| Objectives Discussed the overexpression of wild-type PTEN and its mutant G129 E on activation of focal adhesion kinase(FAK) / p130 Crk-associated substrate(p130Cas) and paxillin signal transduction in activated hepatic stellate(HSC) in vitro.Methods Used AD 293 cells to amplify adenovirus vectors(Ad-PTEN、Ad-G129E、Ad-GFP) and conducted viral titer estimates. Activated HSC were cultivated in vitro and wild-type PTEN gene and G129 E gene transient transfected HSC(HSC-T6). Western blot was used for protein expressions of PTEN, FAK, phosphorylated FAK(Thr397) [p-FAK(Tyr397)], p130 Crk-associated substrate(p130Cas) and paxillin by using western blot. The m RNA expressions of PTEN, FAK, p130 Cas and paxillin were detected by real-time fluorescent quantitation PCR. A database of all materials used Excel 2010 and SPSS 21.0 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, venom in transfection virus steps to join DMEM instead of disease.(2)Ad-GFP group,empty virus carrier with green fluorescent protein(GFP) expression.( 3) Ad-PTEN group, simultaneously with wild-type PTEN gene GFP expression of recombinant adenovirus.(4)Ad-G129 E group, G129 E at the same time with GFP gene expression of recombinant adenovirus.Results 1 By using the method of adenovirus infection repeatedly AD293 cell disease venom required to obtain a experiment,Ad- PTEN drops to 1.3 x 109 pfu/m L, Ad- G129 E drops 1.4 x 109 pfu/m L and Ad- GFP drops 1.6 x 109 pfu/m L. 2 When multiplicity ofinfection(M.O.I) 100 adenovirus infected HSC 48 h, inverted fluorescence microscope counted cells of GFP expression. Ad-GFP, Ad- PTEN and Ad-G129 E groups cells of GFP expression were more than 80%. 3 When adenovirus infected HSC 48 h, applied Real-time fluorescent quantitative PCR detection between groups of HSC PTEN m RNA expression, comparing each group of HSC PTEN m RNA the levels by relative quantitative method(2-△△Ct method), the control group of PTEN m RNA expression quantity was considered 1,AD-GFP,Ad-PTEN and Ad-G129 E groups than the control group of PTEN m RNA relative ratio of 1.03 times,1.90 times and 1.83 times. Control group and Ad-GFP group compared to Ad-PTEN group and Ad-G129 E group PTEN m RNA expression significantly increase, between two groups of Control, Ad-GFP and groups of Ad-PTEN,Ad-G129 E were no statistical significance(P>0.05).Western blot technology to detect each group of HSC PTEN protein expression shows: Ad-PTEN group(1.08±0.07), Ad-G129 E group(0.97±0.04) were statistically higher than those in Control group(0.56±0.05) and AdGFP group(0.69±0.07), P<0.05.between two groups of Control, Ad-GFP and groups of Ad-PTEN,Ad-G129 E were no statistical significance(P>0.05). 4 Applied Western blot and Real-time fluorescent quantitative PCR to detect eachgroup HSC FAK protein and m RNA expression after adenovirus infection 48 h, the result showed that each group FAK protein and m RNA expression had no obvious change(P>0.05).5 Western blot was used to test groups of p-FAK(Tyr397) protein expression, Ad-PTEN group(0.437±0.039)、Ad-G129 E group(0.456±0.042)significantly lower than the Control group(0.701±0.028)and Ad-GFP group(0.694±0.019),there was no significant difference between the two groups of Control, Ad-GFP and groups of Ad- PTEN,Ad-G129E(P>0.05).6 When adenovirus infected HSC 48 h, applied Real-time fluorescent quantitative PCR detection between groups of HSC p130 Cas m RNA expression,as the Control group p130 Cas m RNA expression quantity is 1, compared with the Control group Ad- GFP p130 cas m RNA expression ratio of 1.001 higher than Ad-PTEN group 0.589 times and Ad-G129 E group 0.574 times(P<0.05), between two groups of Control, AdGFP and groups of Ad-PTEN,Ad-G129 E were no statistical significance( P >0.05).Western blot technology to detect each group of HSC p130 Cas protein expression shows: Ad- PTEN group( 0.596 ± 0.041),Ad-G129 E group( 0.534 ± 0.049) were statistically lower than Control group(1.137±0.193)and Ad-GFP group(1.036±0.116), P<0.05, between two groups of Control, Ad-GFP and groups of Ad-PTEN,AdG129 E were no statistical significance(P>0.05). 7 When adenovirus infected HSC 48 h, applied Real-time fluorescent quantitative PCR detection between groups of HSC p130 Cas m RNA expression,as the Control group p130 Cas m RNA expression quantity is 1, compared with the Control group Ad- GFP P130 cas m RNA expression ratio of 0.962 times higher than Ad-PTEN group 0.425 times and Ad-G129 E group 0.437 times(P<0.05), between two groups of Control, Ad-GFP and groups of Ad-PTEN,Ad-G129 E were no statistical significance(P>0.05).Western blot technology to detect each group of HSC paxillin protein expression shows: Ad- PTEN group(0.595±0.054)、AdG129E group(0.667±0.036)were statistically lower than Control group(1.328±0.193) and Ad-GFP group( 1.270 ± 0.114)( P< 0.05), between two groups of Control, Ad-GFP and groups of Ad-PTEN,Ad-G129 E were no statistical significance(P>0.05).Conclusions 1 The overexpression of wild type PTEN and its mutant gene G129 E can remarkably inhibit the FAK/p130 Cas and paxillin signal transduction in activated HSC. 2 There are no significant differences in inhibitory effects on the signal transduction of FAK/p130 Cas and paxillin between wild type PTEN and G129 E in activated HSC. 3 PTEN inhibit the signal transduction of FAK/p130 Cas and paxillin through its protein phosphatase activity in activated HSC. |
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