摘要
目的探究髓核样尿源干细胞(NP-USC)分化程度与分化时间的关系,以及人尿源干细胞外泌体(USC-EXO)对NP-USC增殖及分化的影响。方法提取、鉴定并共培养髓核细胞(NPC)和尿源干细胞(USC)。根据共培养时间不同将细胞进行分组,A组:共培养7 d后,将NP-USC于无外泌体USC培养基中培养14 d;B组:共培养14 d后,将NP-USC于无外泌体USC培养基中培养7 d;C组:共培养21 d;另设USC组(USC单独培养21 d)和NPC组(NPC单独培养21 d)。使用实时荧光定量PCR(RT-qPCR)及Western blot方法检测5组细胞缺氧诱导因子(HIF-1α)、葡萄糖转运蛋白1(GLUT1)、Ⅱ型胶原蛋白(COL2)、蛋白多糖(ACAN)mRNA及蛋白质的表达情况。另将上述5组细胞用无外泌体USC培养基培养28 d,于第7、14、21、28天时采用CCK-8法检测细胞增殖情况。结果RT-qPCR及Western blot方法检测结果显示,USC、A、B、C组中HIF-1α、GLUT1、COL2、ACAN mRNA及蛋白的相对表达量分别依次升高(t=53.36~371.86,P<0.01),但C组与NPC组4种基因及蛋白相对表达量无显著差异(P>0.05);加入USC-EXO前后,5组细胞中4种基因相对表达量无显著差异(P>0.05)。CCK-8法检测结果显示,在各时间点,USC、A、B、C组细胞活力依次下降(F=3.77~103.58,P<0.05),但C组与NPC组无显著差异(P>0.05)。5组细胞中每组均随着培养时间的延长细胞活力逐渐升高(F=9.96~121.68,P<0.05)。结论NPC诱导USC时间越长,所形成的NP-USC分化程度越高、增殖速率越低。USC-EXO具有促NP-USC增殖的能力,但其不具有促分化的能力;NP-USC分化程度越高,USC-EXO的促增殖能力越低。
Objective To explore the relationship between degree of differentiation and time of differentiation of nucleus pulposus-like urine-derived stem cells(NP-USC)and the effects of human urine-derived stem cell exosomes(USC-EXO)on the proliferation and differentiation of NP-USC.Methods Nucleus pulposus cells(NPC)and urine-derived stem cells(USC)were extracted,identified,and cultured.According to the co-cultivation time,the cells were divided into group A(after 7 d of co-culture,the NP-USC were cultured in exosome-free USC medium for 14 d),group B(after 14 d of co-culture,the NP-USC were cultured in exosome-free USC medium for 7 d),group C(USC were cultured alone for 21 d),and NPC group(NPC were cultured alone for 21 d).Quantitative real-time PCR(RT-qPCR)and Western blot were used to measure the mRNA and protein expression of hypoxia-inducible factor 1α(HIF-1α),glucose transporter 1(GLUT 1),type Ⅱ collagen(COL 2),and proteoglycan(ACAN)in the five groups.The cells in the five groups were cultured in exosome-free USC medium for 28 d,and CCK-8 was used to detect the cell proliferation on day 7,14,21,and 28.Results RT-qPCR and Western blot results showed that the relative mRNA and protein expression levels of HIF-1α,GLUT1,COL2,and ACAN increased sequentially in groups USC,A,B,and C(t=53.36-371.86,P<0.01).However,there was no significant difference in the relative expression levels of the four genes and proteins between groups C and NPC(P>0.05).There was no significant difference in the relative expression levels of the four genes in the cells of the five groups after adding USC-EXO(P>0.05).The results of CCK-8 showed that the cell viability decreased sequentially in groups USC,A,B,and C at each time point(F=3.77-103.58,P<0.05),with no significant difference between groups C and NPC(P>0.05).In each of the five groups,the cell viability gradually increased with the prolongation of culture time(F=9.96-121.68,P<0.05).Conclusion The longer the NPC-induced USC time,the higher the degree of NP-USC differentiation and the low
作者
朱有福
李承威
沈娜娜
相宏飞
陈伯华
郭柱
ZHU Youfu;LI Chengwei;SHEN Nana;XIANG Hongfei;CHEN Bohua;GUO Zhu(Department of Spinal Surgery,The Affiliated Hospital of Qingdao University,Qingdao 266035,China)
出处
《精准医学杂志》
2023年第4期283-288,共6页
Journal of Precision Medicine
基金
国家自然科学基金(82172478)
泰山学者青年专家工程资助(tsqn201909190)
中国博士后科学基金项目(2022T150340,2021M701813)
国家骨科与运动康复临床医学研究中心创新基金(2021-NCRC-CXJJ-ZH-02)
山东省自然科学基金青年基金(ZR202111280033)
山东省高等学校“青创科技支持计划”(2021KJ048)。
关键词
干细胞研究
髓核
外泌体
细胞分化
细胞增殖
组织工程
Stem cell research
Nucleus pulposus
Exosomes
Cell differentiation
Cell proliferation
Tissue engineering
