软骨终板通透性对兔退变腰椎间盘Ⅱ型胶原表达的影响

目的探讨软骨终板通透性对兔退变腰椎间盘Ⅱ型胶原表达的影响。方法 6月龄新西兰大白兔14只,建立退变腰椎间盘模型后饲养8周。8周后处死并手术切取腰段椎间盘每只6个,随机分为A组和B组,其中A组为对照组,B组骨蜡封闭上下软骨终板。两组兔退变椎间盘在体外进行整体器官培养。在培养前以及培养7 d和14 d时分别用Mitotracker Green荧光探针、免疫组化SP法和RT-PCR方法对椎间盘中髓核的细胞活力、Ⅱ型胶原及Ⅱ型胶原mRNA的表达情况进行评估。结果经过7 d的体外培养之后,两组的荧光强度、Ⅱ型胶原的灰度值与培养前比较降低不显著(P0.05),而椎间盘髓核Ⅱ型胶原的mRNA表达与与培养前比较差异显著(P0.05),两组间荧光强度、Ⅱ型胶原的灰度值及椎间盘髓核Ⅱ型胶原的mRNA表达对比差异均无统计学意义(P0.05)。经过14 d的培养,两组的荧光强度分别降低了18.6%与31.3%,与培养之前的荧光强度相比以及两组之间相比差异均具有统计学意义(P0.05);两组的Ⅱ型胶原的灰度值均有提升,与培养之前的Ⅱ型胶原的灰度值相比差异具有统计学意义(P0.01),两组间Ⅱ型胶原的灰度值经比较差异显著,具有统计学意义(P0.01);两组Ⅱ型胶原mRNA的表达比培养前以及培养7 d时明显下降(P0.05),两组之间比较差异显著具有统计学意义(P0.05)。结论降低软骨终板的通透性可以在短时间内(14 d)降低细胞活力和Ⅱ型胶原及其mRNA的表达,加速兔腰椎间盘的退变。

Study on the influence of permeability of cartilage endplate on the expression of type II collagen of degenerative lumbar discs in rabbits.

Objective To explore the influence of permeability of cartilage endplate on the expression of type II collagen from degenerative lumbar discs in rabbits Methods A total of 14 New Zealand white rabbits at the age of 6 months were used for the establishment of intervertebral disc degeneration models. At 8 weeks of feeding after operation,each lumbar intervertebral disc had been cut off,and 6 discs were taken from each lumbar vertebral column. They were randomly divided into group A and group B,rabbits in group A were used as control group,and rabbits in B group were used for taken bone wax sealed cartilage end plate. The degenerated intervertebral discs in rabbits of 2 groups were cultured as whole organ in vitro. The activity of nuclear pulposus cells of intervetebral discs,expression of type II collagen and type II collagen mRNA were assessed by Mitotracker Green fluorescence probe,immunohistochemical SP method and RT-PCR in culture and culture for 7 d and 14 d. Results After culture in vitro for 7 days,the gray fluorescence intensity,and values of type II collagen in 2 group were compared with before culture,and they did not significantly reduce（ P 〉0. 05）. The difference in expression of nucleus pulposus of intervertebral disc of type II collagen mRNA between pre-culture and post-culture periods was significant（ P 〈0. 05）. The difference in gray fluorescence intensity of type II collagen value and the expression of type II collagen from nucleus pulposus of intervertebral disc and expression of type II collagen mRNA was not statistically significant （ P 〉0. 05）. After culture for 14 days,the reduction in fluorescence intensity of 2 groups was approximately 18. 6% and 31. 3% respectively, and the difference in fluorescence intensity compared between these 2 groups before culture was statistically significant（ P 〈0. 05）. The values of gray for type II collagen in 2 groups were increased,value of gray of type II collagen after culture compared with before culture was statistically sig-nificant（ P 〈0. 01）,and the difference in gray level of type II collagen between these 2 groups was statistically significant（ P 〈0. 01）;and the decrease in expression of type II collagen mRNA was significant in comparison with before culture and 7d after culture（ P 〈0. 05）,the difference between these 2 groups was statistically significant（ P 〈0. 05）. Conclusion The reduction in permeability of cartilage endplate can decrease the expression of cell viability,type II collagen and mRNA in a short time（14 days）,and accelerate the degeneration of intervertebral discs in rab-bits.