In Vitro And In Vivo Studies Of Adenovirus-mediated PTEN Gene Transduction On Prevention And Treatment Of Rat Liver Fibrosis

Liver fibrosis, and its end stage disease liver cirrhosis, represents a major medical problem worldwide. The hepatic stellate cell (HSCs) plays a critical role in the development and maintenance of liver fibrosis. In the normal liver, HSCs reside in a quiescent state characterized by vitamin A storage, a low proliferative rate, and trace production of ECM components. However, following a fibrogenic stimulus, HSCs undergo a complex activation process associated with morphological changes from a quiescent vitamin A-storing cell to that of an activated myofibroblast-like cell. HSCs activation is also associated with a dramatic increase in the synthesis and deposition of ECM components, of which type I collagen predominates, the appearance of the characteristic activation marker smooth muscleα-actin (α-SMA), and an increase in cellular proliferation.Phosphatase and tensin homolog deleted on chromosome ten (PTEN), the first tumor-suppressing gene found to exhibit phosphatase activity, negatively regulates cell cycle, inhibits the proliferation and promotes the apoptosis of tumor cells. As such, it is reasonable to conclude that dysfunction or absence of PTEN is intimately related to the formation and progression of human tumors.Nevertheless, in recent years, PTEN research has gradually extended beyond cancer, focusing on its role in other disease states. Studies demonstrated that lowered expression and phosphatase activity of PTEN has been found in lung fibroblasts of patients with idiopathic pulmonary fibrosis. Moreover, studies also documented that PTEN inhibits the proliferation and induces the apoptosis of lung fibroblasts cultured in vitro. A study on PTEN and myocardial fibrosis showed that knocking out PTEN in mice leads to an increased ratio of heart to body weight, decreased cardiac contractility and, eventually, interstitial fibrosis. This suggests that the low expression or deactivation of PTEN involves itself in the pathogenesis of lung fibrosis and myocardial fibrosis. In the liver, the absence of PTEN in specific hepatic cells may result not only in hepatocellular carcinoma, but also in non-alcoholic steatohepatitis, a condition closely related to hepatic fibrosis. Currently, though, the expression and function of PTEN in hepatic fibrosis, especially its effect on the proliferation and apoptosis of primary HSCs, remains unclear. So, we set out to determine the effects of PTEN over-expression, via adenoviral transduction of wild type PTEN, its mutant G129E gene, and PTEN shRNA on the proliferation and apoptosis as well as cell cycle of primary and activated HSCs cultured in vitro. Meanwhile, the signaling transduction pathways of PTEN on the proliferation, apoptosis and cell cycle of primary HSCs were studied using the CCl4 induced rat fibrosis model, this study explore the dynamic expression of PTEN in the process of hepatic fibrosis in rats and its relation with the proliferation and apoptosis of HSCs in vivo. And it was designed to provide new viewpoint and defined strategy for preventing and treating hepatic fibrosis.The project contain four parts as below:Part 1:The regulation effects of promotion and inhibition of PTEN expressions on HSCs proliferation, apoptosis and cell cycle phaseObjective: To investigate the influences of over-expression of wild type PTEN, its mutant G129E (exhibiting protein phosphatase, losing lipids phosphatase activity), and RNA interference restructuring adenovirus targeting PTEN on the proliferation, apoptosis and cell cycle phase of freshly isolated hepatic stellate cells and human HSCs LX2 in vitro.Methods: The fresh rat HSCs were isolated with in-situ recirculating perfusion technology and purified by density gradient centrifugation, and observed by fluorescence microscope. Immunocytochemistry was used to detect the activated primary HSCs. The essential adenovirus, such as Ad-GFP, Ad-PTEN, Ad-G129E, and PTEN shRNA were amplified by AD293T cells and transiently transfected into freshly isolated hepatic stellate cells and human LX2 in vitro. The expressions of PTEN mRNA and protein were observed by real-time Q-PCR and Western blot, respectively. MTT and BrdU were used to dectect the proliferation of HSCs; the apoptotic rates of primary rat HSCs and human LX2 were detected by combined marked Annexin-V/PE and TUNEL; Hoechst 33258 was used to show the changes of cell shape; the activity of caspase-3 was investigated by the special kit; the protein expressions of Bcl-2 and Bax were examined by Western blot; while FACs was used to analyze the cell cycle of HSCs; the expressions of CyclinD1, CDK4 and P27kip1 were detected by Western blot, respectively.Cells were grouped as follows: (1) Control group, cells were cultured under the same conditions, until the transient transfection, when the cells were cultured in DMEM (without FBS and antibiotics) replacing the adenovirus; (2) Ad-GFP group, HSCs were transfected with adenovirus expressing green fluorescent protein (GFP) alone; (3) Ad-PTEN group, HSC were transfected with recombinant adenovirus Ad-PTEN with both wild type PTEN and GFP; (4) Ad-G129E group, HSCs were transfected with recombinant adenovirus Ad-G129E with both PTEN mutant G129E and GFP; (5) PTEN shRNA group, HSCs were transfected with RNA interference restructuring adenovirus targeting PTEN.Results: (1) The fresh rat HSCs were successfully isolated with in-situ recirculating perfusion technology, and purified with Nycodenz by density gradient centrifugation, and 1.2×107~2.0×107 cells were obtained for each rat. The survival rate and the purity were higer than 90%. The fresh isolated HSCs were round and rich in fat droplet under inverted microscope. The Spontaneous blue fluorescence of fresh isolated HSCs was also detected using fluorescence microscope and observed by fluorescence microscope under wavelength 328 nm. The primary cells appeared activation state, losing fat droplet, and turned to be fusiform myofibroblasts when cultured for 10 days; (2) Identification of primary HSCs. Theα-SMA expressions from fresh isolated HSCs could not be detected by immunocytochemistry, however, the positive expression increased to all cells after 10 days. The 100% positive dyeing rate revealed that the fresh isolated HSCs were all activated; (3) The essential adenovirus were harvested through infecting AD293T cells repeatedly, and the virus titers of Ad-GFP, Ad-PTEN, Ad-G129E, and PTEN shRNA were 1.6×109, 1.8×109, 1.2×109 and 1.5×109 pfu/ml respectively; (4) At 72 h post-transfection, real time Q-PCR was used to assay relative mRNA expression levels of PTEN in HSCs using the method of fold increase (2-△△Ct method). In primary HSCs, the expression value of control group was arbitrarily assigned an expression value of 1, then the PTEN mRNA relative expression levels in Ad-GFP group, Ad-PTEN group, Ad-G129E group and PTEN shRNA group were 0.994-fold, 1.698-fold, 1.624-fold and 0.357-fold, respectively. Western blot was used to further detect the protein expressions and show a significantly higher expression level in Ad-PTEN group (1.91±0.09), Ad-G129E group (1.74±0.08) than Control group (1.21±0.05) and Ad-GFP group (1.15±0.04), P<0.01, and also showed a significantly lower level in PTEN shRNA group (0.56±0.04) than Control group and Ad-GFP group. There were no significant differences between Control group and Ad-GFP group, Ad-PTEN group and Ad-G129E group. These results confirmed a successful trasnfection, and the similar tendency was also found in experiments of human LX2; (5) The results of MTT showed that wild type PTEN gene and G129 gene were able to inhibit the viability of primary HSCs and human LX2 in a certain period. At 72 h post-transfection, the viability of primary HSCs and human LX2 were significantly reduced to 74.71% and 50.43% compared to control group (100%) by Ad-PTEN; while PTEN shRNA enhanced the viability of primary HSCs and human LX2 to 118.26% and 142.15%, higher than control group(100%); (6)The results of BrdU showed that wild type PTEN gene and G129 gene were able to inhibit the DNA synthesis of primary HSCs and human LX2. At 72 h post-transfection, the DNA synthesis of primary HSCs in Ad-PTEN and G129E group were significantly reduced to 63.32% and 74.75% compared to Ad-GFP group, respectively; while PTEN shRNA enhanced the DNA synthesis of primary HSCs by 35.36%; and the similar tendency was also found in experiments of human LX2; (7) At 72 h post-transfection, the combined marked Annexin-V/PE showed that the apoptotic rates of primary HSCs in Ad-PTEN group and Ad-G129E group (34.85%±2.11%, 25.18%±1.34%) were significantly higher than Control group (3.12%±0.15%) and Ad-GFP group (4.21%±0.35%), P<0.01; while the PTEN shRNA group (1.24%±0.08%) significantly lower than Control group and Ad-GFP. The apoptotic rates of human LX2 in Ad-PTEN group and Ad-G129E group were 41.76%±1.98% and 37.18%±2.13%, significantly higher than Control group (5.68%±1.15%) and Ad-GFP group (7.21%±1.25%), P<0.01; while the PTEN shRNA group (2.25%±1.18%) significantly lower than Control group and Ad-GFP; (8) Using Hoechst 33258, the primary rat HSCs and human LX2 were found apoptotic cells under fluorescence microscope, appearing a light blue swelling of nuclear chromosome in peripheral area, which were of less integrity at 72 h post-transfection in Ad PTEN group and G129E group. However, in GFP group and PTEN shRNA group, the nuclear chromosome were ovalize with a cell peripheral area of integrity, the content of which showed dark blue granules. Five fields of view were randomly selected for each piece, and more than 300 cells were counted for each field. The results showed that the apoptotic rates of primary HSCs significantly increased in Ad-PTEN group and G129E group (18.25%±1.23%, 15.18%±1.15%) than Control group (1.12%±0.15%) and Ad-GFP group (1.78%±0.09%), P<0.01; little apoptotic cells were observed in PTEN shRNA group (0.33%±0.01%),P<0.01; (9) The results of TUNEL showed similar tendency, the apoptotic rates of primary HSCs significantly increased in Ad-PTEN group and G129E group (15.82%±1.53%, 12.23%±0.97%) than Control group (1.22%±0.15%) and Ad-GFP group (1.43%±0.13%), P<0.01; There was significant differences between Ad-PTEN group and G129E group, P<0.01. However, there was no significant differences between Ad-GFP group and Control group, P>0.05; (10)Over-expression of wild type PTEN gene and G129E gene markly promoted the activity of Caspase-3 in primary HSCs for 2.16 folds and 1.73 folds, and PTEN shRNA inhibited the activity of Caspase-3 to 62.13% compared to the Ad-GFP group; for human LX2, the inhibition rates induced by PTEN shRNA was 71.23%, while the activity of Caspase-3 increased to 2.53 folds and 1.96 folds in Ad-PTEN group and Ad-G129E group, respectively; (11)The results of Western blot showed that the apoptosis related protein bcl-2 was decreased and bax was markly promoted by over-expression of wild type PTEN gene and G129E gene in primary HSCs and human LX2, the ratio of Bax/Bcl-2 increased as well; (12)The cell cycle of primary HSCs and human LX2 were arrested in G0/G1 phase and G2/M phase by wild type PTEN gene and G129 gene, while the PTEN shRNA acted as a promoter in the two periods of cell cycle; (13) Wild type PTEN gene and G129 gene inhibited the CyclinD1 and CDK4 protein expressions and promoted P27kip1 protein expression, while the PTEN shRNA played as the opposite role. Conclusions: The fresh rat primary HSCs were successfully isolated and transfected with exogenous wild type PTEN gene, G129 gene and PTEN shRNA recombinant adenovirus as well as human LX2; over-expression of wild type PTEN gene and G129 gene can obviously inhibit proliferation of HSCs; Over-expression of wild type PTEN gene and G129E gene in primary HSCs and human LX2 can induce apoptosis of HSCs, enhance activity of Caspase-3, up-regulate Bax protein expression and down-regulate Bcl-2 protein expression; Over-expression of wild type PTEN gene and G129E gene induce cell cycle arrest in G0/G1 phase, probably through the down-regulation of cyclinD1 and CDK4, up-regulation of P27kip1. The wild type PTEN gene played a more powful role than G129E gene, while the PTEN shRNA acted as an opposite role.Part 2:The regulation effects of promotion and inhibition of PTEN expressions on cell collagen metabolism of HSCsObjective: To investigate the influences of over-expression of wild type PTEN, its mutant G129E (exhibiting protein phosphatase, losing lipids phosphatase activity), and RNA interference restructuring adenovirus targeting PTEN on cell collagen metabolism of freshly isolated hepatic stellate cells and human HSCs LX2 in vitro.Methods: Western blot was used to detect the expressions of collagenⅠ, collagenⅢ, MMP-13, TIMP-1, MMP-2 and TIMP-2 protein before and after transfection. The cells were grouped as part 1.Results: (1) At 72 h post-transfection, Western blot was used to detect the expressions of collagenⅠ. In primary HSCs, the expression of collagenⅠshowed a significantly lower expression level in Ad-PTEN group (0.32±0.05), Ad-G129E group (0.47±0.07) than Control group (0.85±0.05) and Ad-GFP group (0.82±0.09), P<0.01, and showed a significantly higher level in PTEN shRNA group (1.28±0.13) than Control group and Ad-GFP group; the inhibition effects of Ad-PTEN is more notable than the Ad-G129E; there were no significant differences between Control group and Ad-GFP group; and the similar tendency was also found in experiments of human LX2; (2) At 72 h post-transfection, Western blot was used to detect the expressions of collagenⅢ. In primary HSCs, the expression value of collagenⅢshow a significantly lower expression level in Ad-PTEN group (0.18±0.02), Ad-G129E group (0.27±0.03)than Control group (0.41±0.02) and Ad-GFP group (0.38±0.03), P<0.01, and also showed a significantly higher level in PTEN shRNA group (0.72±0.07) than Control group and Ad-GFP group; the inhibition effects of Ad-PTEN is more notable than the Ad-G129E; there were no significant differences between Control group and Ad-GFP group; and the similar tendency was also found in experiments of human LX2; (3) At 72 h post-transfection, Western blot was used to detect the expressions of MMP-13. In primary HSCs, the expression of MMP-13 showed a significantly higher expression level in Ad-PTEN group (0.88±0.06), Ad-G129E group (0.71±0.12) than Control group (0.51±0.03) and Ad-GFP group (0.47±0.02), P<0.01, and also showed a significantly lower level in PTEN shRNA group (0.36±0.03)than Control group and Ad-GFP group; the effects of Ad-PTEN is more notable than the Ad-G129E; there were no significant differences between Control group and Ad-GFP group; and the similar tendency was also found in experiments of human LX2; (4) At 72 h post-transfection, Western blot was used to detect the expressions of TIMP-1. In primary HSCs, the expression value of TIMP-1 showed a significantly lower expression level in Ad-PTEN group (0.17±0.02), Ad-G129E group (0.26±0.03) than Control group (0.38±0.06) and Ad-GFP group (0.42±0.03), P<0.01, and also showed a significantly higher level in PTEN shRNA group (0.73±0.12) than Control group and Ad-GFP group; the inhibition effects of Ad-PTEN is more notable than the Ad-G129E; there were no significant differences between Control group and Ad-GFP group; and the similar tendency was also found in experiments of human LX2; (5) At 72 h post-transfection, Western blot was used to detect the expressions of MMP-2. In primary HSCs, the expression value of MMP-2 showed a significantly higher expression level in Ad-PTEN group (0.85±0.07), Ad-G129E group (0.71±0.02) than Control group (0.35±0.03) and Ad-GFP group (0.38±0.07), P<0.01, and also showed a significantly lower level in PTEN shRNA group (0.16±0.03) than Control group and Ad-GFP group; the effects of Ad-PTEN is more notable than the Ad-G129E; there were no significant differences between Control group and Ad-GFP group; and the similar tendency was also found in experiments of human LX2; (6) At 72 h post-transfection, Western blot was used to detect the expressions of TIMP-2. In primary HSCs, the expression of TIMP-2 showed a significantly lower expression level in Ad-PTEN group (0.34±0.04), Ad-G129E group (0.48±0.08) than Control group (0.88±0.08) and Ad-GFP group (0.84±0.06), P<0.01, and also showed a significantly higher level in PTEN shRNA group (1.13±0.11) than Control group and Ad-GFP group; the inhibition effects of Ad-PTEN is more notable than the Ad-G129E; there were no significant differences between Control group and Ad-GFP group; and the similar tendency was also found in experiments of human LX2;Conclusions: Over-expression of wild type PTEN gene and G129E gene in primary HSCs and human LX2 can inhibit the synthesis of collagenⅠand collagenⅢ, increase the expressions of MMP-13 and MMP-2, decrease the expressions of TIMP-1 and TIMP-2; the wild type PTEN gene played a more powful role than G129E gene, while the PTEN shRNA acted as an opposite role.Part 3:Signaling transduction mechanisms of PTEN on the primary HSCs and human LX2 behaviorsObjective: To investigate the signaling transduction mechanisms of PTEN on the proliferation, apoptosis and cell cycle of primary HSCs and human LX2 in vitro.Methods: The fresh isolated rat HSCs were successfully isolated and transfected with exogenous wild type PTEN gene, G129E gene and PTEN shRNA recombinant adenovirus as well as human LX2. The mRNA expressions of PI3K, Akt, FAK and ERK1 were assayed by real-time Q-PCR; and the protein expressions of Akt, p-Akt (Thr308), FAK, p-FAK (Tyr397), ERK1 and p-ERK1 were detected by Western blot. The experiment groups were the same as Part 1.Results: (1) Exogenous wild type PTEN gene and G129E gene were successfully transfected into HSCs which resulted in higher PTEN expression; lower PTEN expression was also found in PTEN shRNA, these results are consistent with that of Part 1; (2) At 72 h post-transfection, real time Q-PCR was used to assay relative mRNA expression levels of PI3K in HSCs using the method of fold increase (2-△△Ct method). In primary HSCs, the expression value of control group was arbitrarily assigned an expression value of 1, then the PI3K mRNA relative expression levels in Ad-GFP group, Ad-PTEN group, Ad-G129E group and PTEN shRNA group were 0.979-fold, 0.716-fold, 0.962-fold and 1.223-fold, respectively; Western blot showed that, in primary HSCs, PI3K protein expression significantly decreased in Ad-PTEN group (0.49±0.02), compare with Ad-G129E group (0.75±0.06), Control group (0.82±0.07) and Ad-GFP group (0.79±0.03), P<0.01, while significantly increased in PTEN shRNA group (1.12±0.14) than Ad-G129E group and Control group, P<0.01; there were no significant differences between Control group and Ad-GFP group, P>0.05; and the similar tendency was also found in experiments of human LX2; (3) Real time Q-PCR and Western blot showed that, the expressions of Akt mRNA and protein showed no obvious changes in both two cell types, P>0.05; however, in primary HSCs, p-Akt(Thr308) protein expression significantly decreased in Ad-PTEN group (0.28±0.02), compare with Ad-G129E group (0.48±0.03), Control group (0.50±0.02) and Ad-GFP group (0.52±0.05), P<0.01, while significantly increased in PTEN shRNA group (0.62±0.03) than Ad-G129E group and Control group, P<0.01; there were no significant differences between Ad-G129E group, Control group and Ad-GFP group, P>0.05, and the similar tendency was also found in experiments of human LX2; (4) Real time Q-PCR and Western blot showed that, the expressions of FAK mRNA and protein showed no obvious changes in both two cell types, P>0.05; however, for primary HSCs, p-FAK (Tyr397) protein expression significantly decreased in Ad-PTEN group (0.42±0.05) and Ad-G129E group (0.49±0.07) compared with the Control group (0.80±0.09) and Ad-GFP group (0.78±0.05), P<0.01, while significantly increased in PTEN shRNA group (0.92±0.07) than Ad-G129E group and Control group, P<0.01; there were no significant differences between Ad-G129E group, Control group and Ad-GFP group, P>0.05, and the similar tendency was also found in experiments of human LX2; (5) Real time Q-PCR and Western blot showed that, the expressions of ERK1 mRNA and protein showed no obvious changes in both two cell types, P>0.05; however, for primary HSCs, p-ERK1 protein expression significantly decreased in Ad-PTEN group (0.25±0.02) and Ad-G129E group (0.28±0.07), compared with Control group (0.54±0.07) and Ad-GFP group (0.52±0.06), P<0.01, while significantly increased in PTEN shRNA group (0.70±0.05) than Ad-G129E group and Control group, P<0.01; there were no significant differences between Ad-G129E group, Control group and Ad-GFP group, P>0.05, and the similar tendency was also found in experiments of human LX2;.Conclusions: The PTEN phosphatase down-regulated p-Akt(Thr308), p-FAK (Tyr397) and p-ERK1 protein expression. The function of PTEN in negatively regulating cell cycle, inhibiting proliferation and inducing apoptosis of primary HSCs and human LX2, was probably via the phosphoinositol-3-kinase (PI3K)/Akt and FAK/ERk1/2 signal transduction pathways.Part 4:The prevention and treatment role of recombinant PTEN adenovirus in rat liver fibrosis induced by CCl4Objective: To investigate the prevention and treatment role of PTEN over-expression in rat liver fibrosis induced by CCl4.Methods: A total of 128 adult health Wistar male rats were randomly divided into Prevention group (Pre 1 wk, Pre 3 wk, Pre 5 wk and Pre 7 wk, 15 rats for each group), Treatment group (Tre 1 wk, Tre 2 wk, Tre 3 wk and Tre 4 wk, 15 rats for each group) and Control group (8 rats). Moreover, each time point of Prevention group and Treatment group was further divided into 5 groups: CCl4 group; CCl4+Ad-GFP group; CCl4+Ad-PTEN group; CCl4+Ad-G129E group and CCl4+PTEN shRNA group. All rats except that of Control group were received hypodermic injection of CCl4 mixed with olive oil at the concentration of 40% for 7 weeks (2 ml·kg-1, twice a week); the recombinant adenovirus Ad-GFP, Ad-PTEN, Ad-G129E and PTEN shRNA were pushed into rats bodies through tail vein injection (once a week) at the beginning of first week in Prevention group and fourth week in Treatment group. Haematoxylin and eosin staining (H&E staining) and Masson's trichrome staining (MT staining) were used to determine histopathology changes. The expressions of PTEN andα-SMA in liver tissues were measured by immunohistochemical and immunofluorescence staining and western blot. The co-expression between PTEN andα-SMA,α-SMA and TUNEL were observed by confocal laser scanning microscopy. The expressions of CollagenⅠand CollagenⅢin liver tissues were measured by immunohistochemical and Western blot.Results: (1) The experiment animals showed an improvement in mental status, drinking and eating, as well as activities quantitative in the Ad-PTEN and Ad-G129E groups; while the PTEN shRNA acted as an opposite role; (2) The analysis to the biochemical indexes showed that over-expression of Ad-PTEN and Ad-G129E could significantly reduced the level of ALT and AST in rat serum and thus the liver fuction was improved, while the PTEN shRNA acted as an opposite role; (3) H&E and MT staining confirmed that the over-expression of Ad-PTEN and Ad-G129E could reduce the hepatic cells necrosis and decrease the deposition of fibrosis; (4) Immunohistochemical and immunofluorescence staining results showed that the expressions of PTEN were significantly increased after the administration of Ad-PTEN and Ad-G129E recombinant adenovirus in either prevention groups and treatment groups, which indicated that the recombinant adenovirus were introduced into rat liver successfully; Western blot analysis of PTEN protein confimed this result; (5) The co-expression products of PTEN andα-SMA mainly existed in cytoplasm of activated HSCs, and the activated HSCs expressing PTEN accounted for a decreasing percentage of total activated HSCs, which indicates the over-expression of PTEN induced by exogenous wild type PTEN gene and G129E gene can inhibit theα-SMA expressions significantly compared with Ad-GFP group and CCl4 group; (6) Over-expression of PTEN induced by exogenous wild type PTEN gene and G129E gene can induce HSCs apoptosis at each time points compared with Ad-GFP group and CCl4 group; (7) Over-expression of PTEN induced by exogenous wild type PTEN gene and G129E gene can decrease the expressions of collagenⅠand collagenⅢat each time points compared with Ad-GFP group and CCl4 group; (8) The down-regulation of PTEN induced by PTEN shRNA can aggravate the rat liver fibrosis compared with Ad-GFP group and CCl4 group, led to increased expression ofα-SMA and decreased apoptosis of activated HSCs, caused the increased deposition of collagenⅠand collagenⅢ, and developed into liver cirrhosis more easily; (9) Pearson correlation analysis was used to find significant negative correlation between PTEN andα-SMA expression; there were also significant positive correlations between PTEN expression and the apoptotic index of activated HSCs, PTEN expression and the percentage of PTEN-positive apoptotic cells.Conclusions: Hypodermic injection of CCl4 can establish stable model of rat liver fibrosis, PTEN may be involved in this process. Tail vein injection of recombinant adenovirus Ad-GFP, Ad-PTEN, Ad-G129E and PTEN shRNA can infect the rat liver fibrosis model and regulate PTEN expression effectively. Ad-PTEN produced maximum prevention and cure effect on rat liver fibrosis induced by CCl4, promoting it a potential target in prevention and cure on hepatic fibrosis.