The study of improvement of hair follicle stem cells of rats in vitro culture identification and differentiation

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. Key words: Stem cells; Hair follicle; Cell differentiation; Flow cytometry; Immunofluorescence staining; Rats