HLA-G过表达对宫颈癌hela细胞生物学行为的影响

发布时间：2018-02-26 08:45

本文关键词： 宫颈癌 HLA-G 增殖凋亡侵袭　出处：《大连医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:宫颈癌是女性生殖道最常见的恶性肿瘤。人乳头瘤病毒(human papillomaviruses,HPV)的感染是宫颈病变发展过程中的必要步骤,事实上,HPV感染通常是短暂的,宿主免疫系统可以抵抗病毒入侵,导致病变消退,而持续感染的维持还需要其他多种因素的调控。人类白细胞抗原-G(Human leukocyte antigen G,HLA-G)蛋白表达与病毒感染密切相关,研究可知HPV病毒可诱导HLA-G分子的表达,肿瘤或病毒感染细胞的微环境均可上调HLA-G的表达,HLA-G可能与HPV感染(特别是HPV18基因型)和持续易感性相关。从癌前病变进展到癌变,HLA-G抗原的表达量是一个循序渐进的过程,HLA-G与肿瘤或病毒调节的表达之间的平衡可以导致感染的不同结果,促进或消除病毒感染的持续性。显然,HLA-G的表达与HPV病毒的感染之间的关联,对子宫颈肿瘤的发生发展、预测等具有重要意义。HLA-G基因是一种具有免疫抑制功能的非经典的主要组织相容性复合物(major histocompatibility complex,MHC)I类分子,正常生理条件下,HLA-G仅分布在母体与胎儿界面的绒毛膜外滋养层细胞等少数免疫豁免组织,是诱导和维持母胎免疫耐受的一个重要因素。有报道,HLA-G在胃癌、肺癌、乳腺癌等肿瘤细胞和肿瘤微环境中侵入的炎性细胞中有异常表达,可能与肿瘤细胞免疫逃逸相关。另外,现在已经证实,异常的HLA-G在宫颈癌中也有明显表达。然而,关于HLA-G与宫颈癌相互作用的关系仍不明确。本课题旨在通过体外实验,研究HLA-G对宫颈癌HPV18基因型hela细胞增殖活性、凋亡水平及侵袭能力的影响,探究宫颈癌细胞中HLA-G的生物学作用。方法:(1)HLA-G真核表达载体转染及鉴定将构建好的真核表达质粒pc DNA3.1(-)/HLA-G通过lipofectamineTM 2000瞬时转染宫颈癌hela细胞,并设空载体质粒转染组(阴性对照)和未转染组。通过计数镜下带有EGFP荧光标记的细胞比例,计算转染效率。并进行RT-PCR、Western blot法检测癌细胞HLA-G基因在m RNA和蛋白水平上的表达。(2)通过四甲基偶氮哇蓝(MTT)比色法检测3组细胞的生长活性。(3)采用流式细胞术分析3组细胞的凋亡情况。(4)采用transwell细胞体外培养实验检测3组细胞的侵袭能力。结果:(1)转染后,绿色荧光细胞所占比率判断出转染效率约达80%,RT-PCR和Western blot证实转染后宫颈癌hela细胞株中HLA-G m RNA、蛋白表达量增加(p0.05)。(2)MTT法显示:过表达HLA-G组与空质粒组和未转染组相比,细胞增殖最快,差异有统计学意义(p0.01)。而未转染组和空质粒组相比也存在差异(p0.05),考虑可能是由于脂质体和质粒的毒性所致。(3)流式细胞仪检测结果显示:与空质粒组相比,过表达HLA-G组细胞凋亡减少,差异有统计学意义(p0.01)。而未转染组和空质粒组相比也存在差异(p0.05),考虑可能是由于脂质体和质粒的毒性所致。(4)经transwell细胞体外培养,结果显示,空质粒组和过表达HLA-G组细胞体外侵袭能力具有显著差异((p0.01)。结论:过表达HLA-G可能促进宫颈癌hela细胞的增殖和侵袭,减少其凋亡。
[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.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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