不同冻存-解冻法处理小鼠皮肤组织对毛囊干细胞生长的影响

目的观察皮肤组织经不同冻存-解冻法处理后，分离的毛囊于细胞的生长状况，寻找较好的组织冻存方案，为解决无法短期收集足量标本而不能有效获取毛囊干细胞的问题提供实验方法参考。方法获取大鼠有两撮触须的上唇，经不同方法处理后，分离毛囊隆突部细胞。设立对照组：新鲜标本；实验组1：未加冻存保护剂-70cc冻存24h；实验组2：4℃保存24h；实验组3：1．4mol／L乙二醇作为冻存保护剂-70℃冻存24h；实验组4：1．4mol／LDMSO作为冻存保护剂-70℃冻存24h；实验组5：1．4mol／LDMSO作为冻存保护剂-70％冻存3个月；实验组6：0．7mol／LDMSO作为冻存保护剂-70℃冻存24h；冻存各组降温速率为0．5℃／min。其中3～6组分别采用单用培养基漂洗快速复苏法和蔗糖＋培养基漂洗快速复苏法做对比。比较经以上6种方法处理后分离的毛囊隆突部细胞的活力和贴壁、增殖情况。结果经冻存＋单用培养基漂洗快速复苏法，4℃处理组获得的平均细胞活力为90．38％±4．62％，明显高于其他处理组（P值均〈0．05），仅次于对照组94．00％±5．64％。1．4mol／LDMSO作为冻存保护剂无论冻存24h或3个月均可取得较高的细胞活力（56．00％±3．91％和47．25％±3．55％），明显高于其他处理组（P值均〈0．05）。组3-组6经单用培养基漂洗快速复苏法和蔗糖＋培养基漂洗快速复苏法复苏所得平均细胞活力分别是25．63％±2．32％和27．25％±2．56％、56．00％±3．51％和54．88％±4．03％、47．25％±3．01％和44．00％±2．98％、27．88％±2．20％和26．75％±2．26％，差异均不显著（P值均〉0．05）。各组毛囊干细胞生物学特性（贴壁、增殖等能力）与细胞活力呈相关趋势。结论皮肤组织在4℃条件短期内可保持原代培养细胞的活力；皮肤组织以1．4MDMSO为冻存剂-70℃条件下保存可较长时间保持细胞活力，较适合小鼠皮肤组织的保存，为毛囊干细胞的进一步研究提供技术支持。

Morphology and survival of cryopreserved-thawed ovarian tissues after heterotopic autotransplantation

Objective： The aim of our study was to observe the survival and morphological changes of thawed ovarian tis- sues after heterotopic transplantation. Methods： Twenty SPF-SD female rats （5-6 weeks old） were equally randomized into the control group and experimental group. In control group, the freshly isolated ovaries were fixed in formalin. In experimental group, the freshly isolated ovaries were vitrified immediately and cut into thin slices. After stored in liquid nitrogen for 21 days, the tissues of experimental group were rapidly thawed and transplanted into back muscles of rats for 2 or 4 weeks, respectively. After that, all rats in experimental group were sacrificed and the ovarian tissues were collected and fixed in 4% formaldehyde solution. Then the ovarian tissues were stained with HE and observed under the light confocal microscope. Re- suits： With the naked eyes, there was no specific alteration except the size reduction with color changing. Under microscopy, we found normal cortex and medulla in the ovary, and the primordial follicles and follicles in various stages were observed in the cortex. The normal oocytes in ovarian tissues of experimental group were significant decreased than in the control group. Conclusion： The ovarian tissues survive well in experimental group and there is no significant difference in the proportion of follicles between different times （2 and 4 weeks） after grafting. Our results suggest that thawed ovarian tissues could survive after heterotopic transplantation into back muscles of rat models and maintain their morphology and function.