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Objective: Hepatocellular carcinoma (hepatocellular carcinoma, HCC) is one of the most common malignancy in the world, the third largest in malignant cause of death. Hepatitis viruses, aflatoxin, alcohol, water pollution and genetic factors are a major cause of hepatocellular carcinoma, of which the most important cause of the hepatitis B virus (hepatitis B virus, HBV) or hepatitis C virus (hepatitis C virus, HCV) chronic infection. Chronic hepatitis B (chronic hepatitis B, CHB) is one of the most common high incidence and high mortality of liver infection. The global hepatitis B virus infection by about 20 million, of which approximately 350 million hepatitis B virus carriers, each year there are about one million people die from HBV infection secondary to liver failure, cirrhosis and liver cancer. China is a high incidence of hepatitis B, epidemiological data show that the HBsAg positive 7.18% of the total population, about 120 million people. Chronic hepatitis B patients about 2000-3000 people. Liver fibrosis / sclerosis major adverse outcomes as hepatitis B and primary liver cancer also have a higher incidence extracellular matrix extracellular matrix (ECM) characterized by excessive deposition of liver fibrosis is acute, chronic liver disease pathological outcome, and early reversible, but if it is not timely diagnosis and treatment can be further developed into cirrhosis, liver function from compensatory to decompensated patients eventually died of liver failure or concurrent liver cancer affect survival . Hepatic stellate cells (hepatic stellate cell, HSC) is the interstitial cells of the liver, has an important role in the development and progression of the chronic liver disease. Recent studies have shown that hepatic stellate cells as antigen-presenting cells to express a variety of antigen-presenting important molecules also play an important role in the immune function of liver-related. Transforming growth factor β1 (transforming growth factorβ1 TGFβ1) is a potent fibrogenic factor mediated cell tissue damage artery disease in the fiber can activate the fiber-forming cells, promoting extracellular matrix production, changing the matrix metalloproteinase ( matrix metalloproteinase, MMP) and its inhibitor activity, and enhance the resistance of target cells to the cytokine response, hepatitis, hepatic injury, hepatic fibrosis, liver cirrhosis has important initial role. TGFB1 there are at least 10 or more single nucleotide polymorphisms (single nucleotide polymorphism, SNP) locus, the promoter region of -509 locus in TGFβ1 codon10 (Leu GT; Pro) presence of the signal peptide of the precursor molecules on the TGFβ1 , while the signal peptide amino acid sequence changes theoretically may influence the secretion of the corresponding proteins. The TGFβ1 gene -509 sites we design the corresponding experimental detection of eight liver cell lines SNP genotype for codon10 (Leu gt; Pro) to study the impact of the liver cell function. Methods: TGFβ1 gene -509 genotype of the sites in eight liver cell lines detected by PCR-RFLP method. Building containing codon10 (Leu gt; Pro) TGFβ1 (LAP the mature states monomer) eukaryotic expression recombinant CMV-Leu, CMV-Pro. PcDNA3.1 empty vector transfected L02, HepG2, SMMC-7721, LX-2 cells, CMV-Leu, CMV-Pro, after 24 hours of incubation, the ELISA to detect cell the culture supernatant TGFβ1 amount. LO2 and HepG2 cells transfected with three plasmids were cultured in culture 12,24,36,48 hours, MTT assay, to detect plasmid transfection cell proliferation; L02 and HepG2 cells transfected with plasmid After 48 hours, using Annexin Ⅴ and PI double staining, apoptosis was detected by flow cytometry. HepG2 cells transfected with three plasmids, cultured for 24 hours, the cells were labeled with CD105-FITC antibody, detected by flow cytometry the expression of the CD105 molecule. LX-2 cells transfected with three plasmids, cultured for 24 hours, with CD80-FITC, CD83-PC5, Peripheral circulating CD1α-PE fluorescence-labeled antibody was incubated in the dark, and flow cytometry to detect the expression of the three CD molecule. Experimental data statistical software SPSS11.0 (Statistical Package for the Social Science 11.0) with two independent samples t-test for statistical analysis. Results: OK eight liver cell lines TGFβ1 gene -509 sites and codon10 of the genotype. Constructed with codon10 (Leu gt; Pro) TGFβ1 (LAP the mature states monomer) eukaryotic expression recombinant CMV-Leu, CMV-Pro by sequencing to confirm the correct sequence. In the four cell lines were transfected with CMV-Leu, CMV-Pro group TGFβ1 secretion ratio pcDNA3.1 group proved CMV-Leu, CMV-Pro can successfully transfected enters cells and its effect, improve its TGFβ1 secretion. TGFβ1 secretion than cells transfected with CMV-Pro group transfected with CMV-Leu group and was statistically significant (P lt; 0.05). In the LO2 apoptosis experiments, transfection of CMV-Leu, the CMV-Pro apoptosis rate than transfected pcDNA3.1 low rate of apoptosis, which transfected with CMV-Pro apoptosis rate below transfected with CMV- Leu apoptosis rate, and a statistically significant (P lt; 0.01). Can be found in the HepG2 cell apoptosis experiments, the transfection the CMV-Leu group and the group of CMV-Pro apoptosis rate significantly higher than those transfected with pcDNA3.1 group apoptosis rate (P lt; 0.01), which transfection CMV-Leu group apoptosis rate significantly higher than those transfected with CMV-Pro group apoptosis rate (P lt; 0.01). LO2 and HepG2 cell proliferation experiment, the cell proliferation activity of the two types of cells transfected with CMV-Pro group is higher than the corresponding the CMV-Leu-transfected group. HepG2 cells transfected with pcDNA3.1 CMV-Leu, CMV-Pro transfected with CMV-Pro groups CD105 expression rate than transfected CMV-Leu group, and was statistically significant (P lt; 0.05). CMV-Leu, CMV-Pro cells CD1a LX-2 experiment, transfection of CD80 expression, there was no significant difference in Pro group transfected with CMV-CD83 expression was significantly lower than the transfection of CMV-Leu group, there are statistics Differences between (P lt; 0.01). Conclusion: TGFβ1 gene codon10 ratio is Pro to Leu to better promote the secretory activity of the cells of TGFβ1, and can increase the HepG2 cells of CD105 expression. TGFβ1 codon10 than to Leu to Pro to better promote the L02, the proliferation of HepG2 cells. The CMV-Leu, CMV-Pro LO2 cells have a protective effect, TGFβ1 gene codon10 Pro type representing a more protective effect of Leu L02 apoptosis. The CMV-Leu, CMV-Pro can promote apoptosis of HepG2 cells, which codon10 Leu when promoting stronger effect. The TGFβ1 gene codon10 Leu can significantly increase the LX-2 cells CD83 expression, but no significant effect on the expression of CD80, CD1α.
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