低温保存脂肪间充质干细胞的生物学特性评价

目的 探讨低温保存对小鼠脂肪间充质干细胞（ADMSCs)体外培养的生物学特性和分化潜能的影响。方法 分离、培养小鼠ADMSCs，取第3代细胞置于液氮深低温（-196℃）冻存 12个月后复苏。MTT 法测定ADMSCs的增殖活性；β-半乳糖苷酶(SA-β-Gal)染色检测衰老；免疫细胞化学方法检测细胞表面分子CD73、CD90和CD105；条件培养基诱导后，免疫荧光法检测心肌特异性肌钙蛋白（cTnT），茜素红、碱性磷酸酶和油红 O 染色检测成骨、成脂诱导分化潜能，Real time-PCR检测cTnT、Gata4、Ost和Runx2。未经低温保存的第3代ADMSCs为对照。 结果 低温保存的ADMSCs其增殖活性、衰老率与未冻存细胞比较差异无显著性（P>0.05）。诱导后ADMSCs的cTnT阳性表达、茜素红、碱性磷酸酶和油红O染色呈阳性反应,冻存组与对照组比较差异无显著性(P>0.05）。结论 低温保存对ADMSCs体外生长特性和成心肌、成脂肪细胞、成骨细胞的分化潜能差异无显著性。

Biological characteristics of adipose-derived mesenchymal stem cells after cryopreservation in vitro

Objective To explore the effect of cryopreservation on the biological and differentiation potential of mouse adipose-derived mesenchymal stem cells (ADMSCs)in vitro. Methods ADMSCs were isolated from mice fatty tissue and then cultured. The ADMSCs of third passage were cryopreserved in liquid nitrogen (-196℃) for 12 months as the experimental group. After recovery, a MTT method was applied to get ADMSCs growth curve. SA-β-Gal staining was used to detect ADMSCs senescence. Immunocytochemical method was used to detect the expression of cell surface molecules, such as CD73, CD90 and CD105. After induction, immunofluorescence was to applied to detect the expression of cardiac-specific troponin (cTnT). Alizarin red, alkaline phosphatase and oil red O stainings were used to detect the differentiation potential to osteogenic and adipogenic, respectively. Real time-PCR was used to detect the mRNA expression of cTnT, Gata4, Ost and Runx2. ADMSCs without cryopreservation were used as the control group. Results ADMSCs growth and senescence index were no significant difference between cryopreservation group and non-cryopreservation group (P>0.05). After induction, there was no significant difference in the expression of cTnT and the positive reaction of alizarin red, alkaline phosphatase and oil red O staining between two groups(P>0.05). Conclusion Cryopreservation has no significant effects on the biological characteristics of ADMSCs in vitro, including the basic biological properties and differentiation potential into cardiomyocytes, adipocytes and osteoblasts.