| Objective:To observe the biological characteristics of adipose-derived mesenchymal stem cells transfected by rAAV1-EGFP,and detect if ADMSCs transfected by rAAV1-EGFP in vitro could keep on expressing exogenous EGFP in the infarcted myocardium after the homogeneous xenogenic transplantation.To investigate the function of paracrine of ADMSCs transgened with human hepatocyte growth factor in vitro,and probe the feasibility and effectiveness of homogeneous xenogenic transplantation of ADMSCs combined with transgene therapy in acute rat infarcted myocardium.Method:(1)To observe the biological characteristics of adipose-derived mesenchymal stem cells transfected by rAAV1-EGFP.ADMSCs were isolated from adipose tissue near to epididymis of male Wister rats by digestion and segregation,and were cultured,amplified and identified in vitro.We transfected ADMSCs with rAAV1-EGFP,and observed their growth,specific marks of cell surface and potency of multi-directional differentiation.(2)To detect if ADMSCs transfected by rAAV1-EGFP in vitro could keep on expressing exogenous EGFP in the acute infarcted rat myocardium after the homogeneous xenogenic transplantation.Sixty male Wister rats were averagely divided into two groups randomly:the negative control group(n=15),the cell transplantation group for one week(n=15),the cell transplantation group for four weeks(n=15),the cell transplantation group for eight weeks(n=15).Then we prepared the model of acute myocardial infarction by ligating the left anterior descending of coronary artery,and then injected PBS(negative control group)or ADMSCs-EGFP(cell transplantation group)into the part of i(?)farcted myocardium through the epicardium.We executed the rats in the cell transplantation groups at corresponding time(after one week,four weeks and eight weeks respectively) to detect by fluorescence microscope and immunohistochemistry with image analysis if the ADMSCs-EGFP could keep on expressing exogenous EGFP in the acute rat infarcted myocardium after the homogeneous xenogenic transplantation. And we executed the rats in negative control group eight weeks after the operation. (3)At the first we constructed rAAV1-hHGF.Then we inoculated ADMSCs in the six pores plates,and divided these ADMSCs into the non-transfected group (negative control group)and the rAAV1-hHGF group.At the last we determined the changes of the concentration of hHGF in cellular clear supernatant everyday by enzyme linked immunosorbent assay from the first day to the eighth day, and meanwhile counted the cells per well to observe changes of ability of secretion of the cells.(4)The therapy of acute rat myocardial infarction through homogeneous xenogenic transplantation of ADMSCs-hHGF.One hundred male Wister rats were divided averagely four groups randomly:PBS group(n=25), only ADMSCs group(n=25),only hHGF group(n=25)and the ADMSCs-hHGF group(n=25).We ligated the left anterior descending of coronary artery to make model of AMI,and to give corresponding therapy through injection into the epicardium.Eight weeks after the operation we measured their heart function by catheterization and ultrasonic cardiogram.Then we executed the rats to calculate infarct size,SI and DI of left ventricle.Also we observe the typeâ… and typeâ…¢CVF by picrosirius red staining.And we detected the capillary density,MMP-2 and MMP-9 by immunohistochemistry.Further more,we measure the messenger RNA of hHGF in the infarcted myocardium by RT-PCR,and the hHGF at the same part were detected by Western blotting.Result:(1)After transfected by rAAV1-EGFP,the ADMSCs could adhere as normal and proliferate quickly,and the doubling time of the cells was approximately 52.5 hours.These ADMSCs appeared steady after high passage, and the process of transfection didn't influence specific marks of cell surface and the potency of multi-directional differentiation of the cells.(2)After the homogeneous xenogenic transplantation of ADMSCs-EGFP,the green fluorescence and expression of EGFP in one week and four weeks group didn't show conspicuous differences,both exceeding the negative control group significantly.But the green fluorescence nearby the infarcted part weakened obviously in the eight weeks group with thimbleful expression of EGFP.There was no green fluorescence and expression of EGFP in the control group.(3)The rAAV1 was reconstructed successfully with hHGF.The concentration of hHGF in clear supernatant of ADMSCs transfected with hHGF increased significantly,with an enhancement of the ability of secretion of the cells.(4)Eight weeks after myocardial infarction,in the only ADMSCs group and the ADMSCs group with transgenic hHGF,we could see the significant increases of EF,LVSV,LVEDV and +dt/dpmax,-dt/dpmax,+dt/dpmax/LVSP,-dt/dpmax/LVSP,left ventricle relative weight,local capillary density,meanwhile the decreases of typeâ… and typeâ…¢CVF,MMP-2 and MMP-9 compared with control group,with no significant change of apoptotic index(AI).Compared with the only ADMSCs,there were significant increases of local capillary density but more MMP-9 in the ADMSCs-hHGF group.Also the transcription and expression of hHGF could be detected in both only hHGF group and transgenic ADMSCs group eight weeks after cells transplantation.Conclusion:The transfection by rAAV1 did't change the biological characteristics of ADMSCs significantly;the expression of exogenous EGFP could be detected continuously in the part of acute infarcted myocardium four weeks after the transplantation of the ADMSCs-EGFP.The ADMSCs transfected by rAAV1-hHGF could secrete more hHGF in vitro;there was continue expression of hHGF in the part of transplantation gived with ADMSCs with transgenic hHGF.Consequently,the function of ADMSCs in promoting angiopoiesis was strengthened,and the heart function would recover after acute myocardial infarction. |
