The Angiogenic Effects Of Bone Marrow Mesenchymal Stem Cells Transfected With Sonic Hedgehog Gene After Acute Myocardial Infaction

In this experiment,we make good use of BMMSC as carrier cells receiving transfection of SHH gene,with the expectance that BMMSC not only can keep differentiative capacity and favorable biology properties after being transplanted into vivo bue also express effectively the transfected SHH gene. Firstly,We transfect SHH into BMMSC by nucleofector,then transplant those cells into the border of the infracted and normal zones. Because SHH boost angiogenesis by regulating angiogenic growth factors,to prove that BMMSCSHH can boost angiogenesis on the basis of cell thrapy,we then detect changes of SHH downstreams and the angiogenic growth factors such as VEGF、Ang-1at the transplanted zone at the level of molecular biology.At last we demonstrate the actual angiogenesis condition at the border between the infracted and normal zone by vascular density analysis.This experiment is devided into three parts.Firstly,we take advantage of Amaxa’s nuclear transfection instrument to transfect exogenous SHH gene into rat BMMSC,then we test the feasibility of this transfection method by detecting the changes of SHH protein in BMMSCSHH analysising the harms of the transfection method done to BMMSC and so on.Secondly,we obtain samples respectively one,two,four and eight weeks after implantation,and detect the changes of SHH signal conduction pathway as well as angiogenesis growth factors in those samples.At last, we analysis the vascular density difference at the junctional zone of infracted and normal area among teams of different treatment. Part one:transfection Of Sonic hedgehog gene into BMMSCObjective:To evaluate feasibility of transfection of Sonic hedgehog gene into BMMSC.Methods:1.The Shh gene was transfected into BMMSC by electroporation apparatus.we can evaluate the transfection rate by Fluorescence Inverted microscope.2.growth curves of untransfected and transfected BMMSC was drawed;3. The expression of Sonic hedgehog gene in the BMMSCSHH was detected by PCR,RT-PCR,Western-blot analysisResults:Through Fluorescence Inverted microscope,the transfection rate is appropriately30%,PCR show there is a noted increase of SHH expression in transfected cells than that in untransfected cells,This is quantified by appropriately7times by qPCR. Western-blot further demonstrate that the SHH protein also has a distinct increase.However,the exponential phase of BMMSCSHH’growth curve delay.But they overlap12days after transfection.Conclusion:This experiment can transfect exogenous SHH gene into BMMSC sueffciently,and can obtain effective protein expression in BMMSCSHH. This transfection method can meet the needs of this experiment. Part two:BMMSCSHH regulates the downstream of SHH in transplanted zoneObjective:To Investigate effects of BMMSCSHH done to the downstream of SHH in transplanted zone.Methods:160acute ischemic heart animal models were made by left anterior descending coronary artery ligation. The models were divided into4groups.Then we immediately inject those animal models with BMMSCSHH (Transfection group),or equal number of BMMSC(cell group), mixture of same number of BMMSCs and pcDNA3.1-Shh DNA (Mixed group), or equal volume of DMEM(Control group) at the heart infarct periphery zone.1,2,4,8weeks after the transplantation the rat cardiac samples was harvested. RT-PCT was employed to detect the expression of downstream of SHH such as Ptc、Gli-2、COUP-TF II,and the expression of angiogenetic growth factors such as VEGF、Ang-1etc at the transplanted zones.Results:7days after transplantation,there were higher expression of the Ptcl、Gli-2and COUP-TF II at the transplanted zones of transfection group than that of control group、cell grouop and mixed group respectively(Ptcl:P<0.01, P<0.05,P<0.05; COUP-TF II:P<0.01, P<0.05,P<0.05;Gli-2:P<0.01, P<0.01,P<0.01).There is also higher expression of VEGF、Ang-1at the transplanted zones of transfection group than that of control group、cell grouop and mixed group respectively(VEGF:P<0.01,P<0.05,P<0.05;Ang-1:P<0.01,P<0.05,P<0.05).however,there are no notable difference of expression of above-mentioned growth factors of following period’s detections.Conclusions:In this experiment BMMSC SHH can increase the expression of the downstream of SHH as well as angiogenic growth factors in transplanted zone in a limited period,which is the foundation of our following detection of actual angiogenesis condition at transplanted zones. Part three:The actual angiogenic condition of BMMSC SHH in transplanted zoneObjective:Detection of BMMSCSHH’s angiogenic effection in transplanted zone.Methods:Immunohistochemical staining mark the CD34antigen of vascular endothelial cell which display blood vessels of the border between infracted and normal zones. HE staining also can display all levels of vascular.then we can analyze the density difference of blood vessels of those group by vascular density analysis.Results:there were higher density of blood vessels at the border between infracted and normal zones of the transfection group than that of control group、cell grouop and mixed group respectively(P<0.01, P<0.01,P<0.01) regardless of CD34Immunohistochemical staining and HE.Conclusion:Transplantation of BMMSCSHH boost angiogenesis after myocardial infaction,which provide new exploration for therapy towards ischemic heart diseases.