本文主要研究内容
作者周慧（2019）在《PBX1通过ERK促进人皮肤黑色素（干）细胞增殖及其机制初探》一文中研究指出：研究背景:黑色素细胞来源于神经嵴。随着胚胎的发育,神经嵴细胞及其产物通过背外侧、腹外侧途径形成成黑素细胞,向毛囊、表皮、眼葡萄膜、软脑膜和内耳迁移,使毛发和皮肤着色并且参与听力和视力的调节。当黑色素细胞系发育障碍时,会引起听力、视力的丧失,皮肤色素沉着障碍性疾病以及黑色素瘤。环境污染、内在毒素均能影响黑色素细胞的发育。上述疾病的研究及毒理学评价工作的开展均可以黑色素细胞和黑色素干细胞作为种子来源,但其体外传代能力有限,易于分化,满足不了细胞生物学研究的需求。PBX1是细胞转录因子,可维持干细胞的自我更新及干性,且具有促进细胞增殖作用。研究目的:研究过表达PBX1对人皮肤黑色素(干)细胞的增殖作用,探讨过表达PBX1促进人皮肤黑色素(干)细胞增殖的相关机制。研究方法:1.人皮肤黑色素(干)细胞的分离、培养与鉴定取行包皮环切术的成年人、青少年包皮组织,分别把组织剪切成1 mm×1 cm大小,浸泡于DispaseⅡ溶液中,4℃冰箱过夜,分离真、表皮组织。次日,用含EDTA的0.25%胰酶消化表皮组织,获取细胞,离心后用选择培养基培养细胞,RT-PCR、免疫荧光、Western Blot方法检测人源黑色素细胞特异性生物标记物;MTT法检测OD值并绘制人皮肤黑色素(干)细胞的增殖曲线,计算群体倍增时间。2.过表达PBX1人皮肤黑色素(干)细胞的制备将psPAX2、pMD2G和PBX1质粒与EndoFectinTM-MaX转染试剂混合后,转染至293 T细胞。分别收集转染后36h、60h的病毒上清,超滤管内离心、浓缩后,与LV-Enhance混合,加入到人皮肤黑色素(干)细胞。3天后,荧光显微镜下观察细胞荧光表达情况、计数细胞、计算转导效率。采用免疫荧光、Western Blot法检测过表达PBX1的人皮肤黑色素(干)细胞特异性标记物表达情况。3.过表达PBX1对人皮肤黑色素(干)细胞增殖的影响利用MTT检测过表达PBX1对人皮肤黑色素(干)细胞增殖的影响;流式细胞术检测细胞周期并计算增殖指数;Western Blot检测增殖信号通路关键蛋白STAT3,AKT,ERK表达情况;给予ERK抑制剂,利用计数法、Western Blot检测其对人皮肤黑色素干细胞增殖的影响;流式细胞术检测其对人皮肤黑色素干细胞周期的影响。4.过表达PBX1对人皮肤黑色素(干)细胞增殖相关机制探讨Western Blot检测过表达PBX1对人皮肤黑色素(干)细胞周期相关蛋白表达的影响。实验结果:1.人皮肤黑色素(干)细胞的分离、培养与鉴定显微镜下可见:分离、培养的人皮肤黑色素细胞呈两极或三极的形态;人皮肤黑色素干细胞呈纺锤形。RT-PCR结果可见:成年人包皮组织获取的细胞表达基因TYR、TYRP1、MITF、TYRP2、C-KIT、SOX10、PAX3,免疫荧光、Western Blot检测,结果可见:成年人包皮组织获取的细胞表达蛋白TYR、TYRP1、MITF,即为人皮肤黑色素细胞;青少年包皮组织获取的细胞表达基因TYR、TYRP1、MITF、TYRP2、C-KIT、SOX10、PAX3,免疫荧光、Western Blot检测,结果可见:青少年包皮组织获取的细胞表达蛋白MITF,不表达TYR、TYRP1,即为人皮肤黑色素干细胞。MTT的结果计算可知:人皮肤黑色素细胞群体倍增时间为141.2 h;人皮肤黑色素干细胞群体倍增时间为80.8 h。2.过表达PBX1人皮肤黑色素(干)细胞的制备显微镜下可见:转染PBX1的293 T细胞贴壁生长、状态良好,转染效率较高。将收集的病毒转导至人皮肤黑色素(干)细胞。荧光显微镜观察:随着培养时间的延长,荧光百分比逐渐增多,细胞折光度强,细胞状态较好;Cytation3细胞成像多功能微孔板检测系统计数转导的阳性率,结果可见:人皮肤黑色素细胞eGFP组阳性率为94%,过表达PBX1组阳性率为82%。人皮肤黑色素干细胞eGFP组阳性率为83%,过表达PBX1组阳性率为71%;免疫荧光、Western Blot结果可见:过表达PBX1后,人皮肤黑色素细胞仍表达蛋白TYR、TYRP1、MITF。人皮肤黑色素干细胞仍表达蛋白MITF,不表达TYR、TYRP1。3.过表达PBX1对人皮肤黑色素(干)细胞增殖影响同相应eGFP组比较,MTT的结果显示:过表达PBX1组人皮肤黑色素(干)细胞增殖均较快;流式细胞术结果显示:过表达PBX1促进人皮肤黑色素(干)细胞S期细胞比例增加、增殖指数提高;Western Blot结果显示:过表达PBX1可以使人皮肤黑色素(干)细胞p-EKR表达量增加;给予过表达PBX1的人皮肤黑色素干细胞ERK抑制剂后,细胞数减少,细胞周期进程受阻。4.过表达PBX1对人皮肤黑色素(干)细胞周期相关蛋白影响。Western Blot结果显示:过表达PBX1可使人皮肤黑色素细胞的P21表达下调,E2F、CyclinD1表达上调;过表达PBX1可使人皮肤黑色素干细胞P21表达下调,CyclinE1、CDK2表达上调。结论:1.成功在体外分离、培养人皮肤黑色素(干)细胞;2.成功制备过表达PBX1人皮肤黑色素(干)细胞;3.过表达PBX1通过上调磷酸化ERK、影响细胞周期相关蛋白促进人皮肤黑色素(干)细胞增殖。
Abstract
yan jiu bei jing :hei se su xi bao lai yuan yu shen jing ji 。sui zhao pei tai de fa yo ,shen jing ji xi bao ji ji chan wu tong guo bei wai ce 、fu wai ce tu jing xing cheng cheng hei su xi bao ,xiang mao nang 、biao pi 、yan pu tao mo 、ruan nao mo he nei er qian yi ,shi mao fa he pi fu zhao se bing ju can yu ting li he shi li de diao jie 。dang hei se su xi bao ji fa yo zhang ai shi ,hui yin qi ting li 、shi li de sang shi ,pi fu se su chen zhao zhang ai xing ji bing yi ji hei se su liu 。huan jing wu ran 、nei zai du su jun neng ying xiang hei se su xi bao de fa yo 。shang shu ji bing de yan jiu ji du li xue ping jia gong zuo de kai zhan jun ke yi hei se su xi bao he hei se su gan xi bao zuo wei chong zi lai yuan ,dan ji ti wai chuan dai neng li you xian ,yi yu fen hua ,man zu bu le xi bao sheng wu xue yan jiu de xu qiu 。PBX1shi xi bao zhuai lu yin zi ,ke wei chi gan xi bao de zi wo geng xin ji gan xing ,ju ju you cu jin xi bao zeng shi zuo yong 。yan jiu mu de :yan jiu guo biao da PBX1dui ren pi fu hei se su (gan )xi bao de zeng shi zuo yong ,tan tao guo biao da PBX1cu jin ren pi fu hei se su (gan )xi bao zeng shi de xiang guan ji zhi 。yan jiu fang fa :1.ren pi fu hei se su (gan )xi bao de fen li 、pei yang yu jian ding qu hang bao pi huan qie shu de cheng nian ren 、qing shao nian bao pi zu zhi ,fen bie ba zu zhi jian qie cheng 1 mm×1 cmda xiao ,jin pao yu DispaseⅡrong ye zhong ,4℃bing xiang guo ye ,fen li zhen 、biao pi zu zhi 。ci ri ,yong han EDTAde 0.25%yi mei xiao hua biao pi zu zhi ,huo qu xi bao ,li xin hou yong shua ze pei yang ji pei yang xi bao ,RT-PCR、mian yi ying guang 、Western Blotfang fa jian ce ren yuan hei se su xi bao te yi xing sheng wu biao ji wu ;MTTfa jian ce ODzhi bing hui zhi ren pi fu hei se su (gan )xi bao de zeng shi qu xian ,ji suan qun ti bei zeng shi jian 。2.guo biao da PBX1ren pi fu hei se su (gan )xi bao de zhi bei jiang psPAX2、pMD2Ghe PBX1zhi li yu EndoFectinTM-MaXzhuai ran shi ji hun ge hou ,zhuai ran zhi 293 Txi bao 。fen bie shou ji zhuai ran hou 36h、60hde bing du shang qing ,chao lv guan nei li xin 、nong su hou ,yu LV-Enhancehun ge ,jia ru dao ren pi fu hei se su (gan )xi bao 。3tian hou ,ying guang xian wei jing xia guan cha xi bao ying guang biao da qing kuang 、ji shu xi bao 、ji suan zhuai dao xiao lv 。cai yong mian yi ying guang 、Western Blotfa jian ce guo biao da PBX1de ren pi fu hei se su (gan )xi bao te yi xing biao ji wu biao da qing kuang 。3.guo biao da PBX1dui ren pi fu hei se su (gan )xi bao zeng shi de ying xiang li yong MTTjian ce guo biao da PBX1dui ren pi fu hei se su (gan )xi bao zeng shi de ying xiang ;liu shi xi bao shu jian ce xi bao zhou ji bing ji suan zeng shi zhi shu ;Western Blotjian ce zeng shi xin hao tong lu guan jian dan bai STAT3,AKT,ERKbiao da qing kuang ;gei yu ERKyi zhi ji ,li yong ji shu fa 、Western Blotjian ce ji dui ren pi fu hei se su gan xi bao zeng shi de ying xiang ;liu shi xi bao shu jian ce ji dui ren pi fu hei se su gan xi bao zhou ji de ying xiang 。4.guo biao da PBX1dui ren pi fu hei se su (gan )xi bao zeng shi xiang guan ji zhi tan tao Western Blotjian ce guo biao da PBX1dui ren pi fu hei se su (gan )xi bao zhou ji xiang guan dan bai biao da de ying xiang 。shi yan jie guo :1.ren pi fu hei se su (gan )xi bao de fen li 、pei yang yu jian ding xian wei jing xia ke jian :fen li 、pei yang de ren pi fu hei se su xi bao cheng liang ji huo san ji de xing tai ;ren pi fu hei se su gan xi bao cheng fang chui xing 。RT-PCRjie guo ke jian :cheng nian ren bao pi zu zhi huo qu de xi bao biao da ji yin TYR、TYRP1、MITF、TYRP2、C-KIT、SOX10、PAX3,mian yi ying guang 、Western Blotjian ce ,jie guo ke jian :cheng nian ren bao pi zu zhi huo qu de xi bao biao da dan bai TYR、TYRP1、MITF,ji wei ren pi fu hei se su xi bao ;qing shao nian bao pi zu zhi huo qu de xi bao biao da ji yin TYR、TYRP1、MITF、TYRP2、C-KIT、SOX10、PAX3,mian yi ying guang 、Western Blotjian ce ,jie guo ke jian :qing shao nian bao pi zu zhi huo qu de xi bao biao da dan bai MITF,bu biao da TYR、TYRP1,ji wei ren pi fu hei se su gan xi bao 。MTTde jie guo ji suan ke zhi :ren pi fu hei se su xi bao qun ti bei zeng shi jian wei 141.2 h;ren pi fu hei se su gan xi bao qun ti bei zeng shi jian wei 80.8 h。2.guo biao da PBX1ren pi fu hei se su (gan )xi bao de zhi bei xian wei jing xia ke jian :zhuai ran PBX1de 293 Txi bao tie bi sheng chang 、zhuang tai liang hao ,zhuai ran xiao lv jiao gao 。jiang shou ji de bing du zhuai dao zhi ren pi fu hei se su (gan )xi bao 。ying guang xian wei jing guan cha :sui zhao pei yang shi jian de yan chang ,ying guang bai fen bi zhu jian zeng duo ,xi bao she guang du jiang ,xi bao zhuang tai jiao hao ;Cytation3xi bao cheng xiang duo gong neng wei kong ban jian ce ji tong ji shu zhuai dao de yang xing lv ,jie guo ke jian :ren pi fu hei se su xi bao eGFPzu yang xing lv wei 94%,guo biao da PBX1zu yang xing lv wei 82%。ren pi fu hei se su gan xi bao eGFPzu yang xing lv wei 83%,guo biao da PBX1zu yang xing lv wei 71%;mian yi ying guang 、Western Blotjie guo ke jian :guo biao da PBX1hou ,ren pi fu hei se su xi bao reng biao da dan bai TYR、TYRP1、MITF。ren pi fu hei se su gan xi bao reng biao da dan bai MITF,bu biao da TYR、TYRP1。3.guo biao da PBX1dui ren pi fu hei se su (gan )xi bao zeng shi ying xiang tong xiang ying eGFPzu bi jiao ,MTTde jie guo xian shi :guo biao da PBX1zu ren pi fu hei se su (gan )xi bao zeng shi jun jiao kuai ;liu shi xi bao shu jie guo xian shi :guo biao da PBX1cu jin ren pi fu hei se su (gan )xi bao Sji xi bao bi li zeng jia 、zeng shi zhi shu di gao ;Western Blotjie guo xian shi :guo biao da PBX1ke yi shi ren pi fu hei se su (gan )xi bao p-EKRbiao da liang zeng jia ;gei yu guo biao da PBX1de ren pi fu hei se su gan xi bao ERKyi zhi ji hou ,xi bao shu jian shao ,xi bao zhou ji jin cheng shou zu 。4.guo biao da PBX1dui ren pi fu hei se su (gan )xi bao zhou ji xiang guan dan bai ying xiang 。Western Blotjie guo xian shi :guo biao da PBX1ke shi ren pi fu hei se su xi bao de P21biao da xia diao ,E2F、CyclinD1biao da shang diao ;guo biao da PBX1ke shi ren pi fu hei se su gan xi bao P21biao da xia diao ,CyclinE1、CDK2biao da shang diao 。jie lun :1.cheng gong zai ti wai fen li 、pei yang ren pi fu hei se su (gan )xi bao ;2.cheng gong zhi bei guo biao da PBX1ren pi fu hei se su (gan )xi bao ;3.guo biao da PBX1tong guo shang diao lin suan hua ERK、ying xiang xi bao zhou ji xiang guan dan bai cu jin ren pi fu hei se su (gan )xi bao zeng shi 。
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论文作者分别是来自吉林大学的周慧，发表于刊物吉林大学2019-06-25论文，是一篇关于人皮肤黑色素细胞论文,人皮肤黑色素干细胞论文,细胞增殖论文,吉林大学2019-06-25论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自吉林大学2019-06-25论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。