CN107885974A - Transcript profile and proteomic assays method in a kind of liver cancer biological process - Google Patents

Abstract

The invention discloses the transcript profile in a kind of liver cancer biological process and proteomic assays method,（1）Rats With Hepatoma is built and liver（2）Transcript profile is sequenced（3）Differential Proteomic research based on iTRAQ joint LC MALDI（4）Bioinformatic analysis（5）RT‑PCR（6）SABC（7）ELISA result judgements：On ELISA detectors, at 450nm, each hole OD values are surveyed after being returned to zero with blank control wells, are the positive if more than 2.1 times of defined negative control OD value；（8）Data statistical analysis method；The present invention carries out the expression checking of positioning and quantitative using clinical sample to it, finds its evidence with clinical correlation, evaluates clinical value, and new clue is provided for onset of liver cancer and liver cancer Mechanism Study.The liver cancer key molecule of problem screening will establish Research foundation for the exploration liver cancer marker related to early detection, classification, evaluation prognosis, and more efficient, the accurate liver cancer treatment target position of selection.

Description

Transcript profile and proteomic assays method in a kind of liver cancer biological process

Technical field

The present invention relates to genetic transcription group and proteomics field, turn in especially a kind of liver cancer biological process Record group and proteomic assays method.

Background technology

In biology and medical research, it is important that a field be to biosystem and the structure of life process, work( The observation that can and regulate and control.But between the past centuries, biologist focuses on individual gene or protein in biology department always Expression change and function in system, and the change of life system can not be studied from overall situation, the overall angle.With medical science It is progressive, it has been found that the generations of many diseases, particularly cancer is often multifactor, polygenes, multipath synergy cause 's.This just need one can comprehensively, dynamic, the technology and means of systematic research life system, then " group is learned " is general Thought is arisen at the historic moment^[3].But with the completion of the Human Genome Project, it has been found that in only can not be complete from the angle of genomics The shearing that occurs in total correctness predicted gene transcription, splicing and in translation the starting of open reading frame codon, Various modification situations after final position and translation.

In gene expression research, extensive genetic analysis can be relevant to a physiological status either cell phenotype Gene progress system monitoring, high throughput analysis can be utilized in data output and obtain the quick both sides advantage of data, to disease Function candidate gene during disease is identified.The maturation of microarray technology, researcher is sequenced by transcript profile and study, Find marker gene interested.As oncogene expression is to the tissue in various sources and the correlation point of patient's survival outcome Analysis example is the same, and the gene expression analysis research carried out by microarray technology will continue to play the part of in biomarker discovery procedure Important function.

Although the analysis ability of microarray is very powerful, transcription group research platform only includes the change of those Adaptable growth conditions The transcript of cell.In most cells and intercellular Biochemical processes all can by protein-protein or other The influence of protein-substrate interaction.The horizontal gene expression analysis of protein group provides a quickly controllable life The process of thing synthesis, wherein most are regulated and controled by transcription group platform.Meanwhile transcript profile passes through the protein of expression in itself Other changes either under cellular biochemical state, carry out feedback control.

In other words, gene expression is not exclusively from transcript profile to the one-way flow of protein group, but both is mutual Connection.Understanding to this function controlling is generally limited to special signal pathway, or metabolic pathway.It is to be understood that turn Effect of Mutual Regulation between record group and protein group to RNA and protein expression, it is necessary to carry out Integral synchronous monitoring.

The progress of transcription group, proteomics and bioinformatics investigative technique opens for research complex biological system Brand-new approach, the reorganizing research that three is connected together can reveal that the hereditary information carried when disease occurs from gene turns It is changed into that the exception during the entire process of phenotype can be distinguished, its magnanimity information gathered is covered in disease incidence and disease mechanisms Key function node, can be used to identify tumor-related gene and its protein of expression so that thousands of genes and egg The analysis of white matter is possibly realized, for explore early detection, classification, evaluate prognosis tumor markers, and selection it is more efficient, Accurate oncotherapy target position provides reliable guarantee.

Ion proton sequenators of new generation use the technology of semiconductor chips, and sequencing speed is fast, and has high extension Property, by proprietary large-scale parallel semiconductor inductor, ion stream caused by DNA replication dna is realized and directly and in real time examined Survey.When reagent is entered in chip by integrated fluid passage, the reacting hole being clouded on chip immediately becomes up to a million individual micro- Reaction system.The technical combinations of this unique fluid system, the Machine Design of microbody system and semiconductor, enable researcher to exist The pinpoint accuracy sequence more than from 10Mb to 1Gb is obtained in 2 hours.In addition, Ion Proton sequenators and Ion Reporter Analysis software can complete the analysis of individual gene group in an independent server, break current data parsing bottleneck, greatly Research cost is reduced greatly, improves the speed and accuracy of detection, in scientific research and clinically there is good application；To current Untill, in the confluence analysis article delivered, most of LC-MS analyses are used in combination with cold labeling, especially It is iTRAQ reagents.Even with technology it is different, the confluence analysis published so far all indicate transcription group and The importance of protein science.Transcription group or protein science generally only consider regulating system and the net effect of decomposition equilibrium state Should, in fact, the inconsistency occurred is to synthesize a kind of reflection with two kinds of replacement process of degraded, researcher was to changing Mechanism in journey is interested；In addition, transcription group and proteomic assays want successful integration, it is necessary to efficiently and accurately phase Mutually reference.Researcher needs flexibly to define the genome of oneself, it is also possible to needing to select to be directed to using predefined The target figure of protein, when new genome, transcript profile and protein groups sequence occur, researcher needs timely register update, And the information of deletion error.The development of bioinformatics technique is so that genetic transcription, expression during oncobiology are whole Exception during individual is disclosed, and clue is provided for tumour Mechanism Study.

This research is intended, using the sequencing of Ion Proton transcript profiles and LC-MALDI Discrepancy proteome analysis platforms, carrying out liver cancer Transcript profile and proteomic assays in biological process.By building Rats With Hepatoma model, in relatively more normal and liver cancer tissue Genetic transcription and protein expression difference, all occur to transcript profile in liver cancer and protein groups abnormal molecule carry out gene optimization, Alternative splicing analysis, new gene or the screening of new transcript, expression analysis, Differential expression analysis, differential expression cluster analysis and The processing of the bioinformatic analysis such as functional annotation, screens liver cancer key function node and tumor cells, and carry out clinic to it and test Card and clinical value are assessed.This research will provide new clue for onset of liver cancer and liver cancer Mechanism Study.

The technical scheme of transcript profile and proteomic assays method in a kind of liver cancer biological process of the present invention, through retrieval Domestic pharmaceutical industry industry has no identical.

The content of the invention

It is an object of the invention to provide the transcript profile in a kind of liver cancer biological process and proteomic assays method.

Transcript profile and proteomic assays method in this liver cancer biological process,

Comprise the steps of：

（1）Rats With Hepatoma is built and liver

Cleaning grade Wistar male rats 40,150 ± 20g of body weight；It is randomly divided into test group 30, control group 10； Raised by the raising requirement of cleaning grade animal in semi-barrier system；It is 100 μ gPml's with the running water compound concentration of sterilizing Diethylnitrosamine solution (is kept in dark place, Fresh), is freely drunk for rats in test groups, changes once daily, 3 months After be changed in general sterile tap water, continue observation 2 months, whole experiment process 5 totally months；Control group whole experiment process is equal Drink sterile tap water；Overdose of sodium pentobarbital is injected during experiment cut-off and puts to death animal, is opened abdominal cavity, is taken hepatic tissue with 10 % first Aldehyde is fixed, and pathology microscopy is carried out after HE dyeing, determines that rat liver cancer induction is completed；

（2）Transcript profile is sequenced

Extraction normally and Rats With Hepatoma liver total RNA, during pay attention to avoiding RNase, determine concentration of specimens after measuring OD values, divide Not Hun He normal group and liver cancer group rat liver total serum IgE, make the rna contents of two mixing samples pure in more than 100ug, separation Change mRNA, its content is reached 5-400ng, build full transcript profile library, prepare template and determined using Ion proton systems Measure sequence；

（3）Differential Proteomic research based on iTRAQ joints LC-MALDI

Normal group and liver cancer group rat liver total protein liquid are extracted, two groups are mixed respectively, using acetone precipitation by protein It is quantitative after precipitation, make every group containing about 100ug total proteins, carry out carrying out pancreatin enzymolysis, reductive alkylation, iTRAQ marks successively Strictly carried out Deng, process in accordance with iTRAQ kit specifications；Normal group and liver cancer group rat liver enzyme after iTRAQ is marked Product mixed in equal amounts is solved, after carrying out one-dimensional SCX and two-dimentional nano-LC chromatographic isolations, into spectrometer analysis；

（4）Bioinformatic analysis

Data cover degree, comparative information statistics etc. are carried out to transcript profile sequencing and Differential Proteomic data respectively, finds liver cancer Middle transcription and protein expression show the molecule of difference carry out new transcript and annotation, expression analysis and Differential expression analysis, The data mining work such as differential expression cluster analysis, the functional annotation analysis of difference expression gene, screen liver cancer related keyword work( Can node and tumor cells；

（5）RT-PCR

According to the Accession number of liver cancer molecule, obtain its sequence in genebank and design PCR primer；Tissue extraction Total serum IgE, reverse transcription is into cDNA as pcr template；Carry out DNA denaturation, annealing and extension etc. successively, actual conditions with reference to primer and Reagent specification；

（6）SABC

Histotomy dewaxing, after aquation PBS wash 2~3 times it is each 5 minutes；3% H2O2 (80% methanol) is added dropwise on TMA, room temperature Stand 10 minutes；PBS wash 2~3 times it is each 5 minutes；Electric furnace or water-bath heat 0.01 sodium citrate buffer（pH 6.0）To 95 DEG C or so, it is put into the boiling hot reparation of tissue piece heating 10-15 minutes progress；PBS wash 2~3 times it is each 5 minutes；Normal goats are added dropwise Serum block, room temperature 20 minutes；Get rid of surplus liquid；1 anti-50 μ l are added dropwise, being stored at room temperature 1 hour, either 4 DEG C overnight or 37 DEG C 1 hour（Wherein, 4 DEG C overnight after need to be 45 minutes in 37 DEG C of rewarmings）；PBS wash 3 times it is each 5 minutes；Secondary antibody is added dropwise（It can add 0.05% tween-20）40~50 μ l, are stored at room temperature, or 37 DEG C 1 hour;PBS wash 3 times it is each 5 minutes;DAB develops the color 5~10 points Clock, dye levels are grasped under the microscope；PBS or running water rinse 10 minutes；Haematoxylin redyeing 2 minutes, hydrochloride alcohol differentiation； Running water rinses 10~15 minutes；Dehydration, transparent, mounting, microscopy；

（7）ELISA

It is 1~10 μ g/ml that antibody is diluted into protein content with coating buffer solution；In the reacting hole of each XPS Add 0.1ml, 4 DEG C overnight；Next day, solution in hole is discarded, washed 3 times, every time 3 minutes with lavation buffer solution；Add what is necessarily diluted to treat Sample product 0.1ml in the above-mentioned reacting hole being coated with, put 37 DEG C be incubated 1 hour after wash（Pay attention to leaving some space hole, negative right According to hole and Positive control wells）；In each reacting hole, enzyme labelled antibody (dilution factor after titration) 0.1ml of diluted fresh is added； 37 DEG C are incubated 0.5~1 hour, washing；Add the tmb substrate solution 0.1ml of Extemporaneous in each reacting hole, 37 DEG C 10~30 Minute；Terminate liquid 0.05ml is added in each reacting hole；

Result judgement：On ELISA detectors, at 450nm, each hole OD values are surveyed after being returned to zero with blank control wells, if more than rule 2.1 times of fixed negative control OD value, it is the positive；

（8）Data statistical analysis method

Using SPSS19.0 statistical packages, statistical analysis technique is selected by real data type.

Invention beneficial effect：

The present invention carries out the expression checking of positioning and quantitative using clinical sample to it, finds its evidence with clinical correlation, comments Valency clinical value, new clue is provided for onset of liver cancer and liver cancer Mechanism Study.Problem screening liver cancer key molecule will be Explore the liver cancer marker related to early detection, classification, evaluation prognosis, and more efficient, the accurate liver cancer treatment of selection Target position establishes Research foundation.

Embodiment

Embodiment：

Transcript profile and proteomic assays method in liver cancer biological process comprise the steps of：

（1）Rats With Hepatoma is built and liver

Cleaning grade Wistar male rats 40,150 ± 20g of body weight；It is randomly divided into test group 30, control group 10； Raised by the raising requirement of cleaning grade animal in semi-barrier system；It is 100 μ gPml's with the running water compound concentration of sterilizing Diethylnitrosamine solution (is kept in dark place, Fresh), is freely drunk for rats in test groups, changes once daily, 3 months After be changed in general sterile tap water, continue observation 2 months, whole experiment process 5 totally months；Control group whole experiment process is equal Drink sterile tap water；Overdose of sodium pentobarbital is injected during experiment cut-off and puts to death animal, is opened abdominal cavity, is taken hepatic tissue with 10 % first Aldehyde is fixed, and pathology microscopy is carried out after HE dyeing, determines that rat liver cancer induction is completed；

（2）Transcript profile is sequenced

Extraction normally and Rats With Hepatoma liver total RNA, during pay attention to avoiding RNase, determine concentration of specimens after measuring OD values, divide Not Hun He normal group and liver cancer group rat liver total serum IgE, make the rna contents of two mixing samples pure in more than 100ug, separation Change mRNA, its content is reached 5-400ng, build full transcript profile library, prepare template and determined using Ion proton systems Measure sequence；

（3）Differential Proteomic research based on iTRAQ joints LC-MALDI

Normal group and liver cancer group rat liver total protein liquid are extracted, two groups are mixed respectively, using acetone precipitation by protein It is quantitative after precipitation, make every group containing about 100ug total proteins, carry out carrying out pancreatin enzymolysis, reductive alkylation, iTRAQ marks successively Strictly carried out Deng, process in accordance with iTRAQ kit specifications；Normal group and liver cancer group rat liver enzyme after iTRAQ is marked Product mixed in equal amounts is solved, after carrying out one-dimensional SCX and two-dimentional nano-LC chromatographic isolations, into spectrometer analysis；

（4）Bioinformatic analysis

Data cover degree, comparative information statistics etc. are carried out to transcript profile sequencing and Differential Proteomic data respectively, finds liver cancer Middle transcription and protein expression show the molecule of difference carry out new transcript and annotation, expression analysis and Differential expression analysis, The data mining work such as differential expression cluster analysis, the functional annotation analysis of difference expression gene, screen liver cancer related keyword work( Can node and tumor cells；

（5）RT-PCR

According to the Accession number of liver cancer molecule, obtain its sequence in genebank and design PCR primer；Tissue extraction Total serum IgE, reverse transcription is into cDNA as pcr template；Carry out DNA denaturation, annealing and extension etc. successively, actual conditions with reference to primer and Reagent specification；

（6）SABC

Histotomy dewaxing, after aquation PBS wash 2~3 times it is each 5 minutes；3% H2O2 (80% methanol) is added dropwise on TMA, room temperature Stand 10 minutes；PBS wash 2~3 times it is each 5 minutes；Electric furnace or water-bath heat 0.01 sodium citrate buffer（pH 6.0）To 95 DEG C or so, it is put into the boiling hot reparation of tissue piece heating 10-15 minutes progress；PBS wash 2~3 times it is each 5 minutes；Normal goats are added dropwise Serum block, room temperature 20 minutes；Get rid of surplus liquid；1 anti-50 μ l are added dropwise, being stored at room temperature 1 hour, either 4 DEG C overnight or 37 DEG C 1 hour（Wherein, 4 DEG C overnight after need to be 45 minutes in 37 DEG C of rewarmings）；PBS wash 3 times it is each 5 minutes；Secondary antibody is added dropwise（It can add 0.05% tween-20）40~50 μ l, are stored at room temperature, or 37 DEG C 1 hour;PBS wash 3 times it is each 5 minutes;DAB develops the color 5~10 points Clock, dye levels are grasped under the microscope；PBS or running water rinse 10 minutes；Haematoxylin redyeing 2 minutes, hydrochloride alcohol differentiation； Running water rinses 10~15 minutes；Dehydration, transparent, mounting, microscopy；

（7）ELISA

It is 1~10 μ g/ml that antibody is diluted into protein content with coating buffer solution；In the reacting hole of each XPS Add 0.1ml, 4 DEG C overnight；Next day, solution in hole is discarded, washed 3 times, every time 3 minutes with lavation buffer solution；Add what is necessarily diluted to treat Sample product 0.1ml in the above-mentioned reacting hole being coated with, put 37 DEG C be incubated 1 hour after wash（Pay attention to leaving some space hole, negative right According to hole and Positive control wells）；In each reacting hole, enzyme labelled antibody (dilution factor after titration) 0.1ml of diluted fresh is added； 37 DEG C are incubated 0.5~1 hour, washing；Add the tmb substrate solution 0.1ml of Extemporaneous in each reacting hole, 37 DEG C 10~30 Minute；Terminate liquid 0.05ml is added in each reacting hole；

Result judgement：On ELISA detectors, at 450nm, each hole OD values are surveyed after being returned to zero with blank control wells, if more than rule 2.1 times of fixed negative control OD value, it is the positive；

（8）Data statistical analysis method

Using SPSS19.0 statistical packages, statistical analysis technique is selected by real data type.

The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto, Any one skilled in the art the invention discloses technical scope in, technique according to the invention scheme and its Inventive concept is subject to equivalent substitution or change, should all be included within the scope of the present invention.

Claims (1)

1. transcript profile and proteomic assays method in a kind of liver cancer biological process, it is characterised in that：By following steps group Into：

（1）Rats With Hepatoma is built and liver

Cleaning grade Wistar male rats 40,150 ± 20g of body weight；It is randomly divided into test group 30, control group 10； Raised by the raising requirement of cleaning grade animal in semi-barrier system；It is 100 μ gPml's with the running water compound concentration of sterilizing Diethylnitrosamine solution (is kept in dark place, Fresh), is freely drunk for rats in test groups, changes once daily, 3 months After be changed in general sterile tap water, continue observation 2 months, whole experiment process 5 totally months；Control group whole experiment process is equal Drink sterile tap water；Overdose of sodium pentobarbital is injected during experiment cut-off and puts to death animal, is opened abdominal cavity, is taken hepatic tissue with 10 % first Aldehyde is fixed, and pathology microscopy is carried out after HE dyeing, determines that rat liver cancer induction is completed；

（2）Transcript profile is sequenced

Extraction normally and Rats With Hepatoma liver total RNA, during pay attention to avoiding RNase, determine concentration of specimens after measuring OD values, divide Not Hun He normal group and liver cancer group rat liver total serum IgE, make the rna contents of two mixing samples pure in more than 100ug, separation Change mRNA, its content is reached 5-400ng, build full transcript profile library, prepare template and determined using Ion proton systems Measure sequence；

（3）Differential Proteomic research based on iTRAQ joints LC-MALDI

Normal group and liver cancer group rat liver total protein liquid are extracted, two groups are mixed respectively, using acetone precipitation by protein It is quantitative after precipitation, make every group containing about 100ug total proteins, carry out carrying out pancreatin enzymolysis, reductive alkylation, iTRAQ marks successively Strictly carried out Deng, process in accordance with iTRAQ kit specifications；Normal group and liver cancer group rat liver enzyme after iTRAQ is marked Product mixed in equal amounts is solved, after carrying out one-dimensional SCX and two-dimentional nano-LC chromatographic isolations, into spectrometer analysis；

（4）Bioinformatic analysis

Data cover degree, comparative information statistics etc. are carried out to transcript profile sequencing and Differential Proteomic data respectively, finds liver cancer Middle transcription and protein expression show the molecule of difference carry out new transcript and annotation, expression analysis and Differential expression analysis, The data mining work such as differential expression cluster analysis, the functional annotation analysis of difference expression gene, screen liver cancer related keyword work( Can node and tumor cells；

（5）RT-PCR

According to the Accession number of liver cancer molecule, obtain its sequence in genebank and design PCR primer；Tissue extraction Total serum IgE, reverse transcription is into cDNA as pcr template；Carry out DNA denaturation, annealing and extension etc. successively, actual conditions with reference to primer and Reagent specification；

（6）SABC

Histotomy dewaxing, after aquation PBS wash 2~3 times it is each 5 minutes；3% H2O2 (80% methanol) is added dropwise on TMA, room temperature Stand 10 minutes；PBS wash 2~3 times it is each 5 minutes；Electric furnace or water-bath heat 0.01 sodium citrate buffer（pH 6.0）To 95 DEG C or so, it is put into the boiling hot reparation of tissue piece heating 10-15 minutes progress；PBS wash 2~3 times it is each 5 minutes；Normal goats are added dropwise Serum block, room temperature 20 minutes；Get rid of surplus liquid；1 anti-50 μ l are added dropwise, being stored at room temperature 1 hour, either 4 DEG C overnight or 37 DEG C 1 hour（Wherein, 4 DEG C overnight after need to be 45 minutes in 37 DEG C of rewarmings）；PBS wash 3 times it is each 5 minutes；Secondary antibody is added dropwise（It can add 0.05% tween-20）40~50 μ l, are stored at room temperature, or 37 DEG C 1 hour;PBS wash 3 times it is each 5 minutes;DAB develops the color 5~10 points Clock, dye levels are grasped under the microscope；PBS or running water rinse 10 minutes；Haematoxylin redyeing 2 minutes, hydrochloride alcohol differentiation； Running water rinses 10~15 minutes；Dehydration, transparent, mounting, microscopy；

（7）ELISA

It is 1~10 μ g/ml that antibody is diluted into protein content with coating buffer solution；In the reacting hole of each XPS Add 0.1ml, 4 DEG C overnight；Next day, solution in hole is discarded, washed 3 times, every time 3 minutes with lavation buffer solution；Add what is necessarily diluted to treat Sample product 0.1ml in the above-mentioned reacting hole being coated with, put 37 DEG C be incubated 1 hour after wash（Pay attention to leaving some space hole, negative right According to hole and Positive control wells）；In each reacting hole, enzyme labelled antibody (dilution factor after titration) 0.1ml of diluted fresh is added； 37 DEG C are incubated 0.5~1 hour, washing；Add the tmb substrate solution 0.1ml of Extemporaneous in each reacting hole, 37 DEG C 10~30 Minute；Terminate liquid 0.05ml is added in each reacting hole；

Result judgement：On ELISA detectors, at 450nm, each hole OD values are surveyed after being returned to zero with blank control wells, if more than rule 2.1 times of fixed negative control OD value, it is the positive；

（8）Data statistical analysis method

Using SPSS19.0 statistical packages, statistical analysis technique is selected by real data type.