Studies On The Side Population Cells In Huh7 And Their Different Proliferation And Division Ability

Background:The malignant tumors pose a serious threat to the survival of human beings. The existing treatment measures hardly change the status that patients with advanced cancers eventually lead to recurrence and even death. Presently, tumor invasion and drug resistance owing to long-term treatment are still major obstacles to overcome cancer. Therefore, to clarify the molecular mechanism of tumor drug resistance has both important value of basic theory and clinical applications. There are a very small number of stem cell-like tumor cells in tumor tissues based on the theory of cancer stem cells, which have unlimited potential for proliferation, and play a decisive role in tumor formation, drug resistance, and recurrence and metastasis, while the majority of the remaining cells will go to die after a short spell of division. Cancer stem cells are the most fundamental cause of drug resistance, whose tolerance to chemotherapeutic drugs is a main obstacle to cancer treatment.Presently, the isolation of cancer stem cells is mainly based on the markers of surface antigen-specific antibody or detection of the side population cells through FACS. This study aims to explore the the side population cells in hepatocellular carcinoma cell line Huh7 and their special proliferation ability and division ability, and to provide a new way of thinking for the targeted therapy of hepatocellular carcinoma. Objectives:The aim of this study is to explore the isolation of side population cells in Huh7 cell line and their special proliferation ability and division ability compared to non-side population counterparts, to provide a new way of thinking for the targeted therapy of hepatocellular carcinoma.Methods:1. Cell Culture: Huh7 cells were cultured in 10% FBS DMEM medium regularly.2. Preparation of single cell suspension solution: cells growing in the logarithmic phase are used for the study, and the cell concentration adjusts to 2 ~ 3×10~6 / mL. Add 150μmol/L Verapamil to part of the cell suspension solution for the control group, then the cell suspension solution are cultured at 37℃for 20min. 10μg/ml Hoechst33342are added both experimental group and control group, and are incubated in the 37℃water pot and kept in dark for 90min, vibrating them at low speed every 15 minutes. At last, they are washed three times ( 800 round / min, 5min / time) with 10 mmol/L HEPES, 10% FBS Hank's solution, and put on ice . Both experimental group and control group are filtered through 40μm sieve to get single cell suspension solution before sorting, adding PI to a final concentration of 2μg/ml before FACS.3. Isolation of side population cells in Huh7 cell line through FACS.4. Hoechst33342 fluorescence intensity in side population and non-side population cells are detected by fluorescence microscope.5. ABCG2 mRNA expression levels between side population and non-side population cells are detected by semi-quantitative RT-PCR, and the product of RT-PCR is sequenced by sequencing company. GAPDH is an internal control.6. Expression of ABCG2 protein in both side population and non-side population cells are detected by Fluorescent Immunocytochemistry assay, and their different ability of proliferation and asymmetrical division are observed.Results:1. The ratio of side population cells in Huh7 cell lines is about 2.22% through FACS, while in verapamil group, which decreases to 0.74%.2. Hoechst33342 fluorescence intensity in side population cells is significantly weaker than that of non-side population cells under fluorescence microscope.3. ABCG2 mRNA expression level in the side population cells is 4.32-fold (P <0.01) to that of non-side population cells, and the product of RT-PCR is confirmed by sequencing.4. The proliferation ability of side population cells is 2.7-fold (P <0.01) to that of non-side population cells.5. Side population cells can generate both ABCG2 (+) and ABCG2 (-) cells, while non-side population cells can only generate ABCG2 (-) cells.Conclusion:There is a small fraction of side population cells (2.22%) in Huh7 cell line, whose proliferation ability is stronger than that of non-side population cells. Side population cells have asymmetric division ability (self-renewal and directed differentiation), which is the trait of cancer stem cells. So the isolation of side population cells can be used as a method to isolate cancer stem cells, and side population cells may play an important role in tumor formation, drug resistance, even recurrence and metastasis.