本文關(guān)鍵詞： 宮頸癌 HLA-G 增殖 凋亡 侵襲　出處：《大連醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:宮頸癌是女性生殖道最常見的惡性腫瘤。人乳頭瘤病毒(human papillomaviruses,HPV)的感染是宮頸病變發(fā)展過程中的必要步驟,事實上,HPV感染通常是短暫的,宿主免疫系統(tǒng)可以抵抗病毒入侵,導(dǎo)致病變消退,而持續(xù)感染的維持還需要其他多種因素的調(diào)控。人類白細胞抗原-G(Human leukocyte antigen G,HLA-G)蛋白表達與病毒感染密切相關(guān),研究可知HPV病毒可誘導(dǎo)HLA-G分子的表達,腫瘤或病毒感染細胞的微環(huán)境均可上調(diào)HLA-G的表達,HLA-G可能與HPV感染(特別是HPV18基因型)和持續(xù)易感性相關(guān)。從癌前病變進展到癌變,HLA-G抗原的表達量是一個循序漸進的過程,HLA-G與腫瘤或病毒調(diào)節(jié)的表達之間的平衡可以導(dǎo)致感染的不同結(jié)果,促進或消除病毒感染的持續(xù)性。顯然,HLA-G的表達與HPV病毒的感染之間的關(guān)聯(lián),對子宮頸腫瘤的發(fā)生發(fā)展、預(yù)測等具有重要意義。HLA-G基因是一種具有免疫抑制功能的非經(jīng)典的主要組織相容性復(fù)合物(major histocompatibility complex,MHC)I類分子,正常生理條件下,HLA-G僅分布在母體與胎兒界面的絨毛膜外滋養(yǎng)層細胞等少數(shù)免疫豁免組織,是誘導(dǎo)和維持母胎免疫耐受的一個重要因素。有報道,HLA-G在胃癌、肺癌、乳腺癌等腫瘤細胞和腫瘤微環(huán)境中侵入的炎性細胞中有異常表達,可能與腫瘤細胞免疫逃逸相關(guān)。另外,現(xiàn)在已經(jīng)證實,異常的HLA-G在宮頸癌中也有明顯表達。然而,關(guān)于HLA-G與宮頸癌相互作用的關(guān)系仍不明確。本課題旨在通過體外實驗,研究HLA-G對宮頸癌HPV18基因型hela細胞增殖活性、凋亡水平及侵襲能力的影響,探究宮頸癌細胞中HLA-G的生物學作用。方法:(1)HLA-G真核表達載體轉(zhuǎn)染及鑒定將構(gòu)建好的真核表達質(zhì)粒pc DNA3.1(-)/HLA-G通過lipofectamineTM 2000瞬時轉(zhuǎn)染宮頸癌hela細胞,并設(shè)空載體質(zhì)粒轉(zhuǎn)染組(陰性對照)和未轉(zhuǎn)染組。通過計數(shù)鏡下帶有EGFP熒光標記的細胞比例,計算轉(zhuǎn)染效率。并進行RT-PCR、Western blot法檢測癌細胞HLA-G基因在m RNA和蛋白水平上的表達。(2)通過四甲基偶氮哇藍(MTT)比色法檢測3組細胞的生長活性。(3)采用流式細胞術(shù)分析3組細胞的凋亡情況。(4)采用transwell細胞體外培養(yǎng)實驗檢測3組細胞的侵襲能力。結(jié)果:(1)轉(zhuǎn)染后,綠色熒光細胞所占比率判斷出轉(zhuǎn)染效率約達80%,RT-PCR和Western blot證實轉(zhuǎn)染后宮頸癌hela細胞株中HLA-G m RNA、蛋白表達量增加(p0.05)。(2)MTT法顯示:過表達HLA-G組與空質(zhì)粒組和未轉(zhuǎn)染組相比,細胞增殖最快,差異有統(tǒng)計學意義(p0.01)。而未轉(zhuǎn)染組和空質(zhì)粒組相比也存在差異(p0.05),考慮可能是由于脂質(zhì)體和質(zhì)粒的毒性所致。(3)流式細胞儀檢測結(jié)果顯示:與空質(zhì)粒組相比,過表達HLA-G組細胞凋亡減少,差異有統(tǒng)計學意義(p0.01)。而未轉(zhuǎn)染組和空質(zhì)粒組相比也存在差異(p0.05),考慮可能是由于脂質(zhì)體和質(zhì)粒的毒性所致。(4)經(jīng)transwell細胞體外培養(yǎng),結(jié)果顯示,空質(zhì)粒組和過表達HLA-G組細胞體外侵襲能力具有顯著差異((p0.01)。結(jié)論:過表達HLA-G可能促進宮頸癌hela細胞的增殖和侵襲,減少其凋亡。
[Abstract]:Objective: cervical cancer is the most common malignant tumor of the female genital tract. Human papillomavirus (human papillomaviruses, HPV) infection is a necessary step in the development of cervical lesions, in fact, HPV infection is usually transient, the host immune system can resist virus invasion, causing lesions subsided, and persistent infection in maintenance regulation also the need for a variety of other factors. Human leukocyte antigen -G (Human leukocyte antigen G, HLA-G) protein expression is closely related to virus infection, expression of the HPV virus can induce expression of HLA-G molecules, HLA-G can upregulate the microenvironment of tumor or virus infected cells, HLA-G may be associated with HPV infection (especially HPV18 gene type) and continuous susceptibility. Cancer from precancerous lesions to progress, the expression of HLA-G antigen is a gradual process, between the expression of HLA-G and tumor or virus regulating the balance of The different results lead to infection, promote or eliminate persistent viral infection. Obviously, the association between HLA-G expression and HPV virus infection, on the occurrence and development of cervical cancer, the prediction has important significance of.HLA-G gene is a kind of non classical major immunosuppressive function compatibility complex (major histocompatibility complex, MHC) I molecules, under normal physiological conditions, HLA-G is only distributed in the maternal and fetal chorionic trophoblast cells and a few external interface immunity organization, is an important factor in the induction and maintenance of maternal fetal immune tolerance. It is reported that HLA-G in gastric cancer, lung cancer, abnormal expression of invaded cells and tumor of breast tumor microenvironment in inflammatory cells, may be associated with the immune escape of tumor cells. In addition, it has been confirmed that the abnormal HLA-G was also expressed in cervical cancer. However, about The relationship between HLA-G and cervical cancer interactions are still unclear. The purpose of this project is to study the in vitro, HLA-G on cervical carcinoma HPV18 gene HeLa cell proliferation, apoptosis and invasion biology level, exploring the role of HLA-G in cervical cancer cells. Methods: (1) HLA-G eukaryotic expression vector transfection and Identification Construction of eukaryotic expression plasmid PC (-) /HLA-G DNA3.1 built by lipofectamineTM 2000 transiently transfected into cervical cancer cell line HeLa, and empty vector transfected group (negative control) and untransfected group. By counting under microscope with fluorescence labeled EGFP cells proportion, the transfection efficiency was computed. And the RT-PCR, to detect the expression of cancer cells HLA-G Western blot m RNA in gene and protein level. (2) by four methyl thiazolyl blue (MTT) wow than the growth activity of cells in 3 groups were detected. (3) the apoptosis by flow cytometry analysis of 3 groups of cells (4. Transwell) cells in vitro by culture test 3 groups of cells invasion. Results: (1) after transfection, green fluorescent cells accounted for the judgment of the transfection efficiency of about 80%, RT-PCR and Western blot confirmed that the transfection of cervical carcinoma cell line HeLa HLA-G m RNA, an increase in the expression of (P0.05) (2). MTT assay showed that the overexpression of HLA-G group compared with empty plasmid group and non transfection group, cell proliferation is the fastest, the difference was statistically significant (P0.01). Compared with untransfected group and empty plasmid group differences (P0.05), the reason may be due to liposome and plasmid toxicity induced by flow (3). Cytometry showed that: compared with the empty plasmid group, expression of cell apoptosis in HLA-G group decreased, the difference was statistically significant (P0.01). Compared with untransfected group and empty plasmid group differences (P0.05), the reason may be due to toxicity induced by liposome and plasmid. (4) by Transwell fine In vitro culture, the results showed that the invasion ability of empty plasmid group and overexpressing HLA-G group was significantly different (P0.01). Conclusion: over expression of HLA-G may promote the proliferation and invasion of cervical cancer HeLa cells, and reduce its apoptosis.

【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.33

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