The Study On Hepatocellular Carcinoma In Primary Culture In Vitro Chemotherapy Sensitivity Test

BACKGROUNDHepatocellular carcinoma is one of the frequent malignant tumors with high incidence and mortality rate.Chemotherapy is one of the major and auxiliary treatment to Hepatocellular carcinoma.But the treatment significantly can not be enhanced.The research, application of tumor cells with chemotherapeutic drug sensitivity test to guide the study, was gived more and more individualized attention, and as a cancer chemotherapy drug of reference in recent years. This study screen chemotherapy drugs for sensitive of hepatocellular carcinoma by primarily cultured hepatoma cells in vitro chemosensitivity test, and analysis chemotherapy drugs on DLC-1.MTT assay and flow cytometry was screening drug-sensitive method of sensitivity and specificity to compare advantages and disadvantages.At the same time use the peripheral blood lymphocytes in same patients with chemotherapy-sensitive experiments, analysis of hepatoma cells and peripheral blood lymphocytes of the same spectrum whether the chemotherapy drug sensitivity.By hepatocellular carcinoma cells in primary culture and peripheral blood lymphocytes in vitro chemosensitivity test provide a theoretical basis for clinical application.OBJECTIVEWhether the sensitivity of chemotherapeutic drugs in different patients with hepatocellular carcinoma is consistent. Whether the peripheral blood lymphocytes in patients with liver cancer chemotherapeutic drug sensitivity tests can replace the liver cells in primary culture.The relationships between DLC-1 and chemotherapy drugs, Which is the drug resistance mechanisms.METHODS1.Preparation of single hepatocellular carcinoma cell suspension:Choose 9 cases of fresh liver tumor samples. Specimens were confirmed by pathological examination.Under sterile conditions, hepatocellular carcinoma were cut approximately 1.0cm3, washed 2-3 times with PBS,cut into small pieces,filtered after steel mesh sieve which 200 is not the purpose of penetrating, cultured with RPMI1640 medium in the incubator. 2. Separation of peripheral blood lymphocytes:The anticoagulation with peripheral venous blood in patients plus an equal volume of 5ml of lymphocyte separation medium,isolated lymphocytes, washed two times with Hanks and then use the RPMI1640 culture medium prepared into a single cell suspension prepared fluid.3.Inoculation cultured with RPMI1640 medium in incubator.Primary hepato cellular carcinomas was tested by 13 kinds of chemotherapeutic drug to measure sensitivity, choose a high relatively inhibition rate of drugs to test sensitivity of tumor cells and peripheral blood lymphocytes.4. MTT colorimetric drug sensitivity test:The suspension of the above-mentioned three kinds of cancer cells cultured 48h by adding MTT again for 6 hours after adding DMSO to make crystals completely dissolved violet blue A microplate reader at 497nm wavelength absorbance degree of measured absorbance OD values.According to the relative inhibition rate to determine drug sensitivity.The relative inhibition rate of lymphocytes≥20% were as sensitive;the relative inhibition rate of tumor cells>30% were as sensitive.5. Determination of cell apoptosis:cell suspension has been made, washed two times with PBS,cell suspension was fixed by anhydrous ethanol.we adjust the cell concentration is, adding PI on 0.2ml cell suspension staining protect from light 20min,then centrifuged and PBS wash.The cell cycle and apoptosis of cancer cells were detected by the flow cytometer.6. RT-PCR and Westernblot determine the expression of the DLC-1 gene in primary liver cancer cells after adding chemotherapy.7. Analysis:All data are dealed with SPSS software.RULTS1.4 cases were successful cultured in 9 cases of HCC specimens.2.In addition to CTX group all experimental chemotherapy drug can inhibit the growth of hepatocellular carcinoma with varying degrees and increase apoptosis,HEPG-2 cells showed difference degrees of drug resistance to chemotherapeutic drugs of all experiments.5-FU,EADM, MMC, DDP were the highest sensitivity rate of all the sensitivity of primary liver cancer by adding chemothera peutic drugs exist the significant difference,the inhibition of identical drugs in different primary hepatocellular carcinomas were different, it has statistically significance (P<0.05). the sensitivity of different drugs in identical primary hepatocellular carcinomas were different,it has statistically significant (P<0.05). 3.The sensitivity of chemotherapeutic drugs on primary hepatocellular carcinomas and peripheral blood lymphocytes exist an obvious individual differences, the identical chemotherapy drugs on cancer cells and lymphocytes in same patients with inhibition and apoptosis rate was no significant difference(P>0.05);Hepatocellular carcinomas and peripheral blood lymphocytes with the inhibition and apoptosis rate exist relationship (P<0.05).4.Two cases were expressed DLC-1 in four cases of primary hepatocellular carcinomas,compared with the control group were significantly (P<0.05), the expression of DLC-1 was significantly rised after chemotherapy drugs with two cases of primary hepatocellular carcinoma,compared with the original generation of hepatoma cells were significantly (P<0.05).CONCLUSION1.The sensitivity of hepatocellular carcinoma cells by chemotherapy drugs exists an obvious differences. the inhibition of identical drugs in different primary hepatocellular carcinomas were different, the sensitivity of different drugs in identical primary hepatocellular carcinomas were different.2.The sensitivity of peripheral blood lymphocytes and tumor cells to chemoth erapeutic drugs has a good positive correlation. It may be applied to peripheral lymphocyte culture chemotherapeutic drug sensitivity experiments to predicted sens itive drugs.3.The expression of DLC-1 was significantly rised in hepatoma cell culture by adding chemotherapy,DLC-1 gene expression would be play an important role in the early diagnosis of HCC,predict recurrence and metastasis,prognosis in the future.