种属特异基因的筛选及功能的初步研究、microRNA-205与CHN1在宫颈癌中的功能研究及HIF1A与VEGF在月经发

发布时间：2018-05-01 13:34

本文选题：种属特异基因 + 灵长类　；参考：《北京协和医学院》2015年博士论文

【摘要】：第一部分种属特异基因的筛选及功能的初步研究生殖健康,是目前广泛被关注的问题,生殖疾病已经严重危害到我们的生活及后代的健康,并且在全世界范围内生殖疾病的发病率在逐年上升。虽然对生殖疾病的遗传原因及治疗措施等方面已有很多研究,但是仍然不足于满足防治该类疾病的需要。本研究的目的在于通过对人生殖相关的特异基因的研究,为生殖疾病的防治提供分子理论基础。从进化角度看,灵长类和啮齿类是同属灵长总目,但是两者之间的生殖策略却截然不同。分别以人和小鼠为高等灵长类和啮齿类的代表。如,人有血单绒毛膜胎盘、自发蜕膜化及月经行为；而小鼠是血三绒膜胎盘、没有自发蜕膜化及月经行为。生物的性状是由基因控制的。利用同源基因在不同物种间的进化保守性,选择同源基因数据库进行研究。首先,从NCBI网站下载同源基因数据库build67,包含人同源基因18915个,小鼠同源基因20747个,利用Excel软件比较人和小鼠的同源基因并且经过NCBI中FILTERS工具的筛选,获得人特异基因248个,小鼠特异基因680个。在UniGene数据库中,调查了人和小鼠特异基因的组织表达特异性,并利用DAVID在线软件对在子宫和胎盘中有表达的人特异基因进行了初步的功能聚类分析,进一步在the human protein atlas数据库中调查了在女性生殖系统中各基因的蛋白表达水平,可知BTN3A1、IL-32、ZNF222、ZNF649、ZNF773基因在胎盘中的蛋白表达水平很高,而在别的组织中表达量比较弱或者是没有检测到。结合已有相关文献报道,本研究最后选择IL-32、ZNF222、LAIR2作为候选基因,进行下一步的研究验证。用免疫组织化学的方法检测及结合现有研究资料,可知IL-32、ZNF222及LAIR2在人绒毛膜滋养层细胞中有高表达,而在人蜕膜及子宫内膜中表达量相对较弱或没有表达。最后,在人绒毛膜滋养层细胞系HTR8/SVneo中敲降IL-32的表达后,细胞的增殖、迁移及侵袭能力都显著被提高；敲降ZNF222的表达后,细胞的迁移和侵袭能力受到明显的抑制；敲降LAIR2的表达后,细胞的增殖能力被增强了,但是侵袭能力受到明显的抑制。这些结果表明,IL-32、ZNF222及LAIR2可能在妊娠中发挥着一定的作用。由此推测,筛选获得的特异基因在人妊娠过程中可能发挥着重要的功能,可能对生殖的成功具有重要的意义。对人特异基因的研究也为生殖疾病特别是妊娠疾病的防治提供了更深层次的理论基础。第二部分microRNA-205和CHN1在宫颈癌中的功能研究宫颈癌是全球女性癌症死亡的首要原因。但是宫颈癌发生发展的机制仍然不是非常清楚。本部分研究的目的是阐明miR-205和CHN1蛋白在宫颈癌发展中的作用。在研究中发现,与宫颈正常上皮相比较,miR-205和CHN1蛋白的表达水平,在宫颈癌组织中被上调。我们用原位杂交的方法再次证实了在宫颈癌中miR-205的表达被上调。通过生物信息学预测及在HeLa细胞中双荧光素酶基因报告检测, CHN1是miR-205的靶基因。不同寻常的是,在宫颈癌细胞系HeLa、SiHa及C33A中,miR-205的过表达上调了CHN1 mRNA及蛋白水平的表达,相反,miR-205的敲降能够下调CHN1 mRNA及蛋白水平的表达。这些结果表明在宫颈癌中miR-205可能正向调控着CHN1的表达。miR-205的过表达促进了宫颈癌细胞的增殖、凋亡、迁移及侵袭能力,相反,miR-205的敲降则起到了抑制的作用。此外,CHN1的敲降也明显的抑制了miR-205过表达对细胞行为的改变。这也进一步表明miR-205可能通过正向调控CHN1的表达而影响宫颈癌细胞的行为。通过免疫组织化学法检测宫颈癌组织芯片中CHN1的表达,我们发现CHN1表达的强弱与宫颈癌的淋巴结转移有显著性的关联。总之,我们的实验结果表明miR-205通过正向调控CHN1的表达,影响了宫颈癌细胞的生长、凋亡、迁移及侵袭能力。由此推测,miR-205的表达异常及后续的CHN1表达的异常在宫颈癌的发生中发挥着重要的作用。第三部分HIF1A与VEGF在月经发生过程中的表达月经是子宫内膜周期性脱落,脱落物随着经血排出体外的生理现象,主要出现于包括人类的高等灵长类以及少数其它胎盘动物种属中。月经的异常与很多妇科疾病的发生有关。解析子宫内膜周期性崩解出血机制是理解月经异常的关键所在,对女性生殖健康具有重大的意义。本部分研究试图通过建立小鼠月经样模型,研究月经过程相关基因的表达变化。本研究成功的建立了小鼠生理性孕酮撤退月经样模型。在小鼠月经样模型中,在P4撤退后,Hif1a的mRNA与蛋白水平的表达都出现了上调,随着崩解完成又回落到原来的水平,这也提示了HIF1A与子宫内膜的崩解有关。在小鼠月经样模型中,在P4撤退后,Vegf mRNA与蛋白质水平的表达都出现了上调。这些结果提示HIF1A和VEGF在月经的发生过程中可能起着重要的作用。
[Abstract]:The first part of the screening and function of specific genes is a preliminary study of reproductive health. It is a widespread concern at present. Reproductive diseases have seriously endangered the health of our lives and future generations, and the incidence of reproductive diseases in the world is increasing year by year. The purpose of this study is to provide a molecular theoretical basis for the prevention and control of reproductive diseases through the study of specific genes related to human reproduction. From the evolutionary perspective, primates and rodents are the same primate, but the reproductive strategy between them is the same. It is very different. People and mice are the representatives of higher primates and rodents. For example, people have blood monochorionic placenta, spontaneous decidualization and menstrual behavior, and the mice are blood Three fluffy placenta, without spontaneous decidualization and menstrual behavior. Biological characters are controlled by genes. The evolution of the homologous genes in different species is conservative. First, the homologous gene database was selected. First, the homologous gene database build67 was downloaded from the NCBI site, including 18915 homologous genes and 20747 mouse homologous genes. Using Excel software to compare homologous genes of human and mice and screening the FILTERS tool in NCBI, 248 human specific genes were obtained, and the specific gene of mice was 680. In the UniGene database, the specific gene expression specificity of human and mouse genes was investigated, and a preliminary functional cluster analysis of human specific genes expressed in the uterus and placenta was carried out by DAVID online software. The protein of each gene in the female reproductive system was further investigated in the the human protein atlas database. The expression level of BTN3A1, IL-32, ZNF222, ZNF649, ZNF773 gene expression level in the placenta is very high, but the expression in other tissues is weak or not detected. Combined with the relevant literature, this study finally selected IL-32, ZNF222, LAIR2 as candidate genes, and the next step of research and verification. IL-32, ZNF222 and LAIR2 are highly expressed in human chorionic trophoblast cells and are relatively weak or unexpressed in human decidua and endometrium. Finally, the proliferation, migration and invasion of cells in the human chorionic trophoblast cell line, HTR8/SVneo, are knocked down by IL-32 expression. The capacity of the cells was significantly inhibited after the expression of ZNF222, and the proliferation ability of the cells was enhanced after knocking down the expression of LAIR2, but the invasiveness of the cells was significantly inhibited. These results suggest that IL-32, ZNF222 and LAIR2 may play a role in pregnancy. The screening of specific genes may play an important role in the process of human pregnancy and may be of great significance for the success of reproduction. The study of human specific genes also provides a deeper theoretical basis for the prevention and treatment of reproductive diseases, especially pregnancy diseases. Second part of the functional study of microRNA-205 and CHN1 in cervical cancer Cervical cancer is the leading cause of cancer death in women around the world. But the mechanism of the development of cervical cancer is still not very clear. The purpose of this part of this study is to clarify the role of miR-205 and CHN1 in the development of cervical cancer. In the study, it was found that the expression level of miR-205 and CHN1 protein, compared with the normal upper cervix, was in the cervical cancer group. The fabric was up-regulated. We confirmed that the expression of miR-205 in cervical cancer was up-regulated again by in situ hybridization. Through bioinformatics prediction and detection of biluciferase gene in HeLa cells, CHN1 is the target gene for miR-205. It is unusual that the overexpression of miR-205 in the cervical cancer cell line HeLa, SiHa and C33A is up-regulated. On the contrary, the expression of HN1 mRNA and protein levels, on the contrary, the knockdown of miR-205 can down regulate the expression of CHN1 mRNA and protein levels. These results suggest that in cervical cancer, miR-205 may positively regulate the expression of CHN1, the expression of.MiR-205, which promotes the proliferation, apoptosis, migration and invasion of cervical cancer cells. On the contrary, the knockdown of miR-205 is inhibited. In addition, the knockdown of CHN1 also significantly inhibited the change of miR-205 over expression to cell behavior. This further indicated that miR-205 may affect the behavior of cervical cancer cells through the positive regulation of CHN1 expression. We detected the expression of CHN1 in the tissue microarray of cervical cancer by immunohistochemistry. We found that the expression of CHN1 was strong and weak. In conclusion, our experimental results suggest that miR-205 affects the growth, apoptosis, migration and invasion of cervical cancer cells through the positive regulation of the expression of CHN1. Therefore, the abnormal expression of miR-205 and the subsequent abnormal expression of CHN1 play an important role in the occurrence of cervical cancer. Third the expression of HIF1A and VEGF during the period of menstruation is the periodic abscission of the endometrium, the physiological phenomena of exfoliation in vitro, mainly in human primates and a few other placental species. Abnormal menstruation is related to the occurrence of a large number of gynecologic diseases. The mechanism of periodic disintegration and bleeding is the key to understanding the abnormal menstruation and is of great significance for female reproductive health. This part of this study tried to establish a menstrual model to study the changes in the expression of the related genes in the menstrual process. In the model, the expression of mRNA and protein levels of Hif1a increased after P4 withdrawal. As disintegration completed and fell to the original level, it also suggested that HIF1A was associated with the disintegration of the endometrium. In the menses model of mice, the expression of Vegf mRNA and protein level was up to rise after the withdrawal of P4. These results suggest HIF1A and V. EGF may play an important role in the occurrence of menstruation.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.33

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