两种同源间充质干细胞在接触式共培养体系下对退变髓核细胞生物学影响的比较

Comparative study of the biological effects from two kinds of human stromal stem cells to degenerative nucleus pulposus cells under the condition of contact co-culture

中文关键词:  接触式共培养  间充质干细胞  退变髓核细胞  比较研究

英文关键词:Contact co-culture  Mesenchymal stem cells  Degenerative nucleus pulposus cells  Comparative study

中文摘要:

  【摘要】 目的：比较两种人间充质干细胞在接触式共培养体系下对退变髓核细胞生物学功能的影响。方法：取同一例腰椎间盘突出症患者的退变髓核组织、脂肪组织和骨髓组织，分别分离培养退变髓核细胞（NPCs）、脂肪间充质干细胞（ADSCs）和骨髓间充质干细胞（BMSCs）。取3种细胞传代至第3代的细胞进行接触式共培养。共培养前，利用PKH26对第3代退变NPCs进行染色标记，然后按1∶1的细胞比例，将NPCs分别与ADSCs和BMSCs在普通6孔板内进行接触式共培养。实验分为3组，A组为单独培养的NPCs，为对照组；B组为与ADSCs共培养的NPCs；C组为与BMSCs共培养的NPCs。共培养第7天时，利用MoFlo高速流式细胞分选仪将共培养的细胞进行分选，获取各组NPCs。然后提取各组NPCs的总RNA，进行反转录后，再利用Real-Time PCR技术检测各组NPCs的Ⅱ型胶原、蛋白多糖和SOX-9基因的相对表达量。将Ⅱ型胶原、蛋白多糖和SOX-9基因的相对表达量按分组进行组间t检验，P＜0.05为有统计学差异。结果：从退变髓核组织、骨髓组织和脂肪组织中，均成功分离培养出NPCs、BMSCs和ADSCs。待各细胞传至第3代时，成功利用PKH26对NPCs进行染色标记。按实验分组将各细胞进行接触式共培养后第7天，成功利用MoFlo高速流式细胞分选仪将共培养细胞进行分选，获取各组NPCs。经提取各组NPCs的总RNA，反转录并进行Real-time PCR后，获取各组NPCs的Ⅱ型胶原、蛋白多糖和SOX-9基因相对表达量，A组分别为1.03±0.04，1.05±0.07，1.04±0.17；B组分别为5.26±0.24，7.71±0.21，3.84±0.25；C组分别为9.33±0.39，11.07±0.34，5.64±0.26；B、C组NPCs的Ⅱ型胶原、蛋白多糖和SOX-9基因相对表达量与A组比较显著性增加（P<0.05）；C组与B组比较，各指标基因相对表达量显著性增加（P<0.05）。结论：在接触式共培养条件下，BMSCs和ADSCs对退变NPCs均具有营养激活效应；BMSCs对退变NPCs的激活效应比ADSCs更好，对于椎间盘退行性疾病的生物学治疗可能更具优势。

英文摘要:

  【Abstract】 Objectives: To compare the biological effects of degenerative nucleus pulposus cells when co-cultured with two kinds of people’s mesenchymal stem cells under a contact system. Methods: The degenerative nucleus pulposus tissue, dipose tissue and bone marrow were obtained from the same patient who suffered from lumbar disc herniation during the surgery, and then degenerative nucleus pulposus cells(NPCs), adipose- derived mesenchymal stem cells(ADSCs) and bone marrow mesenchymal stem cells(BMSCs) were isolated and cultured. The third generation of cells had the direct cell-cell contact co-culture. First, the third generation of degeneration NPCs was labled by PKH26, and then the NPCs with ADSCs and BMSCs were put in 6-well plates to contact co-culture at the ratio of 1∶1. There were three groups: group A was NPCs cultured alone, as the control group; group B was NPCs co-cultured with ADSCs; group C was NPCs co-cultured with BMSCs. On the seventh day after co-culture, BMSCs, ADSCs and NPCs were seperated by the co-culture flow cytometry, the total RNA of each group was extracted, then the Real-Time PCR technology was used to detect the relative gene expression of type Ⅱ collagen, proteoglycan and SOX-9. The results were statistical analyzed by t test between every two groups, P<0.05 meant statistically significant. Results: After obtained the degenerative nucleus pulposus tissue, fat tissue and bone marrow tissue, the NPCs, ADSCs and BMSCs were successfully isolated and cultured. When cells were transmitted to the third generation, the NPCs labled by PKH26 were got successfully. On the seventh day after co-culture, MoFlo high-speed flow cytometry was used to separate and obtain the NPCs in group B and group C. Total RNA was extracted, after reversing transcription and Real-time PCR, the collagen protein Ⅱ, proteoglycan and SOX-9 gene relative expression of NPCs in each group were obtained. Collagen protein Ⅱ, proteoglycan and SOX-9 of NPCs in group A was 1.03±0.04, 1.05±0.07, 1.04±0.17; 5.26±0.24, 7.71±0.21, 3.84±0.25 respectively in group B; and 9.33±0.39, 11.07±0.34, 5.64±0.26 respectively in group C. Compared with group A, the nucleus pulposus cells of group B and C had a significant increase of collagen protein Ⅱ, proteoglycan and SOX-9 gene expression(P<0.05); The group C had a higher significant increase of gene expression of collagen protein Ⅱ, proteoglycan and SOX-9 than group B(P<0.05). Conclusions: BMSCs and ADSCs can both stimulate and activate the degenerative NPCs under the condition of contact co-culture. Compared with ADSCs, BMSCs have a stronger activation effect on degenerative NPCs, it may be more suitable for degenerative disc diseases′ biological treatment.