【作者】 王秀亮；

【导师】 罗向东；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 硕士

【摘要】 研究背景表皮干细胞(keratinocyte stem cell, KSC)在皮肤生理功能中的作用得到广泛关注。表皮干细胞具有强大增殖能力,可向各个阶段表皮细胞分化以维持皮肤的正常功能。研究KSC调控增殖分化的分子机制,对于研究创面愈合的机制及KSC在组织工程中的应用有重要的意义。KSC高表达整合素β1,是KSC的标志物之一,在KSC增殖和分化中扮演重要角色。整合素β1在表皮基底层及毛囊隆突富含KSC的部位高表达,整合素β1的表达水平在KSC表面比已经分化的快速增殖细胞(transit amplifying cell, TAC)高数倍。KSC通常与基底膜粘附,脱离粘附后,开始走向终末分化[1,2]。整合素β1是被认为是维持KSC未分化状态及其增殖能力的粘附分子之一。Jones等发现表皮细胞表达整合素β1水平的高低,与其增殖能力有关[3];整合素β1小RNA干扰研究证明,下调表达整合素β1蛋白,KSC克隆形成率降低,并促进KSC的分化[4]。这些都提示整合素β1在KSC的增殖/分化调控中发挥重要作用。Piero C等人用MG-63细胞株(人骨肉瘤细胞株)研究证实:整合素β1表达是由近端和远端两个启动子区调控。它们缺少CAAT盒和TATA盒,高GC含量,可作为转录起始的两个独立启动子存在,促进整合素β1基因表达,产生两种mRNA—有相同的编码序列。近端和远端启动子在正常或刺激因素作用下都能促进转录,但具体参与转录水平调控的转录因子及其机制尚不清楚[5]。人表皮细胞株(Human adult skin keratinocytos ,HaCaT)来源于正常成人皮肤,是具有完整表皮分化能力的永生化细胞株。研究表明,移植到裸鼠上,HaCaT可以分化形成上皮组织,表达特异性分化蛋白标志[6];另外,整合素β1小干扰RNA载体转染HaCaT,能够抑制整合素β1蛋白表达,生长曲线右移,HaCaT的增殖受到抑制[7],由此说明HaCaT可以为研究人表皮细胞分化的调控提供一个非常理想的模型。我们已经构建了整合素β1启动子的全长启动序列(-1745bp～+11bp)载体pGL3-1755、近端启动子序列(-250bp～+11)载体pGL3-261、远端启动子序列(-1745bp～-304bp)载体pGL3-1442、远端启动子缺失片段载体(-905bp～-304bp)pGL3-602及(-655bp～-304bp)pGL3-352载体,在HaCat细胞中pGL3-602载体活性明显高于其它启动序列片段载体活性,而pGL3-352载体显著低于pGL3-602,分析在远端启动子-905bp～-655bp序列中有整合素β1的高活性启动序列。研究目的应用整合素β1启动子荧光素酶报告基因系统,以HaCaT细胞为模型,探讨表皮细胞中整合素β1启动子的转录启动活性区域,为进一步研究表皮干细胞增殖与分化的信号转导机制奠定基础。研究方法1.以本实验室前期构建的含整合素β1启动子序列的重组pGL3-602(-905bp～-304bp)载体为模板,用PCR分别扩增并克隆整合素β1启动子区-905bp～-304bp和-655bp～-304bp之间的连续缺失片段载体序列,分别构建荧光素酶报告基因载体pGL3-372(-675bp～-304bp)、pGL3-392(-695bp～-304bp)和pGL3-412(-715bp～-304bp), pGL3-432(-735bp～-304bp)、pGL3-452(-755bp～-304bp)、pGL3-492(-795bp～-304bp)和pGL3-542(-845bp～-304bp)载体,经双酶切、PCR反应及测序鉴定正确后转染HaCaT细胞,检测它们在HaCaT中的启动活性,分析各重组载体在HaCaT中的活性,初步确定整合素β1启动子中的高活性启动序列范围。2.根据第一部分研究结果,用Tfsitescan软件对该启动子的转录因子结合位点进行分析预测,分析远端启动子可能的高活性转录起始位点及可能参与转录调控的转录因子。研究结果1.成功构建了整合素β1启动子区-905bp～-304bp和-655bp～-304bp之间的连续缺失片段载体序列载体pGL3-372(-675bp～-304bp)、pGL3-392(-695bp～-304bp)和pGL3-412(-715bp～-304bp),pGL3-432(-735bp～-304bp)、pGL3-452(-755bp～-304bp)、pGL3-492(-795bp～-304bp)和pGL3-542(-845bp～-304bp)载体,经酶切鉴定及序列测定表明,其序列与GenBank DNA序列数据库对比分析序列一致,且插入方向正确;用此多种质粒转染HaCaT细胞都有启动子活性。2.整合素β1远端启动子连续缺失片段荧光素酶报告基因载体转染HaCaT细胞并检测荧光素酶活性,并进行统计分析表明,远端启动子系列重组质粒pGL3-352(-655bp～-304bp)、pGL3-372(-675bp～-304bp)、pGL3-392(-695bp～-304bp)、pGL3-412(-715bp～-304bp),pGL3-432(-735bp～-304bp)、pGL3-452(-755bp～-304bp)、pGL3-492(-795bp～-304bp)和pGL3-542(-845bp～-304bp)转染HaCaT细胞后,都有明显启动活性,pGL3-352(-655bp～-304bp)、pGL3-372(-675bp～-304bp)、pGL3-392(-695bp～-304bp)明显低于pGL3-412(-715bp～-304bp),pGL3-432(-735bp～-304bp)、pGL3-452(-755bp～-304bp)、pGL3-492(-795bp～-304bp)和pGL3-542(-845bp～-304bp)载体启动活性。研究结论通过构建远端启动子系列缺失片段荧光素酶报告基因载体并转染HaCaT细胞,检测荧光素酶活性(F/R),结果显示:1、pGL3-352(-655bp～-304bp)、pGL3-372(-675bp～-304bp)和pGL3-392(-695bp～-304bp)载体荧光素酶活性相接近;2、pGL3-412(-715bp～-304bp)、pGL3-432(-735bp～-304bp)、pGL3-452(-755bp～-304bp)、pGL3-492(-795bp～-304bp)和pGL3-542(-845bp～-304bp)载体荧光素酶活性相接近;3、pGL3-352(-655bp～-304bp)、pGL3-372(-675bp～-304bp)和pGL3-392(-695bp～-304bp)载体荧光素酶活性显著低于pGL3-412(-715bp～-304bp)、pGL3-432(-735bp～-304bp)、pGL3-452(-755bp～-304bp)、pGL3-492(-795bp～-304bp)和pGL3-542(-845bp～-304bp)载体;4、综上所述,在整合素β1启动子序列中,位于-686bp～-715bp之间的约30bp中可能有在HaCat细胞中让整合素β1高表达的转录因子结合起始位点。更多还原

【Abstract】 Background:At present, the keratinocyte stem cells(KSCs) obtains the widespread attention in theskin physiology function. Keratinocyte stem cells(KSCs) possess the powerfulproliferative capacity, and they also have the ability to differentiate into epidermal cells inevery stage. Investigation of regulative mechanism of KSC proliferation anddifferentiation is vital for wound healing and tissue engineering.Integrinβ1 is one of the generally accepted molecular markers for theundifferentiation of KSCs.Previous studies have indicated that integrinβ1 playedimportant roles in generation and differentiation of KSCs. High expression of Integrinβ1was found in basal body and folliculus pili of epidermal cell(EC), and in this part stemcell is rich. On surface of KSC integrinβ1 expression is several times more than that intransit amplifying cell(TAC). KSC maintain its characteristics of stem cell throughadhering to basal membrane(BM). If KSC cast off , it will turn to terminaldifferentiation[1,2]. Jones found that epidermal cells with high expression of integrinβ1have the capacity to form more clones[3]. In our previous study, we found that expressionof integrinβ1 was down-regulated after the transfection of siIntegrinβ1 recombinant vectorinto KSCs[4]. The clone formation rate of cells after transfection was lower than thatwithout transfection, which showed that integrinβ1 paticipated in the regulation of KSCproliferation and differentiation.Piero C et al[6] have shown that the regulatory region of theβ1-integrin genecontains two promoters, one distal and one proximal tandemly situated. The twotandemly promoter regions are very G + C-rich, and lack both a TATA box and a CAATbox .Each promoter drives the expression of a uniqueβ1 gene, and two mRNAs aresynthesized that share the same coding sequence but diverge in the complete 5′untranslated region. but the regulation mechanism in transcription level was still unclear. HaCaT(Human adult skin keratinocytos ,HaCaT) cell line originates from normalhuman epidermal cells, which have the capacity to proliferate infinitely. Research hasshowed that after injection of HaCaT into subcutaneous skin of the nude mice, it coμlddifferentiate into epithelial tissues, with expression of special markers for differentiation[7].In addition,our previous study indicated that expression of integrinβ1 protein wassupressed by the transfection of siIntegrinβ1 vector. The growth curve of HaCaT cellsafter transfection was moved right forward, indicating that the proliferation of HaCaTcells was inhibited by the transfection[8]. As described above, HaCaT cell line can providean ideal model for the research of regulation of human epidermal cell proliferation.Objective:To explore the mechanism of regulation of integrinβ1 expression. HaCaT cells andluciferase reporter gene system containing integrinβ1 promoter were enrolled in our studyto investigate the influence of integrinβ1 on the proliferation and differentiation of humanepidermal cells.Methods:Dual-luciferase reporter vectors containing distal part and proximal part of integrinβ1 promoter were constructed successfully. Then they were transfected into HaCaT cellsto investigate their activity after transfection. This can help us to understand the role ofintegrinβ1 in the transcriptive regulation of HaCaT cells.The main effect of promotor in transcriptional control was determined in accordingto the result in the first part. Tfsitescan software was used to analyze potential bindingsite of transcription factor in the promotor. Dissection and construction of mutationluciferase report gene vector with series deletion of promotor were carry out according toanalytic result. After sequencing, they were transfected into HaCaT cells, then the activityof luciferase was detected, and the activity of distal promoter of integrinβ1and potentialtranscriptional control factors participating possibly transcriptional control were analyzed.Results:We successfully amplified the whole length of integrinβ1 promoter from humangenome by PCR, and transfected the vector containing proximal ,distal and full part ofintegrinβ1 promoter into HaCaT cells. Analysis of luciferase activity revealed that thedistal part of promoter played an important role in the regulation of integrinβ1 transcripton.In addition, we successfully constructed series deletion mutation luciferase reportgene vector of promotor:pGL3-372,pGL3-392,pGL3-412,pGL3-432,pGL3-452,pGL3-492,p GL3-542, andtransfected the series vector into HaCaT cells. Analysis of luciferase activity revealed thatluciferase activity of pGL3-412,pGL3-432,pGL3-452,pGL3-492,p GL3-542 is higher thanpGL3-352 ,pGL3-372,pGL3-392.Conclusion:1. The luciferase activity of pGL3-352 ,pGL3-372,pGL3-392 is similar in HaCat.2. The luciferase activity of pGL3-412,pGL3-432,pGL3-452,pGL3-492,p GL3-542 issimilar in HaCat .3. The luciferase activity of pGL3-412,pGL3-432,pGL3-452,pGL3-492,p GL3-542 ishigher than pGL3-352 ,pGL3-372,pGL3-392 in HaCat.4. In the integrinβ1 promoter sequences, the transcription factor binding initiationsite possibly is in 30bp located at between -686bp~-715bp in HaCat cells.更多还原