本文關(guān)鍵詞： 卵巢癌 腫瘤干細(xì)胞 CD44 鉑類(lèi)耐藥　出處：《安徽醫(yī)科大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：研究背景:在婦科惡性腫瘤中,卵巢癌有著最高致死率,75%的患者在診斷時(shí)己進(jìn)入腫瘤晚期(Ⅲ-Ⅳ期)。對(duì)于卵巢癌的治療,目前臨床上的主流方案是腫瘤細(xì)胞減滅術(shù)輔以鉑類(lèi)為主的聯(lián)合化療,但是仍有60-80%的病人復(fù)發(fā),如果發(fā)展到卵巢癌晚期階段,平均只有16-22個(gè)月的生存期,5年生存率僅30%左右。導(dǎo)致卵巢癌治療不理想的原因主要是在化療過(guò)程中腫瘤細(xì)胞產(chǎn)生了耐藥性。最近關(guān)于小分子類(lèi)化合物以及靶向的藥物用于卵巢癌的臨床試驗(yàn)研究仍在繼續(xù),但是卵巢癌患者的整體預(yù)后并未得到改善。在獲得相關(guān)的腫瘤基因組圖譜的情況下,對(duì)于卵巢癌研究,多數(shù)學(xué)者仍主要致力于研究表觀遺傳學(xué)以及基因組的變化在卵巢癌臨床預(yù)后中產(chǎn)生的作用,對(duì)于腫瘤復(fù)發(fā)產(chǎn)生的機(jī)理以及在治療過(guò)程中產(chǎn)生的耐藥性的研究尚不清楚,因此使卵巢癌的治療效果進(jìn)展緩慢。由于腫瘤的生長(zhǎng)、復(fù)發(fā)和轉(zhuǎn)移的特點(diǎn)與干細(xì)胞的生物學(xué)特性十分相像,因此,有學(xué)者提出腫瘤干細(xì)胞(cancer stem cell,CSC)理論,這一理論的提出為我們重新認(rèn)識(shí)腫瘤的本質(zhì)和起源,為腫瘤的臨床治療提供了新的視覺(jué)角度和方向。近年來(lái),研究人員為尋找卵巢癌治療的新途徑,把注意力集中到了腫瘤干細(xì)胞上,借鑒其它實(shí)體瘤的研究成果并在此基礎(chǔ)上開(kāi)辟了卵巢癌治療的新方法。對(duì)于卵巢癌的研究,目前主要借鑒其它癌癥相關(guān)領(lǐng)域研究的經(jīng)驗(yàn)進(jìn)行干細(xì)胞標(biāo)記蛋白的選擇,主要采用 CD44、CD133、CD90、CD117、CD24、ALDH1、以及 EPCAM 等作為標(biāo)記物,并取得了一定成果。探尋針對(duì)性的標(biāo)記蛋白尤其重要,對(duì)卵巢癌干細(xì)胞的研究有很重要的意義。多項(xiàng)研究認(rèn)為腫瘤干細(xì)胞以及其表達(dá)的特異分子與惡性腫瘤的發(fā)生、發(fā)展關(guān)系密切,CD44就是其中一個(gè)熱點(diǎn)研究。CD44基因位于人類(lèi)的11號(hào)染色體的短臂上,全長(zhǎng)約為50kb,并由20個(gè)高度保守的外顯子構(gòu)成。CD44為分布廣泛的細(xì)胞表面跨膜糖蛋白,在正常細(xì)胞和腫瘤細(xì)胞上均可表達(dá)。有研究證實(shí),CD44與多種惡性腫瘤的發(fā)生、發(fā)展密切相關(guān)。最新研究發(fā)現(xiàn),腫瘤干細(xì)胞表達(dá)的CD44分子與腫瘤的發(fā)展及預(yù)后密切相關(guān)。目的:從卵巢癌細(xì)胞系和組織中篩選標(biāo)記的CD44+細(xì)胞,對(duì)CD44+細(xì)胞進(jìn)行初步的干細(xì)胞特性的鑒定,通過(guò)體外與體內(nèi)實(shí)驗(yàn),分析CD44+卵巢癌細(xì)胞的sphere形成能力,自身更新能力及分化和致癌能力,并進(jìn)一步驗(yàn)證其對(duì)順鉑的耐藥性,分析CD44的表達(dá)與卵巢癌患者臨床預(yù)后的關(guān)系。方法:1、利用流式細(xì)胞分析法分析卵巢癌細(xì)胞系SKOV3、A2780、ES2、IGROV1、OC3中CD44+細(xì)胞的比例,分選CD44+和CD44-細(xì)胞,驗(yàn)證其在HG-DMEM/10%FBS培養(yǎng)基培養(yǎng)后再次分化亞群的能力;通過(guò)無(wú)血清懸浮培養(yǎng)法檢測(cè)CD44+和CD44-細(xì)胞sphere形成能力以及傳代能力;采用免疫熒光法檢測(cè)SKOV3 sphere對(duì)CD44的富集作用;流式細(xì)胞法分選CD44+和CD44-細(xì)胞,將兩種細(xì)胞按梯度接種于NOD/SCID小鼠皮下,觀察其體內(nèi)成瘤速度,再將瘤體中分選出的兩群細(xì)胞接種到小鼠體內(nèi),觀察成瘤情況;流式細(xì)胞法分析移植瘤中的CD44+細(xì)胞比例,驗(yàn)證CD44+和CD44-細(xì)胞在體內(nèi)的分化能力。2、收集、分離卵巢癌患者癌組織和腹水中癌細(xì)胞,流式細(xì)胞法檢測(cè)原代癌細(xì)胞中CD44+的比例。建立原代癌細(xì)胞sphere培養(yǎng)方法,檢測(cè)原代CD44+和CD44-細(xì)胞的sphere形成和傳代能力。免疫熒光法檢測(cè)SKOV3 sphere對(duì)CD44的富集作用。流式細(xì)胞法分選CD44+和CD44-細(xì)胞,將兩種細(xì)胞按梯度接種于NOD/SCID小鼠皮下,觀察成瘤速度以及兩者的成瘤率和瘤體大小,并進(jìn)行統(tǒng)計(jì)學(xué)分析。小鼠成瘤后,再分選出兩群細(xì)胞并接種到小鼠體內(nèi),再次觀察成瘤情況,流式細(xì)胞法檢測(cè)移植瘤中CD44+的比例,檢測(cè)CD44+和CD44-細(xì)胞在體內(nèi)的分化能力。3、MTT法檢測(cè)SKOV3和ES2細(xì)胞系中CD44+和CD44-細(xì)胞對(duì)順鉑的耐藥性,流式細(xì)胞法檢測(cè)不同濃度順鉑對(duì)SKOV3/CD44+細(xì)胞產(chǎn)生的富集作用。流式細(xì)胞法檢測(cè)化療藥物順鉑對(duì)卵巢癌患者癌組織中CD44+細(xì)胞的富集作用;建立NOD/SCID小鼠卵巢癌移植瘤模型,驗(yàn)證順鉑化療對(duì)于移植瘤中CD44+細(xì)胞的富集作用。結(jié)果:1、五種卵巢癌細(xì)胞系中均有少量的CD44+細(xì)胞群,比率在0.4%-2%。CD44+細(xì)胞亞群可分化出CD44-細(xì)胞,CD44-細(xì)胞卻不能分化出CD44+細(xì)胞。CD44+細(xì)胞形成的sphere多于CD44-細(xì)胞,并且CD44+細(xì)胞形成的Sphere能夠在體外最少傳四代,而CD44-細(xì)胞不能進(jìn)行傳代。免疫熒光和流式細(xì)胞分析均發(fā)現(xiàn)SKOV3 sphere上CD44+細(xì)胞明顯高于貼壁SKOV3細(xì)胞;體內(nèi)實(shí)驗(yàn)發(fā)現(xiàn)CD44+細(xì)胞比CD44-細(xì)胞的成瘤能力強(qiáng),CD44+細(xì)胞移植瘤能夠在小鼠體內(nèi)連續(xù)傳遞,后者的移植瘤則無(wú)法傳遞。HE染色體顯示,CD44+細(xì)胞移植瘤與二代移植瘤組織學(xué)類(lèi)型相同,表明CD44+細(xì)胞在體內(nèi)具有分化能力。2、原代卵巢癌癌組織中的CD44+細(xì)胞可以形成sphere,并可連續(xù)傳代,體內(nèi)實(shí)驗(yàn)發(fā)現(xiàn)CD44+細(xì)胞較CD44-細(xì)胞具有更強(qiáng)的成瘤能力。3、與CD44-細(xì)胞相比,CD44+細(xì)胞對(duì)順鉑有更強(qiáng)的耐藥性;不同濃度的順鉑作用后SKOV3細(xì)胞總數(shù)下降,但CD44的陽(yáng)性率升高。體內(nèi)實(shí)驗(yàn)發(fā)現(xiàn),順鉑化療后CD44+細(xì)胞比例明顯升高;動(dòng)物實(shí)驗(yàn)發(fā)現(xiàn),順鉑化療后小鼠體內(nèi)移植瘤的生長(zhǎng)速度明顯減慢,但移植瘤中的CD44陽(yáng)性率增高,表明順鉑對(duì)于CD44+細(xì)胞具有富集作用,CD44+細(xì)胞對(duì)順鉑具有耐藥性。結(jié)論:1、CD44+細(xì)胞在5種卵巢癌細(xì)胞系中的表達(dá)穩(wěn)定在0.4%-2%,符合干細(xì)胞的比例。CD44+細(xì)胞群具有體內(nèi)外分化能力、sphere形成能力、自身更新能力等腫瘤干細(xì)胞的特點(diǎn)。該類(lèi)細(xì)胞有更強(qiáng)的致瘤性,并可在小鼠體內(nèi)連續(xù)傳代。2、卵巢癌患者的原代癌細(xì)胞中可以分離出CD44+細(xì)胞群,該細(xì)胞群具有體內(nèi)分化能力、sphere形成能力以及自我更新能力等CSC特點(diǎn)。原代癌細(xì)胞中的CD44+細(xì)胞有更強(qiáng)的成瘤能力,可在體內(nèi)進(jìn)行連續(xù)傳代。3、卵巢癌細(xì)胞系、原代卵巢癌細(xì)胞、以及動(dòng)物實(shí)驗(yàn)均證實(shí)順鉑對(duì)CD44+細(xì)胞有富集作用。
[Abstract]:Background: in gynecologic malignant tumors, ovarian cancer has the highest mortality rate, 75% of the patients has entered advanced cancer at diagnosis (III / IV). For the treatment of ovarian cancer, the current mainstream clinical is ovarian cancer cytoreductive surgery combined with chemotherapy with platinum based, but there are still 60-80% the recurrence of patients with advanced ovarian cancer, if the development to the stage, only an average of 16-22 months of survival, 5 year survival rate is only about 30%. The cause of the treatment of ovarian cancer is not ideal in the course of chemotherapy in tumor cells develop resistance. A recent drug small molecular compounds and targeted for use in clinical trials. Study of ovarian cancer continues, but the overall prognosis of patients with ovarian cancer has not been improved. In the tumor genome related to the case for ovarian cancer research, most scholars still focused on epigenetic research Transfer and changes in the genome in the prognosis of ovarian carcinoma, the mechanism of tumor recurrence and drug resistance study produced in the course of treatment is not clear, so that the progress in the treatment of ovarian cancer. Due to slow tumor growth, recurrence and metastasis of the biological characteristics of stem cells is very similar therefore, some scholars have proposed cancer stem cells (cancer stem cell, CSC) theory, this theory is the essence and origin of our understanding of cancer, provides a new visual angle and direction for the clinical treatment of cancer. In recent years, researchers find new ways for the treatment of ovarian cancer, focus to focus on cancer stem cells, drawing lessons from the research results of other solid tumors, and on this basis, it opens up a new way for the treatment of ovarian cancer. The study of ovarian cancer, the main reference for other cancers Study on the related experience in the field of stem cell marker protein, mainly by CD44, CD133, CD90, CD117, CD24, ALDH1, and EPCAM as a marker, and achieved certain results. To explore the specific marker protein is especially important, has very important significance to the study of ovarian cancer stem cell studies. That cancer stem cells and its specific molecular expression and tumorigenesis, development is closely related to the short arm of CD44 is one of the hot research of the human.CD44 gene is located on chromosome 11, the total length of about 50kb, and is composed of 20 highly conserved exon.CD44 is widely distributed cell surface transmembrane glycoprotein can be expressed in normal cells and tumor cells. Studies have confirmed that CD44 with a variety of malignant tumors, is closely related to the development. The latest study found that tumor stem cells expressed CD44 protein and tumor development Development and prognosis. Objective: to screen the marker of CD44+ cells from human ovarian cancer cell lines and tissues, and identify the characteristics of stem cells of CD44+ cells, the in vitro and in vivo experiments, analysis of ovarian cancer cells CD44+ sphere formation ability, self renewing and differentiation and carcinogenic ability, and further verify the cisplatin resistance, analysis of the relationship between CD44 expression and clinical prognosis of patients with ovarian cancer. Methods: 1, using flow cytometric analysis of SKOV3 ovarian cancer cell line A2780, ES2, IGROV1, CD44+ cell proportion of OC3, CD44+ and CD44- cell sorting, verify the medium again after the differentiation of Asia in the HG-DMEM/10%FBS group; through the cultivation method for the detection of CD44+ and CD44- cells sphere formation ability and subculture ability in serum-free suspension; immunofluorescence was used to detect SKOV3 sphere of CD44 enrichment; flow cytometry method CD44+ and CD44- cells, two cells by gradient inoculated into NOD/SCID mice subcutaneously to observe the tumorigenicity rate, then the tumor in the selected two groups of cells were inoculated into mice, the tumor growth was observed; analysis of the proportion of CD44+ cell xenografts in nude mice by flow cytometry, the CD44+ and CD44- collection of cells in vivo differentiation of.2 cell carcinoma, carcinoma from patients with ovarian cancer and ascites. The proportion of flow cytometry was used to detect CD44+ in primary cancer cells. To establish a culture method of sphere primary cancer cells, detection of sphere shape of primary CD44+ and CD44- cells and passage ability. Detection of SKOV3 enrichment sphere immunofluorescence assay of CD44. Flow cytometry sorting CD44+ and CD44- cells, two cells by the gradient inoculated in NOD/SCID mice, observe the tumor growth rate and tumor size and tumor rate of both, and mouse were analyzed. After the tumor cells and then selected two groups inoculated into mice, the tumor growth was observed again, CD44+ transplanted tumor by flow cytometry in proportion, detection of CD44+ and CD44- cells in vivo differentiation ability of.3, CD44+ and CD44- cell drug resistance detection of SKOV3 and MTT in ES2 cell line to cisplatin, enrichment different concentration of cisplatin by flow cytometry in SKOV3/CD44+ cells. The effect of cisplatin by flow cytometry in CD44+ cells in ovarian cancer tissue enrichment; establish NOD/SCID mice xenograft model of human ovarian cancer, cisplatin chemotherapy for verification of enrichment of CD44+ cells in transplanted tumor. Results: 1, there are five kinds of ovarian cancer cell lines in a small amount of CD44+ cell population ratio in the subgroup of 0.4%-2%.CD44+ cells can differentiate into CD44- cells, CD44- cells but not CD44+ cells were differentiated.CD44+ cells to form sphere in CD44- cells The cell, and the formation of Sphere CD44+ cells in vitro can at least four generations, but not CD44- cells were passaged. Immunofluorescence and flow cytometry analysis showed that SKOV3 sphere on CD44+ cells was significantly higher than that of adherent SKOV3 cells; in vivo CD44+ cells than CD44- cells tumorigenic ability, CD44+ cell transplanted tumor can be continuous transfer in mice, the tumor will not be delivered.HE chromosome showed that CD44+ cells transplanted tumor and two tumor histologic types are the same, indicating that CD44+ cells have the differentiation ability of.2 in vivo, the primary ovarian carcinoma CD44+ cells can form sphere, and continuous passage of CD44+ cells in vivo compared with CD44- cells with tumor.3 ability to be stronger, compared with CD44- cells, CD44+ cells are more resistant to cisplatin; cisplatin of different concentration after the total number of SKOV3 cells decreased, but CD44 The positive rate increased. In vivo, the proportion of CD44+ cells after chemotherapy was significantly increased; the animal experiments showed that after chemotherapy in mice transplanted tumor growth rate slowed down, but the positive rate of CD44 in the transplanted tumor increased, showed that cisplatin has enrichment effect for CD44+ cells, CD44+ cells resistant to cisplatin. Conclusion: 1 CD44+, stable cell expression in 5 ovarian cancer cell lines in 0.4%-2%, with stem cell ratio of.CD44+ cells with differentiation ability, the ability to produce sphere, stem cell characteristics of their ability to update tumors. Tumorigenicity of the cells is stronger, and can be continuously passaged in mice.2 primary cancer cells in patients with ovarian cancer can be isolated from CD44+ cells, the cells with in vivo differentiation ability, sphere formation ability and self-renewal characteristics of CSC. The primary cancer cells in CD44+ The cells have stronger tumorigenicity and can be continuously passaged in vivo..3, ovarian cancer cell lines, primary ovarian cancer cells and animal experiments all confirm that cisplatin has enrichment effect on CD44+ cells.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R737.31

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