不同Pfirrmann分级椎间盘内髓核细胞生物学特性的比较
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摘要
椎间盘连接相邻两块椎骨,在脊柱的承重和活动中发挥着重要作用。当椎间盘发生退变时就会导致颈椎病和下腰痛。随着人口逐渐老龄化的进程其发病率越来越高,给社会造成巨大的经济负担。全面的认识椎间盘的退变机制是找到合适治疗措施的前提。椎间盘细胞的培养则是从细胞水平研究椎间盘退变的基础。目前关于人腰椎间盘髓核细胞培养的文献较多。而人颈椎间盘髓核细胞培养的文献报道很少。本研究的第一部分旨在尝试对人颈椎间盘髓核细胞进行培养。磁共振(MRI)在临床上是一种评估椎间盘退变的重要方式。其T2像上的影像特征可以很好的反应椎间盘的退变和老化。Pfirrmann等人根据MRI T2像的信号强度、信号是否均质、髓核与纤维环的分界是否清晰和椎间盘的高度等指标建立了腰椎间盘退变的磁共振分级体系。该体系简单、直观、全面,各级间具有很好的区分性。因此为临床评价和基础研究提供了一种标准化的评估方式。本研究的第二部分目的是通过对比不同Pfirrmann分级椎间盘内髓核细胞的生物学特性检验该分级在细胞水平的效果。
实验一颈椎间盘髓核细胞的体外培养和鉴定
目的:建立人颈椎间盘髓核细胞体外培养体系,并对其细胞表型进行鉴定。方法:采用酶消化法分离人颈椎间盘髓核细胞,进行单层培养,倒置相差显微镜观察细胞生长和形态,流式细胞仪测定细胞周期和凋亡率,并行甲苯胺蓝、Ⅱ型胶原及CK8免疫组化染色对其细胞表型进行鉴定。结果:原代髓核细胞凋亡率6.1±1.4%,S期细胞比例7.3±0.5%。贴壁后形态为多角形或短楔形,传代后生长加速。细胞呈甲苯胺蓝异染性;Ⅱ型胶原免疫组化染色阳性;只有少量椭圆形大细胞CK8免疫组化染色阳性。结论:成功建立人颈椎间盘髓核细胞体外培养模型,细胞鉴定符合髓核细胞特征。并证实成年后人髓核内仍有少量细胞保持脊索细胞表型。
实验二不同Pfirrmann分级椎间盘内髓核细胞生物学特性的比较
目的:比较腰椎间盘退变的Pfirrmann分级各级间的髓核细胞生物学特性。方法:按腰椎间盘退变Pfirrmann分级标准将因腰椎退变性疾病而接受手术病人的髓核组织进行分组,A组为II级,B组为III级,C组为IV级。行组织切片甲苯胺蓝观察比较各组髓核细胞密度。采用酶消化法分离培养髓核细胞。台盼蓝染色计算各组细胞活性比率,光镜观察细胞形态和生长状态,透射电镜观察细胞的超微结构。绘制2代细胞的生长曲线。阿利辛蓝法测蛋白聚糖含量,比较各组差异。结果:组织切片染色显示A组细胞密度最高,B组次之,C组细胞密度最低。A组细胞的活性比率为91.6±4.3%;B组细胞活性比率为83.5±6.7%;C组细胞活性比率为74.8±5.9%。各组间差异具有统计学意义,P﹤0.05。光镜下A组细胞呈短梭形或多角形轮廓清晰饱满,C组细胞轮廓不清,胞突较长。电镜下A组细胞内有大量粗面内质网和线粒体, C组细胞内的粗面内质网和线粒体稀少,存在很多空泡样结构,并且可见到细胞相互吞噬的现象。B组细胞的超微形态介与两者之间。A组细胞生长速率明显快于B组和C组分别为(T=61.5,P<0.05,T=76.4 , P<0.01))。B组细胞生长速率快于C组(T=64.5,P<0.05)。A、B、C组细胞GAG含量分别为408.23±29.25mg/L、273.05±52.44mg/L、91.73±38.06mg/L,各组间均有统计学差异,P﹤0.05。结论:椎间盘退变的Pfirrmann分级,能很好的反应髓核组织在细胞水平的退变程度。是一种简单有效的退变分级系统。
Intervertebral discs play a significant role in the support, durability, and flexibility of the spine. Disc degeneration is a relatively common problem. There is a known strong association between disc degeneration and disc-related pain. As the population to age, the incidence and prevalence of pain related to disc degeneration will undoubtedly increase. This situation causes heavy economic burden on the society. A thorough comprehension of disc degeneration is Fundamental to an appropriate treatment strategy. The culture of the disc cells is the premise to research disc degeneration on the cell level and a source of cells for intervertebral disc engineering. However, there is few article about the culture of human cervical nucleus pulposus cells. So the part one of this article aims to successfully establish a system of the culture of cervical nucleus pulposus cells. Magnetic resonance imaging (MRI) is the most important method for the clinical assessment of intervertebral disc pathology. The signal characteristics of the disc in T2-weighted MRIs reflect changes caused by aging or degeneration. Pfirrmann and his colleagues develop a grading system for lumbar disc degeneration based on MRI signal intensity, disc structure, distinction between nucleus and anulus, and disc height. This grading system is simple and comprehensive, with intra- and inter-observer reliability sufficient to discriminate between the different grades, therefore provides a standardized and reliable assessment method of disc degeneration for research and clinical purposes. the part two of this article aims to compare biological characteristics of nucelus pulposus cells in different grade of this system.
Part one---Cultivation and identification of human cervical nucleus pulposus cells
Objective: To establish the system for culturing the human cervical nucleus pulposus cells and to identify their phenotypes. Methods The human cervical nucleus pulposus cells were isolated by collagenase digestion method and cultured in monolayer by culture solutions of DF12 + 20%FBS. Morphologic changes and growth of the cells were detected by microscope. The cell cycle and apoptosis rate were detected by Flow cytometric. The morphological structure and the phenotype were identified with the toluidine blue staining and immunocytochemistry means. Result The apoptosis rate of primary cells was 6.1±1.4%. The Cells at S phase were 7.3±0.5%. The cells were polygonal or short wedged morphology. The second passage cells grew more fast than that of the primary cells. The cells displayed intense toluidine blue metachromasia. The cells expressed collagen typeⅡ, but only a few elliptic gaint cells expressed CK8. Conclusion The human cervical nucleus pulposus cells were isolated and cultured in monolayer successfully. A few cells of the nucleus pulposus still maintain notochord cells phenotype in adult specimens.
Part two --- Comparison of biological characteristics of nucleus pulposus cells in intervertebral discs with different grade of Pfirrmann grading system
Objective: To compare the biological characteristics of nucleus pulposus cells in intervertebral discs with different grade of Pfirrmann grading system. Method :Human nucleus pulposus cells were isolated by collagenase digestion method and cultured.they were grouped based on the Pfirrmann grading system. Group A represents grade II. Group B represents grade III. Group C represents grade IV. Cell vitality assay were examined with Trypan blue staining. Morphologic changes of the cells were observed by an inverted microscope. Cell ultra-structure morphological changes were examined using transmission electron microscopy. Cell growth curve was determined by cell counting . Proteoglycans expressions were examined by Alican blue chromatometry. These indexes were compared among 3 groups above。Results: Group A has the highest cell density and its cell vitality is 91.6±4.3%. Cell vitality of Group B is 83.5±6.7%. Group C has the lowest cell density and its cell vitality is 74.8±5.9%. There were statistical significances ( P < 0.05). The cells from group A were short spindle or polygonal shaped. They had clear outlines and rich cytoplasm with a lot of rough endoplasmic reticulum and mitochondria. The cells from group C had long processes and vague outlines. Much vacuoles located in their cytoplasm. Additionally, the entosis phenomenon had been observed. Under the same cellular density and culture conditions , the growth rate of passage 2 cells from the three groups were statistically significant respectively. The content of glycosaminoglycan of group A, B and C were 408.23±29.25mg/L、273.05±52.44mg/L、91.73±38.06 mg/L, respectively. There were significant differences among these groups (P﹤0.05). Conclusion:Pfirrmann classification of disc degeneration can be very good reaction in degenerative degree of nucleus pulposus cells. It is a simple and effective disc degeneration grading system.
实验一颈椎间盘髓核细胞的体外培养和鉴定
目的:建立人颈椎间盘髓核细胞体外培养体系,并对其细胞表型进行鉴定。方法:采用酶消化法分离人颈椎间盘髓核细胞,进行单层培养,倒置相差显微镜观察细胞生长和形态,流式细胞仪测定细胞周期和凋亡率,并行甲苯胺蓝、Ⅱ型胶原及CK8免疫组化染色对其细胞表型进行鉴定。结果:原代髓核细胞凋亡率6.1±1.4%,S期细胞比例7.3±0.5%。贴壁后形态为多角形或短楔形,传代后生长加速。细胞呈甲苯胺蓝异染性;Ⅱ型胶原免疫组化染色阳性;只有少量椭圆形大细胞CK8免疫组化染色阳性。结论:成功建立人颈椎间盘髓核细胞体外培养模型,细胞鉴定符合髓核细胞特征。并证实成年后人髓核内仍有少量细胞保持脊索细胞表型。
实验二不同Pfirrmann分级椎间盘内髓核细胞生物学特性的比较
目的:比较腰椎间盘退变的Pfirrmann分级各级间的髓核细胞生物学特性。方法:按腰椎间盘退变Pfirrmann分级标准将因腰椎退变性疾病而接受手术病人的髓核组织进行分组,A组为II级,B组为III级,C组为IV级。行组织切片甲苯胺蓝观察比较各组髓核细胞密度。采用酶消化法分离培养髓核细胞。台盼蓝染色计算各组细胞活性比率,光镜观察细胞形态和生长状态,透射电镜观察细胞的超微结构。绘制2代细胞的生长曲线。阿利辛蓝法测蛋白聚糖含量,比较各组差异。结果:组织切片染色显示A组细胞密度最高,B组次之,C组细胞密度最低。A组细胞的活性比率为91.6±4.3%;B组细胞活性比率为83.5±6.7%;C组细胞活性比率为74.8±5.9%。各组间差异具有统计学意义,P﹤0.05。光镜下A组细胞呈短梭形或多角形轮廓清晰饱满,C组细胞轮廓不清,胞突较长。电镜下A组细胞内有大量粗面内质网和线粒体, C组细胞内的粗面内质网和线粒体稀少,存在很多空泡样结构,并且可见到细胞相互吞噬的现象。B组细胞的超微形态介与两者之间。A组细胞生长速率明显快于B组和C组分别为(T=61.5,P<0.05,T=76.4 , P<0.01))。B组细胞生长速率快于C组(T=64.5,P<0.05)。A、B、C组细胞GAG含量分别为408.23±29.25mg/L、273.05±52.44mg/L、91.73±38.06mg/L,各组间均有统计学差异,P﹤0.05。结论:椎间盘退变的Pfirrmann分级,能很好的反应髓核组织在细胞水平的退变程度。是一种简单有效的退变分级系统。
Intervertebral discs play a significant role in the support, durability, and flexibility of the spine. Disc degeneration is a relatively common problem. There is a known strong association between disc degeneration and disc-related pain. As the population to age, the incidence and prevalence of pain related to disc degeneration will undoubtedly increase. This situation causes heavy economic burden on the society. A thorough comprehension of disc degeneration is Fundamental to an appropriate treatment strategy. The culture of the disc cells is the premise to research disc degeneration on the cell level and a source of cells for intervertebral disc engineering. However, there is few article about the culture of human cervical nucleus pulposus cells. So the part one of this article aims to successfully establish a system of the culture of cervical nucleus pulposus cells. Magnetic resonance imaging (MRI) is the most important method for the clinical assessment of intervertebral disc pathology. The signal characteristics of the disc in T2-weighted MRIs reflect changes caused by aging or degeneration. Pfirrmann and his colleagues develop a grading system for lumbar disc degeneration based on MRI signal intensity, disc structure, distinction between nucleus and anulus, and disc height. This grading system is simple and comprehensive, with intra- and inter-observer reliability sufficient to discriminate between the different grades, therefore provides a standardized and reliable assessment method of disc degeneration for research and clinical purposes. the part two of this article aims to compare biological characteristics of nucelus pulposus cells in different grade of this system.
Part one---Cultivation and identification of human cervical nucleus pulposus cells
Objective: To establish the system for culturing the human cervical nucleus pulposus cells and to identify their phenotypes. Methods The human cervical nucleus pulposus cells were isolated by collagenase digestion method and cultured in monolayer by culture solutions of DF12 + 20%FBS. Morphologic changes and growth of the cells were detected by microscope. The cell cycle and apoptosis rate were detected by Flow cytometric. The morphological structure and the phenotype were identified with the toluidine blue staining and immunocytochemistry means. Result The apoptosis rate of primary cells was 6.1±1.4%. The Cells at S phase were 7.3±0.5%. The cells were polygonal or short wedged morphology. The second passage cells grew more fast than that of the primary cells. The cells displayed intense toluidine blue metachromasia. The cells expressed collagen typeⅡ, but only a few elliptic gaint cells expressed CK8. Conclusion The human cervical nucleus pulposus cells were isolated and cultured in monolayer successfully. A few cells of the nucleus pulposus still maintain notochord cells phenotype in adult specimens.
Part two --- Comparison of biological characteristics of nucleus pulposus cells in intervertebral discs with different grade of Pfirrmann grading system
Objective: To compare the biological characteristics of nucleus pulposus cells in intervertebral discs with different grade of Pfirrmann grading system. Method :Human nucleus pulposus cells were isolated by collagenase digestion method and cultured.they were grouped based on the Pfirrmann grading system. Group A represents grade II. Group B represents grade III. Group C represents grade IV. Cell vitality assay were examined with Trypan blue staining. Morphologic changes of the cells were observed by an inverted microscope. Cell ultra-structure morphological changes were examined using transmission electron microscopy. Cell growth curve was determined by cell counting . Proteoglycans expressions were examined by Alican blue chromatometry. These indexes were compared among 3 groups above。Results: Group A has the highest cell density and its cell vitality is 91.6±4.3%. Cell vitality of Group B is 83.5±6.7%. Group C has the lowest cell density and its cell vitality is 74.8±5.9%. There were statistical significances ( P < 0.05). The cells from group A were short spindle or polygonal shaped. They had clear outlines and rich cytoplasm with a lot of rough endoplasmic reticulum and mitochondria. The cells from group C had long processes and vague outlines. Much vacuoles located in their cytoplasm. Additionally, the entosis phenomenon had been observed. Under the same cellular density and culture conditions , the growth rate of passage 2 cells from the three groups were statistically significant respectively. The content of glycosaminoglycan of group A, B and C were 408.23±29.25mg/L、273.05±52.44mg/L、91.73±38.06 mg/L, respectively. There were significant differences among these groups (P﹤0.05). Conclusion:Pfirrmann classification of disc degeneration can be very good reaction in degenerative degree of nucleus pulposus cells. It is a simple and effective disc degeneration grading system.
引文
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