CN107119142A - A kind of quick detection passes on the kit of stem cell vicious transformation - Google Patents

A kind of quick detection passes on the kit of stem cell vicious transformation Download PDF

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CN107119142A
CN107119142A CN201710483461.2A CN201710483461A CN107119142A CN 107119142 A CN107119142 A CN 107119142A CN 201710483461 A CN201710483461 A CN 201710483461A CN 107119142 A CN107119142 A CN 107119142A
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stem cell
target gene
gene
kit
primers
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喻堃
徐国锋
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CHENGDU GENCELLTECH BIOTECHNOLOGY Co Ltd
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CHENGDU GENCELLTECH BIOTECHNOLOGY Co Ltd
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Priority to CN202010830660.8A priority patent/CN111926079A/en
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

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Abstract

The invention provides the kit that a kind of quick detection passes on stem cell vicious transformation, it is characterised in that the kit includes the corresponding primer of tetra- target gene of P16, RASSF1A, GSTP1 and CDH1 and reference gene β actin primers.The present invention has the advantages that simple to operate, accuracy is good and sensitiveness is high in terms of stem cell vicious transformation is detected.

Description

A kind of quick detection passes on the kit of stem cell vicious transformation
Technical field
The invention belongs to stem cells technology and diagnosing tumor field, and in particular to a kind of quick detection passage stem cell is pernicious The kit of conversion.
Background technology
At present, stem cell development is like a raging fire.The development in stem-cell therapy field, will help the mankind realize repair wound and Pathological tissue, cures the dream of whole Terminal Disease, is expected to solve many significant medical problems of facing mankind.
The mechanism of tumour and the origin of cell are the focus and difficult point of oncology studies now.Tumor stem cell is theoretical Think, tumour originates from the evil of stem cell related to its own tissue or organ and with its own tissue or organ specificity Property conversion.Tumour is likely due to stem cell producer during long-term self-renewing and is mutated, and regulation and control are dry The signal transduction pathway part of cell proliferation and differentiation changes, and stem cell is disturbed during Proliferation, Differentiation by abnormal signal And canceration is tumor stem cell, then hyper-proliferative forms tumour, and stem cell is often the target cell of vicious transformation.But, Which kind of produced actually by stem cell vicious transformation specific to certain tumour, the research of current this respect is also fewer.
DNA methylation is an important epigenetics mechanism, plays important in human cancer develops Effect.The promoter zone methylation of specific gene causes the extensive concern of people extremely, it is considered to be cancer diagnosis it is potential Mark.There was only the independent gene of only a few in cancer, to be proved to be the ratio of methylating very high.Therefore, one group of gene is detected Methylation state, compared with detecting the methylation state of independent gene, diagnostic sensitivity is higher, it is specific more preferably.To moderns Have studied the methylome of kinds cancer.For a preferable methylome, accurate detection method and logic Data analysis be all essential.2012, a kind of method for the methylation level for counting multiple genes is had been set up, Cancer sample and the progressively cumulative analysis of normal sample are carried out according to the methylation level of candidate gene.With former statistical method Compare, this method has taken into full account methylation and the influence of different genes, however, this method is needed to great amount of samples Statistical analysis is carried out, therefore is difficult to apply to the diagnosis of instant single sample or a small amount of sample.
The content of the invention
In view of the shortcomings of the prior art, the invention provides a kind of reagent that simple to operate, accuracy is good and sensitiveness is high Box.The kit includes the corresponding primer of tetra- target gene of P16, RASSF1A, GSTP1 and CDH1 and reference gene β-actin Primer, specific primer sequence is as follows：
Target gene P16 forward primers：TTATTAGAGGGTGGGGCGGATCGC
Target gene P16 reverse primers：GACCCCGAACCGCGACCGTAA
Target gene RASSF1A forward primers：GACCTCTGTGGCGACTTCATCTG
Target gene RASSF1A reverse primers：GACCTAGTCCTCGGGAGCTGTC
Target gene GSTP1 forward primers：TTCGGGGTGTAGCGGTCGTC
Target gene GSTP1 reverse primers：GCCCCAATACTAAATCACGACG
Target gene CDH1 forward primers：TTAGGTTAGAGGGTTATCGCGT
Target gene CDH1 reverse primers：TAACTAAAAATTCACCTACCGAC
Reference gene β-actin forward primers：GTGATGGAGGAGGTTTAG
Reference gene β-actin reverse primers：AAATTACAAAAACCACAA.
Also include the corresponding probe of four target gene P16, RASSF1A, GSTP1 and CDH1 methylation sites and The corresponding probes of reference gene β-actin, specific nucleotide sequence is as follows：
Target gene P16 probes：FAM-AGTAGTATGGAGTCGGCGGCGGG-TAMRA
Target gene RASSF1A probes：FAM-CCCTCTGCCGCGACTTGACCCG-TAMRA
Target gene GSTP1 probes：FAM-CGGGGTGTAGCGGTCG-TAMRA
Target gene CDH1 probes：FAM-TCGCGGGGTTCGTTTTTCGC-TAMRA
Reference gene β-actin probes：FAM-CACCACCCAACACACAAT-TAMRA.
Kit of the present invention also includes PCR reaction solutions, bisulfite.
PCR reaction solutions of the present invention include archaeal dna polymerase, dNTPs, 10 × PCR buffer solutions and Mg2+。
The stem cell includes tumor stem cell, umbilical cord mesenchymal stem cells, placenta mesenchyma stem cell, fat mesenchymal Any one or more in stem cell.
The tumor stem cell includes one in lung cancer stem cell, stomach cancer stem cell, liver-cancer stem cell, colon cancer stem cell Plant or a variety of.
Beneficial effects of the present invention：
The present invention has the advantages that simple to operate, accuracy is good and sensitiveness is high in terms of stem cell vicious transformation is detected.
Brief description of the drawings
Fig. 1 is RT-PCR method target gene and internal memory gene magnification curve map；
Fig. 2 is that cellular morphology after vicious transformation stem cell is cultivated 20 days does not occur；
Cellular morphology after Fig. 3 is cultivated 90 days for generation vicious transformation stem cell.
Embodiment
The present invention is specifically described below by embodiment, it is necessary to it is pointed out here that be following examples be use It is further detailed in the present invention, it is impossible to be interpreted as limiting the scope of the invention, the field is skilled in technique Some nonessential modifications and adaptations that personnel are made according to foregoing invention content, still fall within protection scope of the present invention.
Embodiment 1
The Taq enzyme that the present embodiment is used is purchased from the precious biotech firm (TaKaRa) in Dalian；Cellular genome extracts kit is AxyPrepTM Multisource Genomic DNAMiniprep Kit(Axygen Scientific,Inc.CA,USA)； Sulfiding reagent box is, EZ DNA Methylation kit (Zymo Research, Orange, CA, USA)；Other reagents are state Produce AR.
The stem cell that the stem cell that the present embodiment is used separates for various cancers tissue.
Primer involved by the present embodiment is：
Target gene P16 forward primers：TTATTAGAGGGTGGGGCGGATCGC
Target gene P16 reverse primers：GACCCCGAACCGCGACCGTAA
Target gene RASSF1A forward primers：GACCTCTGTGGCGACTTCATCTG
Target gene RASSF1A reverse primers：GACCTAGTCCTCGGGAGCTGTC
Target gene GSTP1 forward primers：TTCGGGGTGTAGCGGTCGTC
Target gene GSTP1 reverse primers：GCCCCAATACTAAATCACGACG
Target gene CDH1 forward primers：TTAGGTTAGAGGGTTATCGCGT
Target gene CDH1 reverse primers：TAACTAAAAATTCACCTACCGAC
Reference gene β-actin forward primers：GTGATGGAGGAGGTTTAG
Reference gene β-actin reverse primers：AAATTACAAAAACCACAA.
Method：
One, stem cells extract DNA flow (DNA extraction kit, AxyPrepTM Multisource Genomic DNA Miniprep Kit(Axygen Scientific,Inc.CA,USA))
1. centrifuge cell：2000 revs/min of centrifuge cell suspensions 2 minutes, collect cell, outwell supernatant liquid, add 4 DEG C The PBS 200ul of precooling, 2000 revs/min of centrifuge cell suspensions 2 minutes, collect cell, outwell supernatant liquid, add 200ulPBS dissolves cell.
2. adding 25ul OB protease lysates, mix.
3. add 220ul BLBuffer
4.70 DEG C of water-bath 10min.
5. 220ul absolute ethyl alcohols are added to mix
6. being put into post is collected in 2ml collecting pipes, the liquid in step 5 is added and collected in post,
7. maximum (top) speed centrifuges 1min, waste liquid in collecting pipe is outwelled more than or equal to 10000 turns
8. 500ul HBC Buffer maximum (top) speeds (being more than or equal to 10000 turns) centrifugation 30s is added in collecting pipe,
9. add 700ul DNA Wash Buffer maximum (top) speeds (being more than or equal to 10000 turns) centrifugation 30s (2 times)
10. pipe maximum (top) speed (being more than or equal to 10000 turns) centrifugation 2min is collected by centrifugation in sky
11. add 100ul dd water (being heated to 70 DEG C)
12. standing 2min, big rotating speed (being more than or equal to 10000 turns) centrifugation 1min collects DNA, determines concentration.
2nd, DNA vulcanizes flow (DNA sulfiding reagent boxes, the EZ DNAMethylation-GoldTM Kit of ZYMO companies catalog Nos.D5005&D5006)
1. before being applied for CT conversion Reagent, need 900 μ l water of often pipe addition, 300 μ l M-Dilution Buffer, 50 μ l M-Dissolving Buffer to CT conversion Reagent；
2. DNA amount used in vulcanization is 500ng, the DNA volumes according to added by being calculated quality and concentration, 130 μ l are added CT-CR is into 20 μ l DNA samples, if DNA sample volume is less than 20 μ l, and the benefit that adds water flicks test tube to 20 μ l after mixing, Then low-speed centrifugal is by solution to ttom of pipe；
3. the solution prepared is entered into thermal cycle：
a.98℃10min
b.64℃2.5h
4. adding 600 μ l M-Binding Buffer to Zymo-Spin IC Column, Collection is then placed in Tube；
5. the DNA solution obtained by the 2nd step is put into Zymo-Spin IC Column, closes the lid and overturn Column repeatedly For several times；
6. (being more than 10,000g) at a high speed centrifuges 30s, filtrate is abandoned；
7. adding 100 μ l M-Wash Buffer to Column, high speed centrifugation 30s, filtrate is abandoned；
8. 200 μ l M-Desulphonation Buffer to Column are added, (20-30 DEG C) standing 15- of room temperature High speed centrifugation centrifuges 30s after 20min, incubation, abandons filtrate；
9. add 200 μ l M-Wash Buffer to Column, high speed centrifugation 30s；Add 200 μ l M-Wash Buffer to Column, high speed centrifugation 30s, abandons filtrate；
10. by Column to 1.5ml microcentrifuge tube, add 15 μ l M-Elution Buffer extremely Column, high speed centrifugation 30s, eluted dna.
(the fluorescent quantitative detector device used is the 7500fast of Invitrogen companies to three .RT-PCR flows, is used Reagent is the Platinum Taq DNA polymerases of Invitrogen companies, and article No. is C10966-034；Reaction system is 20 μ l)
Shown in the preparation of RT-PCR reaction systems and the following Tables 1 and 2 of reaction condition：
The preparation of table 1RT-PCR reaction systems
The RT-PCR reaction conditions of table 2
4th, Analysis of test results
20 cancer stem cell samples are detected using above-mentioned reaction system, this 20 stem cell samples use methyl Change specific PCR method and verified that corresponding gene all there occurs different journeys to P16, RASSF1A, GSTP1, CDH1 gene Methylating for degree (when four genes while methylating, then assert that stem cell has deteriorated conversion, following " methylating " Four genes are referred both to while methylating), including 7 lung cancer stem cells, (wherein 5 methylate, and 2 without generation first Base), 5 stomach cancer stem cells (wherein 3 methylate, 2 no methylate), 5 liver-cancer stem cells (wherein 4 Example methylates, and 1 no to methylate), 3 colon cancer stem cells (3 all methylate)；Utilize the present invention's Kit testing result is as shown in table 3 and Fig. 1.
The RT-PCR method of table 1 detection cancer stem cell methylates result
It can fast and accurately identify whether passage stem cell occurs vicious transformation using the kit of the present invention, it is thin to do The safe handling of born of the same parents provides technical guarantee.
5th, stem cell long-term cultivation is tested
The authenticity for occurring the stem cell of vicious transformation to verify RT-PCR method to confirm, to above-mentioned 20 stem-like cells This progress long-term in vitro Secondary Culture.Method is as described below：
1 stem cell long-term in vitro Secondary Culture
(1) after where RT-PCR method detection for stem cell length to 80~90% fusions, passage is proceeded by；
(2) old culture medium is suctioned out, 1 × PBS is washed twice, adds 0.25% trypsase/EDTA digestion 30s, treat that cell is returned Contracting is rounded, and fresh culture is added immediately and terminates digestion, is gently blown and beaten；
(3) cell suspension after piping and druming is transferred into 400g after 10ml centrifuge tubes, trim to centrifuge 10 minutes；
(4) take the cell of precipitation with H-DMEM culture mediums dilute after with 1:5 ratio is reached in blake bottle or culture plate, and 2 Change liquid within~3 days, when cell covers with into fine and close individual layer, can again be passed on according to upper method.
2 cell growth curve MTT colorimetric methods
MTT colorimetric methods, are a kind of methods for detecting cell survival and growth.MTT Cleaning Principles is in living cells mitochondrias Succinate dehydrogenase bluish violet crystallization that exogenous MTT can be made to be reduced to water-insoluble first a ceremonial jade-ladle, used in libation and be deposited in cell, it is and dead Cell is without this function.Dimethyl sulfoxide (DMSO) (DMSO) can dissolve the first a ceremonial jade-ladle, used in libation in cell, with enzyme-linked immunosorbent assay instrument at 490nm wavelength Its absorbance value is determined, living cells quantity can be reflected indirectly.In the range of certain cell number, MTT crystallizes the amount to be formed and cell Number is directly proportional.
(1) inoculating cell：0.25% trypsin digestion and cell is used, individual cells suspension is configured to basal medium, With every 104Individual cell is inoculated in 6 piece of 96 orifice plate, per pore volume 200ul.
(2) cell is cultivated：6 pieces of culture plates are put into 37 DEG C, 5%CO2Cultivated under the conditions of saturated humidity.Incubation time is successively It is decremented to 1 day from 6.
(3) colour generation：One piece of 6 orifice plate is taken out daily, 5mg/ml MTT solution 20ul is added per hole, it is small that 37 DEG C of continuation are incubated 4 When terminate culture, careful inhale abandons culture supernatant in hole.150ul DMSO are added per empty, 10min is shaken, fills bluish violet knot thing Divide dissolving.
(4) colorimetric：490nm wavelength is selected, each hole absorbance value is determined on enzyme-linked immunosorbent assay instrument.
(5) draw：With the time (my god) be transverse axis, absorbance value (A) be the longitudinal axis, use Excel Software on Drawing cell growths Curve.
3 stem cell Long Term Passages cultivation results
5 do not methylate stem cell under basal medium long-term in vitro culture, and biological property is basically identical, put down The equal time-to-live is 32 days, and cell growth rate is gradually slack-off with passing on, and the average cell doubling time extended to 3.8 from 1.8 days My god, all stem cells not methylated remain fibroblast-like cellses form and engender that aging is tested, finally Apoptosis.As a result it is as shown in Figure 2.
15 methylate stem cell under basal medium long-term in vitro culture, and mean survival time is substantially elongated (average time is more than 90 days), after culture to 10-20 day after tomorrow, vitro growth rates are substantially accelerated, mean doubling time Foreshortened to from 3.5 days 1.6 days, cell density increase loses normal contact inhibition, in Multi layer Growth, methylated Growth of cancer cells feature is presented in stem cell in incubation, can cross the apoptosis phase, time-to-live, growth rate and cell shape The biological properties such as state change.As a result it is as shown in Figure 3.
Stem cell long-term cultivation result of the test shows occur the dry thin of vicious transformation by what kit of the present invention was detected There are the growth characteristics of cell carcinogenesis during long-term cultivation in born of the same parents, and the detection for further demonstrating kit of the present invention is accurate Property, high efficiency.
Sequence table
SEQUENCE LISTING
<110>Chengdu Rui Jiesen bio tech ltd
<120>A kind of quick detection passes on the kit of stem cell vicious transformation
<130> 2017
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 24
<212> DNA
<213>Target gene P16 forward primers
<400> 1
ttattagagg gtggggcgga tcgc 24
<210> 2
<211> 21
<212> DNA
<213>Target gene P16 reverse primers
<400> 2
gaccccgaac cgcgaccgta a 21
<210> 3
<211> 23
<212> DNA
<213>Target gene RASSF1A forward primers
<400> 3
gacctctgtg gcgacttcat ctg 23
<210> 4
<211> 22
<212> DNA
<213>Target gene RASSF1A reverse primers
<400> 4
gacctagtcc tcgggagctg tc 22
<210> 5
<211> 20
<212> DNA
<213>Target gene GSTP1 forward primers
<400> 5
ttcggggtgt agcggtcgtc 20
<210> 6
<211> 22
<212> DNA
<213>Target gene GSTP1 reverse primers
<400> 6
gccccaatac taaatcacga cg 22
<210> 7
<211> 22
<212> DNA
<213>Target gene CDH1 forward primers
<400> 7
ttaggttaga gggttatcgc gt 22
<210> 8
<211> 23
<212> DNA
<213>Target gene CDH1 reverse primers
<400> 8
taactaaaaa ttcacctacc gac 23
<210> 9
<211> 18
<212> DNA
<213>Reference gene β-actin forward primers
<400> 9
gtgatggagg aggtttag 18
<210> 10
<211> 18
<212> DNA
<213>Reference gene β-actin reverse primers
<400> 10
aaattacaaa aaccacaa 18

Claims (6)

1. a kind of quick detection passes on the kit of stem cell vicious transformation, it is characterised in that the kit include P16, The corresponding primer of tetra- target gene of RASSF1A, GSTP1 and CDH1 and reference gene β-actin primers, specific primer sequence is such as Under：
Target gene P16 forward primers：TTATTAGAGGGTGGGGCGGATCGC
Target gene P16 reverse primers：GACCCCGAACCGCGACCGTAA
Target gene RASSF1A forward primers：GACCTCTGTGGCGACTTCATCTG
Target gene RASSF1A reverse primers：GACCTAGTCCTCGGGAGCTGTC
Target gene GSTP1 forward primers：TTCGGGGTGTAGCGGTCGTC
Target gene GSTP1 reverse primers：GCCCCAATACTAAATCACGACG
Target gene CDH1 forward primers：TTAGGTTAGAGGGTTATCGCGT
Target gene CDH1 reverse primers：TAACTAAAAATTCACCTACCGAC
Reference gene β-actin forward primers：GTGATGGAGGAGGTTTAG
Reference gene β-actin reverse primers：AAATTACAAAAACCACAA.
2. kit according to claim 1, it is characterised in that also including four target gene P16, RASSF1A, The corresponding probe of GSTP1 and CDH1 methylation sites and the corresponding probes of reference gene β-actin, specific nucleotide sequence is such as Under：
Target gene P16 probes：FAM-AGTAGTATGGAGTCGGCGGCGGG-TAMRA
Target gene RASSF1A probes：FAM-CCCTCTGCCGCGACTTGACCCG-TAMRA
Target gene GSTP1 probes：FAM-CGGGGTGTAGCGGTCG-TAMRA
Target gene CDH1 probes：FAM-TCGCGGGGTTCGTTTTTCGC-TAMRA
Reference gene β-actin probes：FAM-CACCACCCAACACACAAT-TAMRA.
3. kit according to claim 1 or 2, it is characterised in that also including PCR reaction solutions, bisulfite.
4. kit according to claim 3, it is characterised in that the PCR reaction solutions include archaeal dna polymerase, dNTPs, 10 × PCR buffer solutions and Mg2+。
5. kit according to claim 1, it is characterised in that the stem cell includes filling between tumor stem cell, umbilical cord Any one or more in matter stem cell, placenta mesenchyma stem cell, fat mesenchymal stem cell.
6. kit according to claim 5, it is characterised in that the tumor stem cell includes lung cancer stem cell, stomach cancer One or more in stem cell, liver-cancer stem cell, colon cancer stem cell.
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CN109097474A (en) * 2018-08-29 2018-12-28 天津智鱼生物科技有限公司 A kind of primer combination of probe and its application of RASSF1A gene and the detection of P16 gene methylation

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