CN104403996A - Human gastric cancer cell line with 5-fluorouracil resistance and establishing method and application thereof - Google Patents
Human gastric cancer cell line with 5-fluorouracil resistance and establishing method and application thereof Download PDFInfo
- Publication number
- CN104403996A CN104403996A CN201410499357.9A CN201410499357A CN104403996A CN 104403996 A CN104403996 A CN 104403996A CN 201410499357 A CN201410499357 A CN 201410499357A CN 104403996 A CN104403996 A CN 104403996A
- Authority
- CN
- China
- Prior art keywords
- cell
- cancer
- tumor
- cell line
- gastric cancer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention discloses a human gastric cancer cell line with 5-fluorouracil resistance and an establishing method and application thereof. The human gastric cancer cell line is preserved in China Center for Type Culture Collection and has a preservation number of CCTCC NO:2013154. The human gastric cancer cell line has the advantages of stable characters, stable multiple passages, high tumor formation rate, short incubation period and good uniformity, and provides a novel experimental material closer to the biological characteristics of clinical tumor for the study of gastric carcinoma. The human gastric cancer cell line has tumorigenicity; the generated tumor has 5-fluorouracil resistance and can be used to analyze the in vitro and in vivo drug sensitivity and resistance correlation, and then establish in vitro and in vivo two related drug screening platforms; and the is cell line is ideal for basic research of gastric cancer and clinical early stage application.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of there is 5 FU 5 fluorouracil resistance SGC-7901 and establishment method and application.
Background technology
Malignant tumour is the principal disease of current serious harm human health, and wherein, cancer of the stomach is one of cancer that global incidence is the highest, is also the 2nd cancer mortality reason in the world, is only second to lung cancer.Report according to the Global Cancer Statistics of 2011, the cancer of the stomach new cases of the current whole world more than 70% and death all occur in developing country, and wherein East Asia ranks first, and sickness rate reaches the male sex 42.4% and women 18.3% respectively.And according to the data that Chinese Anti-Cancer Association clinical tumor cooperation center (CSCO) is enumerated, the annual new cancer of the stomach of sending out in the whole world has 934,000 examples at present, wherein have nearly 400,000 in inland of China, the incidence gastric cancer of China has accounted for global 42%.China dies from cancer of the stomach about 300,000 people every year, and mortality ratio is that 25.2/10 ten thousand (male sex: 32.8/10 ten thousand, women: 17.0/10 ten thousand) account for 23.2% of whole mortality of malignant tumors.And in Shanghai, the sickness rate of male sex's cancer of the stomach is 47.1/10 ten thousand ~ 63.2/10 ten thousand, women is 20.1/10 ten thousand ~ 24.8/10 ten thousand, accounts for the 1st and the 2nd of Cancer Mortality respectively.Wherein, the incidence of adenocarcinoma of stomach accounts for 95% of stomach malignancy.
In recent years, the Therapeutic mode of cancer of the stomach enters from single operative treatment the new Therapeutic mode that complex therapy adds standardization operation, and wherein adjuvant chemotherapy is as one of the method for complex therapy, is more and more paid close attention to.Mainly there are the following problems for current cancer of the stomach clinical chemotherapy: first, and conventional chemotherapy regimen lacks the anticancer susceptibility of objective indicator reaction medicine; Secondly, resistance phenomenon remain the greatest problem existed in chemotherapy of gastric cancer.Given this, set up the gastric carcinoma cell lines of different pathological types, especially there is the clone of resistant characterization, for the treatment dependency effectively instructing preclinical study and clinical different somatotype, there is requisite vital role.Simultaneously owing to using clinical tumor to organize the success ratio directly setting up clone lower, therefore, first animal model is set up by using clinical tumor sample, and then set up people source tumour cell by original cuiture and have more feasibility, simultaneously also close to the Clinical Biological of tumour, to the resistance of medicine and susceptibility, there is good predictability.
The importance that stomach cancer cell ties up to new drug development preclinical applications sets up mouse xenograft model exactly, for predicting the clinical efficacy of cancer therapy drug.But the most of gastric carcinoma cell lines set up at present are limited to the problems such as the low or homogeneity of tumor formation rate is poor, are difficult to be applied to large-scale medicine screening.
Summary of the invention
The technical problem to be solved in the present invention is exactly low for existing SGC-7901 species diversity, differing larger with the biological character of clinical cancer of the stomach waits not enough, provides a kind of new SGC-7901 with 5 FU 5 fluorouracil resistance and establishment method thereof and application.
Therefore, the present invention solves the problems of the technologies described above one of adopted technical scheme and is: a kind of SGC-7901, and it is deposited in China typical culture collection center, and deposit number is CCTCC NO:2013154.
SGC-7901 of the present invention preferably also comprises the daughter cell of SGC-7901 as above.
The present invention solves the problems of the technologies described above two of adopted technical scheme: gastric carcinoma cells as above ties up to the purposes prepared in the reagent making immunodeficient mouse generation cancer of the stomach.
The reagent making immunodeficient mouse produce cancer of the stomach of the present invention is the reagent of this area routine, and the preparation method of this reagent is preferably for be suspended in all kinds of SOLVENTS by SGC-7901 of the present invention and to get final product.Wherein said solvent is this area Conventional solvents, is preferably HBSS damping fluid, and the formula of described HBSS damping fluid is: containing 500U/ml penicillin G, 500 μ g/ml Vetstreps and 1.25 μ g/ml amphotericin Bs.
Reagent of the present invention is preferably for the preparation of animal modal of gastric carcinoma.The preparation method of wherein said animal modal of gastric carcinoma is this area ordinary method, preferably comprises the following steps: the described reagent making immunodeficient mouse produce cancer of the stomach is inoculated in immunodeficient mouse and raises, obtain animal modal of gastric carcinoma.Wherein said immunodeficient mouse is preferably nude mouse or Reconstruction in Sever Combined Immunodeciency Mice (SCID mouse), be preferably Reconstruction in Sever Combined Immunodeciency Mice (SCID mouse), wherein said nude mouse is preferably the nude mouse of NU/NU strain.The animal modal of gastric carcinoma utilizing clone of the present invention to prepare has 5 FU 5 fluorouracil resistance.
The mode of wherein said inoculation is the vaccination ways that this area routine uses.Described vaccination ways preferably comprises subcutaneous puncture inoculation, inoculates in in-situ inoculating or scrotum film, is preferably subcutaneous puncture inoculation.The cell concentration of described inoculation is preferably 1.0 ~ 2 × 10
7individual cell, is preferably 5.0 × 10
6individual cell.
The application method of animal modal of gastric carcinoma of the present invention is preferably, utilizes gained animal modal of gastric carcinoma to detect the suppression cancer of the stomach activity of testing compound, testing compound is applied to animal modal of gastric carcinoma, observes and measure body weight and the tumor growth situation of animal; It is active that the testing compound causing animal modal of gastric carcinoma cancer of the stomach symptom to be improved after using or to cure has suppression cancer of the stomach.
The method that wherein said testing compound is used is the application process of this area routine, preferably comprises: one or more modes in tail vein injection, abdominal injection, gavage, oral and tumor by local medication are applied to the tumor animal of cancer of the stomach.Reference examples in described experiment is preferably: use the solvent application not containing testing compound in the tumor animal experimental group in contrast of cancer of the stomach simultaneously.
The present invention solves the problems of the technologies described above three of adopted technical scheme: a kind of testing compound suppresses the external detection method of gastric cancer cell activity, this external detection method comprises the following steps: be applied to by testing compound in cell model, use rear antiproliferative effect or cause apoptotic testing compound to be have the compound suppressing gastric cancer cell activity, wherein said cell model is SGC-7901 as above.
External detection method of the present invention is the method that this area routine uses, and this external detection method preferably comprises the following steps:
(1) SGC-7901 of the present invention or its daughter cell are inoculated in 96 porocytes to cultivate in plate hole, cultivate 24 hours;
(2) testing compound is diluted to different concns and is applied to cell, compound effects passes through the vigor measuring cell after 72 hours, calculate the Proliferation Ability ability of the compound on intracellular of different concns, computerized compound half-inhibition concentration, in order to judge the ability of the anti-stomach cancer cell of testing compound.
Wherein the inoculum size of step (1) described SGC-7901 or its daughter cell is preferably 5000/ hole.Wherein the described half-inhibition concentration detection method of step (2) preferably comprises ATP biloluminescence method or mtt assay, and detection method of the present invention is preferably ATP biloluminescence method.Described ATP biloluminescence method detects a kind of homogeneous detection method of viable count object in culture by carrying out quantitative assay to the ATP in cell, wherein said ATP is the metabolic important indicator of viable cell, and described ATP biloluminescence method can utilize commercially available detection kit to carry out.
The present invention solves the problems of the technologies described above four of adopted technical scheme: a kind of establishment method of SGC-7901 described above, and this establishment method comprises the following steps:
(1) obtain fresh clinical people's Operative Excision in Gastric Carcinoma sample, cut into fritter, subcutaneous puncture immunoprophylaxis deficient mice;
(2) percutaneous puncture-inoculation is after 70 ~ 90 days, is put to death by tumor animal, takes out tumor tissues, carries out original cuiture and the Secondary Culture of cancer cells.
Be the better quality of the fritter of the fresh clinical people's Operative Excision in Gastric Carcinoma specimen cutting wherein described in step (1) 20 ~ 50mg.Wherein said immunodeficient mouse is preferably Reconstruction in Sever Combined Immunodeciency Mice (SCID mouse) or nude mouse.The processing mode that wherein said fresh clinical people's Operated Specimens of Gastric Carcinoma is better is: with fresh HBSS damping fluid (described HBSS damping fluid preferably comprises 500U/ml penicillin G, 500 μ g/ml Vetstreps and 1.25 μ g/ml amphotericin Bs) rinsing.
Primary culture method wherein described in step (2) is the primary culture method of conventional mammalian cell; Described primary culture method preferably comprises the following steps:
Tumor tissues is cut into fritter, inserts in culturing bottle, in 37 DEG C of incubator 5%CO
2cultivate under condition; Next day, culturing bottle is slowly overturn and keeps flat, in bottle, add RPMI-1640 nutrient solution (containing 5% foetal calf serum, 100U/ml penicillin G, 100 μ g/ml Vetstreps and 0.25 μ g/ml amphotericin B), quiescent culture.
Wherein said Secondary Culture method is the Secondary Culture method of conventional mammalian cell; Described Secondary Culture method preferably comprises the following steps: until the cell of original cuiture spread into 70% full time carry out Secondary Culture and purifying, old nutrient solution is abandoned in suction, 0.05% fresh trypsin solution is added in bottle, after cell detachment, stop digestion, add fresh RPMI-1640 nutrient solution, piping and druming makes it to depart from bottle wall and forms cell suspension; Centrifugally be inoculated in new culturing bottle and continue to cultivate.
The step of cell purification is preferably also comprised in described Secondary Culture method, the method of described cell purification preferably comprises the following steps: the difference utilizing tumour cell and inoblast attachment rate, be inoculated in new culturing bottle and draw supernatant after 20 minutes and transfer to new culturing bottle going down to posterity, repeatedly differential velocity adherent removes inoblast.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is:
1, the SGC-7901 proterties that the present invention sets up is stablized, and Absorbable organic halogens repeatedly goes down to posterity, for cancer of the stomach research provides the new experiment material closer to clinical tumor biological characteristics;
2, under the gastric carcinoma cells that the present invention sets up ties up to the prerequisite retaining Major Clinical biological property, there is tumor formation rate high, latent period is short, the features such as homogeneity is good, successfully can prepare animal modal of gastric carcinoma, obtained animal model may be used for fundamental research and drug screening, for the research carried out based on Chinese population genetic background provides strong scientific research data, also for new drug preclinical study experiment in vivo provides new test materials for the test of clinical anti-cancer drug susceptibility and resistance.
3, the SGC-7901 of the present invention's foundation is by compared with nude mouse interior generation parent tumour, can be used to that analyzing body is outer, the dependency of drug disposition susceptibility and resistance, and then two medicine sorting platforms be associated in external, body can be set up, be the ideal cell line of the fundamental research of people's cancer of the stomach and preclinical phase application.
4, formed by the SGC-7901 set up of the present invention, nude mouse tumor also has resistance to the clinical first-line drug 5 FU 5 fluorouracil of cancer of the stomach, is the good test materials of research cancer of the stomach resistance mechanism, has very high scientific research value and development significance.
5, the cell cording height tumor formation rate of the present invention's foundation, high homogeneity, maintains again clinical cancer of the stomach molecular background, derives from Chinese population simultaneously, meet Chinese hereditary feature, anti-cancer of the stomach new drug research and development being applicable to compatriots has very important theory and clinical meaning.
biomaterial preservation information
SGC-7901 of the present invention, China typical culture collection center (CCTCC) is deposited on November 20th, 2013, preservation address is: China. Wuhan. and Wuhan University's postcode: 430072, culture title is gastric carcinoma cells GAXC066, and deposit number is CCTCC NO:C2013154.
Accompanying drawing explanation
Fig. 1 is the morphological observation (amplifying 100 times of visuals field) of GAXC066 cell.
Fig. 2 is the chromosome analysis of GAXC066 cell.Wherein Fig. 2 (A) is GAXC066 cell; Fig. 2 (B) is mouse cell karyomit(e).
Fig. 3 is GAXC066 Immunohistochemistry coloration result (DAB method).Wherein Fig. 3 (A) is negative control; Fig. 3 (B) is carcinomebryonic antigen (CEA); Fig. 3 (C) is cytokeratin (Cytokeratin); Fig. 3 (D) is vimentin (Vimentin); Fig. 3 (E) is CA19-9 (CA19-9); Fig. 3 (F) is ErbB-2 (HER-2).
Fig. 4 is GAXC066 cell doubling time curve.
Fig. 5 is GAXC066 cell cycle analysis result.
Fig. 6 is the restraining effect result of the multiple cancer therapy drug of vitro test to GAXC066 cell proliferation.Wherein Fig. 6 (A) is for cis-platinum is to the growth-inhibiting effect of GAXC066; Fig. 6 (B) is for irinotecan is to the growth-inhibiting effect of GAXC066; Fig. 6 (C) is for 5 FU 5 fluorouracil is to the growth-inhibiting effect of GAXC066; Fig. 6 (D) is for taxol is to the growth-inhibiting effect of GAXC066; Fig. 6 (E) is for epirubicin is to the growth-inhibiting effect of GAXC066; Fig. 6 (F) is for Docetaxel is to the growth-inhibiting effect of GAXC066.
Fig. 7 is the Tumor formation result of GAXC066 cell.
Fig. 8 is the histopathologic slide (amplify the 10 times visuals field) of GAXC066 cell at formation in nude mice.Wherein Fig. 8 (A) is normal lung tissue; Fig. 8 (B) is corresponding sample transplanted tumor block; Fig. 8 (C) is GAXC066 cell inoculation knurl block.
Fig. 9 is that body build-in test cis-platinum and 5 FU 5 fluorouracil are to the Tumor growth inhibition exercising result figure of GAXC066 cell nude mouse model.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
Cis-platinum is purchased from the gloomy medicine company of Jiangsu person of outstanding talent, and 5 FU 5 fluorouracil is purchased from Tianjin KingYork Amino Acid Co., Ltd., and physiological saline is purchased from Zhejiang Tianrui Pharmaceutical Co., Ltd., and cell culture reagent is all bought from American I nvitrogen company.
The preparation of embodiment 1GAXC066 cell
Fresh clinical Operated Specimens of Gastric Carcinoma (Gender: man, age: 72 years old, nationality: the Chinese, native place: Nantong City is obtained from Nantong tumour hospital.), clinical diagnoses is low differentiation adenocarcinoma of stomach.
After fresh HBSS damping fluid (containing 500U/ml penicillin G, 500 μ g/ml Vetstreps and 1.25 μ g/ml amphotericin Bs) rinsing, be cut into the fritter of 20 ~ 50mg, subcutaneous puncture is inoculated in immunodeficient animals SCID mouse (SCID mouse is bought from Beijing Vital River Experimental Animals Technology Co., Ltd.).Tumor animal, after 70 ~ 90 days, is put to death by percutaneous puncture-inoculation, takes out tumor tissues, carries out original cuiture and the Secondary Culture of cancer cells.
Original cuiture step is: tumor tissues is cut into fritter, inserts in culturing bottle, in 37 DEG C of incubator 5%CO
2cultivate under condition; Next day, culturing bottle is slowly overturn and keeps flat, in bottle, add RPMI-1640 nutrient solution (containing 5% foetal calf serum, 100U/ml penicillin G, 100 μ g/ml Vetstreps and 0.25 μ g/ml amphotericin B), quiescent culture; Until cell spread into 70% full time first go down to posterity, repurity, puts into 37% constant incubator afterwards and cultivates.The method gone down to posterity inhales to abandon old nutrient solution, adds 0.05% fresh trypsin solution in bottle, after cell detachment, stops digestion, add fresh RPMI-1640 nutrient solution, and piping and druming makes it to depart from bottle wall and forms cell suspension; Centrifugally be inoculated in new culturing bottle and continue to cultivate.
The method of purifying is in new culturing bottle by gained cell suspension inoculation, after 20 minutes, draw supernatant and transfer to new culturing bottle, owing to becoming fiber attachment rate very fast, cultivate in new substratum after 20 minutes, the inoblast major part adherent growth in cell suspension, so the tumour cell in cell suspension obtains purifying, by above-mentioned steps repeatedly, the object of purified tumor cell is namely reached.
The present invention will derive from original cuiture and the cultured cell line system called after gastric carcinoma cells GAXC066 of tumor tissues, be deposited in China typical culture collection center (CCTCC) on November 20th, 2013, deposit number is CCTCC NO:C2013154.The clone of preservation is submitted to be the clone gone down to posterity after 30 generations.
The biological characteristics of embodiment 2GAXC066 cell and application
The present invention adopts RPMI1640 to cultivate and purifies GAXC066 cell with differential attachment method, can external long term growth and stablely to go down to posterity.When cell reaches 20 generations more than, cell quality is stablized gradually, the biology carrying out being correlated with, genetics and tissue-derived qualification, until the 50th generation all had identical stable proterties.Through experimental observation and checking, the GAXC066 host cell of growth in vitro is large wicker leaf shape, contactless rejection characteristic.Genetics research confirms that this cell is heteroploid, and Numerical and structural chromsomal aberrations is serious, meets the genetics characteristics of malignant tumour.This cell can form tumour in nude mice, has tumorigenicity.Clinical tumor sample, the nude mouse interior generation parent tumour in this cell and its source form corresponding relation, can be in external, the body of research and the dependency of clinical anti-cancer drug susceptibility and resistance provides new test materials.Specific as follows:
A. morphological observation
Under the culturing bottle cultivating GAXC066 cell is placed in inverted microscope, observe under bright field, the results are shown in Figure 1 (Fig. 1 is the morphological observation 100 × shown of GAXC066 cell), visible GAXC066 host cell is large wicker leaf shape, contactless rejection characteristic.
B. chromosomal qualification
In the GAXC066 cell cultivated, add colchicine, make its final concentration be 0.25 μ g/ml, then in 37 DEG C of incubators, continue cultivation 4 hours.Gather the cell of metaphase, be fixed with stationary liquid, then cell suspension dripped on the microscope slide of precooling, with the dyeing of Giemsa staining fluid, in counted under microscope chromosome number.The results are shown in Figure 2, wherein Fig. 2 (A) is GAXC066 cell; Fig. 2 (B) is mouse cell karyomit(e).Result is visible, after the continuous passage of GAXC066 cell, karyomit(e) still keeps the chromosomal feature of humanized's tumour cell, modal number is 51 ± 2, account for 80.89%, show as hyperdiploid, there is most central authorities and submetacentric chromosome (Fig. 2 (A), 1000 ×); And the chromosome number 2n=40 of nude mouse, and be kinetochore, top (Fig. 2 (B), 1000 ×), can distinguish with human chromosomal accordingly.This cell visible is heteroploid (hyperdiploid), and Numerical and structural chromsomal aberrations, meets the genetics characteristics of malignant tumour.
C. short-movie section tumor-necrosis factor glycoproteins (STR) qualification
STR (short tandem repeat, STR) microsatellite DNA is also called, refer on karyomit(e), by several base pair as core unit (2-6 base pair), the class DNA sequence dna (multiplicity is more than 10 ~ 60 time, and gene fragment is below 400 base pairs) that tandem sequence repeats is formed; The number of times that each core unit repeats there will be individual difference, thus the allelotrope that formation sheet segment length is different.Therefore, the multiplicity of one group of STR sequence is almost unique in Different Individual, is individual gene identities feature, is also that cytobiology is to cell identity and the main method identified of originating.
Collect the GAXC066 cell of fresh culture, with AxyPrep genomic dna small volume of reagent box (purchased from the AxyPrep of Axygen company
tMmultisource Genomic DNA Miniprep Kit, article No. is AP-MN-MS-GDNA) extract cell genomic dna, hold fluorescently-labeled primer to carry out pcr amplification with 5 ', products therefrom is checked order, calculate the repeat number of each short-movie section tumor-necrosis factor glycoproteins.
Extract the genomic dna of cell, specific amplification is carried out to STR site, Amelogenin is comprised by sequencing analysis, THO1, TPOX, D13S317, vWA, D16S539, D5S818, the sequence repeat number in each STR site such as CSF1PO and D7S820, and and ATCC, the database that the cells such as DSMZ preserve storehouse carries out inquiry contrast, do not return identical genetic map, its uniqueness provable, and in original cuiture process, there is not the crossed contamination with other cells, the primer sequence in STR site is as shown in SEQ ID NO:1-SEQ ID NO.18 in sequence table, specifically refer to table 1 and table 2:
The primer sequence in table 1 STR site
The primer sequence in table 2STR site and copy number
D. tissue-derived qualification
The inoculation of GAXC066 cell is cultivated on the cover slip, after cytochrome oxidase isozymes, fixes with 4% formaldehyde, carry out immunohistochemical staining (DAB development process).Result such as Fig. 3 shows, and wherein Fig. 3 (A) is negative control; Fig. 3 (B) is carcinomebryonic antigen (CEA); Fig. 3 (C) is cytokeratin (Cytokeratin); Fig. 3 (D) is vimentin (Vimentin); Fig. 3 (E) is CA19-9 (CA19-9); Fig. 3 (F) is ErbB-2 (HER-2).Cytokeratin (Cytokeratin, Fig. 3 (C), 200 ×) is strong positive, and vimentin (Vimentin, Fig. 3 (D), 200 ×) is strong positive; CA19-9 (Fig. 3 (E), 200 ×) is positive; CEA (Fig. 3 (B), 200 ×) is strong positive, points out it to have suitable grade malignancy; ErbB-2 (Her-2, Fig. 3 (F)) is expressed and is common in gland cancer and rare in squama cancer, is expressed as the weak positive; The clinical information of last combining source tissue and pathological diagnosis result, judge that this cell tissue source is as adenocarcinoma of stomach.
E. cytokinetics
GAXC066 cell is seeded in 96 orifice plates with the density in 2000/ hole, cultivates, use the ATP content in the every porocyte of kit measurement respectively 12 hours, 24 hours, 36 hours, 48 hours, 72 hours and 96 hours.
The calculation formula of doubling time is: doubling time=Log2/ rate of curve; Fig. 4 is GAXC066 cell doubling time curve, and wherein the doubling time (X) with the curvilinear equation of Log fluorescent signal (Y) is: Y=0.0093X+4.9076; Wherein R
2=0.9624.Fig. 4 result shows, and the population doubling time of GAXC066 cell is 32.5 hours.
F. cell cycle distribution
Collect about 10
6cell, in 1.5ml centrifuge tube, centrifugally abandons supernatant.Cell precipitation is resuspended with 1 milliliter of-20 DEG C of 75% ethanol, and room temperature fixes 1 hour.Centrifugally abandon supernatant, add 500 microlitre PI staining fluids.Mixing, incubated at room 30 minutes.The STb gene content (the STb gene content of each cell is proportional with total PI fluorescence intensity of this cell) of every porocyte number and each cell is detected with flow cytometer (BD FACSCalibur); And during according to the different cell cycle, the change of cell STb gene content, calculates the total cellular score of each cell cycle.Detection obtains period profile result (Fig. 5 is GAXC066 cell cycle analysis result) and each period profile ratio (table 3) as shown in Figure 5.
The each period profile schedule of proportion of table 3 GAXC066
G. cell in vitro poison experiment
The clinical Common Chemotherapy medicine of external test cancer of the stomach: Fluracil, cis-platinum, the antiproliferative effect of Irinotecan.Subject cell is seeded in 96 orifice plates with the density in 3000/ hole, to the medicine of different concns after three days, with the cell viability under each drug level of CellTiter Glo kit measurement of Promega company, through XLFit computed in software IC
50(half-inhibition concentration).As shown in Figure 6, wherein Fig. 6 (A) is for cis-platinum is to the growth-inhibiting effect of GAXC066 for result; Fig. 6 (B) is for irinotecan is to the growth-inhibiting effect of GAXC066; Fig. 6 (C) is for 5 FU 5 fluorouracil is to the growth-inhibiting effect of GAXC066; Fig. 6 (D) is for taxol is to the growth-inhibiting effect of GAXC066; Fig. 6 (E) is for epirubicin is to the growth-inhibiting effect of GAXC066; Fig. 6 (F) is for Docetaxel is to the growth-inhibiting effect of GAXC066.The IC of each compound
50in table 4.
The IC of table 46 kinds of compounds
50numerical tabular
H. the Tumor formation of cell
Vitro culture and collection GAXC066 cell, subcutaneous vaccination NU/NU nude mouse (buying from Beijing Vital River Experimental Animals Technology Co., Ltd.) mixes with 1:1 with cell suspension and Matrigel matrigel, and every animal inoculation pvaccination 0.2ml, containing 4.0 × 10
6individual cell, inoculates 5 animals, and bilateral is inoculated, N=10, investigates the weight of animals and tumor size weekly.Inoculate after about 6 weeks, tumour starts to be formed and grows, and tumor formation rate is up to more than 90%.Draw tumor growth curve, wherein gross tumor volume=(long × wide × wide) ÷ 2 (as shown in Figure 7, Fig. 7 is the Tumor formation result of GAXC066 cell to result).Visible GAXC066 cell can form tumour and grow homogeneous in immunodeficient mouse body, has high tumorigenicity.
I. the pathology qualification of tumour
After the subcutaneous one-tenth knurl of GAXC066 cell mouse, take out the fixing rear paraffin embedding of tumour, preparation is cut into slices and is carried out H & E and dyes, and as shown in Figure 8, wherein Fig. 8 (A) is normal lung tissue to its pathological diagnosis result; Fig. 8 (B) is corresponding sample transplanted tumor block; Fig. 8 (C) is GAXC066 cell inoculation knurl block.According to Fig. 8 (C), it is low differentiation adenocarcinoma of stomach, and its tissue morphology height is close to fundamental weave knurl block (Fig. 8 (B)), and Fig. 8 A is corresponding adjacent tissues, i.e. normal gastric mucosa (100 ×).It is low differentiation adenocarcinoma of stomach that result shows this tumour.
J. pharmacodynamic experiment in body
Vitro culture and collection GAXC066 cell, (cell suspension and Matrigel mix with 1:1 subcutaneous vaccination NU/NU nude mouse, and every animal inoculation pvaccination 0.2ml, containing 4.0 × 10
6individual cell).Investigate the weight of animals and tumor size weekly.When gross tumor volume reaches 100-200mm
3shi Jinhang random packet, 30 animals are divided into 3 groups at random, and one group is control group, gives physiological saline, 0.1ml/10g, abdominal injection, twice weekly; One group of cis-platinum giving 5mg/kg, abdominal injection, once in a week; One group of 5 FU 5 fluorouracil giving 40mg/kg, abdominal injection, twice weekly.Investigate the weight of animals and gross tumor volume two to three times weekly, administration, after three weeks, to euthanizing animals, terminates experiment, peels off tumor tissues, weigh fixing.
According to formula gross tumor volume (mm
3)=(is long × wide × wide) ÷ 2, calculate gross tumor volume.Medication effect is evaluated: T/C%=[(T-T with formula T/C%
0)/(C-C
0)] × 100%; If administration group tumor regression, T/C%=[(T-T
0)/C
0] × 100%.Wherein, T is administration group gross tumor volume, T
0for D
0it administration group gross tumor volume; C is control group gross tumor volume, C
0for D
0it control group gross tumor volume.
After administration in 21 days, visible administration group is compared with control group, and the inhibiting rate (T/C ratio) of cis-platinum is 7.08%, has obvious tumor inhibition effect; Fig. 9 is that body build-in test cis-platinum and 5 FU 5 fluorouracil are to the Tumor growth inhibition exercising result figure of GAXC066 cell nude mouse model, show according to Fig. 9 result, 5 FU 5 fluorouracil does not have tumor inhibition effect to SGC-7901 of the present invention, illustrates that this gastric carcinoma cells clone has significant resistant characterization to 5 FU 5 fluorouracil.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a SGC-7901, is characterized in that, it is deposited in China typical culture collection center, and deposit number is CCTCC NO:2013154.
2. the daughter cell of SGC-7901 as claimed in claim 1.
3. gastric carcinoma cells as claimed in claim 1 ties up to the purposes prepared in the reagent making immunodeficient mouse generation cancer of the stomach.
4. purposes as claimed in claim 3, it is characterized in that, the described immunodeficient mouse that makes is SCID mouse or nude mouse.
5. the external detection method of a testing compound suppression gastric cancer cell activity, it is characterized in that, this external detection method comprises the following steps: be applied to by testing compound in cell model, use rear antiproliferative effect or cause apoptotic testing compound to be have the compound suppressing cancer of the stomach activity, wherein said cell model is SGC-7901 according to claim 1.
6. an establishment method for SGC-7901 as claimed in claim 1, it is characterized in that, this establishment method comprises the following steps:
(1) obtain fresh clinical people's Operative Excision in Gastric Carcinoma sample, cut into fritter, subcutaneous puncture immunoprophylaxis deficient mice;
(2) percutaneous puncture-inoculation is after 70 ~ 90 days, is put to death by tumor animal, takes out tumor tissues, carries out original cuiture and the Secondary Culture of cancer cells.
7. establishment method as claimed in claim 6, it is characterized in that, the quality of the fritter described in step (1) is 20 ~ 50mg.
8. establishment method as claimed in claim 6, it is characterized in that, the immunodeficient mouse described in step (1) is SCID mouse or nude mouse.
9. establishment method as claimed in claim 6, it is characterized in that, after fresh clinical people's Operated Specimens of Gastric Carcinoma HBSS damping fluid rinsing described in step (1), carry out subcutaneous puncture inoculation again, described HBSS damping fluid comprises the penicillin G of 500U/ml, the Vetstrep of 500 μ g/ml and the amphotericin B of 1.25 μ g/ml.
10. establishment method as claimed in claim 6, it is characterized in that, the original cuiture of the cell described in step (2) comprises the following steps: tumor tissues is cut into fritter, inserts in culturing bottle, in 37 DEG C of incubator 5%CO
2cultivate under condition; Next day adds RPMI-1640 nutrient solution in bottle, and described RPMI-1640 nutrient solution contains 5% foetal calf serum, 100U/ml penicillin G, 100 μ g/ml Vetstreps and 0.25 μ g/ml amphotericin B, quiescent culture; The Secondary Culture of the cell described in step (2) comprises the following steps: inhale and abandon old nutrient solution, in bottle, add 0.05% fresh trypsin solution, after cell detachment, add fresh DMEM/F12 nutrient solution, careful piping and druming, makes it to depart from bottle wall and forms cell suspension; Collect whole cell, centrifugal, be inoculated in new culturing bottle respectively and continue to cultivate, described per-cent is mass percent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410499357.9A CN104403996B (en) | 2014-09-25 | 2014-09-25 | Human gastric cancer cell line with 5-fluorouracil resistance and establishment method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410499357.9A CN104403996B (en) | 2014-09-25 | 2014-09-25 | Human gastric cancer cell line with 5-fluorouracil resistance and establishment method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104403996A true CN104403996A (en) | 2015-03-11 |
| CN104403996B CN104403996B (en) | 2020-08-07 |
Family
ID=52641665
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410499357.9A Active CN104403996B (en) | 2014-09-25 | 2014-09-25 | Human gastric cancer cell line with 5-fluorouracil resistance and establishment method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104403996B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020206999A1 (en) * | 2019-04-11 | 2020-10-15 | 北京基石生命科技有限公司 | Method for culturing primary cells of gastric cancer and gallbladder and bile duct cancer, and supporting reagents |
| CN113337471A (en) * | 2021-04-02 | 2021-09-03 | 清华-伯克利深圳学院筹备办公室 | 5-fluorouracil-resistant colorectal adenocarcinoma epithelial cells and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102465113A (en) * | 2010-11-12 | 2012-05-23 | 复旦大学附属中山医院 | Human hepatoma carcinoma cell line and application thereof |
| CN103627673A (en) * | 2012-08-21 | 2014-03-12 | 上海睿智化学研究有限公司 | Human brain glioma cell line, and establishing method and application thereof |
-
2014
- 2014-09-25 CN CN201410499357.9A patent/CN104403996B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102465113A (en) * | 2010-11-12 | 2012-05-23 | 复旦大学附属中山医院 | Human hepatoma carcinoma cell line and application thereof |
| CN103627673A (en) * | 2012-08-21 | 2014-03-12 | 上海睿智化学研究有限公司 | Human brain glioma cell line, and establishing method and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| CHENG-PING JIANG ET AL.,: "Overexpression of ECRG4 enhances chemosensitivity to 5-fluorouracil in the human gastric cancer SGC-7901 cell line", 《TUMOR BIOL》 * |
| 张继燃等: "胃癌实验动物模型的研究概况", 《中国普通外科杂志》 * |
| 杨皎娃等: "胃癌多药耐药细胞株BGC823/5-FU的建立及其耐药机制的研究", 《中国病理生理杂志》 * |
| 费洪新等: "顺铂与5 -氟尿嘧啶对体外人胃癌细胞影响的研究", 《大连医科大学学报》 * |
| 郝淑兰等: "人胃癌SGC7901/VCR裸鼠移植瘤模型的建立及耐药性检测", 《世界中西医结合杂志》 * |
| 黎伯培等: "胃癌耐药细胞株SGC－7901／5－FU的建立及其耐药机制的初步探讨", 《消化肿瘤杂志(电子版)》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020206999A1 (en) * | 2019-04-11 | 2020-10-15 | 北京基石生命科技有限公司 | Method for culturing primary cells of gastric cancer and gallbladder and bile duct cancer, and supporting reagents |
| CN113337471A (en) * | 2021-04-02 | 2021-09-03 | 清华-伯克利深圳学院筹备办公室 | 5-fluorouracil-resistant colorectal adenocarcinoma epithelial cells and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104403996B (en) | 2020-08-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103627673B (en) | A kind of human glioma cell line and its method for building up and application | |
| CN102465113B (en) | Human hepatoma carcinoma cell line and application thereof | |
| CN104694476A (en) | Human non-small cell lung cancer cell line, and establishment method and application thereof | |
| CN102250840B (en) | Human pancreatic cancer cell line and its application | |
| CN102719403B (en) | A kind of human pancreas's adenosquamous carcinoma clone and establishment method thereof and application | |
| CN108866000B (en) | Human epidermal growth factor tyrosine kinase inhibitor acquired drug-resistant lung cancer cell line and establishment method and application thereof | |
| CN102067828B (en) | Highly metastatic model of human melanoma, cell subline, creation methods, and dynamic detection of metastasis | |
| CN103333858B (en) | Gleevec-resistant gastrointestinal stromal tumor cell line, method thereof, and nude mouse transplantation tumor model thereof | |
| CN106834486A (en) | Osteosarcoma molecule diagnosis and treatment mark and its application | |
| CN107541494B (en) | Human bile duct cancer cell line and application thereof | |
| CN114908039A (en) | Culture medium for stomach cancer organoid and culture method without bracket thereof | |
| CN110106150A (en) | A kind of preparation method and application of synovial sarcoma cells system hSS-005R | |
| CN102329775B (en) | A kind of human pancreatic cancer cell to gemcitabine resistance and application thereof | |
| CN105255832A (en) | Human bile duct cancer cell line and applications thereof | |
| CN104745530A (en) | Human hepatocellular carcinoma cell line, and establishing method and application thereof | |
| CN106479981B (en) | A kind of people's Endometrial carcinoma cell line and its method for building up | |
| CN103966169A (en) | Chinese people tongue squamous epithelial cell cancer cell line CTSC-1 and establishment method thereof | |
| CN109880902A (en) | A kind of application of long-chain non-coding RP11-499F3.2 in head and neck cancer clinical detection and the treatment of reversing tumor Cetuximab drug resistance | |
| CN104403996A (en) | Human gastric cancer cell line with 5-fluorouracil resistance and establishing method and application thereof | |
| CN115786262B (en) | Human hilar bile duct cancer cell line CBC3T-1 and application thereof | |
| CN106244679A (en) | MiR 100 inhibitor purposes in reducing cancer metastasis | |
| CN113234679B (en) | Crizotinib-resistant human lung adenocarcinoma cell strain and preparation and application thereof | |
| CN104403997A (en) | Human gastric cancer cell line with cisplatin resistance and establishing method and application thereof | |
| CN110172448A (en) | A kind of synovial sarcoma cells system hSS-005R and its progeny cell system | |
| CN109929804B (en) | Human ovarian cancer cell line and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |