| Objective Through the establishment of the scar model of rabbit cleft lip postoperative wound healing,we studied this model by postoperative scar growth time series in order to make clear the time of the scar hyperplasia to the greatest extent.By building p38MAPK gene silencing recombinant adenovirus for interferring with the expression of target genes and blocking p38MAPK signaling pathways,we tested the effects on rabbit model in the different time points to find the best time of gene therapy and to explore the cross-talk between p38MAPK and Smad signaling pathways.To clarify the molecular mechanism of therapeutic effect of adenovirus mediated p38MAPK gene silence on rabbit scar model from the overall level.To lay the foundation for the development of the new ways to the prevention and control of the scar hyperplasia high-risk groups in China.Method 1.We constructed the upper lip scar model of rabbit,using Millard's one-side cleft lip repair method.2.After 2 weeks,3 weeks,4 weeks,5 weeks,we conducted the volume measurement,MFB cell counts and qualitative and quantitative detection of contents of a-SMA in scar tissue using immunohistochemical staining and Western blot.3.To select the most effective shRNA sequences of p38MAPK gene silencing and to construct the adenovirus vector.4.To proceed the adenovirus transfection of p38MAPK gene silence in 0 week,1 week,2 weeks after operation respectively on the scar tissue and then to get specimens in 3 weeks after operation.To detect the relative level of mRNA and protein expression of p38MAPK and scarring related factor(Col?,Col?,MMP1 and TIMP1)with four methods just as Sirius scarlet staining,immunohistochemical staining and Western blot,Real-time PCR qualitatively and quantitatively.5.To test mRNA level and protein expression of Smad2,Smad3,p-Smad2,p-Smad3,p38MAPK and p-p38MAPK using Real-time PCR and Western blot.Results 1.The scar model of rabbit cleft lip was successfully constructed.2.The results of scar volume and MFB cell counts showed that the 2 weeks and 5 weeks after the operation,the 3 weeks and 4 weeks after the operation had no significant difference(P>0.05),but the data of 2 weeks and 3,4 weeks after the operation was significantly different(P<0.01).Immunohistochemical staining results showed that the expression of a-SMA on 3,4 weeks in scar tissue was more than after that on 2 and 5 weeks.The results of Western blot were same to that.3.The most effective shRNA sequences of p38MAPK gene silencing was successfully screened and the adenovirus vector was constructed.4.After local injection of adenovirus,Sirius red staining showed that col? of scar tissue in 1 week after surgery was significantly more than that in 0 week and 2 weeks,but col? of 1 week after surgery was less than that on 0 and 2 weeks.Immunohistochemical staining showed col?,MMP1 expression of scar tissue in lweek after surgery was significantly more than that in 0 week and weeks after surgery but the col?,TEMP1 expression in 1 week after the operation was significantly less than 0 and 2 weeks after surgery.The results of Western blot and Real-time PCR were same to Immunohistochemical staining.5.To inject adenovirus in the scar tissue in lweek after operation,Western blot results showed that the protein expression level of p-Smad2,p-Smad3 and p-p38MAPK in scar tissue in 1 week after operation were decreased,comparing with the control group.The Real-time PCR results was in accordance with Western blot results.Conclusions:We provided car model of rabbit cleft lip in order to study gene therapy of scar.We concluded that the scar reached the maximum proliferation in 3weeks and 4 weeks after operation.We conducted the local injections of adenovirus to the scar tissue and then the target gene silencing effect and treatment effect got best.We also studied the cross-talk between Smad and p38MAPK.The molecular mechanism of therapeutic effect of p38MAPK of adenovirus mediated gene silence on rabbit model of the upper lip scar was clarified from the overall level.The foundation was laid for the development of new ways on early prevention and control of HTS of high-risk groups in China. |
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