本文選題：同型半胱氨酸 + 神經(jīng)干細(xì)胞��； 參考：《天津醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的 研究同型半胱氨酸(homocysteine, Hcy)對體外培養(yǎng)神經(jīng)干細(xì)胞(neural stem cells, NSCs)增殖作用的影響,以及通過建立大腦中動脈栓塞(middle cerebral artrty occlusion, MCAO)模型,觀察Hcy對局灶性腦缺血損傷大鼠神經(jīng)干細(xì)胞增殖的作用,探討Hcy基于DNA甲基化途徑調(diào)控NSCs增殖的作用機(jī)制。 方法 采用無血清培養(yǎng)原代培養(yǎng)新生大鼠NSCs,將細(xì)胞隨機(jī)分成4組：正常對照組(培養(yǎng)液中添加Hcy0μmol/l, Hcy-C組)、Hey低劑量組(培養(yǎng)液中添加Hey30μmol/l, Hcy-L組)、Hcy中劑量組(培養(yǎng)液中添加Hey300μmol/l, Hcy-M組)、Hcy高劑量組(培養(yǎng)液中添加Hey1mmol/l,Hcy-H組,)。采用Cell Dimension軟件對神經(jīng)球直徑進(jìn)行測量；采用免疫熒光(Immunofluorescent, IF)檢測NSCs標(biāo)記物Brdu和Sox2的表達(dá)：采用乳酸脫氫酶(lactate dehydrogenase, LDH)測定試劑盒檢測不同濃度Hcy作用下培養(yǎng)液中LDH的活力；采用DNA甲基化定量試劑盒檢測不同Hcy濃度下細(xì)胞總甲基化水平；采用甲基轉(zhuǎn)移酶活性檢測試劑盒檢測DNMTs總活性以及Western Blot檢測DNA甲基轉(zhuǎn)移酶(DNA methyltransferases, DNMTs)的蛋白表達(dá)；采用高效液相法(HPLC)檢測細(xì)胞內(nèi)S-腺苷甲硫氨酸(s-adenosylmethionine, SAM)、S-腺苷同型半胱氨酸(s-adenosylhomocysteine, SAH)的水平。 成年雄性SD(sprague-nawley)大鼠隨機(jī)分假手術(shù)組(sham operation group, SO組)、大腦中動脈栓塞模型(middle cerebral artery occlusion group, MCAO組)以及大腦中動脈栓塞模型+同型半胱氨酸(middle cerebral artery occlusion+Hcy group, MCAO+Hcy組)3組。SO組和MCAO組腹腔注射生理鹽水5ml/kg·bw·d, MCAO+Hcy組腹腔注射2%Hcy溶液5ml/kg·bw·d,注射28d。通過Morris水迷宮實驗檢測大鼠的空間學(xué)習(xí)記憶能力；采用免疫組化法以及Western blot檢測Sox2蛋白的表達(dá)；采用DNA甲基化定量試劑盒檢測總甲基化水平；采用甲基轉(zhuǎn)移酶活性檢測試劑盒檢測DNMTs總活性；采用Western Blot檢測DNA甲基轉(zhuǎn)移酶的蛋白表達(dá)。采用高效液相法(HPLC)檢測大鼠血漿內(nèi)Hey、SAM、SAH的水平。 結(jié)果 用無血清培養(yǎng)原代培養(yǎng)新生大鼠NSCs6d后,細(xì)胞聚集成球形團(tuán)塊,呈懸浮狀態(tài),Hcy添加組細(xì)胞神經(jīng)球直徑較對照組小,且差異均有統(tǒng)計學(xué)意義(P0.05)。Brdu和Sox2經(jīng)免疫熒光雙標(biāo)記法鑒定均呈雙陽性表達(dá),Hcy添加組Brdu/Sox2雙標(biāo)陽性率較對照組小,且差異均有統(tǒng)計學(xué)意義(P0.05)。經(jīng)LDH測定試劑盒檢測,Hcy添加組細(xì)胞培養(yǎng)液中LDH活性較對照組升高,且差異均有統(tǒng)計學(xué)意義(P0.05)。DNA總甲基化水平檢測結(jié)果顯示：與對照組相比,Hey添加組細(xì)胞總甲基化水平降低,差異均有統(tǒng)計學(xué)意義(P0.05)。DNMTs總活性檢測結(jié)果顯示：與對照組相比,Hcy添加組較對照組DNMTs總活性均下降,差異均有統(tǒng)計學(xué)意義(P0.05)。Western Blot結(jié)果顯示,與對照組相比,Hcy添加組DNMT1蛋白表達(dá)均下降,差異均有統(tǒng)計學(xué)意義(P0.05),Hcy-H組較Hcy-L組和Hcy-M組DNMT1蛋白表達(dá)下降,差異均有統(tǒng)計學(xué)意義(P0.05)；與對照組和Hcy-L組相比,Hcy-M組和Hcy-H組DNMT3a蛋白表達(dá)均下降,差異均有統(tǒng)計學(xué)意義(P0.05)；與對照組和Hcy-L組相比,Hcy-M組和Hcy-H組DNMT3b蛋白表達(dá)均下降,差異均有統(tǒng)計學(xué)意義(P0.05)。HPLC檢測結(jié)果顯示：與對照組和Hcy-L組相比,Hcy-M組和Hcy-H組SAH的水平升高,差異均有統(tǒng)計學(xué)意義(P0.05)；與對照組和Hcy-L組相比,Hcy-M組和Hcy-H組SAM/SAH比值降低,差異均有統(tǒng)計學(xué)意義(P0.05)。 腹腔注射及造模成功后Morris水迷宮實驗顯示,與MCAO組相比,MCAO+Hcy組大鼠逃避潛伏期明顯縮短,差異均有統(tǒng)計學(xué)意義(P0.05)；與MCAO組相比,及MCAO+Hcy組單位時間內(nèi)穿越平臺區(qū)域次數(shù)明顯減少,差異均有統(tǒng)計學(xué)意義(P0.05)。DNA,總甲基化水平檢測結(jié)果顯示：與MCAO組相比,MCAO+Hcy組總甲基化水平下降,差異均有統(tǒng)計學(xué)意義(P0.05)。經(jīng)免疫組化及Western blot檢測：與MCAO組相比,MCAO+Hcy組Sox2蛋白表達(dá)降低,差異均有統(tǒng)計學(xué)意義(P0.05)。DNMTs,總活性檢測結(jié)果顯示：與MCAO組相比,MCAO+Hcy組Sox2蛋白表達(dá)降低DNMTs活性下降,差異均有統(tǒng)計學(xué)意義(P0.05)。Western blot結(jié)果顯示：與MCAO組相比,MCAO+Hcy組DNMT1、 DNMT3a蛋白表達(dá)降低,差異均有統(tǒng)計學(xué)意義(P0.05)。經(jīng)HPLC檢測顯示,與SO組及MCAO組相比,MCAO+Hcy組Hcy濃度下降,差異均有統(tǒng)計學(xué)意義(P0.05)；與SO組及MCAO組相比,MCAO+Hcy組SAM/SAH比值下降,差異均有統(tǒng)計學(xué)意義(P0.05)。免疫熒光染色結(jié)果顯示,Sox2呈陽性表達(dá),且MCAO+Hcy組表達(dá)較SO組及MCAO組降低,差異均有統(tǒng)計學(xué)意義(P0.05)。 結(jié)論 本實驗成功的分離培養(yǎng)大鼠新生鼠NSCs以及建立大鼠大腦中動脈栓塞模型。Hcy抑制NSCs的增殖其機(jī)制可能是：Hcy通過改變甲基化途徑中關(guān)鍵代謝物SAH的濃度,降低SAM/SAH的比值,同時降低甲基化轉(zhuǎn)移酶的活性及蛋白的表達(dá),抑制了甲基化反應(yīng)的發(fā)生,進(jìn)而抑制了NSCs的增殖。
[Abstract]:objective
To investigate the effect of homocysteine (Hcy) on the proliferation of neural stem cells (NSCs) in vitro, and to observe the effect of Hcy on the proliferation of neural stem cells in rats with focal cerebral hemorrhage injury (middle cerebral artrty occlusion, MCAO) by establishing the model of the middle cerebral artery embolism (middle cerebral artrty occlusion, MCAO). Methylation pathway regulates the mechanism of NSCs proliferation.
Method
The primary cultured rat NSCs was cultured in serum-free culture, and the cells were randomly divided into 4 groups: normal control group (Hcy0 mol/l, Hcy-C group), low dose Hey group (Hey30 mol/l, Hcy-L group), Hcy medium dose group (Hey300 mu mol/l, Hcy-M group). Y-H group, Cell Dimension software was used to measure the diameter of the nerve bulb; the expression of NSCs markers Brdu and Sox2 were detected by Immunofluorescent (IF): the assay kit was used to determine the vitality in the incubated liquid with different concentrations in the incubated liquid with the lactic dehydrogenase (lactate dehydrogenase, LDH). The total methylation level of cells under different Hcy concentrations was detected by the agent box; the total activity of DNMTs and the expression of DNA methyltransferase (DNA methyltransferases, DNMTs) were detected by the methyl transferase activity detection kit and Western Blot, and the intracellular S- adenosine methionine (S-adenosylmethionine, SAM) was detected by high performance liquid phase (HPLC) method. The level of S- adenosine homocysteine (S-adenosylhomocysteine, SAH).
The adult male SD (sprague-nawley) rats were randomly divided into 3 groups, including the sham operation group, the SO group, the middle cerebral artery embolism model (middle cerebral artery occlusion group, the MCAO group) and the middle cerebral artery embolism model + homocysteine group. 5ml/kg / BW / D was injected into physiological saline, group MCAO+Hcy was injected with 2%Hcy solution 5ml/kg BW. D, and 28d. was injected by Morris water maze test to detect the spatial learning and memory ability of rats. Immunohistochemistry and Western blot were used to detect the expression of the protein. The total activity of DNMTs was detected by the enzyme activity detection kit and the protein expression of DNA methyltransferase was detected by Western Blot. The level of Hey, SAM and SAH in rat plasma was detected by high performance liquid phase (HPLC).
Result
After the serum-free culture of the primary culture of neonatal rat NSCs6d, the cells were aggregated into spherical mass and suspended. The diameter of the cell nerve ball in the Hcy addition group was smaller than that of the control group, and the difference was statistically significant (P0.05).Brdu and Sox2 were both positive and positive by immunofluorescence double labeling, and the positive rate of Brdu/Sox2 double standard in Hcy addition group was more than that of the control group. The difference was statistically significant (P0.05). The activity of LDH in the Hcy added group was higher than that of the control group by LDH assay, and the difference was statistically significant (P0.05).DNA total methylation level detection results showed that compared with the control group, the total methylation level of Hey added group decreased, the difference was statistically significant (P). 0.05).DNMTs total activity detection results showed that compared with the control group, the total activity of DNMTs in the Hcy addition group decreased compared with the control group, and the difference was statistically significant (P0.05).Western Blot results showed that the expression of DNMT1 protein in Hcy added group decreased, the difference was statistically significant (P0.05), Hcy-H group was more than Hcy-L group and Hcy-M group eggs. The difference of white expression was statistically significant (P0.05). Compared with the control group and the Hcy-L group, the expression of DNMT3a protein in the Hcy-M group and the Hcy-H group decreased, and the difference was statistically significant (P0.05). Compared with the control group and the Hcy-L group, the expression of DNMT3b protein in Hcy-M and Hcy-H groups decreased, and the difference was statistically significant (P0.05).HPLC detection results showed a significant difference. Compared with the control group and the Hcy-L group, the level of SAH in group Hcy-M and group Hcy-H was higher, the difference was statistically significant (P0.05). Compared with the control group and the Hcy-L group, the SAM/SAH ratio of the Hcy-M group and the Hcy-H group decreased, the difference was statistically significant (P0.05).
After the intraperitoneal injection and the success of the Morris water maze test, the escape latency of the MCAO+Hcy group was significantly shorter than that of the MCAO group, and the difference was statistically significant (P0.05). Compared with the MCAO group, the number of crossing platform areas in the unit time of MCAO+Hcy group decreased significantly (P0.05).DNA, and the total methylation level was significant. The results showed that compared with the MCAO group, the total methylation level of MCAO+Hcy group decreased, the difference was statistically significant (P0.05). After immunohistochemistry and Western blot, the Sox2 protein expression in the MCAO+Hcy group decreased, the difference was statistically significant (P0.05).DNMTs, the total activity detection results showed that the MCAO+Hcy group was compared with the MCAO group. The expression of ox2 protein decreased DNMTs activity, and the difference was statistically significant (P0.05).Western blot results showed that the expression of DNMT3a protein in MCAO+Hcy group was lower than that of MCAO group, and the difference was statistically significant (P0.05). 05): compared with group SO and group MCAO, the SAM/SAH ratio of group MCAO+Hcy decreased, and the difference was statistically significant (P0.05). The results of immunofluorescence staining showed that Sox2 was positive, and the expression of MCAO+Hcy group was lower than that of SO and MCAO groups, and the difference was statistically significant (P0.05).
conclusion
The successful separation and cultivation of neonatal rat NSCs and the establishment of rat middle cerebral artery embolization model.Hcy inhibit the proliferation of NSCs may be: Hcy by changing the concentration of the key metabolite SAH in the methylation pathway, reducing the ratio of SAM/SAH, reducing the activity of methyltransferase and the expression of protein, and inhibiting the methylation. The occurrence of the reaction and the inhibition of the proliferation of NSCs.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R741

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相關(guān)期刊論文 前10條

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本文編號：1941326