Effects Of Adenovirus Mediated ShRNA Cut PTEN Expression On Adhesion And Migration In Hepatic Stellate Cells

Objectives To investigate effects of adhesion and migration of activated hepatic stellate cell(HSC)in vitro and the signaling mechanism after down-regulated phosphatase and tensin homology deleted on chromosome Ten(PTEN) expression.Methods Used 293 A cell to amplify adenovirus vectors(Ad-sh RNA PTEN, Ad-GFP) and conducted viral titer estimates. Activated HSC were cultivated in vitro and Ad-sh RNA PTEN transient transfected HSC. Measured the expression of PTEN, ERK1, p-ERK1 protein by western blot, the expression of PTEN and ERK1 m RNA by real time fluorescence quantitative PCR(RT-PCR). Used MTT colorimetric method and toluidine blue staining method to determine the adhesion ability of HSC.Applied scratch repair experiments and the transwell chamber experiment to determine the migration ability of HSC. A database of all materials used Excel 2010 and SPSS 19.0 statistical software for statistical analysis. Mean±standard deviation( x ±s)expressed measurement data.The single factor analysis of variance was compared between groups(one-way ANOVA), and compared the LSD test between groups. When the probability values less than 0.05 for the difference was statistically significant.The experimental group as follows:(1) Control group, at transfection virus steps joined DMEM instead of adenovirus.(2) Ad-GFP group, at transfection virus steps joined adenovirus expressing green fluorescent protein(GFP).(3) Ad-sh RNA PTEN group, at transfection virus steps joined adenovirus taking along mediated sh RNA cut PTEN and expressing GFP.Results 1 Through three rounds repeated infection of 293 A cell method for obtaining experimental adenovirus, Ad-GFP and Ad-sh RNA PTEN viral titers were 1.3 ×109pfu/m L、1.5×109pfu/m L. 2 When multiplicity of infection(M.O.I) 20 adenovirus infected HSC 48 h, inverted fluorescence microscope counted cells of GFP expression. AdGFP and Ad-sh RNA PTEN groups cells of GFP expression were more than 80%. 3 When adenovirus infected HSC 48 h, applied Western blot technology to detect each group of HSC PTEN protein expression shows: Ad-sh RNA PTEN group(1.088±0.036) was statistically lower than Ad-GFP group(1.413±0.058) and control group(1.438±0.038), P﹤0.05, between control group and Ad-GFP group were no statistical significance, P﹥0.05. Detecting each group of HSC PTEN m RNA expression by RT-PCR and comparing each group of HSC PTEN m RNA the levels by relative quantitative method(2-△△Ct method). AdGFP and the Ad- sh RNA PTEN groups than the control group of PTEN m RNA relative ratio of 0.92 times and 0.64 times, the control group of PTEN m RNA expression quantity was considered 1.Control group and Ad-GFP group compared to Ad-sh RNA PTEN group PTEN m RNA expression evident increase, P﹤0.05, there is no statistical significance between control group PTEN m RNA and Ad-GFP group PTEN m RNA, P﹥0.05. 4 At 48 hours after adenoviral infection,toluidine blue stain method was used to determine the adhesion ability of HSC. Compared with the adhesion cell number in control group(495.00±13.23)and Ad-GFP group(500.00±18.03), the adhesion cell number in Adsh RNA/PTEN group(592.00±12.53) significantly incressed(P﹤0.05), but the adhesion cell number between Ad-GFP group and control group were no significant difference(P﹥0.05). 5 At 48 hours after adenoviral infection,the adhesion rate of HSC was determined by MTT colorimetric method. The differences of absorbance value(A value) between control group(0.279±0.015) and Ad-GFP group(0.285±0.011) were not significant(P > 0.05), the adhesion rate of HSC in Ad-GFP group was 102.67±8.29%. A value in Ad-sh RNA/PTEN group(0.335±0.013) significantly increased, compared with control group and Ad-GFP group(P < 0.05), and the adhesion rate of HSC in Adsh RNA/PTEN group was 120.34±7.26%. 6 The result of scratch repair experiments showed:Adenovirus infected in HSC after 12 h, each group of HSC was to be migrated but migration distance was no statistically significant difference between groups, P ﹥0.05.After 24 h control group migration distance was207.78±3.24μm, Ad-GFP group and Ad-sh RNA PTEN group migration distance were 207.95±3.54μm and 225.00±4.48μm;Ad-sh RNA PTEN group significant differences compared with control group and AdGFP group, P﹤0.08, control group and Ad-GFP group were no differences, P﹥0.05. After 48 h control group migration distance was 284.33±2.52μm, Ad-GFP group and Adsh RNA PTEN group migration distance were 281.98±4.34μm and 312.87±2.25μm; Ad-sh RNA PTEN group significant differences compared with control group and Ad-GFP group, P﹤0.05, control group and Ad-GFP group no differences, P﹥0.05. 7 Transwell Chambers across cell membrane migration experiment results showed that: Ad-sh RNA PTEN group(101.67±3.06) compared with control group(69.33±2.52) and Ad-GFP group(67.67±1.53) significantly increased, P﹤0.05, control group and Ad-GFP group were no significant statistical differences, P﹥0.05. 8 Applied Western blot and RT- PCR to detect each group HSC ERK1 protein and m RNA expression after adenovirus infection 48 h, the result showed that each group ERK1 protein and m RNA expression had no obvious change, P﹥0.05. Western blot was used to test groups of p-ERK1 protein expression,compard with control group(0.710±0.023), Ad-sh RNA PTEN group(1.067±0.047) expressed increased significantly, P ﹤ 0.05, Ad-GFP group(0.692 ± 0.037) expressed undifferentiated, P﹥0.05, Ad-sh RNA PTEN group and Ad-GFP group were no discrepancy, P﹥0.05.Conclusions 1 The down-regulation of PTEN expression can promote the adhesion in activated HSC in vitro. 2 The down-regulation of PTEN expression can hasten the plane migration ability and transmembrane migration ability in activated HSC in vitro. 3 ERK signal transduction participate in the regulative effects of PTEN on the adhesion and migration in activated HSC.