| Objective:Hepatic fibrosis is a compensatory response to chronic liver injury.If the potential pathogenic factors are not removed in time,it will develop into cirrhosis and even liver cancer.The proliferation and activation of hepatic stellate cells(HSCs)stimulated by inflammatory factors such as tumor necrosis factor alpha(TNF-?)are central events in the development and progression of liver fibrosis.Single nucleotide polymorphisms(SNPs),TNF-? rs1800620(A94T)and TNF-? rs4645843(P84L)in the TNF-? coding region,which are reported to affect amino acid interactions and the hydrogen bond network reduces the stability of the TNF-? protein,which in turn reduces the level of TNF-?.The aim of this study was to investigate the biology effects of TNF-? SNPs on the proliferation,activation,apoptosis and migration of human hepatic stellate cell line LX-2 by constructing recombinant cell models.Methods: The c DNA of TNF-? was synthesized by PCR amplification,and TNF-?A94T and TNF-? P84 L were synthesized by template point mutation.Subsequently,the linearized viral vector was recombined with the target gene TNF-?/TNF-?A94T/TNF-? P84 L,and transformed in DH5? competent bacteria,and the positive clones were selected respectively.The lentivirus was packaged,transfected into the LX-2 cell line,and screened for 7 days with puromycin to construct a stable cell line.The levels of TNF-? m RNA in each group were detected by real-time quantitative PCR(RTq-PCR).The expression of TNF-? protein in each group was detected by Western blot and enzyme-linked immunosorbent assay(ELISA).The effect of TNF-?SNPs on the proliferation of cells in each group was tested by CCK-8 assay.The m RNA levels of type I collagen,?-smooth muscle actin(?-SMA),and matrix metalloproteinase(MMP)2 and 9 were measured by RTq-PCR.Apoptosis was detected by flow cytometry.The effect of TNF-? SNPs on the migration ability of each group of cells was examined by Transwell chamber method.The expression levels of inflammatory factors IL-6 and TGF-?1 in each group of cells were detected by ELISA.The expression levels of NF-?B signaling pathway proteins P-I?B-?,P-P65,I?B-? and P65 were detected by Western blot.Results: The constructed vector was verified by sequencing.After the puromycin screening,green fluorescent protein(GFP)stably expressed,indicating that the stable cell line of TNF-?/TNF-? A94T/TNF-? P84 L was successfully constructed.RTq-PCR,Western blot and ELISA showed that TNF-? A94T/P84 L significantly down-regulated TNF-? m RNA and protein levels compared with wild-type TNF-? transfection group(WT).CCK8 showed that the number of cells in the WT group was significantly lower than that in the empty plasmid group at 72 h and 96 h.The number of cells in the A94 T and P84 L mutant groups was significantly higher than that in the WT group,indicating that TNF-? SNPs could significantly reduce the growth inhibitory effect of TNF-? on HSCs proliferation.RTq-PCR detection of HSCs activation biomarkers found that WT group significantly down-regulated the m RNA levels of type I collagen and ?-SMA compared with the empty plasmid group,and significantly up-regulated the m RNA level of MMP2/9.Compared with the WT group,the A94 T and P84 L mutant groups up-regulated the level of type I collagen and significantly down-regulated the m RNA level of MMP9.In addition,TNF-? A94 T up-regulated the m RNA content of ?-SMA and down-regulated the MMP2 m RNA level.Flow cytometry to detect apoptosis in each group,and found no significant difference between the groups.The Transwell chamber method found that the number of cells penetrating the pores was significantly reduced in the WT group compared with the empty plasmid group.The A94 T and P84 L mutation groups significantly reversed the inhibitory effect of TNF-? on HSCs cell migration compared with the WT group.ELISA showed that WT group can up-regulate IL-6 expression;TNF-? A94T/P84 L can significantly down-regulate IL-6 expression.In addition,there was no significant difference in the content of TGF-?1 between the groups.Western blot analysis of NF-?B pathway protein found that WT group can up-regulate P-I?B-? and P-P65 protein expression and down-regulate I?B-? level in WT group,while TNF-?A94T/P84 L mutation Compared with the WT group,the group significantly down-regulated the protein expression of P-I?B-? and P-P65 and up-regulated the level of I?B-?.Conclusion: The gene polymorphism of TNF-? A94T/P84 L can down-regulate TNF-? m RNA and protein levels,and can reverse the inhibitory effect of TNF-? on proliferation,activation and migration of HSCs,which may be related to NF-?B signaling pathway. |
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