盐酸甜菜碱对HHcy大鼠脑组织和原代海马细胞损伤的保护作用及机制研究

发布时间：2018-04-27 06:21

本文选题：大鼠海马细胞 + Hhcy　；参考：《泰山医学院》2014年硕士论文

【摘要】：目的研究盐酸甜菜碱（Bet）对高同型半胱氨酸血症（HHcy）大鼠脑组织和原代海马细胞损伤的保护作用及其机制。通过对HHcy大鼠血浆及脑组织匀浆中的SOD活性、LDH活性、MDA、 NO及GSH含量进行测定，HE染色观察HHcy大鼠脑组织形态学等方法研究Bet的保护作用。体外实验通过检测细胞生长抑制率和凋亡率，采用试剂盒测定细胞上清液SOD活性、MDA含量、LDH活性和细胞凋亡，，Western Blot检测细胞凋亡相关蛋白Bcl-2和Bax的表达，探讨Bet拮抗Hcy致大鼠原代培养海马细胞损伤的作用机制。方法 1Bet对HHcy大鼠脑组织的保护作用：采用2%蛋氨酸饲料喂养Wistar大鼠，制备大鼠高同型半胱氨酸血症模型。分组如下：正常对照组（标准饲料，生理盐水）、模型组（HHcy）（高蛋氨酸饲料，生理盐水），盐酸甜菜碱高、中、低浓度组(高蛋氨酸饲料，甜菜碱剂量200、100、50mg/Kg)，大鼠尾部取血离心取血清，检测大鼠血清同型半胱氨酸的含量。灌胃Bet60天后麻醉处死，检测血浆及脑组织匀浆SOD、MDA、NO、GSH、LDH及NO水平，HE染色观察大鼠脑组织形态学变化。 2Bet对原代培养大鼠海马细胞的保护作用：采用低浓度胰蛋白酶消化静置提取海马细胞，高糖DMEM培养原代海马细胞的方法。原代海马细胞分组如下：正常对照组：高糖DMEM培养基；同型半胱氨酸（Hcy）损伤组（模型组）：16mmol/LHcy；盐酸甜菜碱高中低浓度组：16mmol/L Hcy+12.5mmol/L Be（t25mmol/L Bet，50mmol/LBet）。甜菜碱各组在Hcy损伤前Bet预处理12h，Hcy作用12h，采用倒置显微镜观察细胞的形态学变化，MTT法检测细胞的增殖。药物对细胞凋亡的影响采用流式细胞仪检测凋亡率及试剂盒细胞上清液中LDH的释放，氧化应激指标采用比色法检测细胞上清液中SOD活性和MDA含量。Western Blot检测凋亡相关蛋白Bcl-2及Bax的表达。结果 1Bet对HHcy大鼠脑组织的保护作用：利用高蛋氨酸饲料饮食建造大鼠高同型半胱氨酸血症动物模型。利用盐酸甜菜碱灌胃治疗，结果盐酸甜菜碱组血浆及脑组织SOD、MDA、NO、GSH的变化与模型组比较均变化显著(P 0.01)；HE染色结果表明甜菜碱组与模型组相比，模型组脑组织病理状态明显得到改善，明显保护受损的脑组织。 2Bet对原代培养大鼠海马细胞的保护作用：建立体外培养海马细胞的方法并且鉴定成功，同型半胱氨酸损伤浓度为16mmol/L，盐酸甜菜碱的保护浓度分别为12.5、25、50mmol/L，流式细胞仪检测细胞凋亡率甜菜碱组细胞凋亡率明显低于模型组，具有统计学差异（P0.01），MTT法结果与模型组比较，Bet各组细胞死亡率降低,Western-blot法结果与模型组比较，Bet组升高Bcl-2蛋白的表达、降低Bax蛋白的表达，具有显著性差异。结论 1.建立大鼠高同型半胱氨酸血症动物模型。 2.与对照组比较，高同型半胱氨酸血症模型大鼠的大脑皮质和海马组织有明显的病理变化，甜菜碱组的组织病理状态与模型组比较损伤减轻。表明甜菜碱对高同型半胱氨酸所致的脑组织损伤具有保护作用。 3．高同型半胱氨酸血症模型大鼠与正常对照组比较，血浆与脑组织中SOD活性降低，同时GSH含量减少，MDA和NO水平明显增高，甜菜碱组与模型组比较血浆与脑组织匀浆中SOD活性和GSH含量明显提高，MDA和NO水平下降。表明甜菜碱保护同型半胱氨酸损伤的机制可能是通过提高脑组织抗氧化能力途径发挥作用。 4建立体外提取、培养原代海马神经细胞的方法并且鉴定成功。低浓度Hcy可以促进细胞生长，高浓度Hcy可以抑制细胞生长，从而促进细胞凋亡；Bet能够降低Hcy诱导的海马神经元细胞的凋亡率，降低Hcy对神经元造成的损伤。 5Bet可以提高同型半胱氨酸损伤的神经细胞SOD活力，降低同型半胱氨酸损伤的细胞MDA含量，通过降低氧化应激反应来保护高同型半胱氨酸对原代海马细胞的损伤。 6Bet能够升高高同型半胱氨酸损伤的神经细胞中Bcl-2蛋白的表达、降低高同型半胱氨酸损伤的神经细胞中Bax蛋白表达，通过升高神经细胞中Bcl-2/Bax值拮抗Hcy诱导的的原代大鼠海马细胞的凋亡。
[Abstract]:objective
The protective effect and mechanism of betaine hydrochloride (Bet) on the brain tissue and primary hippocampal cells in hyperhomocysteinemia (HHcy) rats were studied. The SOD activity, LDH activity, MDA, NO and GSH in the plasma and brain homogenate of HHcy rats were measured, and the morphology of the brain tissue of HHcy rats was observed by HE staining, and the assurance of Bet was studied. In vitro, by detecting cell growth inhibition rate and apoptosis rate, the cell supernatant SOD activity, MDA content, LDH activity and cell apoptosis were detected by the kit in vitro, and Western Blot was used to detect the expression of apoptosis related protein Bcl-2 and Bax, and the mechanism of Bet to antagonize the damage of primary cultured hippocampal cells in Hcy induced rats was discussed.
Method
The protective effect of 1Bet on the brain tissue of HHcy rats: feeding Wistar rats with 2% methionine feed to prepare the rat model of hyperhomocysteinemia. The groups were grouped as follows: normal control group (standard feed, saline), model group (HHcy) (high methionine feed, saline), high, medium, low concentration (high methionine feed, sweet) The content of serum homocysteine in rats was detected by centrifugation in the tail of 200100,50mg/Kg). The levels of serum homocysteine in rats were detected after Bet60 after gavage. The levels of SOD, MDA, NO, GSH, LDH and NO in the plasma and brain homogenate were detected. The morphological changes of brain tissue were observed in rats by HE staining.
The protective effect of 2Bet on primary cultured rat hippocampal cells: Using low concentration trypsin digestion to extract hippocampal cells and high sugar DMEM to cultivate primary hippocampal cells. The primary hippocampal cells were grouped as follows: normal control group: high glucose DMEM medium; homocysteine (Hcy) injury group (model group):16mmol/LHcy; hydrochloric acid sweet 16mmol/L Hcy+12.5mmol/L Be (t25mmol/L Bet, 50mmol/LBet). Each group pretreated 12h before Hcy injury and 12h on Bet before Hcy damage. The morphological changes of cells were observed by inverted microscope, and cell proliferation was detected by the inverted microscope. The effect of drug on apoptosis was detected by flow cytometry and the reagent box was used to detect the apoptosis. The release of LDH in the cell supernatant and the oxidative stress index were used to detect the activity of SOD and the content of MDA in the supernatant by colorimetry..Western Blot was used to detect the expression of apoptosis related protein Bcl-2 and Bax.
Result
The protective effect of 1Bet on the brain tissue of HHcy rats: the rat model of hyperhomocysteinemia was built with high methionine diet. The changes of SOD, MDA, NO, GSH in the plasma and brain tissues of the betaine hydrochloride group were significantly different from those in the model group (P 0.01). The results of HE staining showed that betaine was found. Compared with model group, the pathological state of brain tissue in model group was obviously improved, and the damaged brain tissue was obviously protected.
The protective effect of 2Bet on the primary cultured rat hippocampal cells: to establish the method of cultured hippocampal cells in vitro and to identify successfully. The concentration of homocysteine was 16mmol/L, and the protective concentration of betaine hydrochloride was 12.5,25,50mmol/L respectively. The apoptosis rate of the apoptosis rate of betaine was significantly lower than that of the model group. There was a statistical difference (P0.01). Compared with the model group, the result of MTT method was lower than that of the model group. The result of Western-blot method was compared with that of the model group. The Bet group increased the expression of Bcl-2 protein and reduced the expression of Bax protein. There was a significant difference in the expression of the Bax protein.
conclusion
1. a rat model of hyperhomocysteinemia was established.
2. compared with the control group, there were obvious pathological changes in the cerebral cortex and hippocampus of the hyperhomocysteinemia model rats. The histopathological state of the betaine group was less than that of the model group. It showed that betaine has protective effect on the brain tissue injury caused by high homocysteine.
3. high homocysteinemia model rats compared with normal control group, the activity of SOD in plasma and brain tissue decreased, and the content of GSH decreased, and the level of MDA and NO increased obviously. The SOD activity and GSH content in plasma and brain homogenates were significantly increased in the betaine group and the level of GSH in the plasma and brain homogenates. The results showed that betaine protected homocysteine. The mechanism of ammonia damage may be mediated by enhancing the antioxidant capacity of brain tissue.
4 the method of extraction and culture of primary hippocampal neurons in vitro is established and identified successfully. Low concentration of Hcy can promote cell growth. High concentration of Hcy can inhibit cell growth and promote cell apoptosis. Bet can reduce the apoptosis rate of Hcy induced hippocampal neurons and reduce the damage caused by Hcy to neurons.
5Bet can improve the SOD activity of the homocysteine injured nerve cells, reduce the MDA content of homocysteine damage, and protect the primary hippocampal cells by reducing the oxidative stress response.
6Bet can increase the expression of Bcl-2 protein in the nerve cells with high homocysteine damage, reduce the expression of Bax protein in the nerve cells with high homocysteine damage, and antagonize the apoptosis of the primary rat hippocampal cells by increasing the Bcl-2/Bax value in the nerve cells to antagonize the Hcy induced rat hippocampus.

【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R589

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