人腹水来源卵巢癌细胞系的建立及其侧群细胞的筛选和生物学特性研究

发布时间：2018-05-06 13:19

本文选题：卵巢癌 + 肿瘤干细胞　；参考：《吉林大学》2016年博士论文

【摘要】：目的:卵巢癌是妇科最常见的致死性肿瘤,五年生存率仅30%左右。虽然卵巢癌对铂制剂等一线化疗药物有较好的应答,然而高复发率和耐药性严重限制了化疗效果,肿瘤干细胞很可能在其中起到了关键作用。因此,我们从卵巢浆液性腺癌患者腹水中分离卵巢癌细胞并建系,建立成熟的卵巢癌侧群细胞分离方法,对侧群细胞进行鉴定和生物学特性研究;比较侧群细胞与非侧群细胞的转录组差异,并对差异基因进行富集分析,为研究卵巢癌干细胞调控肿瘤发生、发展、转移和耐药确立研究方向,并为新的治疗策略提供理论支撑。方法:1.人腹水来源卵巢癌永生化细胞系的建立及其生物学特性研究。采集卵巢浆液性腺癌(stage IIIc,grade 3)患者腹水,分离纯化出其中的卵巢癌细胞,并进行传代培养;观察此细胞系中卵巢癌细胞的生长状态和显微及超微结构;测定卵巢癌细胞的生长曲线,并对其进行染色体核型分析;最后,将卵巢癌细胞接种于NOD-SCID小鼠皮下检测其成瘤性,并对移植瘤进行组织学及免疫组化鉴定。2.人腹水来源卵巢癌细胞系中侧群细胞的分离及鉴定。Hoechst33342预染细胞后,以流式细胞术分选出侧群细胞和非侧群细胞,分别培养于无血清、含血清及软琼脂培养基中,比较两类细胞的自我更新、分化和克隆形成能力;通过免疫细胞化学染色比较两类细胞肿瘤干细胞标记物及干性基因表达差异;再将这两类细胞分别皮下或腹腔注射于NOD-SCID小鼠,比较其成瘤能力的差别,并对移植瘤进行组织学及免疫组化鉴定。3.人腹水来源卵巢癌细胞系中侧群细胞的生物学特性研究。采用划痕和Boydon小室实验检测两者的迁移和侵袭能力;对顺铂的耐药性则由侧群和非侧群细胞在一系列药物浓度下细胞生存率进行评估。4.人腹水来源卵巢癌细胞系中侧群细胞差异表达基因检测与富集分析。应用全基因组芯片检测技术筛选侧群细胞的差异表达基因,对差异基因分别进行GO分析、KEGG pathway分析和PPI分析,并对差异基因进行q PCR验证。结果:1.人腹水来源卵巢癌永生化细胞系的建立及其生物学特性研究。通过分离、纯化和传代培养,成功地由卵巢浆液性腺癌患者腹水的脱落细胞建立了卵巢癌细胞系OVA-W,目前已历经70代,具有永生化特征;该卵巢癌细胞贴壁生长,无接触性抑制,具有细胞异型性、核浆比例增加、核仁增大、细胞器增多等肿瘤细胞结构特征;卵巢癌细胞呈指数生长,群体倍增时间为31.2h,染色体核型均为超二倍体,可见染色体结构异常及衍生染色体;NOD-SCID小鼠移植瘤为卵巢低分化浆液性腺癌,与原发肿瘤无组织学差异,并表达卵巢浆液性腺癌标记物CA125及CK7。2.人腹水来源卵巢癌细胞系中侧群细胞的分离及鉴定。通过荧光标记流式细胞术由此卵巢癌细胞系中分离出的侧群细胞比例为(0.38±0.05)%。在体外实验中,侧群细胞与非侧群细胞经分化培养,前者侧群细胞的比例明显高于后者;经无血清悬浮培养,前者的成球数量显著高于后者;经软琼脂克隆培养,前者克隆形成率也明显高于后者;经免疫细胞化学染色,侧群细胞较非侧群细胞高表达肿瘤干细胞标记物CD133、ALDH及干性基因Nanog;在体内实验中,仅少量侧群细胞(1.0×103)接种于NOD-SCID小鼠皮下及腹腔即可成瘤,瘤组织在组织学特征上与原代肿瘤组织一致,并且表达卵巢浆液性腺癌的分子标志CA125。3.人腹水来源卵巢癌细胞系中侧群细胞的生物学特性研究。划痕实验结果显示侧群细胞较非侧群细胞迁移率高;Boydon小室实验结果显示侧群细胞较非侧群细胞具有更强的侵袭能力;对顺铂的耐药实验中,侧群细胞的IC50值明显高于非侧群细胞及未分选细胞,顺铂可使卵巢癌细胞中的侧群细胞比例显著上升,另外加入维拉帕米后,经顺铂处理的侧群细胞细胞生存率显著下降。4.人腹水来源卵巢癌细胞系中侧群细胞差异表达基因检测与富集分析。通过对侧群与非侧群细胞的全基因组芯片检测,筛选出2倍以上差异表达基因共21个,其中18个基因表达上调,3个基因表达下调;对这些差异表达基因进行富集分析显示TNFAIP3、IL-6和CXCL8基因可能与侧群细胞的特性相关;q PCR结果验证了这些差异基因在侧群细胞中高表达。结论:1.本研究由一高级别晚期卵巢浆液性乳头状囊腺癌患者腹水建立了一个永生化卵巢癌细胞系,经鉴定具有由其分离的肿瘤特征。2.由该细胞系可稳定、持续分离出一个极低比例的侧群细胞亚群,经鉴定该细胞亚群具有自我更新和分化潜能,可高表达肿瘤干细胞标记物及干性基因,以及较强的克隆形成能力和再现原发肿瘤表型的特点,符合肿瘤干细胞的基本特征,可做为卵巢癌干细胞生物学特性研究的细胞模型。3.该细胞系的侧群细胞表现为较强的迁移和侵袭和能力;对卵巢癌一线化疗药物顺铂耐药,且ABC结合盒转运蛋白与其耐药相关。4.根据侧群细胞差异表达基因的富集分析结果,推测TNFAIP3、IL-6和CXCL8基因可能与侧群细胞的肿瘤干细胞特性相关。本研究为卵巢癌干细胞调控肿瘤进展及耐药的分子机制提供了研究思路,并为卵巢癌的特异性治疗提供了可靠的靶点。
[Abstract]:Objective: ovarian cancer is the most common fatal tumor in gynecology, with a five year survival rate of only about 30%. Although ovarian cancer has a good response to platinum preparation and other first-line chemotherapeutic drugs, the high recurrence rate and drug resistance are severely restricted by chemotherapy, and cancer stem cells are likely to play a key role in it. Therefore, we are from ovarian serous adenocarcinoma. To separate the ovarian cancer cells in the ascites and establish the line, establish a mature method for separating the side group cells of the ovarian cancer, identify the side group cells and study the biological characteristics, compare the difference between the side group and the non side group, and enrich the differential genes, in order to study the ovarian cancer stem cells to regulate the tumorigenesis, development and metastasis. To establish the research direction of the drug resistance and provide theoretical support for the new treatment strategy. Methods: the establishment and biological characteristics of the 1. human ovarian cancer immortalized cell lines. The ascites were collected and purified from the ovarian serous adenocarcinoma (stage IIIc, grade 3), and the ovarian cancer cells were isolated and purified, and the cell line was observed. The growth state, microscopic and ultrastructure of the ovarian cancer cells, the growth curve of the ovarian cancer cells, and the karyotype analysis of the ovarian cancer cells. Finally, the ovarian cancer cells were inoculated in NOD-SCID mice to detect their tumorigenicity, and the transplanted tumor was histologically and immunohistochemical identified in the middle side of the ovarian cancer cell line of the.2. human ascites. After cell isolation and identification of.Hoechst33342 prestained cells, side group and non lateral group cells were selected by flow cytometry, and were cultured in serum-free, serum and soft agar medium, to compare the self renewal, differentiation and clone formation ability of the two types of cells, and compared the two types of cell tumor stem cells by immunocytochemical staining. The difference in the expression of marker and dry gene, and the two types of cells were injected subcutaneously or intraperitoneally to NOD-SCID mice, and the difference of their tumor formation ability was compared. The biological characteristics of the lateral group cells in the ovarian cancer cell line of.3. human ascites were identified by histology and immunohistochemistry. The scratch and Boydon laboratory tests were used. Measurement of the migration and invasiveness of the two groups; resistance to cisplatin from the side group and non lateral group cells under a series of drug concentrations to evaluate the differential expression gene detection and enrichment analysis of the side group cells in the ovarian cancer cell line of.4. human ascites. The differential expression of side group cells was screened by the whole genome core detection technique. GO analysis of different genes, KEGG pathway analysis and PPI analysis, and Q PCR verification of the differential genes. Results: the establishment and biological characteristics of 1. human ovarian cancer immortalized cell lines were established and successfully established by separation, purification and passage culture, and the exfoliated cells of the ascites of the serous adenocarcinoma of the ovary were successfully established. The ovarian cancer cell line OVA-W, which has gone through 70 generations, has immortalized characteristics, the ovarian cancer cells are adhered to the cell wall growth, no contact inhibition, cell heteromorphic, the ratio of nucleolus increases, nucleolus increases, organelles increase and other tumor cell structure characteristics; ovarian cancer cells grow exponentially, the population doubling time is 31.2h, chromosome karyotype is super. Diploid, chromosomal structural abnormalities and derived chromosomes are visible; NOD-SCID mice transplantation tumor is a low differentiated serous adenocarcinoma of the ovary, which is different from the primary tumor, and expresses the separation and identification of the lateral group cells in the ovarian cancer cell lines from the ovarian serous adenocarcinoma marker CA125 and CK7.2. human ascites. The proportion of side group cells isolated from the ovarian cancer cell line was (0.38 + 0.05)%. In vitro experiments, the proportion of side group cells and non side group cells was significantly higher than that of the latter; the former was significantly higher than the latter by serum-free suspension culture, and the former was cloned by soft agar clone culture. It was also significantly higher than the latter. By immunocytochemical staining, the side group cells expressed higher expression of tumor stem cell markers CD133, ALDH and Nanog, compared with non lateral group cells. In the experiment, only a small number of side group cells (1 x 103) were inoculated into the subcutaneous and abdominal cavity of NOD-SCID mice, and the tumor tissue was histologically characteristic with the primary tumor tissue. The molecular marker of ovarian serous adenocarcinoma was expressed in CA125.3. human ovarian cancer cell line. The results of scratch test showed that the migration rate of lateral group cells was higher than that of non lateral group cells; Boydon laboratory results showed that lateral group cells had stronger invasion ability than non lateral group cells; cisplatin was more effective than non lateral group cells. In the drug resistance experiment, the IC50 value of side group cells was significantly higher than that of non lateral group cells and undivided cells. Cisplatin could significantly increase the proportion of lateral group cells in ovarian cancer cells. After the addition of Vera Pammy, the survival rate of cisplatin treated side cell cells significantly decreased the differential expression of side group cells in the ovarian cancer cell lines of.4. human abdominal water. Gene detection and enrichment analysis, through the whole genome chip detection of side group and non side group cell, 2 times more than 2 times of differentially expressed genes were screened, of which 18 genes were up-regulated and 3 genes were down regulated. The enrichment analysis of these differentially expressed genes showed that TNFAIP3, IL-6 and CXCL8 genes may be related to the characteristics of side group cells. Q PCR results showed that these differentially expressed genes were highly expressed in lateral group cells. Conclusion: 1. this study established an immortalized ovarian cancer cell line in the ascites of a advanced stage of advanced ovarian serous papillary cystadenocarcinoma. It was identified that the tumor characteristic of.2. was stable by the cell line, and a very low ratio was continuously separated. It is identified that the subgroup of lateral group cells has the potential of self renewal and differentiation, which can highly express the markers and dry genes of tumor stem cells, as well as the characteristics of the strong clone formation ability and the reproduction of the primary tumor phenotype, which conforms to the basic characteristics of the tumor stem cells, and can be used as the cell of the biological characteristics of ovarian cancer stem cells. Model.3. the lateral group cells of the cell line showed strong migration, invasion and ability, and were resistant to cisplatin, a first-line chemotherapy drug for ovarian cancer. The ABC binding cassette transporter and its resistance related.4. were based on the enrichment analysis of differentially expressed genes in the side group cells, suggesting that the TNFAIP3, IL-6 and CXCL8 genes may be associated with the tumor stem cells of the lateral group cells. This study provides a way for ovarian cancer stem cells to regulate the progression of tumor and the molecular mechanism of drug resistance, and provides a reliable target for the specific treatment of ovarian cancer.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.31

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