GPR30在子宫平滑肌瘤细胞增殖中的作用研究

发布时间：2018-01-07 12:29

本文关键词：GPR30在子宫平滑肌瘤细胞增殖中的作用研究　出处：《浙江大学》2015年硕士论文　论文类型：学位论文

【摘要】：研究背景及目的：子宫肌瘤是妇科的常见病,其发病机制尚不清楚。大量的研究表明子宫平滑肌瘤的发生与雌激素的关系密切。传统雌激素核受体ERα、β作为转录调节因子已被广泛接受,但是大量的研究表明子宫肌瘤患者和正常育龄期妇女的循环血液中雌激素的水平没有明显差异,这提示子宫肌瘤的发生与局部的雌激素效应有关。跨膜G蛋白偶联受体(GPR30)(G protein-coupled receptors,G蛋白偶联受体)分子量大小约40KD,基因定位于染色体7p22上,主要位于细胞质的内质网上,是激活ERK-1/2的另一类主要的跨膜信号受体蛋白。GPR30有α、β、γ、和6四个亚基。多项研究证实GPR30可与雌激素特异性结合快速介导雌激素效应,这可能与育龄妇女子宫局部高雌激素效应的表达有关。本研究希望可以进一步探索GPR30在子宫肌瘤发病中的作用。方法： (1)取材,子宫平滑肌瘤原代细胞培养,培养成功后7-8天传代。 (2)平滑肌瘤细胞鉴定,应用HE染色法,在倒置相差显微镜下观察细胞大小、形态、生长特点及排列方式等进行平滑肌瘤细胞鉴定。 (3)免疫荧光法分析GPR30在子宫平滑肌瘤细胞中的表达。 (4)蛋白质印迹法(Western Blot)检测子宫肌瘤细胞中GPR30的表达。 (5)蛋白质印迹法(Western Blot)检测未处理组子宫肌瘤细胞及添加雌二醇的子宫肌瘤细胞在培养24h、48h后细胞中Erk1/2表达的差异。 (6)蛋白质印迹法(Western Blot)检测正常子宫肌瘤细胞及GPR30-siRNA转染的子宫肌瘤细胞在培养24h、48h后细胞中Erkl/2表达的差异。 (7)使用MTT方法并以时间和OD570数值绘制细胞生长曲线。结果： (1)培养1天后,大部分细胞已贴壁。换液后3天贴壁细胞完全伸展。多数呈长梭型,细胞伸出较长的突起并相互接触,部分区域可见典型的“峰一谷”结构,培养7-8天细胞铺满瓶底,并鉴定为平滑肌细胞。 (2) Western Blot检测人子宫肌瘤以及正常子宫平滑肌组织中GPR30蛋白的表达情况,结果显示GPR30在子宫平滑肌瘤和正常子宫平滑肌组织中皆有表达。 (3) Western Blot表明在雌激素的作用下,子宫平滑肌瘤细胞中Erkl/2的表达较未处理组正常子宫平滑肌瘤细胞显著升高；经siRNA干扰GPR30后,干扰组子宫平滑肌瘤细胞中Erk1/2的表达显著低于未处理组平滑肌细胞。 (4)细胞生长曲线分析显示,在雌激素的刺激下,空白载体对照组细胞的生长明显上升(OD数值增加),GPR30干扰组细胞的生长活性和生存率显著下降。结论： GPR30在子宫肌瘤平滑肌瘤细胞的细胞浆中高表达,可能介导雌激素通过ERK通路调节子宫平滑肌瘤细胞的增殖。
[Abstract]:Background and objectives: Uterine leiomyoma is a common gynecological disease, its pathogenesis is not clear. A large number of studies show that the occurrence of uterine leiomyoma is closely related to estrogen. The traditional estrogen receptor ER 伪. Beta as a transcription regulator has been widely accepted, but a large number of studies show that there is no significant difference in the level of estrogen in circulating blood between uterine leiomyoma patients and women of normal reproductive age. This suggests that the occurrence of uterine leiomyoma is related to the local estrogen effect. The transmembrane G protein coupled receptor (GPR30) G protein-coupled receptors is involved in the development of uterine leiomyoma. The molecular weight of G protein coupled receptor is about 40 KD, and the gene is located on chromosome 7p22, mainly located on the endoplasmic reticulum of the cytoplasm. Another major transmembrane signal receptor protein. GPR30, which activates ERK-1/2, has 伪, 尾, 纬. And 64 subunits. Multiple studies have confirmed that GPR30 can specifically bind with estrogen to mediate estrogen effect quickly. This study hopes to further explore the role of GPR30 in the pathogenesis of uterine leiomyoma. Methods: The primary cells of uterine leiomyoma were cultured 7-8 days after successful culture. (2) leiomyoma cells were identified by HE staining. The size, morphology, growth characteristics and arrangement of the cells were observed under inverted phase contrast microscope. The expression of GPR30 in uterine leiomyoma cells was analyzed by immunofluorescence assay. The expression of GPR30 in uterine leiomyoma cells was detected by Western blot. (5) Western blot was used to detect the myoma cells of the untreated group and the fibroid cells supplemented with estradiol for 24 hours. The difference of Erk1/2 expression in cells after 48 h. Western blot was used to detect normal uterine leiomyoma cells and GPR30-siRNA transfected uterine leiomyoma cells for 24 hours. The difference of Erkl/2 expression in cells after 48 h. MTT method and time and OD570 were used to draw cell growth curve. Results: 1) after 1 day of culture, most of the cells were adherent to the wall. 3 days after the fluid exchange, the adherent cells were completely extended. Most of the cells were of long fusiform, with longer protrusions and contact with each other. Typical "peak-valley" structure was found in some areas. After 7-8 days of culture, the cells were covered with flask bottom and identified as smooth muscle cells. Western Blot was used to detect the expression of GPR30 protein in human uterine leiomyoma and normal uterine smooth muscle tissue. The results showed that GPR30 was expressed in both uterine leiomyoma and normal uterine smooth muscle tissues. Western Blot showed that the expression of Erkl/2 in uterine leiomyoma cells was significantly higher than that in normal uterine leiomyoma cells. The expression of Erk1/2 in uterine leiomyoma cells in interference group was significantly lower than that in untreated group after GPR30 was interfered with by siRNA. (4) Cell growth curve analysis showed that under the stimulation of estrogen, the growth of blank vector control group increased significantly and OD value increased. The cell growth activity and survival rate decreased significantly in GPR30 interference group. Conclusion: The high expression of GPR30 in the cytoplasm of leiomyoma cells may mediate estrogen to regulate the proliferation of uterine leiomyoma cells through ERK pathway.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.33

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