| Aim: To investigate the effects of lentivirus- mediated prolyl oligopeptidase overexpression on thioacetamide?TAA?- induced liver fibrosis in rats.?Methods : POP gene sequence?NM031324? was obtained from genebank, then recombined with the vector to construct over-expression vector; the constructed vectors were confirmed by PCR and sequencing; Western Blot was used to determin POP protein overexpression in 293 T cells; Normal Sprague-Dawley?SD? rats were transfected with different dose of lentiverus vector?1.25×10^7TU, 2.5×10^7TU, 5×10^7TU? or empty viral vector?2.5×10^7TU, 5×10^7TU??n=3 each group,portal vein injection?; rats were sacrificed at 2w after injection; relative gene expression of POP m RNA were tested by real-time PC R; another set of rats received the dose of lentivirus vector of 5×10^7TU, and these rats were sacrificed a t 1, 2 or 3w after virus injection and the relative gene expression of POPwas determined by real-time PCR. 40 male SD rats were randomly divided into four groups. 5%TAA was injected intraperitoneally to induce liver fibrosis in rats. After 1-week TAA/saline treatment, rats were given normal saline?normal control group and TAA model group?, empty virus?empty virus group, TAA plus empty vector group?, or lentivirus containing POP gene?POP lentivirus group,TAA plus lentivirus-POP?; after 4 weeks of initial TAA/saline treatment, rats were sacrificed after overnight fasting and tissues?blood, liver? were isolated; Pathological changes and fibrosis of the liver were examed by HE staining and Masson staining, resepctively; hydroxyproline content in liver tissues were detected by chemical colorimetric; quantitative real- time PCR was performed to detect relative gene expression of POP, ?-SMA, TGF-?, MCP-1 and Western Blot were used to detect POP, ?-SMA, TGF-?, p Smad2 protein in liver, respectively; Distribution of C D68-posititive cells in each group were detected by immunohistochemistry staining; serum alanine aminotransferase?ALT?, aspartate transaminase?AST?, albumin?ALB? and total bilirubin?TBIL? were measured by biochemical analyzer.Results: The PCR and sequencing result of lentivirus-POP vector proved successful reconstruction of POP gene, and Western Blot comfirmed POP overexpresion in 293 T cells; the relative POP gene expression levels after 2.5×10^7TU and 5×10^7TU lentivirus vector transfection were 1.38 and 1.3, respectively, which were significantly higher than these of the control group, without significant difference between two lentivirus vector group; no statistical difference was found between empty vector group and normal control group; result of fluorescent of frozen section was consistent with the results of real-time PCR.Relative level of POP protein in model group was?0.123±0.04?, significantly decreased compared with normal control [?0.189±0.052?, P<0.05]; POP protein level in rats received lentivirus-POP was significantly higher than that of other groups?P<0.01?; the changes in POP m RNA expression was similar with POP protein content; the fibrosis grade in TAA model group and empty virus group were mostly of S2, and the semi-quantitative analysis of the fibrotic tissue by Masson staining were?15.2±1.69? and?15.3±4.62?,respectively, which were significantly higher than these of the normal controls and TAA plus lentivirus-POP group [?1.75 ? 0.63? and?7.75±2.71?, P<0.05]; hydroxyproline content in model group and empty virus group were?504.47±111.15?g/g wet liver weight? and?498.32±90.87?g/g wet liver weight?, respectively, significantly higher than that of the normal controls [?298.20±47.47?g/g wet liver weight?, P<0.05], while hydroxyproline content in TAA plus lentivirus-POP group was?383.52±43.49?g/g TAA wet liver weight?, significantly lower than those of model group and empty virus group?P<0.05?; relative level of ?-SMA m RNA or protein in model group and empty virus group was 2-3 times higher respectively than normal control group?P < 0.01?, and?-SMA m RN A in lentivirus-POP group decreased by 50% and protein level by 30%40% compared with that of model group and empty virus group?P<0.05?; serum AST of model group?167.21±41.41?U/L, and empty virus group?169.22±31.46? U/L were 1.4 times of that of the control group[?123.81±23.02? U/L,?P < 0.05?]; serum ALT[?62.79±10.80? U/L,?59.8±15.85? U/L], TBIL[?167.21±41.41??mol/L,?169.22±31.46??mol/L] were 1.7-3.4 t imes of that in the control group[?33.67±2.84? ?mol/L,?2.56±1.17? ?mol/L, P<0.01], and ALB [?20.54±2.02?g/L,?20.19±2.00?g/L] decresed by 20% compared with the control group[?25.01±1.77?g/L,P<0.01]; the serum AST?127.89±32.7?U/L, ALT?41.95±11.75?U/L, TBIL?4.28±1.67? ?mol/L in lentivirus-POP group decreased by 30%70% compared with that of model group and empty virus group?P < 0.05?, but ALB increased by 20%; MCP-1 m RNA in model group and empty virus group was 4-5 times higher respectively than of normal control group?P < 0.01?, but decreased by 50%60% in lentivirus-POP group?P<0.05?; TGF-? m RNA in model group and empty virus group was 6-8 times higher respectively than that of normal control group?P < 0.01?, and decreased by 40%50% in lentivirus-POP group?P<0.05?; protein level lentivirus-POP group decreased by 40% compared with that of model group and empty virus group?P<0.05?; protein level of p Smad2/3 in model group and empty virus group was 1-2 times higher than that of normal control group?P < 0.01?, and protein level of p Smad2/3 by 40% 50%?P<0.05?; CD68 positive cells in model group [?52.75±21.639?/hp] and empty virus[?54±1.19.5?/hp] were significantly higher than that o f normal control [?2.66±1.15?/hp,p<0.01],while transfection of POP gene [?21±13.45?/hp] lowered C D68 positive cells than those of model group and empty virus group [p<0.01].Conclusions: POP protein can be successfully and sustainedly overexpressed in rat liver by lentivirus vector containing POP genes; POP overexpression attenuates TAA- induced liver fibrosis, which is accompanished by suppression of intrahepatic inflammation cytokine?such as MCP-1?, CD68-positive cell infitration, and profibrogenic TGF-?/Smad2/3 signalling. |
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