建立大鼠脊柱功能单位体外培养模型

The establishment of functional spinal unit in rats

目的建立大鼠脊柱功能单位(functional spine unit,FSU)体外培养体系模型。方法选取8~12月龄雄性大鼠,用CO2安乐处死后,解剖大鼠脊柱胸椎和腰椎。保留一侧椎体骨质2 mm,另外一端紧贴椎间盘终板外缘切除,制作骨-椎间盘-骨结构FSU。将FSU用1%PBS清洗后,放入12孔板中并加入培养基连续培养28 d。在不同的时间节点(0、3、5、7、14、21、28 d)用MTT和DAPI(4',6-diamidino-2-phenylindole)染色并冰冻切片分别检测椎间盘纤维环和髓核的细胞活性。提取不同时间点椎间盘髓核组织检测糖胺聚糖(glycosaminoglycan content,GAG)的差异性并做定量评估。Western blot检测不同时间点椎间盘组织聚糖蛋白聚糖降解碎片(aggrecan fragment)中衰老标志基质金属蛋白酶(matrix metalloproteinases,MMPs)及血小板反应蛋白解整合素金属肽酶(a disintegrin and metalloproteinase with thrombospondin motifs,ADAMTS)的表达差异。Q-PCR检测各组相关基因的表达。结果细胞活性检测显示,随着体外培养时间的延长,椎间盘纤维环细胞及髓核细胞活性下降。FSU体外培养7 d后出现髓核细胞(NP)活性明显下降,7 d后细胞活性下降为(58.62±2.77)%,差异有统计学意义(P<0.05),14 d细胞活性为(10.67±2.47)%,差异有统计学意义(P<0.05)。纤维环细胞(AF)相对比较稳定,呈现逐步下降的趋势,28 d后细胞活性仍为(74.01±0.63)%,差异有统计学意义(P<0.05)。GAG检测髓核0、3、5、7 d糖胺聚糖含量差异无统计学意义(P>0.05),但是除以单位含量的DNA后第5天和第7天比值明显升高(P<0.05)。Western blot检测椎间盘组织aggrecan fragment中的MMPs和ADAMTS含量随着培养时间延长出现升高,其中第7天与0 d相比差异有统计学意义(P<0.05)。Q-PCR检测椎间盘成分基因聚糖蛋白聚糖(aggrecan)及CollagenⅠ基因随着培养时间延长表达量减低,并且各组间差异有统计学意义(P<0.05)。结论大鼠脊柱功能单位连续培养7 d后髓核细胞活性急剧降低,体外培养28 d纤维环细胞活性仍较稳定。

Objective To establish a rat functional spinal unit(FSU)model.Methods The Sprague-Dawley(SD)male rats(8-12 months)were selected.CO2 Euthanasia method was used on the rat after which the rat's thoracic and lumbar vertebrae were dissected.2 mm of one side of the vertebral body bone was retained,and the other end attached to the outer edge of the intervertebral disc endplate structure were also collected to form the FSU.After washed with 1%PBS,the FSU was placed in a 12-well plate and cultured for 28 days.Cell viability of the annulus fibrosus and nucleus pulposus were detected by MTT and DAPI staining at different times(Day 0,3,5,7,14,21,28).The glycosaminoglycan(GAG)contents in the nucleus pulposus among different time points were extracted and quantitatively evaluated.Western blot was applied to detect the expression differences of aging markers metalloproteinases MMPs and ADAMTS in aggrecan fragments among different time points.Q-PCR was applied to detect the expression of related genes.Results Cell viability assay showed that the activities of intervertebral disc annulus cells and nucleus pulposus cells were decreased with the prolongation of culture in vitro.On Day 7 of FSU culture,the activity of nucleus pulposus cells(NP)significantly decreased.On Day 7,the NP cell activity decreased to(58.62±2.77)%(P<0.05),and the cell activity at 14 days was(10.67±2.47)%(P<0.05).However,the annulus fibrosus cells(AF)were relatively stable and showed a gradual decline.On Day 28,the AF cell viability was still at(74.01±0.63)%(P<0.05).There was no significant difference in GAG content among on Day 0,3,5,and 7(P>0.05),but the ratios on Day 5 and 7 were significantly increased after dividing by unit content of DNA(P<0.05).The contents of MMPs,and ADAMTS in the intervertebral disc tissue were increased with the prolongation of culture time.There was significant difference between Day 7 and Day 0(P<0.05).The expression of aggrecan and CollagenⅠgenes significantly decreased with in vitro culture time.Conclusion The results of this study suggest the activity of NP cells sharply decreases 7 days after of rat spinal functional unit culture in vitro,the activity of AF cells remains stable 28 days after culture.