细胞周期蛋白CDC50A参与维持卵巢癌干细胞特性及其相关作用机制的研究

发布时间：2018-05-27 18:52

本文选题：卵巢癌 + 肿瘤干细胞　；参考：《北京协和医学院》2016年博士论文

【摘要】：研究背景卵巢癌是女性常见的恶性肿瘤之一,其发生率仅次于宫颈癌和子宫内膜癌,居第三位,然而其致死率高居首位。卵巢癌起病隐匿,大多数患者诊断时已为晚期。在过去的几十年中,对卵巢癌患者的基本治疗方案采取理想的肿瘤细胞减灭术辅助铂类药物为主的联合化疗,虽然取得了一定的疗效,但是仍有70%左右的患者在18个月内复发,晚期卵巢癌患者的5年生存率仅为30%。目前认为肿瘤的耐药及复发是造成卵巢癌高死亡率的主要原因,但是卵巢癌耐药及复发的机制目前仍不清楚,故而严重阻碍了卵巢癌的有效治疗。自上世纪来,肿瘤干细胞(Cancer stem cells, CSCs)理论被提出,认为肿瘤组织中存在少量的干细胞,是导致肿瘤进展、复发及化疗耐药的根源。Bonnet和Dick从白血病患者中分离出CD34+CD38-细胞被证实具有干细胞特性,这是世界上首次发现CSCs的存在。之后大量研究相继发现多种实体肿瘤中也存在极少量的CSCs,如乳腺癌、前列腺癌、结肠癌等。研究表明,CSCs具有分化、自我更新等类似正常干细胞的生物学特性,同时具有致瘤能力且对化疗药物产生耐药性。目前肿瘤干细胞的研究取得了很大的进展,在一些实体肿瘤中已建立了一系列CSCs分离、鉴定的方法,对肿瘤发生、进展、耐药和复发的机制的研究以及探索肿瘤治疗的新领域具有重要意义。近年来,CSCs理论被应用于卵巢癌的研究中取得了一定的成绩。但是,目前对于卵巢癌干细胞的研究仍在借鉴其他实体肿瘤的方法,如对卵巢癌干细胞的识别缺乏较为特异的标记物,目前使用较多的标记物有CD133、CD117、CD44、ALDH1、 EPCAM等,其中CD44被作为乳腺癌、胰腺癌等CSCs的标记物,CD133被作为结肠癌等CSCs的标记物等。其分离获得的方法仍主要依靠SP细胞的分选。因此,寻找特异性的表面标记物,对于卵巢癌干细胞的研究极其重要,对探索卵巢癌新的治疗方法具有重大意义。本课题前期工作中利用稳定同位素标记及质谱为基础的蛋白质组学技术分析比较卵巢癌细胞系SP和非SP细胞膜蛋白的差异表达,筛选出一种跨膜蛋白30A(transmembrane protein 30A, TMEM30A),又称细胞周期蛋白50A(cell division cycle 50A, CDC50A),并通过大量体内外实验对卵巢癌细胞系及原代卵巢癌细胞CDC50A+细胞进行鉴定,证实其具有CSCs生物学特性,是CDC50A有望成为卵巢癌干细胞较为特异的表面标记物。本研究承接前期工作,对CDC50A参与维持卵巢癌干细胞特性及其相关作用机制做进一步研究。研究方法1.构建CDC50A+细胞比例下调的SKOV3细胞。用4段CDC50A shRNA转染293T细胞,运用Western blot方法筛选能有效下调CDC50A基因表达的shRNA,包装慢病毒载体,并利用通过慢病毒感染技术,下调卵巢癌细胞系SKOV3中CDC50A+细胞比例,利用流式细胞学方法检测其下调效率。利用流式分选仪分选得到CDC50A+细胞比例下调组(SKOV3CDC50A-GFP)细胞和阴性对照组(SKOV3 NC-GFP)细胞。采用流式细胞学方法检测SKOV3 CDC50A-GFP及SKOV3 NC-GFP中CDC50A的表达水平。2. CDC50A+细胞比例下调的SKOV3细胞干细胞特性的鉴定。利用含血清的HG-DMEM培养基体外培养,验证SKOV3 CDC50A-GFP及SKOV3 NC-GFP细胞的分化能力。利用无血清悬浮培养方法,检测SKOV3 CDC50A-GFP及SKOV3 NC-GFP细胞sphere形成能力及传代能力,并运用流式细胞学方法验证sphere对CDC50A的富集作用。MTT法检测SKOV3 CDC50A-GFP和NC-GFP细胞对顺铂的耐药性。将SKOV3 CDC50A-GFP和NC-GFP细胞等量接种于NSG小鼠皮下,观察成瘤情况,检测其成瘤能力。3.人卵巢癌CDC50A+和CDC50A细胞RNA测序及其与干细胞功能相关的机制分析。收集3例卵巢癌患者肿瘤组织,分离得到卵巢癌细胞,利用流式细胞分选仪分选获得CDC50A+和CDC50A细胞,分别提取两组细胞RNA,进行RNA测序。获得两组细胞的差异表达基因,并对其进行功能分析和相关信号通路的富集分析。研究结果1.成功构建CDC50A+细胞比例下调的SKOV3细胞。Western方法检测转染4段CDC50A shRNA的293T细胞的CDC50A表达,序列TMEM30A-homo-974沉默CDC50A基因的效率最高,其靶序列为GCCTGTGAACTGGCTTAAACC。将其包装慢病毒载体后,感染293T细胞,Western法检测其能使CDC50A表达降低80%以上。利用慢病毒感染SKOV3细胞,流式检测其感染效率CDC50A-GFP组为87.9%,NC-GFP组为81.6%。分选出成功感染慢病毒的两组细胞后,流式分析SKOV3 NC-GFP细胞中CDC50A+细胞比例为0.48%,而SKOV3 CDC50A-GFP细胞中CDC50A+细胞仅占0.12%。表明慢病毒感染能有效的下调卵巢癌细胞系SKOV3中CDC50A+细胞比例,而含阴性对照序列的慢病毒感染对SKOV3细胞中CDC50A基因的表达基本无影响。2. CDC50A+细胞比例下调的SKOV3细胞株干细胞的生物学特性减弱。在无血清悬浮培养条件下,NC-GFP细胞能形成sphere,且绝大多数能带有绿色荧光；而CDC50A-GFP细胞sphere形成慢,且在荧光显微镜下仅能看到极少带绿色荧光的sphere。SKOV3 CDC50A-GFP细胞形成的sphere个数明显少于SKOV3 NC-GFP细胞,几乎不能传代；而SKOV3 NC-GFP细胞形成的sphere能连续传代至少三代。表明CDC50A基因被沉默的卵巢癌细胞系SKOV3细胞在体外sphere形成能力减弱且丧失自我更新及分化能力。流式检测SKOV3 CDC50A-GFP和NC-GFP细胞所形成的sphere中CDC50A+细胞的比例分别为19.7%和6.7%,均比贴壁生长的细胞中的CDC50A+细胞的比例明显升高,但SKOV3 CDC50A-GFP细胞中CDC50A+比例明显低于NC-GFP细胞。表明sphere对CDC50A+细胞具有富集作用。MTT法测定顺铂对SKOV3 CDC50A-GFP和NC-GFP细胞的IC50均值为1.33±0.14ug/ml,3.05±0.39 ug/ml,差异具有统计学意义(P=0.01)。说明CCDC50A+细胞比例下调的的卵巢癌细胞系SKOV3细胞对顺铂的耐药性减弱。动物实验显示103 NC-GFP细胞能使小鼠成瘤,而同样数量的CDC50A-GFP细胞未能使小鼠成瘤。表明CDC50A基因被沉默的卵巢癌细胞系SKOV3细胞致瘤能力降低。3.通过对CDC50A+细胞与CDC50A-细胞的RNA测序,发现了一些CDC50A参与维持卵巢癌干细胞特性的相关机制。对3原代卵巢癌组织中分选出的CDC50A+细胞和CDC50A-细胞进行RNA测序,分别筛选出92、59和251个差异表达基因(P0.01)。应用RNA谱分析、生物信息学技术对差异基因及其转录本其进行分析分析发现CDC50A可能通过ATPase H+transporting V1 subunit F、MALAT1、MAPK信号及调控核糖体、剪接体功能维持卵巢癌干细胞的特性及功能。结论1.利用CDC50A shRNA慢病毒载体感染技术能有效的下调卵巢癌细胞系SKOV3中CDC50A+细胞比例。2. CDC50A+细胞比例下调的卵巢癌细胞系SKOV3干细胞生物学特性减弱,sphere形成能力、自我更新和分化能力及致瘤能力均减弱,且对顺铂作用更为敏感。3. ATPase H+ transporting V1 subunit F可能是CDC50A参与维持卵巢癌干细胞生物学特性的主要机制基础。
[Abstract]:Background ovarian cancer is one of the most common malignant tumors in women. The incidence of ovarian cancer is second only to cervix cancer and endometrial cancer, which is the third place. However, the death rate of ovarian cancer is the highest. Combined chemotherapy assisted by cytoreduction assisted platinum based chemotherapy has achieved a certain effect, but there are still about 70% of the patients relapsed within 18 months. The 5 year survival rate of advanced ovarian cancer patients is only 30%.. The main reason for the high mortality of ovarian cancer is that the drug resistance and recurrence of the tumor are the main cause, but the drug resistance and recurrence of ovarian cancer The system is still unclear, so it seriously hinders the effective treatment of ovarian cancer. Since last century, the Cancer stem cells (CSCs) theory has been proposed that there is a small amount of stem cells in the tumor tissue, which is the root cause of tumor progression, recurrence and chemotherapy resistance.Bonnet and Dick to separate CD34+CD38- cells from patients with leukemia. CSCs has been found in the world for the first time. After a large number of studies have found that a very small amount of CSCs, such as breast, prostate, and colon, has been found in many kinds of solid tumors. Research shows that CSCs has the biological characteristics such as differentiation, self renewal and similar normal stem cells, and it also has the ability to induce tumorigenesis. There is a great progress in the research of tumor stem cells. A series of CSCs isolation and identification methods have been established in some solid tumors. It is important to study the mechanism of tumor occurrence, progress, drug resistance and recurrence, and to explore new fields of cancer treatment. In recent years, the CSCs theory has been recommended. However, the study of ovarian cancer stem cells is still being used for reference to other solid tumors, such as the lack of specific markers for the identification of ovarian cancer stem cells. There are many markers, such as CD133, CD117, CD44, ALDH1, EPCAM, etc., of which CD44 is used as breast cancer, CSCs marker, such as pancreatic cancer, is used as a marker for CSCs in colon cancer and so on. Its separation method is still mainly dependent on the separation of SP cells. Therefore, finding specific surface markers is very important for the study of ovarian cancer stem cells and is of great significance for the exploration of new treatment methods for ovarian cancer. The differential expression of membrane protein of ovarian cancer cell line SP and non SP cell was analyzed by proteomics technology based on stable isotope labeling and mass spectrometry, and a transmembrane protein 30A (transmembrane protein 30A, TMEM30A), also known as cyclin 50A (cell division cycle 50A), was selected, and through a large number of experiments in vitro and in vitro The identification of ovarian cancer cell lines and primary ovarian cancer cells CDC50A+ cells confirmed that they have CSCs biological characteristics. CDC50A is expected to be a more specific surface marker for ovarian cancer stem cells. This study carried on a further study on the involvement of CDC50A in maintaining the characteristics and mechanism of ovarian cancer stem cells. Method 1. the SKOV3 cells with down regulation of CDC50A+ cells were constructed. 293T cells were transfected with 4 segments of CDC50A shRNA. Western blot method was used to screen the shRNA of CDC50A gene expression effectively. The lentivirus carrier was packaged and the proportion of CDC50A+ cells in SKOV3 of the ovarian cancer cell line was downregulated by the technique of lentivirus infection, and the flow cytology was used. A flow cytometry was used to detect the down regulation of CDC50A+ cells (SKOV3CDC50A-GFP) cells and negative control groups (SKOV3 NC-GFP). Flow cytometry was used to detect the expression level of CDC50A in SKOV3 CDC50A-GFP and SKOV3 NC-GFP by.2. CDC50A+ cells and down regulated SKOV3 cell stem cells. The differentiation ability of SKOV3 CDC50A-GFP and SKOV3 NC-GFP cells was verified by HG-DMEM culture medium containing serum in vitro. The formation and passage ability of SKOV3 CDC50A-GFP and SKOV3 NC-GFP cell sphere were detected by the serum-free suspension culture method, and the enrichment of sphere on CDC50A was verified by flow cytometry. The resistance of SKOV3 CDC50A-GFP and NC-GFP cells to cisplatin was detected. SKOV3 CDC50A-GFP and NC-GFP cells were inoculated subcutaneously in NSG mice, and the tumor formation was observed. The tumor formation ability of.3. human ovarian cancer CDC50A+ and CDC50A cells RNA sequencing and the mechanism related to stem cell function were analyzed. 3 cases of ovarian cancer were collected and the tumor tissues were collected. CDC50A+ and CDC50A cells were isolated from the ovarian cancer cells by flow cytometry. Two groups of RNA were extracted and RNA sequenced. The differential expression genes of two groups of cells were obtained. The function analysis and the enrichment analysis of related signal pathways were carried out. The results 1. successfully constructed the SKOV3 fine of CDC50A+ cells. The cell.Western method detected the CDC50A expression of 293T cells transfected with 4 segments of CDC50A shRNA, and the efficiency of TMEM30A-homo-974 silencing CDC50A gene was the highest. The target sequence was GCCTGTGAACTGGCTTAAACC. to pack the lentivirus carrier and infect 293T cells. Western method could reduce the CDC50A expression by more than 80%. The flow cytometry was used to detect the infection efficiency in the CDC50A-GFP group was 87.9%, and the group NC-GFP was 81.6%. to separate the two groups of the infected lentivirus. The proportion of CDC50A+ cells in the flow analysis SKOV3 NC-GFP cells was 0.48%, while the CDC50A+ cells in SKOV3 CDC50A-GFP cells only accounted for 0.12%. indicating that the slow virus infection could effectively reduce the SKOV3 of the ovarian cancer cell line. The proportion of CDC50A+ cells, but the negative control sequence of lentivirus infection on the expression of CDC50A gene in SKOV3 cells had no effect on the biological characteristics of SKOV3 cell stem cells with down regulation of.2. CDC50A+ cells. In serum-free suspension culture, NC-GFP cells could form sphere, and most of them could have green fluorescence. The formation of sphere in CDC50A-GFP cells is slow, and the number of sphere.SKOV3 CDC50A-GFP cells with little green fluorescence can only be seen under the fluorescence microscope. The number of sphere is obviously less than that of SKOV3 NC-GFP cells, and it is almost impossible to pass the passage; and sphere of SKOV3 NC-GFP cells can be produced for at least three generations. The sphere formation ability of cell line SKOV3 cells was weakened and lost self renewal and differentiation ability. The proportion of CDC50A+ cells in sphere formed by SKOV3 CDC50A-GFP and NC-GFP cells by flow cytometry were 19.7% and 6.7% respectively, which were significantly higher than those of CDC50A+ cells in the cells with adherent growth, but CDC50 in SKOV3 CDC50A-GFP cells The proportion of A+ was significantly lower than that of NC-GFP cells. It showed that the concentration of sphere against CDC50A+ cells was 1.33 + 0.14ug/ml, 3.05 + 0.39 ug/ml, and the difference was statistically significant (P=0.01). The animal experiments showed that the 103 NC-GFP cells could make the mice tumor, and the same number of CDC50A-GFP cells failed to make the mice tumorigenic. It showed that the CDC50A gene was depressed by the silent ovarian cancer cell line SKOV3 cells, and.3. was found by sequencing the RNA of CDC50A+ cells to CDC50A- cells, and some CDC50A participated in the maintenance of ovarian cancer stem. CDC50A+ cells and CDC50A- cells selected from 3 primary ovarian cancer tissues were sequenced by RNA sequencing, and 92,59 and 251 differentially expressed genes (P0.01) were screened. RNA spectrum analysis was used to analyze the differential gene and its transcriptional transcript by bioinformatics. It was found that CDC50A may be through ATPase H+transpo Rting V1 subunit F, MALAT1, MAPK signal and regulation ribosome, splice body function to maintain ovarian cancer stem cells. Conclusion 1. using CDC50A shRNA lentivirus carrier infection technology can effectively reduce the proportion of CDC50A+ cells in ovarian cancer cell line SKOV3 CDC50A+ cells,.2. CDC50A+ cells than the case of ovarian cancer cell lines The ability of sphere formation, self renewal and differentiation and tumorigenesis are weakened, and.3. ATPase H+ transporting V1 subunit F, which is more sensitive to cisplatin, may be the main mechanism of CDC50A participation in the maintenance of biological characteristics of ovarian cancer stem cells.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.31

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