移植骨髓间充质干细胞对兔退变椎间盘髓核细胞凋亡的影响

Effects of mesenchymal stem cells transplantation on the apoptosis after rabbit intervertebral disc degeneration

目的 观察骨髓间充质干细胞（MSCs）移植对兔退变椎间盘髓核细胞凋亡的影响.方法 以各兔L2/3、L3/4、L4/5、L5/6节段分为正常组、退变组、成纤维细胞（SFs）移植对照组、MSCs移植治疗组.MSCs和SFs分别经绿色荧光蛋白（GFP）转染后,注射植入退变椎间盘的髓核.通过透射电镜观察退变椎间盘凋亡髓核细胞形态 用实时定量聚合酶链反应（PCR）检测退变组织中髓核细胞凋亡相关基因bcl-2和box mRNA的表达 免疫荧光法标记髓核细胞凋亡相关蛋白Caspase-3,并通过TUNEL法标记凋亡髓核细胞,激光共聚焦显微镜检测髓核细胞凋亡蛋白表达率和细胞凋亡比率.结果 透射电镜下,退变椎间盘中凋亡髓核细胞呈现出核染色质边集,空泡形成,核膜断裂,凋亡小体形成等变化.MSCs移植治疗组bcl-2 mRNA的表达量高于退变组和SFs移植对照组（P〈0.05）,bax mRNA的表达量与退变组差异无统计学意义（P〉0.05）.MSCs移植治疗组细胞凋亡率和Caspase-3表达率均高于正常组[细胞凋亡率分别为（16.75±2.14）%和（6.86±1.08）% Caspase-3表达率分别为[（20.34±1.03）%和（6.09±0.77）%]（P〈0.05）,低于退变组和SFs移植对照组[细胞凋亡率分别为（31.87±4.16）%和（29.02±2.16）% Caspase-3表达率分别为（31.50±3.78）%和（30.20±4.93）%]（P〈0.05）.结论 髓核细胞凋亡在椎间盘退变过程中起重要作用.MSCs移植能有效抑制椎间盘髓核细胞凋亡,延缓椎间盘退变过程.

Objective To investigate the effects of the mesenchymal stem cells （MSCs） transplantation on the apoptosis of nucleus pulposus cells after rabbit intervertebral disc degeneration. Methods New Zealand white rabbits were used to stimulate allograft. Harvested rabbit MSCs and fibroblasts were cultured , proliferated and labeled in vitro. At the same time, degenerated intervertebral disc models were set up using 21-gauge needle to puncture the annulus. Four weeks later, 2 X 106 MSCs and fibroblasts were injected into the respective disc segments. After 1,2,4 and 8 weeks, specimens from degenerated models were obtained in order. The number of apoptotic nucleus pulposus cells and the expression of Caspase-3 were detected under laser scanning confocal microscopy in every disc from the experimental group and the control groups of the rabbits. The bcl-2 and bax mRNA expression of all discs was assayed by real time reverse transcription-polymerase chain reaction （RT-PCR）. Results The transmission electron microscopy revealed that part of the nucleus pulposus cells became apoptosis in the initial stage in ultramicrostructure. Apoptosis rate was （16.75 ±2. 14）% and （31. 87 ±4. 16）% in the experimental group and the degenerated group, as well as Caspase-3 expression ratio was （20. 34 ± 1. 03）% and （31. 50 ±3. 78）% respectively , demonstrating that MSCs could relieve apoptosis after rabbit intervertebral disc degeneration. The levels of bcl-2 mRNA expression in experimental group were higher than in the degenerated group （P〈0.05）. Conclusion Apoptosis is involved in the development of intervertebral disc degeneration. MSCs transplantation can increase the expression of bcl-2 mRNA in the nucleus pulposus and relieve apoptosis after rabbit intervertebral disc degeneration.