子宫肌瘤中MED12基因突变的研究

发布时间：2018-03-11 16:18

本文选题：子宫肌瘤　切入点：中介体复合物亚基12　出处：《南昌大学》2016年博士论文　论文类型：学位论文

【摘要】：背景与目的:子宫肌瘤是妇科常见的良性肿瘤,子宫肌瘤最主要的临床表现是异常子宫出血、盆腔痛和贫血,其对女性生活质量的影响不容忽视。国外有研究报道子宫肌瘤中中介体复合物亚基12(Mediator complex subunit 12,MED12)基因的高频突变,其中约70%的子宫肌瘤患者病灶组织中存在该基因突变,且大部分(90%)位于MED12基因的第44位甘氨酸(MED12 p.G44)。本研究为了确定子宫肌瘤患者的MED12基因突变谱,对MED12好发突变的区域进行大规模测序;并且构建MED12基因野生型和突变型质粒,通过转染至293T细胞,继而检测293T细胞的活力、侵袭、迁移、凋亡及周期情况,在细胞水平上探索MED12基因突变对子宫肌瘤的发生发展的影响。方法:首先从322名单、多发性子宫肌瘤患者中收取362个肌瘤样本,提取子宫肌瘤组织总DNA,进行PCR扩增,然后进行测序、序列比对,找出潜在突变。然后构建pCMV6-hMED12-DDK质粒,并将质粒转染至293T细胞。分为4组:(1)空载质粒,(2)空白细胞,(3)MED12突变型,(4)野生型,使用Western blotting(WB)检测MED12蛋白的表达;CCK8法检测细胞活力;Transwell检测细胞迁移和侵袭;流式细胞法检测细胞的凋亡水平;PI染色法检测细胞周期。结果:通过测序证明子宫肌瘤患者的362个样本中,158例MED12突变(43.6%);第44位甘氨酸(G)突变为天冬氨酸(D)的比例最高(18.5%)。本研究中并未发现MED12L的突变。MED12的突变与各项临床特征无相关。利用CCK8法检测发现MED12的突变型(G44D)能够提高293T细胞活力,且有统计学差异(P0.01),然而细胞凋亡及细胞周期并无统计学差异。结论:1.本研究中发现子宫肌瘤中存在高频率的MED12基因突变(43.6%,158/362),MED12突变较少发生于相邻的子宫肌层。此外并未发现子宫肌瘤患者中存在MED12L突变。2.MED12突变在多发性子宫肌瘤不同瘤体中具有不同的突变形式,表明多发性子宫肌瘤中不同瘤体的起源不同。3.MED12可能通过影响细胞活力而促进子宫肌瘤的发病。
[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.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.33

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