碱性成纤维细胞生长因子基因真核表达载体转染骨髓间充质干细胞移植治疗糖尿病
Effect of basic fibroblast growth factor gene transfection on bone marrow mesenchymal stem cell transplantation for diabetes mellitus
摘要 背景:研究证实骨髓间充质干细胞可以明显提高糖尿病大鼠胰岛功能恢复及改善高血糖状态,可能与自体胰腺干细胞分化能力增强有关。<br>  目的:验证碱性成纤维细胞生长因子基因真核表达载体(PEGFP-C3-BFGF)转染骨髓间充质干细胞并移植入糖尿病大鼠体内后,对大鼠糖尿病的治疗效果。<br>  方法:通过重组腺病毒(Ad.aFGF)介导PEGFP-C3-BFGF转染入骨髓间充质干细胞,利用荧光显微镜观察PEGFP-C3-BFGF的表达。将80只SD大鼠随机分为正常对照组、糖尿病组、移植组及基因转染组,每组20只,采用链脲佐菌素(STZ)腹腔注射建立大鼠糖尿病模型,模型建立后于对照组给予门静脉注射生理盐水,糖尿病组门静脉注射PBS液,移植组门静脉注射骨髓间充质干细胞悬液1 mL,基因转染组门静脉注射等量转染PEGFP-C3-BFGF的骨髓间充质干细胞悬液。利用RT-PCR法检测各组大鼠胰岛组织中基质金属蛋白酶的mRNA表达差异;检测移植后24 h,3 d,7 d,14 d,21 d各组大鼠血糖水平;移植后3周利用ELISA法检测各组大鼠血浆胰岛素分泌水平;苏木精-伊红染色观察大鼠胰腺组织病理变化。<br>  结果与结论:①荧光显微镜观察到PEGFP-C3-BFGF转染入骨髓间充质干细胞48 h后,呈现出明显的特异性绿色荧光;②移植后2周与对照组比较,糖尿病组的基质金属蛋白酶mRNA表达明显增加(P<0.05),移植组及基因转染组基质金属蛋白酶mRNA的表达较糖尿病组明显下降(P<0.05);③移植后不同时间点各组大鼠血糖比较,基因转染组<移植组<糖尿病组,但基因转染组仍>对照组(均P<0.05);④移植后不同时间点各组大鼠血浆胰岛素分泌水平比较,基因转染组>移植组>糖尿病组,但基因转染组仍<对照组(均P<0.05);⑤各组大鼠胰腺组织病理情况比较,移植组优于糖尿病组,基因转染组又优于移植组,接近于对照组。⑥结果说明,PEGFP-C3-BFGF转染骨髓间充质干细胞移植入糖尿病大鼠体内,可以改善大鼠血糖水平,促进胰岛素的分泌,其可能是通过下调基质金属蛋白酶mRNA的表达从而改善大鼠糖尿病病变程度。
Abstract:
BACKGROUND:Existing studies have shown that bone marrow mesenchymal stem cel s can significantly improve islet function in diabetic rats to decrease excessively high blood glucose level, which may be related to the enhancement of differentiation ability of autologou pancreatic stem cel s. <br> OBJECTIVE:To observe the therapeutic efficacy of basic fibroblast growth factor gene eukaryotic expression vector (PEGFP-C3-BFGF) transfection of bone marrow mesenchymal stem cel s in diabetic rats. <br> METHODS:Recombinant adenovirus (Ad.aFGF) mediated PEGFP-C3-BFGF was transfected into bone marrow mesenchymal stem cel s, and PEGFP-C3-BFGF expression was observed using fluorescence microscopy. Eighty Sprague-Dawley rats were randomly divided into normal control group, diabetes group, transplantation group, gene transfection group, with 20 rats in each group. After modeling, rats in different groups were given portal vein injection of normal saline, PBS, 1 mL of bone marrow mesenchymal stem cel suspension, and 1 mL of PEGFP-C3-BFGF-transfected bone marrow mesenchymal stem cel suspension. RT-PCR method was used to detect mRNA expression of matrix metal oproteinases in pancreatic tissue of rats in each group. Blood glucose levels of rats were detected at 24 hours, 3, 7, 14, 21 days after transplantation. ELISA method was used to detect plasma insulin levels in rats. Pathological changes of the pancreas were observed using hematoxylin-eosin staining. <br> RESULTS AND CONCLUSION:Under the fluorescence microscope, PEGFP-C3-BFGF transfected into cel s after 48 hours showed significant specific red fluorescence. Two weeks after transplantation, matrix metal oproteinases mRNA expression was significantly increased in the diabetes group compared with the control group (P<0.05), while it was decreased in the transplantation and gene transfection groups compared with the diabetes group (P<0.05). After transplantation, the blood glucose levels in rats were ranked as fol ows:control group<gene transfection group<transplantation group<diabetes group (P<0.05), and the plasma insulin levels in rats ranked as fol ows:control group>gene transfection group>transplantation group>diabetes group (P<0.05). Pathological findings of the pancreas showed that the transplantation group was superior to the diabetes group, but inferior to the gene transfection group that was similar to the control group. Al these findings indicate that PEGFP-C3-BFGF-transfected bone marrow mesenchymal stem cel transplantation can improve blood glucose levels and stimulate insulin secretion in diabetic rats, which may improve the severity of diabetes mel itus by decreasing the mRNA expression of matrix metal oproteinases.
作者: 田雪品 刘海英
Author: Tian Xue-pin Liu Hai-ying
作者单位: 承德市中心医院内分泌科,河北省承德市,067000
刊 名 中国组织工程研究 ISTICPKU
年,卷(期): 2016, 20(36)
分类号: R394.2
机标分类号: R73 R65
在线出版日期: 2016年10月11日