软骨终板通透性对体外培养大鼠髓核细胞生物学特性的影响

Effect of vertebral endplates′permeability on the biologic characters of rat nucleus pulposus cells cultured in vitro

中文关键词:  椎间盘退变  器官培养模型  营养通路  终板  大鼠

英文关键词:Disc degeneration  Organ culture model  Nutritional pathway  Endplate  Rat

中文摘要:

  【摘要】 目的：观察软骨终板营养通路通透性对外培养大鼠髓核细胞生物学特性的影响。方法：4周龄雄性SD大鼠20只，处死后立即手术切取腰段椎间盘（包括邻近的软骨终板），每只6个，随机分为A、B、C 3组，其中A组为正常对照组，B组用骨蜡封闭上软骨终板，C组用骨蜡封闭上、下软骨终板，3组椎间盘在体外进行整体器官培养。于培养前和培养7d、14d时，分别用Mitotracker Green荧光探针和RT-PCR方法评估椎间盘髓核细胞的活力和髓核Ⅱ型胶原及蛋白多糖mRNA的表达；于培养前和培养14d时用免疫组化方法观察髓核蛋白多糖、Ⅱ型胶原和基质金属蛋白酶3（MMP-3）的表达。结果：取材后培养前髓核细胞的荧光强度最高；在体外培养7d，3组髓核细胞的荧光强度较培养前变化均不明显，3组之间无显著性差异（P>0.05）；培养14d时A、B、C组荧光强度较培养前分别降低约19%、22%和30%，与培养前比较差异均有显著性（P<0.05），其中C组与A、B两组比较有显著性差异（P<0.05），A、B两组比较差异无显著性（P>0.05）。免疫组织化学观察显示在培养14d时3组髓核蛋白多糖和Ⅱ型胶原的染色强度与培养前比较均有所降低，MMP-3阳性染色增加，蛋白多糖、Ⅱ型胶原和MMP-3染色强度的变化以C组最为明显。3组髓核细胞Ⅱ型胶原和蛋白多糖mRNA的表达在培养7d和14d时均较培养前显著降低（P<0.05），其中7d时3组之间无显著性差异（P>0.05），14d时A、B两组间无显著性差异（P>0.05），C组与A、B组比较有显著性差异（P<0.05）。结论：降低体外培养大鼠椎间盘上下软骨终板的通透性，可在短期内影响髓核细胞的生物学特性，加速椎间盘的退变。

英文摘要:

  【Abstract】 Objective：To observe the effect of vertebral endplates′ permeability on the biologic characters of rat nucleus pulposus cells cultured in vitro.Method：Lumbar intervertebral discs，plus adjacent vertebral endplates，were harvested from 20 male Spragua-Dawley（SD） rats（4 weeks old，n=6/animal）.The intervertebral discs were randomly divided into three groups：control group（group A），superior endplate blocked with bone wax（group B），and superior and inferior endplates blocked with bone wax（group C），all specimens were cultured in vitro. The cell viability，the expression of mRNA of collagen Ⅱ and aggrecan were assessed by using Mitotracker Green probe and RT-PCR before culturing and on the 7th and 14th days after culturing.The expression of collagen Ⅱ，aggrecan and matrix metalloproteinase-3（MMP-3） were assessed by using immunohistochemistry staining methods before culturing and the 14th day after culturing.Result：The fluorescence intensity of fresh intervertebral disc was the maximum.There was no significant difference with respect to fluorescence intensity between three groups and fresh disc on the 7th day（P>0.05），and no significant difference was found among three groups（P>0.05）.There was a significant decrease with respect to fluorescence intensity between three groups（group A：19%，group B：22%，group C：30%） and fresh disc on the 14th day（P<0.05）.A significant downregulated mRNA expression of collagen Ⅱ and aggrecan in three groups on the 7th and 14th days was noted（P<0.05）.No significant difference was found among three groups on the 7th day and between group A and B on the 14th day（P>0.05），but for group C，significant difference was noted compared with group A and B on the 14th day（P<0.05）.Conclusion：Decreasing the permeability of bilateral vertebral endplates of rat intervertebral discs cultured in vitro can accelerate disc degeneration through affecting the biologic characters of nucleus pulposus cells in the short term.