發(fā)布時間：2018-05-01 13:34

  本文選題：種屬特異基因 + 靈長類��； 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：第一部分種屬特異基因的篩選及功能的初步研究生殖健康,是目前廣泛被關(guān)注的問題,生殖疾病已經(jīng)嚴(yán)重危害到我們的生活及后代的健康,并且在全世界范圍內(nèi)生殖疾病的發(fā)病率在逐年上升。雖然對生殖疾病的遺傳原因及治療措施等方面已有很多研究,但是仍然不足于滿足防治該類疾病的需要。本研究的目的在于通過對人生殖相關(guān)的特異基因的研究,為生殖疾病的防治提供分子理論基礎(chǔ)。從進化角度看,靈長類和嚙齒類是同屬靈長總目,但是兩者之間的生殖策略卻截然不同。分別以人和小鼠為高等靈長類和嚙齒類的代表。如,人有血單絨毛膜胎盤、自發(fā)蛻膜化及月經(jīng)行為；而小鼠是血三絨膜胎盤、沒有自發(fā)蛻膜化及月經(jīng)行為。生物的性狀是由基因控制的。利用同源基因在不同物種間的進化保守性,選擇同源基因數(shù)據(jù)庫進行研究。首先,從NCBI網(wǎng)站下載同源基因數(shù)據(jù)庫build67,包含人同源基因18915個,小鼠同源基因20747個,利用Excel軟件比較人和小鼠的同源基因并且經(jīng)過NCBI中FILTERS工具的篩選,獲得人特異基因248個,小鼠特異基因680個。在UniGene數(shù)據(jù)庫中,調(diào)查了人和小鼠特異基因的組織表達特異性,并利用DAVID在線軟件對在子宮和胎盤中有表達的人特異基因進行了初步的功能聚類分析,進一步在the human protein atlas數(shù)據(jù)庫中調(diào)查了在女性生殖系統(tǒng)中各基因的蛋白表達水平,可知BTN3A1、IL-32、ZNF222、ZNF649、ZNF773基因在胎盤中的蛋白表達水平很高,而在別的組織中表達量比較弱或者是沒有檢測到。結(jié)合已有相關(guān)文獻報道,本研究最后選擇IL-32、ZNF222、LAIR2作為候選基因,進行下一步的研究驗證。用免疫組織化學(xué)的方法檢測及結(jié)合現(xiàn)有研究資料,可知IL-32、ZNF222及LAIR2在人絨毛膜滋養(yǎng)層細(xì)胞中有高表達,而在人蛻膜及子宮內(nèi)膜中表達量相對較弱或沒有表達。最后,在人絨毛膜滋養(yǎng)層細(xì)胞系HTR8/SVneo中敲降IL-32的表達后,細(xì)胞的增殖、遷移及侵襲能力都顯著被提高；敲降ZNF222的表達后,細(xì)胞的遷移和侵襲能力受到明顯的抑制；敲降LAIR2的表達后,細(xì)胞的增殖能力被增強了,但是侵襲能力受到明顯的抑制。這些結(jié)果表明,IL-32、ZNF222及LAIR2可能在妊娠中發(fā)揮著一定的作用。由此推測,篩選獲得的特異基因在人妊娠過程中可能發(fā)揮著重要的功能,可能對生殖的成功具有重要的意義。對人特異基因的研究也為生殖疾病特別是妊娠疾病的防治提供了更深層次的理論基礎(chǔ)。第二部分microRNA-205和CHN1在宮頸癌中的功能研究宮頸癌是全球女性癌癥死亡的首要原因。但是宮頸癌發(fā)生發(fā)展的機制仍然不是非常清楚。本部分研究的目的是闡明miR-205和CHN1蛋白在宮頸癌發(fā)展中的作用。在研究中發(fā)現(xiàn),與宮頸正常上皮相比較,miR-205和CHN1蛋白的表達水平,在宮頸癌組織中被上調(diào)。我們用原位雜交的方法再次證實了在宮頸癌中miR-205的表達被上調(diào)。通過生物信息學(xué)預(yù)測及在HeLa細(xì)胞中雙熒光素酶基因報告檢測, CHN1是miR-205的靶基因。不同尋常的是,在宮頸癌細(xì)胞系HeLa、SiHa及C33A中,miR-205的過表達上調(diào)了CHN1 mRNA及蛋白水平的表達,相反,miR-205的敲降能夠下調(diào)CHN1 mRNA及蛋白水平的表達。這些結(jié)果表明在宮頸癌中miR-205可能正向調(diào)控著CHN1的表達。miR-205的過表達促進了宮頸癌細(xì)胞的增殖、凋亡、遷移及侵襲能力,相反,miR-205的敲降則起到了抑制的作用。此外,CHN1的敲降也明顯的抑制了miR-205過表達對細(xì)胞行為的改變。這也進一步表明miR-205可能通過正向調(diào)控CHN1的表達而影響宮頸癌細(xì)胞的行為。通過免疫組織化學(xué)法檢測宮頸癌組織芯片中CHN1的表達,我們發(fā)現(xiàn)CHN1表達的強弱與宮頸癌的淋巴結(jié)轉(zhuǎn)移有顯著性的關(guān)聯(lián)�？傊�,我們的實驗結(jié)果表明miR-205通過正向調(diào)控CHN1的表達,影響了宮頸癌細(xì)胞的生長、凋亡、遷移及侵襲能力。由此推測,miR-205的表達異常及后續(xù)的CHN1表達的異常在宮頸癌的發(fā)生中發(fā)揮著重要的作用。第三部分HIF1A與VEGF在月經(jīng)發(fā)生過程中的表達月經(jīng)是子宮內(nèi)膜周期性脫落,脫落物隨著經(jīng)血排出體外的生理現(xiàn)象,主要出現(xiàn)于包括人類的高等靈長類以及少數(shù)其它胎盤動物種屬中。月經(jīng)的異常與很多婦科疾病的發(fā)生有關(guān)。解析子宮內(nèi)膜周期性崩解出血機制是理解月經(jīng)異常的關(guān)鍵所在,對女性生殖健康具有重大的意義。本部分研究試圖通過建立小鼠月經(jīng)樣模型,研究月經(jīng)過程相關(guān)基因的表達變化。本研究成功的建立了小鼠生理性孕酮撤退月經(jīng)樣模型。在小鼠月經(jīng)樣模型中,在P4撤退后,Hif1a的mRNA與蛋白水平的表達都出現(xiàn)了上調(diào),隨著崩解完成又回落到原來的水平,這也提示了HIF1A與子宮內(nèi)膜的崩解有關(guān)。在小鼠月經(jīng)樣模型中,在P4撤退后,Vegf mRNA與蛋白質(zhì)水平的表達都出現(xiàn)了上調(diào)。這些結(jié)果提示HIF1A和VEGF在月經(jīng)的發(fā)生過程中可能起著重要的作用。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R737.33

【相似文獻】

相關(guān)期刊論文 前3條

1 馬杰;姜寧;王國增;胡傳義;鄭景存;;不同分期及分級膀胱癌組織中microRNA-21、microRNA-205表達變化[J];山東醫(yī)藥;2014年04期

2 余佳;付紅藝;汪曉艷;劉倩;王芳;馮濤;張俊武;;MicroRNA-205對人皮膚上皮細(xì)胞遷移能力的調(diào)控[J];基礎(chǔ)醫(yī)學(xué)與臨床;2010年06期

3 ;[J];;年期

相關(guān)會議論文 前1條

1 趙崇暉;王梅;張斌;張玲;朱瓊芳;錢暉;許文榮;;MicroRNA-205對人胃癌SGC7901細(xì)胞增殖的影響及其機制初步探討[A];中華醫(yī)學(xué)會第七次全國中青年檢驗醫(yī)學(xué)學(xué)術(shù)會議論文匯編[C];2012年

相關(guān)博士學(xué)位論文 前2條

1 陳莉;MicroRNA-205調(diào)控人主動脈平滑肌細(xì)胞鈣化和成骨細(xì)胞樣分化的機制研究[D];山東大學(xué);2014年

2 劉建兵;種屬特異基因的篩選及功能的初步研究、microRNA-205與CHN1在宮頸癌中的功能研究及HIF1A與VEGF在月經(jīng)發(fā)生過程中的表達[D];北京協(xié)和醫(yī)學(xué)院;2015年

，

本文編號：1829597