本文關(guān)鍵詞：RNASET2分子在白癜風(fēng)發(fā)病中的作用及機(jī)制研究　出處：《復(fù)旦大學(xué)》2014年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 白癜風(fēng) RNASET2 HEKs HEMs 應(yīng)激 RNASET2過(guò)表達(dá) HEKs HEMs 凋亡 黑素合成下降 RNASET2過(guò)表達(dá) 催化位點(diǎn) TRAF2的結(jié)構(gòu)域 凋亡

【摘要】：第一部分應(yīng)激對(duì)人表皮角質(zhì)形成細(xì)胞和黑素細(xì)胞RNASET2的表達(dá)影響目的：探討白癜風(fēng)患者皮損區(qū)RNASET2的表達(dá)情況；運(yùn)用改良方法,簡(jiǎn)單、經(jīng)濟(jì)地從人包皮組織中分離擴(kuò)增得到大量高純度原代角質(zhì)形成細(xì)胞(HEKs)和黑素細(xì)胞(HEMs);在體外觀察應(yīng)激因素作用下HEKs和HEMs中RNASET2的表達(dá)情況。方法：利用實(shí)時(shí)PCR和Western blot分析了白癜風(fēng)皮損處RNASET2的表達(dá),并用RNASET2的多克隆抗體進(jìn)行免疫組化研究,以確定白癜風(fēng)樣品中NASET2表達(dá)情況；根據(jù)原代角質(zhì)形成細(xì)胞和黑素細(xì)胞對(duì)胰酶的不同耐受性和成纖維細(xì)胞不能耐受G418的特性可分離出純?cè)谒丶?xì)胞和角質(zhì)細(xì)胞；過(guò)量的紫外線照射(NB-UVB)、氧化應(yīng)激(H2O2)、炎癥(LPS)刺激體外培養(yǎng)的HEKs和HEMs,利用實(shí)時(shí)PCR,Western blot和免疫熒光檢測(cè)技術(shù)鑒定HEKs和HEMs在應(yīng)激作用下RNASET2表達(dá)情況。結(jié)果：與正常對(duì)照組相比較,白癜風(fēng)患者皮損RNASET2在轉(zhuǎn)錄(白癜風(fēng)vs對(duì)照組：4.056±1.115 vs 1.034±0.166；p0.001)和蛋白水平都顯著升高；成功從人包皮組織中分離擴(kuò)增得到大量高純度HEKs和HEMs;與未處理組比較,HEKs和HEMs在一定應(yīng)激作用劑量范圍內(nèi),RNASET2在轉(zhuǎn)錄和蛋白表達(dá)水平都有明顯升高。結(jié)論：白癜風(fēng)患者皮損處RNASET2顯著增加；白癜風(fēng)誘發(fā)因素可以使HEKs和HEMs中RNASET2表達(dá)增加。第二部分RNASET2基因產(chǎn)物對(duì)原代角質(zhì)形成細(xì)胞和黑素細(xì)胞生物學(xué)功能的影響目的：探討體外RNASET2過(guò)表達(dá)對(duì)原代培養(yǎng)的角質(zhì)形成細(xì)胞和黑素細(xì)胞的生物學(xué)功能影響。方法：利用RNASET2過(guò)表達(dá)慢病毒分別感染原代角質(zhì)形成細(xì)胞和黑素細(xì)胞；感染后72h,用熒光顯微鏡觀察鑒定了慢病毒感染細(xì)胞的效率；通過(guò)Western blot技術(shù)對(duì)感染結(jié)果進(jìn)行了鑒定；采用CCK-8細(xì)胞活性實(shí)驗(yàn)、Annexin V-APC/7-AAD雙色標(biāo)記法和colony-forming法對(duì)RNASET2過(guò)表達(dá)原代角質(zhì)形成細(xì)胞和黑素細(xì)胞的增殖凋亡情況進(jìn)行了研究；通過(guò)將適當(dāng)濃度的外源性氧化應(yīng)激因素H202加入RNASET2過(guò)表達(dá)的原代角質(zhì)形成細(xì)胞和黑素細(xì)胞中,觀察氧化應(yīng)激對(duì)RNASET2過(guò)表達(dá)細(xì)胞的影響；采用實(shí)時(shí)PCR、Western blot、黑素含量測(cè)定以及酪氨酸酶活性測(cè)定等技術(shù)檢測(cè)RNASET2過(guò)表達(dá)對(duì)黑素細(xì)胞合成黑素能力的影響情況。結(jié)果：慢病毒成功感染原代角質(zhì)形成細(xì)胞和黑素細(xì)胞72h后,感染效率90%；與轉(zhuǎn)染空載體(VECTOR)細(xì)胞比較,RNASET2過(guò)表達(dá)(OE RNASET2)引起原代角質(zhì)形成細(xì)胞(VECTOR vs OE RNASET2:4.167±0.330 vs 23.467±1.482; p=0.003)和黑素細(xì)胞(VECTOR vs OE RNASET2:5.400±0.455 vs 27.333±1.053; p=0.001)的凋亡具有統(tǒng)計(jì)學(xué)意義；RNASET2過(guò)表達(dá)使原代角質(zhì)形成細(xì)胞和黑素細(xì)胞對(duì)氧化應(yīng)激更敏感；NASET2過(guò)表達(dá)導(dǎo)致黑素細(xì)胞合成黑素能力下降。結(jié)論：RNASET2過(guò)表達(dá)導(dǎo)致原代角質(zhì)形成細(xì)胞和黑素細(xì)胞凋亡；RNASET2過(guò)表達(dá)導(dǎo)致黑素細(xì)胞合成黑素能力下降。第三部分RNASET2基因產(chǎn)物在白癜風(fēng)發(fā)病中的機(jī)制研究目的：探討RNASET2過(guò)表達(dá)導(dǎo)致原代角質(zhì)形成細(xì)胞和黑素細(xì)胞凋亡和黑素合成下降引發(fā)白癜風(fēng)的機(jī)制。方法：為探討RNASET2的促凋亡作用是否依賴于其催化位點(diǎn),采用RNASET2催化位點(diǎn)突變后的過(guò)表達(dá)慢病毒(OE RNASET2-ci)分別轉(zhuǎn)染原代角質(zhì)形成細(xì)胞和黑素細(xì)胞；同時(shí),由于最近研究發(fā)現(xiàn)RNASET2的結(jié)構(gòu)中含有能結(jié)合凋亡因子TRAF2的結(jié)構(gòu)域,為證實(shí)RNASET2分子中TRAF2結(jié)構(gòu)域是否與RNASET2的促凋亡作用相關(guān),我們將RNASET2的TRAF2結(jié)合位點(diǎn)突變后的過(guò)表達(dá)慢病毒(OE RNASET2-ti)分別轉(zhuǎn)染原代角質(zhì)形成細(xì)胞和黑素細(xì)胞；用熒光顯微鏡觀察鑒定了慢病毒感染細(xì)胞的效率；通過(guò)Western blot技術(shù)對(duì)感染結(jié)果進(jìn)行了鑒定；采用CCK-8細(xì)胞活性實(shí)驗(yàn)和Annexin V-APC/7-AAD雙色標(biāo)記法對(duì)RNASET2過(guò)表達(dá)原代角質(zhì)形成細(xì)胞和黑素細(xì)胞的增殖凋亡情況進(jìn)行了研究；為探討RNASET2的促凋亡作用依賴于RNASET2蛋白與TRAF2蛋白的相互結(jié)合作用,采用免疫共沉淀(CO-IP)及Western blot技術(shù)檢測(cè)RNASET2突變前后過(guò)表達(dá)對(duì)細(xì)胞凋亡的影響情況。結(jié)果：RNASET2催化位點(diǎn)突變后的過(guò)表達(dá)慢病毒(OE RNASET2-ci)和RNASET2的TRAF2結(jié)合位點(diǎn)突變后的過(guò)表達(dá)慢病毒(OE RNASET2-ti)分別成功感染原代角質(zhì)形成細(xì)胞和黑素細(xì)胞,感染效率90%；RNASET2基因產(chǎn)物導(dǎo)致細(xì)胞凋亡不依賴于其催化位點(diǎn),而是與凋亡因子TRAF2密切相關(guān)；RNASET2通過(guò)RNASET2-TRAF2-caspases通路引起細(xì)胞凋亡。結(jié)論：RNASET2基因產(chǎn)物引起細(xì)胞凋亡不依賴于其催化位點(diǎn)；RNASET2通過(guò)RNASET2-TRAF2-caspases通路引起細(xì)胞凋亡。
[Abstract]:The first part of the formation stress expression of cells and melanocytes RNASET2 on human epidermal keratinocytesobjective to investigate the expression of RNASET2 in lesional skin of patients with vitiligo; using the improved method, simple and economic separation from human foreskin tissue was obtained with a high purity of primary keratinocytes and melanocytes (HEKs) (HEMs); to observe the expression of RNASET2 HEKs and HEMs in stress factors in vitro. Methods: to analyze the expression of vitiligo lesions using real-time PCR and Western RNASET2 blot, and RNASET2 polyclonal antibodies for immunohistochemical study to determine the expression of NASET2 in vitiligo samples; according to the original generation of keratinocytes and melanocytes on the different cell trypsin tolerance and fibroblasts can not tolerate the characteristics of G418 can be isolated in pure cultured melanocytes and keratinocytes; excessive ultraviolet radiation (NB-UVB), Oxidative stress (H2O2), inflammation (LPS) cultured in vitro HEKs and HEMs using real-time PCR, Western blot and immunofluorescence identification of HEKs and HEMs in RNASET2 under the stress expression. Results: compared with normal control group, the skin lesions of patients with vitiligo vitiligo (RNASET2 in transcription control group: 4.056 + vs 1.115 vs 1.034 + 0.166; p0.001) and the protein level was significantly increased; the successful isolation and amplification of large amounts of highly purified HEKs and HEMs from human foreskin tissue; compared with the non treatment group, HEKs and HEMs in a certain stress dose range, RNASET2 levels are significantly higher in transcription and protein expression. Conclusion: the skin lesions of patients vitiligo at RNASET2 increased significantly; the vitiligo predisposing factors can increase the expression of RNASET2 HEKs and HEMs. The second part of the RNASET2 gene product cells and melanocytes biological function on the formation of primary keratinocytes Objective: To investigate the in vitro effect of RNASET2 overexpression on primary cultured keratinocytes of cells and melanocytes. Methods: the expression of RNASET2 slow virus infection of primary keratinocytes and melanocytes; 72h after infection, observed by fluorescence microscopy and identification of lentivirus infected cells by efficiency; Western blot technology was used to identify the infection; using CCK-8 cell activity experiment, Annexin V-APC/7-AAD double staining method and colony-forming method of RNASET2 primary keratinocytes and Melanocytes Proliferation and apoptosis were studied by H202 expression; exogenous oxidative stress factors in the appropriate concentration of added RNASET2 over expression of the original generation of keratinocytes and melanocytes, observe the oxidative stress on the expression of RNASET2 cells by real-time PCR, Western; blot, melanin containing Determination and tyrosinase activity was measured to detect the effects of RNASET2 overexpression on melanocytes synthesis melanin ability. Results: slow virus infection of primary keratinocytes and melanocytes after 72h infection rate was 90%; and the empty vector transfected (VECTOR) cells compared with expression of RNASET2 (OE RNASET2) from primary keratinocytes (VECTOR vs OE RNASET2:4.167 + 0.330 vs 23.467 + 1.482; p=0.003) and melanocytes (VECTOR vs OE RNASET2:5.400 + 0.455 vs 27.333 + 1.053; p=0.001) with statistical significance of apoptosis; RNASET2 overexpression in primary keratinocytes and melanocytes are more sensitive to oxidative stress; NASET2 the decline of melanocytes synthesis melanin expression ability. Conclusion: RNASET2 in primary keratinocytes and melanocytes apoptosis; RNASET2 melanoma cells led to the synthesis of melanin expression The ability to decline. In the third part, the product of RNASET2 gene in the pathogenesis of vitiligo mechanism research objective: To investigate the RNASET2 leading primary keratinocyte and mechanism of cell apoptosis in melanocytes and melanin synthesis decline caused by vitiligo. Methods: expression for apoptosis of RNASET2 is not dependent on the catalytic site, the overexpression of RNASET2 catalyst the mutation of lentivirus (OE RNASET2-ci) were transfected into primary cultured keratinocytes and melanocytes; at the same time, due to the recent study found that the structure of RNASET2 binding domain containing apoptotic factor TRAF2, whether the TRAF2 domain to RNASET2 molecules associated with apoptosis of RNASET2, we will RNASET2 TRAF2 binding site mutation after overexpression of lentivirus (OE RNASET2-ti) were transfected into primary cultured keratinocytes and melanocytes were observed by fluorescence microscopy; Lentivirus infected cells efficiency; through Western blot technology was used to identify the results of RNASET2 infection; primary keratinocyte proliferation and apoptosis of melanocytes were studied by expression of CCK-8 cell activity assay and Annexin V-APC/7-AAD double staining method; binding interaction for apoptosis of RNASET2 depends on RNASET2 protein and TRAF2 protein, by immunoprecipitation (CO-IP) and RNASET2 Western blot mutation detection before and after expression on apoptosis. Results: overexpression of RNASET2 catalytic site mutated lentivirus (OE RNASET2-ci) and RNASET2 TRAF2 binding site mutation after overexpression of lentivirus (OE RNASET2-ti) respectively. Successful infection of primary keratinocytes and melanocytes, the infection rate was 90%; the product of RNASET2 gene leads to apoptosis independent of its catalytic site, It is closely related to apoptotic factor TRAF2. RNASET2 induces apoptosis through RNASET2-TRAF2-caspases pathway. Conclusion: RNASET2 gene product induces apoptosis and does not depend on its catalytic site. RNASET2 induces apoptosis through RNASET2-TRAF2-caspases pathway.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R758.41

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本文編號(hào)：1366093