CD44在卵巢癌发生发展中作用机制的研究

发布时间：2018-03-02 09:44

本文关键词： 卵巢癌肿瘤干细胞 CD44 铂类耐药　出处：《安徽医科大学》2016年博士论文　论文类型：学位论文

【摘要】：研究背景:在妇科恶性肿瘤中,卵巢癌有着最高致死率,75%的患者在诊断时己进入肿瘤晚期(Ⅲ-Ⅳ期)。对于卵巢癌的治疗,目前临床上的主流方案是肿瘤细胞减灭术辅以铂类为主的联合化疗,但是仍有60-80%的病人复发,如果发展到卵巢癌晚期阶段,平均只有16-22个月的生存期,5年生存率仅30%左右。导致卵巢癌治疗不理想的原因主要是在化疗过程中肿瘤细胞产生了耐药性。最近关于小分子类化合物以及靶向的药物用于卵巢癌的临床试验研究仍在继续,但是卵巢癌患者的整体预后并未得到改善。在获得相关的肿瘤基因组图谱的情况下,对于卵巢癌研究,多数学者仍主要致力于研究表观遗传学以及基因组的变化在卵巢癌临床预后中产生的作用,对于肿瘤复发产生的机理以及在治疗过程中产生的耐药性的研究尚不清楚,因此使卵巢癌的治疗效果进展缓慢。由于肿瘤的生长、复发和转移的特点与干细胞的生物学特性十分相像,因此,有学者提出肿瘤干细胞(cancer stem cell,CSC)理论,这一理论的提出为我们重新认识肿瘤的本质和起源,为肿瘤的临床治疗提供了新的视觉角度和方向。近年来,研究人员为寻找卵巢癌治疗的新途径,把注意力集中到了肿瘤干细胞上,借鉴其它实体瘤的研究成果并在此基础上开辟了卵巢癌治疗的新方法。对于卵巢癌的研究,目前主要借鉴其它癌症相关领域研究的经验进行干细胞标记蛋白的选择,主要采用 CD44、CD133、CD90、CD117、CD24、ALDH1、以及 EPCAM 等作为标记物,并取得了一定成果。探寻针对性的标记蛋白尤其重要,对卵巢癌干细胞的研究有很重要的意义。多项研究认为肿瘤干细胞以及其表达的特异分子与恶性肿瘤的发生、发展关系密切,CD44就是其中一个热点研究。CD44基因位于人类的11号染色体的短臂上,全长约为50kb,并由20个高度保守的外显子构成。CD44为分布广泛的细胞表面跨膜糖蛋白,在正常细胞和肿瘤细胞上均可表达。有研究证实,CD44与多种恶性肿瘤的发生、发展密切相关。最新研究发现,肿瘤干细胞表达的CD44分子与肿瘤的发展及预后密切相关。目的:从卵巢癌细胞系和组织中筛选标记的CD44+细胞,对CD44+细胞进行初步的干细胞特性的鉴定,通过体外与体内实验,分析CD44+卵巢癌细胞的sphere形成能力,自身更新能力及分化和致癌能力,并进一步验证其对顺铂的耐药性,分析CD44的表达与卵巢癌患者临床预后的关系。方法:1、利用流式细胞分析法分析卵巢癌细胞系SKOV3、A2780、ES2、IGROV1、OC3中CD44+细胞的比例,分选CD44+和CD44-细胞,验证其在HG-DMEM/10%FBS培养基培养后再次分化亚群的能力;通过无血清悬浮培养法检测CD44+和CD44-细胞sphere形成能力以及传代能力;采用免疫荧光法检测SKOV3 sphere对CD44的富集作用;流式细胞法分选CD44+和CD44-细胞,将两种细胞按梯度接种于NOD/SCID小鼠皮下,观察其体内成瘤速度,再将瘤体中分选出的两群细胞接种到小鼠体内,观察成瘤情况;流式细胞法分析移植瘤中的CD44+细胞比例,验证CD44+和CD44-细胞在体内的分化能力。2、收集、分离卵巢癌患者癌组织和腹水中癌细胞,流式细胞法检测原代癌细胞中CD44+的比例。建立原代癌细胞sphere培养方法,检测原代CD44+和CD44-细胞的sphere形成和传代能力。免疫荧光法检测SKOV3 sphere对CD44的富集作用。流式细胞法分选CD44+和CD44-细胞,将两种细胞按梯度接种于NOD/SCID小鼠皮下,观察成瘤速度以及两者的成瘤率和瘤体大小,并进行统计学分析。小鼠成瘤后,再分选出两群细胞并接种到小鼠体内,再次观察成瘤情况,流式细胞法检测移植瘤中CD44+的比例,检测CD44+和CD44-细胞在体内的分化能力。3、MTT法检测SKOV3和ES2细胞系中CD44+和CD44-细胞对顺铂的耐药性,流式细胞法检测不同浓度顺铂对SKOV3/CD44+细胞产生的富集作用。流式细胞法检测化疗药物顺铂对卵巢癌患者癌组织中CD44+细胞的富集作用;建立NOD/SCID小鼠卵巢癌移植瘤模型,验证顺铂化疗对于移植瘤中CD44+细胞的富集作用。结果:1、五种卵巢癌细胞系中均有少量的CD44+细胞群,比率在0.4%-2%。CD44+细胞亚群可分化出CD44-细胞,CD44-细胞却不能分化出CD44+细胞。CD44+细胞形成的sphere多于CD44-细胞,并且CD44+细胞形成的Sphere能够在体外最少传四代,而CD44-细胞不能进行传代。免疫荧光和流式细胞分析均发现SKOV3 sphere上CD44+细胞明显高于贴壁SKOV3细胞;体内实验发现CD44+细胞比CD44-细胞的成瘤能力强,CD44+细胞移植瘤能够在小鼠体内连续传递,后者的移植瘤则无法传递。HE染色体显示,CD44+细胞移植瘤与二代移植瘤组织学类型相同,表明CD44+细胞在体内具有分化能力。2、原代卵巢癌癌组织中的CD44+细胞可以形成sphere,并可连续传代,体内实验发现CD44+细胞较CD44-细胞具有更强的成瘤能力。3、与CD44-细胞相比,CD44+细胞对顺铂有更强的耐药性;不同浓度的顺铂作用后SKOV3细胞总数下降,但CD44的阳性率升高。体内实验发现,顺铂化疗后CD44+细胞比例明显升高;动物实验发现,顺铂化疗后小鼠体内移植瘤的生长速度明显减慢,但移植瘤中的CD44阳性率增高,表明顺铂对于CD44+细胞具有富集作用,CD44+细胞对顺铂具有耐药性。结论:1、CD44+细胞在5种卵巢癌细胞系中的表达稳定在0.4%-2%,符合干细胞的比例。CD44+细胞群具有体内外分化能力、sphere形成能力、自身更新能力等肿瘤干细胞的特点。该类细胞有更强的致瘤性,并可在小鼠体内连续传代。2、卵巢癌患者的原代癌细胞中可以分离出CD44+细胞群,该细胞群具有体内分化能力、sphere形成能力以及自我更新能力等CSC特点。原代癌细胞中的CD44+细胞有更强的成瘤能力,可在体内进行连续传代。3、卵巢癌细胞系、原代卵巢癌细胞、以及动物实验均证实顺铂对CD44+细胞有富集作用。
[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.

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.31

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