The Expression Of P16Protein In Human Dermal Papilla Cells And The Influence Of P16for Growth Characteristics

Objective:The hair follicle is not only the bulk of pelage,but also a typicalregeneration system.and the dermal papilla cells(DPCs) play a significant rolein the hair follicle growth and cycle regulation. DPCs are a sort of specializedfibroblasts, and the most significant functional feature is inducing theformation of hair follicle, but the DPCs in vitro cultural do not have thefunction before the occurrence of aggregative growth. With the increasing ofthe passage number, the characteristics of DPCs' aggregative growth graduallyattenuated.P16gene is a variety of tumor suppressor genes which is ocalized in9p21,and the product is P16protein which belongs to the cyclin-dependentkinase inhibitor family, and can be played a inversus regulation role on the cellcycle, which make the cells appearing apoptosis when cell growth chalk acertain period,and containment cells' excessive proliferation, and block theformation of tumors. P16protein also participates in other programs such asaging, invasion, the formation of vascular and red blood cells and HPVinfection etc.Researches have shown that the gene P16was over expressed in agingfibroblasts, but as a specialized fibroblasts, the expression of P16protein inDPCs has not been reported. This essay was to study the expression of P16protein, revealing the aging of possible mechanisms of DPCs in vitro cultural,and provided treatment according for the future clinical pelage diseases.Method:1DPCs' isolation and cultivation. The DPCs which were obtained by two-stepenzymatic digestion were geneticed into DMEM medium (15%fetal bovine,1%double-antibody), and regularly cultivation in the incubator (5%CO2, 37℃).2DPCs' identification and passage. The DPCs were identified by α-smoothmuscle actin staining. The cells were passaged with isolated enzyme (0.25%)when it fusioned to be70%-80%, regularly cultivation.3The expression of P16protein in the4thand8thgeneration DPCs wasmeasured by immunohisto chemistry S-P method.4The expression of P16protein in the4thand8thgeneration DPCs wasmeasured by flow cytometry (FCM).5MTT assay was used to examine the effect of recombinant human P16withdifferent concentrations (5ng/ml~200ng/ml) after72h for the DPCs.6Application of statistical software SPSS13.0: the data were statisticallyhandled and analyzed, using one-way ANOVA analysis and significance ofdifference standard α=0.05.Results:1With the application of "two-step enzymatic digestion" and "differentialcentrifugation", human essay were successfully isolated and extracted morepure DPCs, adherent quickly, and showed the characteristics of aggregativegrowth, which was most significant during3th-5th,and completely disappearedafter7th-8th.2The DPCs displayed filamentous α-smooth muscle actin under the invertedmicroscope after α-SMA immunohisto chemical staining.3The expression of P16protein in the4thand8thgeneration DPCs weremeasured by immunohisto chemistry S-P method.The P16protein were veryweekly positive on cell nuclear in the4thgeneration and weekly positive(±) inthe the8thgeneration DPCs, and weekly positive(±) on endochylema in the4thand8thgeneration DPCs. In the control group, it were negative expression(-)both cell nuclear and endochylema.4The expression of protein P16in the4thand8thgeneration DPCs wasmeasured by FCM. The P16protein's content were respectively-49.32±4.20,-47.38±12.99in the4thand8thgeneration DPCs, and the negative controlgroup were-71.51±19.19. The data were statistically handled and analyzed, which had no significant difference during the groups (P>0.05).5MTT assay was used to examine the effect of exogenous protein P16for theDPCs. The DPCs of logarithmic5thgeneration which cultured with differentconcentrations of recombinant human P16after72h were observed morph-ological difference by light microscope, which had no obvious change.Theresults of MTT showed that P16had the effect of speeding up aging,whichhad statistical significance (P<0.05). The effect was dose-dependent withconcentration of5ng/ml-25ng/ml compared with negative control group, andwas weakened with the concentration' further increased.Conclusion:1The two-step enzymatic digestion could extracted relatively pure hair papillavolantly and effectually. With the increasing of the passage number, thecharacteristics of DPC's aggregative growth gradually attenuated.2In vitro cultural,P16protein content in human DPCs is extremely low.Withthe increasing of the cell generations,the content increased gradually, whichindicated that P16may be play an important role in the process of DPCs'aging and and indirectly confirmed that the DPCs were a specializedfibroblasts.3Exogenous P16protein had promoting effect in DPCs' aging, and it alsoindirectly validated that the P16maybe important in DPCs' aging.