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目的探讨小鼠脂肪源间充质干细胞(ADMSCs)体外原代培养过程中衰老和凋亡变化。方法用差速贴壁法对1月和24月龄小鼠(各30只)腹股沟脂肪组织的ADMSCs进行原代培养,免疫荧光技术检测第3代ADMSCs表面抗原CD73、CD90和CD105的表达,比较两组细胞的形态差异和生长情况,β-半乳糖苷酶(SA-β-Gal)染色检测ADMSCs衰老情况,PI-Hoechst33342双染色观察ADMSCs的凋亡变化。结果两组体外原代培养的ADMSCs均贴壁生长,具有典型干细胞的形态和生长特征,两组ADMSCs CD73、CD90和CD105的表型一致。SA-β-Gal染色显示:原代培养的老年组ADMSCs衰老比例比幼年组多,1月组原代培养2～7d的ADMSCs阳性率分别为(3.87±1.34)%、(4.21±1.22)%、(9.00±0.98)%、(11.20±1.24)%、(9.50±2.19)%和(13.20±2.93)%;24月组分别为(8.67±1.05)%、(10.23±0.98)%、(12.80±0.78)%、(17.10±1.13)%、(19.80±1.59)%和(24.70±1.86)%。PI-Hoechst 33342染液双染显示:1月组原代培养2～7d的ADMSCs凋亡率分别为(22.1±1.4)%、(36.7±1.62)%、(11.7±1.45)%、(38.4±1.57)%、(6.5±1.32)%和(11.3±1.63)%;24月组4～7d的ADMSCs凋亡率分别为(29.4±1.72)%、(37.6±1.64)%、(19.4±1.29)%和(18.9±1.25)%。结论老龄ADMSCs增殖能力下降,衰老、凋亡和坏死的细胞比例增加;随着ADMSCs原代培养时间的延长,其衰老增加,但凋亡和坏死无规律性变化。
Objective To investigate the changes of senescence and apoptosis during primary culture of mouse adipose-derived mesenchymal stem cells (ADMSCs) in vitro. Methods ADMSCs in inguinal adipose tissue from January to 24-month-old mice (30 in each) were primarily cultured by differential adhesion method. The expression of CD73, CD90 and CD105 on the 3rd generation ADMSCs was detected by immunofluorescence staining. The morphological differences and growth of the two groups of cells were observed. The aging of ADMSCs was detected by β-galactosidase (SA-β-Gal) staining. The apoptosis of ADMSCs was observed by PI-Hoechst33342 double staining. Results ADMSCs cultured in vitro were adherently adherent, with the typical morphological and growth characteristics of stem cells. The phenotypes of CD73, CD90 and CD105 of ADMSCs in both groups were consistent. The results of SA-β-Gal staining showed that the percentage of ADMSCs in the primary cultured group was higher than that in the young group (3.87 ± 1.34%, 4.21 ± 1.22%, respectively) , (9.00 ± 0.98)%, (11.20 ± 1.24)%, (9.50 ± 2.19)% and (13.20 ± 2.93)% in the 24th month group and (8.67 ± 1.05)%, ± 0.78%, 17.10 ± 1.13%, 19.80 ± 1.59% and 24.70 ± 1.86%, respectively. PI-Hoechst 33342 staining showed that the apoptotic rates of ADMSCs cultured in primary culture for 2 ~ 7 days were (22.1 ± 1.4)%, (36.7 ± 1.62)%, (11.7 ± 1.45)%, (38.4 ± 1.57)%, (6.5 ± 1.32)% and (11.3 ± 1.63)%, respectively. The apoptotic rates of ADMSCs from 4 to 7 days in the 24 month group were (29.4 ± 1.72)%, (37.6 ± 1.64)% and % And (18.9 ± 1.25)%. CONCLUSION: The proliferation of aged ADMSCs is decreased, while the proportion of aging, apoptotic and necrotic cells is increased. With the prolongation of primary culture of ADMSCs, the aging is increased, but there is no regular change of apoptosis and necrosis.