天堂国产午夜亚洲专区-少妇人妻综合久久蜜臀-国产成人户外露出视频在线-国产91传媒一区二区三区

人CD59基因活性位點突變對腫瘤逃逸相關(guān)分子補體及caspase-3的作用研究

發(fā)布時間:2018-12-25 12:03
【摘要】: 目的構(gòu)建兩種人CD59基因突變的重組體pALTER-MAX-CD59,轉(zhuǎn)染入HeLa細(xì)胞,篩選高表達(dá)人突變CD59的陽性細(xì)胞克隆,檢測HeLa細(xì)胞中突變CD59對腫瘤逃逸相關(guān)分子(caspase-3)的抑制效應(yīng)及突變?nèi)薈D59的補體抑制活性,研究CD59基因突變與腫瘤逃逸的相關(guān)性,進(jìn)一步證實人CD59與腫瘤逃逸相關(guān)的活性位點,為腫瘤逃逸機制的進(jìn)一步闡明提供新的理論依據(jù)。 方法用重組聚合酶鏈反應(yīng)定點誘變技術(shù)構(gòu)建第40位色氨酸缺失的人的CD59突變體(突變1)和第39-41位氨基酸突變?yōu)樯彼岬娜说腃D59突變體(突變2);與pALTER-MAX質(zhì)粒連接后,用陽離子脂質(zhì)體法轉(zhuǎn)染入HeLa細(xì)胞,以新霉素類似物G418篩選陽性細(xì)胞克隆,以免疫熒光、免疫酶標(biāo)、ELISA、Western-blot、流式細(xì)胞術(shù)進(jìn)一步篩選高表達(dá)突變?nèi)薈D59的細(xì)胞株,用免疫組化法檢測轉(zhuǎn)染人突變CD59的細(xì)胞中腫瘤逃逸相關(guān)分子caspase-3的表達(dá)變化,用乳酸脫氫酶(LDH)測試法檢測突變?nèi)薈D59的抗補體活性。 結(jié)果酶切鑒定及序列測定均證實成功構(gòu)建了兩種人CD59基因突變的重組質(zhì)粒;免疫熒光、免疫酶標(biāo)、ELISA、Western-blot、流式細(xì)胞術(shù)篩選出高表達(dá)突變?nèi)薈D59的細(xì)胞株;免疫組化檢測發(fā)現(xiàn)轉(zhuǎn)染突變1后的HeLa細(xì)胞中caspase-3的含量較轉(zhuǎn)染野生型人CD59的HeLa細(xì)胞中的caspase-3的含量明顯增加,轉(zhuǎn)染突變2后的HeLa細(xì)胞中caspase-3的含量較轉(zhuǎn)染野生型人CD59的HeLa細(xì)胞中的caspase-3的含量明顯減少。LDH法檢測轉(zhuǎn)染突變CD59后HeLa細(xì)胞具有抗補體活性,但其活性減弱。 結(jié)論成功構(gòu)建了兩種CD59突變的重組質(zhì)粒,并獲得高表達(dá)兩種突變CD59的HeLa細(xì)胞株,初步研究了CD59基因突變后抗補體活性改變及caspase-3的變化,證實了CD59引起腫瘤逃逸的途徑除了通過調(diào)節(jié)其抗補體活性來影響MAC的形成外還能通過影響caspase-3的表達(dá)來實現(xiàn)。
[Abstract]:Objective to construct two recombinant human CD59 gene mutants pALTER-MAX-CD59, and transfect them into HeLa cells to screen the positive cell clones expressing human mutant CD59. The inhibitory effect of mutated CD59 on tumor escape associated molecule (caspase-3) and complement inhibitory activity of mutant human CD59 in HeLa cells were detected, and the correlation between CD59 gene mutation and tumor escape was studied. The active sites associated with tumor escape were further confirmed by human CD59, which provided a new theoretical basis for further elucidation of the mechanism of tumor escape. Methods Human CD59 mutants (mutant 1) and CD59 mutants (mutagen2) were constructed by site-directed mutagenesis of recombinant polymerase chain reaction (RPCR) for the 40 th position tryptophan deficient human and the 39-41 amino acid mutagenesis for tryptophan. After ligation with pALTER-MAX plasmid, HeLa cells were transfected into HeLa cells by cationic liposome method. Positive cell clones were screened by neomycin analogue G418. Immunofluorescence, immunoenzyme labeling and ELISA,Western-blot, were used to screen the positive cells. Flow cytometry was used to screen the cell lines with high expression of human CD59, and the expression of tumor escape related molecule caspase-3 was detected by immunohistochemical method in the cells transfected with human mutant CD59. The anti complement activity of mutant human CD59 was detected by lactate dehydrogenase (LDH) assay. Results two recombinant plasmids of human CD59 gene mutation were successfully constructed by restriction endonuclease digestion and sequence analysis, immunofluorescence, immunoenzyme labeling and ELISA,Western-blot, flow cytometry were used to screen the cells with high expression of human CD59. Immunohistochemical analysis showed that the content of caspase-3 in HeLa cells transfected with mutant 1 was significantly higher than that in HeLa cells transfected with wild type CD59. The content of caspase-3 in HeLa cells after transfection of mutant 2 was significantly lower than that in HeLa cells transfected with wild type CD59. LDH assay showed that HeLa cells had anti-complement activity after transfection of mutant CD59, but its activity decreased. Conclusion two recombinant plasmids with CD59 mutation were successfully constructed and HeLa cell lines with high expression of CD59 were obtained. The changes of complement resistance and caspase-3 after CD59 gene mutation were preliminarily studied. It is confirmed that the pathway of escape induced by CD59 can not only affect the formation of MAC by regulating its anticomplement activity, but also affect the expression of caspase-3.
【學(xué)位授予單位】:青島大學(xué)
【學(xué)位級別】:碩士
【學(xué)位授予年份】:2008
【分類號】:R392.12;R730.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 楊崇禮,盧學(xué)春,劉慧英;再生障礙性貧血、骨髓增生異常綜合征、陣發(fā)性睡眠性血紅蛋白尿癥演變的問題[J];白血病;2000年06期

2 杜紅俊,惠延年,王雨生,韓泉洪,馬吉獻(xiàn);Caspase-3在柔紅霉素誘導(dǎo)的人視網(wǎng)膜色素上皮細(xì)胞凋亡中作用[J];第四軍醫(yī)大學(xué)學(xué)報;2001年07期

3 高玨華;CD59和陣發(fā)性睡眠性血紅蛋白尿[J];國外醫(yī)學(xué).輸血及血液學(xué)分冊;1998年06期

4 王海濱,郭峰;紅細(xì)胞參與細(xì)胞因子調(diào)控的研究進(jìn)展[J];國外醫(yī)學(xué)(免疫學(xué)分冊);1999年05期

5 張劍波,董巍,江南;貧血患者外周血CD55~-和CD59~-細(xì)胞檢測結(jié)果及其意義[J];臨床檢驗雜志;2003年S1期

6 韓永勝,翟志敏,丁邦勝,吳競生,何曉東;CD59檢測在陣發(fā)性睡眠性血紅蛋白尿癥診斷中的意義[J];臨床輸血與檢驗;2003年03期

7 周珞平,翟志敏,韓永勝,潘理明,朱薇波,蔡曉燕,吳競生,孫自敏;CD59抗原在血液系統(tǒng)惡性腫瘤患者中的表達(dá)及意義[J];臨床輸血與檢驗;2004年01期

8 周鏡然,白云,姜曼,黎萬玲;CD59融合蛋白表達(dá)載體構(gòu)建及其在大腸桿菌中的表達(dá)[J];免疫學(xué)雜志;2000年02期

9 陳政良,朱錫華;天然免疫系統(tǒng)的“分子模式識別作用”及其免疫生物學(xué)意義[J];免疫學(xué)雜志;2000年03期

10 張麗,高美華;突變?nèi)薈D59基因在真核細(xì)胞中的表達(dá)活性[J];免疫學(xué)雜志;2005年04期



本文編號:2391084

資料下載
論文發(fā)表

本文鏈接:http://sikaile.net/yixuelunwen/shiyanyixue/2391084.html


Copyright(c)文論論文網(wǎng)All Rights Reserved | 網(wǎng)站地圖 |

版權(quán)申明:資料由用戶487e2***提供,本站僅收錄摘要或目錄,作者需要刪除請E-mail郵箱bigeng88@qq.com
亚洲五月婷婷中文字幕| 翘臀少妇成人一区二区| 深夜福利亚洲高清性感| 日韩一区二区三区高清在| 欧洲一级片一区二区三区| 伊人欧美一区二区三区| 人妻乱近亲奸中文字幕| 日韩成人h视频在线观看| 久热99中文字幕视频在线| 在线中文字幕亚洲欧美一区| 东京热电东京热一区二区三区| 国产又色又粗又黄又爽| 亚洲第一视频少妇人妻系列| 国产精品日韩欧美一区二区| 欧美多人疯狂性战派对| 精品欧美一区二区三久久| 欧美在线观看视频三区| 日韩和欧美的一区二区三区| 日本不卡视频在线观看| 欧美六区视频在线观看| 亚洲欧美日本国产有色| 精品香蕉国产一区二区三区| 午夜久久精品福利视频| 这里只有九九热精品视频| 亚洲精品一区三区三区| 久久福利视频这里有精品| 国产成人人人97超碰熟女| 日韩av欧美中文字幕| 亚洲欧美黑人一区二区| 最近最新中文字幕免费| 成年午夜在线免费视频| 欧美日韩一区二区午夜| 国产综合一区二区三区av| 国产免费一区二区不卡| 神马午夜福利免费视频| 男女午夜福利院在线观看| 精品人妻一区二区三区四在线| 东京不热免费观看日本| 国产熟女高清一区二区| 99热九九热这里只有精品| 一区二区三区国产日韩|