本文選題：人乳頭瘤病毒 + 宮頸鱗癌　； 參考：《新鄉(xiāng)醫(yī)學(xué)院》2014年碩士論文

【摘要】：背景宮頸癌是女性常見的惡性腫瘤之一,感染高危型人乳頭瘤病毒(high risk-human papillomaviruses,HR-HPVs)是其發(fā)生與演進(jìn)的重要基礎(chǔ),E7的持續(xù)表達(dá)是HR-HPVs致癌的關(guān)鍵,其可通過結(jié)合并降解pRb蛋白等,導(dǎo)致細(xì)胞異常增殖。本課題組經(jīng)Agilent人基因表達(dá)譜芯片分析,發(fā)現(xiàn)沉默E7表達(dá)后,腎細(xì)胞癌下調(diào)基因1(down-regulated in renal cell carcinoma1, DRR1)表達(dá)上調(diào)3-11倍。DRR1廣泛表達(dá)于正常組織,但在許多癌細(xì)胞系和原發(fā)性腫瘤中表達(dá)明顯降低,DRR1作為腫瘤抑制基因可能在癌變過程中發(fā)揮重要作用。 目的以HPV16E7(?)日性和陰性的宮頸癌細(xì)胞系、正常宮頸鱗狀上皮、宮頸上皮內(nèi)瘤變(cervical intraepithelial neoplasias,CIN)和宮頸鱗癌組織為研究對(duì)象,探討DRR1在HPV16相關(guān)宮頸癌發(fā)生與演進(jìn)中的作用,以期豐富HPV16的致癌機(jī)制,為探討HPV16相關(guān)腫瘤新的治療措施提供實(shí)驗(yàn)和理論依據(jù)。 方法 1.以HPV16E7日性宮頸癌CaSki和SiHa細(xì)胞及HPV16E7陰性宮頸癌C33-A細(xì)胞為研究對(duì)象,采用Western blotting方法檢測(cè)E7和DRRl的表達(dá),分析HPV16E7表達(dá)與DRR1表達(dá)的關(guān)系。 2.以HPV16E7陽(yáng)性和陰性的正常宮頸鱗狀上皮、CIN和宮頸鱗癌組織為研究對(duì)象,通過免疫組織化學(xué)(immunohistochemistry, IHC)方法檢測(cè)E7和DRR1的表達(dá),分析HPV16E7與DRR1表達(dá)的關(guān)系。 3.常規(guī)培養(yǎng)HPV16E7陰性的宮頸癌C33-A細(xì)胞至對(duì)數(shù)生長(zhǎng)期,提取總RNA,體外逆轉(zhuǎn)錄合成cDNA第一條鏈,PCR擴(kuò)增DRR1基因編碼序列,構(gòu)建真核表達(dá)重組質(zhì)粒pcDNA3.1-DRR1并鑒定,經(jīng)脂質(zhì)體介導(dǎo)將pcDNA3.1-DRR1重組質(zhì)粒轉(zhuǎn)染對(duì)數(shù)生長(zhǎng)期的宮頸癌CaSki細(xì)胞,通過MTT繪制細(xì)胞生長(zhǎng)曲線、平板克隆形成實(shí)驗(yàn)、細(xì)胞周期分布,Annexin V-FITC/PI結(jié)合流式細(xì)胞術(shù)等方法觀察穩(wěn)定表達(dá)DRR1對(duì)HPV16E7陽(yáng)性宮頸癌CaSki細(xì)胞增殖和凋亡能力的影響。 結(jié)果 1. Western blotting結(jié)果顯示：①C33-A細(xì)胞中HPV16E7無表達(dá),CaSki細(xì)胞和SiHa細(xì)胞中可見HPV16E7表達(dá)。②C33-A細(xì)胞中DRR1的表達(dá)顯著高于CaSki細(xì)胞和SiHa細(xì)胞(P0.01),CaSki細(xì)胞和SiHa細(xì)胞中DRRl的表達(dá)無顯著差異(P0.05)。 2.IHC結(jié)果顯示：HPV16E7陽(yáng)性正常宮頸鱗狀上皮、CIN和宮頸鱗癌組織中DRR1的表達(dá)均分別顯著低于HPV16E7陰性的正常宮頸鱗狀上皮組織、CIN和宮頸鱗癌組織,相關(guān)系數(shù)分別為-0.452,-0.485及-0.444(P0.01)。 3.①M(fèi)TT比色法繪制細(xì)胞生長(zhǎng)曲線的結(jié)果顯示：與CaSki-vect細(xì)胞相比,CaSki-DRR1細(xì)胞生長(zhǎng)速度明顯減慢(P0.01),倍增時(shí)間顯著延長(zhǎng)(P0.01)。②平板克隆形成實(shí)驗(yàn)結(jié)果顯示：CaSki-DRR1細(xì)胞克隆形成均數(shù)明顯低于CaSki-vect細(xì)胞(P0.01),且克隆體積明顯較小。③流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期分布的結(jié)果顯示：與CaSki-vect細(xì)胞相比,CaSki-DRR1細(xì)胞G1期阻滯(P0.01),但S期和G2/M期細(xì)胞百分比均數(shù)均明顯減少(P0.01)。④Annexin V-FITC/PI結(jié)合流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡結(jié)果顯示：CaSki-DRR1細(xì)胞凋亡均數(shù)明顯高于CaSki-vect細(xì)胞(P0.01),凋亡細(xì)胞百分比分別為：12.116%和0.016%。 結(jié)論 1. HPV16E7陽(yáng)性和陰性宮頸癌細(xì)胞系和宮頸組織中,E7表達(dá)與DRRl表達(dá)負(fù)相關(guān)。 2.穩(wěn)定表達(dá)DRR1可顯著抑制HPV16E7P日性宮頸癌CaSki細(xì)胞的增殖能力,促進(jìn)其凋亡。
[Abstract]:Background cervical cancer is one of the most common malignant tumors in women. High risk-human papillomaviruses (HR-HPVs) is an important basis for its occurrence and evolution. The continuous expression of E7 is the key to the carcinogenesis of HR-HPVs, which can lead to abnormal cell proliferation by combining and degrading pRb protein. This group is treated by Agilent people. Gene expression profile chip analysis showed that after the expression of silent E7, the expression of down regulated gene 1 (down-regulated in renal cell carcinoma1, DRR1) was 3-11 times higher than that of normal tissue, but the expression in many cancer cell lines and primary tumors was significantly reduced, and DRR1 as a tumor suppressor gene may play a role in the process of cancer. Important role.
Objective HPV16E7 (?) diurnal and negative cervical cancer cell lines, normal cervical squamous epithelium, cervical intraepithelial neoplasia (cervical intraepithelial neoplasias, CIN) and cervical squamous cell carcinoma were studied to explore the use of DRR1 in the occurrence and evolution of HPV16 related cervical cancer in order to enrich the carcinogenic mechanism of HPV16 and to explore the HPV16 associated swelling. The new treatment of tumor provides experimental and theoretical basis.
Method
1. the expression of E7 and DRRl was detected by Western blotting method, and the relationship between HPV16E7 expression and DRR1 expression was analyzed by Western blotting method with CaSki and SiHa cells of HPV16E7 and C33-A cells of HPV16E7 negative cervical cancer as the research object.
2. HPV16E7 positive and negative normal cervical squamous epithelium, CIN and cervical squamous cell carcinoma were studied. The expression of E7 and DRR1 was detected by immunohistochemistry (immunohistochemistry, IHC), and the relationship between HPV16E7 and DRR1 expression was analyzed.
3. the HPV16E7 negative cervical cancer C33-A cells were cultured to the logarithmic growth period, the total RNA was extracted, the first chain of cDNA was synthesized by reverse transcriptase in vitro, the encoding sequence of DRR1 gene was amplified by PCR, the eukaryotic expression recombinant plasmid pcDNA3.1-DRR1 was constructed and the recombinant plasmid of pcDNA3.1-DRR1 was transfected into the logarithmic long term cervical cancer CaSki cells via liposome. The effect of stable expression of DRR1 on the proliferation and apoptosis of HPV16E7 positive cervical cancer cell CaSki cells was observed by MTT plotting cell growth curve, cell clone formation experiment, cell cycle distribution and Annexin V-FITC/PI combined with flow cytometry.
Result
The results of 1. Western blotting showed that there was no expression of HPV16E7 in C33-A cells, and the expression of HPV16E7 in CaSki and SiHa cells. The expression of DRR1 in C33-A cells was significantly higher than that of CaSki and SiHa cells (P0.01).
2.IHC results showed that the expression of DRR1 in HPV16E7 positive normal cervical squamous epithelium, CIN and cervical squamous cell carcinoma were significantly lower than that of HPV16E7 negative normal cervical squamous epithelium, CIN and cervical squamous cell carcinoma, and the correlation coefficients were -0.452, -0.485 and -0.444 (P0.01), respectively.
3. the results of cell growth curve drawn by MTT colorimetric assay showed that the growth rate of CaSki-DRR1 cells was significantly slower than that of CaSki-vect cells (P0.01), and the doubling time was significantly prolonged (P0.01). The results of the formation of clones in the flat panel showed that the number of CaSki-DRR1 cells was significantly lower than that of CaSki-vect cells (P0.01), and the clone volume was obvious. The results of cell cycle distribution detected by flow cytometry showed that compared with CaSki-vect cells, the G1 phase block of CaSki-DRR1 cells (P0.01), but the percentage of S and G2/M cells decreased significantly (P0.01). 4. The apoptotic results of Annexin V-FITC/PI combined flow cytometry showed that the apoptotic average of CaSki-DRR1 cells was obvious. The percentage of apoptotic cells was higher than that of CaSki-vect cells (P0.01): 12.116% and 0.016%. respectively.
conclusion
1. there was a negative correlation between E7 expression and DRRl expression in HPV16E7 positive and negative cervical cancer cell lines and cervical tissues.
2. stable expression of DRR1 can significantly inhibit the proliferation of HPV16E7P cervical cancer CaSki cells and promote its apoptosis.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.33

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相關(guān)期刊論文 前2條

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本文編號(hào)：2014018