Active Ingredients Of Plastrum Testudinis Inhibited Fetal Rat Epidermal Stem Cells Apoptosis Induced By Ultra-Violet Radiation And Serum-Deprivation Culture

ObjectiveStem cells is one of the most attracting fields in the life sciences in the recent decades and Epidermal Stem Cells (ESCs) is the significant approach to deal with various kinds of skin problems. ESCs is one of stem cells, which has the characteristic that it is easy to separate, proliferate and culture in vitro, has widespreadly applying prospect in clinic. Ultra-Violet (UV) radiation and nutriment deficiency are the significant causes to the skin damage, so it is the most important problem that how to find an safe and effective method to inhibit ESCs apoptosis induced by Ultra-Violet radiation and nutriment deficiency. Based on Traditional Chinese Medicine theory that "the kidney being the origin of congenital constitution, controlling growth and reproduction", we hope to promote ESCs proliferation. and resist apoptosis with the Chinese Medicine PlastrumTestudinis (PT) which can invigorate the kidney.Traditional Chinese Medicine holds that PT has the efficacy of nourishing liver and kidney, so it is often used as an important Traditional Chinese Medicine to clinically treat bone diseases in China. Our previous studies showed that active ingredients of PT had a strong effect on enhancing proliferation and differentiation of Mesenchymal Stem Cells (MSCs) in vitro. What is more, PT was discovered to have a antiapoptosis effect to dopaminergic neuron of Parkinson rats. WhetherPT prompt proliferation of ESCs and against injury which cause ESCs to be apoptotic, together with its mechanism still be unknown. We hope to find a new way for clinical skin damage and exploring skin beauty products through our research. Methods(1) ESCs'cultivation and purification.We used back skin of fatal rats which come from the two-weeks pregnant rats' womb, cut into pieces with scissors, collected cells for cultivation, stained cells from passageⅠtoⅣwith ESCs'special marker keratin19 (K19) andβ1-intergrin, counted double stained cells using fluorescence, flow cytometry (FCM), tested cells, property and purity, and choosen the most purest cells passageⅢto study.(2) Active ingredients of PT inhibited ESCs' apoptosis caused by Ultra-Violet radiation.We divided cells into three groups in light of different radiation time,5 minutes,15 minutes or 30 minutes, based on certain injury dosage of 100mJ/cm2, kept on culturing for 10 hours,20 hours or 36 hours. After that we tested these groups, apoptotic percentage by FCM, and confirmed that ESCs appear to ultraviolet 15 minutes and then culture 20 hours make a stable apoptosis model.Divided cells into normal group, control group and drug groups. Drug groups included 2B group, S6 group (stearic acid ethyl ester), S8 group (tetradecanoic acid sterol ester), and S9 group((+)-4-Cholesten-3-one). No special treatment to the normal group and delt with the control group like the last paragraph. Differences between control group and drug groups are that drug groups added 2B, S6, S8, or S9 in the basic medium after lesion. Comparision of anti-apoptotic effects of different parts of the active ingredients of PT by flow cytometry testing ESCs' apoptotic rate. Immunohistochemical staining of p53, caspase3 and bcl-2 to test positive expression. Western blot method to detect protein expressions of socs7, nck, p53 and caspase3.(3) Active ingredients of PT inhibited ESCs' apoptosis caused by serum-deprivated culture.Planted cells into 96-well-plates. Disposed of the medium of control and drug groups' wells, and washed the wells with D-Hank's after 24h cultivation except the normal group. Added basic medium to the control group, and extra active ingredient of PT to the drug groups, cultured 24h, 48h, and 72h. tested these groups' optical density (OD) values by MTT method to make sure the best time of drug effect. Tested anti-apoptotic effect of the active ingredients of PT by flow cytometry. Western blot method to detect protein expressions of bcl-2 and caspase3.Results(1) ESCs'cultivation and purification.The back skin of fetal rats contain aboundance of ESCs. Results of an examination tested by FCM revealed that the positive rate of ESCs double stained with K19 andβ1-intergrin had a peak value in passage III.(2) Active ingredients of PT inhibited ESCs' apoptosis caused by Ultra-Violet radiation.The normal rate, apoptosis rate and mortality rate of ESCs were more rational under condition that 20h cultivation after 15min radiation damage dosage of 100mJ/c m2,and applicable to be an apoptotic model induced by Ultra-Violet radiation. Immunohistochemistry results showed groups of PTE had more positive cells stained with bcl-2 than the control group and opposite outcomes with caspase.3 and p53. Results of western blot indicated that groups S8 and S9 had a obvious decrease in expression of socs7, nck, p53, and caspase3.(3) Active ingredients of PT inhibited ESCs' apoptosis caused by serum-deprivated culture.As MTT results showed PTE acting on ESCs which cultured in serum deprived medium had a peak value of OD in 48h; results of FCM revealed that drug groups had a better anti-apoptotic effect compared with control group in 48h; western blot outcomes indicated that all the drug groups up-regulated bcl-2 and down-regulated caspase3,2B had a outstanding performance.Conclusion(1) Back skin of fetal rat have aboundance of ESCs' and the passageⅢcells reach a biggest percentage of ESCs.(2) Active ingredients of PT inhibit ESCs' apoptosis caused by Ultra-Violet radiation, S8 and S9 have obviously anti-apoptotic effect.(3) Active ingredients of PT inhibited ESCs' apoptosis caused by serum-deprivated culture, and 2B has a notable activity.