| 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.
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