EXPERIMENTAL STUDY ON VASCULAR ENDOTHELIAL GROWTH FACTOR 165 GENE-MODIFIED RAT HAIR FOLLICLE STEM CELLS MEDIATED BY LENTIVIRAL VECTOR_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHENGXuan , XUShichao ,  QUANRenfu

Department of Orthopedics, Zhejiang Xiaoshan Traditional Chinese Medicine Hospital, Hangzhou Zhejiang, 310009, P. R. China;

Corresponding?author：

QUANRenfu, Email: quanrenfu@126.com

Keywords：

Skin tissue engineering; Hair follicle stem cells; Vascular endothelial growth factor 165; Lentivirus; Gene modification; Rat

DOI：

10.7507/1002-1892.20140035

Video：

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Objective To obtain rat hair follicle stem cells (rHFSCs) which can constantly and highly express vascular endothelial growth factor 165 (VEGF₁₆₅), and to observe the expression of VEGF₁₆₅ gene in rat HFSCs. Methods The cirri skin of 1-week-old Sprague Dawley rat was harvested and digested by using combination of Dispase and type IV collagenases. The bulge was isolated under microscope. The rHFSCs were cultured by tissue block method. After purified by rapid adhering on collagen type IV, the growth curve of different generations rHFSCs was drawn. The cells were identified by immunofluorescence staining and real time quantitative PCR (RT-qPCR) analysis that tested the expression level of correlated genes. Lentivirus of pLV-internal ribosome entry site (IRES)-VEGF₁₆₅-enhanced green fluorescent protein (EGFP) (experimental group) and pLV-IRES-EGFP empty vector (control group) was packaged by calcium transfected method and the rHFSCs were transfected. The green fluorescent protein expression was observed by inverted fluorescence microscope, and VEGF₁₆₅ mRNA and protein expressions were detected using RT-PCR and Western blot. Results The rHFSCs which were isolated, cultured, and purified were like the "slabstone", and had strong adhesion ability and colony formation ability. The purified cells were in latent growth phase at 2-3 days; they were in exponential growth phase at 5-6 days. The expressions of cytokeration 15 (CK15), integrin α₆, and integrin β₁ (markers of HFSCs) were positive by immunocytochemistry. The RT-qPCR analysis showed that CK15, CK19, integrin α₆, and integrin β₁ expressed highly, but CD34 (a marker of epidermal stem cells) and CK10 (a marker of keratinocyte) expressed lowly. After 14 days, the transfection efficiency was up to 85.76%±1.91%. RT-PCR analysis and Western blot showed that VEGF₁₆₅ mRNA and protein expressions were positive in experimental group, and were negative in control group. Conclusion The rHFSCs with high purity and strong proliferation ability can be obtained by using microscope combined with tissue cultivation and rapid cell adhesion on collagen type IV. The rHFSCs with high expression of VEGF₁₆₅ can be successfully obtained by lentiviral transfection. This method provides good seeding cells for tissue engineering to construct artificial hair follicles, blood vessels, and skins.

Citation： ZHENGXuan, XUShichao, QUANRenfu. EXPERIMENTAL STUDY ON VASCULAR ENDOTHELIAL GROWTH FACTOR 165 GENE-MODIFIED RAT HAIR FOLLICLE STEM CELLS MEDIATED BY LENTIVIRAL VECTOR. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(2): 155-161. doi: 10.7507/1002-1892.20140035 Copy

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2.	B?ttcher-Haberzeth S,Biedermann T,Reichmann E.Tissue engineering of skin.Burns,2010,36(4):450-460.
3.	Wang X,Li Q,Hu X,et al.Fabrication and characterization of poly (L-lactide-co-glycolide) knitted mesh-reinforced collagen-chitosan hybrid scaffolds for dermal tissue engineering.J Mech Behav Biomed Mater,2012,8:204-215.
4.	Novosel EC,Kleinhans C,Kluger PJ.Vascularization is the key challenge in tissue engineering.Adv Drug Deliv Rev,2011,63(4-5):300-311.
5.	Cassell OC,Hofer SO,Morrison WA,et al.Vascularisation of tissue-engineered grafts the regulation of angiogenesis in reconstructive surgery and in disease states.Br J Plast Surg,2002,55(8):603-610.
6.	Cotsarelis G,Sun TT,Lavker RM.Label-retaining cells reside in the bulge area of pilosebaceous unit:Implications for follicular stem cells,hair cycle,and skin carcinogenesis.Cell,1990,61(7):1329-1337.
7.	Cotsarelis G.Epithelial stem cells:a folliculocentric view.J Invest Dermatol,2006,126(7):1459-1468.
8.	Rochat A,Kobayashi K,Barrandon Y.Location of stem cells of human hair follicles by clonal analysis.Cell,1994,76(6):1063-1073.
9.	Taylor G,Lehrer MS,Jensen PJ,et al.Involvement of follicular stem cells in forming not only the follicle but also the epidermis.Cell,2000,102(4):451-461.
10.	Yu H,Fang D,Kumar SM,et al.Isolation of a novel population of multipotent adult stem cells from human hair follicles.Am J Pathol,2006,168(6):1879-1888.
11.	Liu JY,Peng HF,Andreadis ST.Contractile smooth muscle cells derived from hair-follicle stem cells.Cardiovasc Res,2008,79(1):24-33.
12.	Neufeld G,Cohen T,Gengrinovitch S,et al.Vascular endothelial growth factor (VEGF) and its receptors.FASEB J,1999,13(1):9-22.
13.	Kafri T,Bl?mer U,Peterson DA,et al.Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors.Nat Genet,1997,17(3):314-317.
14.	Yamane J,Nakamura M,Iwanami A,et al.Transplantation of galectin-1-expressing human neural stem cells into the injured spinal cord of adult common marmosets.J Neurosci Res,2010,88(7):1394-1405.
15.	Amoh Y,Kanoh M,Niiyama S,et al.Human hair follicle pluripotent stem (hfPS) cells promote regeneration of peripheral-nerve injury:An advantageous alternative to ES and iPS cells.J Cell Biochem,2009,107(5):1016-1020.
16.	Nowak JA,Fuchs E.Isolation and culture of epithelial stem cells.Methods Mol Biol,2009,482:215-232.
17.	楊珂,姜自林,楊恬.大鼠毛囊干細胞的體外分離培養及增強型綠色熒光蛋白示蹤.中國組織工程研究與臨床康復,2008,12(8):1481-1484.
18.	王聯群,藍蔚,黃培信,等.Ⅳ型膠原快速黏附法體外分選人胎兒表皮干細胞.中國組織工程研究與臨床康復,2008,12(43):8535-8538.
19.	趙蕊,呂學詵.人胚胎干細胞建系培養及體外誘導分化的研究進展.國際遺傳學雜志,2010,33(1):12-15.
20.	韓冰,付小兵.間充質干細胞的研究進展與臨床應用前景.中國修復重建外科雜志,2006,20(12):1257-1261.
21.	Cao L,Yang F,Liu G,et al.The promotion of cartilage defect repair using adenovirus mediated Sox9 gene transfer of rabbit bone marrow mesenchymal stem cells.Biomaterials,2011,32(16):3910-3920.
22.	Charruyer A,Ghadially R.What's new in dermatology:epid ermal stem cells.G Ital Dermatol Venereol,2011,146(1):57-67.
23.	魯彥瑋,王志軍.組織工程皮膚種子細胞和支架材料的研究現狀.中國美容整形外科雜志,2011,22(6):373-376.
24.	楊平,尹爍,崔磊,等.脂肪干細胞向血管平滑肌細胞誘導的實驗研究.中國修復重建外科雜志,2008,22(4):481-486.
25.	Lavker RM,Sun TT.Epidermal stem cells:properties,markers and location.Proc Natl Acad Sci U S A,2000,97(25):13473-13475.
26.	Jones PH,Watt FM.Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.Cell,1993,73(4):713-724.
27.	張群,楊光輝,叢笑倩,等.差速貼壁法分選人毛囊干細胞的研究.中華實驗外科雜志,2006,23(6):755-757.
28.	譚挺,胡志奇,周洪軍.顯微分離培養與免疫磁珠法分離純化人毛囊干細胞.中國修復重建外科雜志,2008,22(2):202-205.
29.	Kloepper JE,Tiede S,Brinekmann J,et al.Immunophenotyping of the human bulge region:the quest to define useful in situ markers for human epithelial hair follicle stem cells and their niche.Exp Dermatol,2008,17(7):592-609.
30.	Ohyama M,Terunuma A,Tock CL,et al.Characterization and isolation of stem cell-enriched human hair follicle bulge cells.J Clin Invest,2006,116(1):249-260.
31.	Amoh Y,Li L,Katsuoka K,et al.Multipotent nestin-positive,keratin-negative hair-follicle bulge stem cells can form neurons.Proc Natl Acad Sci U S A,2005,102(15):5530-5534.
32.	Lavker RM,Sun TT.Heterogeneity in epidermal basal keratinocytes:morphological and functional correlations.Science,1982,215(4537):1239-1241.
33.	陳少華,李揚秋,楊力建,等.實時定量RT-PCR檢測TCR V γ亞家族基因表達水平.細胞與分子免疫學雜志,2009,25(2):166-168.
34.	司徒鎮強.細胞培養.西安:世界圖書出版公司,1996:115.
35.	Buchschacher GL Jr,Wong-Staal F.Development of lentiviral vectors for gene therapy for human Diseases.Blood,2000,95(8):2499-2504.
36.	李光輝,紀華,陳正堂,等.Lentivirus介導RNAi抑制人膠質瘤干細胞STAT3基因生物學效應觀察.中華神經外科疾病研究雜志,2008,7(6):498-501.
37.	Shima DT,Deutsch U,D'Amore PA.Hypoxic induction for vascular endothelial growth factor (VEGF) in human epithelial cells is mediated by increases in mRNA stability.FEBS Lett,1995,370(3):203-208.
38.	李傳勇,李曉強,姜坤,等.綠色熒光蛋白基因體外轉染骨髓源性血管內皮祖細胞.中國組織工程研究與臨床康復,2009,13(14):2641-2644.
39.	張向榮,劉德伍,郭光華,等.轉基因骨髓間充質干細胞移植與組織工程皮膚的血管化.中國組織工程研究與臨床康復,2009,13(15):2837-2840.

1. Groeber F,Holeiter M,Hampel M,et al.Skin tissue engineering in vivo and in vitro applications.Adv Drug Deliv Rev,2011,63(4-5):352-366.
2. B?ttcher-Haberzeth S,Biedermann T,Reichmann E.Tissue engineering of skin.Burns,2010,36(4):450-460.
3. Wang X,Li Q,Hu X,et al.Fabrication and characterization of poly (L-lactide-co-glycolide) knitted mesh-reinforced collagen-chitosan hybrid scaffolds for dermal tissue engineering.J Mech Behav Biomed Mater,2012,8:204-215.
4. Novosel EC,Kleinhans C,Kluger PJ.Vascularization is the key challenge in tissue engineering.Adv Drug Deliv Rev,2011,63(4-5):300-311.
5. Cassell OC,Hofer SO,Morrison WA,et al.Vascularisation of tissue-engineered grafts the regulation of angiogenesis in reconstructive surgery and in disease states.Br J Plast Surg,2002,55(8):603-610.
6. Cotsarelis G,Sun TT,Lavker RM.Label-retaining cells reside in the bulge area of pilosebaceous unit:Implications for follicular stem cells,hair cycle,and skin carcinogenesis.Cell,1990,61(7):1329-1337.
7. Cotsarelis G.Epithelial stem cells:a folliculocentric view.J Invest Dermatol,2006,126(7):1459-1468.
8. Rochat A,Kobayashi K,Barrandon Y.Location of stem cells of human hair follicles by clonal analysis.Cell,1994,76(6):1063-1073.
9. Taylor G,Lehrer MS,Jensen PJ,et al.Involvement of follicular stem cells in forming not only the follicle but also the epidermis.Cell,2000,102(4):451-461.
10. Yu H,Fang D,Kumar SM,et al.Isolation of a novel population of multipotent adult stem cells from human hair follicles.Am J Pathol,2006,168(6):1879-1888.
11. Liu JY,Peng HF,Andreadis ST.Contractile smooth muscle cells derived from hair-follicle stem cells.Cardiovasc Res,2008,79(1):24-33.
12. Neufeld G,Cohen T,Gengrinovitch S,et al.Vascular endothelial growth factor (VEGF) and its receptors.FASEB J,1999,13(1):9-22.
13. Kafri T,Bl?mer U,Peterson DA,et al.Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors.Nat Genet,1997,17(3):314-317.
14. Yamane J,Nakamura M,Iwanami A,et al.Transplantation of galectin-1-expressing human neural stem cells into the injured spinal cord of adult common marmosets.J Neurosci Res,2010,88(7):1394-1405.
15. Amoh Y,Kanoh M,Niiyama S,et al.Human hair follicle pluripotent stem (hfPS) cells promote regeneration of peripheral-nerve injury:An advantageous alternative to ES and iPS cells.J Cell Biochem,2009,107(5):1016-1020.
16. Nowak JA,Fuchs E.Isolation and culture of epithelial stem cells.Methods Mol Biol,2009,482:215-232.
17. 楊珂,姜自林,楊恬.大鼠毛囊干細胞的體外分離培養及增強型綠色熒光蛋白示蹤.中國組織工程研究與臨床康復,2008,12(8):1481-1484.
18. 王聯群,藍蔚,黃培信,等.Ⅳ型膠原快速黏附法體外分選人胎兒表皮干細胞.中國組織工程研究與臨床康復,2008,12(43):8535-8538.
19. 趙蕊,呂學詵.人胚胎干細胞建系培養及體外誘導分化的研究進展.國際遺傳學雜志,2010,33(1):12-15.
20. 韓冰,付小兵.間充質干細胞的研究進展與臨床應用前景.中國修復重建外科雜志,2006,20(12):1257-1261.
21. Cao L,Yang F,Liu G,et al.The promotion of cartilage defect repair using adenovirus mediated Sox9 gene transfer of rabbit bone marrow mesenchymal stem cells.Biomaterials,2011,32(16):3910-3920.
22. Charruyer A,Ghadially R.What's new in dermatology:epid ermal stem cells.G Ital Dermatol Venereol,2011,146(1):57-67.
23. 魯彥瑋,王志軍.組織工程皮膚種子細胞和支架材料的研究現狀.中國美容整形外科雜志,2011,22(6):373-376.
24. 楊平,尹爍,崔磊,等.脂肪干細胞向血管平滑肌細胞誘導的實驗研究.中國修復重建外科雜志,2008,22(4):481-486.
25. Lavker RM,Sun TT.Epidermal stem cells:properties,markers and location.Proc Natl Acad Sci U S A,2000,97(25):13473-13475.
26. Jones PH,Watt FM.Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.Cell,1993,73(4):713-724.
27. 張群,楊光輝,叢笑倩,等.差速貼壁法分選人毛囊干細胞的研究.中華實驗外科雜志,2006,23(6):755-757.
28. 譚挺,胡志奇,周洪軍.顯微分離培養與免疫磁珠法分離純化人毛囊干細胞.中國修復重建外科雜志,2008,22(2):202-205.
29. Kloepper JE,Tiede S,Brinekmann J,et al.Immunophenotyping of the human bulge region:the quest to define useful in situ markers for human epithelial hair follicle stem cells and their niche.Exp Dermatol,2008,17(7):592-609.
30. Ohyama M,Terunuma A,Tock CL,et al.Characterization and isolation of stem cell-enriched human hair follicle bulge cells.J Clin Invest,2006,116(1):249-260.
31. Amoh Y,Li L,Katsuoka K,et al.Multipotent nestin-positive,keratin-negative hair-follicle bulge stem cells can form neurons.Proc Natl Acad Sci U S A,2005,102(15):5530-5534.
32. Lavker RM,Sun TT.Heterogeneity in epidermal basal keratinocytes:morphological and functional correlations.Science,1982,215(4537):1239-1241.
33. 陳少華,李揚秋,楊力建,等.實時定量RT-PCR檢測TCR V γ亞家族基因表達水平.細胞與分子免疫學雜志,2009,25(2):166-168.
34. 司徒鎮強.細胞培養.西安:世界圖書出版公司,1996:115.
35. Buchschacher GL Jr,Wong-Staal F.Development of lentiviral vectors for gene therapy for human Diseases.Blood,2000,95(8):2499-2504.
36. 李光輝,紀華,陳正堂,等.Lentivirus介導RNAi抑制人膠質瘤干細胞STAT3基因生物學效應觀察.中華神經外科疾病研究雜志,2008,7(6):498-501.
37. Shima DT,Deutsch U,D'Amore PA.Hypoxic induction for vascular endothelial growth factor (VEGF) in human epithelial cells is mediated by increases in mRNA stability.FEBS Lett,1995,370(3):203-208.
38. 李傳勇,李曉強,姜坤,等.綠色熒光蛋白基因體外轉染骨髓源性血管內皮祖細胞.中國組織工程研究與臨床康復,2009,13(14):2641-2644.
39. 張向榮,劉德伍,郭光華,等.轉基因骨髓間充質干細胞移植與組織工程皮膚的血管化.中國組織工程研究與臨床康復,2009,13(15):2837-2840.

Chinese Journal of Reparative and Reconstructive Surgery

EXPERIMENTAL STUDY ON VASCULAR ENDOTHELIAL GROWTH FACTOR 165 GENE-MODIFIED RAT HAIR FOLLICLE STEM CELLS MEDIATED BY LENTIVIRAL VECTOR

Abstract Full text Figures/Tables Video References Cited by

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