Establishment And Identification Of Primary Hepatocellular Carcinoma Cell Lines

The mortality rate of Liver cancer is particular high, because of the pathogenesis remaining unclear, developing more quickly, finding difficulty, high probability of recurrence after resection, etc. Recent years, many scientists had been put forward the theory of tumor heterogeneity and tumor stem cell, which making us seeking live cancer from a new angle. Additionally, they also indicated that live tumor stem cell markers would provide a new target for tumor immunity therapy.Aim :1. Isolating tumor cells from liver cancer tissue and pericarcinomatous tissue, stable cell strains were obtained by purification. 2. Identifying biological characteristics of these 8 cell strains, including cell morphology, cell growth cycle, migration and invasion ability, ability of single cell cloning, growth cycle, Karyological Characteristic, molecular markers, tumorigenic ability and others.Methods:1. Separation and culture of the tumor cells: retrieved the fresh tissues of the cell by cutting up method; Purifying tumor cell by repeated differential digested, differential adherent method and then expanding the culture.2.Identifying the biological characteristics of tumor cells: Using Incu Cyte Zoo M to detect cellular morphology, cell migration, invasion ability and tracking single cell cloning because it can dynamic observation and analyzing for a long time. HE staining for calculating nuclear mass ratio; Giemsa stain modal for analyzing of chromosomes mode and karyotype. Tumorigenesis of Nude mice in vivo. Collecting the supernatants to detect the copy of HBV-DNA by Q-PCR. Detecting the expression of AFP, GPC3, Hep Par-1, CK18, CK19, PCNA, Vimentin in the tumor cells by immunofluorescence technology; Detecting the expression of Ep CAM, CD13, CD44, CD90, CD24, CD47, CD133, DLK1 and cell cycle in the tumor stem cell by flow cytometer. Employing three drugs( Plasmocin, BM- Cyclin, MRA) to treat mycoplasma contamination cells and using three ways(CLARK one-step reagents, Biotool,PCR detection) to detect mycoplasma.Results: Separation and purification tumor cells from different sources and establish 8 stable strain cells. The cellular morphology of these 8 strain cells was different and the nucleus mass ratio was abnormal. Compared with MTT, Incu Cyte Zoom,fluorescence, cell growth curve which drew based on cell confluence could reflect the proliferation these 8 strain cells exactly, of which 177 T was the fastest for proliferation and it's doubling time was 29.70±0.84 h. The followed sequenced based on double time was 216T3, 78 T, 83 N, 216T2, 216 N, 216T1, 92 N. What's more, 216T2 was the strongest for their migrate ability and it's wound healing time was 45±0.82 h. Through matrix time was 22±0.45 h. All of these 8 strains tumor cell can form cloning in vitro detected by Incu Cyte Zoo M. The ability of 177 T was strongest and 78 T was weakest. Compared these cells cycle, G2 phase of 216T1 was shorter than others. Among these cells, 216 N, 216T1 were the tetraploid and the rest were 6 times, excepting 216 N,216T1,216T2, the others were all HBV-NDA related. All of these strain cells were not expressed of AFP, 8 Hep Par – 1 and high expression of CK18, GPC3, while low expression of CK19, some cells expressing PCNA, Vimentin. 216T1 expressed more vimentin than the others 7 strain cells. The different of PCNA in these cells reflected the differentiated degree and 216T2 was the lowest degree.CD47 were all expressed on these 8 strain cells at high levels and DLK1 do not expressed. In addition, these eight markers were also expressed in that 8 strains cells we sorted and could be expressed stably in continued cell line, but the ratios of these markers were expressed inconsistently. To verify the tumorigenic ability, we found that excepting 177 and 216T3, all of the others could induce tumor and these tumor were not transfer. These 8 strains of liver cancer stem cells were polluted by mycoplasma in the process of cultivation. Using three methods for clearing up and three methods for testing. One-step reagent test showed that Plasmocin need 14 d to treat mycoplasma, rapid detection reagent showed BM need 21 d to eradicate mycoplasma, and MRA by PCR detection found eliminated mycoplasma thoroughly need 14 d; In addition, three kinds of drugs used interchangeably to remove more significant effect of mycoplasma.Conclutions: 1. Obtaining and establishing 8 strains stable liver cancer cell lines in vitro and grasping the biological characteristics of these 8 strain accurately through repeated experiments. Related markers expressed differently indicated that the heterogeneous of their origin is different. At the same time, the differences of singlecells on expression stem cell markers reflected individual differences.The research of Liver cancer stem cell would provide a new research direction for the study of heterogeneous and can help us to seek new target for cancer immunity therapy.