| Alopecia is a common disease in clinic which has few effective treatments. With the elevation of people's living standard, everyone wants to attain beautiful and healthy hair. To produce new hairs, existing follicles undergo cycles of growth (anagen), regression (catagen) and rest (telogen). Androgenetic alopecia is an extremely common disorder affecting both men and women. Patients have a reduction in the terminal-to-vellus hair ratio. Following miniaturization of the follicles, fibrous tracts remain. A variety of genetic and environmental factors likely play a role in causing androgenic alopecia. Researchers have determined that this form of hair loss is related to hormones called androgens. It will be an effective treatment if we could topically reduce the expression of androgen receptor of hair follicle.In this study, the murine hair cycle was induced by depilation. The protein of murine skin were separated by two-dimensional gel electrophoresis and identified by mass spectrometry which is verified by western blot and immunohistochemistry. Further more, several molecules of cell signal transduction in hair cycle werer defined by western blot and the expression level of micro RNAs were detected using micro RNA array. RNA inference expression vectors targeting to androgen receptor were constructed and transfected into human dermal papilla cell (DPC), human hair follicle and murine skin by lipofectin. The main results are as following:1. Comparative proteome profile of murine skin during hair cycleThe hair cycle of murine was induced by depilation. To better understand the molecular mechanisms involved in skin and hair development, 2-D PAGE and MALDI-TOF/TOF technologies were used to construct a comparative proteome profile of the murine skin at specific time points (days 0, 8, 20). A total of 45 differential protein spots such as myosin light chain, Tropomyosin 1, Annexin A, Lamin A/C, elongation factor 1, cytochrome b and Glyceraldehyde phosphate dehydrogenase were definitively identified between any two time points. Further cluster analysis showed four expression patterns, and each pattern correlated with specific cell processes that occur during hair cycle. Ten proteins were randomly selected to verify expression patterns using western blotting, and subsequently immunohistochemistry was performed to further investigate their localization.2. Molecules of cell signal transduction in hair cycle and micro RNA array Cyclin A, Cyclin B, Cyclin D, Cyclin E related to cell cycle, P53, P21, P38, BCL2, Bax, Rb related to apoptosis, and TGF-β1 related to transcription were detected by western blot. Micro RNA array was carried out and miR-690,miR-1308,miR-291a-5p,miR-212,miR-31 were found differentially expressed during hair cycle.3. Basic research of inhibition of the expression of androgen receptor by RNA interference and gene therapy of androgenic alopeciaThe eukaryotic expression vectors RNA interference targeting to AR were constructed and were transfected into human DPCs, human follicles and murine skin. The results of western blot showed that the vectors decreased the level of AR expression in DPCs(P<0.05)The results of MTT assay, 3H-TdR incorporation and flow cytometry suggested that blocking the AR with the RNAi expression vector could neither improve the proliferation of DPC, nor affect on their apoptosis. They could not influence the growth of hair follicle in vitro and they could be transfected into murine skin successfully.In conclusion, we obtained the proteome profile and the micro RNA profile in murine hair cycle. Several differentially expressed proteins and miRNAs were found and some molecules related to cell signal transduction were detected. The eukaryotic expression vectors of RNA interference targeting to AR were constructed and were transfected into human DPCs, human follicles and murine skin successfully. It might be significant for the gene therapy of androgenic alopecia. |
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