本文選題：低氧　切入點：支持細胞　出處：《青島大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：觀察低氧對原代大鼠睪丸支持細胞生長活性和occludin蛋白表達量的影響,探討低氧導(dǎo)致不育機制。 方法：18～22d齡Wistar大鼠,取出睪丸,通過兩步酶消化法,建立體外支持細胞培養(yǎng)體系,使用油紅0、HE染色和免疫熒光法鑒定。經(jīng)胰蛋白酶消化傳代后隨機分為以下氧濃度組進行培養(yǎng)：20%、15%、10%、5%和1%,分別培養(yǎng)至6h、12h、24h、48h和72h,CCK-8方法測定細胞增值活性,倒置顯微鏡下觀察細胞的形態(tài)變化,Western blot測定occludin蛋白表達量,并進行差異性分析。 結(jié)果：原代支持細胞的體外生長周期為14d左右。油紅0染色可見胞質(zhì)內(nèi)脂滴染成紅色,細胞胞體寬大；HE染色見細胞貼壁片狀生長,細胞核可見“雙極小體”；免疫熒光可見Fasl蛋白表達陽性,支持細胞的純度95%。CCK-8結(jié)果顯示：與20%氧濃度相比,細胞在15%和10%氧濃度下增值率逐漸降低,5%和1%氧濃度下,細胞存活率明顯下降(P0.01)；倒置顯微鏡下可見細胞胞體明顯回縮,細胞間隙增大,胞質(zhì)內(nèi)吞噬顆粒增多,細胞膜層結(jié)構(gòu)缺失,凋亡細胞增多。Western blot結(jié)果顯示：細胞培養(yǎng)6小時,各低氧組的occludin表達量沒有明顯性差異(P0.05)；培養(yǎng)12小時后,隨著時間延長和氧濃度降低,occludin蛋白的表達量逐漸降低,呈現(xiàn)明顯時間和濃度依賴性變化,差異有顯著性(P0.01)。 結(jié)論：本研究結(jié)果顯示,低氧體外培養(yǎng)對大鼠睪丸支持細胞的生長具有明顯抑制作用,減少occludin蛋白表達量。我們推測低氧環(huán)境會對睪丸支持細胞參與形成的血睪屏障產(chǎn)生不良影響甚至破壞作用,使支持細胞之間的緊密連接發(fā)生變化,因而影響睪丸正常的生精過程的微環(huán)境,導(dǎo)致雄性動物生育能力降低或不育。
[Abstract]:Aim: to investigate the effects of hypoxia on the growth activity and occludin protein expression of primary rat testicular Sertoli cells. Methods the testis were removed from the testis of Wistar rats at the age of 22 days. The culture system of Sertoli cells in vitro was established by two-step enzyme digestion. After digestion and passage with trypsin, the cells were randomly divided into the following oxygen concentration groups: 10 5% and 1% respectively. The cell proliferation activity was measured by CCK-8 method, which was cultured to 6 h, 12 h, 24 h, 24 h, 48 h, and 72 h, respectively. The morphological changes of cells were observed under inverted microscope. The expression of occludin protein was detected by Western blot and the difference was analyzed. Results: the growth cycle of primary Sertoli cells in vitro was about 14 days. "Bipolar bodies" were found in the nucleus, Fasl protein was positive in immunofluorescence, the purity of Sertoli cells was 95. CCK-8. The results showed that compared with the oxygen concentration of 20%, the proliferation rate of the cells at 15% and 10% oxygen concentration decreased gradually by 5% and 1% oxygen concentration, respectively. The cell survival rate was significantly decreased (P 0.01), the cell body was obviously retracted, the intercellular space was enlarged, the number of phagocytic granules in the cytoplasm was increased, the structure of cell membrane layer was missing, and the number of apoptotic cells was increased. Western blot showed that the cells were cultured for 6 hours. There was no significant difference in the expression of occludin in different hypoxia groups (P 0.05). After 12 hours of culture, the expression of occludin protein gradually decreased with the prolongation of time and the decrease of oxygen concentration, showing a significant time and concentration-dependent change, and the difference was significant (P 0.01). Conclusion: the results showed that hypoxic culture could inhibit the growth of rat testicular Sertoli cells in vitro. We speculate that hypoxia may have a negative effect on or even destroy the blood-testis barrier in which testicular Sertoli cells are involved, resulting in changes in the tight connections between Sertoli cells. This affects the microenvironment of the normal spermatogenesis process in the testis, leading to a decrease in fertility or infertility in male animals.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363

【參考文獻】

相關(guān)期刊論文 前3條

1 呂偉宏;田懷軍;;低氧對大鼠睪丸支持細胞形態(tài)結(jié)構(gòu)與存活率的影響[J];第三軍醫(yī)大學(xué)學(xué)報;2007年01期

2 段秀慶,宋純,許評,宋春芳;大鼠FasL~+Sertoli細胞的分離與鑒定[J];基礎(chǔ)醫(yī)學(xué)與臨床;2004年01期

3 金毅;呂麗飛;李德超;;精索內(nèi)靜脈高位結(jié)扎術(shù)聯(lián)合高壓氧治療精索靜脈曲張伴精液異常的療效觀察[J];中華男科學(xué)雜志;2007年01期

，

本文編號：1627208