本文選題：宮頸癌 + 腫瘤微環(huán)境�。� 參考：《復(fù)旦大學(xué)》2014年碩士論文

【摘要】：[背景與目的] 已有研究證實(shí),在腫瘤發(fā)生的微環(huán)境中,間質(zhì)成纖維細(xì)胞的衰老可能促進(jìn)上皮性腫瘤的發(fā)生,但宮頸癌的發(fā)生是否與其組織中間質(zhì)成纖維細(xì)胞的衰老相關(guān)有待深入研究。檢測(cè)宮頸腫瘤微環(huán)境中間質(zhì)成纖維細(xì)胞的GRO-α表達(dá)水平,分析GRO-α誘導(dǎo)宮頸間質(zhì)成纖維細(xì)胞衰老研究的信號(hào)傳導(dǎo)通路,可能為宮頸癌的早期診斷及治療提供指導(dǎo)和幫助。[方法]宮頸癌組織和正常宮頸組織HE染色以及間質(zhì)細(xì)胞和上皮細(xì)胞鑒定；分離純化正常宮頸成纖維細(xì)胞(normal fibroblasts, NFs)和宮頸癌相關(guān)成纖維細(xì)胞(cancer-associated fibroblasts,CAFs),用ELISA的方法檢測(cè)NFs和CAFs中GRO-α的表達(dá)水平,同時(shí)檢測(cè)細(xì)胞因子白介素6(IL-6)和血管內(nèi)皮生長(zhǎng)因子(VEGF)的分泌水平；用衰老相關(guān)β-半乳糖甘酶染色(SA-β-gal)、細(xì)胞計(jì)數(shù)和蛋白檢測(cè)法(Western Blot)分別鑒定細(xì)胞衰老、繪制細(xì)胞生長(zhǎng)曲線(xiàn)和NFs與CAFs中VEGF和p16蛋白表達(dá)；用RT-PCR的方法檢測(cè)GRO-α的受體CXCR2的表達(dá)情況；用人重組GRO-α細(xì)胞因子處理正常宮頸成纖維細(xì)胞后,再通過(guò)β-半乳糖苷酶染色法觀察其衰老情況；用蛋白免疫印跡法探討GRO-α對(duì)間質(zhì)成纖維細(xì)胞作用的可能機(jī)理：用CAF-CM (conditioned medium), CAF-CM+ GRO-aAb,或GRO-α分別處理NFs,檢測(cè)細(xì)胞中相關(guān)信號(hào)分子的表達(dá)變化。[結(jié)果]在宮頸癌相關(guān)間質(zhì)成纖維細(xì)胞的條件培養(yǎng)基中GRO-α的分泌量較正常宮頸成纖維細(xì)胞中的明顯增多；宮頸癌相關(guān)成纖維細(xì)胞分泌的細(xì)胞因子IL-6和VEGF也比正常宮頸成纖維細(xì)胞要高2倍以上(P0.05)；宮頸癌相關(guān)成纖維細(xì)胞中SA-β-gal的活性要比正常宮頸成纖維細(xì)胞高；宮頸正常成纖維細(xì)胞生長(zhǎng)速度明顯快于宮頸癌相關(guān)成纖維細(xì)胞生長(zhǎng)速度；宮頸癌相關(guān)成纖維細(xì)胞中p16的表達(dá)水平比正常宮頸成纖維細(xì)胞中要高；宮頸癌相關(guān)成纖維細(xì)胞表面受體CXCR2較正常宮頸成纖維細(xì)胞中明顯增強(qiáng)；用人重組GRO-α細(xì)胞因子處理正常宮頸成纖維細(xì)胞后,間質(zhì)細(xì)胞呈現(xiàn)明顯的衰老跡象,進(jìn)一步的機(jī)理研究顯不：GRO-α在腫瘤微環(huán)境中主要是通過(guò)PI3K/AKT、MAPK、NF-κB等信號(hào)傳導(dǎo)通路來(lái)促進(jìn)間質(zhì)細(xì)胞的衰老。[結(jié)論]本實(shí)驗(yàn)證實(shí)宮頸癌的發(fā)生伴有間質(zhì)成纖維細(xì)胞的衰老,細(xì)胞因子GRO-a可能通過(guò)PI3K/AKT、MAPK、NF-κB信號(hào)通路介導(dǎo)宮頸癌間質(zhì)衰老,從而可能對(duì)宮頸癌的發(fā)生和發(fā)展具有促進(jìn)作用。
[Abstract]:[background and objective] it has been demonstrated that in the microenvironment of tumorigenesis, the aging of mesenchymal fibroblasts may promote the development of epithelial tumors. However, whether the occurrence of cervical cancer is related to the senescence of fibroblasts in tissue needs further study. To detect the expression of GRO- 偽 in the mesenchymal fibroblasts of cervical neoplasms, and to analyze the signal transduction pathway of GRO- 偽 induced senescence of cervical stromal fibroblasts, which may provide guidance and help for the early diagnosis and treatment of cervical cancer. [methods] HE staining and identification of interstitial cells and epithelial cells in cervical carcinoma and normal cervical tissues; Normal fibroblasts (NFS) and cancer-associated fibroblasts (Cafs) were isolated and purified from normal cervical fibroblasts (NFS) and cancer-associated fibroblast cells (Cafs). The expression of GRO- 偽 in NFs and CAFs was detected by ELISA, and the secretion levels of interleukin 6 (IL 6) and vascular endothelial growth factor (VEGF) were detected. Senescence related 尾 -galactosidase staining, cell count and protein detection were used to identify cell senescence, cell growth curve and VEGF and p16 protein expression in NFs and CAFs, and CXCR2 expression of GRO- 偽 receptor was detected by RT-PCR method. After the normal cervical fibroblasts were treated with recombinant GRO- 偽 cytokines, the aging of normal cervical fibroblasts was observed by 尾 -galactosidase staining. The possible mechanism of effect of GRO- 偽 on mesenchymal fibroblasts was investigated by Western blotting. NFS were treated with CAF-CM conditioned mediums, CAF-CM GRO-aAbor GRO- 偽, respectively, and the expression of related signal molecules was detected. [results] the secretion of GRO- 偽 in the conditioned medium of cervical cancer associated mesenchymal fibroblasts was significantly higher than that in normal cervical fibroblasts. The cytokines IL-6 and VEGF secreted by cervical cancer associated fibroblasts were more than 2 times higher than those of normal cervical fibroblasts, and the activity of SA- 尾 -gal in cervical cancer associated fibroblasts was higher than that in normal cervical fibroblasts. The growth rate of normal cervical fibroblasts was significantly faster than that of cervical cancer related fibroblasts, and the expression level of p16 in cervical cancer associated fibroblasts was higher than that in normal cervical fibroblasts. The surface receptor CXCR2 of cervical cancer associated fibroblasts was significantly enhanced than that of normal cervical fibroblasts, and the interstitial cells showed obvious signs of senescence after treatment of normal cervical fibroblasts with human recombinant GRO- 偽 cytokines. The further study on the mechanism of GRO- 偽 in tumor microenvironment is to promote the senescence of interstitial cells through signal transduction pathways such as PI3K / AKTK MAPK- 魏 B and so on. [conclusion] this study confirmed that cervical cancer is accompanied by the senescence of interstitial fibroblasts. The cytokine GRO-a may mediate the interstitial senescence of cervical cancer through the signal pathway of PI3K / AKTK MAPK- 魏 B, which may promote the occurrence and development of cervical cancer.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.33

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