ANKH基因與強(qiáng)直性脊柱炎異位骨化的相關(guān)性研究
發(fā)布時(shí)間：2019-05-23 08:36
【摘要】：一、AS患者與脊柱損傷患者的韌帶組織在成骨礦化程度和ANKH基因含量間的比較[目的]比較AS實(shí)驗(yàn)組與JK對(duì)照組韌帶組織間的成骨礦化差異。探討ANKH是否參與了AS異位骨化的形成。[方法]分別取AS韌帶做實(shí)驗(yàn)組,脊柱損傷患者的韌帶組織做正常對(duì)照組,利用HE染色、茜素紅染色和馮庫(kù)薩染色觀察各組織的大體形態(tài)和礦化程度。同時(shí)行免疫組織化學(xué)技術(shù)對(duì)ANKH的表達(dá)進(jìn)行定位分析,以及行RT-PCR技術(shù)檢測(cè)兩組織間ANKH表達(dá)是否存在差異。[結(jié)果]兩種組織HE染色均可見含有較大量的成纖維細(xì)胞,且細(xì)胞形態(tài)未見明顯差異。茜素紅染色和馮庫(kù)薩染色結(jié)果顯示AS關(guān)節(jié)韌帶附著端有明顯的礦化,而正常人未見礦化。免疫組化檢測(cè)發(fā)現(xiàn)ANKH主要定位在細(xì)胞膜,且其在韌帶成纖維細(xì)胞中有較高表達(dá)。RT-PCR檢測(cè)發(fā)現(xiàn)：ANKH基因在AS實(shí)驗(yàn)組與JK對(duì)照組間有明顯差異,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。[結(jié)論]AS患者韌帶異位骨化起始于關(guān)節(jié)韌帶附著點(diǎn)處,且韌帶骨化組織間存在較大量的成纖維細(xì)胞。AS患者的韌帶組織中ANKH表達(dá)較少,ANKH基因可能在AS患者韌帶組織的異位成骨過程中起到重要作用。二、原代成纖維細(xì)胞的培養(yǎng)及兩種組織的成纖維細(xì)胞間成骨礦化能力和ANKH基因含量的比較[目的]掌握簡(jiǎn)單高效的原代培養(yǎng)成纖維細(xì)胞的方法。比較兩組成纖維細(xì)胞間的成骨礦化能力,研究ANKH基因是否參與了AS異位骨化的形成及其可能的參與機(jī)制。[方法]兩組韌帶組織的原代成纖維細(xì)胞均采用組織塊貼壁法培養(yǎng)獲得,并鏡下觀察兩組原代細(xì)胞的形態(tài)。取第三代細(xì)胞行免疫熒光技術(shù)鑒定成纖維細(xì)胞的含量。分別取兩組的第三代成纖維細(xì)胞進(jìn)行礦化誘導(dǎo)培養(yǎng),并通過檢測(cè)ALP活力變化及茜素紅染色來對(duì)比兩組成纖維細(xì)胞成骨分化、礦化能力的大小。同時(shí)行RT-PCR檢測(cè)兩組韌帶成纖維細(xì)胞間ANKH表達(dá)是否存在差異。[結(jié)果]通過組織塊貼壁培養(yǎng)法成功培養(yǎng)出韌帶成纖維細(xì)胞,鏡下觀察細(xì)胞形態(tài)未見差異,經(jīng)波形蛋白免疫熒光鑒定成纖維細(xì)胞含量在99%以上。堿性磷酸酶(ALP)活性檢測(cè)及茜素紅染色結(jié)果均顯示,兩組細(xì)的胞外ALP分泌均增加,并形成一定數(shù)量的礦化小結(jié)節(jié),但AS實(shí)驗(yàn)組的胞外ALP分泌量及礦化小結(jié)節(jié)數(shù)量均較JK對(duì)照組高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。RT-PCR檢測(cè)發(fā)現(xiàn),AS患者韌帶成纖維細(xì)胞ANKH基因表達(dá)明顯少于JK對(duì)照組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。[結(jié)論]組織塊貼壁培養(yǎng)法可以簡(jiǎn)單高效的培養(yǎng)出高純度的韌帶成纖維細(xì)胞,韌帶成纖維細(xì)胞具有成骨潛能,可經(jīng)誘導(dǎo)分化為成骨細(xì)胞,從而發(fā)揮成骨礦化功能。體外培養(yǎng)條件下,AS患者韌帶成纖維細(xì)胞的成骨趨勢(shì)強(qiáng)于正常對(duì)照組,即成骨礦化能力更強(qiáng),表明AS患者異位骨化與成纖維細(xì)胞的成骨分化存在某種關(guān)聯(lián)。AS患者成纖維細(xì)胞的ANKH表達(dá)明顯低于正常對(duì)照組,表明其可能參與了AS患者韌帶組織的異位骨化形成,但其具體機(jī)制仍需要進(jìn)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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R593.23
本文編號(hào)：2483752
[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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R593.23
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