CN105274057A - Oxaliplatin-resistant human high-metastasis hepatoma cell line and building method thereof - Google Patents
Oxaliplatin-resistant human high-metastasis hepatoma cell line and building method thereof Download PDFInfo
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Abstract
The invention discloses an oxaliplatin-resistant human high-metastasis hepatoma cell line and a building method thereof. The oxaliplatin-resistant human high-metastasis hepatoma cell line is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number of the oxaliplatin-resistant human high-metastasis hepatoma cell line is C2014164. The building method is characterized in that a human high-metastasis hepatoma cell line LM3 is used as an induction object, and a continuous induction method is used to build the oxaliplatin-resistant human high-metastasis hepatoma cell line LM3-OR. The built oxaliplatin-resistant human high-metastasis hepatoma cell line can be used for building in-vivo and in-vitro tumor drug resistance models, and an experiment basis is provided for disclosing of a liver cancer drug resistance mechanism, appearance of drug resistance reversing and development of new anti-cancer drugs.
Description
Technical field
The invention belongs to cell engineering field, particularly a kind of Oxaliplatin-resistant people height transfer hepatoma cell strain and establishment method thereof.
Background technology
Hepatocellular carcinoma is the world the 5th, the third-largest common malignant tumour of China, and the whole world has every year more than 500,000 liver cancer new cases, wherein occurs in China more than 50%.
Due to early, mid-term liver cancer without obvious clinical manifestation, the liver cancer patient of more than 60% first medical time just missed best opportunity of operation.EACH research confirms first, and the FOLFOX chemotherapy new departure containing oxaliplatin makes advanced liver cancer death risk reduce by 20%, and relapse and metastasis risk reduces by 38%, and tumour objective remission rate significantly improves arrival 8.2%.Although oxaliplatin clear curative effect, many liver cancer patients produce resistance to oxaliplatin after treating continuously, thus cause chemotherapy failure.Therefore, to set up the human hepatoma cell strain of Oxaliplatin-resistant thus research resistance mechanism is very important for raising targeted therapy effect.
In recent years, tumor drug resistance clone has become the important tool of research tumor drug resistance mechanism and reversing tumor resistance.Set up on basis at drug-resistant cell strain, the deployable a series of research of researchist, as disclosed resistance mechanism, reversing drug resistance occurs, develop new cancer therapy drug, response etc. is treated in assessment.The method at present setting up tumor drug resistance clone by drug screening is divided into two kinds, i.e. successive induction method and heavy dose of ballistic method.And the former has the advantages such as resistance produces more easily, stability is stronger.Therefore, the present invention selects people's height transfer hepatoma cell strain LM3 to be induction object, and people's height transfer hepatoma cell strain LM3, after the plantation of nude mice hepatocyte in situ, has highly spontaneous transfer ability.Wherein, Lung metastases rate is 100%, and Abdominal wall metastasis rate is 100%, and the diaphragm rate of transform is 70%.
Up to the present, the research of setting up the human hepatoma cell strain LM3-OR of resistance to oxaliplatin with people's height transfer hepatoma cell strain LM3 for induction object rarely has report.
Summary of the invention
The object of this invention is to provide a kind of people to Oxaliplatin-resistant height transfer hepatoma cell strain and establishment method thereof, this cell strain can be used for the inside and outside tumor drug resistance model of construct.
In order to achieve the above object, the invention provides a kind of people's height transfer hepatoma cell strain of Oxaliplatin-resistant, Classification And Nomenclature is hepatoma cell strain LM3, be preserved in China typical culture collection center (CCTCC) on September 24th, 2014, deposit number is CCTCCNO:C2014164, preservation address is: Wuhan, China, Wuhan University, postcode: 430072.
Present invention also offers the establishment method of above-mentioned Oxaliplatin-resistant people height transfer hepatoma cell strain, the method for induction object, adopts successive induction method to set up Oxaliplatin-resistant people height transfer hepatoma cell strain LM3-OR with people's height transfer hepatoma cell strain LM3.
Preferably, the concrete steps of described successive induction method comprise:
The first step: after people's height transfer hepatoma cell strain LM3 recovery, logarithmic phase is cultured to nutrient solution, induce with oxaliplatin pharmaceutical, the method of described induction is: add oxaliplatin when the cell nutrient solution of logarithmic phase is cultured to 40%-60% adherent rate, starting point concentration is 0.4-0.6 μ g/mL, continue cultivate, and maintain this drug level until cell can stable growth, go down to posterity;
Second step: passage cell nutrient solution is cultured to logarithmic phase, repeat to adopt the induction method described in the first step to induce, but progressively improve add the starting point concentration of oxaliplatin, until cell can in the nutrient solution of the oxaliplatin containing 8.0-12.0 μ g/mL concentration stable growth, go down to posterity and recover, cell strain is now Oxaliplatin-resistant people height transfer hepatoma cell strain LM3-OR.
Preferably, the nutrient solution in the first step and second step is the DMEM nutrient solution containing 10-15% foetal calf serum.
More preferably, also penicillin 100-200U/mL and Streptomycin sulphate 100-200 μ g/mL is contained in described DMEM nutrient solution.
Preferably, the cell culture temperature in the first step and second step is 37 DEG C, cultivates CO in atmosphere
2concentration is 5%.
Preferably, the progressively raising mode of the starting point concentration of oxaliplatin described in second step is: the concentration first increasing oxaliplatin in the mode increasing 0.5-1 μ g/ml at every turn; When oxaliplatin concentration is increased to 4-6 μ g/ml, then increase the concentration of oxaliplatin in the mode increasing 1-1.5 μ g/ml at every turn.
In a kind of optimal way of the present invention, the progressively raising mode of the starting point concentration of oxaliplatin described in second step is: the starting point concentration of oxaliplatin is changed successively into 1.5 μ g/ml, 2.5 μ g/ml, 3.5 μ g/ml, 4.5 μ g/ml, 5.5 μ g/mL, 7 μ g/mL, 8.5 μ g/mL, 10 μ g/mL.
Compared with prior art, Oxaliplatin-resistant people height transfer hepatoma cell strain LM3-OR drug-resistant performance provided by the invention is good, can be used for the inside and outside tumor drug resistance model of construct, for liver cancer disclose resistance mechanism, reversing drug resistance occurs, develops new cancer therapy drug and provide experiment basis.
Accompanying drawing explanation
Fig. 1 is that the cell of LM3, LM3-OR is containing Colony forming result under different oxaliplatin effective concentration;
Fig. 2 is basis of microscopic observation LM3 (A), LM3-OR (B) clone is containing the form (100 ×) in 10.0 μ g/ml oxaliplatin effective concentration nutrient solutions;
Fig. 3 is the cell growth curve figure of LM3, LM3-OR;
Fig. 4 is LM3 (A), LM3-OR (B) cell invasion experimental result (400 ×);
Fig. 5 is LM3, LM3-OR cytogene cluster analysis figure.
Embodiment
The present invention is further illustrated below by embodiment.Embodiments of the invention are only used for the present invention is described, instead of limitation of the present invention, under concept thereof of the present invention, all belong to the scope of protection of present invention to the simple modifications of the inventive method.
Embodiment 1: the induction of Oxaliplatin-resistant people height transfer hepatoma cell strain LM3-OR is set up
A kind of people's height transfer hepatoma cell strain of Oxaliplatin-resistant, name is called Oxaliplatin-resistant people height transfer hepatoma cell strain LM3-OR, be deposited in China typical culture collection center (CCTCC), deposit number is C2014164, its establishment method is: adopt people's height transfer hepatoma cell strain LM3 to be induction object, adopt successive induction method to set up Oxaliplatin-resistant people height transfer hepatoma cell strain LM3-OR, described successive induction method is:
(1) get commercially available people's height transfer hepatoma cell strain LM3, after recovery, use DMEM nutrient solution (containing 10% foetal calf serum, penicillin 100U/mL, Streptomycin sulphate 100 μ g/mL) in 5%CO
2, be cultured to logarithmic phase under 37 DEG C of conditions;
(2) to take the logarithm LM3 cell in vegetative period, renew fresh DMEM nutrient solution (containing 10% foetal calf serum, penicillin 100U/mL, Streptomycin sulphate 100 μ g/mL), induce with oxaliplatin pharmaceutical, described induction method is: add oxaliplatin when the cell nutrient solution of logarithmic phase is cultured to 40%-60% adherent rate, starting point concentration is 0.5 μ g/mL, at 5%CO
2, continue to cultivate under 37 DEG C of conditions, change liquid in good time, maintain 0.5 μ g/mL drug level until cell can in the survival of this concentration and slowly growth;
(3) passage cell nutrient solution is cultured to logarithmic phase, repeat to adopt the induction method in step (2) to induce, the starting point concentration of oxaliplatin is changed successively into 1.5 μ g/mL, 2.5 μ g/mL, 3.5 μ g/mL, 4.5 μ g/mL, 5.5 μ g/mL, 7 μ g/mL, 8.5 μ g/mL and 10 μ g/mL, so repeatedly induce, change liquid, go down to posterity, last about 6 months, final cell can grow and go down to posterity in the nutrient solution of the oxaliplatin containing 10.0 μ g/mL concentration, and cell strain is now Oxaliplatin-resistant people height transfer hepatoma cell strain LM3-OR;
(4) above-mentioned LM3 and LM3-OR cell is inoculated in 6 orifice plates (1000, every hole cell), adds the oxaliplatin (5-10 μ g/mL) of different concns gradient after 24 hours, fix after 10 days, take pictures after Giemsa dyeing.As shown in Figure 1, LM3 cell in 5 μ g/mL concentration without Clone formation; LM-OR cell all can grow 5-10 μ g/mL oxaliplatin and form colony, illustrates that LM3-OR cell can tolerate oxaliplatin preferably.
Embodiment 2: drug-resistant cell strain morphological observation
The people's height transfer hepatoma cell strain LM3 taken the logarithm vegetative period and Oxaliplatin-resistant people height transfer hepatoma cell strain LM3-OR, grow 72 hours in 10 μ g/mL nutrient solutions after, observation of cell form under inverted microscope, result is as shown in Figure 2: compared to LM3 cell, LM3-OR Growth of Cells is good, in fusiformis, the even one-tenth wire had.
Embodiment 3: cell growth curve measures
People's height transfer hepatoma cell strain LM3 and Oxaliplatin-resistant people height transfer hepatoma cell strain LM3-OR is digested respectively, counts, 96 orifice plates are inoculated according to 3000/hole, after cultivating 24,48,72,96 hours respectively, every hole adds CCK8 (the green skies, No.C0037), 2 hours are hatched.570nm microplate reader (Model680, Bio-Rad) surveys absorbancy.LM3 and LM3-OR cell growth curve is shown in Fig. 3.
Embodiment 4: cell invasion measuring
With Matrigel (BD company, No.356234) by after 1:7 dilution, get 75 μ L and wrap by room on Transwell, at being placed in 37 DEG C 4 hours.People's height transfer hepatoma cell strain LM3 and Oxaliplatin-resistant people height transfer hepatoma cell strain LM3-OR is used respectively trysinization, centrifugal, add DMEM substratum and make containing 2 × 10
4cell suspension 500 μ L, adds in the upper room of Transwell (Corning company, No.3422), adds containing 10% foetal calf serum nutrient solution 500 μ L in lower room.Put into 5%CO
2, 37 DEG C of incubator cellar cultures are fixing after 12 hours, Giemsa dyes, and takes pictures under inverted microscope.LM3-OR comparatively LM3 cell invasion ability strengthens (the results are shown in Figure 4).
Embodiment 5 cellular gene expression chip detection
Agilent people's chip of expression spectrum 4 × 44K (designID:014850) is selected in this experiment, and chip contains more than 41,000 Human genome and transcript.After RNA quality inspection is qualified, carry out reverse transcription with reference to Afilent chip of expression spectrum normal process and become double-strand cDNA, synthesize the cRNA with Cy3 mark more further, chip hybridization, utilize AgilentScanner to scan after wash-out and obtain original image, adopt FeatureExtraction software processes original image to extract raw data, utilize Genespring software to carry out quantile standardization.
Express spectra variance analysis result shows, LM3 and LM3-OR cell is compared, and find differences (P005FC2) gene 264 altogether, wherein raises (LM3-OR/LM3) gene 46, down-regulated gene 218.Test the selective analysis gene relevant to resistance, find abc transport body (ABCtransporters), cell cycle (Cellcycle), apoptosis (Apoptosis), epithelial cell interstitial transition (Epithelia-mesenchymetransition), drug metabolism-lipid synthesis (Drugmetabolism-lipidsynthesis) path generation significant enrichment, especially abc transport body pathway gene generation considerable change, present rise (administration group/control group) trend, ABCB, CASP, MAPK genes involved is in important regulating and controlling status in path, demonstrate the important mechanisms that oxaliplatin kills and wounds cells of resistant tumors.
More than describe preferred embodiment of the present invention in detail.Should be appreciated that the ordinary skill of this area just design according to the present invention can make many modifications and variations without the need to creative work.Therefore, all technician in the art, all should by the determined protection domain of claims under this invention's idea on the basis of existing technology by the available technical scheme of logical analysis, reasoning, or a limited experiment.
Claims (9)
1. an Oxaliplatin-resistant people height transfer hepatoma cell strain, name is called people's height transfer hepatoma cell strain LM3-OR of Oxaliplatin-resistant, and be deposited in China typical culture collection center (CCTCC), deposit number is C2014164.
2. the establishment method of Oxaliplatin-resistant people height transfer hepatoma cell strain as claimed in claim 1, it is characterized in that, with people's height transfer hepatoma cell strain LM3 for induction object, successive induction method is adopted to set up people's height transfer hepatoma cell strain LM3-OR of Oxaliplatin-resistant.
3. the establishment method of Oxaliplatin-resistant people height transfer hepatoma cell strain as claimed in claim 2, it is characterized in that, the concrete steps of described successive induction method comprise:
The first step: after people's height transfer hepatoma cell strain LM3 recovery, logarithmic phase is cultured to nutrient solution, induce with oxaliplatin pharmaceutical, the method of described induction is: add oxaliplatin when the cell nutrient solution of logarithmic phase is cultured to 40%-60% adherent rate, starting point concentration is 0.4-0.6 μ g/mL, continue to cultivate, and maintain this drug level until cell energy stable growth, to go down to posterity;
Second step: passage cell nutrient solution is cultured to logarithmic phase, repeat to adopt the induction method described in the first step to induce, but progressively improve add the starting point concentration of oxaliplatin, until cell can in the nutrient solution of the oxaliplatin containing 8.0-12.0 μ g/mL concentration stable growth, go down to posterity and recover, cell strain is now Oxaliplatin-resistant people height transfer hepatoma cell strain LM3-OR.
4. the establishment method of Oxaliplatin-resistant people height transfer hepatoma cell strain as claimed in claim 3, is characterized in that, the described nutrient solution in the first step and second step is the DMEM nutrient solution containing 10-15% foetal calf serum.
5. the establishment method of Oxaliplatin-resistant people height transfer hepatoma cell strain as claimed in claim 4, is characterized in that, also containing penicillin 100-200U/mL and Streptomycin sulphate 100-200 μ g/mL in described DMEM nutrient solution.
6. the establishment method of Oxaliplatin-resistant people height transfer hepatoma cell strain as claimed in claim 3, is characterized in that, the cell culture temperature in the first step and second step is 37 DEG C, cultivates CO in atmosphere
2concentration is 5%.
7. the establishment method of Oxaliplatin-resistant people height transfer hepatoma cell strain as claimed in claim 3, it is characterized in that, the progressively raising mode of the starting point concentration of the oxaliplatin described in second step is: the concentration first increasing oxaliplatin in the mode increasing 0.5-1 μ g/mL at every turn; When oxaliplatin concentration is increased to 4-6 μ g/mL, then increase the concentration of oxaliplatin in the mode increasing 1-1.5 μ g/mL at every turn.
8. the establishment method of Oxaliplatin-resistant people height transfer hepatoma cell strain as claimed in claim 3, it is characterized in that, the progressively raising mode of the starting point concentration of oxaliplatin described in second step is: the starting point concentration of oxaliplatin is changed successively into 1.5 μ g/mL, 2.5 μ g/mL, 3.5 μ g/mL, 4.5 μ g/mL, 5.5 μ g/mL, 7 μ g/mL, 8.5 μ g/mL, 10 μ g/mL.
9. the application of Oxaliplatin-resistant people height transfer hepatoma cell strain according to claim 1 in tumor research.
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| CN108315303A (en) * | 2018-01-30 | 2018-07-24 | 复旦大学附属中山医院 | A method of preparing Human gallbladder carcinoma gemcitabine medicine-resistant cell line |
| CN108384757A (en) * | 2018-01-30 | 2018-08-10 | 复旦大学附属中山医院 | A method of preparing Human gallbladder carcinoma oxaliplatin resistant cell line |
| CN114015654A (en) * | 2021-10-21 | 2022-02-08 | 复旦大学附属中山医院 | Double-fluorescence labeled oxaliplatin human hepatoma cell strain and construction method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315303A (en) * | 2018-01-30 | 2018-07-24 | 复旦大学附属中山医院 | A method of preparing Human gallbladder carcinoma gemcitabine medicine-resistant cell line |
| CN108384757A (en) * | 2018-01-30 | 2018-08-10 | 复旦大学附属中山医院 | A method of preparing Human gallbladder carcinoma oxaliplatin resistant cell line |
| CN114015654A (en) * | 2021-10-21 | 2022-02-08 | 复旦大学附属中山医院 | Double-fluorescence labeled oxaliplatin human hepatoma cell strain and construction method and application thereof |
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