A New Method Of Promoting DPCs Proliferation: P21Gene Transcriptional Silencing

Objective： The hair follicle， a unique characteristic organ ofmammals, represents a stem cell-rich, prototypicneuroectodermal-mesodermal interaction system which is a typicalregeneration system. The dermal papilla cells (DPCs) play a crucial role inhair follicle formation, development and periodic cycles. Thehair-inductivity is the major biological property of DPCs in vivo and vitro,which are located at the bottom of hair follicles. But the high-passagedDPCs are detected to lose the property in vitro, which is thought to berelated to the cell senescence. The cyclin-dependent kinase inhibitor(CDKI) containing p21is the one of the primary cellsenescence-associated genes. The P21, located in the downstream of p53,as a kind of tumor suppressor genes, can inhibit all kinds of the cyclin-dependent kinase. In recent years, the inhibitory effect of p21onproliferation, apoptosis and the progression of cell-cycle in varieties ofcells is became the research focus about CDKI. However, the effect of p21in human hair follicle DPCs has not been reported. In this study, therelationship between p21and the biological property of DPCs is analysedthrough the transcriptional silencing of the p21by adenovirus transfection.And it is detected a new method of promoting the proliferation in DPCs.Methods:1The experimental group:Normal group: The cells with conventional subculture.Negative control group: The cells with adenovirus HK-1p2shRNAtransfection.Positive control group: The cells with adenovirus CDKN1A-1p2shRNAtransfection. 2The culture of the DPCs and the adenovirus transfection.The three groups of DPCs were cultured in the MSCM medium, andplaced in the incubator. And the transfection efficiency of adenovirus wasdetected by the fluorescent microscope.3Comparison the morphological changes and biological functions ofthe three groups of DPCs.4The morphology of DPCs in three different groups was observedby the scanning electron microscopy.5The growth curves of the different generations DPCs and the DPCswith adenovirus in the same passage were determined by MTT.6The cell cycle and the apoptosis of DPCs were determined by flowcytometry.7The expression of p21protein in DPCs were detected by threemethods containing flow cytometry, immunohistochemistry andimmunofluorescence.8The expression of p21gene mRNA in DPCs were detected by thereverse transcription PCR and the real-time fluorescence quantificationPCR.9The experimental data was statistically analysed by the softwareSPSS16.0on the docimastic significance level of ’a=0.05’.Results:1The DPCS were successfully cultivated to the thirteenth generationin vitro, and the adenovirus transfection efficiency of the DPCs withHK-1p2shRNA and CDKN1A-1p2shRNA were about80%-90%.2The normal group: The early passaged DPCs were show obviousagglutinative growth characteristics which was disappearing began to theeighth generation. And the cell volume was biger and biger, themorphology was irregular, and the cytoplasmic granules were inceeasingwith the increasing passages of DPCs; It was not detected obviouslychange of the agglutinative growth characteristics and the cellmorphology in the same passage DPCs in the negative group.And it was not detected obviously change of the agglutinatice growth chatacteristicsin the same passage DPCs in the Positive control group, But the DPCs ofpositive control group were obvisered a bit biger cell volume and morecytoplasmic granules.3The karyolobism in the DPCs with the silencing of p21wereobserved to increase by the scanning electron microscope。4The growth curves of the different generations DPCs and the DPCswith adenovirus：The growth curve of the4th,7thand10th-passaged DPCsin the normal group was declined with the increasing passages, while thegrowth rate of DPCs with p21silencing was increased.5The results about cell cycle and apoptosis in the DPCs by Flowcytometry.The proportion of G1-phase was expressed by percentage, and theapoptosis ratio was expressed by%Gated. The results of flow cytometryshow that: in the4th-passaged,7th-passaged and10th-passaged DPCs inthe normal group, the percentages of G1-phase were39.7667%±6.7575%,53.9667%±3.0271%and61.4667%±0.6028%, the apoptosis ratios were:2.12±0.4232,5.6433±1.3503and10.54±2.1345; in the5th-passagedDPCs of the normal, negative and positive group, the percentages ofG1-phase were47.9333%±9.7285%,51.7%±7.494%and40.1%±6.3095,the apoptosis ratios were3.3767±0.8769,7.3567±0.4387and12.3233±1.769; in the8th-passaged DPCs of the normal, negative andpositive group, the percentages of G1-phase were55.5333%±1.9858%,58.86667%±4.6801%and44.9667%±7.9689%, the apoptosis ratios were9.4067±0.9463,13.6967±0.8838and17.9567±3.2875.6The results of p21protein by flow cytometry was showed that:The FI of p21protein expression in4th-passaged,7th-passaged and10th-passaged DPCs in normal group were2.0825±0.1923,2.4945±0.135and2.8298±0.071; the FI of p21protein expression in5th-passaged DPCs of the three groups were2.2546±0.0363,2.35±0.127and2.0278±0.1443; the FI of p21protein expression in8th-passaged DPCs of the three groups were2.5947±0.1184,2.683±0.1984and2.4019±0.1048.And the localizations of p21protein in different passages DPCs innormal and negative control groups were nucleus and cytoplasm, while thelocalizations of p21in positive DPCs were nucleus and perinuclear region.Finally the level of p21protein in10th-passaged DPCs of normalgroup was more than the one in the4thand7th-passaged DPCs of nomalgroup.7The expression of P21gene mRNA in the DPCs with p21silencingwere detected to obviously decline by reverse transcription PCR;The results of the real-time fluorescent quantitative PCR wereshowed that: the p21gene mRNAexpression in the4th,7thand10th-passaged DPCs in normal group were1,4.8787±0.8342and33.2018±2,536; the p21gene mRNAexpression in the5th–passagedDPCs in the normal,negative and positive groups were1,2.7617±0.9544and0.0609±0.0342; the p21gene mRNAexpression in the8th-passagedDPCs in the normal,negative and positive groups were1，9.3697±4.9706and0.0640±0.0305.Conclusions:1P21may be taken part in regulating the cell senescence of theDPCs.2P21can inhibit DPCs proliferation, which can lead DPCs togrowth arrest.3P21can lead DPCs to cell-cycle arrest, which can inhibit thetransition from G-phase to S-phase.4P21may inhibit the apoptosis of DPCs.5The silencing of P21gene may be acted as a new method ofpromotingDPCs proliferation.