雄激素调节星形胶质细胞免疫的机制研究

发布时间：2018-06-01 10:21

本文选题：雄激素 + 星形胶质细胞　；参考：《西南大学》2012年硕士论文

【摘要】：雄激素(Androgen)是体内一类碳-19固醇物质的总称,包括睾酮(Testosterone, T)、双氢睾酮(Dihydrotestosterone DHT)等。脑是雄激素作用的重要靶器官,雄激素不仅对脑的记忆与认知功能有一定的影响,还对神经系统有保护调节作用。目前雄激素对神经系统的保护作用的研究多以神经元为研究对象。星形胶质细胞作为脑内数量最多的一种胶质细胞,对神经元具有营养、保护、支持、调节等重要的作用,对神经系统的稳定和功能的止常发挥有着重要的作用。研究雄激素对星形胶质细胞的免疫调节机制,不仅为雄激素对神经系统的保护调节作用提供理论依据,还对一些神经性疾病,尤其是与雄激素相关的一些老年性疾病的治疗和预防有重要的理论价值。为了更有效的研究雄激素对体外星形胶质细胞的免疫学机制的作用,需要构建合理的星形胶质细胞体外培养体系。本实验的原代星形胶质细胞取自1-3日龄的SD大鼠的新生鼠,然后经过原代细胞的分离培养,并经多次纯化和传代获得所需细胞,所得细胞的纯化率达99%以上。可为体外研究提供符合要求的星形胶质细胞。本实验通过采用10μg/ml的LPS激发培养AST10h后,再分别用浓度为OnM、10nM、100nM的5α-DHT处理激发培养的AST24h。结果发现随5a-DHT浓度增加细胞的活性下降,其中LPS+10nM5α-DHT组细胞活性较LPS+0nM5α-DHT组下降且差异显著(P0.05),而与对照组相比差异不显著(P0.05)。当采用10μg/ml的LPS激发培养AST10h后,再用浓度为10nM的5a-DHT处理激发培养的AST24h后,分别检测0h、2h、4h、8h、12h、24h、36h、48h时星形胶质细胞的活性,结果显示24h时只加LPS组比LPS+5a-DHT组活性高14.03%,差异显著(P0.05),其它组差异不显著。细胞的免疫功能与细胞周期有一定相关性,本实验用10μg/ml LPS激发培养AST10h后,加入含浓度为OnM、10nM5α-DHT的培养液,继续培养24h后收集细胞进行细胞周期检测。经流式细胞技术检测分析,结果发现10nM5a-DHT能显著抑制10μg/ml LPS诱导的S+G2期细胞比率的升高(P0.05)。星形胶质细胞发生炎症反应时,会发生超微结构的改变,为了解雄激素对其超微结构的调节作用,本试验采用10μg/ml LPS激发AST10h后,试验组用10nM5α-DHT继续处理24h,对照组加0nM5α-DHT处理,利用常规培养细胞电镜制备技术制作电镜标本。结果表明10μg/ml LPS使AST粗面内质网扩张,部分线粒体肿胀,甚至空泡化,而LPS+10nM5α-DHT组异常的粗面内质网和线粒体明显减少。为了了解雄激素对星形胶质细胞免疫介质分泌水平的影响,试验用10μg/ml LPS激发培养AST10h后,加入含浓度为0nM、10nM5α-DHT的培养液,继续培养24h后测定AST NO、 IL-1β和TNF-α水平。发现,随着雄激素浓度升高,由LPS诱导的AST NO、IL-1β和TNF-α的分泌水平下降,其中10nM5α-DHT可以显著抑制LPS诱导的NO、IL-1β和TNF-α的分泌(P0.05)。为了进一步确定AST NO、IL-1β和TNF-α的分泌水平下降是雄激素作用的结果,实验用10μg/ml LPS激发培养AST10h后,加入含浓度为0nM5α-DHT、10nM5α-DHT、10nM5α-DHT+100nM氟他胺的培养液,继续培养24h后测定AST NO、IL-1β和TNF-α水平。结果发现,加氟他胺组雄激素失去对LPS的抑制作用。同时,检测10μg/ml LPS+10nM5α-DHT组和10μg/ml LPS+10nM5α-DHT+100Nm氟他胺组0.5h、1h、2h、8h、12h、24h时AST NO、 IL-1β和TNF-α水平,发现10μg/ml LPS+10nM5α-DHT组ASTNO、IL-1β和TNF-α的分泌逐渐下降,10μg/ml LPS+10nM5α-DHT100Nm氟他胺组AST NO、IL-1β和TNF-α的分泌逐渐升高。为了了解雄激素作用的信号转导路径,试验用10μg/ml LPS激发培养AST10h后,添加10nM5α-DHT,在加5α-DHT前30min分别加20μM p38抑制物SB203580、20μM ERK-1/-2抑制物U0126,对照组加0μM SB203580和U0126,继续培养10min后测定AST NO、IL-1β和TNF-α水平。结果发现雄激素可以通过ERK-1/-2转导路径调节AST免疫反应。检测20μM SB203580和20μM U0126组5min、10min、20min、30min时AST NO、IL-1β和TNF-α水平,结果显示20μM SB203580组AST NO和IL-1β水平变化不显著,而TNF-α水平逐渐下降,20μM U0126组AST NO、IL-1β和TNF-α水平均无显著变化。综上所述,本试验可以得到如下结论： 1、本试验构建了雄激素调节AST功能的体外研究模型。 2、雄激素可以显著抑制10μg/ml LPS引起的AST细胞活性升高及AST的过度增殖,降低S+G2期细胞比率,保护AST超微结构正常。 3、雄激素通过ERK-1/-2转导路径抑制由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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

【参考文献】