CN105200114A - Method for predicting chemicotherapy sensitivity by using cervical carcinoma side population cell - Google Patents
Method for predicting chemicotherapy sensitivity by using cervical carcinoma side population cell Download PDFInfo
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Abstract
A disclosed method for predicting chemicotherapy sensitivity by using cervical carcinoma side population cell comprises the following steps: preparing cervical carcinoma cell, and performing in-vitro adherent culture on the cervical carcinoma cell; collecting cervical carcinoma cell at logarithmic phase, and adding the cell into Hoechst33342; sorting SP cells by using a superspeed flow sorting system, and applying SP cell and NSP cell obtained through sorting to further experiment research; and then adding cis-platinum with different concentrations respectively into NSP cell and SP cell, staining by using an Annexin V FITC cell apoptosis detection kit, performing detection by using a flow cytometer, performing different doses of radiotherapy on SP cell and NSP cell, staining by using an Annexin V FITC cell apoptosis detection kit, and performing detection by using a flow cytometer. The cell apoptosis rate is equal to UR% plus LR%. Cervical cancer stem cell screening is performed by taking SP cell as an entry point, chemoradiotherapy sensibility difference and possible mechanism are analyzed, and theoretical basis is provided for researching and developing tumor stem cell resisting medicines, some cytokines interdicting signal path and chemical substances.
Description
Technical field
The present invention relates to a kind of medicine technology field, be specifically related to a kind of method applying cervical cancer side population cell prediction Concurrent Chemoradiotherapy Sensitivity.
Background technology
The sickness rate of cervical cancer accounts for the second of global women's malignant tumour, is only second to mammary cancer.China has new cases about 100,000 every year, accounts for 1/5 of whole world cervical cancer new cases.In developing country, the mortality ratio of woman uterus cancer has exceeded mammary cancer, occupies first of the female malignant cause of the death.The M & M of China in this year partial area cervical cancer has rising tendency, and ill rejuvenation trend also appears in some areas.The treatment of cervical cancer adopts based on operation and radiotherapy, and chemotherapy is auxiliary comprehensive therapeutic plan.The result for the treatment of of early cervical carcinoma is better, but 5 years survival rates of IV phase and Recurrent Cervical Cancer are only 3.2% ~ 13%, and recurrence great majority occur after diagnosis in 2 years, and poor prognosis, median overall survival is 7 months.Therefore cervical cancer is one of disease of serious threat whole world WomanHealth and life.
Tumor stem cell (cancerstemcell, CSC) theory is the study hotspot in current cancer field, this theory is thought: the cell in tumour has heterogeneity, most of tumour cell does not have infinite multiplication power, minority tumour cell is only had to have the infinite multiplication potential similar to stem cell and multinomial point of voltinism feature, all tumour cells all derive from these tumor stem cells, these tumor stem cells accounting for tumour cell quantity 0.1%-1% are progenitor cells of all tumour cells, to chemotherapeutics and radiotherapy tolerance, cause tumor drug resistance, the root of recurrence and transfer.Therefore, seek the origin of cell of cervical cancer, study their biological characteristics, new thinking can be provided for the prevention of cervical cancer, examination, for the treatment of recurrence and transitivity cervical cancer patient indicates new direction.
The Isolation and Identification of tumor stem cell is the first step of tumor stem cell research.With a high credibility by CSC surface specific mark sieve method separate stem cells, be the method that qualification CSC is the most authoritative, but, up to now, only have the Stem cell surface marker thing of Partial tumors out identified.Goodell in 1996 etc. with DNA dyestuff be hemopoietic stem cell Hoechst33342 dye and carry out Fluorescence Activated Cell sorting (fluorescenceactivatedcellsorting, FACS) time find that there is a group dyeing on the low side, with the different cell colony of other most cells.This part cell utilizing this characteristic to be separated is called as side population cell (Sidepopulation, SP), and this characteristic can getting rid of Hoechest33342 is called as SP phenotype.Along with going deep into SP cell research, find that the function of SP cell is similar to normal stem cell, Asymmetric division can occur, carry out self etc., so SP phenotype has become one of common method of sorting tumor stem cell at present.Existing research prompting cervical cancer SP phenotype possesses the biological characteristics of Multidrug-resistant in recent years, can be considered as the effective ways of sorting cervical cancer stem cell.The reported first such as domestic scholars Feng Ding celebratings in 2010 are separated single cell suspension eight examples after tumour cell ball nutrient solution (TSM) is cultivated obtained from the cervical cancer tissues of 19 example different clinical stagess has the tumour cell ball of suspension to be formed, and experiment confirms that these cells meet cells and characteristic of stem.But related experiment is few, and current research in recent years also mainly concentrates on the qualification aspect being separated and carrying out it biological function, and has no report about the correlative study of Concurrent Chemoradiotherapy Sensitivity.
Now generally acknowledged, a large characteristic of tumor stem cell is exactly resist chemotherapeutics.ABCG2 albumen is that ABC is in conjunction with box (ATP-bindingcassette, ABC) one of superfamily member, a kind of P glycoprotein, at important role such as maintenance cell homeostasis and body normal physiological function etc., the expression being ABCG2 by ABCG2 expression and the decline of the known cell of directly related property to drug susceptibility of SP phenotype causes.Along with to other molecules of abc transport protein family as ABCB1, ABCC2 study day by day deep, investigators think, other abc transport albumen also may be the reason forming SP phenotype, is thus also the molecular basis of SP cell multidrug resistance.
Radiotherapy occupies critical role in the treatment of cervical cancer.According to statistics, the cervical cancer patient of nearly 80% needs radiotherapy as one of means for the treatment of or complex therapy separately.Radiotherapy is for each other cervical cancer of clinical phase, but the raising of its curative effect is unsatisfactory in recent years, and Partial tumors cell is very inresponsive to radiotherapy, and after clinical I, II phase Patients Treated by Radiotherapy, 5 years survival rates are 65% ~ 85%; III, IV phase patient about 20% ~ 50%.The mechanism of tumor stem cell Radioresistence may with the reparation of damage dna, affect the transduction signal paths such as cell cycle regulating, apoptosis, propagation and change relevant.Evidence show that the noumenal tumour such as mammary cancer, glioma is relevant with the existence of CSCs to the insensitivity of radiotherapy, can infer audaciously, the irradiation for cervical cancer insensitivity of late period or relapse and metastasis is resisted relevant with the radioactive rays of SP cell.The radiosensitivity of tumour inherence can be understood by detecting the responsive correlation factor of radiotherapy, research shows in malignant tumour, suppressor gene p53 process LAN is a universal phenomenon, DNA reparation can be carried out or start apoptosis program making necrocytosis, play an important role in Radiation damage repair, and have certain relation with cellular radiosensitivity.Epithelial growth factor receptor (epidermalgrowthfactorreceptor, EGFR) be the expression product of proto-oncogene of activation, an acceptor for transmembrane tyrosine kinase somatomedin, all has expression in a lot of tumour cell, has close relationship with the prognosis of tumour.It is relevant to tumor radiotherapy curative effect that research also thinks that EGFR raises, EGFR signal pathway is the important factor affecting tumour cell radiation sensitivity, its overexpression can cause tumour cell to the opposing of radioactive rays, can think that EGFR is one of important indicator of prediction radiation sensitivity.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of method applying cervical cancer side population cell prediction Concurrent Chemoradiotherapy Sensitivity.
For solving the problem, a kind of method applying cervical cancer side population cell prediction Concurrent Chemoradiotherapy Sensitivity of the present invention, is characterized in that, comprise the steps:
(1) patient's cervical cancer tissues is obtained by operation source;
(2) primary cervical cancer cell suspension is prepared:
Get the fresh Cervical Tumor tissue of excision, stroke-physiological saline solution is rinsed well, is cut into 1mm
3the fragment of size, adds the collagenase that final concentration is 0.1%, 0.01% Unidasa, the DNA enzymatic of 0.002%, 37 DEG C digest 2 hours, 100 order nylon net filters, the centrifugal 10min of 500rpm, abandon supernatant, add appropriate Hanks liquid, after mixing, row discontinuous density gradient is centrifugal, 2000rpm20min, collect the suspension being rich in tumour cell, after washing 2 times, for subsequent use to testing by the DMEM nutrient solution adjustment cell count containing 10% serum;
(3) by cervical cancer cell in DMEM substratum, in 37 DEG C, 5%CO
2, conventional adherent culture in incubator under 95% humidity condition;
(4) cervical cancer cell of logarithmic phase is collected, adjustment cell count to 1 × 10
6individual/mL, is divided into two groups by cell, one group adds Hoechst33342 to final concentration is 5mg/L, and another group adds 50 μm of ol/L reserpines in contrast simultaneously; Mixing cell is resuspended in the ice-cold DMEM substratum containing 2%FBS, and with hypervelocity fluidic cell separation system sorting SP cell, the SP cell obtained after sorting and NSP cell are for further experimental study;
(5) collect SP and the NSP cell of logarithmic phase, be prepared into single cell suspension after digestion, adjustment cell concn is 2 × 10
5individual/mL, is inoculated in culture plate, changes liquid after cultivating 24h, NSP cell and SP cell add the cis-platinum of different concns respectively, digest after cultivating 24h, and PBS washs, AnnexinVFITC cell apoptosis detection kit dyes, flow cytomery, apoptosis rate=UR%+LR%;
(6) collect SP and the NSP cell of logarithmic phase, be prepared into single cell suspension after digestion, adjustment cell concn is 2 × 10
5individual/mL, is inoculated in culture plate, changes liquid after cultivating 24h, and the radiotherapy that SP cell and NSP cell give various dose is irradiated, and digest after cultivating 24h, PBS washs 1 time; AnnexinVFITC cell apoptosis detection kit dyes, flow cytomery, apoptosis rate=UR%+LR%.
In the preferred technical solution of the present invention, described step (5) can be exchanged with step (6) tandem.
In the preferred technical solution of the present invention, in described step (5), NSP cell and SP cell add cis-platinum 0 μ g/mL, 0.475 μ g/mL, the 0.95 μ g/mL and 1.9 μ g/mL of different concns respectively.
In the preferred technical solution of the present invention, in described step (6), NSP cell and SP cell give the radiotherapy irradiation that various dose 0,2,4,8Gy is irradiated respectively.
In described step (3), DMEM substratum contains 10%FBS, 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates.
In embodiments of the invention scheme, described cervical cancer cell is selected from HeLa cell.
The Hoechst33342 dyeing process of the classics that the present invention adopts Goodell etc. to report combines with flow cytometer, sorting is carried out to the SP cell in s and NSP cell, observation of cell growth and body outer clone formational situation, carry out chemotherapy and radiation sensitivity experiment further, both observations are to the reactive difference of chemotherapeutic drugs Cisplatin and radioactive rays, explore possible related mechanism simultaneously, for further investigation cervical cancer stem cell provides research model and basis, for clinical application provides foundation.
Advantage of the present invention comprises as follows:
1, tumor stem cell and tumour occur, development and transfer in close relations, receive increasing concern, these study the qualification aspect mainly concentrating on sorting cervical cancer stem cell and it is carried out to biological function, but have no report about the correlative study of Concurrent Chemoradiotherapy Sensitivity so far.
2, the present invention have studied the dependency that tumor stem cell and chemicotherapy are resisted, and the center of gravity for the treatment of is turned to tumor stem cell, makes every effort to tumor eradication stem cell, the multiplication capacity of tumour entirety is declined, and tumour is degenerated atrophy gradually, thus really cures tumour.
3, the present invention carries out cervical cancer stem cell screening using SP cell as point of penetration, analyzing the difference of Concurrent Chemoradiotherapy Sensitivity and possible mechanism thereof, providing theoretical foundation for developing resisting tumour stem cells medicine, some cytokines of disabling signal path and chemical substance.For the individualized treatment of recurrence and transitivity cervical cancer patient indicates new direction.
Accompanying drawing explanation
Fig. 1 is SP cell flow cytometer detection figure in s.
Fig. 2 is SP and NSP cell proliferation in vitro ability comparison diagram.
Fig. 3 is the body outer clone test chart of SP and NSP cell.
Fig. 4 is the body outer clone Forming ability histogram of SP and NSP cell.
Fig. 5 is the DDP flow cytomery figure that SP and NSP cell adds 0.95 μ g/ml concentration.
Fig. 6 is that SP and NSP cell adds apoptosis rate after the cis-platinum of different concns.
Fig. 7 is that SP and NSP cell gives flow cytomery figure after 2Gy dose irradiation.
Fig. 8 is that SP and NSP cell gives apoptosis rate after different radiation doses.
Fig. 9 is the expression of ABCG2, ABCC2 gene in SP and NSP cell before intervening.
Figure 10 is the expression of P53, EGFR gene in SP and NSP cell before intervening.
Figure 11 is the expression of ABCG2, ABCC2 gene after SP and NSP cell chemotherapy.
Figure 12 is the expression contrast histogram of P53 after the radiotherapy of SP and NSP cell, EGFR gene.
Embodiment
Below in conjunction with accompanying drawing, the specific embodiment of the invention is described.
One, material and equipment
1. laboratory animal or material source and process
Human cervical carcinoma Hela cell is purchased from the American Type Culture Collection council of Chinese Academy of Sciences cell bank (life science institute cellular resources center, Shanghai).
2. key instrument: CO2 constant temperature cell culture incubator, 96 orifice plates, FACS flow cytometer, inverted microscope, Biohazard Safety Equipment, ultrapure water system, whizzer, water bath, spectrophotometer, cell counting box-8 (CCK-8), full-automatic quantitative fluorescent PCR diagnositc system, linear accelerator.
3. main agents: 10% foetal calf serum (FBS), DMEM/F121:1 substratum, mycillin mixed solution, trypsinase, Hochest33342, PI (propidium iodide), phosphate buffered saline buffer, Ji's nurse Sa dye liquor, Trizol reagent, RNA enzyme inhibitors, MMLV ThermoScript II.
Two, experimental technique
1. s vitro culture
HeLa cell containing in the DMEM substratum of 10%FBS, 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates, in 37 DEG C, 5%CO
2, conventional adherent culture in incubator under 95% humidity condition.
2. selected by flow cytometry apoptosis goes out SP cell and NSP cell
Collect the HeLa cell of logarithmic phase, be prepared into single cell suspension after digestion, centrifugal, PBS washes twice, resuspended with the DMEM substratum containing 2%FBS, adjustment cell count to 1 × 106/mL.Cell is divided into two groups, one group adds Hoechst33342 to final concentration is 5mg/L, and another group adds 50 μm of ol/L reserpines in contrast simultaneously; Mixing cell, in 37 DEG C of water bath with thermostatic control shaking tables, lucifuge hatches 90min.Wash 1 time with the PBS containing 2%FBS, be resuspended in the ice-cold DMEM substratum containing 2%FBS.According to the operation of cell apoptosis detection kit specification sheets, with hypervelocity fluidic cell separation system sorting HeLa cell.The SP cell obtained after sorting and NSP cell are for further experimental study.
3. compare the multiplication capacity of SP and NSP cell, body outer clone Forming ability
3.1 multiplication capacities (ATP-TCP method) comparing SP and NSP cell:
Collect SP and the NSP cell of logarithmic phase, be prepared into single cell suspension after digestion, often kind of cell is all seeded to 96 orifice plates according to every hole 100 μ L containing 1000, cell, and often kind of cell does 4 parallel holes, do 8 groups altogether, DMEM (10%FBS) is as blank group.According to the operation of ATP detection agent box specification sheets, obtain and detect numerical value.The proliferation rate of cell is calculated, the read value * 100% of read value/1st of proliferation rate=the n-th of n-th day cell day day according to the read value of each group of cell every day.Take proliferation rate as ordinate zou, the time is that X-coordinate draws cell proliferation curve.
The 3.2 body outer clone Forming ability (plates of cells clone forming method) comparing SP and NSP cell:
Collect SP and the NSP cell of logarithmic phase, be prepared into single cell suspension after digestion, often kind of cell is all seeded to 6 orifice plates according to every hole 5mL containing 100, cell, and often kind of cell does 3 parallel holes.When there is macroscopic clone, stop cultivating.After abandoning liquid, PBS washes 2 times.1mL4% paraformaldehyde fixed cell, 15 minutes.Add 1mLgiemsa dyeing after abandoning liquid, after 30 minutes, flowing water slowly washes away, 37 DEG C of dryings 30 minutes.Counting is greater than clone's number of 50 cells under the microscope.Lg cloning efficiency=lg (clone's number/inoculating cell number) × 100%.
4. detect the differential expression of genes involved in SP cell and NSP cell
4.1RNA extract
Collect about 107, SP and NSP cell, put into grinding pipe, add Biozol1mL, piping and druming mixing, after incubated at room 7min, adds 200 μ L chloroform.4 DEG C, the centrifugal 15min of 12000g, absorption supernatant enters new ep and manages, and adds the pre-cold isopropanol of equal-volume, blows even, incubated at room 20min.4 DEG C, the centrifugal 10min of 12000g, removes supernatant, adds 1mL75% pre-cooled ethanol, resuspended washing.4 DEG C, the centrifugal 5min of 10000g, removes supernatant.Add 50 μ LDEPC water dissolution ,-80 DEG C of preservations after mensuration concentration.
4.2cDNA synthesis
Get in clean ep pipe and add 1 μ Lrandom6mers, 1 μ LdNTP, 7 μ LRNasefreedH
2o, 1 μ LTempRNA, blow even, put into PCR instrument, setting program: 65 DEG C, 5min.Add 4 μ L5*buffer again, 0.5 μ LRNaseinhibitor, 1 μ LRTase, 4.5 μ LRNasefreedH2O, blow even, put into PCR instrument, setting program: 30 DEG C, 10min → 42 DEG C, 50min → 95 DEG C, 5min.Reversion terminates ,-80 DEG C of preservations after mensuration concentration.
4.3RT-QPCR reaction
30 μ L systems: 15 μ LSYBRMIX+0.6 μ Lprimer-F+0.6 μ Lprimer-R+11.8 μ LddH2O+2 μ LcDNA.Standard two-step method response procedures is: 95 DEG C of denaturation 10min → 40cycles95 DEG C 15s → 60 DEG C 1min.In table 1 primer sequence
Differential expression multiple=△ CT (SP)/△ CT (NSP); △ CT=CT (goal gene)-CT (GAPDH)
5. Concurrent Chemoradiotherapy Sensitivity experiment
5.1 chemosensitivity experiments
Collect SP and the NSP cell of logarithmic phase, be prepared into single cell suspension after digestion, adjustment cell concn is 2 × 105/mL, is inoculated in 6 well culture plates.Change liquid after cultivating 24h, NSP cell and SP cell add cis-platinum 0 μ g/mL, 0.475 μ g/mL, the 0.95 μ g/mL and 1.9 μ g/mL of different concns respectively.Digest after cultivating 24h, PBS washs 1 time.AnnexinVFITC cell apoptosis detection kit dyes, flow cytomery.Apoptosis rate=UR%+LR%.
5.2 radiation sensitivity experiments
Collect SP and the NSP cell of logarithmic phase, be prepared into single cell suspension after digestion, adjustment cell concn is 2 × 105/mL, is inoculated in 6 well culture plates.Change liquid after cultivating 24h, SP cell and NSP cell give 0,2,4,8Gy respectively and irradiate.Digest after cultivating 24h, PBS washs 1 time.AnnexinVFITC cell apoptosis detection kit dyes, flow cytomery.Apoptosis rate=UR%+LR%
6 statistical procedures
Adopt SPSS17.0 software data processing.Data acquisition is used
represent, adopt t to check between dosage, enumeration data adopts x2 inspection, and P<0.05 has statistical significance.
Three, experimental result
(1) separation results of SP cell in cancer Hela cells
Cultured cell is through Hoechest33342 fluorescent dye, show through flow cytometry analysis detected result, exist containing a small amount of SP cell in human cervical carcinoma Hela cell system, its sorting ratio is 1.47 ± 0.85%, and after verapamil antagonism, the content of SP cell is down to 0.02%.(see Fig. 1).
(2) the multiplication capacity result of SP and NSP cell is compared
Through the isolated two strain cell strains of Hela, respectively through the cultivation of 8 days, proliferative differences when finding the 5th day, the 6th day between cell has statistical significance, be respectively p=0.0057, p=0.0163, but along with the prolongation of incubation time, the difference between cell does not have significant difference.SP cell speeds at the 5th day obvious comparatively NSP of cell proliferation rate, illustrates that the cytoactive of SP cell is obviously better than NSP cell.But along with the prolongation of incubation time, remain constantly reducing and the continuous consumption of nutritive medium of living space in culture dish, the speed of cell proliferation obviously slows down.Therebetween obvious difference reduces, and sees Fig. 2.
(3) the body outer clone Forming ability of SP and NSP cell is compared
Collect SP and the NSP cell of logarithmic phase, be prepared into single cell suspension after digestion, often kind of cell all contains cell 100 according to every hole 5mL, when there is macroscopic clone, stops cultivating.Formaldehyde fixed cell, giemsa dyeing, drying.Counting is greater than clone's number of 50 cells under the microscope.Lg cloning efficiency=lg (clone's number/inoculating cell number) × 100%.The results are shown in Figure 3, Fig. 4.
The clonality of in vitro tests research SP and NSP cell, observe two kinds after 5 days dissimilar cell clone situations, relative to NSP, SP cell showed increased, bacterium colony formation efficiency lg calculates, find that SP cell and NSP cell have statistical significance through statistical analysis (2.407 ± 0.1509vs1.160 ± 0.1989, p=0.009).
(4) chemosensitivity experiment
Find after giving the DDP chemotherapy of different concns, find that the apoptosis rate of SP cell under different pharmaceutical concentration is all lower than NSP cell, all there is statistical significance, the difference maximum (p=0.005) wherein between the apoptosis rate SP of the DDP of 1.9 μ g/ml concentration and NSP cell.Analyze and find that the apoptosis rate of cell also raises gradually along with the increase of drug level, but faster compared with SP cell of the apoptosis speedup of NSP cell, and visible NSP cell is more responsive to chemotherapeutics DDP relative to SP cell.See Fig. 5, Fig. 6, table 2.
Table 2SP and NSP cell adds apoptosis rate after the cis-platinum of different concns
TheRateofApoptosisbyDDP
p<0.05
(5) radiation sensitivity experiment
Not accept the SP cell of radiotherapy and NSP cell for contrast, analyze the apoptosis situation accepting cell after different Radiotherapy dosimetry.Result of study shows, after giving different radiation doses, under 2Gy dose irradiation SP and NSP apoptosis rate between there is statistical significance (p=0.003), in other dose irradiation situations, the two does not have significant difference.See Fig. 7, Fig. 8,
Table 3.
Table 3SP and NSP cell gives apoptosis rate after different radiation doses
TheRateofApoptosisbyRT
p<0.05
(6) differential expression of related neoplasms gene in SP cell and NSP cell is detected
1, the expression of genes involved in front SP cell and NSP cell is intervened
Without the expression level of ABCG2, ABCC2, P53, EGFR gene in the lower SP cell of extraneous factor impact and NSP cell.Result shows, in SP cell and NSP cell, the differential expression multiple of ABCG2, ABCC2, P53, EGFR gene is respectively 1.2,1.0,1.9,2.0.Cell SP and NSP is under affecting without extraneous factor, the gene expression amount of SP is higher than NSP cell, and ABCG2, ABCC2 and P53, EGFR ratio do not have statistical significance, have no notable difference at molecule parting, illustrate that two strain cells are with root homology, derive from same Hela cell.See Fig. 9, Figure 10, table 4, table 5.
The expression of ABCG2, ABCC2 gene in front SP and NSP cell intervened by table 4
Geneexpression
p<0.05
The expression of P53, EGFR gene in front SP and NSP cell intervened by table 5
Geneexpression
p<0.05
2, ABCG2 and ABCC2 genetic expression in SP and NSP after chemotherapy
We choose NSP cell to the IC50 value 0.95 μ g/ml of DDP as observation concentration, further the changing conditions of analysis goal gene under the effect of same concentration chemotherapeutics.After 0.95 μ g/mLDDP and Hela co-culture of cells 24h, RT-PCR detects the expression of the gene of ABCG2, ABCC2 of rear SP and NSP for the treatment of, find that the expression of ABCG2 gene in SP cell and NSP have significant difference (p=0.013) between expressing, in SP cell, the expression of ABCC2 gene and NSP have significant difference (p=0.024) between expressing.Illustrate that in SP cell, the relative high expression level of ABCG2, ABCC2 gene has certain resistant function to chemotherapeutic DDP.See Figure 11, table 6.
The expression of ABCG2, ABCC2 gene after table 6SP and NSP cell chemotherapy
GeneexpressionafterchemotherapybyDDP
p<0.05
3, SP and NSP correlative P 53 and EFGR genetic expression after radiotherapy
The Hela cell cultivated is after the radiation exposure of 2Gy, the expression of the gene of P53, EGFR of SP and NSP after RT-PCR detection radiotherapy, find that the expression of P53 gene in SP cell and NSP have significant difference (p=0.044) between expressing, in SP cell, the expression of VEGF gene and NSP have significant difference (p=0.036) between expressing.Illustrate that in SP cell, the relative high expression level of P53, VEGF gene has certain resistant function to radioactive rays.See Figure 12, table 7.
The expression of P53, EGFR gene after the radiotherapy of table 7SP and NSP cell
GeneexpressionafterRTby2Gy
p<0.05
Cervical cancer SP cell has the biological characteristics of tumor stem cell
Recurrence and transfer are two hang-ups for the treatment of malignant tumour, and in recent years along with going deep into malignant tumour research, it is found that in tumor tissues, to there is sub-fraction and the similar oncocyte of stem cell properties, is the reason of metastases and recurrence.Subsequently, tumor stem cell theory is proposed.This theory is thought, not every tumour cell can be unlimited growth and just sub-fraction wherein can infinitely self, hyperproliferation, Multidirectional Differentiation, be referred to as tumor stem cell, this small set of cell is swollen neoplastic initiator cell, and maintains the growth of tumour.This part of cell is resisted chemicotherapy by number of mechanisms simultaneously, is the root of tumor recurrence and transfer.So the existence of tumor stem cell is the major cause of Endodontic failure.The research of cervical cancer stem cell is still still in the starting stage.SP separating method has become a kind of common method of tumor stem cell research.Profit in this way isolated cell is called as SP cell.A lot of scholar thinks, SP cell has that stronger oneself is more capable, Multidirectional Differentiation, hyperproliferation and chemicotherapy are resisted compared with NSP cell, and similar to tumor stem cell, SP cell can as the ideal model of stem-cell research.SP cell is the most separated in hematological system tumor, confirmation, and along with going deep into of research, current SP cell is found to be present in widely in kinds of tumor cells system and tumor tissues.
Adopt FACS method to sub-elect a small amount of SP cell herein from human cervical carcinoma Hela cell system, sorting ratio is 1.47 ± 0.85%, similar to bibliographical information.Whether there is to inquire into sub-elected SP cell the feature of tumor stem cell, utilizing the SP cell sub-elected to carry out the research of ability of cell proliferation and body outer clone Forming ability.Confirmed by these two experiments, the SP cell sub-elected from cancer Hela cells proliferation activity in vitro and clonality are all better than NSP cell, meet the feature of tumor stem cell hyperproliferation and Clone formation, tentative confirmation SP cell has Partial tumors stem cell properties really, can be used as the model of cervical cancer stem-cell research, for the further research of cervical cancer stem cell provides experimental basis.
2. cervical cancer side population cell is relevant to chemoresistance
One large characteristic of tumor stem cell is exactly resist chemotherapeutics, research is had to adopt As2O3 to reverse human cervical carcinoma cell persister SiHa/CDDP, found that along with its stem cell properties weakens, the resistance of SiHa/CDDP cell significantly declines, the expression of the ABCG2 simultaneously in SiHa/CDDP cell significantly reduces, and prompting cervical cancer cell has close relationship to the resistance of CDDP and stem cell properties.Whether have chemoresistance for understanding SP cell in cancer Hela cells, the present invention utilizes the SP cell that sorts out from s and NSP cell to carry out chemosensitivity testing test.Find after giving the DDP chemotherapy of SP cell and NSP cell different concns that the apoptosis rate of SP cell under different pharmaceutical concentration is all lower than NSP cell, all there is statistical significance, wherein the rear apoptosis rate difference of the DDP effect of 1.9 μ g/ml concentration is maximum, and analyze the increase found along with drug level, the apoptosis rate of cell also raises gradually, but the apoptosis speedup of NSP cell comparatively SP cell is faster, and visible NSP cell is more responsive to chemotherapeutics DDP relative to SP cell.Our result of study is consistent with bibliographical information.
3. cervical cancer side population cell is resisted relevant to radiotherapy
Tumor stem cell may be relevant to some reason following to the opposing of radiotherapy: 1, tumor stem cell effectively can promote the injury repairing of cell DNA after radiotherapy
[10]; 2, tumor stem cell Cycle Arrest; 3, the interaction of tumor stem cell and its surrounding microenvironment
[20-21].Bao etc.
[22]find that glioblastoma stem cell shows Radioresistence, likely cause clinical radiotherapy failure.Can infer that part cervical cancer patient also may be resisted relevant with the radiation of tumor stem cell to radiotherapy is insensitive.Before the present invention finds not give roentgen radiation x, the apoptosis rate of SP cell is lower than NSP cell, and after 2Gy dose irradiation, the apoptosis rate of SP and NSP all raises, both have statistical significance at difference, after 4Gy, 8Gy dose irradiation, apoptosis rate raises further, but both no significant difference.Illustrate that the radiotherapy giving high dosage can cause two kinds of cells all cannot tolerate, apoptosis increases, and the apoptosis rate given compared with the radiotherapy SP cell of low dosage is lower than NSP cell, and there is significant difference, can illustrate that SP cell possesses stronger capability of resistance to radiation further, can survive better in radiotherapy, may be the root of tumour progression, recurrence and transfer.
The expression of 4.SP cell and NSP cell ABCG2, ABCC2 and P53 and EGFR
Study the expression level exception of chemicotherapy sensitive factor and the product thereof finding some tumour cells relevant with the chemotherapy of tumour cell and radiotherapeutic response.
Most expression abc transport body families membranin on tumor stem cell cytolemma, especially ABCG2, it can transport and discharge the material such as meta-bolites, medicine, makes many antineoplastic chemotherapeutics not play lethal effect on tumor stem cell, or effect obviously weakens.The research such as ChenZ then demonstrates ABCG-2 except transport function, also has vital role regulating in the propagation of SP cell, differentiation and Cell protection.Along with to other molecules of abc transport protein family as ABCB1, ABCC2 study day by day deep, investigators think, other abc transport albumen also may be the reason forming SP phenotype, is thus also the molecular basis of SP cell multidrug resistance.Result display SP and NSP cell of the present invention is under affecting without extraneous factor, and ABCG2, ABCC2 gene expression amount of SP cell is higher than NSP cell, but ratio does not have statistical significance.Find after DDP chemotherapy that the expression of ABCG2, ABCC2 gene in SP cell is all higher than NSP cell, has significant difference (p=0.013, P=0.024).Illustrate that the high expression level of ABCG2, ABCC2 in SP cell has certain resistant function to chemotherapeutic DDP.Gene expression results before DDP of the present invention treatment is reported consistent with external, but in SP cell after HeLa and the SiHa cell sorting shown with the beautiful research report of Qi Wen, ABCG2 is high expression level, then expresses inconsistent in NSP cell hardly.Thinking that SP and NSP cell has no notable difference at molecule parting herein, illustrate that two strain cells are with root homology, may be that under the effect of DDP medicine, between both, subtle difference causes two strain cells to create diametrically opposite phenomenon.We find that the speed of growth of SP cell is faster than NSP cell before, and Level of Apoptosis is lower than NSP cell, under DDP drug effect, cell is constantly dead, the difference of ABCG2 and ABCC2 expression amount result in the relative expression quantity of ABCG2 and ABCC2 in SP cell gradually higher than NSP cell, finally result in qualitative change to occur, thus result in the generation of drug-resistant cell strain.We courageously guess, along with constantly carrying out of the Chemotherapy in Patients course for the treatment of, the SP cell that ABCG2 and ABCC2 expression amount is high constantly accumulates in tumor tissues, and final quantitative change result in qualitative change, thus the generation of drug-resistant cell strain, perhaps this be that tumour patient is to one of reason that chemotherapeutics tolerates.
At present, the research for suppressor gene p53 is comparatively extensive and go deep into, and the reparation after display wild-type p 53 gene inhibition tumor cell radiotherapy damage, promotes the apoptosis of tumour cell, have Apoptosis.But, once P53 gene is undergone mutation, be then conducive to tumor cell proliferation, canceration, progress, and produce radioresistant.The reason that EGFR (epithelial growth factor receptor) process LAN causes tumour radiotherapy to be resisted may have by stimulating misgrowth, the repair of apoptosis inhibit and the DNA to radiation-induced damage.The research of Myllynen etc. finds, EGFR regulates the reparation of double-strand break (DSB) and had both related to nonhomologous end connection (NHEJ) and homologous recombination repair (HRR), after EGFR is activated, the repairing effect of whole DSB significantly improves, thus produces radiation opposing.This result of study display SP and NSP cell is under affecting without extraneous factor, and P53, VEGF gene expression amount of SP cell is higher than NSP cell, but ratio does not have statistical significance.Find after 2Gy irradiates that there were significant differences compared with the expression of P53, VEGF in SP cell is expressed with NSP.We think that SP and NSP cell has no notable difference at molecule parting before radiotherapy, illustrate that two kinds of cells are with root homology, after 2GyX line irradiates, gene level there occurs obvious change, the radiotherapy of P53 after inference radiotherapy, difference that EGFR gene is expressed and SP cell can resist certain relation, SP cell more tolerates radioactive rays, thus result in failure and the recurrence of oncotherapy.
In sum, the Hoechst33342 dyeing process of the classics that the present invention adopts Goodell etc. to report combines with flow cytometer, sorting is carried out to the SP cell in s and NSP cell, observation of cell growth and body outer clone formational situation, carry out chemotherapy and radiation sensitivity experiment further, the SP cell sub-elected has stronger proliferation activity and clonality in vitro, meets the biological characteristics of tumor stem cell; Under different pharmaceutical concentration, the apoptosis rate of SP cell is significantly all lower than NSP cell, and along with the increase of drug level, the apoptosis rate of cell raises gradually, but comparatively SP cell is faster for the apoptosis speedup of NSP cell; Give the apoptosis rate of SP cell after the radiotherapy compared with low dosage significantly lower than NSP cell, and the radiotherapy of high dosage can cause two kinds of cells all cannot tolerate, apoptosis increases; In cervical cancer SP cell chemicotherapy sensitive factor ABCG2, ABCC2P53, EGFR under without the impact of extraneous factor slightly higher than NSP cell, but without significant difference, after DDP chemotherapy and radiation exposure, in SP cell, the genetic expression of above-mentioned correlation factor is apparently higher than NSP cell, result causes tumour cell all to create resistance to chemotherapy and radiotherapy, SP cell to the susceptibility of chemicotherapy not as good as NSP cell.
More than show and describe ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not by the restriction of above-mentioned example; what describe in above-mentioned example and specification sheets just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (5)
1. apply a method for cervical cancer side population cell prediction Concurrent Chemoradiotherapy Sensitivity, it is characterized in that, comprise the steps:
(1) patient's cervical cancer tissues is obtained by operation source;
(2) primary cervical cancer cell suspension is prepared:
Get the fresh Cervical Tumor tissue of excision, stroke-physiological saline solution is rinsed well, is cut into 1mm
3the fragment of size, adds the collagenase that final concentration is 0.1%, 0.01% Unidasa, the DNA enzymatic of 0.002%, 37 DEG C digest 2 hours, 100 order nylon net filters, the centrifugal 10min of 500rpm, abandon supernatant, add appropriate Hanks liquid, after mixing, row discontinuous density gradient is centrifugal, 2000rpm20min, collect the suspension being rich in tumour cell, after washing 2 times, for subsequent use to testing by the DMEM nutrient solution adjustment cell count containing 10% serum;
(3) by cervical cancer cell in DMEM substratum, in 37 DEG C, 5%CO
2, conventional adherent culture in incubator under 95% humidity condition;
(4) cervical cancer cell of logarithmic phase is collected, adjustment cell count to 1 × 10
6individual/mL, is divided into two groups by cell, one group adds Hoechst33342 to final concentration is 5mg/L, and another group adds 50 μm of ol/L reserpines in contrast simultaneously; Mixing cell is resuspended in the ice-cold DMEM substratum containing 2%FBS, and with hypervelocity fluidic cell separation system sorting SP cell, the SP cell obtained after sorting and NSP cell are for further experimental study;
(5) collect SP and the NSP cell of logarithmic phase, be prepared into single cell suspension after digestion, adjustment cell concn is 2 × 10
5individual/mL, is inoculated in culture plate, changes liquid after cultivating 24h, NSP cell and SP cell add the cis-platinum of different concns respectively, digest after cultivating 24h, and PBS washs, AnnexinVFITC cell apoptosis detection kit dyes, flow cytomery, apoptosis rate=UR%+LR%;
(6) collect SP and the NSP cell of logarithmic phase, be prepared into single cell suspension after digestion, adjustment cell concn is 2 × 10
5individual/mL, is inoculated in culture plate, changes liquid after cultivating 24h, and the radiotherapy that SP cell and NSP cell give various dose is irradiated, and digest after cultivating 24h, PBS washs 1 time; AnnexinVFITC cell apoptosis detection kit dyes, flow cytomery, apoptosis rate=UR%+LR%.
2. a kind of method applying cervical cancer side population cell prediction Concurrent Chemoradiotherapy Sensitivity according to claim 1, it is characterized in that, described step (5) can be exchanged with step (6) tandem.
3. a kind of method applying cervical cancer side population cell prediction Concurrent Chemoradiotherapy Sensitivity according to claim 1, it is characterized in that, in described step (5), NSP cell and SP cell add cis-platinum 0 μ g/mL, 0.475 μ g/mL, the 0.95 μ g/mL and 1.9 μ g/mL of different concns respectively.
4. a kind of method applying cervical cancer side population cell prediction Concurrent Chemoradiotherapy Sensitivity according to claim 1, is characterized in that, in described step (6), NSP cell and SP cell give the radiotherapy irradiation that various dose 0,2,4,8Gy is irradiated respectively.
5. a kind of method applying cervical cancer side population cell prediction Concurrent Chemoradiotherapy Sensitivity according to claim 1, it is characterized in that, in described step (3), DMEM substratum contains 10%FBS, 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates.
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