叶酸诱导骨髓间充质干细胞的体内外实验研究

发布时间：2018-05-07 23:29

  本文选题：叶酸 + 骨髓间充质干细胞　； 参考：《山西医科大学》2010年硕士论文

【摘要】： 目的：本实验旨在研究叶酸对骨髓基质干细胞(Human Bone Marrow Stromal Cells, HBMSCs)诱导分化为神经细胞的影响及其移植治疗大鼠脑缺血模型神经细胞的增值分化作用,探讨HBMSCs的移植机制,主要包括以下两个方面：①研究叶酸对HBMSCs向神经细胞诱导的影响；探讨HBMSCs在传代培养和向神经细胞分化过程中的分泌作用及其机制。②观察叶酸诱导后HBMSCs经尾静脉注射治疗大鼠脑缺血后七天原位神经前体细胞的增殖、分化情况,评价HBMSCs治疗价值并探讨其移植入大鼠体内作用机制。 方法：HBMSCs由正常成年人骨髓经密度梯度离心加贴壁离心获得,取第5代HBMSCs以1mM二巯基乙醇(BME)预诱导24h后,用羟基茴香醚(BHA)、2%二甲基亚砜(DMSO)、和3种不同浓度的叶酸(4mg/L、40 mg/L、400mg/L)诱导分化,采用免疫细胞化学法检测神经细胞特异性抗原标志神经元特异性烯醇化酶NSE、胶原纤维酸性蛋白GFAP的表达,MTT(四唑盐比色)法检测不同时间段对HBMSCs增殖的影响。选取16只健康Wistar大鼠分为假手术组、缺血对照组、低浓度HBMSCs组、高浓度HBMSCs组,后三组采用接扎双侧颈总动脉法建立大鼠大脑中动脉缺血再灌注模型(MCAO),模型制作24h后将叶酸诱导后不同浓度HBMSCs经尾静脉注射入大鼠体内。各组腹腔注射5-溴脱氧尿嘧啶(bromodeoxyuridine, BrdU)用以标记处于增殖状态的神经前体细胞,应用免疫组织化学染色检测缺血后7d脑组织BrdU、BrdU+NSE、BrdU+GFAP阳性细胞数,结果进行统计学分析。 结果：叶酸诱导后3h细胞胞体成圆形,折光性强,多级细长的突起,随着时间的延长突起变的更多更长,并相互连接交织成网。诱导后8h细胞免疫化学显示,对照组约70%细胞呈阳性染色,GFAP阳性为主,叶酸低剂量组阳性细胞数与对照组相近,叶酸中高剂量组阳性细胞数占90%,以NSE为主,统计学显示,对照组,叶酸低剂量组与叶酸中高剂量组比较差异有显著性(P0.01)。使用40 mg／L叶酸诱导HBMSCs后干预治疗大鼠脑缺血,脑梗死后7d侧脑室室管膜下区、海马齿状回区BrdU、BrdU+NSE、BrdU+GFAP阳性细胞数开始增多,且高浓度组的上述阳性细胞数明显多于对照组(P0.05)和低浓度组(P0.05)。 结论：①不同剂量的叶酸均能促进HBMSCs向神经细胞增值分化,以40 mg／L中等剂量的叶酸诱导HBMSCs向神经细胞增值分化最为理想。②叶酸诱导HBMSCs干预治疗后,可以促进脑梗死大鼠损伤原位内源性神经前体细胞的增殖、分化,具有潜在的治疗价值。
[Abstract]:Objective: to study the effect of folic acid on the differentiation of bone marrow stromal cells (BMSCs) into neural cells induced by human Bone Marrow Stromal Cells, HBMSCs) and the effect of transplantation on the proliferation and differentiation of neural cells in rat model of cerebral ischemia, and to explore the mechanism of HBMSCs transplantation. The effects of folic acid on the induction of HBMSCs into nerve cells were studied in the following two aspects: 1. To investigate the secretory role of HBMSCs in the process of passage culture and differentiation into nerve cells and its mechanism .2 to observe the proliferation and differentiation of in situ neural precursor cells in rats treated with folic acid-induced HBMSCs via caudal vein for 7 days after cerebral ischemia. To evaluate the therapeutic value of HBMSCs and to explore the mechanism of its transplantation into rats. Methods BMSCs were obtained from normal adult bone marrow by density gradient centrifugation and adherent centrifugation. The fifth passage of HBMSCs was preinduced by 1mM dimercaptoethanol for 24 hours, then induced by 2% dimethyl sulfoxide DMSOL and 3 different concentrations of folate 4 mg / L 40 mg / L 4 mg 路L ~ (-1) 路L ~ (400) mg 路L ~ (-1). The immunocytochemistry method was used to detect the expression of neuron-specific enolase (NSEs) and the expression of collagen fibrillary acidic protein (GFAP) to detect the effect on the proliferation of HBMSCs in different time periods. Sixteen healthy Wistar rats were divided into sham operation group, ischemic control group, low concentration HBMSCs group and high concentration HBMSCs group. The model of middle cerebral artery ischemia-reperfusion was established by ligating bilateral common carotid artery in the latter three groups. 24 hours after folic acid was induced, different concentrations of HBMSCs were injected into rat caudal vein. 5-bromodeoxyuridine (BrdU) was injected intraperitoneally in each group to label the proliferating neural precursor cells. The number of BrdU BrdU NSE-BrdU GFAP positive cells in brain tissue 7 days after ischemia was detected by immunohistochemical staining. The results were statistically analyzed. Results: three hours after folic acid induction, the cell bodies became round, the refraction was strong, the multistage slender protrusions became more and longer with the prolongation of time, and the cells interlinked with each other to form a network. At 8 h after induction, 70% of the cells in the control group showed positive staining for GFAP, and the number of positive cells in the low dose group was similar to that in the control group. The number of positive cells in the middle and high dose group of folic acid accounted for 90%, mainly in NSE, and the statistics showed that the number of positive cells in the low dose group was similar to that in the control group. In the control group, there was a significant difference between the low dose folic acid group and the middle and high dose group (P 0.01). After 40 mg/L folic acid induced HBMSCs, the number of BrdU NSEN BrdU GFAP positive cells in the subependymal and hippocampal dentate gyrus began to increase 7 days after cerebral infarction in rats with cerebral ischemia. The number of positive cells in high concentration group was significantly higher than that in control group (P 0.05) and low concentration group (P 0.05). Conclusion different doses of folic acid can promote the proliferation and differentiation of HBMSCs into neural cells, and 40 mg/L folic acid at medium dose can induce the differentiation of HBMSCs into neuronal cells. The most ideal treatment is that folic acid can induce the differentiation of HBMSCs into neuronal cells after the intervention of HBMSCs. 2 folic acid. It can promote the proliferation and differentiation of in situ endogenous neural precursor cells in rats with cerebral infarction, and has potential therapeutic value.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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