bBMP对体外培养人胎儿关节软骨细胞分化的促进作用

为了解运动创伤中肌肉、肌腱、关节囊中异位化骨的机制及软骨细胞在此过程中所起的作用，本研究用牛骨形态发生蛋白（ｂＢＭＰ）诱导体外不传代培养的胎儿关节软骨细胞，发现：在ｂＢＭＰ的诱导下，不传代培养的软骨细胞逐渐变为成骨细胞样形态，它们表达的碱性磷酸酶明显增加，细胞周围的钙沉积和基质沉积也明显增加。在ｂＢＭＰ的诱导下，关节软骨细胞开始表达具有成骨细胞特异性和骨基质特异性的骨钙素。这些结果表明，在ｂＢＭＰ的诱导下，胎儿关节软骨细胞已开始表达成骨细胞表型，并向成骨细胞样细胞方向分化。本研究提示，在异位化骨和各种软骨内化骨过程中，软骨细胞可能具有较活跃的参与骨形成的作用，可能不像以前所假设的那样仅分化为肥大软骨细胞，然后变性，最终坏死。     

骨形态发生蛋白和转化生长因子—β及碱性成纤维细胞生长因？…

观察骨形态发生蛋白（ＢＭＰ）、转化生长因子－β、碱性成纤维细胞生长因子在骨折修复中的表达及分布情况,进而探讨其作用机制。方法以成年雄性新西兰兔为研究对象,制作桡骨骨折愈合模型。伤后不同时期处死取材,分别进行这和ＢＭＰ、ＴＧＦ－β和ｂＦＧＦ免疫组化染色观察。结果（１）伤后３ｄ开始形成原始骨痂。１周时肉芽组织中的间质细胞开始分化为软骨细胞,软骨形成后再进行软骨内化骨。４周时形成连接骨折端的桥接骨痂。２     

骨折愈合过程中BMP2和TGFβ1的基因表达及分析

目的：探讨骨形成蛋白（ＢＭＰ）和转化生长因子β（ＴＧＦβ）在骨生长愈合中的组织细胞来源、作用方式及二者的关系．方法：建立颌骨骨折模型，利用原位杂交方法检测与骨组织关系较为密切的ＢＭＰ２和ＴＧＦβ１ｍＲＮＡ在骨折愈合不同时期的表达．结果：处于分化状态的未分化间充质细胞内ＢＭＰ２ｍＲＮＡ的水平较高，处于功能活跃期的成骨细胞和软骨细胞内ＴＧＦβ１ｍＲＮＡ的水平较高．ＢＭＰ２的基因表达水平在骨折愈合的膜内成骨阶段较高；ＴＧＦβ１的基因表达水平在骨折愈合的软骨形成期和软骨骨化阶段较高．结论：未分化间充质细胞与ＢＭＰ的合成有关，成骨细胞和软骨细胞与ＴＧＦβ的合成有关．ＢＭＰ在骨折愈合早期的骨诱导中发挥重要作用，使未分化间充质细胞向骨细胞系分化，而ＴＧＦβ则主要在骨折愈合后期调节成骨细胞和软骨细胞的增殖，并增强其合成基质的能力．     

PDGF-B基因表达及其对成纤维细胞增生和胶原合成的促进作用

目的建立稳定表达人血小板衍生生长因子－ＢＢ（ｐｌａｔｅｌｅｔ－ｄｅｒｉｖｅｄｇｒｏｗｔｈｆａｃｔｏｒ－ＢＢ，ＰＤＧＦ－ＢＢ）的细胞株，并考察该重组蛋白与损伤修复有关的生物学作用。方法将含ＰＤＧＦ－Ｂ基因的真核表达载体ｐｃＤＮＡ３转染小鼠成纤维细胞ＮＩＨ３Ｔ３中，在Ｇ４１８选择压力下得到抗性克隆，收集扩增的抗性克隆培养液并制备细胞膜上ＰＤＧＦ－ＢＢ蛋白质，３Ｈ－ＴｄＲ掺入法表达产物的生物学活性，并以３Ｈ－Ｐｒｏ掺入测胶原合成率。结果免疫荧光染色法证实抗性细胞中ＰＤＧＦ－ＢＢ的表达，膜上ＰＤＧＦ－ＢＢ显著促进ＮＩＨ３Ｔ３细胞ＤＮＡ合成，活性可达约３１．２Ｕ／１０６细胞，而培养液的致丝裂活性低于可检测水平。抗性细胞的胶原合成率显著增高。结论ＰＤＧＦ－ＢＢ的真核表达系统已得以成功构建，该重组蛋白可促进成纤维细胞增生和胶原合成，提示其在损伤修复中有潜在应用价值     

IL1,TGFβ和BMP在骨性关节炎的关节软骨中的表达

采用免疫组织化学染色技术，检测白细胞介素１（ＩＬ１），转化生长因子β（ＴＧＦβ）和骨形态发生蛋白（ＢＭＰ）在骨性关节炎的关书软骨中的表达。结果显示：（１）ＩＬ１与ＴＧＦβ在病损的关节软骨表浅层细胞中表达，ＴＧＦβ还在病损的关节软骨中由发生族集的软骨细胞表达。（２）在关节软骨病损达软骨下骨部位，ＢＭＰ，ＴＣＦβ和ＩＬ１在新生的软骨组织的一些细胞中表达。我们的结果提示，ＩＬ１，ＴＧＦβ和ＢＭＰ以自分泌和（或）旁分泌的形式，在骨性关节炎的病理过程中起重要作用，涉及关节软骨的病损及修复。     

诱导性人工骨膜成骨效应与骨形态发生蛋白的量效关系

目的 研制一种新型的胶原－骨形态发生蛋白（ＢＭＰ）人工骨膜，探讨它在肌内诱导成骨中的生物效应和吸附ＢＭＰ的最佳含量，以求得最佳的量效关系。方法 先从猪皮中提取纯化胶原，制成厚约１ｍｍ的白色、柔软易折叠的胶原膜，以每１０ｍｇ胶原膜为载体，分别吸附不同含量的猪ＢＭＰ。     

行气消肿类中药对体外培养的软骨细胞代谢的影响

采用家兔关节软骨细胞体外培养的方法，观察４种中药黄柏、木香、续断和骨碎补不同浓度对培养的软骨细胞的影响，并采用同位素示踪观察软骨细胞ＤＮＡ、胶原和蛋白多糖的合成变化。结果显示，所有４种中药的不同浓度对软骨细胞ＤＮＡ合成均起抑制作用，除低浓度木香溶液外，对胶原合成亦呈抑制作用，１％和５％的黄柏和骨碎补对蛋白多糖合成有促进作用。认识培养软骨细胞对４种中药的特殊反应，为正确使用这些药物提供了新的参考依据。     

降解材料隔膜引导性骨再生的实验研究

目的 观察长管骨降解材料隔膜引导性骨再生现象,探讨其作用机制。方法 采用胶原隔膜、制做长管骨引导性骨再生动物模型。术后进行Ｘ线观察;不同时期处死取材,进行组织学观察及碱性成纤维细胞生长因子,转移生长因子－β（ＴＧＦ－β）,骨形态发生蛋白（ＢＭＰ）免疫组化染色,并对３种骨诱导因子的含量进行测量。结果 （１）胶原隔膜能够成功引导骨再生、１２周时修复骨缺损。（２）术后１周时,胶原隔膜即在骨缺损处形成密闭     

逆转录病毒介导的基因转移方法的建立

将人促红细胞生成素（ｈＥＰＯ）、人粒细胞集落刺激因子（ｈＧ－ＣＳＦ）、细胞程序死亡抑制基因（ＢＣＬ－２）重组逆转录病毒质粒ＤＮＡ转染病毒包装细胞ＰＡ３１７后，经Ｇ４１８抗性筛选，其单克隆细胞株培养上清液均具有感染ＮＩＨ３Ｔ３及大鼠原代成纤维细胞、成肌细胞并形成Ｇ４１８抗性克隆的能力。其感染细胞的染色体ＤＮＡＰＣＲ鉴定结果均为阳性，表明其染色体中均成功地整合了目的基因；感染的靶细胞均能表达外源基因编码蛋白，表明已成功地建立了逆转录病毒介导的基因转移技术。     

骨形态发生蛋白复合纤维蛋白黏合剂临床应用16例

目的　用纤维蛋白黏合剂（ｆｉｂｒｉｎ ｇｌｕｅ,ＦＧ）作为骨形态发生蛋白（ＢＭＰ） 的缓释放载体,以增强诱导成骨效果。方法　粗制ｂＢＭＰ２００ ｍｇ,纤维蛋白原１ ．０ ｇ,凝血酶１００ Ｕ,混合溶解液２０ｍｌ 组成复合物（ｂＢＭＰ／ＦＧ） ;临床应用于股骨干骨折加压钢板内固定１６ 例,并进行ｂＢＭＰ／ＦＧ的超微结构、Ｘ线片观察及４ 种病毒的安全监控。结果　ｂＢＭＰ／ＦＧ结构致密厚实;术后１ 个月出现新骨,３个月形成“生物接骨板”效应;１６ 例均未发现病毒感染。结论　ＦＧ 是ＢＭＰ 理想的缓释放载体,ＢＭＰ／ＦＧ可形成“生物接骨板”效应,而且其病毒感染可以监控     

hBMP7基因转染对兔骨髓间充质干细胞增殖及胶原合成的影响

使用含hBMP7基因的重组逆转录病毒液感染兔骨髓间充质干细胞（BMSc）,MTT法检测细胞增殖能力,流式细胞仪检测细胞周期,^3H脯氨酸掺故法检测I型胶原合成和表达情况。结果显示,经hBMP7基因转染的BMSc与空载体转染及未转染的BMSc在细胞增殖、细胞周期表现方面无显著性差异,经转染的BMSc合成胶原显著高于空载体转染和未转染者。提示采用逆转录病毒介导转染BMSc后能促进细胞的胶原合成,对BMSc增殖和细胞周期无显著影响,有利于进一步 应用于构建组织工程化骨组织。     

Comparison of biochemical characteristics of cultured fibrochondrocytes isolated from the inner and outer regions of human meniscus

In order to evaluate the ability of fibrochondrocytes to synthesize collagen and proteoglycan, human medial meniscal cells were cultured in a monolayer. Meniscal cells were prepared from the two regions (outer 1/3 and inner 2/3) in consideration of the difference in vascular supply, and articular chondrocytes were also obtained from the same knee joint. Regarding total collagen synthesis, regional differences were not found, but age-related differences were found in human medial meniscus. In contrast, proteoglycan synthesis revealed significant regional differences; meniscal cells from the inner 2/3 synthesized a greater amount of proteoglycan. After long-term monolayer culturing, proteoglycan synthesis by meniscal cells decreased in a time-dependent manner, and morphological changes to fibroblast-like cells were found. In the presence of transforming growth factor (TGF)-β, proteoglycan synthesis increased in a dose-dependent manner. These findings suggest that the inner regions of the human meniscus contain cells with a chondrocytic phenotype. Received: 11 February 1998 Accepted: 14 September 1998     

4种中药对体外培养兔关节软骨细胞代谢的影响

采用家兔关节软骨细胞体外培养的方法，通过同位素示踪探讨当归、丹参、黄芪、川芎嗪４种中药对软骨细胞ＤＮＡ、蛋白多糖以及胶原合成的影响。实验结果显示：１％当归、１％黄芪、１％川芎嗪能促进ＤＮＡ合成，１０％当归无促进作用，１％丹参、１０％丹参，１０％黄芪、１０％川芎嗪则能起抑制作用；１％或１０％当归、黄芪、川芎嗪可促进胶原合成，１０％丹参无影响，１％丹参有抑制作用；１％当归、１０％当归、１％丹参、１０％川芎嗪能促进蛋白多糖的合成，１０％丹参、１％黄芪、１０％黄芪、１％川芎嗪则无影响。本研究证明：当归、黄芪、川芎嗪能显著地促进体外培养的兔关节软骨细胞代谢，有可能促进软骨损伤的修复。这对探讨中药制剂对关节软骨损伤修复的作用机理和寻找有效的治疗药物有一定意义。     

转染转化生长因子-β3对前软骨干细胞增殖及向成软骨方向分化的影响

目的 探讨稳定表达重组转化生长因子-β3(hTGF-β3)对前软骨干细胞(precartilaginous stem cells,PSCs)增殖及向成软骨方向定向分化的诱导作用.方法 免疫磁珠法分离纯化得到新生大鼠PSCs后,用线性化的聚乙烯亚胺转染pcDNA3.1(+)-hTGF-β3到体外单层培养的PSCs中,抗生素筛选使其稳定表达.采用四甲基偶氮唑盐比色法(MTT法)、流式细胞术(FCM)测定转染对PSCs增殖和DNA合成的影响,实时定量RT-PCR、免疫组化及Western blot方法比较转染后目的基因和软骨特异性标志物表达的情况.结果 hTGF-β3在分离纯化的PSCs中稳定表达.与未转染组细胞相比,转染后细胞DNA合成增多,增殖速度加快.PCR和免疫组化学结果表明,转染后软骨标志物基因上调明显,分泌软骨多糖基质及Ⅱ型胶原蛋白也明显增加.结论 通过hTGF-β3基因强化的PSCs可以稳定表达高效的hTGF-β3蛋白,从而促进PSCs的增殖并向成软骨方向分化,为软骨缺损的高效修复提供了新的思路.     

Osteochondritis dissecans (OCD), an endoplasmic reticulum storage disease?: a morphological and molecular study of OCD fragments

Osteochondritis dissecans (OCD) fragments, cartilage and blood from four patients were used for morphological and molecular analysis. Controls included articular cartilage and blood samples from healthy individuals. Light microscopy and transmission electron microscopy (TEM) showed abnormalities in chondrocytes and extracellular matrix of cartilage from OCD patients. Abnormal type II collagen heterofibrils in “bundles” and chondrocytes with abnormal accumulation of matrix proteins in distended rough endoplasmic reticulum were typical findings. Further, Von Kossa staining and TEM showed empty lacunae close to mineralized “islands” in the cartilage and hypertrophic chondrocytes containing accumulated matrix proteins. Immunostaining revealed: (1) that types I, II, VI and X collagens and aggrecans were deposited intracellulary and (2) co‐localization within the islands of types I, II, X collagens and aggrecan indicating that hypertrophic chondrocytes express a phenotype of bone cells during endochondral ossification. Types I, VI and X collagens were also present across the entire dissecates suggesting that chondrocytes were dedifferentiated. DNA sequencings were non‐conclusive, only single nucleotide polymorphism was found within the COL2A1 gene for one patient. We suggest that OCD lesions are caused by an alteration in chondrocyte matrix synthesis causing an endoplasmic reticulum storage disease phenotype, which disturbs or abrupts endochondral ossification.     

逆转录病毒介导hBMP7基因转染兔骨髓间充质干细胞的表达

采用粘－粘端连接方法构建hBMP7逆转录病毒载体,重组质粒转染包装细胞PT67后,制备含目的基因的重 逆转录病毒液感染兔骨髓间充质干细胞。限制性内切酶切分析筛选插入方向正确的重组质粒并进行基因测序。结果显示,hBMP7逆转录病毒载体中外源基因插入方向正确、无碱基错误和缺失。原位杂交和免疫组织化学检测表明,感染后2天经基因转染的BMSc原位杂交和免疫组化检测结果呈阳性,未经转染的细胞检测结果呈阴性。转染的BMSc经G418筛选至4周仍有外源BMP蛋白的表达。提示采用逆转录病毒介导方法转染的BMSc中可有源性BMP7mRNA和蛋白的表达。     

低浓度人血清对兔关节软骨细胞培养的影响

关于软骨细胞的体外培养旨在为创伤性骨关节病的临床治疗提供有效的方法,本文实验结果表明:用5-10%的低浓度人血清较之10%小牛血清对体外培养的关节软骨细胞有明显的促细胞生成作用,但在保持软骨细胞合成硫酸软骨素蛋白的生物特性方面,人血清明显不如小牛血清。     

Transplanted articular chondrocytes co-overexpressing IGF-I and FGF-2 stimulate cartilage repair in vivo

Purpose

The combination of chondrogenic factors might be necessary to adequately stimulate articular cartilage repair. In previous studies, enhanced repair was observed following transplantation of chondrocytes overexpressing human insulin-like growth factor I (IGF-I) or fibroblast growth factor 2 (FGF-2). Here, the hypothesis that co-overexpression of IGF-I and FGF-2 by transplanted articular chondrocytes enhances the early repair of cartilage defects in vivo and protects the neighbouring cartilage from degeneration was tested.

Methods

Lapine articular chondrocytes were transfected with expression plasmid vectors containing the cDNA for the Escherichia coli lacZ gene or co-transfected with the IGF-I and FGF-2 gene, encapsulated in alginate and transplanted into osteochondral defects in the knee joints of rabbits in vivo.

Results

After 3 weeks, co-overexpression of IGF-I/FGF-2 improved the macroscopic aspect of defects without affecting the synovial membrane. Immunoreactivity to type-I collagen, an indicator of fibrocartilage, was significantly lower in defects receiving IGF-I/FGF-2 implants. Importantly, combined IGF-I/FGF-2 overexpression significantly improved the histological repair score. Most remarkably, such enhanced cartilage repair was correlated with a 2.1-fold higher proteoglycan content of the repair tissue. Finally, there were less degenerative changes in the cartilage adjacent to the defects treated with IGF-I/FGF-2 implants.

Conclusion

The data demonstrate that combined gene delivery of therapeutic growth factors to cartilage defects may have value to promote cartilage repair. The results also suggest a protective effect of IGF-I/FGF-2 co-overexpression on the neighbouring articular cartilage. These findings support the concept of implementing gene transfer strategies for articular cartilage repair in a clinical setting.     

Chondrogenesis in a hyaluronic acid scaffold: comparison between chondrocytes and MSC from bone marrow and adipose tissue

Treatment of focal lesions of the articular cartilage of the knee using chondrocytes in a hyaluronic acid (HA) scaffold is already being investigated in clinical trials. An alternative may be to use mesenchymal stem cells (MSC). We have compared articular chondrocytes with MSC from human bone marrow (BM) and adipose tissue (AT), all cultured in HA scaffolds, for their ability to express genes and synthesize proteins associated with chondrogenesis. The cells were expanded in monolayer cultures. After seeding into the scaffold, the chondrocytes were maintained in medium, while the two MSC populations were given a chondrogenic differentiation medium. Chondrogenesis was assessed by real-time RT-PCR for chondrocyte-associated genes, by immunohistochemistry and by ELISA for collagens in the supernatant. Redifferentiation of the dedifferentiated chondrocytes in the HA scaffold was shown by a modest increase in type II collagen mRNA (COL2A1) and reduction in COL1A1. BM-MSC expressed 600-fold higher levels of COL2A1 than chondrocytes after 3 weeks in the scaffold. The levels of aggrecan (AGC1) and COL1A1 were similar for chondrocyte and BM-MSC scaffold cultures, while COL10A1 was higher in the BM-MSC. AT-MSC expressed levels of COL2A1 and COL1A1 similar to chondrocytes, but less AGC1 and COL10A1. Surprisingly, little collagen II protein was observed in the scaffold. Instead, collagen II was found in the culture medium. Chondrogenesis in HA scaffolds was more efficient using BM-MSC than AT-MSC or chondrocytes. Some of the secreted collagen II escaped entrapment in the extracellular space and was detected in the culture medium.