人解整合素样金属蛋白酶22在前列腺癌中的表达及其意义探讨
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摘要
在发达国家,前列腺癌是临床上排名第二的常见肿瘤,同时也是男性肿瘤死亡率排第三的疾病。我国前列腺癌的发病率虽明显低于欧美国家,但近年随着人均寿命的延长、膳食结构的改变和诊断技术的提高,发病率有明显增高的趋势,在男性泌尿生殖系统恶性肿瘤中发病率跃居第三位。前列腺癌的发病机制未明,但是其局部微环境理应有重要作用,ADAM分子是近年新发现的具有重要作用的金属蛋白酶分子,因此,本课题拟探讨其中一种分子ADAM22在前列腺癌中的表达和意义。
目的:探讨ADAM22在前列腺癌中的分布和意义。制备其特异性抗体。判断细胞因子对其调节及分子机理。其对肿瘤凋亡的影响,从而从新的角度探讨前列腺癌的发病及病理情况,为前列腺癌的诊治和预后建立新的评判角度。
方法:RT-PCR法克隆ADAM22 cDNA,将其胞膜外区插入载体,诱导表达并纯化,免疫小鼠制备单克隆抗体并利用流式细胞术、western blot和免疫组织化学进行鉴定。探讨TNF-α诱导前列腺癌细胞ADAM22转录,表达及其相关信号转导途径。观察重组ADAM22对重组Fas的水解作用以及抗体对这种作用的封闭,将ADAM22与Fas基因共转染或进行基因干涉,判断其对Fas诱导凋亡的影响。
结果:成功克隆ADAM22 cDNA并表达融合蛋白。获得3株ADAM22特异性单克隆抗体,其中一株能够用于流式鉴定,一株能够用于免疫组织化学鉴定。ADAM22在前列腺癌中阳性分布,主要集中在腺样上皮和间质细胞中。TNF-α通过活化p38MAPK和NF-κB而促进ADAM22的转录和表达。ADAM22能够水解Fas,这种水解过程能被我们制备的单抗阻断。ADAM22对Fas的水解降低肿瘤细胞对Fas介导凋亡的敏感性。
结论:1. ADAM22在前列腺癌细胞和组织中阳性表达;2. TNF-α能够有效增强ADAM22表达;3. ADAM22通过水解Fas而介导肿瘤对凋亡的耐受。
Prostate cancer is the second ranked carcinoma in developed countries an is the third mortal disease among the male. The morbidity of prostate cancer is obviously lower than the developed countries but with the increased tendency because of increased life span, westernized diet and improvement of diagnosis technology and it ranked the third in male urine malignancy. The etiology of prostate cancers was not clear. The microenvironments of tumor should be vital. A disintegrin and metalloproteinases, abbreviated as ADAMs were nearly identified and with vital function. This study was aimed to explore ADAM22, one member of the family distribution and role in prostate carcinomas.
Aim: To explore the distribution and function of ADAM22 in prostate cancers. To prepare its specific monoclonal antibodies. To discuss the modification of cytokines targeting ADAM22 and find its role in potential roles during tumor apoptosis. All these will help us to understand etiology and pathology of prostate cancer, which will supply new evidences for therapy and prognosis for prostate cancers.
Methods: ADAM22 cDNA was cloned with RT-PCR and its extracellular domain subcloned and inserted into plasmid. The recombinant protein was induced and purified. Mice were immunized with the recombinant protein and the corresponding monoclonal antibodies were prepared and characterized by flow cytometry, western blot and immunohistochemistry. Transcription and expression of ADAM22 induced by TNF-αwere identified by real-time RT-PCR and flow cytomety and its involved signaling were tested by specific inhibitors. Cleavage of Fas by ADAM22 and the potential role of resistance to apoptosis were identified.
Results: ADAM22 cDNA was cloned successfully and its recombinant proteins were expressed. Three strains of mAb specific for ADAM22 were prepared. One of them was applicable in flow cytometry and the other was usable in IHC. ADAM22 was positive in prostate cancer tissues and most of it was located in gland-like and interstitial cells. TNF-αincreased transcription and expression of ADAM22 by activation of p38MAPK and NF-κB。ADAM22 sheded Fas and induced apoptosis resistance, which was blocked by the mAb we prepared previously.
Conclusion: 1. ADAM22 was positive in prostate cancer tissues and cells. 2. TNF-αincrease ADAM22 expression. 3. ADAM22 induced apoptosis resistance of tumor cells by shedding of Fas.
目的:探讨ADAM22在前列腺癌中的分布和意义。制备其特异性抗体。判断细胞因子对其调节及分子机理。其对肿瘤凋亡的影响,从而从新的角度探讨前列腺癌的发病及病理情况,为前列腺癌的诊治和预后建立新的评判角度。
方法:RT-PCR法克隆ADAM22 cDNA,将其胞膜外区插入载体,诱导表达并纯化,免疫小鼠制备单克隆抗体并利用流式细胞术、western blot和免疫组织化学进行鉴定。探讨TNF-α诱导前列腺癌细胞ADAM22转录,表达及其相关信号转导途径。观察重组ADAM22对重组Fas的水解作用以及抗体对这种作用的封闭,将ADAM22与Fas基因共转染或进行基因干涉,判断其对Fas诱导凋亡的影响。
结果:成功克隆ADAM22 cDNA并表达融合蛋白。获得3株ADAM22特异性单克隆抗体,其中一株能够用于流式鉴定,一株能够用于免疫组织化学鉴定。ADAM22在前列腺癌中阳性分布,主要集中在腺样上皮和间质细胞中。TNF-α通过活化p38MAPK和NF-κB而促进ADAM22的转录和表达。ADAM22能够水解Fas,这种水解过程能被我们制备的单抗阻断。ADAM22对Fas的水解降低肿瘤细胞对Fas介导凋亡的敏感性。
结论:1. ADAM22在前列腺癌细胞和组织中阳性表达;2. TNF-α能够有效增强ADAM22表达;3. ADAM22通过水解Fas而介导肿瘤对凋亡的耐受。
Prostate cancer is the second ranked carcinoma in developed countries an is the third mortal disease among the male. The morbidity of prostate cancer is obviously lower than the developed countries but with the increased tendency because of increased life span, westernized diet and improvement of diagnosis technology and it ranked the third in male urine malignancy. The etiology of prostate cancers was not clear. The microenvironments of tumor should be vital. A disintegrin and metalloproteinases, abbreviated as ADAMs were nearly identified and with vital function. This study was aimed to explore ADAM22, one member of the family distribution and role in prostate carcinomas.
Aim: To explore the distribution and function of ADAM22 in prostate cancers. To prepare its specific monoclonal antibodies. To discuss the modification of cytokines targeting ADAM22 and find its role in potential roles during tumor apoptosis. All these will help us to understand etiology and pathology of prostate cancer, which will supply new evidences for therapy and prognosis for prostate cancers.
Methods: ADAM22 cDNA was cloned with RT-PCR and its extracellular domain subcloned and inserted into plasmid. The recombinant protein was induced and purified. Mice were immunized with the recombinant protein and the corresponding monoclonal antibodies were prepared and characterized by flow cytometry, western blot and immunohistochemistry. Transcription and expression of ADAM22 induced by TNF-αwere identified by real-time RT-PCR and flow cytomety and its involved signaling were tested by specific inhibitors. Cleavage of Fas by ADAM22 and the potential role of resistance to apoptosis were identified.
Results: ADAM22 cDNA was cloned successfully and its recombinant proteins were expressed. Three strains of mAb specific for ADAM22 were prepared. One of them was applicable in flow cytometry and the other was usable in IHC. ADAM22 was positive in prostate cancer tissues and most of it was located in gland-like and interstitial cells. TNF-αincreased transcription and expression of ADAM22 by activation of p38MAPK and NF-κB。ADAM22 sheded Fas and induced apoptosis resistance, which was blocked by the mAb we prepared previously.
Conclusion: 1. ADAM22 was positive in prostate cancer tissues and cells. 2. TNF-αincrease ADAM22 expression. 3. ADAM22 induced apoptosis resistance of tumor cells by shedding of Fas.
引文
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