| Objectives To investigate effects of over-expression of wild type phosphatase and tensin homology deleted on chromosome Ten(PTEN)and its mutant G129E(only exhibit protein phosphatase and lose lipids phosphatase activity)on the adhesion and migration of activated hepatic stellate cell(HSC)in vitro.Methods Amplifications of adenoviral vectors(Ad-PTEN、Ad-G129E、Ad-GFP)were performed in 293 A cells and viral titer estimates were conducted. The wild type PTEN and its mutant G129 E gene were transduced into activated HSC(HSC-T6)in vitro via adenoviral vector.Detecting the expression of PTEN protein by western blot.Determining the ability of HSC adhesion by counting cells stained with toluidine blue and MTT colorimetric method. Detecting the ability of HSC plane migration by the scarification and plerosis experiments. Detecting the ability of HSC transmembrane migration by the transwell chamber experiment. All data was put into a database in Excel and was analyzed by software of SPSS 21.0,and the measurement data was showed as mean±standard deviation( x ±s). Additionally, single-factor analysis of variance method(one-way ANOVA) was utilized to compare more groups, and LSD test was used to check every two groups. If the probability values was less than 0.05,then which was considered statistically significant. Cells were grouped as follows:(1)Control group,cells were cultured simply in DMEM with 8% FBS,and viral midium was replaced by DMEM without FBS or antibacterial.( 2) Ad-GFP group, HSC were infected with adenovirus expressing green fluorescent protein(GFP) alone.(3)Ad-PTEN group: HSC were infected with adenovirus harboring genes for both wild type PTEN and GFP.(4)Ad-G129 E group, HSC were infected with adenovirus harboring genes for both PTEN mutant G129 E and GFP.Results 1 Adenoviral vectors viral titers of Ad-GFP,Ad-PTEN and Ad-G129E:1.6×109pfu/ml, 1.3×109pfu/ml, 1.4×109pfu/ml, respectively for experiment were obtained via performing repeated amplifications of virus in 293 A cells. 2 At 48 hours after adenovirus infection in HSC at Multiplicity of infection(M.O.I) 100, the infection rate of each group including Ad-GFP, Ad-PTEN and Ad-G129 E were measured by fluorescent microscopic counting. All of which were more than 80%. 3 Western blot analysis recapitulated data by showing that expression levels of PTEN protein in Ad-PTEN group(1.08±0.07)and Ad-G129 E group(0.97±0.04)were statistically higher than the control group(0.56±0.05)and Ad-GFP group(0.69±0.07),P<0.05,Furthermore,no significant differences were observed in the expressions of PTEN protein between AdPTEN group and Ad-G129 E group or between control group and Ad-GFP group,P>0.05. 4 At 48 hours after adenoviral infection, the cell adhesion was measured by counting cells stained with toluidine blue. Compared with cuntrol, adhesion inhibitionrate in Ad-GFP group(1.89±0.88)% had no significant differences,P>0.05,while adhesion inhibition rate in Ad-PTEN group( 20.38 ± 4.37) % and Ad-G129 E group(19.57±3.78)% obviously reduced,P<0.05,The adhesion inhibition rate of HSC had no statistical difference between Ad-PTEN group and Ad-G129 E group,P>0.05. 5At 48 hours after adenoviral infection,the adhesion inhibition rate of HSC was measured by MTT colorimetric method. Compared with cuntrol, adhesion inhibition rate in AdGFP group(6.17±4.98)% had no significant differences,P>0.05,while adhesion inhibition rate in Ad-PTEN group(38.13±6.93)% and Ad-G129 E group(35.83±4.24) % obviously reduced, P < 0.05, The adhesion inhibition rate of HSC had no statistical difference between Ad-PTEN group and Ad-G129 E group, P > 0.05. 6 The scarification and plerosis experiments showed that: At 12 hours after adenoviral infection HSC in each group transferred,but the distances showed little difference. At 24 hours after adenoviral infection,the distances changed obviously. Compared with control group(391.34±10.41μm), transfered distance in Ad-GFP group(392.66±10.41μm)had no significant differences,P>0.05,while which in Ad-PTEN group(332.35±6.23μm)and Ad-G129 E group(336.77±5.25μm)obviously reduced(P<0.05).At 48 hours after adenoviral infection,the distances obviously changed. Compared with control group(551.68±8.91μm), transfered distance in Ad-GFP group(550.81±11.55μm)had no significant differences,P>0.05,while which in Ad-PTEN group(468.74±19.29μm)and Ad-G129 E group(462.48±13.47μm)significantly reduced,P<0.05. 7 Transwell chamber experiment showed that: Compared with control group( 67.67 ± 6.28), the transmembrane cell numbers in Ad-GFP group( 64.67 ± 5.53) had no significant differences,P>0.05,which in Ad-PTEN group(38.67±4.50)and Ad-G129 E group(33.33±3.83)obviously reduced,P<0.05,but numbers in Ad-PTEN group and AdG129 E groups showed no difference,P>0.05.Conclusions 1 The overexpression of wild type PTEN can remarkably inhibit the adhesion and migration of activated HSC. 2 There are no significant differences in inhibitory effect on the adhesion and migration of activated HSC between wild type PTEN and its mutant gene G129 E. 3 PTEN gene inhibit the adhesion and migration of activated HSC through the protein phosphatase activity. |
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