CN109628593A - A kind of osteosarcoma stem cell molecular marker CD24 and its application - Google Patents
A kind of osteosarcoma stem cell molecular marker CD24 and its application Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
A kind of osteosarcoma stem cell molecular marker CD24 and its application, the molecular marked compound include CD24 gene or CAD24 albumen.Using molecular marker CD24 as osteosarcoma stem cell molecular labeling, utilize CD24 gene expression dose in the product testing osteosarcoma tissue sample to be measured of detection CD gene expression, and compared with CD24 gene expression dose in normal cancer beside organism, to diagnose osteosarcoma progression of disease, assess prognosis, with preferable specificity, higher sensitivity, a kind of completely new approach is provided for diagnosing and/or treating for osteosarcoma.
Description
Technical field
The invention belongs to field of biomedicine, it is related to a kind of osteosarcoma stem cell molecular marker CD24 and its application.
Background technique
Osteosarcoma is one of most common bone malignant tumour, accounts for about the 20-35% of Primary Malignant Bone Tumor, sends out the age well
At 10-25 years old.The grade malignancy of osteosarcoma is high, and early stage may occur in which hematogenous spread, and about 10% patient is medical for the first time
When just shifted, be particularly easy to occur that Lung metastases, osteosarcoma is poor to various anti-tumor drug sensibility in addition,
Prognosis is also poor, and survival rate is low within 5 years, and disability rate is high, and life quality is low.In general, osteosarcoma is from diagnosing to Lung metastases are occurring
Time average out to 10 months, being transferred to the death time from discovery is 6 months, death in 1-2 after most of diagnosis.Although as
Osteosarcoma progress and complex treatment it is universal, therapeutic effect increases.But the correlations such as osteosarcoma generation, recurrence and transfer
Mechanism is unclear, still lacks effective special target therapeutic agent, it would be highly desirable to further study from gene with molecular level.
Tumor stem cell hypothesis is that most one of the theory of attraction, the theory think tumour in the research of oncobiology
It is made of the cell of a group functional heterogeneity, only there is sub-fraction the cancer cell of stem cell properties just to have self-renewing
With the ability for being divided into different tumour cells, maintain the malignant proliferation of tumour, invasion, transfer and in terms of in play certainly
Qualitative effect.The earliest report of tumor stem cell sees leukaemia.There was only CD34 in most of acute granulocytic leukemias+/
CD38-Cell can form leukemic clone in immunodeficient mouse marrow, it was demonstrated that this cell subset has self-renewing
Ability.Since blood cell originates from candidate stem cell, therefore there is certain particularity compared with other solid tissues.In recent years
Come, with the research and development of stem cell surface antigen, the research of solid tumor stem cell also achieves large development.Successfully divide
From and identify solid tumor stem cell further include breast cancer, brain tumor, prostate cancer, melanoma and cancer of pancreas etc., lung cancer
The research of tumor stem cell also achieves huge progress.But osteosarcoma tumor stem-cell research report is actually rare.
Cancer stem-cell hypothesis is that tumor research opens a new visual field, this controls the past for thoroughly overturning tumour
Treat strategy.The theory thinks that the generation of tumour, development are played a leading role by the tumor stem cell for accounting for fraction ratio, and common
Tumour cell compare, this fraction tumor stem cell to chemotherapy, radiotherapy have higher repellence.In following oncotherapy
In, it should be emphasised that for the treatment of tumor stem cell, make every effort to all remove tumor stem cell.
Although osteosarcoma is the most common malignant tumour, the system research about osteosarcoma stem cell is actually rare.
And so far, it there is no and reported in relation to CD24 gene and albumen and any correlation of osteosarcoma stem cell,
Summary of the invention
The purpose of the present invention is to provide a kind of osteosarcoma stem cell molecular marker CD24 and its applications, using osteosarcoma
Stem cell molecular marker diagnoses osteosarcoma disease, Index for diagnosis, has very high specificity and sensitivity.
In order to achieve the above objectives, the technical scheme is that
The present invention has found that the generation of CD24 gene and osteosarcoma is closely related, wherein CD24 by further investigation for the first time+
Cell subsets has the characteristic of self-renewing, Multidirectional Differentiation, has tumor stem cell phenotype.In addition, through detecting, CD24+Subgroup
The expression of the stemness gene such as OCT4, NANOG, SOX2, BMI1 is all remarkably higher than CD24 in cell-Cell subset expresses water
It is flat；CD24 in two plants of osteosarcoma cells+Proliferative capacity, invasive ability, the transfer ability of cell subsets are all significantly stronger than CD24-Carefully
Born of the same parents group.
The present invention provides a kind of osteosarcoma stem cell molecular marker CD24, includes CD24 gene or CAD24 albumen.
The present invention provides osteosarcoma stem cell molecular marker CD24 gene as the molecular marker of detection osteosarcoma
Effect.
The present invention provides osteosarcoma stem cell molecular marker CD24 albumen as the molecular marker of detection osteosarcoma
Effect.
The answering in the tool of preparation diagnosis or prognosis evaluation osteosarcoma the present invention also provides CD24 gene or CAD24 albumen
With the product for providing detection osteosarcoma stem cell molecular marker CD24 expression diagnoses tool or the prognosis of osteosarcoma in preparation
The application assessed in the tool of osteosarcoma utilizes inspection using CD24 gene or CD24 albumen as osteosarcoma stem cell molecular labeling
CD24 gene or CD24 protein expression level in the product testing osteosarcoma tissue sample to be measured of CD24 gene or protein expression are surveyed,
And compared with CD24 gene in normal cancer beside organism or CD24 protein expression level, to diagnose osteosarcoma progression of disease, assessment
Prognosis.
CD24 gene or CD24 the albumen of the present invention high expression in the more normal cancer beside organism of osteosarcoma tissue, especially exist
CD24+Overexpression in cell subset.
Further, the product of the detection osteosarcoma stem cell molecular marker CD24 expression includes RT-PCR, determines in real time
The expression of PCR, immune detection, chip or high-flux sequence detection of platform CD24 gene or CD24 albumen is measured to diagnose bone and flesh
The product of tumor.
Product of the present invention with RT-PCR detection osteosarcoma stem cell molecular marker CD24 expression at least wraps
Include the primer of a pair of of specific amplification CD24 gene；The product with real-time quantitative PCR detection CD24 gene expression dose is extremely
Few includes the primer of a pair of of specific amplification CD24 gene；The product with immune detection CD24 protein expression level includes:
Antibody in conjunction with CD24 protein-specific；The product with chip detection CD24 expression includes: genetic chip and egg
White chip, wherein genetic chip includes the probe with CD24 gene recombination, and the protein chip includes and CD24 protein-specific
In conjunction with antibody.
Preferably, the product with RT-PCR detection osteosarcoma stem cell molecular marker CD2 expression includes one
To the primer of specific amplification CD24 gene, the primer includes: upstream primer: TGAAGAACATGTGAGAGGTTTGAC；Under
Swim primer: GAAAACTGAATCTCCATTCCACAA.
Preferably, the product with RT-PCR detection osteosarcoma stem cell molecular marker CD24 expression further includes
PCR reaction solution, PCR reaction solution is by SYBR Green dyestuff, dNTP, Mg2+, Taq enzyme and Buffer composition.In the PCR reaction solution
Each component and dosage are common knowledge for one of ordinary skill in the art.
The tool of the diagnosis osteosarcoma or the tool of prognosis evaluation osteosarcoma pass through CD24 gene/CD24 in detection sample
The expression of albumen diagnoses osteosarcoma disease.
The tool of diagnosis osteosarcoma of the present invention or the tool of prognosis evaluation osteosarcoma include kit, reagent, chip
Or high-flux sequence platform.The kit includes gene detecting kit and protein immunization detection kit, the gene inspection
Test agent box includes the reagent for detecting CD24 gene transcription level, and the protein immunization detection kit includes CD24 albumen
Specific antibody；The reagent includes the reagent for detecting CD24 gene transcription level；The chip includes genetic chip
And protein-chip, the genetic chip include solid phase carrier and the oligonucleotide probe for being fixed on solid phase carrier, the widow
Nucleotide probe includes the oligonucleotide probe for CD24 gene for detecting CD24 gene transcription level；The protein
Chip include solid phase carrier and be fixed on solid phase carrier CD24 albumen specific antibody；The high-flux sequence platform packet
Include the reagent for detecting CD24 gene transcription level.
Further, the reagent or kit are enzyme linked immunological ELISA kit, chemoluminescence method kit, solid-state
Or liquid chip kit, or other kits prepared according to the method that antibody is coated with detection antigen.
Turn in the tool of diagnosis osteosarcoma of the present invention or the tool of prognosis evaluation osteosarcoma for detecting CD24 gene
The reagent of record level includes the primer of a pair of of specific amplification CD24 gene, and the primer includes: upstream primer:
TGAAGAACATGTGAGAGGTTTGAC；The downstream primer for expanding CD24 gene is GAAAACTGAATCTCCATTCCACAA.
The products application of detection CD24 protein expression of the present invention is in preparation osteosarcoma reagent for clinical diagnosis or reagent
It is the specific antibody (antibody of anti-CD24 albumen) for preparing CD24 albumen in conventional manner when in box, establishes detection CD24 egg
White qualitative or quantitative method and matched reagent or kit.The specific antibody of used CD24 albumen is with conventional side
Method preparation, conventional method refers to that the CD24 albumen for using heterogenous expression or chemically synthesized CD24 polypeptide are immune as antigen
The antibody of experimental animal preparation.The specific antibody of the CD24 albumen includes monoclonal antibody or polyclonal antibody.Described
The qualitative or quantitative method of detection CD24 albumen and matched reagent or kit, are particularly used for vitro detection osteosarcoma tissue
Whether the expression of middle CD24 albumen is abnormal.
Whether the expression of CD24 genes/proteins is abnormal in vitro detection osteosarcoma tissue, is detection osteosarcoma to be measured first
The expression quantity of CD24 albumen in cell tissue is followed by compared with the expression quantity of the CD24 albumen in normal cancer side, finally
Judge this protein molecule marker in osteosarcoma tissue to be measured with the presence or absence of up-regulated expression；Or detection osteosarcoma to be measured
CD24 gene expression dose in CD24 gene expression dose in cell tissue, with normal cancer side carries out statistical with SPSS software
Analysis judges this CD24 gene molecule marker object in osteosarcoma tissue to be measured with the presence or absence of up-regulated expression.
The present invention detects its expression using CD24 genes/proteins as osteosarcoma stem cell molecular marker,
It can be used for instructing the Index for diagnosis of Patients with Osteosarcoma.For example, paraffin section is made to osteosarcoma pathological anatomy, HE dye is being carried out
Color, light microscopic observation osteosarcoma tissue staining conditions carry out histochemistry's scoring；Standards of grading are staining cell number: 0 point
(0%), 1 point (1-10%), 2 points (11-50%) and 3 points (> 50%)；Staining power: 0 point (negative), 1 point (weak), 2
Divide (moderate) and 3 points (strong), 2 kinds of standards of grading institute score value is added to score to the end, finally score range
It is 0-6 point, finally scoring is CD24 low expression group lower than 3 points, and Patients with Osteosarcoma prognosis is preferably postoperative Overall survival and without tumor
Life cycle is longer；It finally scores higher than 3 points for CD24 high expression group, Patients with Osteosarcoma poor prognosis, that is, postoperative Overall survival and without tumor
Life cycle is short.
The present invention also provides CD24 genes to prepare the application in anti-bone and flesh tumor medicine or the anti-bone and flesh tumor medicine of screening.It is described
CD24 gene is screening application in anti-bone and flesh tumor medicine, in particular to candidate drug is being used in osteosarcoma cell, detects
Whether the expression of CD24 gene declines.
The present invention also provides CD24 albumen to prepare the application in anti-bone and flesh tumor medicine or the anti-bone and flesh tumor medicine of screening.It is described
CD24 albumen is screening application in anti-bone and flesh tumor medicine, in particular to candidate drug is being used in osteosarcoma cell, detects
Whether the expression quantity of CD24 albumen declines.
The present invention provides the osteosarcoma stem cell molecular marker CD24 and expands in osteosarcoma stem cell quick separating
Or the application in the method for induction differentiation, the application are using CD24 gene or CD24 albumen as point of osteosarcoma stem cell
Sub- marker isolates CD24 positive osteosarcoma stem cell in conjunction with flow-sorting methods from osteosarcoma tissue.
The present invention also provides the osteosarcoma stem cells of the CD24 positive (i.e. CD24+ cell subsets) to prepare anti-bone and flesh tumor medicine
Or the application in the anti-bone and flesh tumor medicine of screening.
Further, the osteosarcoma stem cell of the CD24 positive be CD24 positive osteosarcoma stem cell primary cell and its
Break up the functional progeny cell generated.
Again, the functional progeny cell that the primary cell of the CD24 positive osteosarcoma stem cell and its differentiation generate is to use
What following methods obtained: by osteosarcoma stem cell root marker CD24, dividing from osteosarcoma in conjunction with the method for airflow classification
Separate out the primary cell of CD24 positive osteosarcoma stem cell；The primary cell of CD24 positive osteosarcoma stem cell further breaks up production
Raw functionality progeny cell.
Beneficial effects of the present invention are as follows:
CD24 gene and albumen provided by the invention are the marks with biological function, under the expression of CD24 genes/proteins
After tune, CD24+Cell subsets is substantially reduced in the invasion of osteosarcoma cell and transfer ability, therefore, CD24 gene and albumen with
Osteosarcoma stem cell is closely related, and can be used CD24 gene and albumen dry to osteosarcoma as osteosarcoma stem cell molecular marked compound
Cell carries out quick separating amplification or induction differentiation, and diagnosis and Index for diagnosis can be carried out to osteosarcoma disease, has specificity
Well, the features such as high sensitivity, and patient can be made according to prognosis evaluation situation, prevents and treats measure using corresponding, is bone and flesh
Diagnosing and/or treating for tumor provides a kind of completely new approach.
The present invention divides osteosarcoma stem cell using CD24 gene and albumen as osteosarcoma stem cell molecular marked compound
From CD24 positive osteosarcoma stem cell i.e. CD24+ cell subsets is obtained after amplification or induction differentiation, this method has quick, special
Property it is good the advantages that, the CD24+ cell subsets isolated has the ability of self-renewing, Multidirectional Differentiation, compares CD24-Subgroup has more
Strong proliferation and invasive ability has stronger tumorigenesis ability, and is a functional surface marker, with Patients with Osteosarcoma
Prognosis it is related, can be used as the pre-warning mark of osteosarcoma early stage Preventive, and can be used as osteosarcoma early stage Preventive
Pre-warning mark can simultaneously serve as the molecular target of osteosarcoma, accelerate the progress of osteosarcoma lung cancer individuation targeted therapy.
Detailed description of the invention
Fig. 1 be in the embodiment of the present invention 1 in 4 plants of osteosarcoma cell lines in parental cell and sphere cells CD24 expression knot
Fruit.
Fig. 2 is osteosarcoma MG-63 and MNNG/HOS cell strain CD24 in the embodiment of the present invention 1+And CD24-Stemness in cell
The expression of results of gene.
Fig. 3 is osteosarcoma MG-63 and MNNG/HOS cell strain CD24 in the embodiment of the present invention 2+And CD24-The proliferation of cell
Situation.
Fig. 4 is osteosarcoma MG-63 and MNNG/HOS cell strain CD24 in the embodiment of the present invention 2+And CD24-The invasion of cell
Situation；A is CD24 under light microscopic+And CD24-The HE violet staining photo of invasion cell, B are that statistic analysis result prompts CD24+
Invasive ability and CD24-Cell, which is compared, has statistical difference.
Fig. 5 is osteosarcoma MG-63 and MNNG/HOS cell strain CD24 in the embodiment of the present invention 2+And CD24-The migration of cell
Situation；A is CD24 under light microscopic+And CD24-The violet staining photo of migrating cell, B are that statistic analysis result prompts CD24+It moves
Shifting ability and CD24-Cell, which is compared, has statistical difference.
Fig. 6 is the invasion situation of osteosarcoma cell after striking low CD24 in the embodiment of the present invention 3；A is that CD24 does not strike under light microscopic
Except the violet staining photo of group (Vector) and CD24 knockout group (CD24-sh1 and CD24-sh2) invasion cell；B is statistics
Analysis result prompt CD24 does not knock out group (Vector) cell invasion ability and CD24 knockout group (CD24-sh1 and CD24-sh2)
Cell, which is compared, has statistical difference.
Fig. 7 is the migration situation of osteosarcoma cell after striking low CD24 in the embodiment of the present invention 3；A is that CD24 does not strike under light microscopic
Except the violet staining photo of group (Vector) and CD24 knockout group (CD24-sh1 and CD24-sh2) migrating cell；B is statistics
Analysis result prompt CD24 does not knock out group (Vector) cell migration ability and CD24 knockout group (CD24-sh1 and CD24-sh2)
Cell, which is compared, has statistical difference.
Fig. 8 is the overall survival phase curve of 87 osteosarcoma CD24 high expression groups and low expression group in the embodiment of the present invention 4
Figure.
Fig. 9 is the Sulfurless fixative curve of 87 osteosarcoma CD24 high expression groups and low expression group in the embodiment of the present invention 4
Figure.
Specific embodiment
The technical scheme of the invention is further explained by means of specific implementation.Those skilled in the art should be bright
, the described embodiments are merely helpful in understanding the present invention, should not be regarded as a specific limitation of the invention.Made in following embodiments
Experimental method is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
1 osteosarcoma CD24 of embodiment+The expression of stemness gene in cell subset
Experimental method:
(1) cell sorting: immunological magnetic bead sorting and flow cytometer showed
Four plants of osteosarcoma cell line MG-63, MNNG/HOS, U-2OS and OSC228 parental cells and corresponding sphere cells
Carry out immunological magnetic bead sorting, the operating guidance Miltenyi Biotec MACS that magnetic bead sorting step is provided according to manufacturer
Cell Separation Kit is carried out, and cell is repeatedly at least pure to reach 95% or more sorting cell three times by sorting column
Degree, final sorting obtain the high-purity C D24 in four plants of osteosarcoma cell line parental cells and corresponding sphere cells+。
Fluidic cell sorting is then carried out using BD FACSAria.It collects cell and removes culture solution, pancreatin digests about 3 points
De- wall is patted in clock, digestion to cell, and 5ml culture solution is added and neutralizes, and fully dispersed cell (it is allowed to as far as possible in single cell suspension, it can
Judged by micro- sem observation, take machine on 100ul cell, measures transfection efficiency, remaining cell numeration, centrifugation.It is imitated according to transfection
Rate, sorting mode (general to select exclusion mode), adjusts cell concentration, reaches 1000-3000/second of loading rate, mesh
300/second of cell (cell concentration (a/ul)=300/ aim cell percentage) on machine, sort cell.Flow cytometer point
Selecting the standard of cell to delimit is 10%, i.e. 10% the brightest cell is selected in positive cell sorting, and negative cells sorting is selected
10% cell of most dark-part.The cell purity that cell after sorting is sorted using the detection of FCM analysis instrument.Use Flowjo
It analyzes software and data analysis is carried out to detection.It is as shown in Figure 1 that fluidic cell sorts testing result.
(2) extraction of total serum IgE
It is cleaned cell 2 times to be measured with the sterile PBS of pre-cooling；After blotting raffinate, by 1mL/cm2Add Trizol lytic cell；
Cell pyrolysis liquid is moved in 2mL centrifuge tube, stands 5min at room temperature；200 μ L chloroforms are added into cell pyrolysis liquid, acutely shake
After swinging, 3min is stood at room temperature；4 DEG C, 12000r/min, it is centrifuged 15min；The upper strata aqueous phase containing RNA is sucked out, moves to another centrifugation
Pipe；Addition and the isometric isopropanol of water, mix gently, stand 10min at room temperature；4 DEG C, 12000r/min, it is centrifuged 10min,
Enable RNA precipitate；Supernatant is abandoned, 75% ethyl alcohol 1mL is added, washs RNA；4 DEG C, 7500r/min, it is centrifuged 8min, abandons supernatant；
RNA precipitate dries 10-20min in air, is dissolved with 20 μ LDEPC water；1 μ LRNA solution is taken, is diluted to 100 μ with distilled water
L, detect its A260nm and A280nm through UV detector and estimate its RNA concentration (C=A260 × 40 × extension rate ×
10-3, unit: μ g/ μ L)；RNA concentration is adjusted to 2 μ g/ μ L with DEPC water, is set -70 DEG C of refrigerators and is saved.
(3) reverse transcription synthesis cDNA (RT)
1 μ L, DEPC H2O of sample total serum IgE 1 μ L, Oligo (dT) primer, 10 μ is sequentially added in 200 μ L centrifuge tubes
L, light to mix, 70 DEG C of 5min, ice-water bath is cooling, is added after centrifugation: 2 μ L, AMV reverse of 5 × Buffer4 μ L, 10mmo/L dNTP
It records enzyme 15U (1 μ L), 1 μ L of RNase inhibitor；37 DEG C, 5min；42 DEG C, 60min；70 DEG C, 10min；Ice bath 5min；From
After the heart, -20 DEG C of preservations.
(4) polymerase chain reaction (PCR)
CDNA template 2.5 μ L, 10mmol/L dNTP0.5 μ L, 25mmol/L are sequentially added in 200 μ L centrifuge tubes
MgCl2 1.5 μ L, 10 × Buffer0.5 μ L, 0.5 μ L of upstream primer, 0.5 μ L of downstream primer, 0.5 μ L of Taq enzyme, sterile water 16.5
μL.Reaction condition: 94 DEG C, 5min；30 circulations: 94 DEG C, 45s, 58 DEG C, 30s, 72 DEG C, 1min；72 DEG C, 7min；4 DEG C of preservations.It takes
10 μ L PCR reaction product electrophoresis on 1.5% agarose, are as a result analyzed by gel imaging system.
Experimental result
As shown in Figure 1, Flow cytometry is as the result is shown: osteosarcoma MG-63 cell strain parental cell and corresponding sphere
The expression rate of CD24 gene is respectively 6.5%, 64.4% in cell, osteosarcoma MNNG/HOS cell strain parental cell and corresponding ball
The expression rate of CD24 is respectively 6.16%, 30% in body cell, U-2OS cells strain parental cell and corresponding sphere cells
The expression rate of middle CD24 is respectively 9%, 30.4%, CD24 in osteosarcoma OSC228 cell strain parental cell and corresponding sphere cells+Expression rate be respectively 1.5%, 65.1%, it is seen then that in four plants of osteosarcoma cell line parental cells and corresponding sphere cells
There are significant difference, expression of the CD24 in osteosarcoma cell line sphere cells is obviously raised for expression in CD24.
The CD24 that MG-63 and MNNG/HOS cell strain is sorted out by qRT-PCR method+Cell subset, CD24-It is sub-
Group's cell has carried out the contrasting detection of the stemness gene level such as OCT4, NANOG, SOX2, BMI1, and testing result is as shown in Fig. 2, knot
Fruit shows osteosarcoma CD24+The expression of OCT4, NANOG, SOX2, BMI1 gene is all remarkably higher than it in CD24 in cell-Carefully
Expression in born of the same parents.
2 osteosarcoma CD24 of embodiment+Subgroup has stronger proliferation, invasion and transfer ability
Experimental method:
(1) cell sorting
With embodiment 1
CD24 is sub-elected from MG-63 and MNNG/HOS cell strain+Subgroup and CD24-Cell subset.
(2) ability of cell proliferation is tested
The CD24+ subgroup and CD24- cell subset suspension of 100 μ L are separately added into 96 orifice plates.Culture plate is being cultivated
Case preculture 24 hours (37 DEG C, 5%CO2).It can choose in the periphery of 96 orifice plates and PBS be added, prevent liquid during the cultivation process
Evacuator body is excessive.Culture plate is incubated for different periods (1,2,3,4,5day) in incubator.Discard culture medium, and with culture
Base washs cell twice, and new 100ul culture medium is then added, and 10 μ LCCK8 solution are added to every hole (preferably will according to 1:10
CCK8 reagent and culture medium are added after mixing).Culture plate is incubated for 1-4h in incubator.It is measured with microplate reader in 450nm
The absorbance at place.
Experimental result
The proliferative capacity of CD24+ cell subsets is had detected in MG63 and MNNG cell, as a result as shown in Figure 3.It can by Fig. 3
Know, the proliferative capacity of CD24+ cell subsets is all significantly stronger than CD24- cell mass in osteosarcoma cell MG63 and MNNG.
(3) cell invasion and migration experiment
Transwell, 24 well culture plates, Matrigel, 1640 culture medium of serum-free and suction nozzle be placed in 4 DEG C of refrigerator overnights by
The ratio mixing Matrigel and 1640 culture medium of serum-free of 1:3 is to prepare artificial basement membrane；Transwell is placed in the training of 24 holes
It supports in plate, the mixed liquor of 50 μ L is respectively added in upper chamber, 37 DEG C of incubation 2h are so that it is solidified；Upper and lower room be separately added into 100 μ L and
1640 culture medium of serum-free of 600 μ L, 37 DEG C of incubation 8h；By 1 × 106The concentration of/ml will be in the G1 and G2 of logarithmic growth phase
Cell is prepared into single cell suspension with 1640 culture medium of serum-free；The training liquid for discarding each room up and down is added 100 μ L's in upper chamber
Cell suspension (1 × 105A cell), serum-free 1640 culture medium of the 600 μ L containing 5%fibronectin, every room is added in lower room
If 3 controls；In 37 DEG C, 5%CO2Condition in cultivate for 24 hours；Upper chamber is taken out, after the cell for failing invasion with cotton swab erasing,
30min, conventional H E dyeing, as a result referring to Fig. 5 A are fixed with neutral formalin；Under 200 times of light microscopics, randomly selects 5 visuals field and count
Infiltrating cells are averaged and are compared, as a result referring to Fig. 5 B.
Cell invasion is very close with migration experimental material method, and the Matrigel that do not exist together uniquely is on polycarbonate membrane
Room side spreads a floor Matrigel matrigel, to parody extracellular matrix.
Experimental result
The CD24 that MG-63 and MNNG/HOS cell strain is sorted out+Subgroup and CD24-Cell subset carried out invasion and
Transfer ability detection, as a result respectively as shown in fig. 4-5.By Fig. 4 A, Fig. 4 B it is found that in MG-63 and MNNG cell, CD24+
The invasive ability of cell is significantly stronger than CD24-Subgroup (MG63:P < 0.05；MNNG:P < 0.001).By Fig. 5 A, Fig. 5 B it is found that this
CD24 in two kinds of cells+The transfer ability of subgroup is equally significantly higher than CD24-Subgroup (P < 0.001).
3 CD24 of embodiment influences the biological function of osteosarcoma cell as a target spot
Experimental method:
(1) slow virus carrier building, slow virus packaging and cell infection
The sequence for going out CD24 with PCR amplification from human gene group DNA is cloned into Lentiviral PLVX (limitation
Property restriction enzyme site be BamH and EcoR I), recombinant plasmid is named as PLVX-CD24.The recombinant plasmid of interference CD1 is named as
PLVX-CD24-shRNA.The present embodiment devises two sh sequences and is respectively as follows: CD24sh-1:TACTGGAACATCTAAGCAT；
CD24sh-2:TTAATATTGGCATCCATCA.It will with Lipofectamine2000 (Invitrogen) when slow virus is packed
PLVX-CD1/PLVX-CD1-shRNA, packaging plasmid psPAX2 and envelope plasmid pMD2.G corotation
It contaminates in 293T cell, obtains virion.The titre of virus is measured in HEK 293T cell with serial dilution.With measurement
Virus infected cell afterwards adds 6 μ g/mL polybrene, then determines infectious effect according to fluorescence intensity.
(2) cell invasion and migration experiment
Transwell, 24 well culture plates, Matrigel, 1640 culture medium of serum-free and suction nozzle be placed in 4 DEG C of refrigerator overnights by
The ratio mixing Matrigel and 1640 culture medium of serum-free of 1:3 is to prepare artificial basement membrane；Transwell is placed in the training of 24 holes
It supports in plate, the mixed liquor of 50 μ L is respectively added in upper chamber, 37 DEG C of incubation 2h are so that it is solidified；Upper and lower room be separately added into 100 μ L and
1640 culture medium of serum-free of 600 μ L, 37 DEG C of incubation 8h；By 1 × 106The concentration of/ml (walks the cell in logarithmic growth phase
Suddenly the virus infected cell that (1) obtains) with 1640 culture medium of serum-free it is prepared into single cell suspension；Discard the training of each room up and down
The cell suspension (1 × 10 of 100 μ L is added in liquid in upper chamber5A cell), 600 μ L are added in lower room containing 5%fibronectin
1640 culture medium of serum-free, every room sets 3 controls；In 37 DEG C, 5%CO2Condition in cultivate for 24 hours；Upper chamber is taken out, cotton swab is used
After son erasing fails the cell of invasion, 30min, conventional H E dyeing are fixed with neutral formalin；Under 200 times of light microscopics, 5 are randomly selected
A visual field counts infiltrating cells, is averaged and is compared.
Cell invasion is very close with migration experimental material method.The Matrigel that do not exist together uniquely is on polycarbonate membrane
Room side spreads a floor Matrigel matrigel, to parody extracellular matrix.
Experimental result:
In order to observe, CD24 subgroup invades entire osteosarcoma cell line and the influence of transfer ability, the present invention interfere downward
The expression of CD24 in MG63 and MNNG, after CD24 expression is lowered, in the invasion of MG63 and MNG cell and transfer ability detection knot
Fruit difference is as shown in Figure 6, Figure 7.
By Fig. 6, Fig. 7 it is found that the invasion of MG63 and MNNG cell and transfer ability obviously drop after CD24 expression downward
It is low.Although CD24 in this explanation MG63 and MNNG cell+Not high (the CD24 in MG63 and MNNG cell of cell subsets proportion
Expression rate is respectively 6.5%, 6.16%), but this sub-fraction CD24+Cell subsets is to the invasion of osteosarcoma cell line and moves
Move ability orientation important role.And CD24 is the functional surface marker of tool, strikes meeting after low or knockout CD24
The change for leading to osteosarcoma cell biological function can be used for the target spot of clinical treatment osteosarcoma.
The expression of embodiment 4CD24 is related to clinical Patients with Osteosarcoma prognosis
Experimental method:
(1) sample paraffin section immunohistochemistry and histology
Typical 4 μm of serial section of osteosarcoma pathological tissue paraffin mass, conventional dewaxing to water；Distilled water flushing slice, PBS
5min is impregnated, 50 μ L normal serum working solutions are added dropwise, 15min is incubated at room temperature, discards, 50 μ L diluted one are added dropwise in every slice
It is anti-, 37 DEG C of incubation 1-2h；PBS is rinsed, 3min × 3 time；The secondary antibody of 50 μ L biotin labelings, 37 DEG C of incubation 10-15min are added dropwise；
PBS is rinsed, 3min × 3 time；50 μ L horseradish enzymes label streptomysin avidin working solution, 37 DEG C of incubation 10-15min are added dropwise；PBS punching
It washes, 3min × 3 time；The DAB solution of every slice plus 100 μ L Fresh, microscopically observation 3-10min；Tap water is abundant
It rinses, haematoxylin is redyed, neutral gum mounting.Negative control replaces primary antibody with PBS.
Paraffin section is placed in 62 DEG C of baking ovens and bakes 2h；Dimethylbenzene dewaxing, ethyl alcohol aquation, PBS wash 3 × 3min；It will slice
It is placed in 0.01mol/L sodium citrate antigen retrieval buffers (pH6.0) and carries out hot repair again (95 DEG C, 15min), keep the temperature 15min, it is cooling
To room temperature, PBS washes 3 × 3min；3%H2O2Endogenous peroxydase is inhibited to be incubated at room temperature 20min；PBS washes 3 × 3min；Serum
37 DEG C of incubation 30min；The CD24 with FITC mark is added, room temperature is incubated for 30min in the place of being protected from light；PBS washes 3 × 3min；DAPI
Dye 20min；PBS washes 3 × 3min；Anti- fluorescence quenching mounting.It is observed under fluorescence microscope.OL image software photographic analysis.
The routine techniques such as histologic analysis is commonly fixed, embedded, being sliced and HE is dyed and bone tissue embed at preceding decalcification
Reason is carried out with reference to the method discussed in existing document.
(2) CD24 expression and patient's prognostic analysis in Patients with Osteosarcoma tissue samples
Histochemistry's scoring (histochemistry score) is to handle a kind of histological score of ImmunohistochemistryResults Results
Method converts corresponding numerical value for cell quantity and its staining power positive in every slice, reaches to tissue staining half
Quantitative purpose.
Each case paraffin section of selected survival analysis takes 10 high power fields (× 400) to take pictures at random, root
Analysis scoring is carried out using IPP software according to CD24 staining cell number and dyeing power.Standards of grading are staining cell number: 0 point
(0%), 1 point (1-10%), 2 points (11-50%) and 3 points (> 50%).Staining power: 0 point (negative), 1 point (weak), 2
Divide (moderate) and 3 points (strong), 2 kinds of standards of grading institute score value is added to score to the end, finally score range
It is 0-6 points, finally scoring is CD24 low expression group lower than 3 points；It is CD24 high expression group that finally scoring, which is higher than 3 points,.
Experimental result
The present invention analyzes pass of the expression with other Clinical symptoms and life cycle of CD24 in Patients with Osteosarcoma sample
Overall survival phase curve graph (as shown in Figure 8) is drawn and without tumor according to the clinical data to above-mentioned 87 patient's Follow-up Afters by system
Life cycle curve graph (as shown in Figure 9),
Present invention discover that operation excision sample in CD24 express high case there will be it is higher recurrence or transfer wind
Danger, and CD24 and 3 years Overall survivals and 3 years Sulfurless fixatives are significant related, by Fig. 8-Fig. 9 it is found that CD24 expression is high
Case Overall survival and Sulfurless fixative be substantially less than CD24 and express low case.
Therefore, CD24 is marked as osteosarcoma specific diagnosis with the detection of this kit, by with normal in kit
Control sample, i.e. standard items most compare, and can clearly distinguish the Patients with Osteosarcoma of the CD24 positive, and examine as clinic offer
Break rope.The characterization of molecules of tumour is introduced into osteosarcoma survival region appraisement system, it can be more accurate on the basis of this
It assesses patient to recur in the future and the risk of transfer, whether to postoperative chemotherapy, radiotherapy sensitive, thus more if more accurately assessing the patient
It targetedly proposes therapeutic scheme, improves the prognosis of Patients with Osteosarcoma.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent defines.
Claims (10)
1. a kind of osteosarcoma stem cell molecular marker CD24, which is characterized in that the osteosarcoma stem cell molecular marker
CD24 is CD24 gene or CD24 albumen.
2. osteosarcoma stem cell molecular marker CD24 as described in claim 1 is as the molecular marker for detecting osteosarcoma
Using.
3. osteosarcoma stem cell molecular marker CD24 as described in claim 1 is in preparation diagnosis or prognosis evaluation osteosarcoma
Application in tool.
4. application according to claim 3, which is characterized in that detect osteosarcoma stem cell molecule described in claim 1
Application of the product of marker CD24 expression in the tool of preparation diagnosis or prognosis evaluation osteosarcoma.
5. application according to claim 4, which is characterized in that detection osteosarcoma stem cell described in claim 1
The product of molecular marker CD24 expression includes RT-PCR, real-time quantitative PCR, immune detection, chip or high-flux sequence platform
The expression of detection molecules marker CD24 is to diagnose the product of osteosarcoma.
6. application according to claim 5, which is characterized in that described to express water with RT-PCR detection molecules marker CD24
Flat product includes at least the primer of a pair of of specific amplification CD24 gene；It is described to use real-time quantitative PCR detection molecules marker
The product of CD24 expression includes at least the primer of a pair of of specific amplification CD24 gene；It is described to use immune detection molecular labeling
The product of object CD24 expression includes: the antibody in conjunction with CD24 protein-specific；It is described to use chip detection molecules marker
The product of CD24 expression includes: genetic chip and protein chip, wherein genetic chip includes the spy with CD24 gene recombination
Needle, the protein chip include the antibody in conjunction with CD24 protein-specific.
7. application according to claim 6, which is characterized in that described to express water with RT-PCR detection molecules marker CD24
Flat product includes the primer of a pair of of specific amplification CD24 gene, and the primer includes: upstream primer:
TGAAGAACATGTGAGAGGTTTGAC；Downstream primer: GAAAACTGAATCTCCATTCCACAA.
8. application according to claim 3, which is characterized in that the tool of the diagnosis or prognosis evaluation osteosarcoma passes through inspection
The expression of molecular marked compound CD24 diagnoses osteosarcoma disease in test sample sheet.
9. the application according to claim 3 or 8, which is characterized in that the kit of the diagnosis or prognosis evaluation osteosarcoma
Include kit, reagent, chip or high-flux sequence platform.
10. application according to claim 9, which is characterized in that the kit includes gene detecting kit and albumen
Immunity detection reagent, the gene detecting kit include the reagent for detecting CD24 gene transcription level, the albumen
Immunity detection reagent includes the specific antibody of CD24 albumen；The reagent includes for detecting CD24 gene transcription level
Reagent；The chip includes genetic chip and protein-chip, and the genetic chip includes solid phase carrier and is fixed on solid phase
The oligonucleotide probe of carrier, the oligonucleotide probe include for detect CD24 gene transcription level for CD24 gene
Oligonucleotide probe；The protein-chip include solid phase carrier and be fixed on solid phase carrier CD24 albumen specificity
Antibody；The high-flux sequence platform includes the reagent for detecting CD24 gene transcription level.
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Inventor after: Zhou Zhenhua Inventor after: Cao Jiashi Inventor after: Xiao Jianru Inventor after: Hu Shuo Inventor after: Wang Xudong Inventor after: Zhang Weiwei Inventor after: Kuang Muyu Inventor after: Gong Dejun Inventor after: Jia Qi Inventor after: Li Yan Inventor before: Zhou Zhenhua Inventor before: Cao Jiashi Inventor before: Xiao Jianru Inventor before: Hu Shuo Inventor before: Wang Xudong Inventor before: Zhang Weiwei Inventor before: Kuang Muyu Inventor before: Gong Dejun Inventor before: Jia Qi Inventor before: Li Yan |
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Application publication date: 20190416 |