【摘要】：目的 通過(guò)體外培養(yǎng)胚胎期SD大鼠神經(jīng)干細(xì)胞(neural stem cells, NSCs),添加同型半胱氨酸(homocysteine, Hcy)進(jìn)行干預(yù),觀察Hcy對(duì)神經(jīng)干細(xì)胞增殖能力的影響,并進(jìn)一步觀察其增殖期蛋白表達(dá)譜的變化,探討Hcy對(duì)神經(jīng)干細(xì)胞增殖影響的機(jī)制；建立HuSH shRNA Plasmid介導(dǎo)的RNA干擾技術(shù)體系。 方法 采用無(wú)血清體外細(xì)胞培養(yǎng)方法,培養(yǎng)胚胎期大鼠NSCs,將NSCs分為四組：①正常對(duì)照組(normal control group)培養(yǎng)基中葉酸含量為4μg/mL,同型半胱氨酸含量為0μg/mL;②葉酸缺乏組(Folic acid-D group)培養(yǎng)基中葉酸含量為0.65μg/mL,同型半胱氨酸含量為0μg/mL;③同型半胱氨酸中度增高組(Hey-medium group)培養(yǎng)基中葉酸含量為4μg/mL,同型半胱氨酸含量為100μg/mL;④同型半胱氨酸高度增高組(Hcy-high group)培養(yǎng)基中葉酸含量為4μg/mL,同型半胱氨酸含量為300μg/mLc采用MTT法檢測(cè)神經(jīng)干細(xì)胞增殖能力,繪制生長(zhǎng)曲線(xiàn)；計(jì)數(shù)神經(jīng)球的數(shù)量及直徑以檢測(cè)神經(jīng)球的結(jié)球能力；采用臺(tái)盼藍(lán)染色法檢測(cè)神經(jīng)干細(xì)胞活力；培養(yǎng)6天后收集增殖期細(xì)胞,提取細(xì)胞蛋白,進(jìn)行雙向電泳,經(jīng)考馬斯亮藍(lán)染色后使用PD-Quest軟件比對(duì)蛋白差異點(diǎn),將篩選出的蛋白點(diǎn)進(jìn)行質(zhì)譜鑒定。采用Real-time PCR法檢測(cè)經(jīng)質(zhì)譜鑒定蛋白mRNA表達(dá)水平。采用試劑盒檢測(cè)神經(jīng)干細(xì)胞內(nèi)ATP含量、SOD活力、MDA水平、ROS含量；測(cè)定神經(jīng)干細(xì)胞線(xiàn)粒體呼吸鏈酶復(fù)合物NADH-Q還原酶(CⅠ)、琥珀酸-Q還原酶(CⅡ)、細(xì)胞色素還原酶(CⅢ)及細(xì)胞色素氧化酶(CⅣ)的活性。采用HuSH shRNA Plasmid為載體建立RNA干擾技術(shù)體系。 結(jié)果 添加Hcy后,NSCs增殖水平受到抑制。MTT結(jié)果顯示,添加Hcy后各時(shí)間點(diǎn)細(xì)胞活力較正常對(duì)照組有所降低,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。在Hcy添加組,神經(jīng)球直徑明顯減少,與對(duì)照組比較差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；但神經(jīng)球數(shù)目各組間差異不明顯(P0.05)。將神經(jīng)球裂解后,計(jì)數(shù)神經(jīng)干細(xì)胞數(shù)目發(fā)現(xiàn),在兩組Hcy添加組,細(xì)胞數(shù)目較對(duì)照組有所減少,且呈濃度依賴(lài)性趨勢(shì)(P0.05)。經(jīng)臺(tái)盼藍(lán)染色,發(fā)現(xiàn)各組間神經(jīng)干細(xì)胞活力差異不明顯(P0.05)。蛋白質(zhì)組學(xué)結(jié)果顯示,添加Hcy后神經(jīng)干細(xì)胞增殖期蛋白表達(dá)譜發(fā)生了明顯變化；我們從差異蛋白中挑選出7種蛋白進(jìn)行質(zhì)譜鑒定,成功鑒定6種蛋白；其中細(xì)胞色素b-c1復(fù)合體-亞基2和烏頭酸水合酶為線(xiàn)粒體呼吸鏈和三羧酸循環(huán)涉及蛋白,提示Hcy可能是通過(guò)影響線(xiàn)粒體的功能而降低神經(jīng)干細(xì)胞增殖的。Real-time PCR結(jié)果顯示,細(xì)胞色素b-c1復(fù)合體-亞基2和烏頭酸水合酶mRNA水平同樣被Hcy抑制。采用試劑盒檢測(cè)ATP含量發(fā)現(xiàn)Hcy降低了神經(jīng)干細(xì)胞的能量合成(P0.05)；降低了SOD活性(P0.05),升高了MDA和ROS水平(P0.05)；且線(xiàn)粒體呼吸鏈復(fù)合酶Ⅰ～Ⅳ的活性均受到不同程度的抑制(P0.05)。成功建立經(jīng)HuSH shRNA Plasmid介導(dǎo)的RNA干擾技術(shù)體系。 結(jié)論 本實(shí)驗(yàn)成功分離、培養(yǎng)了體外胚胎期SD大鼠NSCs,研究結(jié)果顯示同型半胱氨酸可以抑制NSCs的增殖,降低神經(jīng)球的結(jié)球能力,影響NSCs增殖期蛋白表達(dá)譜的水平；在mRNA和蛋白水平均降低細(xì)胞色素b-c1復(fù)合體-亞基2和烏頭酸水合酶的表達(dá)；減少細(xì)胞內(nèi)ATP的含量,增加活性氧自由基生成,降低其抗氧化能力,降低線(xiàn)粒體呼吸鏈復(fù)合酶Ⅰ～Ⅳ的活性。建立經(jīng)HuSH shRNA Plasmid介導(dǎo)的RNA干擾技術(shù)體系。
[Abstract]:Purpose The effects of Hcy on the proliferation ability of neural stem cells were observed by the culture of neural stem cells (NSCs) in the embryonic stage SD rats in vitro, and the change of the expression profile of the protein in the proliferative phase was further observed. The mechanism of the effect of Hcy on the proliferation of neural stem cells was discussed, and the RNA interference technique mediated by HuSH shRNA Plasmaid was established. Department. Methods The non-serum in-vitro cell culture method was used to culture the NSCs in the embryonic stage, and the NSCs were divided into four groups: the content of the folic acid in the normal control group was 4.mu. g/ mL and the content of the same cysteine was 0. The folic acid content in the Folic acid-D group culture medium was 0.65 & mu; g/ mL, the content of the same cysteine was 0. mu. g/ mL, and the folic acid content in the medium-high group (Hy-medium group) medium of the same type of cysteine was 4. m u.g/ mL and the same cysteine content was 100. The content of folic acid in Hcy-high group (Hcy-high group) medium was 4.mu. g/ mL, the content of the same cysteine was 300. m u.g/ mLc, the proliferation ability of neural stem cells was detected by MTT method, the growth curve was drawn, and the number and diameter of the nerve ball were counted to detect the nerve ball. the activity of the neural stem cells is detected by a trypan blue staining method, the proliferation period cells are collected after 6 days of culture, the cell protein is extracted, two-way electrophoresis is carried out, the protein difference points are compared with the protein difference points by using the PD-Quest software ratio after the Coomassius brilliant blue staining, and the screened protein points are The identification of mRN by mass spectrometry was detected by real-time PCR. A. The level of ATP in the neural stem cells, the activity of SOD, the level of MDA and the content of ROS were detected by using the kit. The mitochondrial respiratory chain of the neural stem cells, NADH-Q reductase (C I), and the succinic acid-Q reduction, were determined. Enzyme (C II), Cytochrome Reductase (C 鈪，

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