The Effect Of G16Gene Silencing To Human Dermal Papilla Cells

Objective: Gene Silencing is one of the most important methods foreukaryotes to change its gene expression. We called the gene silencingafter the transcription RNA interference, RNAi, that is double-strandedRNA，dsRNA in or out induced the homology mRNA degrade specificity.Dermal papilla cells (DPCs) as a cluster of specialized fibroblasts are themain mesenchymal cells which anchored at the bottom of the hair folliclecomposing dermal papilla. The salient biological characteristics of DPCsare able to induce hair follicle formation in vivo and in vitro. The growthpattern of DPCs is aggregation property significantly, but there is studiesshowed that aggregation property of DPCs gradually disappeared whenthe DPCs passage to7、8、9generations,then the DPCs lost its ability tocontrol the growth of hair follicles. Then some hair diseases such asalopecia areata occur. Gene P16as cell cycle regulatory factor arrests thecell cycle from G1to S, through preventing the role of CDK4.Somearticles suggest that the protein P16is higher in high generation DPCsthan the low generations, and the P16gene is over-expression in agingfibroblasts. We also find that the aggregation property disappeared in highgeneration DPCs which cell cycle is longer and its expression protein P16is higher than the low generations. So we can imagine that if we make theP16gene silence, can we slow down the DPCs aging or recover itsaggregation property. So the DPCs can able to control the growth of hairfollicles again. Can we provide new treatment for hair disease especiallyfor alopecia areata?Methods:1Divide the cells we get into groups. Divide the cells into threegroups that gene silencing group, negative control group and normal group at random.2RNAi in DPCs. After cell recovering, we cultured the cells inDMEM medium supplemented with15%heat inactivated fetal bovineserum and in a humidified incubator at37℃,95%air,5%CO2,selectingthe aiming cells, joining supplement without serum but concludingcalculated virus particles when the cell fusion rate is60％-70％.4hourslater, change the supplement to common one as usual, then cells was putinto thermostat to culter48hours. Place the cells under the fluorescencemicroscope and view its transfection rate.3Using inverted phase contrast microscope and electron microscopy(sem) observe the different of different groups.4MTT measure absorbance of different groups changing over time.At the same time, we check the influence of the supplement, the virusparticles without double-stranded RNA and so on to absorbance.5PCR detection the amount of mRNA of P16gene in differentgroups. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as theinternal control.6Immunofluorescence and flow cytometry instrument measure theexpression of protein P16in veriety groups.7Flow cytometry instrument detect the cell cycle an cell apoptosisrate in different groups.8Statistic analyses: Data was analyzed by spss13.0for windows.Results:1Cell transfection rate is about70%-80%and the result can be usedin the experimental(Fig.2-2).2Under the inverted phase contrast microscope and electronmicroscopy (sem),we can find more nuclear fission and particles in thegroup P16gene silencing and negative control group(Fig.2-3,Fig.2-4).3The result of MTT tells us the change in absorbance from the firstday to the fifth day is0.196±0.030、0.266±0.048、0、325±0.027、0.369±0.045、0.465±0.057in gene silencing group;0.164±0.039、0.283± 0.063、0.314±0.064、0.406±0.075、0.437±0.0700.164、0.283、0.314、0.406、0.437in negative control group；0.206±0.047、0.253±0.033、0.359±0.081、0.519±0.079、0.536±0.052in normal group(asFig.2-5,Table2-3).4The result of common PCR shows the brightness of gene silencinggroup is less than the others(as Fig.2-5).The Q-PCR shows that the RQ ofmRNA of gene P16is0.04±0.007in gene silencing group;0.66±0.356innegative control group；and1in normal group（as Fig.2-8、Table2-2).5The result of immunofluorescence shows that the percentage ofgreen fluorescence in gene silencing group is about5%,50%in negativecontrol group and60%in normal group(as Fig.2-9).6Flow cytometry instrument suggests that the FI which instead ofexpression of protein P16is2.43±0.02,2.04±0.06in gene silencinggroup；2.25±0.03,1.91±0.07in negative control group;1.27±0.31,1.2±0.05in normal group（as Fig.2-10、Table2-4）.7Flow cytometry instrument measure the cell cycle and cellapoptosis rate of different groups.The result shows that the cell apoptosisrate is8.92±0.61in the group P16gene silencing; is4.35±0.05innegative control group and2.86±0.07in normal group;the percentage ofG1is57.8±4.38in the group P16gene silencing; is68±1.27in negativecontrol group and82.1±0.99in normal group and the percentage of S is45.1±1.27in the group P16gene silencing; is26.7±0.71in negativecontrol group and8.6±4.45in normal group(Fig.2-11、Table2-5).Conclusions:1P16gene may restrain the growth of cells through regulatingcell cycle.2P16gene has somethings with cell aging.3We can regulate the cell cycle by means of changing the expressionof P16gene and provide new treatment to the people who is alopeciaareate.