亚低温上调蛋白SUMO化修饰促进BMSCs在缺氧环境中存活

发布时间：2018-08-26 07:56

【摘要】：目的:建立体外培养骨髓间充质干细胞(BMSCs)并加以鉴定,利用相关化学试剂的诱导分化作用,诱导BMSCs向神经细胞方向分化。以BMSCs为研究对象,实验验证亚低温通过上调BMSCs中SUMO化修饰水平增加细胞本身对缺血缺氧的耐受性。制备动物脑梗死模型,给予亚低温联合缺血半暗带BMSCs移植,验证亚低温通过上调细胞内SUMO化修水平饰改善脑梗死模型大鼠的神经功能。本研究旨在实验验证亚低温通过上调SUMO化修饰发挥细胞保护作用,旨在为探索亚低温联合干细胞移植治疗脑梗死打下理论和实验基础。方法:选择改良后的原代细胞贴壁法对提取的SD大鼠BMSCs进行培养,然后再用流式细胞术检测鉴定细胞表面标记蛋白,利用化学试剂法体外诱导其向神经细胞分化;在33℃和37℃两种温度条件下培养BMSCs,之后运用Western blotting方法对在不同的温度培养下BMSCs内的SUMO1和SUMO 2/3蛋白表达情况进行检测,细胞免疫荧光染色法检测SUMO1和SUMO2/3蛋白在细胞内的定位变化;利用RNA干扰沉默技术(siRNA)制备UBC9敲低的BMSCs(SiUBC9BMSCs),建立野生BMSCs和SiUBC9 BMSCs的氧糖剥夺(OGD)模型并分别给予常温37℃和亚低温33℃干预,Western blotting方法检测不同细胞分组和不同温度培养条件下SUMO1和SUMO2/3蛋白表达水平变化,检测每组细胞凋亡率变化和培养基中LDH浓度变化;线栓法建立SD大鼠大脑中动脉栓塞(MCAO)模型,亚低温33℃联合BMSCs或SiUBC9 BMSCs梗死缺血半暗带区移植,观察大鼠神经功能恢复情况,盲法实施神经功能评价。结果:利用改良后的细胞贴壁法获取到体外培养大鼠BMSCs后,流式细胞术检测所培养细胞满足BMSCs相关特征,在体外化学试剂诱导下,体外培养的大鼠BMSCs能够表达神经胶质细胞标记蛋白以及神经元表面标记蛋白;Western blotting检测发现亚低温培养条件下SUMO1和SUMO2/3结合蛋白表达水平升高,细胞免疫荧光染色发现亚低温干预后SUMO1和SUMO2/3出现明显的细胞核内转移;OGD处理BMSCs后,Western blotting检测发现亚低温可明显促进SUMO1和SUMO2/3结合蛋白表达水平的升高,减少细胞凋亡率和LDH分泌,发挥出神经保护作用,这种细胞保护作用为SUMO依赖性的;成功制备大鼠脑梗死模型,亚低温条件下可增加移植到缺血半暗带BMSCs的存活率,亚低温联合BMSCs移植治疗可明显促进脑梗死后大鼠神经功能的恢复。结论:1.BMSCs在细胞条件培养基诱导下,可部分地表达神经元特异性蛋白NSE,大部分细胞表达神经胶质细胞标志蛋白GFAP。2.亚低温增加BMSCs中SUMO1和SUMO2/3与靶蛋白的结合,诱发SUMO浆核移位;SiUBC9抑制SUMO1和SUMO2/3在细胞乏氧状态下与靶蛋白的结合;亚低温可降低BMSCs在乏氧状态下的细胞凋亡比率和LDH分泌水平,发挥细胞保护作用,且这种作用为SUMO依赖性。3.亚低温联合BMSCs缺血半暗带移植能够提高移植区细胞的存活率,保护缺血半暗带区受损神经元,改善受损的神经功能,且这种作用为SUMO依赖性。4.亚低温对乏氧状态下BMSCs的保护作用与细胞内大量蛋白的SUMO化修饰密不可分,由此推测,增强靶蛋白SUMO化修饰可能是亚低温脑保护作用的分子机制之一。
[Abstract]:AIM: To establish and identify bone marrow mesenchymal stem cells (BMSCs) cultured in vitro, and to induce BMSCs to differentiate into neural cells by inducing differentiation with related chemical reagents. Cerebral infarction model was induced by mild hypothermia combined with ischemic penumbra BMSCs transplantation to verify that mild hypothermia can improve the neurological function of cerebral infarction model rats by up-regulating the level of intracellular SUMO modification. Methods: BMSCs of SD rats were cultured by modified primary cell adherence method, then identified by flow cytometry, and induced to differentiate into neurons by chemical reagent method in vitro. BMSCs were cultured at 33 C and 37 C. Western blotting was used to detect the expression of SUMO1 and SUMO2/3 proteins in BMSCs cultured at different temperatures, immunofluorescence staining was used to detect the localization of SUMO1 and SUMO2/3 proteins in cells, and RNA interference silencing (siRNA) was used to prepare UBC9 knockdown BMSCs (SiUBC9BMSCs) to establish wild BMSCs and SiUBC9BMs. The expression of SUMO1 and SUMO2/3 protein was detected by Western blotting. The apoptosis rate and LDH concentration in the medium of SD rats were detected by thread embolization method. Embolization (MCAO) model was established by mild hypothermia combined with BMSCs or SiUBC9 BMSCs transplantation in ischemic penumbra to observe the recovery of nerve function in rats and evaluate the nerve function blindly. Western blotting showed that the expression of SUMO1 and SUMO2/3 binding proteins was elevated under mild hypothermia, and the expression of SUMO1 and SUMO2/3 binding proteins was detected by immunofluorescence staining. After OGD treatment of BMSCs, Western blotting showed that mild hypothermia could significantly promote the expression of SUMO1 and SUMO2/3 binding protein, reduce the apoptosis rate and LDH secretion, and play a neuroprotective role, which was SUMO-dependent. The rat model of cerebral infarction was successfully prepared with mild hypothermia strips. BMSCs transplanted into ischemic penumbra can increase the survival rate of BMSCs, mild hypothermia combined with BMSCs transplantation can significantly promote the recovery of neural function in rats after cerebral infarction. Conclusion: 1. BMSCs can partially express neuron-specific protein NSE under the induction of cell-conditioned medium, and most cells express glial cell marker protein GFAP.2. Hypothermia increased the binding of SUMO 1 and SUMO 2/3 to target proteins in BMSCs and induced SUMO nucleus translocation; SiUBC9 inhibited the binding of SUMO 1 and SUMO 2/3 to target proteins under hypoxic conditions; and mild hypothermia decreased the apoptosis rate and LDH secretion level of BMSCs under hypoxic conditions, and this effect was SUMO-dependent.3. Temperature combined with BMSCs transplantation in ischemic penumbra can improve the survival rate of transplanted cells, protect the damaged neurons in ischemic penumbra and improve the impaired nerve function. This effect is SUMO-dependent. 4. The protective effect of mild hypothermia on BMSCs in hypoxic state is closely related to the SUMO modification of a large number of intracellular proteins, which may enhance the target. Protein SUMO modification may be one of the molecular mechanisms of mild hypothermia brain protection.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R743.3

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