本文選題：大鼠海馬細(xì)胞 + Hhcy��； 參考：《泰山醫(yī)學(xué)院》2014年碩士論文

【摘要】：目的 研究鹽酸甜菜堿（Bet）對高同型半胱氨酸血癥（HHcy）大鼠腦組織和原代海馬細(xì)胞損傷的保護(hù)作用及其機(jī)制。通過對HHcy大鼠血漿及腦組織勻漿中的SOD活性、LDH活性、MDA、 NO及GSH含量進(jìn)行測定，HE染色觀察HHcy大鼠腦組織形態(tài)學(xué)等方法研究Bet的保護(hù)作用。體外實(shí)驗(yàn)通過檢測細(xì)胞生長抑制率和凋亡率，采用試劑盒測定細(xì)胞上清液SOD活性、MDA含量、LDH活性和細(xì)胞凋亡，，Western Blot檢測細(xì)胞凋亡相關(guān)蛋白Bcl-2和Bax的表達(dá)，探討B(tài)et拮抗Hcy致大鼠原代培養(yǎng)海馬細(xì)胞損傷的作用機(jī)制。 方法 1Bet對HHcy大鼠腦組織的保護(hù)作用：采用2%蛋氨酸飼料喂養(yǎng)Wistar大鼠，制備大鼠高同型半胱氨酸血癥模型。分組如下：正常對照組（標(biāo)準(zhǔn)飼料，生理鹽水）、模型組（HHcy）（高蛋氨酸飼料，生理鹽水），鹽酸甜菜堿高、中、低濃度組(高蛋氨酸飼料，甜菜堿劑量200、100、50mg/Kg)，大鼠尾部取血離心取血清，檢測大鼠血清同型半胱氨酸的含量。灌胃Bet60天后麻醉處死，檢測血漿及腦組織勻漿SOD、MDA、NO、GSH、LDH及NO水平，HE染色觀察大鼠腦組織形態(tài)學(xué)變化。 2Bet對原代培養(yǎng)大鼠海馬細(xì)胞的保護(hù)作用：采用低濃度胰蛋白酶消化靜置提取海馬細(xì)胞，高糖DMEM培養(yǎng)原代海馬細(xì)胞的方法。原代海馬細(xì)胞分組如下：正常對照組：高糖DMEM培養(yǎng)基；同型半胱氨酸（Hcy）損傷組（模型組）：16mmol/LHcy；鹽酸甜菜堿高中低濃度組：16mmol/L Hcy+12.5mmol/L Be（t25mmol/L Bet，50mmol/LBet）。甜菜堿各組在Hcy損傷前Bet預(yù)處理12h，Hcy作用12h，采用倒置顯微鏡觀察細(xì)胞的形態(tài)學(xué)變化，MTT法檢測細(xì)胞的增殖。藥物對細(xì)胞凋亡的影響采用流式細(xì)胞儀檢測凋亡率及試劑盒細(xì)胞上清液中LDH的釋放，氧化應(yīng)激指標(biāo)采用比色法檢測細(xì)胞上清液中SOD活性和MDA含量。Western Blot檢測凋亡相關(guān)蛋白Bcl-2及Bax的表達(dá)。 結(jié)果 1Bet對HHcy大鼠腦組織的保護(hù)作用：利用高蛋氨酸飼料飲食建造大鼠高同型半胱氨酸血癥動(dòng)物模型。利用鹽酸甜菜堿灌胃治療，結(jié)果鹽酸甜菜堿組血漿及腦組織SOD、MDA、NO、GSH的變化與模型組比較均變化顯著(P 0.01)；HE染色結(jié)果表明甜菜堿組與模型組相比，模型組腦組織病理狀態(tài)明顯得到改善，明顯保護(hù)受損的腦組織。 2Bet對原代培養(yǎng)大鼠海馬細(xì)胞的保護(hù)作用：建立體外培養(yǎng)海馬細(xì)胞的方法并且鑒定成功，同型半胱氨酸損傷濃度為16mmol/L，鹽酸甜菜堿的保護(hù)濃度分別為12.5、25、50mmol/L，流式細(xì)胞儀檢測細(xì)胞凋亡率甜菜堿組細(xì)胞凋亡率明顯低于模型組，具有統(tǒng)計(jì)學(xué)差異（P0.01），MTT法結(jié)果與模型組比較，Bet各組細(xì)胞死亡率降低,Western-blot法結(jié)果與模型組比較，Bet組升高Bcl-2蛋白的表達(dá)、降低Bax蛋白的表達(dá)，具有顯著性差異。 結(jié)論 1.建立大鼠高同型半胱氨酸血癥動(dòng)物模型。 2.與對照組比較，高同型半胱氨酸血癥模型大鼠的大腦皮質(zhì)和海馬組織有明顯的病理變化，甜菜堿組的組織病理狀態(tài)與模型組比較損傷減輕。表明甜菜堿對高同型半胱氨酸所致的腦組織損傷具有保護(hù)作用。 3．高同型半胱氨酸血癥模型大鼠與正常對照組比較，血漿與腦組織中SOD活性降低，同時(shí)GSH含量減少，MDA和NO水平明顯增高，甜菜堿組與模型組比較血漿與腦組織勻漿中SOD活性和GSH含量明顯提高，MDA和NO水平下降。表明甜菜堿保護(hù)同型半胱氨酸損傷的機(jī)制可能是通過提高腦組織抗氧化能力途徑發(fā)揮作用。 4建立體外提取、培養(yǎng)原代海馬神經(jīng)細(xì)胞的方法并且鑒定成功。低濃度Hcy可以促進(jìn)細(xì)胞生長，高濃度Hcy可以抑制細(xì)胞生長，從而促進(jìn)細(xì)胞凋亡；Bet能夠降低Hcy誘導(dǎo)的海馬神經(jīng)元細(xì)胞的凋亡率，降低Hcy對神經(jīng)元造成的損傷。 5Bet可以提高同型半胱氨酸損傷的神經(jīng)細(xì)胞SOD活力，降低同型半胱氨酸損傷的細(xì)胞MDA含量，通過降低氧化應(yīng)激反應(yīng)來保護(hù)高同型半胱氨酸對原代海馬細(xì)胞的損傷。 6Bet能夠升高高同型半胱氨酸損傷的神經(jīng)細(xì)胞中Bcl-2蛋白的表達(dá)、降低高同型半胱氨酸損傷的神經(jīng)細(xì)胞中Bax蛋白表達(dá)，通過升高神經(jīng)細(xì)胞中Bcl-2/Bax值拮抗Hcy誘導(dǎo)的的原代大鼠海馬細(xì)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.

【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R589

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