本文選題：子宮肌瘤　切入點(diǎn)：中介體復(fù)合物亞基12　出處：《南昌大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：背景與目的:子宮肌瘤是婦科常見的良性腫瘤,子宮肌瘤最主要的臨床表現(xiàn)是異常子宮出血、盆腔痛和貧血,其對女性生活質(zhì)量的影響不容忽視。國外有研究報(bào)道子宮肌瘤中中介體復(fù)合物亞基12(Mediator complex subunit 12,MED12)基因的高頻突變,其中約70%的子宮肌瘤患者病灶組織中存在該基因突變,且大部分(90%)位于MED12基因的第44位甘氨酸(MED12 p.G44)。本研究為了確定子宮肌瘤患者的MED12基因突變譜,對MED12好發(fā)突變的區(qū)域進(jìn)行大規(guī)模測序;并且構(gòu)建MED12基因野生型和突變型質(zhì)粒,通過轉(zhuǎn)染至293T細(xì)胞,繼而檢測293T細(xì)胞的活力、侵襲、遷移、凋亡及周期情況,在細(xì)胞水平上探索MED12基因突變對子宮肌瘤的發(fā)生發(fā)展的影響。方法:首先從322名單、多發(fā)性子宮肌瘤患者中收取362個肌瘤樣本,提取子宮肌瘤組織總DNA,進(jìn)行PCR擴(kuò)增,然后進(jìn)行測序、序列比對,找出潛在突變。然后構(gòu)建pCMV6-hMED12-DDK質(zhì)粒,并將質(zhì)粒轉(zhuǎn)染至293T細(xì)胞。分為4組:(1)空載質(zhì)粒,(2)空白細(xì)胞,(3)MED12突變型,(4)野生型,使用Western blotting(WB)檢測MED12蛋白的表達(dá);CCK8法檢測細(xì)胞活力;Transwell檢測細(xì)胞遷移和侵襲;流式細(xì)胞法檢測細(xì)胞的凋亡水平;PI染色法檢測細(xì)胞周期。結(jié)果:通過測序證明子宮肌瘤患者的362個樣本中,158例MED12突變(43.6%);第44位甘氨酸(G)突變?yōu)樘於彼?D)的比例最高(18.5%)。本研究中并未發(fā)現(xiàn)MED12L的突變。MED12的突變與各項(xiàng)臨床特征無相關(guān)。利用CCK8法檢測發(fā)現(xiàn)MED12的突變型(G44D)能夠提高293T細(xì)胞活力,且有統(tǒng)計(jì)學(xué)差異(P0.01),然而細(xì)胞凋亡及細(xì)胞周期并無統(tǒng)計(jì)學(xué)差異。結(jié)論:1.本研究中發(fā)現(xiàn)子宮肌瘤中存在高頻率的MED12基因突變(43.6%,158/362),MED12突變較少發(fā)生于相鄰的子宮肌層。此外并未發(fā)現(xiàn)子宮肌瘤患者中存在MED12L突變。2.MED12突變在多發(fā)性子宮肌瘤不同瘤體中具有不同的突變形式,表明多發(fā)性子宮肌瘤中不同瘤體的起源不同。3.MED12可能通過影響細(xì)胞活力而促進(jìn)子宮肌瘤的發(fā)病。
[Abstract]:Background & objective: uterine leiomyoma is a common benign tumor in gynecology. The main clinical manifestations of uterine leiomyoma are abnormal uterine bleeding, pelvic pain and anemia. The influence on the quality of life of women can not be ignored. Some foreign studies have reported the high frequency mutation of mediator complex subunit 12MED12) gene in uterine leiomyoma, and about 70% of the patients with uterine leiomyoma have this gene mutation. In order to determine the mutation profile of MED12 gene in patients with uterine leiomyoma, we sequenced the region of MED12 mutation on a large scale, and constructed the wild-type and mutant plasmids of MED12 gene. After transfection to 293T cells, the activity, invasion, migration, apoptosis and cell cycle of 293T cells were detected, and the effect of MED12 gene mutation on the occurrence and development of uterine leiomyoma was explored at the cell level. A total of 362 myoma samples were collected from multiple uterine leiomyoma patients. The total DNA of uterine leiomyoma tissue was extracted, amplified by PCR, sequenced, sequenced, sequenced, and potential mutations were identified. Then pCMV6-hMED12-DDK plasmid was constructed. The plasmids were transfected into 293T cells and divided into 4 groups: 1) empty plasmids were divided into 4 groups. (1) empty cells were divided into 4 groups. (2) the wild type of empty cells was identified. The expression of MED12 protein was detected by Western blotting and MED12 protein expression was detected by Western blotting.Transwell assay was used to detect cell migration and invasion. Flow cytometry was used to detect the level of apoptosis and Pi staining to detect cell cycle. Results: the proportion of MED12 mutation in 158 patients with uterine leiomyoma was confirmed by sequencing. The 44th GG mutation was aspartic acid D). The mutation of MED12L, MED12, was not related to the clinical characteristics. The mutational type G44D of MED12 was detected by CCK8 method, and the activity of 293T cells was increased. However, there was no significant difference in cell apoptosis and cell cycle. Conclusion: 1. In this study, we found that there is a high frequency mutation of MED12 gene in uterine leiomyoma. The mutation of MED12 in 1588 / 362 is less common in the myometrium than in the adjacent myometrium. No MED12L mutation was found in patients with uterine leiomyoma. 2. MED12 mutation had different mutation forms in different tumors of multiple uterine leiomyoma. It is suggested that the origin of different tumors in multiple uterine leiomyomas is different. 3. MED12 may promote the pathogenesis of uterine leiomyomas by affecting cell viability.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R737.33

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1 吳娟;子宮肌瘤中MED12基因突變的研究[D];南昌大學(xué);2016年

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本文編號：1598932