S100β基因修饰的嗅鞘细胞在炎性环境中的作用

发布时间：2018-03-06 02:32

本文选题：嗅鞘细胞　切入点：S100β　出处：《宁夏医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的利用S100β基因修饰的嗅鞘细胞体外炎症模型,探讨S100β对炎症环境中嗅鞘细胞的作用及机制。方法(1)嗅球取自新生7天SD乳鼠,嗅鞘细胞(OECs)的培养采用差速贴壁改良法联合胰酶限时消化法纯化;(2)利用慢病毒载体LV5(EF-1a F/GFPPuro)分别将S100β或空载体转染嗅鞘细胞并分组为S100β-OECs组和空载体-OECs组;采用流式细胞仪验证转染效率。(3)利用CCK-8法在1、3、5、7d分别检测病毒转染OECs(S100β-OECs组/空载体-OECs组)和未转染OECs对照组细胞增殖情况。(4)通过在两组培养体系添加重组IFN-γ建立嗅鞘细胞的体外炎症模型,在诱导炎症0、3、6、12、24h后,通过TUNNEL染色法观察各组OECs凋亡状况;(5)采用实时荧光定量PCR检测S100β-OECs组和空载体-OECs组的S100β、凋亡因子Bax、Puma、抗凋亡因子Bcl-2和炎症相关因子INOS、IL-1β、TNF-α的基因表达水平;利用蛋白免疫印迹法检测两组在诱导炎症后不同时间点的S100β、p65、p38、ERK1/2、P-JNK等蛋白含量,探讨S100β对炎症环境中嗅鞘细胞的作用机制。结果1.采用改良方法原代培养的大鼠嗅鞘细胞纯度为82.7%。2.采用LV5载体构建的S100β过表达载体平均转染效率为74%。3.采用CCK-8法绘制细胞生长曲线显示,三组间细胞增殖能力无统计学差异(P0.05)。4.在加入INF-γ后6、12和24h,S100β-OECs组的抗凋亡因子Bcl-2表达水平较空载体-OECs组高(P0.05),S100β-OECs组的炎症因子INOS、IL-1β、TNF-α和凋亡因子Bax、Puma表达水平较空载体-OECs组低(P0.05)。5.TUNNEL染色结果显示,在INF-γ诱导后0、3、6h和12h,两组TUNNEL阳性细胞数目无差异(P0.05);在致炎24h后,空载体-OECs组TUNNEL阳性细胞数目明显多于S100β-OECs组(P0.05)。6.在INF-γ诱导后3h,两组中S100β表达均上升(P0.05),但致炎6h、12h及24h后,两组中S100β表达均持续下降(P0.05);同一时间点内,S100β-OECs组S100β蛋白表达均高于空载体-OECs组(P0.05)。7.空载体-OECs组中NFκB p65蛋白表达在致炎后3h开始出现上调(P0.05),在6h上调达到峰值;而S100β-OECs组NFκB p65蛋白表达在致炎后3h出现下调(P0.05),在6h后出现上调趋势。致炎后3h、6h、12h后S100β-OECs组NFκB p65蛋白表达水平均明显低于同一时间点空载体-OECs组(P0.05)。8.致炎后3h,两组中p38蛋白表达水平均出现下调(P0.05),致炎后6h,空载体-OECs组中p38蛋白表达水平出现上调并达到顶峰(P0.05)。致炎后3h、6h、12h,S100β-OECs组的p38蛋白表达低于空载体-OECs组(P0.05)。致炎后不同时间点,两组中ERK1/2蛋白及P-JNK蛋白均无明显变化(P0.05)。结论1.在体外细胞实验S100β能够抑制OECs释放炎症相关因子。2.S100β可降低炎症条件下OECs凋亡率,从而提高其存活率。3.S100β蛋白可能通过介导NFκB p65/MAPK p38通路调控炎症环境中OECs的炎症反应机制。
[Abstract]:Objective to investigate the effect and mechanism of S100 尾 on olfactory ensheathing cells (OECs) in vitro by using S100 尾 gene modified olfactory ensheathing cells. The culture of olfactory ensheathing cells (OECs) was purified by differential adherence method and trypsin time-limited digestion method. S100 尾 or empty vector was transfected into olfactory ensheathing cells and divided into S100 尾 -OECs group and empty vector -OECs group using lentivirus vector LV5(EF-1a F / GFP Puro. The efficiency of transfection was verified by flow cytometry. (CCK-8 method was used to detect the cell proliferation of OECs(S100 尾 -OECs group / empty vector OECs group) and untransfected OECs control group respectively for 7 days by adding recombinant IFN- 纬 to establish olfactory ensheathing by adding recombinant IFN- 纬 into the culture system of the two groups. The inflammatory model of cells in vitro, 24 hours after inducing inflammation, the apoptosis of OECs in each group was observed by TUNNEL staining. The expression of S100 尾 in S100 尾 -OECs group and empty vector -OECs group, apoptosis factor Bax-Puma, anti-apoptotic factor Bcl-2 and inflammatory related factor INOSIL-1 尾 TNF- 伪 were detected by real-time quantitative PCR. The protein contents of S100 尾 -p65-p38-ERK1 / 2 and P-JNK at different time points after inflammation were detected by Western blotting. To investigate the effect of S100 尾 on olfactory ensheathing cells in inflammatory environment. Results 1. The purity of the primary cultured rat olfactory ensheathing cells by modified method was 82.7.2. the average transfection efficiency of S100 尾 overexpression vector constructed by LV5 vector was 74. 3. CCK-8 method was used. The cell growth curve shows that, There was no significant difference in cell proliferation ability among the three groups. The expression level of anti-apoptotic factor Bcl-2 in S100 尾 -OECs group was lower than that in empty vector -OECs group (P 0.05 尾 -OECs group), and the expression level of inflammatory factor INOSIL-1 尾 TNF- 伪 and apoptosis factor Bax-Puma was lower than that in empty vector OECs group (P 0.05n.5.TUNNEL staining results showed that the expression level of anti-apoptotic factor Bcl-2 was lower than that in empty vector OECs group after the addition of INF- 纬 to S100 尾 -OECs. The expression level of anti-apoptotic factor Bcl-2 in S100 尾 -OECs group was higher than that in empty vector -OECs group. There was no difference in the number of TUNNEL positive cells between the two groups at 6 h and 12 h after exposure to INF- 纬, but 24 h after inflammation, the number of TUNNEL positive cells in empty vector OECs group was significantly higher than that in S100 尾 -OECs group (P0.05 路6). At 3 h after INF- 纬 induction, the expression of S100 尾 in both groups was increased (P 0.05), but the expression of S100 尾 was increased at 6 h, 12 h and 24 h, respectively. At the same time, the expression of S100 尾 protein in S100 尾 -OECs group was higher than that in empty vector -OECs group (P0.05). The expression of NF- 魏 B p65 protein in empty vector -OECs group began to up-regulate at 3 h after inflammation and reached its peak at 6 h. However, the expression of NF 魏 B p65 in S100 尾 -OECs group was down-regulated at 3 h after inflammation and up-regulated at 6 h. The expression of NF 魏 B p65 protein in S100 尾 -OECs group was significantly lower than that in S100 尾 -OECs group at the same time point at 3 h after inflammation, and the expression level of p38 protein in S100 尾 -OECs group was significantly lower than that in S100 尾 -OECs group at 3 h after inflammation, 3 h after inflammation, and 3 h after inflammation, p38 protein in both groups was significantly lower than that in S100 尾 -OECs group. The expression of p38 protein in empty vector OECs group was up-regulated and reached its peak level at 6 h after inflammation. The expression of p38 protein in S100 尾 -OECs group was lower than that in empty vector OECs group at different time points after inflammation, and the expression level of p38 protein in S100 尾 -OECs group was lower than that in empty vector OECs group at different time points after inflammation 3 h after inflammation, the expression of p38 protein in S100 尾 -OECs group was lower than that in empty vector OECs group at different time points after inflammation. Conclusion 1. In vitro, S100 尾 can inhibit the release of inflammatory cytokines by OECs. S100 尾 can reduce the apoptosis rate of OECs under the condition of inflammation. Therefore, the increase of survival rate. 3. S100 尾 protein may regulate the inflammatory response mechanism of OECs in inflammatory environment by mediating NF 魏 B p65 / MAPK p38 pathway.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R364.5

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