朱敏，张跃，汤健，杜森杰，华裕，傅大林，陆芬，李红英，赵晓科.慢病毒介导EPhrinB2基因转染骨髓间充质干细胞对脑瘫大鼠脑损伤的保护作用[J].中华物理医学与康复杂志,2018,40(11):814-820
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| 慢病毒介导EPhrinB2基因转染骨髓间充质干细胞对脑瘫大鼠脑损伤的保护作用 |
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| DOI： |
| 中文关键词: EPhrinB2 骨髓间充质干细胞 神经元细胞 脑瘫 大鼠 |
| 英文关键词: EPhrinB2 Bone marrow Mesenchymal stem cells Neurons Cerebral palsy |
| 基金项目:国家自然科学基金(81401864) |
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| 中文摘要: |
| 目的 观察慢病毒介导EPhrinB2基因转染骨髓间充质干细胞（BMSCs）对脑瘫大鼠脑损伤的保护作用及相关机制。 方法 从大鼠中分离、培养BMSCs，以慢病毒为载体介导EPhrinB2转染BMSCs。采用随机数字表法将96只大鼠分为假手术组、溶剂对照组（简称PBS组）、空载慢病毒组（简称EGFP/BMSCs组）及EPhrinB2重组慢病毒组（简称EPhrinB2/BMSCs组）。选用改良缺氧缺血脑病（HIE）造模方法将PBS组、EGFP/BMSCs组及EPhrinB2/BMSCs组大鼠制成脑瘫模型，于术后7 d时分别将PBS溶液、BMSCs或慢病毒介导EPhrinB2基因转染BMSCs注射到PBS组、EGFP/BMSCs组、EPhrinB2/BMSCs组大鼠侧脑室内。于脑损伤28 d后采用免疫组织化学法检测各组大鼠海马组织EPhrinB2蛋白表达；采用HE染色方法观察细胞移植后大鼠海马CA1区神经元密度；采用TUNEL法检测大鼠海马神经元凋亡情况；采用免疫荧光法检测大鼠海马区Nestin及CD31表达水平。于脑损伤28 d后采用Morris水迷宫检测各组大鼠学习、记忆能力变化。 结果 脑损伤28 d后，发现EPhrinB2/BMSCs组海马组织EPhrinB2蛋白表达均显著高于假手术组、PBS 组及EGFP/BMSCs组，组间差异均具有统计学意义（P<0.05）；另外EPhrinB2/BMSCs组海马CAl区病理改变明显轻于PBS 组及EGFP/BMSCs组；EPhrinB2/BMSCs组海马区神经元凋亡率明显低于其它各组(P<0.05)。另外免疫荧光检查显示EPhrinB2/BMSCs组海马Nestin及CD31阳性率均显著高于其它各组。远期行为学测试结果显示EPhrinB2/BMSCs组Morris水迷宫实验平均潜伏期明显优于PBS 组及EGFP/BMSCs组(P<0.05)。 结论 慢病毒介导EPhrinB2转染BMSCs移植到脑瘫大鼠侧脑室海马区能高效表达EPhrinB2基因，有助于神经元细胞分化及血管新生，抑制细胞凋亡，加速受损神经功能恢复。 |
| 英文摘要: |
| Objective To investigate any protective effect of transplanting EPhrinB2-modified bone marrow mesenchymal stem cells (BMSCs) with a rat model of cerebral palsy. Methods BMSCs were isolated and cultured, then further modified by lentivirus-mediated transfection of the EPhrinB2 gene. Ninety-six Sprague-Dawley rats were randomly divided into a sham group, a solvent control group (PBS group), an empty lentivirus group (EGFP group) and an EPhrinB2 recombinant lentivirus group (EPhrinB2 group), each of 24. A model of cerebral palsy was established in the rats of the PBS, EGFP and EPhrinB2 groups using hypoxic-ischemic encephalopathy. Seven days after the operation, the lateral ventricles of the PBS, EGFP and EPhrinB2 group mice were injected with phosphate-buffered saline solution, BMSCs or EPhrinB2-modified BMSCs respectively. EPhrinB2 protein expression in the hippocampus was detected using immunohistochemistry 28 days after the operation. The neuron density in the CA1 region of the hippocampus was observed using hematoxylin and eosin staining, and any apoptosis of hippocampal neurons was detected using terminal deoxynucleotidyl transferase dUTP nick end labeling. The expression of nestin and CD31 in the hippocampus was observed using immunofluorescence assays. Morris water maze testing was also conducted to evaluate changes in learning and memory ability. Results Compared with the other 3 groups, a significant increase in the expression of protein EPhrinB2 was observed in the hippocampuses of the EPhrinB2 group rats. The pathological changes in the hippocampus among the EPhrinB2 group were significantly less severe than those in the PBS and EGFP groups. The rate of apoptosis in the hippocampuses of the EPhrinB2 group was significantly lower than that of the other groups. Immunofluorescence showed that nestin- and CD31-positive cells were significantly more numerous in the EPhrinB2 group than in the others. In the water maze the average latency of the EPhrinB2 group was significantly shorter than those of the other groups. Conclusion Lentiviral-mediated EPhrinb2 transfection of BMSCs into the hippocampus can promote EPhrinB2 gene expression, promote angiogenesis and neuron differentiation, inhibit apoptosis and accelerate the repair of injured nerves. |
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