Application of controllable gene expression system in hair growth

基因轉移到毛囊是一種有潛力的技術來治療皮膚疾病。因為皮膚有非常低的滲透性，所以在活體的皮膚毛囊細胞上，非病毒基因轉殖才被認為是一種困難的方法。在此，我們利用聚乙烯亞胺 (PEI)來包覆人類的端粒酶逆轉錄酶基因 (hTERT)，然後利用塗抹在皮膚表面的方式，可以成功的將hTERT這個基因送到正常大鼠皮膚組織和受傷過的皮膚組織的毛囊細胞，讓這些細胞表現hTERT這個基因。我們發現，在正常皮膚組織的毛囊中，hTERT的表達會使毛囊幹細胞增生，進而加速毛髮的生長。另一方面，在受傷的皮膚組織的毛囊中，hTERT的表達則是會造成毛囊的再生。本研究利用創新的基因轉移的方法，並提供了一種實際的解決方法治療皮膚疾病。Gene transfer to hair follicles is a potential technology to treat skin disease. Non-viral transfection is considered a difficult method to deliver genes into hair follicles in vivo because of the extremely low permeability of skin. We utilized polyethylenimine (PEI) to transfect the human telomerase reverse transcriptase (hTERT) in hair follicle cells in normal and wounding rat skin tissues. We found that expression of hTERT in normal skin tissue could increase the number of proliferating stem cells in hair follicles. On the other hand, expression of hTERT in wounding skin tissue can promote hair follicle de novo formation. These studies might not only reveal an innovative method for gene transfer, but also provide a practical solution for skin disease therapy.目 錄試委員會審定書………………………………………………………………… i謝………………………………………………………………………………….. ii文摘要……………………………………………………………………….. iii文摘要…………………………………………………………………………….. iv. Introduction：…………………………………………………………………. 1~2. Material and method：…………………………………………..…………. 3~13 .1 Antibodies and Chemicals. ………………………………………………. 3~4.2 Magnetic Labeling and Separation of Cell………………………………. 4?5.3 Flow cytometry…………………………………………………………... 5~6.4 Cell culture of hair follicle stem cell…………………………………… 6~8.5 Immunofluorescent staining ………………………...………………… 8~9.6 Colony formation…………………………………………………………... 9.7 Transfection in vitro……………………………………………………… 9~10.8 Cytotoxicity - MTT assay………………………………………………. 10~11.9 Histochemical assay of ß-galactosidase activity…………………………… 11.10 In vivo mouse hair follicle transfection………………………………………. 11.11 Immunohistochemistry staining and H&E staining……………………… 12.12 Counting of hair length……………………………………………………12~13.13 Whole-mount hair follicle neogenesis assay. ………………………… 13. Result……………………………………………………………………… 13~22 .1 Purification of hair follicle stem cells…………………………………… 13~15.2 In vitro transfection efficiency and cytotoxicity of PEI…………………..15~16.3 The function of hTERT in vitro in rat keratinocyte……………………… 17~18.4 In vivo transfection efficiency of PEI………………………… 18~19 .5 The function of hTERT in vivo in rat skin tissue…………….…………...… 19~21.6 hTERT promote de novo follicle formation. ………...…………21~22. Discussions……………………………………………………………… 22~25.1 PEI serves as a promising vector for gene therapy in skin disease……… 22~23.2 TERT serve as a hair growth enhancer…………………………………..23~24.3 TERT and Wnt-signaling pathway………………………………24~25. Conclusion …………………………………………………………………25. Reference ……………………………………………………………… 26~27錄……………………………………………………………………….….…. 28~42 目錄igure 1. Hair follicle stem cells were sorted by MACS………….………28igure 2. The expression of integrin α6…………………………...…………………29igure 3. The expression of integrin ß1……………………………………………30igure 4. The expression of CD34…………………………………………………38igure 5. Clonal growth assays of keratinocytes…………………………………….39igure 6. EGFP reported gene expression in hair follicle stem cells…………….…40igure 7. hTERT reported gene expression in hair follicle stem cells………....41~42igure 8. Cytotoxicity test and transfection efficiency in hair follicle stem cells.…43.igure 9. In vivo transfection of rat hair follicles…………………………………….44igure 10. H&E stain and length of hair after in vivo transfection………………….45igure 11 Immunofluorescent staining for PCNA………………………………….46igure 12. Expression of hTERT potentiates de novo follicle formation at day10......47igure 13. Expression of hTERT potentiates de novo follicle formation at day 40…………………………………………………………………………………48~4