大鼠骨髓间充质干细胞与髓核细胞共培养对白介素1β致髓核细胞退变的影响

The effect of rat bone marrow mesenchymal stem cells on IL-1β induced degenerative nucleus pulposus cells under the condition of co-culture

中文关键词:  骨髓间充质干细胞  椎间盘退行性变  髓核细胞  凋亡  共培养

英文关键词:Mesenchymal stem cells  Disc degeneration  Nucleus pulposus cell  Apoptosis  Co-culture system

中文摘要:

  【摘要】 目的：观察大鼠骨髓间充质干细胞（BMSCs）与髓核细胞（NPCs）共培养对白介素1β（IL-1β）致NPCs退变的影响。方法：在体外分别分离培养大鼠NPCs和BMSCs，分别传至第3代，采用流式细胞仪鉴定BMSCs纯度，分别测定CD29、CD31、CD45、CD90表型。取第3代NPCs分为3组：A组，无IL-1β干预，不与BMSCs共培养，设为对照组；B组，用IL-1β干预NPCs后不与BMSCs共培养；C组，用IL-1β干预NPCs后与BMSCs细胞借由transwell小室间接共培养。IL-1β干预时间和BMSCs共培养时间均为24h，之后取出transwell，将3组NPCs通过实时定量荧光PCR（RT-PCR）观察其解聚蛋白样金属蛋白-4（ADAMTS-4）、 解聚蛋白样金属蛋白酶-5（ADAMTS-5）、基质金属蛋白酶-13（MMP-13）基因表达量，同时通过Annexin V-FITC/propidium iodide（PI）凋亡试剂盒和半胱氨酸天冬氨酸蛋白酶-3（Caspase-3）试剂盒观察3组NPCs凋亡情况。结果：流式细胞鉴定结果显示90%以上的BMSCs表现为CD29、CD90阳性，小于5%的BMSCs表现为CD31、CD45阳性，细胞纯度较好。3组NPCs中ADAMTS-4、ADAMTS-5、MMP-13的基因相对表达量：A组为0.98±0.19、1.10±0.08、1.21±0.24，B组为5.23±0.25、6.92±2.33、23.39±0.09，C组为2.31±0.26、3.33±1.52、12.68±0.11，与A组比较，B、C组均明显升高（P<0.05），C组与B组比较均明显降低（P<0.05）。Caspase-3活性相对表达量A组为1.20±0.18，B组为5.92±0.93，C组为2.33±0.52，与A组比较，B、C组明显升高（P<0.05），C组与B组比较明显降低（P<0.05）。细胞凋亡率A组为4.2±2.2，B组为17.1±3.7，C组为10.5±2.4，与A组比较，B、C组明显升高（P<0.05），C组与B组比较明显降低（P<0.05）。结论：与BMSCs共培养可有效降低IL-1β致NPCs退变相关因子的基因表达，减少细胞凋亡；BMSCs可作为种子细胞对椎间盘炎性环境起到“治疗”作用。

英文摘要:

  【abstract】 Objectives: To investigate the effect of rat bone marrow mesenchymal stem cells(BMSCs) on IL-1β induced degenerative nucleus pulposus cells(NPCs) under the condition of co-culture. Methods: NPCs and BMSCs were isolated from SD rat and cultured to the 3rd passage respectively. BMSCs were taken for flow cytometry to analyze the phenotypes of CD29, CD31, CD45, CD90. Experiments were divided into three groups by using the 3rd passage NPCs. Group A, without IL-1β intervention and without BMSCs co-culture were regarded as control group; group B, IL-1β induced NP cells but without BMSCs co-culture; group C, IL-1β induced NP cells and then co-cultured with BMSCs by indirect co-culture transwell. Both IL-1β intervention time and BMSCs co-culture time were set at 24h, then the transwell was removed. The gene(ADAMTS-4, ADAMTS-5, MMP-13) expressions of NPCs through real-time fluorescent quantitative PCR(RT-PCR) were detected in each group, at the same time the Annexin V-FITC/propidium iodide(PI) apoptosis kit and Caspase 3 kits were used to observe the apoptosis in each group. Results: BMSCs flow results showed that more than 90% of BMSCs presented as CD29, CD90 positive, less than 5% of BMSCs presented as CD31 and CD45 positive, which showed good uniform. ADAMTS-4, ADAMTS-5, MMP-13 gene relative expressions in each group were obtained by RT-PCR, 0.98±0.19, 1.10±0.08, 1.21±0.24 respectively in group A, 5.23±0.25, 6.92±2.33, 23.39±0.09 respectively in group B, 2.31±0.26, 3.33±1.52, 12.68±0.11 respectively in group C. Compared with group A, the NP cell in group B and group C showed significant increase of ADAMTS-4, ADAMTS-5, MMP-13 gene expression(P<0.05), and compared with group B, the rise of ADAMTS-4, ADAMTS-5, MMP-13 gene expression showed obvious inhibition in group C(P<0.05). In detection of apoptosis, the relative expression of Caspase 3 activity was 1.20±0.18 in group A, 5.92±0.93 in group B, 2.33±0.52 in group C. Compared with group A, the relative expression quantity of Caspase 3 in group B and group C showed significant increase(P<0.05), and compared with group B, the rise of Caspase 3 activity was suppressed obviously in group C(P<0.05). Annexin V-FITC/propidium iodide(PI) by flow cytometry indicated the increase of apoptotic cells in group B and group C compared with that in group A(P<0.05). Conculsion: BMSCs effectively decrease the degeneration and apoptosis of NP cells induced by IL-1β under the condition of co-culture. As seed cells, BMSCs have therapeutical effect on intervertebral disc inflammation.