Changes Of Immune Equilibrium In Th17 / Treg Cells In The Treatment Of Acute Myocardial Infarction By Transplanted Skeletal Muscle Myoblasts

Skeletal myoblasts transplantation in the treatment of myocardial infarction is the most potential treatment measure, a number of studies have shown that the survival of allogeneic skeletal myoblasts transplanted into the myocardial infarction region is very low, generally, the survival rate after24hours is lower than20%, which is already an indisputable fact and seriously impacts on the effect of skeletal myoblasts treatment. It may be associated with the inflammation and immune responses induced allogeneic skeletal myoblasts transplanted into infarcted myocardium. Th17/Treg cells immunomodulatory balance may play an important role in the process of inflammation and immune responses, therefore, our study clearly showed that the imbalance of Th17/Treg cells exists in the process of skeletal myoblasts transplantation for AMI.VEGF is not only a major regulatory factor of angiogenesis,but also play an important role in regulating the inflammatory response. The new and novel nano-particles--hyperbranched polyamidoamine(hPAMAM) is a non-viral, low toxicity and efficient vector, which produce low inflammatory response, and can protect the genes from the protease digestion. Therefore, we choosed hPAMAM as a vector carrying VEGF165gene to transfect skeletal myoblasts, and then which were transplanted into the infarcted myocardial region, VEGF165was slowly and continuously released in the infarcted myocardial region to achieve long-lasting effect, which was conducive to the formation and growth of the microvessel and the mitigation of inflammation, thus alleviated the imbalance of immune regulation of Th17/Treg cells. Chapter ISkeletal myoblasts separation, culture and identification, and the VEGF165gene transfection Objective:To establish the method of separation of C57BL/6mouse skeletal myoblasts, which were cultured, purified and identified in vitro. The expression vectors hyperbranched polyamidoamine (hPAMAM) nanoparticles were combined with cDNA-VEGF165, then were transfected into skeletal myoblasts, and detected the optional transfection efficiency.Methods:WE injected0.1ml bupivacaine in3-4w old mice for48h for the proliferation and activation of skeletal myoblasts, then skeletal myoblasts were extracted from by using one-step digestion(II collagenase+dispease) method and the discontinuous Percoll density gradient and then purified by repeated differential adhesion time method after48h. Skeletal myoblasts was identified by specific antigen desmin and a-sarcomeric actin. The0.4ug^0.6ug、0.8ug cDNA-VEGF165were respectively combined with hPAMAM according to1:4,1:6,1:8,1:10,1:12proportion, and then respectively co-cultured with the2×104skeletal myoblasts for4h, we used the living cells Monitor Cell-IQ cell culture platforms to observe transfected hPAMAM-VEGF Skeletal myoblasts growth and cell number changes in real time, using the freely distribution image software analysis of monitoring data.Results:Skeletal myoblasts showed the growth of adhesion three days after the separation, and gradual fusion growth after7-9days, and were like fusiform, spindle-shaped, and were in linear growth, and formed muscle tube-like structures. Skeletal myoblasts specific antigen, desmin and a-sarcomeric actin were positive expression, desmin-positive cells accounted for82.34±2.96%and a-sarcomeric actin positive cells accounted for80.1±3.39%using flow cytometry, desmin and a-sarcomeric actin double-positive skeletal myoblasts accounted for81.22±3.23%. The optional transfection conditions of the hPAMAM-VEGF165transfected skeletal myoblasts was: when the VEGF165was plasmid0.4ug, V:N is1:8, the proliferation of skeletal myoblasts were closest to the normal cells growth, and the secretion of VEGF reached maximum value. Conclusion:One-step digestion method, discontinuous Percoll density gradient method and the repeatedly differential adhesion time method can get more quantity and higher purity of skeletal myoblasts. hPAMAM vector can combined with VEGF165to transfect skeletal myoblasts and can be used as a new type of low toxicity and non-viral vector. Chapter ⅡEtablishment of an animal model of AMI andTransplantation of allogeneic skeletal myoblast for treatment of AMIObjective:We established the C57BL/6mouse model of acute myocardial infarction using coronary artery ligation, and pure skeletal myoblasts and skeletal myoblasts transfected hPAMAM-VEGF were respectively transplanted to treat acute myocardial infarction for observing feasibility and effectiveness.Methods:C57BL/6mice were had a left thoracotomy with the gas anesthesia, and we ligated the proximal left anterior descending artery directly under the amplifier and formed acute anterior myocardial infarction. Then, we used the microinjector to inject skeletal myoblasts into the surrounding points of myocardial infarction. We divided mice into acute myocardial infarction group (AMI, n=50), simple Skeletal cell transplantation group (AMI-S group, n=50) and skeletal myoblast transfected hPAMAM-VEGF165transplantation group (AMI-V group, n=50). body weights of mice were observe before surgery,1d after surgery,1w after surgery and4w after surgery. The ultrasound measurement of cardiac function was made before surgery, immediately after surgery,1w and4w after surgery to mainly observe the changes in EF values. Evans Blue was direct injected into the renal vein to observe the area of myocardial infarction in vivo, the depth and the scope of myocardial infarction was observed by the TTC method. CD4+T cells infiltration around the infarction myocardial was obseerved by immunohistochemical staining.Results:The rate of success was about86%to establish of mice acute myocardial infarction model. Before surgery, there was no significant difference in the body weight of mice among the AMI group, AMI-S group and AMI-V group.1d after surgery, there also was no significant difference in the body weight of mice among three groups. However,1w after surgery, compared with the AMI group(16.58±0.35g), body weights of mice in AMI-S group(17.66±0.30g) and in AMI-V group(18.34±.28grams of) significantly increased (P<0.05), compared with body weights of mice in AMI-S group, those in AMI-V group significantly increased (P<0.05). After4w, compared with body weights of mice in AMI group (16.50±0.35g) those in AMI-S group (18.23±0.34g) and in AMI-V group (18.97±0.18g) obviously increased (P<0.01), compared with body weights of mice in AMI-S group, those in AMI-V group significantly increased (P<0.05). The location of myocardial infarction were left ventricular anterior wall and apex appropriately in line with the requirements of the experiment, and can clearly be seen under direct vision. The TTC test showed that the depth of myocardial infarction is through the whole layer. Preoperative left ventricular EF in the three groups of normal mice were no significant differences (P>0.05). In the immediate postoperative left ventricular EF in the three groups of mice were no significant differences too (P>0.05). However, lw after ligation, compared with EF in AMI group (31.54±3.23%), the EF in AMI-S group (37.94±2.29%) and AMI-V group (46.28±2.44%) was significantly increased (P<0.05), and compared with EF in AMI-S group, EF in AMI-V group was significantly increased (P<0.05).4w after surgery, compared with EF in AMI group (33.67±3.18%), EF in AMI-S-group (56.28±2.83%) and AMI-V group (63.73±3.33%) was significantly increased (P<0.05), furthermore, compared with in AMI-S-group, that in AMI-V group was significantly increased (P<0.05). In addition, Myocardial tissue by immunohistochemical staining showed CD4+T lymphocyte infiltration around myocardial infarction, which is more obvious in AMI-S-group.Conclusion:The coronary artery ligation under direct vision can effectively control the scope of myocardial infarction, myocardial infarction location and extent were appropriate and in line with the requirements of the experiment. Single skeletal myoblasts transplantation and skeletal myoblast transfected hPAMAM-VEGF treatment of acute myocardial infarction can be effective in restoring cardiac function. Chapter ⅢThe shift of Th17/Treg cell immunomodulatory balanceObjective:To investigate Th17/Treg imbalance in transplanting skeletal myoblasts for treatment of AMI, and whether or not Th17/Treg imbalance can be alleviated to transplant transfected hPAMAM-VEGF165skeletal myoblasts for treatment AMI.Methods:In the1st d,2nd d,4th d,7th d respectively, we counted the percentage of Treg cells and Th17cells in the spleen using flow cytometry, analyzed the transcription factor Foxp3and RORyt mRNA levels in the spleen and heart using real-time PCR, and analyzed the concentration of Treg cells related regulatory cytokine TGF-β1, IL-2, IL-10and Th17cells related proinflammatory cytokine IL-1β, IL-6, IL-17A in serum, as well as VEGF in serum using the Bio-plex suspension array.Results:In AMI, AMI-S, AMI-V groups, the percentage of CD4+CD25+Foxp3+Tregs, the transcription factor Foxp3mRNA levels, the concentration of related regulatory cytokine TGF-β1, IL-2, IL-10after transplantation showed a gradual downward trend from the1st day and reached the minimum in th7th day. The percentage of Treg, the transcription factor Foxp3mRNA level and the concentrations of related regulatory cytokines TGF-β1, IL-2, IL-10in AMI-S group were lower than those in AMI group(P<0.05or P<0.01). However, the percentage of Treg cells, transcription factor Foxp3mRNA level and related regulatory cytokine TGF-β1in IL-2, IL-10concentrations in AMI-V group were apperently higher than those in AMI group (P<0.05or P<0.01). On the contrary, the percentage of CD4+IL-17+Th17cells, the mRNA level of transcription factor RORyt, the concentrations of related proinflammatory cytokine IL-1β, IL-6, IL-17A gradually increased, and compared with AMI group, the percentage of Th17cells, transcription factor RORyt mRNA levels, the concentrations of related regulatory cytokine TGF-β1, IL-2, IL-10in AMI-S group were obviously higher (P<0.05or P <0.01), and the percentage of Thl7cells, the mRNA level of transcription factor RORyt, the concentrations of related cytokine IL-1β, IL-6, IL-17Ain AMI-V group were significantly lower (P<0.05or P<0.01).Conclusion:Transplantation of skeletal myoblast for treatment AMI can induce the Thl7/Treg imbalance mediated inflammatory response, which may decline the survival of transplanted skeletal myoblasts, however, Transplantation of transfected hPAMAM-VEGF165skeletal myoblast for treatment AMI may alleviate the Th17/Treg imbalance.