COX-2及钙离子通道蛋白与子宫肌瘤发病机制间的关系研究
发布时间：2018-09-02 05:36
【摘要】：研究背景 子宫肌瘤(uterine fibroids)是育龄妇女生殖器官最常见的良性肿瘤,发病率呈逐年上升趋势,临床发病率为20%-40%。子宫肌瘤的发病机制至今尚不明了。子宫肌瘤是类固醇激素依赖肿瘤,其发生与遗传、基因突变等多种因素有关。异常表达的基因主要涉及细胞信号和传递蛋白、离子通道运输蛋白等。子宫平滑肌和内膜组织中具有丰富的前列腺素合成分泌,通过自分泌和旁分泌机制作用于平滑肌细胞,参与妊娠和非妊娠期的子宫生理效应。既往,人们对子宫肌瘤的发病机制的研究中很少涉及前列腺素系统的作用。 前列腺素(Prostaglandins, PG)是多不饱和脂肪酸--花生四烯酸(Arachidonic acid,aa)的衍生物。前列腺素是调节子宫收缩的重要激素,而环氧合酶(Cyclooxygenase-2,COX-2)则是催化aa产生PG途径当中的限速酶,也是非甾体类抗炎药(Non-steroidal anti-inflammatory drugs,NSAIDs)作用的靶点,它不仅调节前列腺素的合成,而且广泛参与肿瘤的发生发展过程。COX-2是COX(Cyclooxygense)的一个同工酶,一个即刻早期反应基因,通常在大多数的组织和细胞中是没有表达的,但是在炎性反应、激素和肿瘤启动子中被高度诱导,而COX-1(Cyclooxygense-1)的含量并无明显的变化。肿瘤组织中COX-2的高度表达,抑制COX-2活性与抗肿瘤作用直接相关,而抗COX-2选择性抑制剂有抗增殖作用。COX-2衍生的前列腺素E2(prostaglandin E2,PGE2)是一种促生物活性脂质,是主要的前列腺素。PGE2可促进肿瘤生长。研究表明,在人类多种恶性肿瘤如乳腺癌、肠癌、前列腺癌、肺癌、肝癌、头颈部肿瘤等,COX-2及PGE2表达明显上调,活性增加。鉴于COX-2以及PGE2在肿瘤发生发展中的作用,但是目前对于COX-2和PGE2在子宫肌瘤组织中的表达及其与子宫肌瘤的发生发展的生物学关系并不十分清楚。 既往研究表明子宫肌瘤与雌激素和异常细胞增殖有关,但并不清楚其确切的病理生理机制。有学者发现子宫肌瘤患者的子宫出现异常收缩。最近的研究表明,子宫深层组织,即内膜下肌层组织,出现“蠕变收缩”,且平滑肌细胞运动机能下降但扩散增强。所有这些研究提示异常的平滑肌收缩可能在子宫肌瘤病理生理过程中起着重要的作用,而其潜在的分子生物学机制尚不清楚,有待进一步研究。钙离子是启动细胞收缩的重要的上游信号分子。已知,子宫平滑肌兴奋-收缩偶联受双重信号转导通路的调节,其中钙离子信号转导通路在细胞应激过程中起着十分重要的作用。钙通道蛋白是信号转导过程中的分子开关,他们不仅调节细胞收缩还参与细胞周期的调节,这与众多肿瘤和致癌路径紧密相关。瞬时钙离子通道蛋白(calcium transient receptor potential channel,TRPC)是介导细胞Ca2+内流的跨膜钙通道蛋白,不仅调控细胞收缩运动,而且参与细胞增殖周期调控,与多种肿瘤的发生发展关系密切。近年来,钙离子通道蛋白的研究领域取得显著成绩。研究表明,TRPC与异常的子宫收缩有关,但其是否能促进子宫肌瘤的增殖尚不清楚。 目前,人们对存在子宫肌瘤组织中的前列腺素和钙通道的认识十分有限,一些重要的环节需要我们进一步求证。我们在本研究中,拟开展COX-2在子宫肌瘤组织及正常平滑肌组织中的表达及其对子宫肌瘤平滑肌细胞的增殖相关性研究,以明确COX-2与子宫肌瘤发病的相关性,并研究子宫肌瘤细胞钙离子通道蛋白的表达情况,及其对肌瘤细胞增殖和PGE2分泌的影响、钙离子的变化。该研究结果可能对进一步加深我们对子宫肌瘤的发生发展机制有重要意义。 第一部分子宫肌瘤组织中COX-2的表达及PGE2的分泌研究 [研究目的] 观察子宫肌瘤肌组织中COX-2的表达和PGE2的分泌情况,探讨COX-2介导的信号通路与子宫肌瘤发病机制的关系。 [研究方法] (1)收集30例腹腔镜下手术的子宫肌瘤患者的肌瘤组织标本和正常瘤旁平滑肌组织标本；体外分离和培养子宫肌瘤细胞和正常瘤旁平滑肌细胞。 (2)免疫组化、Western blo、RT-PCR法检测子宫肌瘤组织和正常瘤旁平滑肌组织中COX-2的表达。 (3)免疫荧光检测观察COX-2在正常瘤旁平滑肌细胞和肌瘤细胞中的表达。 (4)MTT检测子宫肌瘤细胞和正常瘤旁平滑肌细胞增殖情况,及COX-2的选择性抑制剂NS-398和celecoxib对肌瘤细胞增殖的影响。 (5)ELISA法定量测定子宫肌瘤细胞的PGE2分泌,及NS-398和celecoxib对肌瘤细胞PGE2分泌的影响。 [结果] (1)我们已经掌握了人子宫肌瘤平滑肌细胞及正常人子宫平滑肌细胞的体外培养方法,建立了成熟及稳定的临床样本的子宫肌瘤平滑肌细胞的分离及体外培养技术。 (2)免疫组化、Western blot实验及荧光定量PCR实验：免疫组化的方法检测子宫平滑肌组织有少量的COX-2表达,而子宫肌瘤组织表达显著增加,表达部位主要见于细胞核和细胞浆。子宫平滑肌中COX-2的阳性指数=11.90,子宫肌瘤中COX-2的阳性指数=46.50,差异有显著统计学意义,*P0.05。Western blot结果同样显示COX-2在子宫肌瘤中的表达(0.872±0.035)明显高于子宫平滑肌组织(0.202±0.056),*P0.05。荧光定量PCR显示COX-2mRNA在子宫肌瘤中的表达高于正常平滑肌组织,*P0.05。 (3)子宫肌瘤平滑肌细胞的增殖率约为正常平滑肌细胞的1.48±0.09倍,组间比较*P0.05。 (4)分别向体外培养的子宫肌瘤平滑肌细胞及正常子宫组织的平滑肌细胞添加10μM的NS-398或celecoxib后孵育24小时,加入MTT检测细胞的增殖情况,结果发现子宫肌瘤平滑肌细胞的增殖均受到显著抑制,但正常子宫平滑肌细胞增殖则未受到显著影响。 (5)子宫肌瘤细胞的PGE2分泌显著高于正常平滑肌细胞,*P0.05。 (6)分别向体外培养的子宫肌瘤平滑肌细胞及正常子宫组织的平滑肌细胞分别添加10μM的NS-398或celecoxib后孵育24小时,子宫肌瘤平滑肌细胞的PGE2的分泌均受到显著抑制,但正常子宫平滑肌细胞PGE2的分泌则未受到显著影响。 [结论及意义] 子宫肌瘤组织中的COX-2显著高表达,PGE2分泌显著增加,体外培养子宫肌瘤细胞增殖速度显著高于瘤旁正常子宫平滑肌细胞的增殖速度；而上述现象均可被COX-2的特异性抑制剂显著抑制。这说明肌瘤细胞COX-2过表达与肌瘤细胞增殖间有相关性,提示COX-2可能在子宫肌瘤的发病机制中起着某种重要作用。 第二部分子宫肌瘤细胞钙离子通道蛋白的表达及其与COX-2诱导的PGE2分泌的关系研究 [研究目的] 观察比较子宫肌瘤细胞与子宫平滑肌细胞的钙离子水平浓度和钙离子通道蛋白的表达情况,采用分别针对TRPC1及TRPM7的shRNA慢病毒载体干扰相应蛋白表达后,观察子宫肌瘤细胞的增殖、PGE2的分泌及钙离子的变化,从而鉴定钙离子信号转导通路与子宫肌瘤间的关系。 [研究方法] (1)收集30例腹腔镜下手术的子宫肌瘤患者的子宫肌瘤组织和相应的瘤旁正常平滑肌组织,子宫肌瘤细胞和正常瘤旁平滑肌细胞的分离及体外培养。 (2)激光扫描共聚焦显微镜检测细胞内游离钙离子浓度。 (3) RT-PCR及Western blot法检测子宫肌瘤组织和子宫平滑肌组织中钙通道亚型的表达。 (4)采用携带有TRPC1shRNA和TRPM7shRNA的GIPZ慢病毒载体行RNA干预实验。 (5)MTT检测RNA干扰TRPC1或TRPM7表达后对肌瘤细胞增殖的影响。 (6) ELISA检测RNA干扰TRPC1或TRPM7表达后对子宫肌瘤细胞分泌PGE2的影响。 (7)激光扫描共聚焦显微镜检测RNA干扰TRPC1和TRPM7后钙离子的变化。 [结果] (1)激光共聚焦显微镜检测胞内游离钙离子浓度显示,肌瘤组的钙浓度值25.443土3.203(n=30)显著高于癌旁正常平滑肌组13.223±1.450(n=30),*P0.05。 (2)PCR定量方法检测并比较子宫肌瘤组织和瘤旁正常平滑肌组织间的钙通道亚型mRNA表达显示,子宫肌瘤组的TRPM7和TRPC1表达显著高于正常瘤旁平滑肌组,组间比较*P0.05,而两组间钙通道的其他亚型的表达无显著差异,P0.05。免疫印迹分析检测并比较子宫肌瘤组织和瘤旁正常平滑肌组织间的钙通道亚型mRNA表达显示,子宫肌瘤组的TRPM7和TRPC1表达显著高于正常瘤旁平滑肌组,组间比较*P0.05,而两组间钙通道的其他亚型的表达无显著性差异,P0.05。 (3)采用shRNA病毒显著降低肌瘤细胞TRPC1的表达后,发现子宫肌瘤细胞增殖受到显著抑制,且子宫肌瘤细胞PGE2的分泌受到显著抑制,钙离子水平降低,*P0.05。 (4)采用shRNA病毒显著降低肌瘤细胞TRPM7的表达后,发现子宫肌瘤细胞增殖受到显著抑制,且子宫肌瘤细胞的PGE2的分泌受到显著抑制,钙离子水平降低,*P0.05。 [结论及意义] 子宫肌瘤细胞内的游离钙离子浓度显著较高；子宫肌瘤组织的钙离子通道蛋白TRPC1和TRPM7显著高表达；采用RNA干扰技术沉默TRPC1和TRPM7表达后,子宫肌瘤细胞增殖明显受抑制,PGE2的产生分泌明显降低,并且钙离子水平下降。 本研究的创新点 1.我们从前列腺素代谢入手,研究COX2、PGE2在子宫肌瘤发生发展中的作用,为子宫肌瘤的发病机制研究提供新思路。 2.从NSAIDs着手,试图从抑制剂阻断子宫肌瘤的发展,为子宫肌瘤的防治提供新思路。 3.本研究发现了子宫肌瘤细胞的钙离子通道蛋白TRPC1和TRPM7表达显著增高,并通过RNA干扰技术分别将两种基因沉默后发现,肌瘤细胞的增殖均受到显著抑制,其PGE2分泌亦均受到显著抑制,并且钙离子水平下降。这说明TRPC1和TRPM7可能参与了子宫肌瘤发生发展过程,而两者所介导的钙离子的转运路径可能与COX2诱导的PGE2的分泌路径存在信号交互作用。
[Abstract]:Research background
Uterine fibroids are the most common benign tumors in reproductive organs of women of childbearing age. The incidence of uterine fibroids is increasing year by year. The clinical incidence is 20%-40%. The pathogenesis of uterine fibroids is still unknown. Uterine fibroids are steroid-dependent tumors, which are related to many factors, such as heredity, gene mutation and so on. In the past, the pathogenesis of uterine leiomyoma has been studied in the past. The role of prostaglandin system is rarely involved in the study.
Prostaglandins (PG) are derivatives of polyunsaturated fatty acids, arachidonic acid (aa). Prostaglandins are important hormones that regulate uterine contraction, while cyclooxygenase-2 (COX-2) is the rate-limiting enzyme in the pathway of PG production by AA and is also a non-steroidal anti-inflammation drug. COX-2 is an isoenzyme of COX (Cyclooxygense), an immediate early-response gene that is not normally expressed in most tissues and cells, but is involved in inflammatory reactions, hormones, and tumor promoters. High expression of COX-2 and inhibition of COX-2 activity are directly related to the anti-tumor effect, while anti-COX-2 selective inhibitors have anti-proliferative effect. COX-2-derived prostaglandin E2 (PGE2) is a bioactive lipid and is the main one. Prostaglandin-PGE2 promotes tumor growth. Studies have shown that COX-2 and PGE2 are up-regulated and their activities are increased in many human malignant tumors such as breast, bowel, prostate, lung, liver, head and neck tumors. The expression and its biological relationship with the occurrence and development of uterine leiomyoma are not very clear.
Previous studies have shown that uterine leiomyoma is associated with estrogen and abnormal cell proliferation, but the exact pathophysiological mechanism is unclear. Some scholars have found abnormal contraction of the uterus in patients with uterine leiomyoma. Recent studies have shown that "creep contraction" occurs in the deep uterine tissue, i.e. the subendometrial myometrium, and smooth muscle cell motility. All these studies suggest that abnormal smooth muscle contraction may play an important role in the pathophysiological process of uterine leiomyoma, and the underlying molecular biological mechanisms remain unclear and need further study. Calcium ion is an important upstream signaling molecule that initiates cell contraction. Condensation coupling is regulated by dual signal transduction pathways, in which calcium ion signal transduction pathways play an important role in cell stress. Calcium channel proteins are molecular switches in signal transduction. They not only regulate cell contraction but also participate in cell cycle regulation, which is closely related to many tumors and carcinogenic pathways. Calcium transient receptor potential channel (TRPC) is a transmembrane calcium channel protein that mediates the influx of Ca2+ into cells. It not only regulates cell contraction, but also participates in the regulation of cell proliferation cycle. TRPC is closely related to the occurrence and development of various tumors. Studies have shown that TRPC is associated with abnormal uterine contractions, but whether TRPC can promote the proliferation of uterine leiomyomas remains unclear.
At present, the understanding of prostaglandins and calcium channels in uterine leiomyoma is very limited, and some important links need further confirmation. In this study, we intend to study the expression of COX-2 in uterine leiomyoma tissue and normal smooth muscle tissue and its correlation with the proliferation of uterine leiomyoma smooth muscle cells. To clarify the relationship between COX-2 and the pathogenesis of uterine leiomyoma, and to study the expression of calcium channel protein in uterine leiomyoma cells, its effect on the proliferation of leiomyoma cells, the secretion of PGE2, and the changes of calcium ions.
Part one COX-2 expression and PGE2 secretion in uterine fibroids
[research purposes]
To observe the expression of COX-2 and the secretion of PGE2 in uterine leiomyoma, and to explore the relationship between COX-2-mediated signaling pathway and the pathogenesis of uterine leiomyoma.
[research methods]
(1) Samples of leiomyoma tissue and normal paraneoplastic smooth muscle tissue were collected from 30 patients with uterine leiomyoma undergoing laparoscopic surgery, and myoma cells and normal paraneoplastic smooth muscle cells were isolated and cultured in vitro.
(2) Immunohistochemistry, Western Blo and RT-PCR were used to detect the expression of COX-2 in uterine leiomyoma tissues and normal paraneoplastic smooth muscle tissues.
(3) immunofluorescence assay was used to observe the expression of COX-2 in normal tumor adjacent smooth muscle cells and myoma cells.
(4) MTT assay was used to detect the proliferation of uterine leiomyoma cells and normal paraneoplastic smooth muscle cells, and the effects of COX-2 selective inhibitors NS-398 and celecoxib on the proliferation of leiomyoma cells.
(5) Quantitative determination of PGE2 secretion by ELISA and the effects of NS-398 and celecoxib on PGE2 secretion by hysteromyoma cells.
[results]
(1) We have mastered the culture methods of human leiomyoma smooth muscle cells and normal human leiomyoma smooth muscle cells in vitro, and established the isolation and culture techniques of mature and stable clinical samples of leiomyoma smooth muscle cells in vitro.
(2) Immunohistochemistry, Western blot and fluorescence quantitative PCR: Immunohistochemistry showed that there was a small amount of COX-2 expression in uterine leiomyoma tissues, but the expression of COX-2 in uterine leiomyoma tissues was significantly increased, mainly in the nucleus and cytoplasm. The positive index of COX-2 in uterine leiomyoma was 11.90, and the positive index of COX-2 in uterine leiomyoma tissues was 11.90. The expression of COX-2 in uterine leiomyoma was significantly higher than that in uterine leiomyoma (0.872 (+ 0.035)), * P 0.05. The expression of COX-2 mRNA in uterine leiomyoma was higher than that in normal uterine leiomyoma, * P 0.05.
(3) The proliferation rate of leiomyocytes in uterine leiomyoma was 1.48 65507
(4) The proliferation of uterine leiomyoma smooth muscle cells and normal uterine smooth muscle cells was detected by MTT after incubation for 24 hours. The results showed that the proliferation of leiomyoma smooth muscle cells was significantly inhibited, but the proliferation of normal uterine smooth muscle cells was not. Significant impact.
(5) PGE2 secretion of uterine leiomyoma cells was significantly higher than that of normal smooth muscle cells, *P0.05.
(6) The secretion of PGE2 in uterine leiomyoma smooth muscle cells was significantly inhibited 24 hours after incubation with NS-398 or celecoxib of 10 mu M respectively, but the secretion of PGE2 in normal uterine smooth muscle cells was not significantly affected.
[Conclusion and significance]
The expression of COX-2 and the secretion of PGE2 were significantly increased in uterine leiomyoma tissues, and the proliferation rate of cultured uterine leiomyoma cells was significantly higher than that of normal uterine leiomyoma cells in vitro. Correlation suggests that COX-2 may play an important role in the pathogenesis of uterine leiomyoma.
Part 2 Expression of calcium channel protein in uterine leiomyoma cells and its relationship with COX-2-induced PGE2 secretion
[research purposes]
To observe and compare the level of calcium ion and the expression of calcium channel protein in uterine leiomyoma cells and uterine smooth muscle cells, the proliferation of uterine leiomyoma cells, the secretion of PGE2 and the change of calcium ion were observed after the expression of corresponding protein was interfered by shRNA lentiviral vector targeting TRPC1 and TRPM7 respectively, and the calcium ion signal transduction was identified. The relationship between conduction pathways and uterine fibroids.
[research methods]
(1) Isolation and culture of uterine leiomyoma tissues and normal paraneoplastic smooth muscle tissues, uterine leiomyoma cells and normal paraneoplastic smooth muscle cells from 30 patients with uterine leiomyoma undergoing laparoscopic surgery.
(2) intracellular free calcium concentration was detected by laser scanning confocal microscope.
(3) The expression of calcium channel subtypes was detected by RT-PCR and Western blot.
(4) TRPM7shRNA lentiviral vectors carrying TRPC1shRNA and GIPZ were used for RNA intervention experiment.
(5) MTT detected the effect of RNA interference on the proliferation of myoma cells after the expression of TRPC1 or TRPM7.
(6) ELISA can detect the effect of RNA interference on TRPC1 and TRPM7 expression in the secretion of PGE2 from uterine leiomyoma cells.
(7) laser scanning confocal microscopy was used to detect the changes of calcium ions after RNA interferes with TRPC1 and TRPM7.
[results]
(1) Laser confocal microscopy showed that the intracellular free calcium concentration in leiomyoma group was 25.443 soil 3.203 (n=30), which was significantly higher than that in normal smooth muscle group (13.223 + 1.450) (n = 30), * P 0.05.
(2) The expression of TRPM7 and TRPC1 mRNA in uterine leiomyoma tissues and normal smooth muscle tissues adjacent to uterine leiomyoma were detected and compared by PCR quantitative method. The expression of TRPM7 and TRPC1 mRNA in uterine leiomyoma group was significantly higher than that in normal smooth muscle tissues adjacent to uterine leiomyoma group, and the expression of other subtypes of calcium channel between the two groups was not significantly different, P 0.05. The expression of TRPM7 and TRPC1 was significantly higher in uterine leiomyoma group than in normal paraneoplastic leiomyoma group (P 0.05). There was no significant difference in the expression of other subtypes of calcium channel between the two groups (P 0.05).
(3) After the expression of TRPC1 was significantly decreased by shRNA virus, the proliferation of uterine leiomyoma cells was significantly inhibited, the secretion of PGE2 was significantly inhibited, and the level of calcium was decreased, * P 0.05.
(4) After the expression of TRPM7 was significantly decreased by shRNA virus, the proliferation of uterine leiomyoma cells was significantly inhibited, the secretion of PGE2 was significantly inhibited, and the level of calcium was decreased, * P 0.05.
[Conclusion and significance]
The free calcium concentration in uterine leiomyoma cells was significantly higher; the expression of calcium channel proteins TRPC1 and TRPM7 was significantly higher in uterine leiomyoma tissues; the proliferation of uterine leiomyoma cells was significantly inhibited, the production and secretion of PGE2 were significantly decreased, and the level of calcium ion was significantly decreased after TRPC1 and TRPM7 were silenced by RNA interference.
The innovation of this research
1. Starting with prostaglandin metabolism, we studied the role of COX2 and PGE2 in the occurrence and development of uterine leiomyoma, and provided new ideas for the pathogenesis of uterine leiomyoma.
2. Starting with NSAIDs, we try to block the development of uterine fibroids from inhibitors, and provide new ideas for the prevention and treatment of uterine fibroids.
3. This study found that the expression of calcium channel protein TRPC1 and TRPM7 in uterine leiomyoma cells was significantly increased, and the proliferation of leiomyoma cells was significantly inhibited, the secretion of PGE2 was significantly inhibited, and the level of calcium was decreased by RNA interference. The development of uterine leiomyoma may be mediated by calcium transport pathway and COX2-induced PGE2 secretion pathway.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.33
本文编号：2218413
[Abstract]:Research background
Uterine fibroids are the most common benign tumors in reproductive organs of women of childbearing age. The incidence of uterine fibroids is increasing year by year. The clinical incidence is 20%-40%. The pathogenesis of uterine fibroids is still unknown. Uterine fibroids are steroid-dependent tumors, which are related to many factors, such as heredity, gene mutation and so on. In the past, the pathogenesis of uterine leiomyoma has been studied in the past. The role of prostaglandin system is rarely involved in the study.
Prostaglandins (PG) are derivatives of polyunsaturated fatty acids, arachidonic acid (aa). Prostaglandins are important hormones that regulate uterine contraction, while cyclooxygenase-2 (COX-2) is the rate-limiting enzyme in the pathway of PG production by AA and is also a non-steroidal anti-inflammation drug. COX-2 is an isoenzyme of COX (Cyclooxygense), an immediate early-response gene that is not normally expressed in most tissues and cells, but is involved in inflammatory reactions, hormones, and tumor promoters. High expression of COX-2 and inhibition of COX-2 activity are directly related to the anti-tumor effect, while anti-COX-2 selective inhibitors have anti-proliferative effect. COX-2-derived prostaglandin E2 (PGE2) is a bioactive lipid and is the main one. Prostaglandin-PGE2 promotes tumor growth. Studies have shown that COX-2 and PGE2 are up-regulated and their activities are increased in many human malignant tumors such as breast, bowel, prostate, lung, liver, head and neck tumors. The expression and its biological relationship with the occurrence and development of uterine leiomyoma are not very clear.
Previous studies have shown that uterine leiomyoma is associated with estrogen and abnormal cell proliferation, but the exact pathophysiological mechanism is unclear. Some scholars have found abnormal contraction of the uterus in patients with uterine leiomyoma. Recent studies have shown that "creep contraction" occurs in the deep uterine tissue, i.e. the subendometrial myometrium, and smooth muscle cell motility. All these studies suggest that abnormal smooth muscle contraction may play an important role in the pathophysiological process of uterine leiomyoma, and the underlying molecular biological mechanisms remain unclear and need further study. Calcium ion is an important upstream signaling molecule that initiates cell contraction. Condensation coupling is regulated by dual signal transduction pathways, in which calcium ion signal transduction pathways play an important role in cell stress. Calcium channel proteins are molecular switches in signal transduction. They not only regulate cell contraction but also participate in cell cycle regulation, which is closely related to many tumors and carcinogenic pathways. Calcium transient receptor potential channel (TRPC) is a transmembrane calcium channel protein that mediates the influx of Ca2+ into cells. It not only regulates cell contraction, but also participates in the regulation of cell proliferation cycle. TRPC is closely related to the occurrence and development of various tumors. Studies have shown that TRPC is associated with abnormal uterine contractions, but whether TRPC can promote the proliferation of uterine leiomyomas remains unclear.
At present, the understanding of prostaglandins and calcium channels in uterine leiomyoma is very limited, and some important links need further confirmation. In this study, we intend to study the expression of COX-2 in uterine leiomyoma tissue and normal smooth muscle tissue and its correlation with the proliferation of uterine leiomyoma smooth muscle cells. To clarify the relationship between COX-2 and the pathogenesis of uterine leiomyoma, and to study the expression of calcium channel protein in uterine leiomyoma cells, its effect on the proliferation of leiomyoma cells, the secretion of PGE2, and the changes of calcium ions.
Part one COX-2 expression and PGE2 secretion in uterine fibroids
[research purposes]
To observe the expression of COX-2 and the secretion of PGE2 in uterine leiomyoma, and to explore the relationship between COX-2-mediated signaling pathway and the pathogenesis of uterine leiomyoma.
[research methods]
(1) Samples of leiomyoma tissue and normal paraneoplastic smooth muscle tissue were collected from 30 patients with uterine leiomyoma undergoing laparoscopic surgery, and myoma cells and normal paraneoplastic smooth muscle cells were isolated and cultured in vitro.
(2) Immunohistochemistry, Western Blo and RT-PCR were used to detect the expression of COX-2 in uterine leiomyoma tissues and normal paraneoplastic smooth muscle tissues.
(3) immunofluorescence assay was used to observe the expression of COX-2 in normal tumor adjacent smooth muscle cells and myoma cells.
(4) MTT assay was used to detect the proliferation of uterine leiomyoma cells and normal paraneoplastic smooth muscle cells, and the effects of COX-2 selective inhibitors NS-398 and celecoxib on the proliferation of leiomyoma cells.
(5) Quantitative determination of PGE2 secretion by ELISA and the effects of NS-398 and celecoxib on PGE2 secretion by hysteromyoma cells.
[results]
(1) We have mastered the culture methods of human leiomyoma smooth muscle cells and normal human leiomyoma smooth muscle cells in vitro, and established the isolation and culture techniques of mature and stable clinical samples of leiomyoma smooth muscle cells in vitro.
(2) Immunohistochemistry, Western blot and fluorescence quantitative PCR: Immunohistochemistry showed that there was a small amount of COX-2 expression in uterine leiomyoma tissues, but the expression of COX-2 in uterine leiomyoma tissues was significantly increased, mainly in the nucleus and cytoplasm. The positive index of COX-2 in uterine leiomyoma was 11.90, and the positive index of COX-2 in uterine leiomyoma tissues was 11.90. The expression of COX-2 in uterine leiomyoma was significantly higher than that in uterine leiomyoma (0.872 (+ 0.035)), * P 0.05. The expression of COX-2 mRNA in uterine leiomyoma was higher than that in normal uterine leiomyoma, * P 0.05.
(3) The proliferation rate of leiomyocytes in uterine leiomyoma was 1.48 65507
(4) The proliferation of uterine leiomyoma smooth muscle cells and normal uterine smooth muscle cells was detected by MTT after incubation for 24 hours. The results showed that the proliferation of leiomyoma smooth muscle cells was significantly inhibited, but the proliferation of normal uterine smooth muscle cells was not. Significant impact.
(5) PGE2 secretion of uterine leiomyoma cells was significantly higher than that of normal smooth muscle cells, *P0.05.
(6) The secretion of PGE2 in uterine leiomyoma smooth muscle cells was significantly inhibited 24 hours after incubation with NS-398 or celecoxib of 10 mu M respectively, but the secretion of PGE2 in normal uterine smooth muscle cells was not significantly affected.
[Conclusion and significance]
The expression of COX-2 and the secretion of PGE2 were significantly increased in uterine leiomyoma tissues, and the proliferation rate of cultured uterine leiomyoma cells was significantly higher than that of normal uterine leiomyoma cells in vitro. Correlation suggests that COX-2 may play an important role in the pathogenesis of uterine leiomyoma.
Part 2 Expression of calcium channel protein in uterine leiomyoma cells and its relationship with COX-2-induced PGE2 secretion
[research purposes]
To observe and compare the level of calcium ion and the expression of calcium channel protein in uterine leiomyoma cells and uterine smooth muscle cells, the proliferation of uterine leiomyoma cells, the secretion of PGE2 and the change of calcium ion were observed after the expression of corresponding protein was interfered by shRNA lentiviral vector targeting TRPC1 and TRPM7 respectively, and the calcium ion signal transduction was identified. The relationship between conduction pathways and uterine fibroids.
[research methods]
(1) Isolation and culture of uterine leiomyoma tissues and normal paraneoplastic smooth muscle tissues, uterine leiomyoma cells and normal paraneoplastic smooth muscle cells from 30 patients with uterine leiomyoma undergoing laparoscopic surgery.
(2) intracellular free calcium concentration was detected by laser scanning confocal microscope.
(3) The expression of calcium channel subtypes was detected by RT-PCR and Western blot.
(4) TRPM7shRNA lentiviral vectors carrying TRPC1shRNA and GIPZ were used for RNA intervention experiment.
(5) MTT detected the effect of RNA interference on the proliferation of myoma cells after the expression of TRPC1 or TRPM7.
(6) ELISA can detect the effect of RNA interference on TRPC1 and TRPM7 expression in the secretion of PGE2 from uterine leiomyoma cells.
(7) laser scanning confocal microscopy was used to detect the changes of calcium ions after RNA interferes with TRPC1 and TRPM7.
[results]
(1) Laser confocal microscopy showed that the intracellular free calcium concentration in leiomyoma group was 25.443 soil 3.203 (n=30), which was significantly higher than that in normal smooth muscle group (13.223 + 1.450) (n = 30), * P 0.05.
(2) The expression of TRPM7 and TRPC1 mRNA in uterine leiomyoma tissues and normal smooth muscle tissues adjacent to uterine leiomyoma were detected and compared by PCR quantitative method. The expression of TRPM7 and TRPC1 mRNA in uterine leiomyoma group was significantly higher than that in normal smooth muscle tissues adjacent to uterine leiomyoma group, and the expression of other subtypes of calcium channel between the two groups was not significantly different, P 0.05. The expression of TRPM7 and TRPC1 was significantly higher in uterine leiomyoma group than in normal paraneoplastic leiomyoma group (P 0.05). There was no significant difference in the expression of other subtypes of calcium channel between the two groups (P 0.05).
(3) After the expression of TRPC1 was significantly decreased by shRNA virus, the proliferation of uterine leiomyoma cells was significantly inhibited, the secretion of PGE2 was significantly inhibited, and the level of calcium was decreased, * P 0.05.
(4) After the expression of TRPM7 was significantly decreased by shRNA virus, the proliferation of uterine leiomyoma cells was significantly inhibited, the secretion of PGE2 was significantly inhibited, and the level of calcium was decreased, * P 0.05.
[Conclusion and significance]
The free calcium concentration in uterine leiomyoma cells was significantly higher; the expression of calcium channel proteins TRPC1 and TRPM7 was significantly higher in uterine leiomyoma tissues; the proliferation of uterine leiomyoma cells was significantly inhibited, the production and secretion of PGE2 were significantly decreased, and the level of calcium ion was significantly decreased after TRPC1 and TRPM7 were silenced by RNA interference.
The innovation of this research
1. Starting with prostaglandin metabolism, we studied the role of COX2 and PGE2 in the occurrence and development of uterine leiomyoma, and provided new ideas for the pathogenesis of uterine leiomyoma.
2. Starting with NSAIDs, we try to block the development of uterine fibroids from inhibitors, and provide new ideas for the prevention and treatment of uterine fibroids.
3. This study found that the expression of calcium channel protein TRPC1 and TRPM7 in uterine leiomyoma cells was significantly increased, and the proliferation of leiomyoma cells was significantly inhibited, the secretion of PGE2 was significantly inhibited, and the level of calcium was decreased by RNA interference. The development of uterine leiomyoma may be mediated by calcium transport pathway and COX2-induced PGE2 secretion pathway.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.33
【参考文献】
相关期刊论文 前3条
1 康华峰;王西京;刘小旭;代志军;薛锋杰;薛兴欢;;塞来昔布预防大鼠乳腺癌发生及其机制[J];南方医科大学学报;2006年11期
2 冷金花;张震宇;段华;王立杰;姚书忠;陈春林;金力;杨佳欣;樊庆泊;;子宫肌瘤诊治的热点问题[J];现代妇产科进展;2007年05期
3 Vibeke Andersen;Jonas Halfvarson;Ulla Vogel;;Colorectal cancer in patients with inflammatory bowel disease:Can we predict risk?[J];World Journal of Gastroenterology;2012年31期
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