Bone marrow mesenchymal stem cell transplantation inhibits apoptosis in the rat spinal cord injured by acrylamide

doi:10.3969/j.issn.2095-4344.0435

Abstract

Abstract:

BACKGROUND: Until now, there is no effective treatment for peripheral neuropathy caused by acrylamide. Therefore, it is necessary to explore new treatment methods.
OBJECTIVE: To explore the protection role and its mechanism of bone marrow mesenchymal stem cells (BMSCs) against acrylamide-induced intoxication in the spinal cords of rats.
METHODS: BMSCs were cultured by the whole bone marrow adherence method and identified by morphological observation and flow cytometry detection. Thirty Sprague-Dawley rats, clean grade, were randomly divided into three groups (n=10 for each group): normal control group, acrylamide group and BMSCs transplantation group. The latter two groups received acrylamide by gavage, 50 mg/(kg•d), 5 days per week, for 2 weeks with an interval of 2 days. Then, in the BMSCs transplantation group, 3×10⁶ BMSCs were transplanted by the caudal vein, 5 days per week, for 3 consecutive weeks. Hematoxylin-eosin staining was utilized to observe the morphological changes of the spinal cord. Tunel assay was used to detect cell apoptosis. Western blot assay was adopted to detect the expression levels of Bcl-2 and Bax.
RESULTS AND CONCLUSION: In the acrylamide-exposed rats, the damage to the structure was found in the spinal cords by morphological observation, which was significantly alleviated after BMSCs transplantation. The disturbed expression levels of Bax and Bcl-2 were also significantly inversed after BMSCs transplantation (P < 0.05). These results suggest that BMSCs transplantation can inhibit cell apoptosis in the spinal cords of acrylamide-intoxicated rats, probably by up-regulating expression of Bcl-2 and down-regulating expression of Bax.

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

Key words: Bone Marrow, Mesenchymal Stem Cell Transplantation, Acrylamide, Spinal Cord, Apoptosis

CLC Number:

R394.2

Sun Jing-song, Zhou Xue-ying, Qu Shu-xian, Bu Tong-jing, Li Shuang-yue. Bone marrow mesenchymal stem cell transplantation inhibits apoptosis in the rat spinal cord injured by acrylamide[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(5): 680-685.

Figures/Tables 1

2.1 骨髓间充质干细胞的形态原代培养细胞呈圆形，大小不一，折光性强，大多数漂浮在培养液中。培养6-8 h后细胞开始逐渐贴壁，换新的培养液除去未贴壁的细胞，在培养24 h时可见细胞形态不一，呈多角形改变，见图1A。3 d后细胞集落呈放射状排列，伸出长短不等、粗细不一的突起。胞浆丰富，核仁清晰可见，见图1B。在10-12 d细胞之间融合成片，细胞相互贴附生长，细胞排列呈螺旋状或漩涡状，见图1C。细胞生长旺盛，四至五天可传代1次，细胞形态和生长速度均未发现有明显异常，大多数呈不规则的三角形或者四边形，见图1D。 2.2 骨髓间充质干细胞表面标志物表达通过流式细胞仪检测骨髓间充质干细胞表面标记物的表达，结果显示，第3代骨髓间充质干细胞的CD44、CD90阳性率均大于90%，而CD45的阳性率不足5%，见图2。 2.3 骨髓间充质干细胞分化能力骨髓间充质干细胞经成骨诱导21 d后，茜素红染色显示，实验组细胞呈现广泛的橙红色，有明显的矿化结节形成，见图3A。骨髓间充质干细胞经成脂诱导18 d后，油红O染色显示，实验组出现较多强阳性成脂细胞，其特征为细胞体积较大、呈圆形、内含鲜红色串珠样或大的融合脂滴，见图3B。 2.4 实验动物数量分析 30只SD健康大鼠，在模型制备过程中无死亡。每组3只大鼠脊髓组织用于苏木精-伊红染色，每组2只大鼠脊髓组织用于Tunel染色；每组5只大鼠脊髓组织用于Western-Blotting检测。 2.5 脊髓组织苏木精-伊红染色结果正常对照组镜下可见大量的神经元细胞且形态正常，尼氏体清晰，细胞膜完整，细胞质染色均一，细胞核可见，见图4A。丙烯酰胺中毒组镜下可见神经元细胞数量较少，尼氏体消失，细胞膜破坏，细胞核固缩、消失，见图4B。骨髓间充质干细胞移植组镜下可见大量新生的神经元细胞，尼氏体清晰，细胞膜结构完整，细胞核清晰可见，见图4C。 2.6 Tunel法检测细胞凋亡情况如图5所示，对照组几乎未观察到TUNEL阳性细胞，丙烯酰胺中毒组大鼠脊髓TUNEL阳性细胞显著增多，而骨髓间充质干细胞移植后脊髓TUNEL阳性细胞明显减少。 2.7 Western-Blotting检测脊髓组织Bcl-2和Bax表达水平如图6所示，与对照组相比，丙烯酰胺中毒组大鼠脊髓组织Bcl-2的表达水平明显下降、Bax的表达水平明显升高，差异有显著性意义(P < 0.05)。骨髓间充质干细胞移植后，脊髓组织Bcl-2的表达水平升高、Bax的表达水平降低，与丙烯酰胺中毒组相比，差异有显著性意义(P < 0.05)。与对照组相比，骨髓间充质干细胞移植组脊髓组织Bcl-2表达几乎恢复至正常水平，而Bax表达略高于正常水平。"

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