低氧体外培养对支持细胞活性和occludin蛋白表达的影响

发布时间：2018-03-18 00:30

本文选题：低氧　切入点：支持细胞　出处：《青岛大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：观察低氧对原代大鼠睾丸支持细胞生长活性和occludin蛋白表达量的影响,探讨低氧导致不育机制。方法：18～22d龄Wistar大鼠,取出睾丸,通过两步酶消化法,建立体外支持细胞培养体系,使用油红0、HE染色和免疫荧光法鉴定。经胰蛋白酶消化传代后随机分为以下氧浓度组进行培养：20%、15%、10%、5%和1%,分别培养至6h、12h、24h、48h和72h,CCK-8方法测定细胞增值活性,倒置显微镜下观察细胞的形态变化,Western blot测定occludin蛋白表达量,并进行差异性分析。结果：原代支持细胞的体外生长周期为14d左右。油红0染色可见胞质内脂滴染成红色,细胞胞体宽大；HE染色见细胞贴壁片状生长,细胞核可见“双极小体”；免疫荧光可见Fasl蛋白表达阳性,支持细胞的纯度95%。CCK-8结果显示：与20%氧浓度相比,细胞在15%和10%氧浓度下增值率逐渐降低,5%和1%氧浓度下,细胞存活率明显下降(P0.01)；倒置显微镜下可见细胞胞体明显回缩,细胞间隙增大,胞质内吞噬颗粒增多,细胞膜层结构缺失,凋亡细胞增多。Western blot结果显示：细胞培养6小时,各低氧组的occludin表达量没有明显性差异(P0.05)；培养12小时后,随着时间延长和氧浓度降低,occludin蛋白的表达量逐渐降低,呈现明显时间和浓度依赖性变化,差异有显著性(P0.01)。结论：本研究结果显示,低氧体外培养对大鼠睾丸支持细胞的生长具有明显抑制作用,减少occludin蛋白表达量。我们推测低氧环境会对睾丸支持细胞参与形成的血睾屏障产生不良影响甚至破坏作用,使支持细胞之间的紧密连接发生变化,因而影响睾丸正常的生精过程的微环境,导致雄性动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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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