本文選題：雄激素 + 星形膠質(zhì)細胞��； 參考：《西南大學》2012年碩士論文

【摘要】：雄激素(Androgen)是體內(nèi)一類碳-19固醇物質(zhì)的總稱,包括睪酮(Testosterone, T)、雙氫睪酮(Dihydrotestosterone DHT)等。腦是雄激素作用的重要靶器官,雄激素不僅對腦的記憶與認知功能有一定的影響,還對神經(jīng)系統(tǒng)有保護調(diào)節(jié)作用。目前雄激素對神經(jīng)系統(tǒng)的保護作用的研究多以神經(jīng)元為研究對象。星形膠質(zhì)細胞作為腦內(nèi)數(shù)量最多的一種膠質(zhì)細胞,對神經(jīng)元具有營養(yǎng)、保護、支持、調(diào)節(jié)等重要的作用,對神經(jīng)系統(tǒng)的穩(wěn)定和功能的止常發(fā)揮有著重要的作用。研究雄激素對星形膠質(zhì)細胞的免疫調(diào)節(jié)機制,不僅為雄激素對神經(jīng)系統(tǒng)的保護調(diào)節(jié)作用提供理論依據(jù),還對一些神經(jīng)性疾病,尤其是與雄激素相關(guān)的一些老年性疾病的治療和預(yù)防有重要的理論價值。 為了更有效的研究雄激素對體外星形膠質(zhì)細胞的免疫學機制的作用,需要構(gòu)建合理的星形膠質(zhì)細胞體外培養(yǎng)體系。本實驗的原代星形膠質(zhì)細胞取自1-3日齡的SD大鼠的新生鼠,然后經(jīng)過原代細胞的分離培養(yǎng),并經(jīng)多次純化和傳代獲得所需細胞,所得細胞的純化率達99%以上�？蔀轶w外研究提供符合要求的星形膠質(zhì)細胞。 本實驗通過采用10μg/ml的LPS激發(fā)培養(yǎng)AST10h后,再分別用濃度為OnM、10nM、100nM的5α-DHT處理激發(fā)培養(yǎng)的AST24h。結(jié)果發(fā)現(xiàn)隨5a-DHT濃度增加細胞的活性下降,其中LPS+10nM5α-DHT組細胞活性較LPS+0nM5α-DHT組下降且差異顯著(P0.05),而與對照組相比差異不顯著(P0.05)。當采用10μg/ml的LPS激發(fā)培養(yǎng)AST10h后,再用濃度為10nM的5a-DHT處理激發(fā)培養(yǎng)的AST24h后,分別檢測0h、2h、4h、8h、12h、24h、36h、48h時星形膠質(zhì)細胞的活性,結(jié)果顯示24h時只加LPS組比LPS+5a-DHT組活性高14.03%,差異顯著(P0.05),其它組差異不顯著。 細胞的免疫功能與細胞周期有一定相關(guān)性,本實驗用10μg/ml LPS激發(fā)培養(yǎng)AST10h后,加入含濃度為OnM、10nM5α-DHT的培養(yǎng)液,繼續(xù)培養(yǎng)24h后收集細胞進行細胞周期檢測。經(jīng)流式細胞技術(shù)檢測分析,結(jié)果發(fā)現(xiàn)10nM5a-DHT能顯著抑制10μg/ml LPS誘導(dǎo)的S+G2期細胞比率的升高(P0.05)。 星形膠質(zhì)細胞發(fā)生炎癥反應(yīng)時,會發(fā)生超微結(jié)構(gòu)的改變,為了解雄激素對其超微結(jié)構(gòu)的調(diào)節(jié)作用,本試驗采用10μg/ml LPS激發(fā)AST10h后,試驗組用10nM5α-DHT繼續(xù)處理24h,對照組加0nM5α-DHT處理,利用常規(guī)培養(yǎng)細胞電鏡制備技術(shù)制作電鏡標本。結(jié)果表明10μg/ml LPS使AST粗面內(nèi)質(zhì)網(wǎng)擴張,部分線粒體腫脹,甚至空泡化,而LPS+10nM5α-DHT組異常的粗面內(nèi)質(zhì)網(wǎng)和線粒體明顯減少。 為了了解雄激素對星形膠質(zhì)細胞免疫介質(zhì)分泌水平的影響,試驗用10μg/ml LPS激發(fā)培養(yǎng)AST10h后,加入含濃度為0nM、10nM5α-DHT的培養(yǎng)液,繼續(xù)培養(yǎng)24h后測定AST NO、 IL-1β和TNF-α水平。發(fā)現(xiàn),隨著雄激素濃度升高,由LPS誘導(dǎo)的AST NO、IL-1β和TNF-α的分泌水平下降,其中10nM5α-DHT可以顯著抑制LPS誘導(dǎo)的NO、IL-1β和TNF-α的分泌(P0.05)。 為了進一步確定AST NO、IL-1β和TNF-α的分泌水平下降是雄激素作用的結(jié)果,實驗用10μg/ml LPS激發(fā)培養(yǎng)AST10h后,加入含濃度為0nM5α-DHT、10nM5α-DHT、10nM5α-DHT+100nM氟他胺的培養(yǎng)液,繼續(xù)培養(yǎng)24h后測定AST NO、IL-1β和TNF-α水平。結(jié)果發(fā)現(xiàn),加氟他胺組雄激素失去對LPS的抑制作用。同時,檢測10μg/ml LPS+10nM5α-DHT組和10μg/ml LPS+10nM5α-DHT+100Nm氟他胺組0.5h、1h、2h、8h、12h、24h時AST NO、 IL-1β和TNF-α水平,發(fā)現(xiàn)10μg/ml LPS+10nM5α-DHT組ASTNO、IL-1β和TNF-α的分泌逐漸下降,10μg/ml LPS+10nM5α-DHT100Nm氟他胺組AST NO、IL-1β和TNF-α的分泌逐漸升高。 為了了解雄激素作用的信號轉(zhuǎn)導(dǎo)路徑,試驗用10μg/ml LPS激發(fā)培養(yǎng)AST10h后,添加10nM5α-DHT,在加5α-DHT前30min分別加20μM p38抑制物SB203580、20μM ERK-1/-2抑制物U0126,對照組加0μM SB203580和U0126,繼續(xù)培養(yǎng)10min后測定AST NO、IL-1β和TNF-α水平。結(jié)果發(fā)現(xiàn)雄激素可以通過ERK-1/-2轉(zhuǎn)導(dǎo)路徑調(diào)節(jié)AST免疫反應(yīng)。檢測20μM SB203580和20μM U0126組5min、10min、20min、30min時AST NO、IL-1β和TNF-α水平,結(jié)果顯示20μM SB203580組AST NO和IL-1β水平變化不顯著,而TNF-α水平逐漸下降,20μM U0126組AST NO、IL-1β和TNF-α水平均無顯著變化。 綜上所述,本試驗可以得到如下結(jié)論： 1、本試驗構(gòu)建了雄激素調(diào)節(jié)AST功能的體外研究模型。 2、雄激素可以顯著抑制10μg/ml LPS引起的AST細胞活性升高及AST的過度增殖,降低S+G2期細胞比率,保護AST超微結(jié)構(gòu)正常。 3、雄激素通過ERK-1/-2轉(zhuǎn)導(dǎo)路徑抑制由10μg/ml LPS引起的AST TNF-α水平升高。
[Abstract]:Androgen (Androgen) is the general name of a class of carbon -19 sterol substances in the body, including testosterone (Testosterone, T), and dihydrotestosterone (Dihydrotestosterone DHT). The brain is an important target organ for androgen action. Androgen not only has a certain effect on the memory and cognitive function of the brain, but also protects the nervous system. Androgen is present to the God at present. The study of the protective effect of the system is mostly based on the study of neurons. Astrocytes are the most important glial cells in the brain. They have important roles in nutrition, protection, support and regulation of neurons. It plays an important role in the stability and function of the nervous system. The study of androgens to astrocytes The immunomodulatory mechanism not only provides a theoretical basis for the regulation of the androgens for the protection of the nervous system, but also has important theoretical value for the treatment and prevention of some neuropathic diseases, especially some old androgen related diseases.
In order to more effectively study the role of androgens in the immunological mechanism of astrocytes in vitro, a reasonable in vitro culture system of astrocytes is needed. The primary astrocytes of this experiment were taken from the newborn rats of the SD rats of 1-3 days old, and then were isolated and cultured from the primary cells, and were obtained by multiple purification and generation. The purity of the cells was more than 99%, which could provide astrocytes for the in vitro study.
In this experiment, AST10h was stimulated with 10 g/ml LPS, and the results of AST24h. with a concentration of OnM, 10nM, and 100nM in the 5 alpha -DHT treatment showed that the cell activity decreased with the increase of 5a-DHT concentration, and the activity of LPS+10nM5 alpha -DHT group decreased and the difference was significant compared with those of the control group, but the difference was compared with the control group. The activity of astrocytes in 0h, 2h, 4h, 8h, 12h, 8h, 8h, 12h, 4h, 8h, 4h, 8h, 4h, 8h, 8h, 12h, g/ml were detected, and the differences in other groups were not significant when the AST10h was stimulated with 10 - g/ml LPS.
The immune function of the cell has a certain correlation with the cell cycle. In this experiment, the culture of AST10h was stimulated with 10 mu g/ml LPS, and the culture solution containing OnM, 10nM5 alpha -DHT was added. After continuing to cultivate 24h, cells were collected for cell cycle detection. The result of flow cytometry analysis showed that 10nM5a-DHT could significantly inhibit the S+G2 10 g/ml LPS induced S+G2. The increase of cell ratio (P0.05).
In order to solve the ultrastructural changes of astrocytes in the inflammatory reaction, in order to solve the regulation of the ultrastructure of androgen, the test group used 10 g/ml LPS to stimulate AST10h, the experimental group continued to treat 24h with 10nM5 alpha -DHT, and the control group was treated with 0nM5 alpha -DHT, and the electron microscope preparation technique was used to produce electron microscope specimens. The results showed that 10 g/ml LPS dilated the rough endoplasmic reticulum of AST, partly mitochondria swelled and even vacuolated, while the abnormal rough endoplasmic reticulum and mitochondria in the LPS+10nM5 alpha -DHT group decreased significantly.
In order to understand the effect of androgens on the secretory level of the astrocyte immune media, the experiment was stimulated with 10 g/ml LPS to stimulate AST10h, adding a medium containing a concentration of 0nM, 10nM5 alpha -DHT. After continuing to cultivate 24h, AST NO, IL-1 beta and TNF- alpha levels were measured. The level of secretion decreased, and 10nM5 alpha -DHT could significantly inhibit the secretion of NO, IL-1 beta and TNF- alpha induced by LPS (P0.05).
In order to further determine AST NO, the decrease of the secretion level of IL-1 beta and TNF- alpha is the result of androgen action. After the experiment was stimulated by 10 mu g/ml LPS, the culture medium containing 0nM5 a -DHT, 10nM5 alpha -DHT and 10nM5 alpha fluorodiamine was added. At the same time, 10 g/ml LPS+10nM5 alpha -DHT group and 10 g/ml LPS+10nM5 alpha -DHT+100Nm flutamines group 0.5h, 1H, 2h, 8h, 12h. The secretion of -1 beta and TNF- alpha was increased gradually.
In order to understand the signal transduction pathway of androgens, the experiment was stimulated with 10 mu g/ml LPS to stimulate AST10h, and 10nM5 alpha -DHT was added to 30min before adding 20 mu M p38 inhibitor SB203580,20 mu M ERK-1/-2 inhibitor before adding 5 alpha -DHT. Androgen could regulate the AST immune response through the ERK-1/-2 pathway. The levels of 5min, 10min, 20min, 30min AST NO, 10min, 20min and 30min were detected in the 5min, 10min, 20min and 30min groups of the group of AST, 5min, 10min, 20min, and 30min. The results showed that the level of 20 mu was not significant, but the level of alpha and alpha decreased gradually, and the level of beta and alpha was not obvious. Change.
To sum up, this experiment can get the following conclusions:
1, we constructed an in vitro model of androgen regulating AST function.
2, androgen can significantly inhibit the activity of AST cells induced by 10 micron g/ml LPS and the excessive proliferation of AST, reduce the ratio of S+G2 phase cells, and protect the ultrastructure of AST.
3, androgen inhibits elevated AST TNF- alpha level induced by 10 mu g/ml LPS through ERK-1/-2 transduction pathway.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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