本文選題：卵巢癌 + 腫瘤干細(xì)胞��； 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：研究背景卵巢癌是女性常見(jiàn)的惡性腫瘤之一,其發(fā)生率僅次于宮頸癌和子宮內(nèi)膜癌,居第三位,然而其致死率高居首位。卵巢癌起病隱匿,大多數(shù)患者診斷時(shí)已為晚期。在過(guò)去的幾十年中,對(duì)卵巢癌患者的基本治療方案采取理想的腫瘤細(xì)胞減滅術(shù)輔助鉑類藥物為主的聯(lián)合化療,雖然取得了一定的療效,但是仍有70%左右的患者在18個(gè)月內(nèi)復(fù)發(fā),晚期卵巢癌患者的5年生存率僅為30%。目前認(rèn)為腫瘤的耐藥及復(fù)發(fā)是造成卵巢癌高死亡率的主要原因,但是卵巢癌耐藥及復(fù)發(fā)的機(jī)制目前仍不清楚,故而嚴(yán)重阻礙了卵巢癌的有效治療。自上世紀(jì)來(lái),腫瘤干細(xì)胞(Cancer stem cells, CSCs)理論被提出,認(rèn)為腫瘤組織中存在少量的干細(xì)胞,是導(dǎo)致腫瘤進(jìn)展、復(fù)發(fā)及化療耐藥的根源。Bonnet和Dick從白血病患者中分離出CD34+CD38-細(xì)胞被證實(shí)具有干細(xì)胞特性,這是世界上首次發(fā)現(xiàn)CSCs的存在。之后大量研究相繼發(fā)現(xiàn)多種實(shí)體腫瘤中也存在極少量的CSCs,如乳腺癌、前列腺癌、結(jié)腸癌等。研究表明,CSCs具有分化、自我更新等類似正常干細(xì)胞的生物學(xué)特性,同時(shí)具有致瘤能力且對(duì)化療藥物產(chǎn)生耐藥性。目前腫瘤干細(xì)胞的研究取得了很大的進(jìn)展,在一些實(shí)體腫瘤中已建立了一系列CSCs分離、鑒定的方法,對(duì)腫瘤發(fā)生、進(jìn)展、耐藥和復(fù)發(fā)的機(jī)制的研究以及探索腫瘤治療的新領(lǐng)域具有重要意義。近年來(lái),CSCs理論被應(yīng)用于卵巢癌的研究中取得了一定的成績(jī)。但是,目前對(duì)于卵巢癌干細(xì)胞的研究仍在借鑒其他實(shí)體腫瘤的方法,如對(duì)卵巢癌干細(xì)胞的識(shí)別缺乏較為特異的標(biāo)記物,目前使用較多的標(biāo)記物有CD133、CD117、CD44、ALDH1、 EPCAM等,其中CD44被作為乳腺癌、胰腺癌等CSCs的標(biāo)記物,CD133被作為結(jié)腸癌等CSCs的標(biāo)記物等。其分離獲得的方法仍主要依靠SP細(xì)胞的分選。因此,尋找特異性的表面標(biāo)記物,對(duì)于卵巢癌干細(xì)胞的研究極其重要,對(duì)探索卵巢癌新的治療方法具有重大意義。本課題前期工作中利用穩(wěn)定同位素標(biāo)記及質(zhì)譜為基礎(chǔ)的蛋白質(zhì)組學(xué)技術(shù)分析比較卵巢癌細(xì)胞系SP和非SP細(xì)胞膜蛋白的差異表達(dá),篩選出一種跨膜蛋白30A(transmembrane protein 30A, TMEM30A),又稱細(xì)胞周期蛋白50A(cell division cycle 50A, CDC50A),并通過(guò)大量體內(nèi)外實(shí)驗(yàn)對(duì)卵巢癌細(xì)胞系及原代卵巢癌細(xì)胞CDC50A+細(xì)胞進(jìn)行鑒定,證實(shí)其具有CSCs生物學(xué)特性,是CDC50A有望成為卵巢癌干細(xì)胞較為特異的表面標(biāo)記物。本研究承接前期工作,對(duì)CDC50A參與維持卵巢癌干細(xì)胞特性及其相關(guān)作用機(jī)制做進(jìn)一步研究。研究方法1.構(gòu)建CDC50A+細(xì)胞比例下調(diào)的SKOV3細(xì)胞。用4段CDC50A shRNA轉(zhuǎn)染293T細(xì)胞,運(yùn)用Western blot方法篩選能有效下調(diào)CDC50A基因表達(dá)的shRNA,包裝慢病毒載體,并利用通過(guò)慢病毒感染技術(shù),下調(diào)卵巢癌細(xì)胞系SKOV3中CDC50A+細(xì)胞比例,利用流式細(xì)胞學(xué)方法檢測(cè)其下調(diào)效率。利用流式分選儀分選得到CDC50A+細(xì)胞比例下調(diào)組(SKOV3CDC50A-GFP)細(xì)胞和陰性對(duì)照組(SKOV3 NC-GFP)細(xì)胞。采用流式細(xì)胞學(xué)方法檢測(cè)SKOV3 CDC50A-GFP及SKOV3 NC-GFP中CDC50A的表達(dá)水平。2. CDC50A+細(xì)胞比例下調(diào)的SKOV3細(xì)胞干細(xì)胞特性的鑒定。利用含血清的HG-DMEM培養(yǎng)基體外培養(yǎng),驗(yàn)證SKOV3 CDC50A-GFP及SKOV3 NC-GFP細(xì)胞的分化能力。利用無(wú)血清懸浮培養(yǎng)方法,檢測(cè)SKOV3 CDC50A-GFP及SKOV3 NC-GFP細(xì)胞sphere形成能力及傳代能力,并運(yùn)用流式細(xì)胞學(xué)方法驗(yàn)證sphere對(duì)CDC50A的富集作用。MTT法檢測(cè)SKOV3 CDC50A-GFP和NC-GFP細(xì)胞對(duì)順鉑的耐藥性。將SKOV3 CDC50A-GFP和NC-GFP細(xì)胞等量接種于NSG小鼠皮下,觀察成瘤情況,檢測(cè)其成瘤能力。3.人卵巢癌CDC50A+和CDC50A細(xì)胞RNA測(cè)序及其與干細(xì)胞功能相關(guān)的機(jī)制分析。收集3例卵巢癌患者腫瘤組織,分離得到卵巢癌細(xì)胞,利用流式細(xì)胞分選儀分選獲得CDC50A+和CDC50A細(xì)胞,分別提取兩組細(xì)胞RNA,進(jìn)行RNA測(cè)序。獲得兩組細(xì)胞的差異表達(dá)基因,并對(duì)其進(jìn)行功能分析和相關(guān)信號(hào)通路的富集分析。研究結(jié)果1.成功構(gòu)建CDC50A+細(xì)胞比例下調(diào)的SKOV3細(xì)胞。Western方法檢測(cè)轉(zhuǎn)染4段CDC50A shRNA的293T細(xì)胞的CDC50A表達(dá),序列TMEM30A-homo-974沉默CDC50A基因的效率最高,其靶序列為GCCTGTGAACTGGCTTAAACC。將其包裝慢病毒載體后,感染293T細(xì)胞,Western法檢測(cè)其能使CDC50A表達(dá)降低80%以上。利用慢病毒感染SKOV3細(xì)胞,流式檢測(cè)其感染效率CDC50A-GFP組為87.9%,NC-GFP組為81.6%。分選出成功感染慢病毒的兩組細(xì)胞后,流式分析SKOV3 NC-GFP細(xì)胞中CDC50A+細(xì)胞比例為0.48%,而SKOV3 CDC50A-GFP細(xì)胞中CDC50A+細(xì)胞僅占0.12%。表明慢病毒感染能有效的下調(diào)卵巢癌細(xì)胞系SKOV3中CDC50A+細(xì)胞比例,而含陰性對(duì)照序列的慢病毒感染對(duì)SKOV3細(xì)胞中CDC50A基因的表達(dá)基本無(wú)影響。2. CDC50A+細(xì)胞比例下調(diào)的SKOV3細(xì)胞株干細(xì)胞的生物學(xué)特性減弱。在無(wú)血清懸浮培養(yǎng)條件下,NC-GFP細(xì)胞能形成sphere,且絕大多數(shù)能帶有綠色熒光；而CDC50A-GFP細(xì)胞sphere形成慢,且在熒光顯微鏡下僅能看到極少帶綠色熒光的sphere。SKOV3 CDC50A-GFP細(xì)胞形成的sphere個(gè)數(shù)明顯少于SKOV3 NC-GFP細(xì)胞,幾乎不能傳代；而SKOV3 NC-GFP細(xì)胞形成的sphere能連續(xù)傳代至少三代。表明CDC50A基因被沉默的卵巢癌細(xì)胞系SKOV3細(xì)胞在體外sphere形成能力減弱且喪失自我更新及分化能力。流式檢測(cè)SKOV3 CDC50A-GFP和NC-GFP細(xì)胞所形成的sphere中CDC50A+細(xì)胞的比例分別為19.7%和6.7%,均比貼壁生長(zhǎng)的細(xì)胞中的CDC50A+細(xì)胞的比例明顯升高,但SKOV3 CDC50A-GFP細(xì)胞中CDC50A+比例明顯低于NC-GFP細(xì)胞。表明sphere對(duì)CDC50A+細(xì)胞具有富集作用。MTT法測(cè)定順鉑對(duì)SKOV3 CDC50A-GFP和NC-GFP細(xì)胞的IC50均值為1.33±0.14ug/ml,3.05±0.39 ug/ml,差異具有統(tǒng)計(jì)學(xué)意義(P=0.01)。說(shuō)明CCDC50A+細(xì)胞比例下調(diào)的的卵巢癌細(xì)胞系SKOV3細(xì)胞對(duì)順鉑的耐藥性減弱。動(dòng)物實(shí)驗(yàn)顯示103 NC-GFP細(xì)胞能使小鼠成瘤,而同樣數(shù)量的CDC50A-GFP細(xì)胞未能使小鼠成瘤。表明CDC50A基因被沉默的卵巢癌細(xì)胞系SKOV3細(xì)胞致瘤能力降低。3.通過(guò)對(duì)CDC50A+細(xì)胞與CDC50A-細(xì)胞的RNA測(cè)序,發(fā)現(xiàn)了一些CDC50A參與維持卵巢癌干細(xì)胞特性的相關(guān)機(jī)制。對(duì)3原代卵巢癌組織中分選出的CDC50A+細(xì)胞和CDC50A-細(xì)胞進(jìn)行RNA測(cè)序,分別篩選出92、59和251個(gè)差異表達(dá)基因(P0.01)。應(yīng)用RNA譜分析、生物信息學(xué)技術(shù)對(duì)差異基因及其轉(zhuǎn)錄本其進(jìn)行分析分析發(fā)現(xiàn)CDC50A可能通過(guò)ATPase H+transporting V1 subunit F、MALAT1、MAPK信號(hào)及調(diào)控核糖體、剪接體功能維持卵巢癌干細(xì)胞的特性及功能。結(jié)論1.利用CDC50A shRNA慢病毒載體感染技術(shù)能有效的下調(diào)卵巢癌細(xì)胞系SKOV3中CDC50A+細(xì)胞比例。2. CDC50A+細(xì)胞比例下調(diào)的卵巢癌細(xì)胞系SKOV3干細(xì)胞生物學(xué)特性減弱,sphere形成能力、自我更新和分化能力及致瘤能力均減弱,且對(duì)順鉑作用更為敏感。3. ATPase H+ transporting V1 subunit F可能是CDC50A參與維持卵巢癌干細(xì)胞生物學(xué)特性的主要機(jī)制基礎(chǔ)。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.31

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1 李曉瑩;細(xì)胞周期蛋白CDC50A參與維持卵巢癌干細(xì)胞特性及其相關(guān)作用機(jī)制的研究[D];北京協(xié)和醫(yī)學(xué)院;2016年

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