ANKH基因与强直性脊柱炎异位骨化的相关性研究

发布时间：2019-05-23 08:36

【摘要】：一、AS患者与脊柱损伤患者的韧带组织在成骨矿化程度和ANKH基因含量间的比较[目的]比较AS实验组与JK对照组韧带组织间的成骨矿化差异。探讨ANKH是否参与了AS异位骨化的形成。[方法]分别取AS韧带做实验组,脊柱损伤患者的韧带组织做正常对照组,利用HE染色、茜素红染色和冯库萨染色观察各组织的大体形态和矿化程度。同时行免疫组织化学技术对ANKH的表达进行定位分析,以及行RT-PCR技术检测两组织间ANKH表达是否存在差异。[结果]两种组织HE染色均可见含有较大量的成纤维细胞,且细胞形态未见明显差异。茜素红染色和冯库萨染色结果显示AS关节韧带附着端有明显的矿化,而正常人未见矿化。免疫组化检测发现ANKH主要定位在细胞膜,且其在韧带成纤维细胞中有较高表达。RT-PCR检测发现：ANKH基因在AS实验组与JK对照组间有明显差异,差异具有统计学意义(P0.05)。[结论]AS患者韧带异位骨化起始于关节韧带附着点处,且韧带骨化组织间存在较大量的成纤维细胞。AS患者的韧带组织中ANKH表达较少,ANKH基因可能在AS患者韧带组织的异位成骨过程中起到重要作用。二、原代成纤维细胞的培养及两种组织的成纤维细胞间成骨矿化能力和ANKH基因含量的比较[目的]掌握简单高效的原代培养成纤维细胞的方法。比较两组成纤维细胞间的成骨矿化能力,研究ANKH基因是否参与了AS异位骨化的形成及其可能的参与机制。[方法]两组韧带组织的原代成纤维细胞均采用组织块贴壁法培养获得,并镜下观察两组原代细胞的形态。取第三代细胞行免疫荧光技术鉴定成纤维细胞的含量。分别取两组的第三代成纤维细胞进行矿化诱导培养,并通过检测ALP活力变化及茜素红染色来对比两组成纤维细胞成骨分化、矿化能力的大小。同时行RT-PCR检测两组韧带成纤维细胞间ANKH表达是否存在差异。[结果]通过组织块贴壁培养法成功培养出韧带成纤维细胞,镜下观察细胞形态未见差异,经波形蛋白免疫荧光鉴定成纤维细胞含量在99%以上。碱性磷酸酶(ALP)活性检测及茜素红染色结果均显示,两组细的胞外ALP分泌均增加,并形成一定数量的矿化小结节,但AS实验组的胞外ALP分泌量及矿化小结节数量均较JK对照组高,差异具有统计学意义(P0.05)。RT-PCR检测发现,AS患者韧带成纤维细胞ANKH基因表达明显少于JK对照组,差异具有统计学意义(P0.05)。[结论]组织块贴壁培养法可以简单高效的培养出高纯度的韧带成纤维细胞,韧带成纤维细胞具有成骨潜能,可经诱导分化为成骨细胞,从而发挥成骨矿化功能。体外培养条件下,AS患者韧带成纤维细胞的成骨趋势强于正常对照组,即成骨矿化能力更强,表明AS患者异位骨化与成纤维细胞的成骨分化存在某种关联。AS患者成纤维细胞的ANKH表达明显低于正常对照组,表明其可能参与了AS患者韧带组织的异位骨化形成,但其具体机制仍需要进一步研究。
[Abstract]:1. Comparison of osteogenic mineralization and ANKH gene content between AS patients and spinal cord injury patients [objective] to compare the difference of osteogenic mineralization between AS experimental group and JK control group. To explore whether ANKH is involved in the formation of ectopic ossification of AS. [methods] the ligaments of AS were taken as the experimental group and the ligaments of the patients with spinal injury as the normal control group. The gross morphology and mineralization degree of the tissues were observed by HE staining, alizalin red staining and Fengkusa staining. At the same time, Immunohistochemical technique was used to analyze the expression of ANKH, and RT-PCR technique was used to detect the difference of ANKH expression between the two tissues. [results] HE staining showed a large number of fibroblasts in both tissues, and there was no significant difference in cell morphology between the two tissues. The results of alizalin red staining and Feng Kusa staining showed that there was obvious mineralization at the attached end of AS joint ligament, but no mineralization was found in normal subjects. Immunohistochemical analysis showed that ANKH was mainly located on the cell membrane and highly expressed in ligament fibroblasts. RT-PCR showed that ANKH gene was significantly different between AS experimental group and JK control group. The difference was statistically significant (P 0.05). [conclusion] ectopic ossification of ligament in patients with AS begins at the attachment point of joint ligament, and there are a large number of fibroblasts between ossification tissues of ligament. the expression of ANKH in ligament tissue of patients with as is less. ANKH gene may play an important role in ectopic osteogenesis of ligament tissue in AS patients. Second, the culture of primary fibroblasts and the comparison of osteogenic mineralization ability and ANKH gene content between the two kinds of fibroblasts [objective] to master a simple and efficient method of primary cultured fibroblasts. The osteogenic mineralization ability between the two fiber cells was compared to study whether ANKH gene was involved in the formation of ectopic ossification of AS and its possible mechanism. [methods] the primary fibroblasts of ligaments in both groups were cultured by tissue block adherent method, and the morphology of primary fibroblasts in the two groups was observed under microscope. The content of fibroblasts was identified by immunofluorescence technique. The third generation fibroblasts of the two groups were cultured for mineralization induction, and the osteogenic differentiation and mineralization ability of the two groups were compared by detecting the changes of ALP activity and alizalin red staining. At the same time, RT-PCR was performed to detect the difference of ANKH expression between the two groups. [results] ligamentous fibroblasts were successfully cultured by tissue block adherent culture. There was no difference in cell morphology under microscope. The content of fibroblasts was more than 99% by vimentin immunofluorescence. The results of alkaline phosphatase (ALP) activity and alizalin red staining showed that the secretion of fine extracellular ALP increased and a certain number of small mineralization nodules were formed in both groups. However, the extracellular ALP secretion and the number of small mineralization nodules in AS group were significantly higher than those in JK control group (P 0.05). RT-PCR showed that the expression of ANKH gene in ligament fibroblasts of AS patients was significantly lower than that of JK control group. The difference was statistically significant (P 0.05). [conclusion] High purity ligament fibroblasts can be cultured simply and efficiently by tissue adherent culture. Ligament fibroblasts have osteogenic potential and can be induced to differentiate into osteoblasts, thus giving full play to osteogenic mineralization. Under the condition of in vitro culture, the osteogenic trend of ligament fibroblasts in AS patients was stronger than that in normal controls, that is, the osteogenic mineralization ability was stronger. The results showed that there was a certain correlation between ectopic ossification and osteogenic differentiation of fibroblasts in patients with AS. The expression of ANKH in fibroblasts of patients with as was significantly lower than that of the normal control group, indicating that it may be involved in the formation of ectopic ossification of ligaments in patients with AS. However, its specific mechanism still needs to be further studied.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.23

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