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.	Böttcher-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,Blömer 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.	杨珂,姜自林,杨恬.大鼠毛囊干细胞的体外分离培养及增强型绿色荧光蛋白示踪.中国组织工程研究与临床康复,2008,12(8):1481-1484.
18.	王联群,蓝蔚,黄培信,等.Ⅳ型胶原快速黏附法体外分选人胎儿表皮干细胞.中国组织工程研究与临床康复,2008,12(43):8535-8538.
19.	赵蕊,吕学诜.人胚胎干细胞建系培养及体外诱导分化的研究进展.国际遗传学杂志,2010,33(1):12-15.
20.	韩冰,付小兵.间充质干细胞的研究进展与临床应用前景.中国修复重建外科杂志,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.	鲁彦玮,王志军.组织工程皮肤种子细胞和支架材料的研究现状.中国美容整形外科杂志,2011,22(6):373-376.
24.	杨平,尹烁,崔磊,等.脂肪干细胞向血管平滑肌细胞诱导的实验研究.中国修复重建外科杂志,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.	张群,杨光辉,丛笑倩,等.差速贴壁法分选人毛囊干细胞的研究.中华实验外科杂志,2006,23(6):755-757.
28.	谭挺,胡志奇,周洪军.显微分离培养与免疫磁珠法分离纯化人毛囊干细胞.中国修复重建外科杂志,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.	陈少华,李扬秋,杨力建,等.实时定量RT-PCR检测TCR V γ亚家族基因表达水平.细胞与分子免疫学杂志,2009,25(2):166-168.
34.	司徒镇强.细胞培养.西安:世界图书出版公司,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.	李光辉,纪华,陈正堂,等.Lentivirus介导RNAi抑制人胶质瘤干细胞STAT3基因生物学效应观察.中华神经外科疾病研究杂志,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.	李传勇,李晓强,姜坤,等.绿色荧光蛋白基因体外转染骨髓源性血管内皮祖细胞.中国组织工程研究与临床康复,2009,13(14):2641-2644.
39.	张向荣,刘德伍,郭光华,等.转基因骨髓间充质干细胞移植与组织工程皮肤的血管化.中国组织工程研究与临床康复,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. Böttcher-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,Blömer 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. 杨珂,姜自林,杨恬.大鼠毛囊干细胞的体外分离培养及增强型绿色荧光蛋白示踪.中国组织工程研究与临床康复,2008,12(8):1481-1484.
18. 王联群,蓝蔚,黄培信,等.Ⅳ型胶原快速黏附法体外分选人胎儿表皮干细胞.中国组织工程研究与临床康复,2008,12(43):8535-8538.
19. 赵蕊,吕学诜.人胚胎干细胞建系培养及体外诱导分化的研究进展.国际遗传学杂志,2010,33(1):12-15.
20. 韩冰,付小兵.间充质干细胞的研究进展与临床应用前景.中国修复重建外科杂志,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. 鲁彦玮,王志军.组织工程皮肤种子细胞和支架材料的研究现状.中国美容整形外科杂志,2011,22(6):373-376.
24. 杨平,尹烁,崔磊,等.脂肪干细胞向血管平滑肌细胞诱导的实验研究.中国修复重建外科杂志,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. 张群,杨光辉,丛笑倩,等.差速贴壁法分选人毛囊干细胞的研究.中华实验外科杂志,2006,23(6):755-757.
28. 谭挺,胡志奇,周洪军.显微分离培养与免疫磁珠法分离纯化人毛囊干细胞.中国修复重建外科杂志,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. 陈少华,李扬秋,杨力建,等.实时定量RT-PCR检测TCR V γ亚家族基因表达水平.细胞与分子免疫学杂志,2009,25(2):166-168.
34. 司徒镇强.细胞培养.西安:世界图书出版公司,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. 李光辉,纪华,陈正堂,等.Lentivirus介导RNAi抑制人胶质瘤干细胞STAT3基因生物学效应观察.中华神经外科疾病研究杂志,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. 李传勇,李晓强,姜坤,等.绿色荧光蛋白基因体外转染骨髓源性血管内皮祖细胞.中国组织工程研究与临床康复,2009,13(14):2641-2644.
39. 张向荣,刘德伍,郭光华,等.转基因骨髓间充质干细胞移植与组织工程皮肤的血管化.中国组织工程研究与临床康复,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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