不同培养体系对鼠表皮干细胞增殖和分化的影响

Effects of different culture systems on proliferation and differentiation of epidermal stem cells in rats

目的探讨不同培养体系对表皮干细胞增殖、分化的影响，建立理想的调控表皮干细胞增殖、分化的培养体系。方法酶消化和Ⅳ型胶原快速黏附法获取鼠表皮干细胞。分别在普通培养皿培养、与几丁质膜生物支架材料共培养及以几丁质膜生物支架材料作为载体植入裸鼠体内培养等不同培养体系下观察表皮干细胞生长情况。普通培养和与几丁质膜生物支架材料共培养4周后，对比表皮干细胞克隆形成率的差异。免疫组织化学染色观察表皮干细胞以几丁质膜为载体植入裸鼠体内后4周表皮干细胞的增殖、分化情况。结果表皮干细胞在普通培养皿培养3d左右，细胞开始克隆增殖；12d左右融合成片；传代培养后增殖能力逐渐减低，融合成片时间逐渐延长，传代培养3～4代后细胞终末分化，失去增殖能力。几丁质膜生物支架材料培养表皮干细胞，2周后呈棋盘式集落生长，几丁质膜生物支架材料上有大量的表皮干细胞小集落，集落上有大量的增殖细胞附着生长，扫描电镜下见几丁质膜生物支架材料纤维直径约10μm，以纤维为主，上下两层呈纵横排列成十字孔，孔间有大量表皮干细胞集落。几丁质膜生物支架材料培养表皮干细胞4周后，其克隆形成率明显高于普通培养皿培养[（12．6±2．7）％比（5．7±1．1）％，P〈0．05]。表皮干细胞几丁质膜生物支架材料植入裸鼠体内培养4周后，细胞大量增殖形成巢状排列，在表皮干细胞巢周围，可见有类似皮肤附件结构。结论表皮干细胞在体外普通培养皿培养可增殖生长，但维持增殖时间较短；与几丁质膜生物支架材料共培养，可较长时间地维持表皮干细胞的增殖特性；植入体内后表皮干细胞大量增殖。

Objective To explore the effects of different culture systems on proliferation and differentiation of epidermal stem cells and to establish an optimal culture system which can regulate and control the proliferation and differentiation of epidermal stem cells. Methods The rat epidermal stem cells obtained by enzyme digestion and type IV collagen rapid adherence were cultured in different culture systems （culture on dish, co-culture with biological chitin scaffold material, and culture with chitin membrane as the carrier in nude mice）. The growth of epidermal stem cells was then observed. After 4 weeks, the colony- formation rates of epidermal stem cells were compared among two systems （co-culture with biological chitin scaffold material and culture on dish）. Immunohistochemistry was used to study the proliferation and differentiation of the epidermal stem cells with chitin membrane as a carrier in nude mice at 4 weeks after implantation. Results In routine culture dish, the epidermal stem cells began to proliferate and clone at around day 3 and fused into patches at around day 12. However, the proliferation gradually decreased and the time to fusing into patches gradually became longer after passage, until terminal differentiation and loss of proliferation after passages 3 to 4. In co-culture with biological chitin scaffold materials, the epidermal stem cells grew in a chessboard-like colony after 2 weeks, and a great number of colonies could be seen on the biological chitin scaffold materials, with plenty of proliferating cells adhering to the colonies. Under scanning electronic microscope （SEM）, the biological chitin scaffold materials were found to mainly consist of fibers （10 μm in diameter） arranged in two crisscrossing layers with massive colonies of epidermal stem cells between the X-shaped holes. The epidermal stem cells cultured with biological chitin scaffold materials had a significantly higher colony forming efficiency as compared with that in culture dish after 4 weeks [ （12.6 ± 2.7）% vs （5.7 ± 1.1）%, P〈0.05 ]. The epidermal stem cells implanted into the nude mice showed vigorous proliferation with formation of ＂epidermal nests＂ at 4 weeks after implantation. Structure of skin appendages could be seen around the nests of epidermal stem cells. Conclusions Epidermal stem cells can proliferate in the culture dish in vitro but remain proliferating only for a relatively short time. Co-culture with biological chitin scaffold materials may allow for longerlasting proliferation of the epidermal stem cells. Massive proliferation can be found in the epidermal stem cells implanted into nude mice.