本文關(guān)鍵詞：GPR30在子宮平滑肌瘤細(xì)胞增殖中的作用研究　出處：《浙江大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： GPR30 ERK-1/2 MTT 子宮平滑肌瘤 細(xì)胞增殖

【摘要】：研究背景及目的： 子宮肌瘤是婦科的常見病,其發(fā)病機(jī)制尚不清楚。大量的研究表明子宮平滑肌瘤的發(fā)生與雌激素的關(guān)系密切。傳統(tǒng)雌激素核受體ERα、β作為轉(zhuǎn)錄調(diào)節(jié)因子已被廣泛接受,但是大量的研究表明子宮肌瘤患者和正常育齡期婦女的循環(huán)血液中雌激素的水平?jīng)]有明顯差異,這提示子宮肌瘤的發(fā)生與局部的雌激素效應(yīng)有關(guān)�？缒蛋白偶聯(lián)受體(GPR30)(G protein-coupled receptors,G蛋白偶聯(lián)受體)分子量大小約40KD,基因定位于染色體7p22上,主要位于細(xì)胞質(zhì)的內(nèi)質(zhì)網(wǎng)上,是激活ERK-1/2的另一類主要的跨膜信號(hào)受體蛋白。GPR30有α、β、γ、和6四個(gè)亞基。多項(xiàng)研究證實(shí)GPR30可與雌激素特異性結(jié)合快速介導(dǎo)雌激素效應(yīng),這可能與育齡婦女子宮局部高雌激素效應(yīng)的表達(dá)有關(guān)。本研究希望可以進(jìn)一步探索GPR30在子宮肌瘤發(fā)病中的作用。 方法： (1)取材,子宮平滑肌瘤原代細(xì)胞培養(yǎng),培養(yǎng)成功后7-8天傳代。 (2)平滑肌瘤細(xì)胞鑒定,應(yīng)用HE染色法,在倒置相差顯微鏡下觀察細(xì)胞大小、形態(tài)、生長特點(diǎn)及排列方式等進(jìn)行平滑肌瘤細(xì)胞鑒定。 (3)免疫熒光法分析GPR30在子宮平滑肌瘤細(xì)胞中的表達(dá)。 (4)蛋白質(zhì)印跡法(Western Blot)檢測子宮肌瘤細(xì)胞中GPR30的表達(dá)。 (5)蛋白質(zhì)印跡法(Western Blot)檢測未處理組子宮肌瘤細(xì)胞及添加雌二醇的子宮肌瘤細(xì)胞在培養(yǎng)24h、48h后細(xì)胞中Erk1/2表達(dá)的差異。 (6)蛋白質(zhì)印跡法(Western Blot)檢測正常子宮肌瘤細(xì)胞及GPR30-siRNA轉(zhuǎn)染的子宮肌瘤細(xì)胞在培養(yǎng)24h、48h后細(xì)胞中Erkl/2表達(dá)的差異。 (7)使用MTT方法并以時(shí)間和OD570數(shù)值繪制細(xì)胞生長曲線。 結(jié)果： (1)培養(yǎng)1天后,大部分細(xì)胞已貼壁。換液后3天貼壁細(xì)胞完全伸展。多數(shù)呈長梭型,細(xì)胞伸出較長的突起并相互接觸,部分區(qū)域可見典型的“峰一谷”結(jié)構(gòu),培養(yǎng)7-8天細(xì)胞鋪滿瓶底,并鑒定為平滑肌細(xì)胞。 (2) Western Blot檢測人子宮肌瘤以及正常子宮平滑肌組織中GPR30蛋白的表達(dá)情況,結(jié)果顯示GPR30在子宮平滑肌瘤和正常子宮平滑肌組織中皆有表達(dá)。 (3) Western Blot表明在雌激素的作用下,子宮平滑肌瘤細(xì)胞中Erkl/2的表達(dá)較未處理組正常子宮平滑肌瘤細(xì)胞顯著升高；經(jīng)siRNA干擾GPR30后,干擾組子宮平滑肌瘤細(xì)胞中Erk1/2的表達(dá)顯著低于未處理組平滑肌細(xì)胞。 (4)細(xì)胞生長曲線分析顯示,在雌激素的刺激下,空白載體對照組細(xì)胞的生長明顯上升(OD數(shù)值增加),GPR30干擾組細(xì)胞的生長活性和生存率顯著下降。 結(jié)論： GPR30在子宮肌瘤平滑肌瘤細(xì)胞的細(xì)胞漿中高表達(dá),可能介導(dǎo)雌激素通過ERK通路調(diào)節(jié)子宮平滑肌瘤細(xì)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 偽. 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 偽, 尾, 緯. 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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R737.33

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