發(fā)布時間：2018-04-27 21:32

  本文選題：乳腺癌 + HPV　； 參考：《中國疾病預(yù)防控制中心》2016年博士論文

【摘要】：乳腺癌已經(jīng)成為全球范圍女性最常見的惡性腫瘤之一,其發(fā)病率逐年增高,數(shù)據(jù)顯示,在美國女性中2014年新增癌癥病例數(shù)中乳腺癌占比達到29%。我國近年來乳腺癌發(fā)病率正以每年3%的速度遞增,在所有癌癥中乳腺癌的發(fā)病率位居第一,成為城市中死亡率增長最快的癌癥。乳腺癌的發(fā)病原因復(fù)雜,至今尚不能用已知的單因素或多因素模型來完全解釋其發(fā)生原因與發(fā)展過程。流行病學(xué)研究認(rèn)為乳腺癌的發(fā)生與生活環(huán)境、生活方式,膳食結(jié)構(gòu),內(nèi)分泌等因素有關(guān)。近年來,對病毒感染與乳腺癌病因關(guān)系的研究成為研究的熱點。1995年國際癌癥組織發(fā)布高風(fēng)險的人乳頭瘤病毒(Human papillomavirus簡稱HPV)16型和18型具有導(dǎo)致人類癌癥的作用,有研究證實HPV的E6和E7基因?qū)肴橄賹?dǎo)管上皮細(xì)胞導(dǎo)致細(xì)胞永生化或癌變。由于HPV亞型眾多,不同的亞型致病力不同,以及檢測方法、標(biāo)本類型、人群地域分布等差異致使文獻報道檢測結(jié)果陽性率的差異,使HPV感染與乳腺癌發(fā)生的相關(guān)性存在著爭議。小鼠乳腺腫瘤病毒(Mouse mammary tumor virus簡稱MMTV)是一種p逆轉(zhuǎn)錄病毒,含有9kb的RNA基因組,其中包含GAG基因、POL基因以及編碼病毒進入宿主細(xì)胞時需要的包膜蛋白的ENV基因。研究證實90%以上的小鼠乳腺腫瘤與MMTV相關(guān)。Indik等學(xué)者發(fā)現(xiàn)MMTV可感染人類乳腺癌細(xì)胞系,這一研究證實了MMTV跨物種傳播的可能性。但是,至今沒有MMTV感染人類乳腺細(xì)胞致瘤的直接證據(jù),更沒有以HPVE6和E7病毒基因為靶點的特異性基因治療的先例。明確病毒基因在乳腺癌組織中的存在以及表達狀況,探索以病毒基因為靶點的乳腺癌基因治療和疫苗研究的新策略,對于預(yù)防乳腺癌的發(fā)生降低乳腺癌的死亡率具有重要的意義。本研究分為兩部分：第一部分是對乳腺癌石蠟包埋組織標(biāo)本以及乳腺癌細(xì)胞系SKBR3進行了人乳頭瘤病毒即HPV和小鼠乳腺腫瘤病毒即MMTV的基因篩查；第二部分在篩查結(jié)果的基礎(chǔ)上應(yīng)用RNA干擾技術(shù)沉默HPV致癌基因,探索以病毒基因為靶點對乳腺癌細(xì)胞株的抑制作用。第一部分乳腺癌細(xì)胞中人乳頭瘤病毒和小鼠乳腺腫瘤病毒基因的篩查收集76例病理學(xué)確診的乳腺癌石蠟包埋組織提取DNA,以乳腺癌細(xì)胞系SKBR3為對照,分別設(shè)計合成HPV L1、HPV16 E6、HPV16 E7、HPV18 E6、HPV18 E7以及MMTV-ENV基因的引物,聚合酶鏈?zhǔn)椒磻?yīng)(PCR)擴增基因片段。在76例乳腺癌石蠟包埋組織樣本中,HPV16型E6、E7皆為陰性。HPV L1陽性7例(9.21%)。HPV18 E6陽性5例(6.58%),HPV18 E7陽性18例(23.68%),在SKBR3細(xì)胞系中HPV16E6、E7均為陰性,HPV18E6、E7為陽性,并有HPV18E6、E7的基因表達。對照樣本的臨床病理資料顯示HPV18E6和E7在乳腺癌細(xì)胞基因的檢出率與年齡無關(guān),而與乳腺癌分級以及乳腺細(xì)胞的分化程度有關(guān)；在19例HPV18E6、E7陽性的患者中均為浸潤性導(dǎo)管癌,隨著浸潤程度級別的增加,HPV18E6、E7的檢出率增加。其中6例為低分化的乳腺細(xì)胞癌。小鼠乳腺腫瘤病毒基因的篩查陽性23例,陽性率為30.26%。這項研究為人乳頭瘤病毒感染與乳腺癌的發(fā)生存在相關(guān)性提供了依據(jù),為探索以病毒基因為靶點的乳腺癌基因治療和疫苗研究的新策略提供依據(jù),從HPV18E6、E7基因在不同類型乳腺癌中的檢出率可以預(yù)測以病毒基因為靶點的基因治療對不同類型乳腺癌的有效性,為臨床的應(yīng)用提供了依據(jù)。第二部分探索以病毒基因為靶點對乳腺癌細(xì)胞株抑制作用的研究在第一部分研究結(jié)果的基礎(chǔ)上,以HPV18E6 E7為靶點應(yīng)用bioinformatics research computing提供的在線siRNA設(shè)計工具設(shè)計合成siRNA,構(gòu)建入pSUPER RNAi System,篩選出最優(yōu)的siRNA,檢測以HPV18 E6、E7為靶點的RNA干擾對乳腺癌細(xì)胞的生長活力、克隆形成能力、侵襲能力、細(xì)胞周期以及細(xì)胞周期調(diào)控基因的表達情況分析對乳腺癌細(xì)胞的抑制作用。細(xì)胞生長活力的檢測試驗顯示,與對照組相比48h時針對HPV18 E6、E7的RNA干擾組抑制率分別是12%和9%(差異顯著,P0.05)。克隆形成試驗結(jié)果顯示,與對照組相比針對HPV18E6 E7的RNA干擾組抑制率分別是61%和64%(差異顯著,P0.01)。細(xì)胞侵襲試驗顯示,與對照組相比針對HPV18 E6、E7的RNA干擾組抑制率分別是59%和65%(差異顯著,P0.01)；應(yīng)用流式試驗測定細(xì)胞周期變化,與對照組相比HPV18 E6E7的RNA干擾組的S%降低65%和80%(差異顯著,P0.01),GO/G1升高26%和35%(差異顯著,P0.01),G2/M降低50%和72%(差異顯著,P0.01)；應(yīng)用熒光定量PCR測定細(xì)胞株中HPV18 E6、 E7靶基因以及細(xì)胞周期調(diào)控基因的表達差異,與對照組相比,HPV18 E6的RNA干擾組對E6的抑制率為35%,HPV18 E7的RNA干擾組對E7的抑制率為78%,與對照組相比HPV18 E6、E7的RNA干擾組的TP53-mutant表達降低55%和39%, MDM2表達降低63%和36%, CCNA1表達降低7%和15%, CCND1表達降低18%和23%,BCL-2表達降低39%和77%,RB表達增高94%和109%, VEGFA表達沒有發(fā)生明顯變化。以HPV18E6 E7為靶點的RNA干擾對乳腺癌細(xì)胞的抑制作用明顯,為乳腺癌基因治療提供了新的靶點。
[Abstract]:Breast cancer has become one of the most common malignant tumors in the world. The incidence of breast cancer is increasing year by year. The data show that in 2014, the number of new cases of cancer in the United States has reached 29%., the incidence of breast cancer in China is increasing at a rate of 3% in recent years, and the incidence of breast cancer is the first in all cancers. It has become the fastest growing cancer in the city. The causes of breast cancer are complex. So far, a known single factor or multi factor model can not be used to fully explain the cause and development process. Epidemiological studies suggest that the occurrence of breast cancer is related to the living environment, lifestyle, dietary structure, endocrine and other factors. The research on the relationship between virus infection and the cause of breast cancer has become a hot spot of research in.1995..1995 international cancer organization published high risk human papillomavirus (Human papillomavirus for short HPV) type 16 and type 18 have the effect on human cancer. It has been proved that HPV's E6 and E7 genes are introduced into mammary gland ductal epithelial cells to lead to cell immortalization or cancer Because of the diversity of HPV subtypes, different subtypes of pathogenicity, detection methods, specimen types, and regional distribution of the population, the difference in the positive rate of the results of the literature report results in the correlation between the HPV infection and the incidence of breast cancer. The Mouse mammary tumor virus (MMTV) is a P inverse. The transcriptional virus, which contains the RNA genome of 9KB, contains the GAG gene, the POL gene, and the ENV gene of the envelope protein needed to encode the virus into the host cell. The study confirmed that more than 90% of the mice breast tumor and MMTV related.Indik have found that MMTV can infect human breast cancer cell lines. This study confirmed the spread of MMTV across species. But there is no direct evidence of MMTV infection in human breast cells, and there is no precedent for specific gene therapy targeting HPVE6 and E7 virus genes. The strategy is of great significance for preventing the occurrence of breast cancer and reducing the mortality of breast cancer. This study is divided into two parts: the first part is the screening of paraffin embedded tissue specimens and breast cancer cell line SKBR3 for human papillomavirus (HPV) and mouse mammary tumor virus (MMTV) gene screening; the second part is screened. On the basis of the results, the RNA interference technique was used to silence the HPV oncogene and explore the inhibitory effect of the virus gene on the breast cancer cell lines. In part 1, the screening of human papillomavirus and mouse breast tumor virus gene in breast cancer cells was used to collect 76 cases of pathological diagnosis of breast cancer with paraffin embedded tissue and extract DNA for breast cancer. HPV L1, HPV16 E6, HPV16 E7, HPV18 E6, HPV18 E7 and MMTV-ENV gene primers, polymerase chain reaction (PCR) amplification gene fragment were designed, respectively. In 76 cases of paraffin embedded tissue samples of breast cancer, 7 cases (9.21%) positive were positive (6.58%), 18 cases (23) positive (23). .68%), in the SKBR3 cell line, HPV16E6, E7 were negative, HPV18E6, E7 were positive, and HPV18E6, E7 gene expression. The clinicopathological data of the control samples showed that the detection rate of HPV18E6 and E7 in the breast cancer cell genes was not related to age, but related to the classification of breast cancer and the degree of differentiation of mammary cells; in 19 HPV18E6, E7 positive patients All of them were infiltrative ductal carcinoma. With the increase of degree of infiltration, the detection rate of HPV18E6 and E7 increased. 6 of them were low differentiated breast cancer. 23 cases of mouse breast tumor virus gene screening were positive, and the positive rate was 30.26%.. The study provided the basis for the correlation of human papillomavirus infection and breast cancer. Explore new strategies for gene therapy and vaccine research that target virus genes. The detection rate of HPV18E6 and E7 gene in different types of breast cancer can predict the effectiveness of gene therapy targeting different types of breast cancer for different types of breast cancer, and provide the basis for clinical application. The second part explores the disease. On the basis of the results of the first part of the study, the research on the inhibitory effect of the target on the breast cancer cell line is based on the results of the first part of the study. HPV18E6 E7 is used as an on-line siRNA design tool provided by the bioinformatics research computing as a target. The pSUPER RNAi System is constructed and the optimal siRNA is screened. The inhibitory effects of interference on breast cancer cell growth activity, clonogenic ability, invasion ability, cell cycle and cell cycle regulation gene expression analysis on breast cancer cells. Detection test of cell growth activity showed that the inhibition rates of RNA interference group for HPV18 E6 compared with the control group were 12% and 9% respectively (the difference was 12% and 9%, respectively). Significant, P0.05). The results of clone formation test showed that the inhibition rates of RNA interference group for HPV18E6 E7 were 61% and 64% respectively compared with the control group (significant difference, P0.01). Cell invasion test showed that the inhibition rates of RNA interference group of E7 were 59% and 65%, respectively compared with the control group, compared with the control group (the difference was significant, P0.01); the flow test was used to determine the cells. Compared with the control group, the S% of the RNA interference group in the HPV18 E6E7 group decreased by 65% and 80% (the difference was significant, P0.01), the GO/G1 increased by 26% and 35% (P0.01), the G2/M decreased by 50% and 72% (the difference was significant, P0.01). The differential expression of the HPV18 E6, the target gene and the cell cycle regulation gene in the cell lines was measured by the fluorescent quantitative PCR, and the control group was compared with the control group. Compared with the RNA interference group of HPV18 E6, the inhibition rate of E6 was 35%, and the inhibition rate of RNA interference group of HPV18 E7 was 78%, compared with the control group, the expression decreased by 55% and 39%, the expression decreased by 63% and 36%, the expression decreased by 7% and 15%, the expression decreased 18% and 23%, and the expression decreased 39% and 77% expression. The expression of VEGFA was increased by 94% and 109%, and there was no obvious change in expression. The inhibitory effect of RNA interference targeting HPV18E6 E7 on breast cancer cells was obvious, which provided a new target for the gene therapy of breast cancer.

【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R737.9;R450
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本文編號：1812405