Abstract:Objective To improve the method to isolate, culture, proliferate and purify the Sprague-Dawley rat hair follicle stem cells (rHFSCs) and the identification of immunization, ultrastructure in vitro, to research their biological characteristics, and to explore their adipogenic and osteogenic differentiation potentials. Methods From January to June 2015, six cleaning stage Sprague-Dawley rats(1 week-old) was selected , and the cirri skin of one-week-old Sprague-Dawley rat was cut in aseptic condition and digested by using intermixture of Dispase and type Ⅳ collagenase enzyme, the bulge part of hair follicle was isolated under the microscope, the rHFSCs were cultured by gradient plus medium method and tissue adherence method, passaged by two-step enzyme digestion method and purified by collagen Ⅳ anchorage velocity-dependent separation method. Finally, the 3rd generation rHFSCs was collected, identified by combined flow cytometry instrument detection with cell immunofluorescence staining, and observed the internal structure by transmission electron microscope. The first to 10th generation rHFSCs were collected to test their viability, and test the proliferation of the 3rd, 5th, 7th, 9th generation rHFSCs from first day to eighth day, the 3rd generation rHFSCs were collected, which were divided to induced group and control group according to different medium, then target gene, which were the relative expression of PPAR-γ, C/EBPa, OPG, Runx2 of two groups. Finally , OD value after dyeing was compared to explore their adipogenic and osteogenic differentiation potentials. Results The rHFSCs which were isolated, cultured and purified by improved methods above, showed typical “pavement”-like, growth curve was “S” type, had strong capacity of growth proliferation and forming clones. Transmission electron microscopy (sem) showed that cells in the original condition. The detection of flow cytometry indicated integrin β1, integrin α6 and P63 were high expression, and CK15 was moderate expression. It subjectsed to the identification of the rHFSCs . Moreover, the results of immunofluorescence and transmission electron microscopy showed that the cells were rHFSCs. Seven days after adipogenesis induced, RFqPCR results showed that the relative expression of target gene (PPAR-γ, C/EBPa ) of induced group were significantly higher than that of control group, the differences were statistically significant[(5.598±0.168, 4.757±0.416) vs (1.119±0.344, 1.126±0.355), t=34.955, 20.266, all P values<0.01)]. And 14 day later, the cells with oil red staining showed positive, and the OD value was obvious differ significantly with control group (2.472±0.091 vs 0.817±0.003), t=114.641, P<0.01). Fourteen days after osteogenesis induced, RFqPCR results showed that the relative expression of target gene(OPG, Runx2 ) of induced group (1.921±0.275,3.892±0.265) were significantly higher than control group (1.085±0.288,1.046±0.216), the differences were statistically significant(t=4.667, 17.332, all P values<0.01). And 21 day later, the cells with alizarin red staining showed positive, and two groups of OD values had significant difference (0.716±0.016 vs 0.076±0.002, t=14.078, P<0.01). Conclusions After the improvement, we can successfully establish the system of isolate, culture, proliferate and purify rHFSCs in vitro, prove that it has a high purity, high proliferation efficiency, multi-directional differentiation potential and other characteristics.So good seeding cells for the stem cells in tissue engineering can be provided related development.
杜伟斌,全仁夫,郑宣,李强,曹国平,庄伟,邵荣学,杨迪生. 改良大鼠毛囊干细胞体外培养鉴定及诱导分化的研究[J]. 中华解剖与临床杂志, 2016, 21(6): 558-565.
Du Weibin, Quan Renfu, Zheng Xuan, Li Qiang, Cao Guoping, Zhuang Wei, Shao Rongxue, Yang Disheng. The study of improvement of hair follicle stem cells of rats in vitro culture identification and differentiation. Chinese Journal of Anatomy and Clinics, 2016, 21(6): 558-565.
Najafzadeh N, Nobakht M, Pourheydar B, et al. Rat hair follicle stem cells differentiate and promote recovery following spinal cord injury[J]. Neural Regen Res, 2013, 8(36): 3365-3372. DOI:10.3969/j.issn.1673-5374.2013.36.001.
[4]
Najafzadeh N, Esmaeilzade B, Dastan Imcheh M. Hair follicle stem cells: In vitro and in vivo neural differentiation[J]. World J Stem Cells, 2015, 7(5): 866-872. DOI:10.4252/wjsc.v7.i5.866.
[5]
Aoki H, Hara A, Motohashi T, et al. Keratinocyte stem cells but not melanocyte stem cells are the primary target for radiation-induced hair graying[J]. J Invest Dermatol, 2013 ,133(9): 2143-2151.DOI: 10.1038/jid.2013.155.
[6]
Xu ZC, Zhang Q, Li H. Human hair follicle stem cell differentiation into contractile smooth muscle cells is induced by transforming growth factor-β1 and platelet-derived growth factor BB[J]. Mol Med Rep, 2013, 8(6): 1715-1721. DOI:10.3892/mmr.2013.1707.
[7]
Xu ZC, Zhang Q, Li H. Differentiation of human hair follicle stem cells into endothelial cells induced by vascular endothelial and basic fibroblast growth factors[J]. Mol Med Rep, 2014, 9(1): 204-210. DOI:10.3892/mmr.2013.1796.
Kloepper JE, Tiede S, Brinckmann 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[J]. Exp Dermatol, 2008, 17(7): 592-609. DOI:10.1111/j.1600-0625.2008.00720.x.
[13]
Blanpain C, Lowry WE, Geoghegan A, et al. Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche[J]. Cell, 2004, 118(5): 635-648. DOI:10.1016/j.cell.2004.08.012.
[14]
Quan R, Zheng X, Xu S, et al. Gelatin-chondroitin-6-sulfate-hyaluronic acid scaffold seeded with vascular endothelial growth factor 165 modified hair follicle stem cells as a three-dimensional skin substitute[J]. Stem Cell Res Ther, 2014, 5(5): 118. DOI:10.1186/scrt508.
[15]
Jaks V, Barker N, Kasper M, et al. Lgr5 marks cycling, yet long-lived, hair follicle stem cells[J]. Nat Genet, 2008, 40(11): 1291-1299. DOI:10.1038/ng.239.
[16]
Jiang J, Miao Y, Xiao S, et al. DAPT in the control of human hair follicle stem cell proliferation and differentiation[J]. Postepy Dermatol Alergol, 2014, 31(4): 201-206. DOI:10.5114/pdia.2014.44002.
[17]
Wang Y, Liu ZY, Zhao Q, et al. Future application of hair follicle stem cells: capable in differentiation into sweat gland cells[J]. Chin Med J(Engl), 2013, 126(18): 3545-3552.
[18]
Yucel G, Van Arnam J, Means PC, et al. Partial proteasome inhibitors induce hair follicle growth by stabilizing β-catenin. [J]. Stem Cells, 2014, 32(1): 85-92. DOI:10.1002/stem.1525.
[19]
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]. J Cell Biochem, 2009, 107(5): 1016-1020. DOI:10.1002/jcb.22204.
Ohyama M, Terunuma A, Tock CL, et al. Characterization and isolation of stem cell-enriched human hair follicle bulge cells[J]. J Clin Invest, 2006, 116(1): 249-260. DOI:10.1172/JCI26043.
[22]
Hwang J, Mehrani T, Millar SE, et al. Dlx3 is a crucial regulator of hair follicle differentiation and cycling[J]. Development, 2008, 135(18): 3149-3159. DOI:10.1242/dev.022202.
[23]
Kai-Hong J, Jun X, Kai-Meng H, et al. P63 expression pattern during rat epidermis morphogenesis and the role of p63 as a marker for epidermal stem cells[J]. J Cutan Pathol, 2007, 34(2): 154-159. DOI:10.1111/j.1600-0560.2006.00585.x.
Polisetti N, Chaitanya VG, Babu PP, et al. Isolation, characterization and differentiation potential of rat bone marrow stromal cells[J]. Neurol India, 2010, 58(2): 201-208. DOI:10.4103/0028-3886.63789.
[26]
Hu DH, Zhang ZF, Zhang YG, et al. A potential skin substitute constructed with hEGF gene modified HaCaT cells for treatment of burn wounds in a rat model[J]. Burns, 2012, 38(5): 702-712. DOI:10.1016/j.burns.2011.12.014.