Investigation Of Stem Cell Transplantation Combined With Heme Oxygenase-1 And Cardiotrophin-1 Gene Transfer For The Treatment Of Acute Myocardial Infarction In Rats

Objective: To investigate the transplantation effects of bone marrow derived- mesenchymal stem cells(BMSCs) transferred with heme oxygenase-1(HO-1) and cardiotrophin-1(CT-1) on functional recovery of rats hearts after acute myocardial infarction.Methods: (1) Total mRNA was extracted from Sprague-Dawley(SD) rat spleen cells by Trizol method and HO-1 gene was cloned through RT-PCR reaction and connected with PIRES2-EGFP vector. Consequently the recombinant eukaryon expression vector PIRES2-EGFP/HO-1 was established. PIRES2-EGFP/CT-1 expression vector was constructed and preserved by our own lab. (2) Mesenchymal stem cells(BMSCs) were isolated from SD rats'bone marrow and cultured in vitro, followed by the investigation of growth characteristics, phenotypes and in vitro proliferation abilities through inverted microscope, flow cytometry and MTT method respectively. (3) Plasmid DNA was extracted by plasmid large extract kit and quantified; Albumin microbubbles were prepared and mixed with plasmid DNA gently, set at 4℃for 1 hour and then were added into culture fluid; The cells were exposed to ultrasound for 20 seconds with 0.75 W/cm2 pulsed energy, which was repeated after setting at 37℃for 5 minutes; 6 hours later culture medium was replaced. Continue culturing for 48 hours, the expression of EGFP was analyzed by fluorescent microscope and flow cytometry; the activity of cells with transfection was detected through trypan blue dye. (4) Acute rat myocardial infarction(AMI) model was established by left coronary artery ligation in anesthetized rats, and then was identified by electrocardiogram and serum treponin T. (5) 40 myocardial infarcted rats were divided into four groups randomly,①control group(BMSCs group), injected with BMSCs, n=10;②HO-1 + BMSCs group, injected with BMSCs transfered by HO-1; n=10;③CT-1 + BMSCs group, injected with BMSCs transfered by CT-1; n=10;④HO-1 + BMSCs and CT-1 + BMSCs group, injected with BMSCs transfered by HO-1 and CT-1 respectively; n=10. 20 days after operations, the effects were evaluated by ultrasonic test and statistical analysis. (6) the heart tissues under the ligation point were generated, fixed, dehydration, embed, slice, HE dyeing and histopathological observation; The frozen section was observed using fluorescent microscope to search transplanted cells with EGFP expression.Results: (1) HO-1 gene was cloned from rat spleen cells and used for the construction of eukaryon expression vector PIRES2-EGFP/HO-1. The positive clones with correct sequence were stored for future. (2) BMSCs showed fusiform and the growth characteristics of adherence to the wall, Trypan blue dye showed the percentages of living cells exceeded 95%; Proliferation curve measured by MTT method indicated that cells entered logarithmic growth phase at third day. Flow cytometer showed that those cells expressed high levels of CD29(97.6%), CD44(96.5%), CD90(98.2%), and low levels of CD34(3.7%), CD45(6.8%). (3) The density of albumin microbubble exceeded 1.5×1011/ml and the diameter was 2.0-4.0μm; Through Trypan blue dye, the percentages of living cells with the treatment of ultrasound exceeded 95%; 48 hours later after transfection, EGFP could be observed by fluorescent microscope, and the transfection rate was 30.8% using flow cytometry; Total mRNA was extracted from transfected cells and analysis by RT-PCR, the results showed that the specific strap was detected only in transfected group but not in the control group. (4) Identified the myocardial infracted model: electrocardiogram showed ST segment raise, echocardiogram showed evident myocardial infracted area and serum treponin T reflect masculine. (5) 20 days after operation, each heart functional parameter tested in echocardiogram showed: each transfered group was compared with control group, P<0.05; HO-1 group compared with CT-1 group, P>0.05; transfered twice gene group compared with HO-1 group and CT-1 group, P<0.05.(6) After 20 days, cardiac muscle histopathological slide showed: normal myocardial cells have transverse striation and branch, caryon situate in the center of the cell. We could find evident coagulation necrosis in the infarct tissues, nuclear fragmentation,solution and disappear, endochylema vermilion homogen, interstitial edema, and a large number of inflammatory cell infiltration. We detected some trachychromatic blue new formed cells in every treated group and we also found scar tissues grew downwards. (7) The frozen section was observed under the fluorescent microscope and a small quantity cells expressed EGFP were found.Conclusions: BMSCs have the potential ability to differentiate into cardiomyocyte-like cells,especially their conveniences for obtaining and proliferating, and can be auto- graftted without immunological rejection. The ultrasound-target microbubble destruction method could enhance the exogenous gene transfection and expression efficiency. Deligate rats'aeteria coronaria can establish myocardial infracted models can be established by coronary artery ligation in rats; it is simple and reliable. Contrast with merely transplant BMSCs, we found that transfected BMSCs have more positive effects on the recovery of heart function, and combined two types of transfected BMSCs are better.