骨髓间充质干细胞重复静脉移植治疗脑梗死大鼠的实验研究

发布时间：2018-07-01 17:13

本文选题：骨髓间充质干细胞 + 重复移植　；参考：《华北理工大学》2015年硕士论文

【摘要】：目的本研究通过动物实验初步探索骨髓间质干细胞重复移植治疗脑梗死是否可改善其神经功能预后,减少实验动物脑梗死面积,是更为有效的细胞疗法,以及可能作用机制,为临床应用细胞疗法治疗脑梗死提供理论依据。方法1取幼龄SD大鼠长骨骨髓进行培养,利用全骨髓贴壁筛选法培养、分离、纯化BMSCs。取第3或4代细胞进行移植,移植前24h以终浓度10umol/L的Brd U标记。2用改良的Longa线栓法建立大鼠脑梗死模型,根据m NSS选出60只大鼠模型,随机分为对照组,单次组,重复组,每组20只,在建模后24h,对照组注射1ml生理盐水,单次组和重复组注射3×106个BMSCs,建模后7d,对照组和单次组注射1ml生理盐水,重复组注射3×106个BMSCs,均通过尾静脉注射。3建模后7、14d,运用m NSS对大鼠进行神经功能评定,然后每组各取5只大鼠进行TTC染色比较各组相对梗死体积。4建模后14d处死大鼠,进行免疫组织化学染色观察Brdu标记率,单染定性观察VEGF、Olig-2、Bcl-2、Caspase-3蛋白情况,观察新生血管内皮、少突胶质细胞增生情况、细胞凋亡。5 Western blot检测Bcl-2、Caspase-3的蛋白含量。结果1 mNSS结果:建模后3d,单次组及重复组比对照组mNSS低,差异有统计学意义(P0.05),重复组与单次组m NSS低,差异有统计学意义(P0.05);建模后7d,单次组和重复组比对照组m NSS低,差异有统计学意义(P0.01);建模后14d,单次组、重复组m NSS均显著低于对照组,差异有统计学意义(P0.01),重复组比单次组m NSS低,差异有统计学意义(P0.05)。2建模后7d,单次组及重复组相对梗死体积小于对照组,差异有统计学意义(P0.05),单次组与重复组之间无差异;建模后14d,单次组与对照组相对梗死体积比较,差异有统计学意义(P0.05),重复组与对照组比较,差异有统计学意义(P0.01),重复组与单次组比较,差异有统计学意义(P0.05)。3免疫组织化学染色结果,对照组脑VEGF表达水平低于单次组及重复组,差异有统计学意义(P0.05),单次组与重复组比较有统计学意义(P0.05);对照组脑Olig-2表达低于单次组及重复组,单次组与对照组比较(P0.01),单次组与重复组比较(P0.01),差异有统计学意义;对照组脑Bcl-2表达水平低于单次组及重复组,差异有统计学意义(P0.05),单次组与重复组比较有统计学意义(P0.05);对照组Caspase-3蛋白表达高于单次组及重复组,差异有统计学意义(P0.05)。4 Western blot结果提示:对照组脑组织Bcl-2蛋白表达低于单次组及重复组,差异有统计学意义(P0.01),单次组与重复组比较有统计学意义(P0.05);对照组脑组织Caspase-3蛋白表达高于单次组及重复组,差异有统计学意义(P0.01),单次组与重复组比较有统计学意义(P0.05),提示BMSCs可能通过促进血管内细胞增生、刺激少突胶质细胞再生、减少凋亡治疗脑梗死。结论1全骨髓贴壁法培养BMSCs,可迅速增殖、分化、纯化。2同种异体BMSCs对大鼠脑梗死有治疗作用,且重复移植治疗效果优于单次移植。3 BMSCs远期治疗效果较好。4线栓法建立大鼠永久性脑梗死模型操作相对简单,可重复,损伤较小。5 Brd U标记BMSCs,操作简单,标记率高,对细胞形态、生长等无影响。6尾静脉移植BMSCs,可迁移至脑缺血区域,且未发现不良反应。7 BMSCs可能通过刺激VEGF蛋白分泌,促进血管再生,改善脑组织血液循环,增加Olig-2蛋白分泌,刺激少突胶质细胞增生,提高轴突传导能力,并上调Bcl-2蛋白,下降Caspase-3蛋白抑制细胞凋亡,减少梗死体积等机制治疗脑梗死。
[Abstract]:Objective to explore the effect of repeated transplantation of bone marrow mesenchymal stem cells in the treatment of cerebral infarction to improve the prognosis of cerebral infarction and reduce the area of cerebral infarction in experimental animals. It is a more effective cell therapy and possible mechanism to provide a theoretical basis for the clinical application of cell therapy in the treatment of cerebral infarction. Method 1 the young age SD was taken. The bone marrow of the long bone of the rat was cultured. The third or 4 generation cells were isolated and purified by the full bone marrow adherence screening method. The BMSCs. Brd U marker of the final concentration of 10umol/L was used to establish the rat model of cerebral infarction with the modified Longa thread thrombus method before transplantation. 60 rat models were selected according to m NSS, and were randomly divided into the control group, the single group and the repeat group. 20 rats in each group were established after modeling 24h, the control group was injected with 1ml physiological saline, the single group and the repeated group were injected with 3 x 106 BMSCs. After modeling 7d, the control group and the single group were injected with 1ml physiological saline, and the repeated group was injected with 3 x 106 BMSCs. The.3 modeling 7,14d was injected through the tail vein, and the rats were evaluated with m NSS, then 5 rats in each group were given 5 rats. The rats were stained with TTC to compare the relative infarct volume in each group, and the rats were killed after the.4 modeling. The Brdu labeling rate was observed by immunohistochemical staining. The VEGF, Olig-2, Bcl-2, Caspase-3 protein were observed by single staining, and the proliferation of oligodendrocytes was observed, and the apoptotic.5 Western blot detected Bcl-2, and the protein contained the protein content. Results 1 mNSS results: after modeling, 3D, single group and repeated group were lower than the control group mNSS, the difference was statistically significant (P0.05). The difference was statistically significant (P0.05) in the repeated group and the single group M NSS; the model 7d, the single group and the repeat group were lower than the control group M NSS, and the difference was statistically significant (P0.01); after modeling, the 14d, single group, and repeated groups were all obvious. The difference was significantly lower than that of the control group (P0.01), the repeat group was lower than the single group M NSS, the difference was statistically significant (P0.05).2 modeling 7d, the relative infarct volume in the single and repeated groups was less than the control group, the difference was statistically significant (P0.05), there was no difference between the single group and the repeated group; after modeling 14d, the single group and the control group were relative infarct bodies. The difference was statistically significant (P0.05). The difference was statistically significant (P0.01) compared with the control group (P0.01). The difference was statistically significant (P0.05) with statistical significance (P0.05), and the level of VEGF expression in the control group was lower than that of the single group and the repeated group, and the difference was statistically significant (P0.05). The comparison group had statistical significance (P0.05); the brain Olig-2 expression in the control group was lower than the single group and the repeated group, the single group was compared with the control group (P0.01), the single group and the repeated group were compared (P0.01), the difference was statistically significant; the level of Bcl-2 expression in the control group was lower than that of the single group and the repeated group (P0.05), the single group and the repeat group ratio were statistically significant (P0.05). The expression of Caspase-3 protein in the control group was higher than that of the single group and the repeated group, the difference was statistically significant (P0.05).4 Western blot results suggested that the expression of Bcl-2 protein in the brain tissue of the control group was lower than that of the single group and the repeat group, the difference was statistically significant (P0.01), and there was a statistical significance (P0.05) in the single group and the repeat group (P0.05); the difference was statistically significant (P0.05) in the control group (P0.05); and the difference was statistically significant (P0.05) in the single group and the repeat group (P0.05); The expression of Caspase-3 protein in the brain tissue was higher than that of the single group and repeated group. The difference was statistically significant (P0.01). There was a significant difference between the single group and the repeated group (P0.05). It suggested that BMSCs could stimulate the proliferation of the cells in the blood vessels, stimulate the regeneration of oligodendrocytes and reduce the apoptosis in the treatment of cerebral infarction. Conclusion 1 full bone marrow adherent method can be used to cultivate BMSCs. Rapid proliferation, differentiation, and purification of.2 allogeneic BMSCs have therapeutic effect on cerebral infarction in rats, and the effect of repeated transplantation is better than that of single transplant.3 BMSCs. It is relatively simple to operate the model of permanent cerebral infarction by.4 line thrombus method, and can be repeated, and the damage is smaller than.5 Brd U, the operation is simple, the labeling rate is high, and the cell mark rate is high, and the cell mark rate is high, and the cell mark rate is high. Morphology, growth and so on without influence of.6 tail vein graft BMSCs, can migrate to cerebral ischemia area, and no adverse reaction.7 BMSCs can stimulate the secretion of VEGF protein, promote vascular regeneration, improve blood circulation of brain tissue, increase the secretion of Olig-2 protein, stimulate oligodendrocyte proliferation, improve the axon conduction ability, and up regulation of Bcl-2 protein and decrease C Aspase-3 protein inhibits apoptosis and reduces infarct volume to treat cerebral infarction.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R743.33

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