蛋白质SUMO化修饰在低温与高热应激条件下的细胞保护功能研究

发布时间：2018-06-04 23:09

本文选题：小泛素样修饰蛋白 + 骨髓间充质干细胞　；参考：《天津医科大学》2017年博士论文

【摘要】：目的:研究神经元中小泛素样修饰蛋白在低温及热应激条件下的变化特点和规律,并将该规律应用于中枢神经系统疾病中,发挥其神经保护作用,达到救治与温度应激相关的中枢神经系统疾病的目的。方法:(1)(1)原代分离培养小鼠源性BMSCs,流式细胞术鉴定其细胞表面标记蛋白的表达;(2)分别将其置于37°C(正常体温)、33°C(亚低温)及18°C(深低温)培养环境中,MTT方法检测BMSCs细胞增殖活性,流式细胞术检测BMSCs细胞周期变化,细胞免疫荧光方法检测其在不同温度条件下向神经元和神经胶质细胞的分化潜能改变情况;(3)建立神经元OGD模型,细胞原位凋亡方法检测BMSCs的细胞凋亡情况,ELISA实验检测LDH含量;western blot方法检测共价结合状态及游离状态的SUMO1和SUMO2/3的蛋白表达情况;细胞免疫荧光方法检测SUMO1和SUMO2/3在BMSCs中的细胞定位变化;(4)si RNA技术沉默BMSCs中SUMO1/2/3的基因表达,利用壮观霉素B1抑制Ubc9的基因表达,并将其置于不同温度下观察其细胞形态、细胞增殖活性、向神经细胞分化以及耐受OGD环境能力的变化;western blot方法检测PCNA、Oct4、P53、HIF-1α的蛋白表达水平,分析低温诱导SUMO活化保护BMSCs耐受乏氧不利环境的可能机制。(2)(1)免疫荧光双染方法和western blot方法鉴定SUMO1/2/3基因敲除及SUMO1/2/3转基因小鼠的基因型;(2)原代分离和培养小鼠胎鼠大脑皮层神经元细胞,将其分别置于37°C(正常体温)、42°C(高热)及47°C(超高热)培养环境中,western blot方法检测SUMO1和SUMO2/3的蛋白表达水平,免疫荧光方法检测SUMO1和SUMO2/3蛋白在神经元中的细胞定位;(3)细胞原位凋亡方法检测野生型、SUMO1/2/3基因敲除型及SUMO1/2/3转基因型神经元在上述温度下的细胞凋亡情况,ELISA方法检测上清液中LDH含量;(4)制作小鼠热惊厥模型,五点量表法进行惊厥发作评分,记录小鼠惊厥百分率、潜伏时间和惊厥持续时间。结果:(1)(1)BMSCs高表达间充质干细胞特异性蛋白CD71和CD105,而不表达造血干细胞特异蛋白CD34和CD4;(2)BMSCs在低温下生长缓慢,随着温度的降低其分化为神经细胞的能力逐渐降低;BMSCs在OGD条件下可见大量细胞凋亡,伴随着高水平的乳酸脱氢酶释放,低温能够改善上述情况;(3)低温可以诱导大量的蛋白质被SUMOs修饰,表现为共价结合态SUMO1和SUMO2/3水平的明显升高,而游离SUMO1和SUMO2/3水平的下降,同时伴随着其从细胞质向细胞核的移位;(4)沉默SUMO1/2/3基因表达后BMSCs形态开始老化,细胞生长速度明显减慢,细胞周期停滞,并向神经细胞发生终末分化;壮观霉素B1能够在一定程度上抑制Ubc9的表达,进而降低共价结合状态的SUMO1和SUMO2/3的蛋白水平,BMSCs增殖速度减缓,低温能够增加BMSCs对乏氧不利环境的耐受能力;壮观霉素B1能够降低PCNA、Oct4、P53、HIF-1α的蛋白表达水平。(2)(1)免疫荧光双染方法和western blot方法鉴定SUMO1/2/3基因敲除及SUMO1/2/3转基因小鼠的基因型在转基因小鼠神经元中稳定表达,性状稳定;(2)小鼠胎鼠大脑皮层神经元细胞置于37°C、42°C及47°C培养环境中时共价及游离状态的SUMO1蛋白表达水平没有明显变化,但共价结合状态的SUMO2/3的蛋白表达水平明显升高,相应地其游离状态SUMO2/3的蛋白表达水平下降;免疫荧光结果显示SUMO2/3蛋白存在由细胞浆向细胞核的移位;上述现象在自42°C回复到37°C后能够得到恢复,但自47°C回复到37°C后不能完成恢复;(3)与野生型小鼠源性的神经元相比较,SUMO1/2/3基因敲除神经元在高热应激条件下具有更高的细胞凋亡和LDH水平,而SUMO1/2/3转基因神经元能够明显增加其对高热应激的耐受程度;(4)小鼠热惊厥模型检测结果显示,与野生型小鼠相比较,SUMO1/2/3基因敲除小鼠的惊厥评分、惊厥发作百分率、潜伏时间和惊厥持续时间均最高,而SUMO1/2/3转基因小鼠的惊厥评分、惊厥发作百分率、潜伏时间和惊厥持续时间均最低。结论:(1)低温能够抑制BMSCs的增殖能力,降低其向神经细胞的分化潜能,同时能够增加其对乏氧微环境的耐受程度;(2)低温能够增加BMSCs中大量蛋白质的SUMO化修饰水平,并诱导SUMO1和SUMO2/3从细胞浆向细胞核移位;(3)SUMOs通路的活化对于BMSCs的增殖和干性维持是必不可少的;(4)壮观霉素B1能够通过抑制Ubc9基因表达抑制SUMOs通路,进而降低BMSCs对乏氧微环境的耐受能力。(5)低温能够增加PCNA、Oct4、P53、HIF-1α蛋白的SUMO化修饰水平,上述机制共同参与BMSCs在低温环境下的增殖能力、干性维持能力以及对乏氧不利微环境的耐受能力;(6)SUMO2/3(而非SUMO1)参与介导神经元热应激早期反应;(7)短时间的高热应激(42°C)诱导的SUMO2/3共价修饰反应是可逆的,而超高温度的热应激(47°C)诱导的SUMO2/3共价修饰反应是不可逆的;(8)增加神经元中SUMOs过表达能够使其耐受更高程度的热应激环境,降低其发生热惊厥的几率。
[Abstract]:Objective: To study the characteristics and rules of small ubiquitin like modified neurons in neurons under low temperature and heat stress, and to apply this rule to central nervous system disease, and to play its neuroprotective role in the treatment of central nervous system diseases related to temperature stress. Methods: (1) (1) primary isolation and culture of the mouse derived BM SCs, flow cytometry identified the expression of the cell surface marker protein; (2) in the culture environment of 37 degree C (normal body temperature), 33 degree C (mild hypothermia) and 18 degree C (deep hypothermia), MTT method was used to detect the proliferation activity of BMSCs cells. Flow cytometry was used to detect the changes of BMSCs cell cycle, and cell immunofluorescence method was used to detect its direction under different temperature conditions. The differentiation potential of neurons and glial cells was changed; (3) the neuron OGD model was established, the apoptosis of BMSCs was detected by the cell in situ apoptosis method, the LDH content was detected by ELISA test, the Western blot method was used to detect the protein expression of SUMO1 and SUMO2 /3 in the covalent binding state and free state, and the cell immunofluorescence method was used to detect SU. The cell localization of MO1 and SUMO2/3 in BMSCs; (4) the Si RNA technique silenced the gene expression of SUMO1/2/3 in BMSCs, inhibited the gene expression of Ubc9 using splendin B1, and observed the cell morphology, cell proliferation activity, the differentiation of the cells to the nerve cells and the tolerance to the OGD environment at different temperatures; Western blot method was detected. The protein expression level of PCNA, Oct4, P53, HIF-1 alpha and the possible mechanism of SUMO activation to protect BMSCs from the adverse environment of hypoxia tolerance. (2) (1) immunofluorescence double staining method and Western blot method to identify the genotype of SUMO1/2/3 knockout and SUMO1/2/3 transgenic mice; (2) primary isolation and Cultivation of cerebral cortex neurons in mouse fetal mice Cells were placed in 37 degree C (normal body temperature), 42 C (high fever) and 47 C (hyperthermia) culture environment. Western blot was used to detect the protein expression level of SUMO1 and SUMO2/3. Immunofluorescence method was used to detect the cell location of SUMO1 and SUMO2/3 protein in neurons; (3) in situ apoptosis method was used to detect the wild type and SUMO1/2/3 gene knockout type And the apoptosis of SUMO1/2/3 transgenic neurons at the above temperature, ELISA method to detect the LDH content in the supernatant; (4) the model of thermal convulsion in mice was made and the five point scale was used for the seizure score, the percentage of convulsion in mice, the latency time and the duration of convulsion were recorded. The results were (1) (1) the specificity of the high expression of mesenchymal stem cells in BMSCs. Protein CD71 and CD105, but not expression of hematopoietic stem cell specific protein CD34 and CD4; (2) BMSCs grows slowly at low temperature, and the ability to differentiate into nerve cells gradually decreases with the decrease of temperature; BMSCs can be seen in a large number of cell apoptosis under OGD conditions. With the release of high level lactate dehydrogenase, low temperature can improve the above situation; (3) hypothermia It can induce a large number of proteins to be modified by SUMOs, showing a significant increase in covalent binding state SUMO1 and SUMO2/3 levels, while the decrease of free SUMO1 and SUMO2/3 levels and the shift from the cytoplasm to the nucleus; (4) after the silence of SUMO1/2/3 gene expression, the BMSCs morphology begins to grow, the growth speed of the cells is significantly slowed and the cell cycle is slow. Stagnation and terminal differentiation to the nerve cells; splendin B1 can inhibit the expression of Ubc9 to a certain extent, and then reduce the protein level of SUMO1 and SUMO2/3 in covalent binding state, BMSCs proliferation slows, and low temperature can increase the tolerance of BMSCs to the hypoxic adverse environment; splendamycin B1 can reduce PCNA, Oct4, P53, HIF-1 alpha. (2) (2) (1) the double staining method of immunofluorescence and Western blot method identified that the genotype of SUMO1/2/3 gene knockout and SUMO1/2/3 transgenic mice were stable and stable in transgenic mice neurons. (2) the cerebral cortical neurons of mice were covalent and free when they were placed in 37 degrees C, 42 degree C and 47 degree C culture environment. There was no obvious change in the expression level of SUMO1 protein in the state, but the protein expression level of the covalent binding state of SUMO2/3 was significantly increased, and the protein expression level of the free state of SUMO2/3 decreased accordingly. The immunofluorescence results showed that the SUMO2/3 protein was shifted from the cytoplasm to the nucleus, and the above phenomenon could be obtained from 42 degree C to 37 C. The recovery could not be recovered from 47 degree C to 37 C. (3) compared with the wild type mice, the SUMO1/2/3 gene knockout neurons had higher apoptosis and LDH level under high heat stress, while SUMO1/2/3 transgenic neurons could significantly increase their tolerance to high heat stress; (4) the heat shock of mice. The convulsive score, the rate of convulsion, the latent time and the duration of convulsion were the highest in the SUMO1/2/3 gene knockout mice compared with the wild type mice. The convulsion score, the percentage of convulsions, the latent time and the duration of convulsion were the lowest in the SUMO1/2/3 transgenic mice. Conclusion: (1) low temperature can be suppressed. The proliferation ability of BMSCs, reducing its differentiation potential to neural cells, and increasing its tolerance to the hypoxia microenvironment; (2) low temperature can increase the SUMO modification level of a large number of proteins in BMSCs and induce SUMO1 and SUMO2/3 to shift from the cytoplasm to the nucleus; (3) the activation of the SUMOs pathway is for the proliferation and dry maintenance of BMSCs. It is essential: (4) granomycin B1 can inhibit the SUMOs pathway by inhibiting the expression of Ubc9 gene, and then reduce the tolerance of BMSCs to hypoxia microenvironment. (5) low temperature can increase the SUMO modification level of PCNA, Oct4, P53, HIF-1 alpha protein, and the mechanisms mentioned above jointly participate in the proliferation and dry maintenance ability of BMSCs in the low temperature environment. Tolerance to the hypoxic adverse microenvironment; (6) SUMO2/3 (rather than SUMO1) was involved in mediating early response to heat stress in neurons; (7) a short time hyperthermia (42 C) induced SUMO2/3 covalent modification was reversible, while the SUMO2/3 covalent reaction induced by hyperthermia (47 C) was irreversible; (8) increasing the SUMOs in neurons Overexpression can make them tolerate a higher degree of heat stress and reduce the risk of febrile seizures.
【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】：R741

【相似文献】