Therapeutic effect of erythropoietin gene-modified bone marrow mesenchymal stem cell transplantation on rat cerebral infarction

doi:10.3969/j.issn.2095-4344.2014.23.010

Abstract

Abstract:

BACKGROUND: Previous studies have shown that erythropoietin can protect neurons and promote nerve regeneration.

OBJECTIVE: To explore the therapeutic effect of erythropoietin gene-modified bone marrow mesenchymal stem cell transplantation via caudal vein on rat cerebral infarction.

METHODS: Western blot assay was used to identify the expression of exogenous erythropoietin in bone marrow mesenchymal stem cells. A model of middle cerebral artery occlusion was established in Wistar rats using thread method. And then, model rats were randomly divided into model group (PBS injection via the caudal vein), transplantation group (transplantation of bone marrow mesenchymal stem cell suspension), erythropoietin group (transplantation of erythropoietin-transfected bone marrow mesenchymal stem cell suspension). Neurologic function was assessed at 3 days, 1, 2, 3, 4 weeks after cell transplantation. Four weeks after transplantation, the rats were decapitated after anesthesia to take brain tissues for RT-PCR detection of Bcl-2/Bax gene expression. Cell apoptosis was measured by TUNEL. Hematoxylin-eosin staining and fluorescence microscopy were employed to observe the survival and distribution of PKH26-labeled bone marrow mesenchymal stem cells.

RESULTS AND CONCLUSION: Western blot results showed that erythropoietin-transfected bone marrow mesenchymal stem cells could express the erythropoietin in vitro. At 1, 2, 3, 4 weeks after transplantation, the neurological defect scores in the transplantation group and erythropoietin group were significantly lower than those in the model group (P < 0.05, P < 0.01). The expression of bcl-2 gene in the infarct region was significantly higher in the erythropoietin group than the transplantation and model groups (P < 0.05), but the expression of bax was significantly decreased (P < 0.05). In the erythropoietin group, the number of apoptotic cells was reduced, and the number of PKH26 positive cells was increased as compared with the other two groups (P < 0.05). These findings indicate that the transplantation of erythropoietin-modified bone marrow mesenchymal stem cells via caudal vein can significantly improve the neurological function in the rats with cerebral infarction.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: stem cells, transplantation, bone marrow, mesenchymal stem cells

CLC Number:

R394.2

Li Jin-yan, Wang Liang. Therapeutic effect of erythropoietin gene-modified bone marrow mesenchymal stem cell transplantation on rat cerebral infarction[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(23): 3664-3669.

Figures/Tables 2

2.1 实验动物数量分析纳入大鼠100只，无死亡和感染，均进入结果分析。 2.2 大鼠骨髓间充质干细胞形态培养第5天时培养瓶底骨髓间充质干细胞集落数目明显增多，传3代的骨髓间充质干细胞增殖活跃。见图1A。流式细胞仪检测结果显示CD29、CD44、CD105等表达阳性，CD34、CD80、CD86阴性，骨髓间充质干细胞均一性好，纯度达96%以上。PKH-26标记的骨髓间充质干细胞在荧光显微镜下呈红色荧光，见图1B。Western blot鉴定结果显示，在转染了pcDNA3-促红细胞生成素质粒的骨髓间充质干细胞中可检测到促红细胞生成素蛋白的表达，见图1C，其蛋白条带位置(相对分子质量：34 000)与阳性对照即人胎肝细胞LO2一致，而未经转染的骨髓间充质干细胞中无促红细胞生成素蛋白的表达。 2.3 大鼠神经功能变化移植后第3天，促红细胞生成素-骨髓间充质干细胞组与骨髓间充质干细胞组、模型组比较差异无显著性意义；在移植后2-4周开始，与模型组和骨髓间充质干细胞组比较，促红细胞生成素-骨髓间充质干细胞组大鼠神经学缺损评分均降低(P < 0.01，P < 0.05)，骨髓间充质干细胞组神经学缺损评分低于模型组(P < 0.05)，见表1。 2.4 大鼠脑梗死区组织bcl-2和bax基因的表达将PCR产物条带进行扫描统计分析显示bcl-2和bax mRNA在模型组中相对表达量分别为0.348±0.060，0.962±0.102；在骨髓间充质干细胞组中相对表达量分别为0.692±0.093，0.586±0.132；在促红细胞生成素-骨髓间充质干细胞组中相对表达量分别为1.135±0.146，0.273±0.028。与骨髓间充质干细胞组及模型组相比，促红细胞生成素-骨髓间充质干细胞组bcl-2的表达明显增高(P < 0.05)，bax的表达明显降低(P < 0.05)，见图2。 2.5 大鼠脑梗死区组织细胞凋亡情况细胞核内存在棕黄色颗粒者为凋亡细胞。光镜下细胞散布于整个缺血区内，病灶边缘可见成堆的阳性细胞，有染色质边集及核固缩现象，某些区域可见凋亡小体。TUNEL法测定，促红细胞生成素-骨髓间充质干细胞组、骨髓间充质干细胞组和模型组凋亡细胞数(400倍视野下)分别为(16.46±1.67)，(25.4± 1.63)，(36.67±2.69)个，促红细胞生成素-骨髓间充质干细胞组棕黄色颗粒的凋亡细胞明显少于骨髓间充质干细胞组及模型组(P < 0.05)。见图3。 2.6 大鼠脑梗死区组织形态及的PKH26的表达苏木精-伊红染色显示，移植4周后，模型组可见梗死处脑组织被胶质细胞和胶质纤维充填，形成胶质瘢痕，局部脑萎缩，软化灶形成(图4A)。骨髓间充质干细胞组见在移植部位出现典型的神经细胞样形态学改变，组织瘢痕和软化灶小于模型组，而大于促红细胞生成素-骨髓间充质干细胞组(图4B)。促红细胞生成素-骨髓间充质干细胞组出现典型的神经细胞样形态学改变且软化灶消失，有少量瘢痕(图4C)。荧光显微镜观察结果显示大鼠损伤灶脑组织中的PKH26 阳性细胞数：模型组(图4D)为(0±0.00)个/高倍视野，骨髓间充质干细胞组(图4E)为(23.28±3.52)个/高倍视野，促红细胞生成素-骨髓间充质干细胞组(图4F)为(31.47±3.46)个/高倍视野，各组之间均差异有显著性意义(P < 0.05)。 "

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