不同Miyazaki分级颈椎间盘内髓核细胞的生物学特性

Biological characteristics of nucleus pulposus cells in cervical intervertebral discs with different Miyazaki grades

目的:比较不同Miyazaki分级颈椎间盘内髓核细胞生物学特性的差异,评估该分级系统反映颈椎间盘退变程度的可靠性与一致性。方法:收集我院2017年12月~2018年7月36例行颈前路手术患者术中切除的颈椎间盘髓核组织。患者男21例,女15例,年龄为41.2±4.5岁,手术节段为单节段27例,两节段9例。将收集到的45个髓核组织按照术前颈椎MRI T2像上的Miyazaki分级分为4组,依次为Ⅱ级(n=6),Ⅲ级(n=10),Ⅳ级(n=14),Ⅴ级(n=15)。组织切片后行甲苯胺蓝染色,观察髓核组织内细胞的密度以及分布情况。免疫组织化学染色和蛋白免疫印迹实验检测不同分级组织内聚集蛋白聚糖(Aggrecan)和Ⅱ型胶原蛋白(typeⅡcollagen,ColⅡ)的表达量。采用酶消化法分离不同分级髓核组织内的细胞并进行体外培养,光镜下观察髓核细胞的形态,台盼蓝染色测定各组髓核细胞的活性比率,CCK-8法绘制各组1代髓核细胞的生长曲线。结果:Ⅱ级组髓核组织内细胞密度显著高于Ⅲ级组,Ⅲ级组高于Ⅳ级组,Ⅴ级组的细胞密度最低。Aggrecan和ColⅡ在Ⅱ级组织中表达量最高,Ⅲ级组高于Ⅳ级组,Ⅴ级组中表达量最低,组间比较均有统计学差异(P<0.05)。Ⅱ级组椎间盘内髓核细胞多呈三角形、短梭形,折光性较好;Ⅲ级组髓核细胞出现突起,轮廓不清晰;Ⅳ级组细胞形态多呈长梭形,细胞扁平且突起明显;Ⅴ级组髓核细胞形态各异,轮廓模糊,折光度差。Ⅱ、Ⅲ、Ⅳ、Ⅴ级组的细胞活性比率分别为(94.8±2.8)%、(84.3±2.6)%、(75.1±4.8)%和(66.1±3.3)%,其中Ⅱ级组高于Ⅲ级组,Ⅳ级组低于Ⅲ级组,而Ⅴ级组最低,组间比较均有统计学差异(P<0.05)。Ⅱ级组髓核细胞生长速率比Ⅲ级组快,Ⅲ级组比Ⅳ级组快,Ⅴ级组生长速率最低,组间比较均有统计学差异(P<0.05)。结论:颈椎间盘Miyazaki分级系统可以较好地反映髓核细胞的生物学特性和颈椎间盘的退变程度,可作为临床治疗和诊断的重要参考。

Objectives:To compare the biological characteristics of cervical nucleus pulposus cells in different Miyazaki grades in order to evaluate the reliability of the grading system.Methods:These 45 nucleus pulposus were collected from 36 patients(aged 41.2±4.5 years)undergone anterior cervical operation from December 2017 to July 2018 in our hospital,including 21 males and 15 females,and 27 cases of single segment surgery,and 9 cases of two segments surgery.The 45 nucleus pulposus were divided into 4 groups according to Miyazaki classification,gradeⅡ(n=6),gradeⅢ(n=10),gradeⅣ(n=14)and gradeⅤ(n=15).Tissue sections were stained with toluidine blue to observe the density and distribution of nucleus pulposus cells.Immunohistochemistry and western blotting were performed to determine the expression levels of typeⅡcollagen and aggrecan in different Miyazaki grades of nucleus pulposus tissue.The nucleus pulposus cells were isolated by trypsin and collagenaseⅡand cultured to observe cells morphology under light microscope.The activity ratio of nucleus pulposus cells was determined by trypan blue staining and the growth curve of nuclear cells was drawn by CCK-8 kit.Results:The nucleus pulposus cells density in gradeⅡwas higher than that in gradeⅢandⅣ,followed by that in gradeⅤ.The expression of typeⅡcollagen and aggrecan was the highest in gradeⅡ,followed by that in gradeⅢ,Ⅳand gradeⅤ,with significant differences between every two groups(P<0.05).In addition,the cells in gradeⅡwere triangular and short spindle in shape,with pretty well refractive index.While the cells in gradeⅢhad long processes,vague outline and pseudopodium.For the gradeⅣandⅤ,these cells had many different shapes,longer processes,blurred outlines and poor refractive index.The cell activity ratio was(94.8±2.8)%in gradeⅡgroup,(84.3±2.6)%in gradeⅢgroup,(75.1±4.8)%in gradeⅣgroup and(66.1±3.3)%in gradeⅤgroup,with significant differences between every two groups(P<0.05).The cell growth rate in gradeⅡof passage 1 cells were markedly faster than that of gradeⅢ(P<0.05),and that of gradeⅢwas faster than that of gradeⅣ(P<0.05),so was that of gradeⅣthan gradeⅤ(P<0.05).Conclusions:The Miyazaki grading system could reflect biological characteristics of nucleus pulposus cells and the degree of disc degeneration well and provide an important reference for clinical treatment and diagnosis.