發(fā)布時(shí)間：2018-05-06 13:19

  本文選題：卵巢癌 + 腫瘤干細(xì)胞��； 參考：《吉林大學(xué)》2016年博士論文

【摘要】：目的:卵巢癌是婦科最常見(jiàn)的致死性腫瘤,五年生存率僅30%左右。雖然卵巢癌對(duì)鉑制劑等一線化療藥物有較好的應(yīng)答,然而高復(fù)發(fā)率和耐藥性嚴(yán)重限制了化療效果,腫瘤干細(xì)胞很可能在其中起到了關(guān)鍵作用。因此,我們從卵巢漿液性腺癌患者腹水中分離卵巢癌細(xì)胞并建系,建立成熟的卵巢癌側(cè)群細(xì)胞分離方法,對(duì)側(cè)群細(xì)胞進(jìn)行鑒定和生物學(xué)特性研究;比較側(cè)群細(xì)胞與非側(cè)群細(xì)胞的轉(zhuǎn)錄組差異,并對(duì)差異基因進(jìn)行富集分析,為研究卵巢癌干細(xì)胞調(diào)控腫瘤發(fā)生、發(fā)展、轉(zhuǎn)移和耐藥確立研究方向,并為新的治療策略提供理論支撐。方法:1.人腹水來(lái)源卵巢癌永生化細(xì)胞系的建立及其生物學(xué)特性研究。采集卵巢漿液性腺癌(stage IIIc,grade 3)患者腹水,分離純化出其中的卵巢癌細(xì)胞,并進(jìn)行傳代培養(yǎng);觀察此細(xì)胞系中卵巢癌細(xì)胞的生長(zhǎng)狀態(tài)和顯微及超微結(jié)構(gòu);測(cè)定卵巢癌細(xì)胞的生長(zhǎng)曲線,并對(duì)其進(jìn)行染色體核型分析;最后,將卵巢癌細(xì)胞接種于NOD-SCID小鼠皮下檢測(cè)其成瘤性,并對(duì)移植瘤進(jìn)行組織學(xué)及免疫組化鑒定。2.人腹水來(lái)源卵巢癌細(xì)胞系中側(cè)群細(xì)胞的分離及鑒定。Hoechst33342預(yù)染細(xì)胞后,以流式細(xì)胞術(shù)分選出側(cè)群細(xì)胞和非側(cè)群細(xì)胞,分別培養(yǎng)于無(wú)血清、含血清及軟瓊脂培養(yǎng)基中,比較兩類細(xì)胞的自我更新、分化和克隆形成能力;通過(guò)免疫細(xì)胞化學(xué)染色比較兩類細(xì)胞腫瘤干細(xì)胞標(biāo)記物及干性基因表達(dá)差異;再將這兩類細(xì)胞分別皮下或腹腔注射于NOD-SCID小鼠,比較其成瘤能力的差別,并對(duì)移植瘤進(jìn)行組織學(xué)及免疫組化鑒定。3.人腹水來(lái)源卵巢癌細(xì)胞系中側(cè)群細(xì)胞的生物學(xué)特性研究。采用劃痕和Boydon小室實(shí)驗(yàn)檢測(cè)兩者的遷移和侵襲能力;對(duì)順鉑的耐藥性則由側(cè)群和非側(cè)群細(xì)胞在一系列藥物濃度下細(xì)胞生存率進(jìn)行評(píng)估。4.人腹水來(lái)源卵巢癌細(xì)胞系中側(cè)群細(xì)胞差異表達(dá)基因檢測(cè)與富集分析。應(yīng)用全基因組芯片檢測(cè)技術(shù)篩選側(cè)群細(xì)胞的差異表達(dá)基因,對(duì)差異基因分別進(jìn)行GO分析、KEGG pathway分析和PPI分析,并對(duì)差異基因進(jìn)行q PCR驗(yàn)證。結(jié)果:1.人腹水來(lái)源卵巢癌永生化細(xì)胞系的建立及其生物學(xué)特性研究。通過(guò)分離、純化和傳代培養(yǎng),成功地由卵巢漿液性腺癌患者腹水的脫落細(xì)胞建立了卵巢癌細(xì)胞系OVA-W,目前已歷經(jīng)70代,具有永生化特征;該卵巢癌細(xì)胞貼壁生長(zhǎng),無(wú)接觸性抑制,具有細(xì)胞異型性、核漿比例增加、核仁增大、細(xì)胞器增多等腫瘤細(xì)胞結(jié)構(gòu)特征;卵巢癌細(xì)胞呈指數(shù)生長(zhǎng),群體倍增時(shí)間為31.2h,染色體核型均為超二倍體,可見(jiàn)染色體結(jié)構(gòu)異常及衍生染色體;NOD-SCID小鼠移植瘤為卵巢低分化漿液性腺癌,與原發(fā)腫瘤無(wú)組織學(xué)差異,并表達(dá)卵巢漿液性腺癌標(biāo)記物CA125及CK7。2.人腹水來(lái)源卵巢癌細(xì)胞系中側(cè)群細(xì)胞的分離及鑒定。通過(guò)熒光標(biāo)記流式細(xì)胞術(shù)由此卵巢癌細(xì)胞系中分離出的側(cè)群細(xì)胞比例為(0.38±0.05)%。在體外實(shí)驗(yàn)中,側(cè)群細(xì)胞與非側(cè)群細(xì)胞經(jīng)分化培養(yǎng),前者側(cè)群細(xì)胞的比例明顯高于后者;經(jīng)無(wú)血清懸浮培養(yǎng),前者的成球數(shù)量顯著高于后者;經(jīng)軟瓊脂克隆培養(yǎng),前者克隆形成率也明顯高于后者;經(jīng)免疫細(xì)胞化學(xué)染色,側(cè)群細(xì)胞較非側(cè)群細(xì)胞高表達(dá)腫瘤干細(xì)胞標(biāo)記物CD133、ALDH及干性基因Nanog;在體內(nèi)實(shí)驗(yàn)中,僅少量側(cè)群細(xì)胞(1.0×103)接種于NOD-SCID小鼠皮下及腹腔即可成瘤,瘤組織在組織學(xué)特征上與原代腫瘤組織一致,并且表達(dá)卵巢漿液性腺癌的分子標(biāo)志CA125。3.人腹水來(lái)源卵巢癌細(xì)胞系中側(cè)群細(xì)胞的生物學(xué)特性研究。劃痕實(shí)驗(yàn)結(jié)果顯示側(cè)群細(xì)胞較非側(cè)群細(xì)胞遷移率高;Boydon小室實(shí)驗(yàn)結(jié)果顯示側(cè)群細(xì)胞較非側(cè)群細(xì)胞具有更強(qiáng)的侵襲能力;對(duì)順鉑的耐藥實(shí)驗(yàn)中,側(cè)群細(xì)胞的IC50值明顯高于非側(cè)群細(xì)胞及未分選細(xì)胞,順鉑可使卵巢癌細(xì)胞中的側(cè)群細(xì)胞比例顯著上升,另外加入維拉帕米后,經(jīng)順鉑處理的側(cè)群細(xì)胞細(xì)胞生存率顯著下降。4.人腹水來(lái)源卵巢癌細(xì)胞系中側(cè)群細(xì)胞差異表達(dá)基因檢測(cè)與富集分析。通過(guò)對(duì)側(cè)群與非側(cè)群細(xì)胞的全基因組芯片檢測(cè),篩選出2倍以上差異表達(dá)基因共21個(gè),其中18個(gè)基因表達(dá)上調(diào),3個(gè)基因表達(dá)下調(diào);對(duì)這些差異表達(dá)基因進(jìn)行富集分析顯示TNFAIP3、IL-6和CXCL8基因可能與側(cè)群細(xì)胞的特性相關(guān);q PCR結(jié)果驗(yàn)證了這些差異基因在側(cè)群細(xì)胞中高表達(dá)。結(jié)論:1.本研究由一高級(jí)別晚期卵巢漿液性乳頭狀囊腺癌患者腹水建立了一個(gè)永生化卵巢癌細(xì)胞系,經(jīng)鑒定具有由其分離的腫瘤特征。2.由該細(xì)胞系可穩(wěn)定、持續(xù)分離出一個(gè)極低比例的側(cè)群細(xì)胞亞群,經(jīng)鑒定該細(xì)胞亞群具有自我更新和分化潛能,可高表達(dá)腫瘤干細(xì)胞標(biāo)記物及干性基因,以及較強(qiáng)的克隆形成能力和再現(xiàn)原發(fā)腫瘤表型的特點(diǎn),符合腫瘤干細(xì)胞的基本特征,可做為卵巢癌干細(xì)胞生物學(xué)特性研究的細(xì)胞模型。3.該細(xì)胞系的側(cè)群細(xì)胞表現(xiàn)為較強(qiáng)的遷移和侵襲和能力;對(duì)卵巢癌一線化療藥物順鉑耐藥,且ABC結(jié)合盒轉(zhuǎn)運(yùn)蛋白與其耐藥相關(guān)。4.根據(jù)側(cè)群細(xì)胞差異表達(dá)基因的富集分析結(jié)果,推測(cè)TNFAIP3、IL-6和CXCL8基因可能與側(cè)群細(xì)胞的腫瘤干細(xì)胞特性相關(guān)。本研究為卵巢癌干細(xì)胞調(diào)控腫瘤進(jìn)展及耐藥的分子機(jī)制提供了研究思路,并為卵巢癌的特異性治療提供了可靠的靶點(diǎn)。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【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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