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目的:建立体外分离培养、扩增、鉴定大鼠毛囊干细胞(rat hair follicle stem cells,rHFSCs)的方法,观察其生物学特性。方法:切取1周龄SD大鼠触须部皮肤,用Dispase酶和IV型胶原酶混合液消化,显微镜下分离毛囊隆突部,用组织块法培养rHFSCs,培养基为DMEM/F12基础培养基,加不同浓度的KSR血清替代物、青链霉素混合液、L-谷氨酰胺、非必需氨基酸、EGF、bFGF、羟基乙醇和氢化可的松。用IV型胶原差速贴壁法纯化rHFSCs,再通过细胞免疫荧光染色及Q-PCR检测相关基因来联合鉴定,分别取不同代rHFSCs检测细胞增殖能力和活力。结果:以上方法分离、培养、纯化的HFSCs呈典型的铺路石状,贴壁较牢,克隆形成能力强。免疫细胞化学鉴定可见角蛋白15(Keratin-15,Krt15)、整合素α6(Integrin-α6,Itgα6)和整合素β1(Integrin-β1,Itgβ1)抗体表达呈阳性。纯化后的细胞最初2~3d细胞处于生长的潜伏期,5~6d时细胞处于对数生长期;从第7代开始,随着传代次数的增加,细胞活力也明显下降。Q-PCR检测发现,经过全新的培养体系得到的干细胞活性大,拥有较强的增殖能力,几乎不含有表皮角质形成细胞。结论:改进的显微镜联合组织块法分离培养rHFSCs,经IV型胶原差速贴壁法筛选后,可得到高纯度的rHFSCs,细胞增殖能力强,可以为干细胞组织工程学构建人工毛囊、血管及皮肤等提供良好的种子细胞。
OBJECTIVE: To establish a method for isolating, culturing, amplifying and identifying rat hair follicle stem cells (rHFSCs) in vitro and observe their biological characteristics. Methods: The skin of the 1-week-old SD rats was cut out and digested with Dispase and collagenase IV. The hair folds of the hair follicle were separated under the microscope. The rHFSCs were cultured by tissue block method. The medium was DMEM / F12 basal medium, Different concentrations of KSR serum replacement, a mixture of penicillin, L-glutamine, non-essential amino acids, EGF, bFGF, hydroxyethanol and hydrocortisone were added. RHFSCs were purified by differential adhesion method of type IV collagen, and then identified by cell immunofluorescence staining and Q-PCR to detect related genes. Different generations of rHFSCs were used to detect cell proliferation and viability. Results: The isolated, cultured and purified HFSCs showed typical paving stone shape, more firmly adhered and strong clonogenic capacity. Immunocytochemistry showed that Keratin-15 (Krt15), integrin-α6 (Itgα6) and Integrin-β1 (Itgβ1) antibodies were positive. Cells in the first 2 to 3d days of incubation were in a latent period of growth, and in the period of 5 to 6 days, cells were in a logarithmic growth phase. From the 7th generation onwards, the viability of cells was also significantly decreased with the increase of passage times. Q-PCR detection found that after a new culture system obtained stem cell activity, has a strong proliferative capacity, almost no epidermal keratinocytes. Conclusion: The improved rHFSCs were isolated and cultured by microscopy combined with tissue block method. After being screened by differential adhesion method of type IV collagen, high purity rHFSCs can be obtained with good cell proliferation ability, which can be used to construct artificial hair follicles, blood vessels and skin for stem cell tissue engineering Etc. to provide good seed cells.