Effects of brain homogenate on the differentiation of rat bone mesenchymal stem cells into neuron-like cells following traumatic brain injury

Abstract

AIM：The bone mesenchymal stem cells can transform into neuron-like cells in vitro induced by brain homogenate. There are growth factors secreted from brain tissue extracts and bone mesenchymal stem cells in the cultural liquids, which can induce the bone mesenchymal stem cells into neuron-like cells. In this study, the different effects between traumatic brain tissue extracts and normal brain tissue extracts on the differentiation of rat bone mesenchymal stem cells were observed.

METHODS： Experiments were conducted in the Cell Laboratory of Department of Anatomy of Hebei Medical University from March to August in 2007. ①One healthy clean SD rat weighting 100-150 g aged 4-6 weeks was provided by Animal Experimental Center of Hebei Medical University. Experimental procedures were accorded with the Animal Ethical Standards. ②One SD rats were anaesthetized and tibias and femurs were dissected out, and then bone marrow was flushed out with L-DMEM. After it was centrifuged, the supernatant was discarded. The extracted cells were resuspended and seeded in L-DMEM supplemented with fetal bovine serum of 0.10 volume fraction. The bone mesenchymal stem cell morphologies were observed under an inverted phase microscope. Rat models of moderate brain injury were established by Modified Gruncr Method. The traumatic brain tissue extracts acquired on the point about 24 hours after the injury and normal brain tissue extracts were used to induce 3rd passage of bone mesenchymal stem cells in vitro. ③The morphological changes of the stem cells were observed with the inverted phase microscope. The expression of neuron-specific enolase was identified by immunocytochemical technique. The different effects between traumatic brain tissue extracts and normal brain tissue extracts on the differentiation of bone mesenchymal stem cells were observed.

RESULTS: After 24-hours induction with traumatic brain tissue extracts, the cellular bodies changed large, 36 hours later, part of the bone mesenchymal stem cells body contracted into round or spindle shape. 48 hours later, neuron-like cells with two or more prominence could be seen. The immunocytochemical method showed that the ratio of neuron-specific enolase expressing was (54.28±6.03)%. The differential ratio of bone mesenchymal stem cells induced with normal brain tissue extracts was lower, and ratio of neuron-specific enolase expressing was (32.76±3.25)%.

CONCLUSION:Bone mesenchymal stem cells can be induced differentiating into neuron-like cells. The induction proportion induced with traumatic brain tissue extracts is higher than with normal brain tissue extracts.

Song YZ, Cui HX, Lü Z, Wang XS, Wang ZX, Shi ZL.Effects of brain homogenate on the differentiation of rat bone mesenchymal stem cells into neuron-like cells following traumatic brain injury.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(3):461-464(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-3/3k-461(ps).pdf]

摘要
目的：骨髓间充质干细胞在脑组织匀浆诱导环境下可以转化为神经元样细胞，损伤脑组织匀浆的骨髓间充质干细胞培养液中，不仅有脑组织中提取的生长因子，而且有骨髓间充质干细胞分泌的多种生长因子，共同刺激骨髓间充质干细胞向神经元样细胞的分化。实验观察了创伤后24 h和正常脑组织匀浆诱导大鼠骨髓间充质干细胞向神经元样细胞分化的差别。
方法：实验于2007-03/08在河北医科大学解剖教研室细胞培养中心完成。①实验材料：体质量100~150 g的健康SD大鼠由河北医科大学实验动物中心提供，4~6周龄,清洁级。实验过程中对动物处置符合动物伦理学标准。②实验方法：取1只 SD大鼠，麻醉后分离股骨和胫骨，用培养基冲洗骨髓腔，离心弃上清液，加入含体积分数为0.10胎牛血清的L-DMEM培养基重悬，接种于培养瓶培养并传代，于倒置显微镜下观察细胞形态。采用Gruncr改良法制作中度脑损伤大鼠模型，取伤后 24 h和正常大鼠脑组织匀浆，对体外培养的第3代骨髓间充质干细胞进行诱导。③实验评估：在倒置显微镜下观察细胞形态学变化，并应用免疫细胞化学技术检测细胞内神经元特异性烯醇化酶的表达，比较创伤后和正常脑组织匀浆两组诱导率差别。
结果：骨髓间充质干细胞经创伤性脑组织匀浆培养基诱导24 h后，细胞的胞体变大，36 h后部分细胞分化，回缩成圆形或梭形，48 h后部分细胞可见两个或多个突起伸出，类似神经元。免疫细胞化学技术检测显示，创伤性脑组织匀浆培养基诱导组神经元特异性烯醇化酶阳性表达为（54.28±6.03）%，正常脑组织匀浆诱导分化率较前者低，神经元特异性烯醇化酶阳性表达为（32.76±3.25）%，细胞生长状态略差。
结论：脑组织匀浆可诱导大鼠骨髓间充质干细胞向神经元样细胞分化，创伤性脑组织匀浆可明显促进其分化。
关键词：脑组织匀浆；骨髓间充质干细胞；神经元样细胞

宋永周，崔慧先，吕哲，王新生，王振显，史正亮.创伤性脑组织匀浆对大鼠骨髓间充质干细胞分化为神经元样细胞的影响[J].中国组织工程研究与临床康复，2008，12(3):461-464
[www.zglckf.com/zglckf/ejournal/upfiles/08-3/3k-461(ps).pdf]