Enhancement of cardiac function of chronic heart failure rats by marrow stromal cell-based sarcoplasmic reticulum Ca2+ adenosine triphosphatase gene therapy
- VernacularTitle:钙离子ATP酶2a基因修饰骨髓间充质干细胞移植改善慢性心力衰竭大鼠的心功能
- Author:
Yutao GUO
;
Xiaoying LI
;
Xiaochun LU
;
Di WU
;
Kequn YAO
;
Ping CHEN
;
Kangtao MA
;
Chunyan ZHOU
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2008;12(8):1550-1557
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: There are still few effective methods to repair injured myocardium after myocardial failure and pathologically rebuild reverral myocardium. As a new therapy, normal myocytes and therapeutic gene to interfere injured myocardium have advantageous effects in improving heart function.OBJECTIVE: To observe the efficiency and stability of adenovirus-medicated gene transferred into different passages of bone marrow mesenchymal stem cell (MSC) and investigate the effect of MSC-based sarcoplasmic reticulum Ca2+ ATPase gene (SERCA2a) gene therapy for rats with chronic heart failure. To compare the effects of gene therapy, cell transplantation and MSC-based SERCA2a gene therapy for chronic heart failure. DESIGN: Randomized controlled study.SETTING: Department of Senile Angiocardiopathy, General Hospital of Chinese PLA; Department of Biochemistry, Beijing Medical University. MATERIALS: Male Sprague-Dawley (SD) rats with 4 weeks old, clean grade and weighing 45-50 g provided by the Animal Experimental Center, Peking Medical University were used as donators of bone marrow. Other female SD rats of 12 weeks old, clean grade and weighing 200-250 g were used as receptors of cell transplantation and gene therapy. Sry gene of Y chromosome in male rats was used to evaluate whether transplanted cells of donators lived in myocardium of receptor rats. Ad-SERCa2a and Ad-EGFP were constructed by Doctor Lu Xiao-chun; MSC in the 3rd and 8th generations was isolating cultured on its own. METHODS: The experiment was carried out in the Zhou CY Laboratory (BSL-2), Department of Biochemistry, Beijing Medical University from July 2004 to December 2005. Thirty female SD rats received ligation at the left coronary artery to make models with chronic cardiac failure following acute myocardial infarction. And then, 29 rats were randomly divided into four groups, including gene therapy group (n=7), MSC group (n=7), gene-modified MSC group (n=8) and control group (n=7). Rats in the four groups were given the interventions of SERCA2a gene, MSC transplantation, MSC+Ad/SERCa2a and empty adenoviral vector, respectively. MSCs were separated and cultured, and then Ad-SERCA2a-GFP was used to transfer MSC in the 3rd and 8th generations.MAIN OUTCOME MEASURES: Ad-SERCA2a-GFP transfection rate of MSC was measured by using flow cytometer. Before and at 14 and 21 days after treatment, cardiac function was evaluated by ultrasonic echocardiogram. Expression of cytokine Ⅷ was tested by immunohistochemical staining. SERCA2a gene and protein expression were evaluated by RT-PCR and Western blot respectively, as well as SERCA2a enzyme activity. RESULTS: ① Transfection rate: The infection efficiency of adenovirus-medicated gene into different passages of MSC was over 80%, and there was no difference between passage three (P3) MSC and P8 MSC (P > 0.05). ② Heart function: Left ventricle wall was thickened obviously in group MSC and group MSC+Ad/SERCa2a on the 21st day after treatment, while volume was shortened and gradually rounded. Compared to control group, ejection fraction (EF) and shortening fraction (FS) of group Ad-SERCa2a, group MSC and group MSC+Ad/SERCa2a were elevated significantly on the 14th day after therapy (P < 0.01). While the elevation values of EF and FS began to reduce in group Ad-SERCa2a on 14th day after therapy, it continued to increase in both group MSC and group MSC+Ad/SERCa2a (P < 0.01). Improvement rate of EF at 21 days after therapy (EF D21) increased in group MSC and group MSC+Ad/SERCa2a respectively, but decreased in group Ad-SERCa2a. Compared to group Ad-SERCa2a, peak systolic flow velocity of anterior wall and interventricular septum in group MSC+Ad/SERCa2a increased significantly on the 21st day after therapy, and peak diastolic flow velocity of anterior wall and interventricular septum elevated in group MSC+Ad/SERCa2a, too (P < 0.01). ③ SERCA2a gene, protein expression and enzyme activity in group MSC+Ad/SERCa2a were significantly stronger in group MSC and control group. Parts of MSC transplanted into scar zone expressed Ⅷ.CONCLUSION: ① MSC is an effective platform for the targeted delivery of therapeutic gene. It suggests that different passages of MSC from P3 MSC to P8 MSC are regarded as high-effectively gene vehicles. MSC-based SERCA2a gene therapy showed much strong and lasting beneficial effect on exhausted myocardium. ② Effect of MSC transplantation on improving heart function may be related to promoting vascular neogenesis.
