本文選題：嗅鞘細(xì)胞　切入點(diǎn)：S100β　出處：《寧夏醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的利用S100β基因修飾的嗅鞘細(xì)胞體外炎癥模型,探討S100β對炎癥環(huán)境中嗅鞘細(xì)胞的作用及機(jī)制。方法(1)嗅球取自新生7天SD乳鼠,嗅鞘細(xì)胞(OECs)的培養(yǎng)采用差速貼壁改良法聯(lián)合胰酶限時消化法純化;(2)利用慢病毒載體LV5(EF-1a F/GFPPuro)分別將S100β或空載體轉(zhuǎn)染嗅鞘細(xì)胞并分組為S100β-OECs組和空載體-OECs組;采用流式細(xì)胞儀驗(yàn)證轉(zhuǎn)染效率。(3)利用CCK-8法在1、3、5、7d分別檢測病毒轉(zhuǎn)染OECs(S100β-OECs組/空載體-OECs組)和未轉(zhuǎn)染OECs對照組細(xì)胞增殖情況。(4)通過在兩組培養(yǎng)體系添加重組IFN-γ建立嗅鞘細(xì)胞的體外炎癥模型,在誘導(dǎo)炎癥0、3、6、12、24h后,通過TUNNEL染色法觀察各組OECs凋亡狀況;(5)采用實(shí)時熒光定量PCR檢測S100β-OECs組和空載體-OECs組的S100β、凋亡因子Bax、Puma、抗凋亡因子Bcl-2和炎癥相關(guān)因子INOS、IL-1β、TNF-α的基因表達(dá)水平;利用蛋白免疫印跡法檢測兩組在誘導(dǎo)炎癥后不同時間點(diǎn)的S100β、p65、p38、ERK1/2、P-JNK等蛋白含量,探討S100β對炎癥環(huán)境中嗅鞘細(xì)胞的作用機(jī)制。結(jié)果1.采用改良方法原代培養(yǎng)的大鼠嗅鞘細(xì)胞純度為82.7%。2.采用LV5載體構(gòu)建的S100β過表達(dá)載體平均轉(zhuǎn)染效率為74%。3.采用CCK-8法繪制細(xì)胞生長曲線顯示,三組間細(xì)胞增殖能力無統(tǒng)計學(xué)差異(P0.05)。4.在加入INF-γ后6、12和24h,S100β-OECs組的抗凋亡因子Bcl-2表達(dá)水平較空載體-OECs組高(P0.05),S100β-OECs組的炎癥因子INOS、IL-1β、TNF-α和凋亡因子Bax、Puma表達(dá)水平較空載體-OECs組低(P0.05)。5.TUNNEL染色結(jié)果顯示,在INF-γ誘導(dǎo)后0、3、6h和12h,兩組TUNNEL陽性細(xì)胞數(shù)目無差異(P0.05);在致炎24h后,空載體-OECs組TUNNEL陽性細(xì)胞數(shù)目明顯多于S100β-OECs組(P0.05)。6.在INF-γ誘導(dǎo)后3h,兩組中S100β表達(dá)均上升(P0.05),但致炎6h、12h及24h后,兩組中S100β表達(dá)均持續(xù)下降(P0.05);同一時間點(diǎn)內(nèi),S100β-OECs組S100β蛋白表達(dá)均高于空載體-OECs組(P0.05)。7.空載體-OECs組中NFκB p65蛋白表達(dá)在致炎后3h開始出現(xiàn)上調(diào)(P0.05),在6h上調(diào)達(dá)到峰值;而S100β-OECs組NFκB p65蛋白表達(dá)在致炎后3h出現(xiàn)下調(diào)(P0.05),在6h后出現(xiàn)上調(diào)趨勢。致炎后3h、6h、12h后S100β-OECs組NFκB p65蛋白表達(dá)水平均明顯低于同一時間點(diǎn)空載體-OECs組(P0.05)。8.致炎后3h,兩組中p38蛋白表達(dá)水平均出現(xiàn)下調(diào)(P0.05),致炎后6h,空載體-OECs組中p38蛋白表達(dá)水平出現(xiàn)上調(diào)并達(dá)到頂峰(P0.05)。致炎后3h、6h、12h,S100β-OECs組的p38蛋白表達(dá)低于空載體-OECs組(P0.05)。致炎后不同時間點(diǎn),兩組中ERK1/2蛋白及P-JNK蛋白均無明顯變化(P0.05)。結(jié)論1.在體外細(xì)胞實(shí)驗(yàn)S100β能夠抑制OECs釋放炎癥相關(guān)因子。2.S100β可降低炎癥條件下OECs凋亡率,從而提高其存活率。3.S100β蛋白可能通過介導(dǎo)NFκB p65/MAPK p38通路調(diào)控炎癥環(huán)境中OECs的炎癥反應(yīng)機(jī)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- 緯 to establish olfactory ensheathing by adding recombinant IFN- 緯 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- 偽 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- 偽 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- 緯 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- 緯, 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- 緯 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.
【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R364.5

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