The Role Of Integrinβ1 In Regulating Proliferation And Differentiation Of Keratinocyte Stem Cell In Vitro

Background: Human epidermis, a continuously renewing tissue, is maintained throughout life by keratinocyte stem cells(KSCs) in the basal layer. KSCs have pluripotentiality and plasticity, they could produce multiple cell lineages in the skin and could form irrelative tissue in the body. KSCs play a central role in homeostasis and wound repair and also represent a major target of tumour initiation and gene therapy. However, purifying KSCs is a challenging task in the experiment. Although integrin β₁ has been suggested to be one of the stem cell markers, in fact, lack of specific molecular markers was a major obstacle for stem cell research. Therefore, further studies should be carried out in isolating, cultivating, purifying and characterizing of KSCs in vitro.Quite a number of evidences suggest that integrin (31 is required to maintain KSCs in an undifferentiated state. When keratinocytes are detached from basal layer into suspension, terminal differentiation takes place. This phenomenon can be inhibited by ligand of integrin β₁. The suspension-induced differentiation signal is mediated by integrin β₁. When KSCs exit from the stem cell compartment, the level of integrin β₁ is decreased. However, whether down-regulation of integrin β₁ could induce KSCs differentiation remains to be elucidated .RNA interference(RNAi) has become a powerful and widely used tool for the analysis of gene function in mammalian cells recently. Introduction of 19-21 nt double-stranded RNA(dsRNA) known as short-interfering RNA (siRNA) into these cells leads to the sequence-specific destruction of endogenous RNAs that match the dsRNA. Vector-based delivery siRNA was rapidly developed within the last two years. In our study, a plasmid-based siRNA delivery strategy was used.Objective: To isolate and culture KSCs and investigate their proliferation and differentiation in vitro. To study the role of integrin β₁ in regulating proliferation anddifferentiation of keratinocyte stem cell in vitro.Methods:Primary human keratinocytes were isolated from young foreskins and placed onto plastic dishes which coated by type IV collagen(20ug/ml) for 10 minutes, then the adhesion cells were harvested and replated onto a mitomycin C-treated NIH3T3 cell as the feeder with clony density using the medium as previously described. After 14 days culture, the clones were isolated, cultured and passaged consecutively in the presence of a feeder-layer of 3T3 cells, child's or mouse embryonic fibroblasts. The colony forming efficiency(CFE) was determinated by stained with 1% rhodanile blue. Meanwhile the expression of integrin pi and involucrin were detected by immunofluorescence stain.Construction of recombinant siRNA expression plasmids: (D Choose appropriate 19bp sequence on the transcript of target gene integin pi and blast to eliminate any target sequences with significant homology to other coding sequences. The oligonucleotides were synthesized by Shanghai Bioasia corporation. ?Annealing of each pair of oligonucleotides ?Double digestion of vector by endonuciease @Ligation of annealed oligonucleotide inserts to linearized vector.?Transformation of DH5a.(§)Identification of recombinant plasmids by sequencing.Western blotting: Optimizing the transfection condition according to manufacture's instruction. Two or three days after transfection, cells were washed with PBS and collected by scraping. They were lysed in ice-cold RIPA buffer and centrifuged. The supernatant was used for protein concentration determination by BCA-200 method. The proteins were resolved on 10% SDS-polyacrylamide gels, transferred onto nitrocellulose membranes, and incubated with the appropriate antibodies. The peroxidase-based detection was performed with Chemiluminescence Reagent (Pierce) according to the manufacturer's instruction.RT-PCR: Total RNA was prepared from keratinocyte stem cells 48⁷2h after transfection using the Trizol reagent according to the manufacturer's instruction. RT-PCR amplification products were resolved by 1% agarose gel.cell cycle analysis: The keratinocyte stem cells were seeded in 6.0 cm dishes and tansfected with silntegrinpi vector by using Dotap reagent. The cells were trypsinized 48 72h after transfection and washed by PBS for three times.Results: Keratinocyte stem cells could be isolated and rapidly adhere to type IV collagen-coated plates. CFE of keratinocytes that adhere to type IV collagen was from15.9% to 18.7% . The purity of KSCs screening from holoclones could be as much as >95%.The isolated human KSCs grew on the mitomycin C-treated 3T3 or child's or mouse embryonic fibroblasts feeder. Three types of colony, namely, holoclone, paraclone and meroclone, were observed in our experiments, most of those colonies were holoclones with high amplification potential and maintained a high level of integrin (31 expression. Meraclone and paraclone was formed by transit amplifying cell and characterized by expression of involucrin partly or fully.Plasmid based siRNA expression could inhibit endogenous target genes. Several repeated experiments showed that silntegrinpi transfection could specially inhibit integrin pi mRNA and protein expression. Our data also indicated that HI promoter drived siRNA could inhibit integrin pi expression by 60-70%.Cell cycle analysis showed that 96.38% of KSCs were in G0/G1 stage and only 3.62% in S stage. With the knockdown of integrin pi in KSCs, the rate of G0/G1 stage was 74.45% and the proliferation index (PI) was 21.55% contrast to 93% and 5% separately(P<0.05). Further study showed that down-regulated integrin pi in KSCs, CFE of KSCs was sharply decreased compare to the control(P<0.01).These experiments indicated that integrin pi down-regulation in KSCs could lead to differentiation.Summary: The KSCs could be isolated successfully from keratinocytes by adhering to type IV collagen and selecting holoclone in vitro. The KSCs isolated by this method could be cultured, passaged consecutively.silntegrin P 1 transfection can specially inhibit integrin P 1 mRNA and protein expression by 60-70%.Down-regulation of integrin pi could lead to differentiation in KSCs.