摘要
目的:运用特异性探针观察不同年龄组黄韧带Ⅰ和Ⅱ型胶原基因表达分布、强度及随增龄而发生的变化,采用客观图像分析验证黄韧带退行性变和骨化增龄性变假说。方法:实验于2003-08/2004-08在第二军医大学长征医院中心实验室完成。21块腰椎黄韧带标本取自椎间盘突出症和椎管狭窄行后路椎板减压术患者,对照组标本取自4例青少年腰椎骨折行后路椎板减压术患者。提供标本患者均知情同意。椎间盘突出症和椎管狭窄患者的腰椎黄韧带标本按年龄分为4组:<20岁组、20～40岁组、41～60岁组和>60岁组。用Ⅰ和Ⅱ胶原cDNA探针,对不同年龄组的黄韧带冰冻切片进行原位杂交和图像分析,观察其Ⅰ型胶原表达(成纤维细胞基因表型)和Ⅱ型胶原表达(软骨细胞基因表型)。结果:①原位杂交组织切片观察:<20岁组黄韧带附着点处和体部纤维细胞排列规则,细胞呈梭形细胞数目较多,有明显的Ⅰ胶原表达,无Ⅱ型胶原表达信号;20～40岁组黄韧带细胞排列尚规则,细胞的数量相对减少,出现轻度纤维化,在黄韧带的附着点处有少量的Ⅰ和Ⅱ型胶原阳性表达信号;41～60岁组黄韧带体部细胞排列不规则,细胞的数量相对减少,出现明显纤维化,黄韧带体部无明显的阳性表达,黄韧带的附着点处有较弱的的Ⅰ型胶原阳性表达,阳性表达细胞分布不均匀,但Ⅱ型胶原的表达较41岁以下组表达增强;>60岁组黄韧带出现纤维化,未见Ⅰ型胶原阳性表达信号,黄韧带的附着点处Ⅱ型胶原阳性表达信号增强。②Ⅰ型胶原基因表达:<20岁组、20～40岁组、41～60岁组和>60岁组均显著高于对照组(1.53±0.18,1.34±0.16,0.86±0.13,0.71±0.21,0.51±0.11,P<0.01),随年龄增长表达逐渐减弱(P<0.05),但41～60岁组和>60岁组无显著差异(P>0.05)。Ⅱ型胶原基因表达:<20岁组、20～40岁组、41～60岁组和>60岁组均显著高于对照组(0.73±0.16,1.14±0.14,1.46±0.13,1.65±0.16,0.53±0.12,P<0.01),随年龄增长表达逐渐增强(P<0.05)。结论:Ⅰ和Ⅱ型胶原cDNA探针具有高度的特异性,随着年龄的增长,ⅠI型胶原表达逐渐减弱,在附着点处出现Ⅱ型胶原的表达增强,提示成纤维细胞基因表达逐渐减弱,而软骨细胞的基因表达逐渐活跃,为黄韧带退行性变和骨化提供了分子生物学依据。
AIM:To observe the distribution,intensity and changes with age of gene expression of typeⅠand Ⅱcollagen of human lumbar ligamentum flavum of patients at different age by using the specific probe and verify the increase-with-age hypothesis of degeneration and ossification of ligamentum flavum by objective image analysis. METHODS:The study was completed in the central laboratory of Changzheng Hospital,Second Military Medical University from August 2003 to August 2004.Twenty-one ligamentum flava were separated from the patients with lumbar disc herniation(LDH) or with spinal stenosis following vertebral posterior decompression of vertebral lamina.The control specimens were separated from 4 young patients with lumbar spine fracture also following posterior decompression of vertebral lamina.The specimens were all supplied by voluntary patients.The ligamentum flavum specimens from patients with LDH or spinal stenosis were divided into 4 groups based on their age:under 20 years group,20 to 40 years group,41 to 60 years group and over 60 years group.TypeⅠand Ⅱcollagen probes were labeled to perform in situ hybridization and image analysis of frozen sections of ligamentum flavum specimens in the above groups followed by the observation of the expression of typeⅠcollagen(gene phenotypes of fibroblasts) and type Ⅱcollagen(gene phenotypes of chondrocytes). RESULTS:①Observation on tissue sections by in situ hybridizatio:In the under 20 years group,fibrocytes in the insertion site and body of ligamentum flavum arranged orderly,most of which were fusiform,and expression type Ⅰcollagen expressed was obvious,but there was no expression of type Ⅱcollagen.In the 20 to 40 years group,most cells in the ligamentum flavum arranged orderly,but the number of which were decreased relatively,slight fibrosis was found,and a few expression signals of type Ⅰand Ⅱcollagen were observed on the insertion sites of ligamentum flavum.In the 41 to 60 years group,the cells in the body of ligamentum flavum arranged disorderly,the number of which decreased relatively,obvious fibrosis was found;No positive expression was found in body of ligamentum flavum,but weakly positive expression of type Ⅰcollagen was found in insertion site of ligamentum flavum,and the positive cells distributed unevenly;The expression of type Ⅱcollagen was higher than that in the groups under 41 years.In the above 60 years group,fibrosis was found in ligamentum flavum,but no expression signal of type Ⅰcollagen,and the expression signal of type Ⅱcollagen was enhanced in the insertion site of ligamentum flavum.②The expression of type Ⅰcollagen in the groups under 20 years,from 20 to 40 year,from 41 to 60 years,and over 60 years(1.53±0.18,1.34±0.16,0.86±0.13,0.71 ±0.21 respectively) were significantly higher than that in the control group(0.51±0.11)(P< 0.1),and it was decreased with age(P< 0.05),but it was insignificantly different between the groups from 41 to 60 years and over 60 years.The exprssion of type Ⅱcollagen was also higher in the above four groups of age than in the control group(0.73±0.16,1.14±0.14,1.46 ±0.13,1.65±0.16 vs 0.53±0.12,P< 0.01),and it was increased with age(P< 0.05). CONCLUSION:The type Ⅰand Ⅱcollagen cDNA probes are of high specificity.Along with the increasing age,the expression of type Ⅰcollagen is suppressed gradually,but the expression of type Ⅱcollagen is enhanced,indicating that the gene expression of fibroblasts is weakened gradually,and the expression of chondrocytes is activated gradually,so it provides molecular biological basis for the degeneration and ossification of ligamentum flavum.
出处
《中国临床康复》
CSCD
北大核心
2005年第22期82-83,共2页
Chinese Journal of Clinical Rehabilitation
