强直性脊柱炎病理性成骨的分子机制研究
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摘要
强直性脊柱炎(ankylosing spondylitis,AS)是一种全身性免疫性疾病,其病变最终结局是脊柱周围韧带和椎间盘骨化,中轴关节出现纤维化及骨性强直,导致关节运动功能丧失。可见病理性成骨是AS患者致残的主要原因,强直性脊柱炎的治疗最终必须解决正常韧带及滑膜骨化强直问题。
BMP2、bFGF和Cbfa1均为具有明确成骨作用的细胞因子。骨形态发生蛋白(BMP)又称为转化生长因子蛋白酶,主要位于骨髓基质细胞、骨膜细胞和骨细胞内。BMP具有诱导成骨与促进成骨细胞分化的能力,能够诱导血管、肌肉和筋膜周围游离的和未分化的间充质细胞以及纤维细胞,转化为不可逆的骨系细胞,从而促进骨折愈合,也可在骨骼以外产生软骨和骨组织。其中BMP2成骨作用最强,也是唯一能单独诱导成骨的因子,在骨组织修复中最常用到。碱性成纤维细胞生长因子(bFGF)是一种能广泛促进来源于中胚层及神经外胚层细胞增殖的生物活性蛋白。bFGF可以促进成骨细胞增殖,它与生物材料联合植入动物模型体内能明显促进骨形成,在骨组织工程中被广泛应用。同时它还是一种强大的毛细血管增殖刺激剂,能为骨痂生长提供充足的血供和营养。bFGF主要由成骨细胞合成,储存于骨细胞外基质中,通过自分泌和旁分泌发挥作用。核心蛋白结合因子(Cbfa1)作为成骨分化关键性的转录因子,具有明确的成骨细胞分化和成骨效应。基因敲除实验证实Cbfa1缺陷的纯合小鼠无骨的形成,杂合鼠则出现成骨能力下降。Cbfa1也可与其它成骨因子如骨形成蛋白等相互协同作用,促进成骨。强直性脊柱炎存在着病理性成骨过程,其局部成骨因子很可能异常表达。
成纤维细胞来源于间充质细胞,具有成骨特性,可向成骨细胞定向分化。它可以认为是一种原始的成骨祖细胞。已有学者体外培养强直性脊柱炎棘上韧带成纤维细胞,观察到其基质中沉积了大量的钙盐颗粒,成功转化为成骨细胞,证明关节韧带中成纤维细胞是强直性脊柱炎病理性成骨的靶向细胞。
本研究以股骨颈骨折患者为对照,通过免疫组化及原位杂交技术检测强直性脊柱炎患者骶髂关节与对照组髋关节滑膜组织中BMP2、bFGF和Cbfa1的表达情况。同时体外培养腰椎骨折患者棘上韧带成纤维细胞,培养基中加入BMP2、bFGF和Cbfa1,观察其向成骨细胞转化情况,以探讨BMP2、bFGF和Cbfa1在AS患者病理性成骨过程中的表达及意义。
主要实验结果及结论如下:
1.免疫组化检查结果提示:AS病人骶髂关节滑膜组织标本中Cbfa1、BMP2、bFGF蛋白阳性表达。而对照组股骨颈骨折患者髋关节滑膜组织标本中Cbfa1、BMP2、bFGF蛋白阴性表达。
2.原位杂交检查结果提示:AS病人骶髂关节滑膜组织标本中Cbfa1、BMP2、bFGFmRNA阳性表达。而对照组股骨颈骨折患者髋关节滑膜组织标本中Cbfa1、BMP2、bFGFmRNA阴性表达。
3.体外培养腰椎骨折患者脊上韧带成纤维细胞,通过培养基中加入Cbfa1,BMP2和bFGF,观察到成纤维细胞向成骨细胞转化。免疫组化技术检测转化后的细胞,转化后的细胞成骨细胞标志物碱性磷酸酶(AKP)阳性表达,证实成纤维细胞顺利转化为成骨细胞。
Ankylosing spondying was a kind of general immune disease.Vertebral column surrounding ligaments and discus intervertebralis ossification were the final pathological changes.Central axis joints emerged fibrous degeneration and tetanization,then movement function of joints were lost.It’s thus clear that pathological bone formation was the most important cause for mutilation of patients with AS.If we wanted to treat AS,we must solve the ossification tetanization of the ligaments and synovium tissues.
BMP2、bFGF and Cbfa1 were all definite osteogenesis. Bone morphogenetic protein(BMP) were also called transforming growthfactor protease,They often situated marrow stroma cells、periosts cells and frame cells.BMP could induce bone formation and encourage osteoblast differentiation.They transformed mesenchymals and fibrocytes of emissary、muscle and fascia into frame systema cells,so BMP were able to promote the union of fracture.BMP2 was the most powerful factor and could solo induce bone formation.Basic fibroblast growth factor(bFGF) was one kind of biological activity protein,it could hasten scleroblast proliferation.bFGF and biomaterial could be implanted into the animal model and obviously promote skeleton formation.So it often was used in bone tissue engineering.bFGF also could propagate blood capillary and provid sufficient blood supply and nourishment for osteotylus.bFGF was principally synthetized by skeltogenous cells and saved in extracellular matrix.Core-binding factor a1(Cbfa1) was one of critical transcription factor in bone formation and differentiation.Gene knock-out experiments confirmed that homozygosis mice without Cbfa1 formed no bone,heterozygosis mice emerged degression of bone formation.Cbfa1 and other bone formation factors had synergism action to encourage bone formation.
MES cells were the source of the fibroblasts.Fibroblasts could be differentiated into osteoblasts,they’re more archaic osteogenitor cells.Somebody cultured in vitro the fibroblast from supraspinal ligament of AS and observed that a lot of calcium salts deposited in the base material.
In our experiment,we used in situ hybridization and immunohistochemical analysis to detect the expression of BMP2、Cbfa1 and bFGF in the synovial tissues of patients’cacroiliac joint with active AS,compared the results with those in the patients with femoral neck fracture.At the same time,we collected fibroblasts from patients’supraspinal ligament with fracture of lumbar vertebra and cultured in vitro.In experiment groups,we added BMP2、Cbfa1 and bFGF into the substratum.As control groups,we didn’t add any osteogenesis cytokines.We observed them through microscope and used immunohistochemistry to detect the expressions of Alkali phosphatase (AKP). The important results and conclusions are as follow:
1.Immunohistochemical analysis showed that BMP2、Cbfa1 and bFGF in the synovial tissues of patient’s cacroiliac joint with active AS were positive expressions and the control groups were negative expressions.
2.In situ hybridization showed the same results as immunohistochemical analysis.
3.We cultured in vitro the fibroblasts from supraspinal ligament of fracture of lumbar vertebra and added BMP2、Cbfa1 and bFGF into the substratum.The collagenoblasts transformed into osteoblasts successfully. Alkali phosphatase(AKP) were positive expressions in the variant cells.
BMP2、bFGF和Cbfa1均为具有明确成骨作用的细胞因子。骨形态发生蛋白(BMP)又称为转化生长因子蛋白酶,主要位于骨髓基质细胞、骨膜细胞和骨细胞内。BMP具有诱导成骨与促进成骨细胞分化的能力,能够诱导血管、肌肉和筋膜周围游离的和未分化的间充质细胞以及纤维细胞,转化为不可逆的骨系细胞,从而促进骨折愈合,也可在骨骼以外产生软骨和骨组织。其中BMP2成骨作用最强,也是唯一能单独诱导成骨的因子,在骨组织修复中最常用到。碱性成纤维细胞生长因子(bFGF)是一种能广泛促进来源于中胚层及神经外胚层细胞增殖的生物活性蛋白。bFGF可以促进成骨细胞增殖,它与生物材料联合植入动物模型体内能明显促进骨形成,在骨组织工程中被广泛应用。同时它还是一种强大的毛细血管增殖刺激剂,能为骨痂生长提供充足的血供和营养。bFGF主要由成骨细胞合成,储存于骨细胞外基质中,通过自分泌和旁分泌发挥作用。核心蛋白结合因子(Cbfa1)作为成骨分化关键性的转录因子,具有明确的成骨细胞分化和成骨效应。基因敲除实验证实Cbfa1缺陷的纯合小鼠无骨的形成,杂合鼠则出现成骨能力下降。Cbfa1也可与其它成骨因子如骨形成蛋白等相互协同作用,促进成骨。强直性脊柱炎存在着病理性成骨过程,其局部成骨因子很可能异常表达。
成纤维细胞来源于间充质细胞,具有成骨特性,可向成骨细胞定向分化。它可以认为是一种原始的成骨祖细胞。已有学者体外培养强直性脊柱炎棘上韧带成纤维细胞,观察到其基质中沉积了大量的钙盐颗粒,成功转化为成骨细胞,证明关节韧带中成纤维细胞是强直性脊柱炎病理性成骨的靶向细胞。
本研究以股骨颈骨折患者为对照,通过免疫组化及原位杂交技术检测强直性脊柱炎患者骶髂关节与对照组髋关节滑膜组织中BMP2、bFGF和Cbfa1的表达情况。同时体外培养腰椎骨折患者棘上韧带成纤维细胞,培养基中加入BMP2、bFGF和Cbfa1,观察其向成骨细胞转化情况,以探讨BMP2、bFGF和Cbfa1在AS患者病理性成骨过程中的表达及意义。
主要实验结果及结论如下:
1.免疫组化检查结果提示:AS病人骶髂关节滑膜组织标本中Cbfa1、BMP2、bFGF蛋白阳性表达。而对照组股骨颈骨折患者髋关节滑膜组织标本中Cbfa1、BMP2、bFGF蛋白阴性表达。
2.原位杂交检查结果提示:AS病人骶髂关节滑膜组织标本中Cbfa1、BMP2、bFGFmRNA阳性表达。而对照组股骨颈骨折患者髋关节滑膜组织标本中Cbfa1、BMP2、bFGFmRNA阴性表达。
3.体外培养腰椎骨折患者脊上韧带成纤维细胞,通过培养基中加入Cbfa1,BMP2和bFGF,观察到成纤维细胞向成骨细胞转化。免疫组化技术检测转化后的细胞,转化后的细胞成骨细胞标志物碱性磷酸酶(AKP)阳性表达,证实成纤维细胞顺利转化为成骨细胞。
Ankylosing spondying was a kind of general immune disease.Vertebral column surrounding ligaments and discus intervertebralis ossification were the final pathological changes.Central axis joints emerged fibrous degeneration and tetanization,then movement function of joints were lost.It’s thus clear that pathological bone formation was the most important cause for mutilation of patients with AS.If we wanted to treat AS,we must solve the ossification tetanization of the ligaments and synovium tissues.
BMP2、bFGF and Cbfa1 were all definite osteogenesis. Bone morphogenetic protein(BMP) were also called transforming growthfactor protease,They often situated marrow stroma cells、periosts cells and frame cells.BMP could induce bone formation and encourage osteoblast differentiation.They transformed mesenchymals and fibrocytes of emissary、muscle and fascia into frame systema cells,so BMP were able to promote the union of fracture.BMP2 was the most powerful factor and could solo induce bone formation.Basic fibroblast growth factor(bFGF) was one kind of biological activity protein,it could hasten scleroblast proliferation.bFGF and biomaterial could be implanted into the animal model and obviously promote skeleton formation.So it often was used in bone tissue engineering.bFGF also could propagate blood capillary and provid sufficient blood supply and nourishment for osteotylus.bFGF was principally synthetized by skeltogenous cells and saved in extracellular matrix.Core-binding factor a1(Cbfa1) was one of critical transcription factor in bone formation and differentiation.Gene knock-out experiments confirmed that homozygosis mice without Cbfa1 formed no bone,heterozygosis mice emerged degression of bone formation.Cbfa1 and other bone formation factors had synergism action to encourage bone formation.
MES cells were the source of the fibroblasts.Fibroblasts could be differentiated into osteoblasts,they’re more archaic osteogenitor cells.Somebody cultured in vitro the fibroblast from supraspinal ligament of AS and observed that a lot of calcium salts deposited in the base material.
In our experiment,we used in situ hybridization and immunohistochemical analysis to detect the expression of BMP2、Cbfa1 and bFGF in the synovial tissues of patients’cacroiliac joint with active AS,compared the results with those in the patients with femoral neck fracture.At the same time,we collected fibroblasts from patients’supraspinal ligament with fracture of lumbar vertebra and cultured in vitro.In experiment groups,we added BMP2、Cbfa1 and bFGF into the substratum.As control groups,we didn’t add any osteogenesis cytokines.We observed them through microscope and used immunohistochemistry to detect the expressions of Alkali phosphatase (AKP). The important results and conclusions are as follow:
1.Immunohistochemical analysis showed that BMP2、Cbfa1 and bFGF in the synovial tissues of patient’s cacroiliac joint with active AS were positive expressions and the control groups were negative expressions.
2.In situ hybridization showed the same results as immunohistochemical analysis.
3.We cultured in vitro the fibroblasts from supraspinal ligament of fracture of lumbar vertebra and added BMP2、Cbfa1 and bFGF into the substratum.The collagenoblasts transformed into osteoblasts successfully. Alkali phosphatase(AKP) were positive expressions in the variant cells.
引文
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2. Collantes-Estevez E,Cisnal DMA,Munoz-Gmariz E.A ssessment of 2 systems of spondylarthropathy diagnostic and cliassification criteria (Amor and ESSG) by a spanish muiticenter study.Rheumatol,1995,22:246-251.
3.施桂英.幼年脊柱关节病的早期特征[J].中华风湿病学杂志,1999,3:131-132.
4.陈国敏,王东辰.人白细胞抗原-B27在强直性脊柱炎中的意义[J].检验医学与临床,2007,4(10):931-933.
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6. Marks D J; Mitchison N A; Segal A W; Sieper J. Can unresolved infection precipitate autoimmune disease? Curr-Top-Microbiol-Immunol. 2006; 305: 105-25.
7. Rehakova Z, Capkova J, Stepankova R, etal.Germ-free mice do not develop ankylosing enthesopathy,a spontaneous joint disease. Hum Immunol 2000,61:555–558.
8.张秀华,徐少丽.强直性脊柱炎免疫学指标的检测及临床意义[J].中国社区医师(医学专业半月刊),2008,08(10):123-124.
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