高纯度猪软骨Ⅱ型胶原的制备与检测

探讨以猪透明软骨为原材料制备高纯度Ⅱ型胶原的方法,为批量化生产提供依据。以猪透明软骨为原料,采用盐酸胍抽提蛋白多糖,酸性条件下酶解,中间过程经多步纯化去除杂胶原、降解及变性产物,最后经Sepharose H.P.阴离子柱层析纯化制备出产品并以Sigma公司产品作对照。SDS－PAGE电泳、氨基酸成份分析和紫外最大吸收光谱的结果表明,所提取的Ⅱ型胶原性质符合文献报导,纯度比Sigma公司的产品高。所提取的Ⅱ型胶原为高纯度的Ⅱ型胶原,由于采用肉用猪作原料,来源丰富,成本低,适合批量化的产品开发。     

猪软骨Ⅱ型胶原的制备及理化性能分析

目的建立一套完善的提取、纯化、检测Ⅱ型胶原的生产系统,供临床和科研应用.方法选取新鲜的猪透明软骨,经盐酸胍、胃蛋白酶等多步抽提,以及DEAE纤维素的柱层析,制备出高纯度的Ⅱ型胶原.结果通过SDS-PAGE电泳、氨基酸成分分析及羟脯氨酸的测定、紫外最大吸收光谱、电镜检测证实本法所制备的Ⅱ型胶原比Sigma(9007-34-5)公司的产品纯度高.结论用此方法制备的Ⅱ型胶原,材料来源广泛,程序简便,产品纯度高,易于大批量生产.     

检测体内异种II型胶原异体移植引起的细胞毒反应

检测用猪II型胶原免疫新西兰大白兔的异种异体细胞免疫反应。用II型胶原免疫新西兰大白兔60 d,定期抽取血浆检测抗II型胶原抗体;第60 d取兔的外周血淋巴细胞、兔脾细胞、淋巴结分别分离淋巴细胞,进行体外二次II型胶原刺激,检测由此引起的反应性的细胞增殖规律。实验分为二组,第一组加入不同浓度植物血凝素(PHA)作阳性对照,并测定非特异性免疫;第二组加入不同浓度II型胶原,检测特异性免疫。正常兔的淋巴细胞在PHA剌激下发生增殖,但对II型胶原的第一次剌激不发生增殖,而免疫兔对PHA和II型胶原的剌激均能发生显著的增殖。表明异种II型胶原在一定浓度下,可以引起免疫兔的抗II型胶原抗体的升高,并可引起兔脾、外周血淋巴细胞增殖,异种II型胶原能在体内引起细胞免疫反应。     

鸡胶原II型诱导大鼠类风关模型的建立

用鸡胶原II型 (chickencollagenII,CCII)和完全弗氏佐剂免疫Wistar大鼠诱导胶原性关节炎 ,建立自身免疫性类风湿性关节炎 (RA)实验动物模型。以ELISA法检测实验组和对照组动物血清中的CCII抗体的水平 ,同时采用组织病理学和X射线摄片的方法显示关节炎大鼠的发病程度和病理学特征 ,并对其血液和滑膜液进行微生物学鉴定。结果显示 ,用CCII和完全弗氏佐剂免疫的实验组大鼠关节炎的发病率为 80 %,同时在免疫后 2 5d ,关节炎指数为最高 (AI=7 8)。ELISA法检测抗CCII抗体结果显示 ,实验组大鼠血清中抗CCII抗体的水平均较各对照组大鼠明显增高 (P <0 0 0 1)。组织病理学和X 射线摄片结果显示 ,关节软骨组织、骨组织和滑膜组织呈典型的关节炎病变。微生物鉴定结果均为阴性。本实验成功建立了胶原诱导的大鼠关节炎模型 ,且其病理特征和人类RA极为相似 ,为进一步深入研究人类RA的发病机制及其临床治疗提供了有价值的实验材料。     

口服鸡II型胶原治疗大鼠实验性类风湿性关节炎的研究

本文研究口服鸡II型胶原 (CCII)治疗大鼠类风湿性关节炎的作用。以CCII和完全弗氏佐剂免疫Wistar大鼠 ,建立大鼠的类风湿性关节炎 (RA )模型。用CCII进行口服 ,观察实验组和对照组动物关节炎的发病程度。并以ELISA法对发病大鼠血清中的抗CCII抗体的水平进行检测。结果表明 ,口服CCII可以减轻大鼠关节炎的发病程度。其中口服 6μg、 60 μgCCII的实验组大鼠关节炎指数均较对照组明显减低 ,且差异有显著性意义 (P <0 0 1;P <0 0 5 )。而口服 60 0 μgCCII的实验组大鼠关节炎指数虽较对照组大鼠降低 ,但二者相比 ,无显著性差异 (P >0 0 5 )。ELISA检测结果显示 :口服低剂量 (6μg和 60μg )可溶性CCII对大鼠体内抗CCII抗体的产生有一定的抑制作用。口服CCII对关节炎大鼠的发病程度有较明显的抑制作用 ,为应用口服耐受治疗RA等自身免疫性疾病提供了实验基础     

利用II型胶原变构肽抑制类风湿关节炎患者T细胞活化的研究

为了研究II型胶原(CII)变构肽对类风湿性关节炎(RA)患者外周血T细胞激活的抑制作用,以固相法合成CII263-272原型肽及3条变构肽,计算机模拟分析变构肽与HLA-DR4分子的结合能力3。H掺入法检测61例RA患者外周血单个核细胞对CII263-272原型肽及变构肽的T细胞增殖反应。ELISA法检测CII263-272原型肽及变构肽刺激下外周血T细胞IL-2及IFN-γ的分泌。竞争抑制试验观察CII变构肽对原型肽介导T细胞激活的抑制作用。计算机模拟结果发现,CII263-272原型肽可与HLA-DR4分子结合,在此基础上替换TCR结合位点267位谷氨酰胺、270位赖氨酸和271或265位甘氨酸为丙氨酸,不影响该多肽与HLA-DR4分子的结合能力。CII变构肽在RA患者外周血T细胞具增殖中有明显低反应性,下调IL-2和IFN-γ的产生,并且可特异性抑制CII原型肽诱导的T细胞活化(P<0.05或P<0.01)。以上结果提示,替换CII263-272多肽中与T细胞受体结合的氨基酸形成的CII变构肽可抑制RA患者外周血T细胞活化,可能在本病的免疫治疗中有重要意义。     

抗Ⅲ型前胶原肽单克隆抗体的制备及初步应用

制备出两株分泌抗ＰⅢＰ（Ⅲ型前胶原肽）单克隆抗体的杂交瘤细胞株。竞争抑制ＥＬＩＳＡ试验表明：两株单抗与ＰＣＩ（Ⅰ型前胶原）及ＣⅢ（Ⅲ型胶原）无交叉反应。Ｄｏｔ－ＥＬＩＳＡ示两株单抗钧针对ＰⅢＰ构象决定簇。应用所制备的单抗对人肝细胞癌组织及大鼠、小鼠成纤维母细胞进行免疫定位。结果显示两种成纤维母细胞呈明显膜阳性；肝癌细胞浆内呈粗颗粒状染色，提示肝癌细胞具有合成Ⅲ型前胶原的能力。     

ELISA定量检测Ⅳ型胶原方法的建立

利用双抗体夹心法研制Ⅳ型胶原ELISA诊断试剂，检测血清中的Ⅳ型胶原。结果表明，标准曲线工作范围为16-1000ng／mL，灵敏度可达8ng／mL，批内及批间变异系数小于12％。本法灵敏、准确和特异性好。     

肝病患者血清Ⅲ型胶原抗体的检测分析

肝病患者血清Ⅲ型胶原抗体的检测分析陈黎明李跃旗辛绍杰程云张士兰张晓峰赵军董漪张鸿飞肝纤维化是肝硬变的早期，对肝纤维化的全面了解，将有助于肝硬变防治或逆转〔１〕。肝纤维化发生时，Ⅲ型胶原和其它胶原在肝脏内大量沉积，同时血清中也有一定量的胶原游离，与肝脏...     

鱿鱼软骨Ⅱ型胶原蛋白提取方法及结构分析

目的从鱿鱼软骨中分离纯化非变性II型胶原蛋白,并对其进行结构表征。方法将鱿鱼软骨冻干后粉碎成200目软骨粉,用4%(w/w)Na OH溶液去除多糖、0.5 M EDTA溶液脱灰对软骨进行前处理,采用胃蛋白酶水解提取,Na Cl盐析用纯水透析后冷冻干燥得到II型胶原蛋白样品。通过氨基酸组成分析、SDS-PAGE电泳、紫外吸收光谱、电镜扫描等对制备的样品鉴定。结果制备的II型胶原蛋白具有3条α1链,分子量112 k Da,在224.5 nm波长处有最大紫外吸收,具有三螺旋结构特征。     

Cyclic Loading Can Denature Type II Collagen in Articular Cartilage

We sought to explain previously reported associations between osteoarthritis, cartilage collagen denaturation, and repetitive mechanical loading. Eighty specimens of cartilage-on-bone were obtained from bovine patella grooves. After soaking in protease inhibitors, specimens were subjected to 3,600 cycles of compressive loading by means of a flat indenter. Loaded cartilage was supported by adjacent cartilage and subchondral bone. Peak stress ranged between 3.5 MPa and 14 MPa. Collagen denaturation was assessed by an inhibition ELISA assay using the col2–3/4 m antibody. Results showed that denaturation increased with loading severity, from 2% in control (unloaded) cartilage to 7.5% at 14 MPa (p <. 0003). Collagen and glycosaminoglycan content of cartilage were unchanged after loading. Denaturation was largely unaffected by the absence of protease inhibitors or when chondrocytes were killed by repeated freeze-thaw cycles prior to loading. We conclude that vigorous cyclic mechanical loading causes an immediate dose-related increase in collagen denaturation in bovine articular cartilage.     

胶原纤维束对软骨力学性能的影响

目的 研究胶原纤维束对软骨力学性能的影响,为临床医生指导早期软骨损伤患者的康复运动提供参考。方法 建立一种纤维增强的多孔黏弹性二维数值模型,考虑纤维分布、弹性模量、孔隙率和渗透率随软骨深度的变化关系。研究纤维束局部断裂和从表面渐进断裂以及纤维束尺寸对软骨力学性能的影响,获得软骨基质的最大主应变。结果 基质的最大主应变出现在软骨中层靠上某个位置,此位置不受纤维断裂模式和纤维束尺寸的影响。含较粗纤维束软骨的应变降低。结论 软骨中层易发生力学损伤,纤维束增粗可以降低基质的最大主应变,一旦纤维束发生断裂,较粗纤维束的软骨的基质最大主应变更大,使软骨更易发生损伤演化情况。     

Immunohistochemical Demonstration of Type II Collagen in the Chondroid Tissue of Pleomorphic Adenomas of the Salivary Glands

A study was done to investigate the presence of type II collagen and elastin in the metaplastic chondroid tissue of 21 pleornorphic adenomas of the major and minor salivary glands. Type II collagen was detected with anti-bovine type II collagen antibody after double digestion of histological sections with trypsin and hyaluronidase. The immunoreaction was positive in the chondrocytic cells and intercellular matrix. Elastic fibers in the chondroid tissue were found by orcein staining; they were scarce and randomly distributed. Although the presence of type II collagen and elastin in the metaplastic chondroid tissue is not directly implicated in the genesis of the tumor, it reveals a unique and high grade of cellular differentiation in comparison with true cartilage.     

牛骨胶原基质的制备和性能研究

研究了从牛密质骨制备用作生物医学异种移植物的加工方法。将从屠宰场获得的牛腿骨骨干中部密质骨切割成一定的形状,然后经过脱脂和脱蛋白,制得可加工成任意形状的材料。用脱脂剂、非胶原蛋白除剂和蛋白酶消化剂等进行处理,脱除非胶原蛋白并切除胶原端肽,骨块经干燥,γ射线消毒后,密封包装。并进行各种理化性能测试,包括红外光谱、X射线衍射光谱、差热扫描分析(DSC)、单轴拉伸力学性能测试以及扫描电镜观察。结果表明,经过一系列处理后,获得的骨胶原基质(BBCM)是一种白色、具有一定孔隙结构的材料,保留了天然骨的无机成分和大部分胶原蛋白,具有良好的力学性能和较低的免疫原性,是一种潜在的骨移植材料和组织工程基质材料。     

Localization of Carboxy-terminal Type II Procollagen Peptide (pCOL-II-C) and Type II Collagen in the Repair Tissue of Full-thickness Articular Cartilage Defect

It is well established that a full-thickness articular cartilage defect is repaired with a fibrocartilaginous tissue of which cells are derived from undifferentiated mesenchymal stem cells in the bone marrow. To characterize the repair tissue immunohistochemically, full-thickness defects were created in rabbit knee joints, and the repair tissues immunostained at 3, 6, and 12 weeks after surgery. Well characterized polyclonal antibody against carboxy-terminal type II procollagen peptide (pCOL-II-C) and monoclonal antibody against type II collagen were used to evaluate the repair tissue with regard to the metabolism of type II collagen. lmmunohistochemistry revealed that pCOL-II-C was localized in or around most of the repair cells obtained at 3 and 6 weeks after surgery, while type II collagen distributed mainly in the pericellular matrix of metaplastic round-shaped repair cells. The results suggest that the repair cells taken at the early stage of the repair process of the defect could originally have more activity of type II collagen synthesis.     

N-Telopeptide of Type II Collagen Interacts with Annexin V on Human Chondrocytes

Type II collagen binds to chondrocytes through integrins and annexin V. While the potential integrin binding sites have been identified, it is unclear which domains bind to annexin V. Proteolytic fragments of collagen are known to modulate cell signaling pathways resulting in degradation of articular cartilage; it is unknown whether annexin V binds to the fragments. The focus of our study was to determine the binding of type II collagen and its fragments to chondrocytes using flow cytometry and fluorescence microscopy. The N-telopeptide binds to annexin V, whereas the C-telopeptide and triple helical peptides do not. These data suggest that the binding of the N-telopeptide of type II collagen is through annexin V, whereas binding of the C-telopeptide and the triple helical peptide to the surface of chondrocytes are potentially facilitated through other collagen receptors, such as integrins or cell-associated matrix proteins.