摘要
目的观察着色性干皮病C组基因(XPC)缺失对间充质干细胞(MSCs)衰老的影响,并探讨其机制。方法分离培养XPC敲除(XPC-/-)小鼠及野生型(WT)小鼠骨髓MSCs,流式细胞仪检测培养细胞表面标志表达,CCK8法检测细胞增殖情况,茜素红染色以及油红O染色观察成骨分化及成脂分化能力,衰老相关β半乳糖苷酶(SA-β-gal)染色检测细胞衰老情况,实时定量聚合酶链式反应(Real-time PCR)检测衰老相关基因P16、P21 mRNA表达情况。结果分离培养的两组小鼠骨髓来源的MSCs(m BMSCs)均高表达阳性表面抗原标志物CD44、CD29,基本不表达阴性表面抗原标志物CD11b、CD45,但均具有成骨及成脂分化能力。体外培养至20 PD(群体倍增水平)时,XPC-/-m BMSCs中衰老细胞比例(44.41±5.49)%,明显高于WT m BMSCs的(13.17±1.54)%(P<0.01);与WT m BMSCs相比,XPC-/-m BMSCs增殖能力明显下降(4 d时OD值:0.18±0.04 vs 0.36±0.04,P<0.05;5 d时OD值:0.27±0.04 vs 0.56±0.05,P<0.01);XPC-/-m BMSCs中P16 mRNA相对表达量与WT细胞相比无明显差异(P>0.05),而XPC-/-m BMSCs中P21 mRNA相对表达量明显高于WT细胞(3.30±0.23 vs 1.00±0.09,P<0.01);XPC敲除组细胞生长缓慢,出现衰老表型,增殖能力明显下降,SA-β-gal染色阳性率明显高于野生型细胞(P<0.01),衰老相关基因P21表达与野生型相比明显上调(P<0.01)。结论 XPC敲除促进MSCs衰老,其机制可能是由于DNA损伤累积引起的P21/P53信号通路的激活。
Objective To observe the effect of xeroderma pigmentosum group C（ XPC） deletion on the senescence of mesenchymal stem cells（ MSCs） and explore its mechanism. Methods Bone marrow-derived MSCs from XPC knockout（ XPC-/-） mice and wild-type（ WT） mice were isolated and cultured in vitro. Flow cytometry was used to detect the expression of surface markers of cultured cells. Cell counting kit-8（ CCK-8） was used to detect cell proliferation. Alizarin red staining and oil red O staining were used to respectively observe the abilities of osteogenic differentiation and adipogenic differentiation of MSCs. Senescence-associated β-galactosidase（ SA-β-gal） staining was used to detect cell senescence situation. Real-time quantitative polymerase chain reaction（ Real-time PCR） was used to detect the expressions of agingrelated genes P16 and P21 mRNAs. Results Bone marrow-derived MSCs of mice（ m BMSCs） in both two groups presented high expressions of positive surface markers CD44 and CD29 and basically had no expressions of negative surface markers CD11 b and CD45,but they had the abilities of osteogenic differentiation and adipogenic differentiation. During being cultured to 20 PDs（ population doubling level） in vitro,the proportion of senescent cells in XPC-/-m BMSCs was significantly higher than that in WT m BMSCs [（ 44. 41 ± 5. 49） % vs（ 13. 17 ± 1. 54） %,P〈0. 01]. Compared with WT m BMSCs,the proliferation ability of XPC-/-m BMSCs decreased significantly（ OD value at 4-day： 0. 18 ± 0. 04 vs 0. 36 ±0. 04,P〈0. 05; OD value at 5-day： 0. 27 ± 0. 04 vs 0. 56 ± 0. 05,P〈0. 01）. There was no significant difference in the relative expression level of P16 mRNA between XPC-/-m BMSCs and WT m BMSCs（ P〈0. 05）,while the relative expression level of XPC-/-P21 mRNA was significantly higher than that of WT m BMSCs（ 3. 30 ± 0. 23 vs 1. 00 ± 0. 09,P〈0. 01）. m BMSCs in XPC-/-mice grew slowly and appeared aging phenotype,and their proliferation ability decreased signif
作者
余瑾
黄亚琴
杨劲
YU Jin;HUANG Ya-qing;YANG Jin(Department of Cell Biology Teaching and Research,Army Medical University,Chongqin 400038,China)
出处
《中国临床研究》
CAS
2018年第7期865-869,共5页
Chinese Journal of Clinical Research
基金
国家自然科学基金项目(81402102)
重庆市科技计划项目(CSTC
2014jcyj A10090)
