RNASET2分子在白癜风发病中的作用及机制研究

发布时间：2018-01-01 20:30

本文关键词：RNASET2分子在白癜风发病中的作用及机制研究　出处：《复旦大学》2014年博士论文　论文类型：学位论文

【摘要】：第一部分应激对人表皮角质形成细胞和黑素细胞RNASET2的表达影响目的：探讨白癜风患者皮损区RNASET2的表达情况；运用改良方法,简单、经济地从人包皮组织中分离扩增得到大量高纯度原代角质形成细胞(HEKs)和黑素细胞(HEMs);在体外观察应激因素作用下HEKs和HEMs中RNASET2的表达情况。方法：利用实时PCR和Western blot分析了白癜风皮损处RNASET2的表达,并用RNASET2的多克隆抗体进行免疫组化研究,以确定白癜风样品中NASET2表达情况；根据原代角质形成细胞和黑素细胞对胰酶的不同耐受性和成纤维细胞不能耐受G418的特性可分离出纯原代黑素细胞和角质细胞；过量的紫外线照射(NB-UVB)、氧化应激(H2O2)、炎症(LPS)刺激体外培养的HEKs和HEMs,利用实时PCR,Western blot和免疫荧光检测技术鉴定HEKs和HEMs在应激作用下RNASET2表达情况。结果：与正常对照组相比较,白癜风患者皮损RNASET2在转录(白癜风vs对照组：4.056±1.115 vs 1.034±0.166；p0.001)和蛋白水平都显著升高；成功从人包皮组织中分离扩增得到大量高纯度HEKs和HEMs;与未处理组比较,HEKs和HEMs在一定应激作用剂量范围内,RNASET2在转录和蛋白表达水平都有明显升高。结论：白癜风患者皮损处RNASET2显著增加；白癜风诱发因素可以使HEKs和HEMs中RNASET2表达增加。第二部分RNASET2基因产物对原代角质形成细胞和黑素细胞生物学功能的影响目的：探讨体外RNASET2过表达对原代培养的角质形成细胞和黑素细胞的生物学功能影响。方法：利用RNASET2过表达慢病毒分别感染原代角质形成细胞和黑素细胞；感染后72h,用荧光显微镜观察鉴定了慢病毒感染细胞的效率；通过Western blot技术对感染结果进行了鉴定；采用CCK-8细胞活性实验、Annexin V-APC/7-AAD双色标记法和colony-forming法对RNASET2过表达原代角质形成细胞和黑素细胞的增殖凋亡情况进行了研究；通过将适当浓度的外源性氧化应激因素H202加入RNASET2过表达的原代角质形成细胞和黑素细胞中,观察氧化应激对RNASET2过表达细胞的影响；采用实时PCR、Western blot、黑素含量测定以及酪氨酸酶活性测定等技术检测RNASET2过表达对黑素细胞合成黑素能力的影响情况。结果：慢病毒成功感染原代角质形成细胞和黑素细胞72h后,感染效率90%；与转染空载体(VECTOR)细胞比较,RNASET2过表达(OE RNASET2)引起原代角质形成细胞(VECTOR vs OE RNASET2:4.167±0.330 vs 23.467±1.482; p=0.003)和黑素细胞(VECTOR vs OE RNASET2:5.400±0.455 vs 27.333±1.053; p=0.001)的凋亡具有统计学意义；RNASET2过表达使原代角质形成细胞和黑素细胞对氧化应激更敏感；NASET2过表达导致黑素细胞合成黑素能力下降。结论：RNASET2过表达导致原代角质形成细胞和黑素细胞凋亡；RNASET2过表达导致黑素细胞合成黑素能力下降。第三部分RNASET2基因产物在白癜风发病中的机制研究目的：探讨RNASET2过表达导致原代角质形成细胞和黑素细胞凋亡和黑素合成下降引发白癜风的机制。方法：为探讨RNASET2的促凋亡作用是否依赖于其催化位点,采用RNASET2催化位点突变后的过表达慢病毒(OE RNASET2-ci)分别转染原代角质形成细胞和黑素细胞；同时,由于最近研究发现RNASET2的结构中含有能结合凋亡因子TRAF2的结构域,为证实RNASET2分子中TRAF2结构域是否与RNASET2的促凋亡作用相关,我们将RNASET2的TRAF2结合位点突变后的过表达慢病毒(OE RNASET2-ti)分别转染原代角质形成细胞和黑素细胞；用荧光显微镜观察鉴定了慢病毒感染细胞的效率；通过Western blot技术对感染结果进行了鉴定；采用CCK-8细胞活性实验和Annexin V-APC/7-AAD双色标记法对RNASET2过表达原代角质形成细胞和黑素细胞的增殖凋亡情况进行了研究；为探讨RNASET2的促凋亡作用依赖于RNASET2蛋白与TRAF2蛋白的相互结合作用,采用免疫共沉淀(CO-IP)及Western blot技术检测RNASET2突变前后过表达对细胞凋亡的影响情况。结果：RNASET2催化位点突变后的过表达慢病毒(OE RNASET2-ci)和RNASET2的TRAF2结合位点突变后的过表达慢病毒(OE RNASET2-ti)分别成功感染原代角质形成细胞和黑素细胞,感染效率90%；RNASET2基因产物导致细胞凋亡不依赖于其催化位点,而是与凋亡因子TRAF2密切相关；RNASET2通过RNASET2-TRAF2-caspases通路引起细胞凋亡。结论：RNASET2基因产物引起细胞凋亡不依赖于其催化位点；RNASET2通过RNASET2-TRAF2-caspases通路引起细胞凋亡。
[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.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R758.41

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