 2 | Mm.271992 |
| 333. | RIN1 | Ras and Rab interactor 1 | Cell signal transmits | 2 | Mm.271922 |
| 334. | NRP1 | Neuropilin | Cell adhesion | 2 | Mm.271745 |
| 335. | ITG | Integrin | Unknown Function | 2 | Mm.271674 |
| 336. | AGT | Angiotensin II Platelet-derived growth factor A | The cell physiological metabolism | 2 | Mm.2679 |
| 337. | PDGFA | chain | Cell signal transmits | 2 | Mm.2675 |
| 338. | Epo | Erythropoietin Protein synthesis initiation factor | Neural defence protection | 2 | Mm.2653 |
| 339. | EIF4A2 | 4AII | Albumen is synthetic | 2 | Mm.260084 |
| 340. | NF-kb-p105 | NF-kappa B p105 subunit | Cell transcription is regulated | 2 | Mm.256765 |
| 341. | TIMP | Tissue inhibitor of metalloproteinase | Albumen is synthetic | 2 | Mm.255607 |
| 342. | AChE | Acetylcholinesterase | Cell signal transmits | 2 | Mm.255464 |
| 343. | Vav | Vav | Cell adhesion | 2 | Mm.248172 |
| 344. | PKCe | Protein kinase C epsilon | Cell signal transmits | 2 | Mm.24614 |
| 345. | Scya4 | Smail inducible cytokine A4 | The inflammatory reaction factor | 2 | Mm.244263 |
| 346. | Cd52 | CD52 antigen | Epicyte protein | 2 | Mm.24130 |
| 347. | SYNJ | Synaptojanin Macrophage migration inhibitory | Cell signal transmits | 2 | Mm.236068 |
| 348. | MIF | factor | Unknown Function | 2 | Mm.2326 |
| 349. | RA | Retinoic acid | Cell transcription is regulated | 2 | Mm.227484 |
| 350. | Xpo1 | Exportin 1 | Cell signal transmits | 2 | Mm.217547 |
| 351. | Hsp27a | Heat shock protein，27kDa A | The trauma stress reaction | 2 | Mm.21549 |
| 352. | Cd9 | CD9 antigen | The trauma stress reaction | 2 | Mm.210676 |
| 353. | TUBA2 | Tubulin alpha 2 | The cell tissue structure | 2 | Mm.209290 |
| 354. | Apod | apolipoprotein D Platelet-activating factor | The cell physiological metabolism | 2 | Mm.2082 |
| 355. | PAFAH | acetylhydrolase | Cell growth circulation | 2 | Mm.200859 |
| 356. | SLC | Vesicular monoamine transporter Nerve growth factor-induted protein | Cell signal transmits | 2 | Mm.19301 |
| 357. | EGR1 | A | Cell transcription is regulated | 2 | Mm.181959 |
| 358. | Egr1 | Early growth response protein 1 | The wound early response factor | 2 | Mm.181959 |
| 359. | IAP | Inhibitor of apoptosis protein | Apoptosis | 2 | Mm.181824 |
| 360. | Rph3a | Rabphilin 3A | Albumen is synthetic | 2 | Mm.181166 |
| 361. | Fas | Apoptosis antigen 1 | Apoptosis | 2 | Mm.181033 |
| 362. | Hsp72 | Heat shock protein，72kDa | The trauma stress reaction | 2 | Mm.1777 |
| 363. | Hsp40 | Heat shock protein，40kDa | The trauma stress reaction | 2 | Mm.1777 |
| 364. | Hsp32 | Heat shock protein，32kDa | The trauma stress reaction | 2 | Mm.1777 |
| 365. | Hsp25 | Heat shock protein，25kDa Mini chromosome maintenance | The trauma stress reaction | 2 | Mm.1777 |
| 366. | Mcmd | deficient | Apoptosis | 2 | Mm.16711 |
| 367. | Cd68 | CD68 antigen | Epicyte protein | 2 | Mm.15819 |
| 368. | Lag | Leukemia-associated gene cAMP response element binding | Cell signal transmits | 2 | Mm.148748 |
| 369. | CREB | protein 1 | Cell transcription is regulated | 2 | Mm.1376 |
| 370. | CAMKA | calcium/calmodulin dependent protein kinase，alpha | Cell signal transmits | 2 | Mm.131530 |
| 371. | MCP1 | Membrane cofactor protein 1 | Neural defence protection | 2 | Mm.12884 |
| 372. | CXCR3 | chemokine(C-X-C motif)receptor 3 | The inflammatory reaction factor | 2 | Mm.12876 |
| 373. | SAPK3 | SAP kinase 3 | Cell signal transmits | 2 | Mm.12775 |
| 374. | PON | Paraoxonase | The peroxidation stress reaction | 2 | Mm.126984 |
| 375. | Neuna60 | NeuN Nuclear receptor subfamily 4 group | Cell transcription is regulated | 2 | Mm.125874 |
| 376. | NR4A1 | A，member 1 | Cell receptor | 2 | Mm.119 |
| 377. | Hsp10 | Heat shock protein，10kDa | The trauma stress reaction | 2 | Hs.558338 |
| 378. | MEG3 | Maternally expressed gene 3 | Unknown Function | 2 | Hs.525589 |
| 379. | Narp | Narp | Cell transcription is regulated | 2 | Hs.489287 |
| 380. | HSPA8 | Heat shock protein cognate 70kDa | The trauma stress reaction | 2 | Hs.180414 |
| 381. | Gfp | Green fluorescent protein | Unknown Function | 2 | At.48658 |
Claims (10)
1, a kind of functional central nervous system injury diagnostic detection gene chip, immobilized oligonucleotide gene probe array on the chip, the oligonucleotide biomolecules that it is characterized in that described oligonucleotide gene probe array is the specific gene that plays a crucial role in multiple central nervous system injury generation evolution simultaneously, relate separately to nerve cell death and tissue necrosis, inflammation and stress reaction, ionic equilibrium imbalance in the cell, the signal conduction, cell cycle regulating is unusual, cellularstructure unusual and neurodegeneration damage pathologic, physiologic and molecular biology active gene.
2, functional central nervous system injury diagnostic detection gene chip according to claim 1 is characterized in that these specific genes comprise: the reaction of (1), trauma stress; (2), wound early response; (3), apoptosis and anti-apoptotic; (4), peroxidation stress reaction; (5), redox reaction; (6), inflammatory reaction; (7), cell cycle regulation; (8), neurodegeneration; (9), neural defence protection; (10), albumen is synthetic; (11), ion path and ionic equilibrium are regulated; (12), cell growth circulation; (13), cell transcription is regulated; (14), intracellular transport; (15), cell receptor; (16), cell physiological metabolism; (17), cell adhesion; (18), cell signal transmission; (19), cell tissue structure; (20), the gene of epicyte protein function.
3, the combination that functional central nervous system injury diagnostic detection gene chip according to claim 1, the oligonucleotide arrays that it is characterized in that gene chip are to use photomask technology and traditional DNA synthetic chemistry is with the randomly ordered manufacturing of oligonucleotide probe array.
4, functional central nervous system injury diagnostic detection gene chip according to claim 1, the length that it is characterized in that oligonucleotide probe is 70-mer.
5, functional central nervous system injury diagnostic detection gene chip according to claim 1 is characterized in that each gene on the chip all is two point samples.
6,, it is characterized in that the oligonucleotide gene probe array is the total gene oligonucleotide gene probe of central nervous system injury of listed 381 kinds of keys in the table 1 according to the described functional central nervous system injury diagnostic detection gene chip of one of claim 1-5.
7, functional central nervous system injury diagnostic detection gene chip according to claim 6, it is characterized in that described oligonucleotide gene probe adopts coupling/mismatch probe right, promptly when special Oligo of design mates, design a non-specific Oligo probe simultaneously, this probe only is equipped with a base in interposition and replaces, and the difference between usefulness coupling and the mismatch is as strength of signal.
8, a kind of functional central nervous system injury diagnostic detection gene chip manufacture method comprises that the structure of DNA square formation, original position are synthesized, the point sample step of synthetic back probe, it is characterized in that:
The structure of A.DNA square formation: adopt the method for surface chemistry or the method for combinatorial chemistry to handle the carrier-pellet base when preparing oligonucleotide chip earlier, the specific biological gene factor that will play a crucial role in multiple central nervous system injury generation evolution simultaneously relates separately to nerve cell death and tissue necrosis then, inflammation and stress reaction, ionic equilibrium imbalance in the cell, the signal conduction, cell cycle regulating is unusual, cellularstructure is unusual, the oligonucleotide probe random alignment of neurodegeneration damage pathologic, physiologic and molecular biology active gene factor is on chip;
B. original position is synthetic: adopt original position photoetching route of synthesis to make the probe of oligonucleotide gene chip;
C. point sample: utilize automatic spot sample device with the Oligo cDNA probe for preparing in advance among the step B by put at a high speed model machine directly point on the carrier-pellet base, behind the point sample chip and probe are cured.
9, functional central nervous system injury diagnostic detection gene chip manufacture method according to claim 8 is characterized in that in the steps A that the specific biological factor comprises: the reaction of (1), trauma stress; (2), wound early response; (3), apoptosis and anti-apoptotic; (4), peroxidation stress reaction; (5), redox reaction; (6), inflammatory reaction; (7), cell cycle regulation; (8), neurodegeneration; (9), neural defence protection; (10), albumen is synthetic; (11), ion path and ionic equilibrium are regulated; (12), cell growth circulation; (13), cell transcription is regulated; (14), intracellular transport; (15), cell receptor; (16), cell physiological metabolism; (17), cell adhesion; (18), cell signal transmission; (19), cell tissue structure; (20), the gene of epicyte protein function.
10. functional central nervous system injury diagnostic detection gene chip manufacture method according to claim 8 is characterized in that the oligonucleotide gene probe array is the total gene oligonucleotide gene probe of central nervous system injury of listed 381 kinds of keys in the table 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610002921 CN101008033A (en) | 2006-01-27 | 2006-01-27 | Gene chip for detecting functional central nerve damage and its production method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610002921 CN101008033A (en) | 2006-01-27 | 2006-01-27 | Gene chip for detecting functional central nerve damage and its production method |
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| CN101008033A true CN101008033A (en) | 2007-08-01 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200610002921 Pending CN101008033A (en) | 2006-01-27 | 2006-01-27 | Gene chip for detecting functional central nerve damage and its production method |
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| Country | Link |
|---|---|
| CN (1) | CN101008033A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101760530B (en) * | 2008-12-26 | 2012-05-23 | 上海基康生物技术有限公司 | Method for detecting stroke related locus |
| CN104519891A (en) * | 2012-05-16 | 2015-04-15 | 桑比欧公司 | Methods and compositions for treatment of traumatic brain injury and for modulation of migration of neurogenic cells |
| CN111621525A (en) * | 2020-06-18 | 2020-09-04 | 山东如戴生物科技有限公司 | Application of STX1B gene in promoting growth and differentiation of human adipose-derived mesenchymal stem cells |
| CN113462769A (en) * | 2021-08-05 | 2021-10-01 | 中国医学科学院医药生物技术研究所 | inhibitor/CaMKII system and application thereof as biomarker |
| CN116879564A (en) * | 2023-09-06 | 2023-10-13 | 暨南大学附属第一医院（广州华侨医院） | Spinal cord injury biomarker based on proteomics and phosphorylated protein modification histology and application thereof |
-
2006
- 2006-01-27 CN CN 200610002921 patent/CN101008033A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101760530B (en) * | 2008-12-26 | 2012-05-23 | 上海基康生物技术有限公司 | Method for detecting stroke related locus |
| CN104519891A (en) * | 2012-05-16 | 2015-04-15 | 桑比欧公司 | Methods and compositions for treatment of traumatic brain injury and for modulation of migration of neurogenic cells |
| CN104519891B (en) * | 2012-05-16 | 2019-03-22 | 桑比欧公司 | Method and composition for treating traumatic brain injury and for adjusting neurogenic cell migration |
| CN111621525A (en) * | 2020-06-18 | 2020-09-04 | 山东如戴生物科技有限公司 | Application of STX1B gene in promoting growth and differentiation of human adipose-derived mesenchymal stem cells |
| CN111621525B (en) * | 2020-06-18 | 2021-04-23 | 中赛干细胞基因工程有限公司 | Application of STX1B gene in promoting growth and differentiation of human adipose-derived mesenchymal stem cells |
| CN113462769A (en) * | 2021-08-05 | 2021-10-01 | 中国医学科学院医药生物技术研究所 | inhibitor/CaMKII system and application thereof as biomarker |
| CN116879564A (en) * | 2023-09-06 | 2023-10-13 | 暨南大学附属第一医院（广州华侨医院） | Spinal cord injury biomarker based on proteomics and phosphorylated protein modification histology and application thereof |
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