Green fluorescent protein as marker in chondrocytes overexpressing human insulin-like growth factor-l for repair of articular cartilage defects in rabbits

Objective： To label the primary articular chondrocytes overexpressing human insulin-like growth factor （ IdGF-1 ） with green fluorescent protein （GFP） for repair of articular cartilage defects in rabbits. Methods： GFP cDNA was inserted into PeDNA3.1- hlGF-1 to label the expression vector. The recombinnnt vector, pcGI, a mammalian expression vector with multiple cloning sites under two respective cytomegalovirus promoters/enhancers, was transfected into the primary articular chondrocytes with the help of lipofectamine. After the positive cell clones were selected by G418, G418- resistant chondrocytes were cultured in medium for 4 weeks. The stable expression of hlGF-1 in the articular chondrocytes was determined by in situ hybridization and immunocytochemical analysis and the GFP was confirmed under a fluorescence microscope. Methyl thiazolyl tetrazolium （MTT） and flow cytometer methods were employed to determine the effect of transfection on proliferation of chondrocytes. Gray value was used to analyze quantitatively the expression of type lI collagen. Results： The expression of hlGF-1 and GFP was confirmed in transfected chondrocytes by in situ hybridization, immunocytochemical analysis and fluorescence microscope observation. Green articular chondrocytes overexpressing hlGF-1 could expand and maintain their chondrogenic phenotypes for more than 4 weeks. After the transfectton of IGF-1, the proliferation of chondrocytes was enhanced and the chondrocytes could effectively maintain the expression of type lI collagen. Conclusions： The hlGF-1 eukaryotic expression vector containing GFP marker gene has been successfully constructed. GFP, which can be visualized in real time and in situ, is stably expressed in articular chondrocytes overexpressing hlGF-1. The labeled articular chondrocytes overexpressing hlGF-1 can be applied in cell-mediated gene therapy as well as for other biomedical purposes of transgenic chondrocytes.     

Labeling of human insulin-like growth factor-I eukaryotic expression vector with green fluorescent protein

Articularcartilageinjuriesinadultsarecommon.Theintrinsicrepairisgenerallyminimalandcartilageinjuriescanfurtherdevelopintoosteoarthritis.Insulin likegrowthfactor I(IGF I)is regardedasoneofthemostimportantgrowthfactorsin cartilagedevelopmentandhomeostasis.Theadditionof IGF Itochondrocytesinvitroenhanceschondrocyte metabolism,maintainsadifferentiatedchondrocyte morphologyandpromotessynthesisofmajorcartilage matrixproteins,includingtype IIcollagenand proteoglycans.1,2IGF Iinlowconcentrationi…     

外源性人胰岛素样生长因子-1基因在关节软骨细胞中的表达

目的探讨含有绿色荧光蛋白(GFP)的外源性人胰岛素样生长因子(hIGF)-1基因真核表达载体在关节软骨细胞内稳定表达的可行性，建立IGF-1基因工程化的关节软骨细胞。方法利用基因重组技术，将GFPcDNA片段插入pcDNA3．1-hIGF-1载体中，构建重组载体pcGI。然后采用脂质体方法，转染原代关节软骨细胞，经G418筛选后，继续体外单层培养4周。原位杂交和免疫细胞化学检测hIGF-1的表达，荧光显微镜观察GFP的表达，Ⅱ型胶原免疫细胞化学检测软骨细胞表型，四甲基偶氮唑蓝(MTT)比色法检测软骨细胞增殖能力。结果根据重组载体pcGI内的酶切位点，设计采用BamHI酶切显示为线性化，XbaI酶切显示为512bp、1768bp、5915bD三个片段，HindⅢ则酶切下3023bp、5172bp二个片段，证明所构建的质粒方向及大小正确，含有GFP和hIGF-1cDNA片段。稳定转染pcGI的软骨细胞原位杂交、免疫细胞化学和荧光显微镜证实了hIGF-1和GFP的表达，同时MTT显示转染后的软骨细胞增殖能力增强，并能维持Ⅱ型胶原的表达。结论外源性hIGF-1和GFP基因在关节软骨细胞内获得稳定表达，体外单层培养的转染关节软骨细胞增殖能力增强，同时维持软骨细胞表型。     

生长因子对成人关节软骨细胞的促增殖作用

目的观察不同生长因子对成人关节软骨细胞（adult human articular chondrocytes，AHAC）增殖的影响，探索AHAC体外大量扩增的方法。方法以酶消化法从成人关节软骨分离细胞，条件培养基培养；传2代细胞分别用不同浓度成纤维细胞生长因子2（fibroblast growth factor-2，FGF-2）、转化生长因子β1（transforming growth factor β1，TGF—β1）、血小板衍生因子bb（platelet derived growth factor-bb，PDGF-hb）、肝细胞生长因子（hepatocyte growth factor，HGF）或其不同组合作用。用MTT法比较细胞增殖情况，用组化和免疫组化检测观察细胞表型变化。结果FGF-2、TGF—β1、PDGF—bb、HGF均有促AHAC增殖的作用，其最大效应剂量分别是50ng/ml、1ng／ml、1ng／ml、20ng／ml。5ng／ml FGF-2＋1ng/ml TGF-β1有最强的促增殖作用，继续加用PDGF—bb和（或）HGF无进一步促进作用；用这一因子组合培养AHAC，可以传10代以上，细胞扩增2000倍以上，且传9代细胞仍弱表达Ⅱ型胶原和aggrecan。结论FGF-2、TGF-β1、PDGF—bb、HGF均对AHAC有一定的促增殖作用；5ng／ml FGF-2＋1ng/ml TGF-β1有最大的促增殖效应，细胞短期内大量扩增，且在大量扩增的同时维持了一定的软骨细胞表型，因此是合适的AHAC体外大量扩增促进剂。     

补骨脂多糖局部填充修复家兔关节软骨全层缺损的实验研究

目的探讨中药提取物补骨脂多糖局部运用修复兔关节软骨全层态发生蛋白／明胶复合物组（B组）、空白组（C组）,每组24只,48个关节。分别给予A缺损的可行性和部分机理。方法将72只成年健康日本大耳白兔随机分为补骨脂多糖组（A组）、人骨形组补骨脂多糖凝胶、B组骨形态发生蛋白／明胶复合物填充膝关节股骨内髁滑车面软骨全层缺损区,c组只做钻孔处理,空白对照。术后取标本行大体观察、HE染色、改良Pinsda法组织学评分的方法进行评估。结果补骨脂多糖和rhBMP!明胶复合物在2、4、8周缺损区填充均优于空白组,为乳白色半透明质软类透明软骨组织;组织学评分与空白组存在极显著性差异（P〈0．01）,用药组之间没有统计学意义（P〉0．05）。结论补骨脂多糖局部运用可以修复家兔关节软骨缺损,经组织学观察修复组织以透明软骨为主,接近正常关节软骨。其修复关节软骨缺损是可行的,具有很好的实用价值。     

转化生长因子-β1和胰岛素样生长因子-1对人髓核细胞体外增殖活性的影响

目的：研究转化生长因子-β1（TGF—β1）和胰岛素样生长因子-1（IGF—1）单独及联合应用对人髓核细胞体外增殖活性的影响，并观察其量效和时效关系。方法：体外分离培养人髓核细胞．将传2代细胞种于96孔板，采用噻唑蓝（MTT）比色法，观察TGF—β1和IGF—1在1％和10％血清浓度下对人髓核细胞体外增殖的调节作用及其剂量、时间与作用效果的关系。结果：在1％血清条件下，IGF—1的作用不显著，TGF—B1具有促增殖作用。在10％血清条件下，TGF—B1和IGF—1均能提高细胞的增殖活性，并且在有效浓度范围内呈剂量效应关系，TGF—B1的作用强于IGF—1。二者联合应用效果更显著。结论：TGF—B1和IGF—1均能不同程度地促进入髓核细胞的体外增殖，其效应在一定范围内与剂量和时间呈正相关．联合应用促进增殖作用更显著。     

肝细胞生长因子对关节软骨缺损修复作用的实验研究

目的 探讨肝细胞生长因子＊ｈｅｐａｔｏｃｙｌｅ ｇｒｏｗｔｈ ｆａｃｔｏｒ,ＨＧＦ）对创伤性关节软骨损伤的修复作用。方法 选 用大耳白 兔２５只,随机分成５组,每组５只。在两侧髌股关节股骨侧的关节面中心分别造成直径３．５ｍｍ的全层关节软骨缺损。造反右侧为实验组,自术后起,膝关节内注射１０μｇ／ｍｌ的ＨＧＦ０．１ｍｌ,每周３次,共４周;左侧为对照组,与实验组同一时间注射等量生理盐水。于术后４、６、８     

Growth hormone stimulates insulin-like growth factor I actions on adult articular chondrocytes

We report effects of adding insulin-like growth factor I (IGF-I) and methionyl human growth hormone (GH), alone or in combination, to adult bovine articular chondrocytes plated at high density. Purified human and synthetic IGF-I stimulated chondrocyte DNA and proteoglycan synthesis. GH had no effect on either process. However, GH added in combination with IGF-I increased proteoglycan, cell-associated proteoglycan, and keratan sulfate synthesis over levels observed with IGF-I alone. IGF-I and GH did not alter the hydrodynamic size of proteoglycans or synthesis of collagen. Our results show that GH and IGF-I act together to stimulate adult chondrocyte extracellular matrix synthesis.     

Signaling through the small G-protein Cdc42 is involved in insulin-like growth factor-I resistance in aging articular chondrocytes.

During aging, chondrocytes become unresponsive to insulin-like growth factor-I (IGF-I). This study examined the role of Cdc42 (cell-division-cycle 42) in IGF-I signaling during aging. Experiments were performed using cartilage and chondrocytes isolated from horses ages 1 day-25 years. Northern analysis was used to examine expression of the small GTPases Cdc42, Rac, and RhoA. Western analysis was utilized to assess total Cdc42 (GTP + GDP-bound); active, GTP-Cdc42 was assessed using a pulldown assay with Western analysis. GTP-Cdc42 was also measured following IGF-I treatment. Gene expression for Cdc42 and Rac were decreased in mature samples, but there was no difference in total Cdc42 (GTP + GDP-bound) protein expression due to age. GTP-Cdc42 was significantly greater in prepubescent samples compared to other age groups. IGF-I diminished the GTP-bound state of Cdc42 in prepubescent chondrocytes; however, this effect was lost during aging. No differences in results were observed due to sample type; that is, cartilage tissues versus isolated chondrocytes. These studies suggest that loss of IGF-I-mediated regulation of Cdc42 activation may be a mechanism for the chondrocyte unresponsive state during aging. Further, the activation state of Cdc42, measured in native and IGF-I-treated cartilage tissue for the first time, is similar to that of isolated chondrocytes, indicating that the activation state of small G-proteins is not affected by isolation of chondrocytes from the extracellular matrix. Continued studies will identify the upstream regulators of Cdc42, which will further elucidate the molecular mechanism of IGF-I resistance during aging thereby providing insight into targeted strategies for age-related osteoarthritis.     

Endogenous versus exogenous growth factor regulation of articular chondrocytes

Anabolic growth factors that regulate the function of articular chondrocytes are candidates for articular cartilage repair. Such factors may be delivered by pharmacotherapy in the form of exogenous proteins, or by gene therapy as endogenous proteins. It is unknown whether delivery method influences growth factor effectiveness in regulating articular chondrocyte reparative functions. We treated adult bovine articular chondrocytes with exogenous recombinant insulin‐like growth factor‐I (IGF‐I) and transforming growth factor‐beta1 (TGF‐β1), or with the genes encoding these growth factors for endogenous production. Treatment effects were measured as change in chondrocyte DNA content, glycosaminoglycan production, and aggrecan gene expression. We found that IGF‐I stimulated chondrocyte biosynthesis similarly when delivered by either exogenous or endogenous means. In contrast, exogenous TGF‐β1 stimulated these reparative functions, while endogenous TGF‐β1 had little effect. Endogenous TGF‐β1 became more bioactive following activation of the transgene protein product. These data indicate that effective mechanisms of growth factor delivery for articular cartilage repair may differ for different growth factors. In the case of IGF‐I, gene therapy or protein therapy appear to be viable options. In contrast, TGF‐β1 gene therapy may be constrained by a limited ability of chondrocytes to convert latent complexes to an active form. Published 2013 by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 32:54–60, 2014.     

Prognostic value of insulin-like growth factor 1 and insulin-like growth factor binding protein 3 blood levels in breast cancer

High circulating insulin-like growth factor 1 (IGF-1) levels are firmly established as a risk factor for developing breast cancer, especially estrogen positive tumors. The effect of circulating IGF-1 on prognosis once a tumor is established is unknown. The authors explored the effect of IGF-1 blood levels and of it's main binding protein, IGFBP-3, on overall survival and occurrence of second primary breast tumors in breast cancer patients, as well as reproductive and lifestyle factors that could modify this risk. Patients were accrued from six hospitals in the Netherlands between 1998 and 2003. Total IGF-1 and IGFBP-3 were measured in 582 plasma samples.No significant association between IGF-1 and IGFBP-3 plasma levels and overall survival was found. However, in a multivariate Cox regression model including standard prognostic variables high IGF-1 levels were related to worse overall survival in patients receiving endocrine therapy (HR = 1.37, 95% CI: 1.11, 1.69, P 0.004). These data at least indicate that higher IGF-1 levels, and as a consequence most likely IGF-1-induced signaling, are related to a less favorable overall survival in breast cancer patients treated with endocrine therapy. Interventions aimed at reducing circulating levels of IGF-1 in hormone receptor positive breast cancer may improve survival.     

Alterations in endogenous osteogenic protein-1 with degeneration of human articular cartilage.

A synchronized balance between synthesis and breakdown of extracellular matrix (ECM) molecules in normal articular cartilage is disturbed in osteoarthritis (OA). The focus of our study is the anabolic factor, osteogenic protein-1 (OP-1) that is expressed in articular cartilage and is able to induce the synthesis of ECM components. The major aim was to investigate both qualitatively and quantitatively endogenous OP-1 in normal, degenerative, and OA cartilage. Normal and degenerative cartilage was obtained at autopsies from femoral condyles of human organ donors with no documented history of joint disease; OA cartilage was obtained from patients undergoing joint arthroplasty. Appearance of donor cartilage was evaluated by Collins scale, where normal cartilage is assigned grades 0-1, and degenerated cartilage is assigned grades 2-4. OP-1 mRNA expression was assessed by RT-PCR; OP-1 protein (pro- and active forms) was qualitatively analyzed by Western blotting and quantified by OP-1 ELISA. The highest levels of OP-1 expression (mRNA and protein) were detected in normal cartilage of grade 0. The concentration of OP-1 protein was about 50 ng per gram cartilage dry weight. With the progression of cartilage degeneration (increased Collins grades and OA) OP-1 protein was down-regulated up to 9-fold. These changes affected primarily the active form of OP-1. OP-1 message also declined in cartilages with the increase of degenerative changes. In conclusion, an overall decrease in endogenous OP-1 in degenerated and OA tissue suggests that OP-1 could be one of the factors responsible for normal homeostasis and matrix integrity in cartilage.     

骨软骨移植联合rhBMP -2/bFGF修复软骨缺损

[目的]探讨采用冷冻同种异体骨软骨移植,配合骨形态发生蛋白(rhBMP-2)/碱性成纤维细胞生长因子(bFGF)修复软骨缺损的效果,为进一步应用于临床提供理论依据。[方法]48只日本大耳白兔,96个关节,随机分A、B、C、D组。A组采用骨软骨移植联合rhBMP-2/bFGF,B组单纯应用骨软骨移植,C组单纯局部注射rh-BMP-2/bFGF混悬液,D组用作空白对照。无菌条件下制作骨软骨缺损模型。术后第4、8、12周作解剖学观察、磁共振检查、组织学检查及软骨细胞记数、免疫组化检查。[结果]A组软骨缺损修复面光滑,呈瓷白色、半透明,B、C组未完全修复,D组无明显修复。A组修复软骨组织学评分与其他组比较差异有统计学意义(P<0.05),图像分析仪软骨细胞记数与其他组比较差异有统计学意义(P<0.05)。修复软骨Ⅱ型胶原免疫组化染色阳性。[结论]联合应用冷冻同种异体骨软骨移植、rhBMP-2/bFGF,能促进新生软骨的形成,提高软骨缺损修复的质量。     

Insulin-like growth factor-1, growth hormone-dependent insulin-like growth factor-binding protein and growth in children with chronic renal failure

Insulin-like growth factor-binding protein-3 (IGFBP-3) and insulin-like growth factor-1 (IGF-1) levels were measured by specific radioimmunoassays in children with all degrees of chronic renal failure (CRF). Study group 1 comprised 29 children (10 on dialysis) who had been studied one to four times over 2 years to determine whether IGF-1 and IGFBP-3 levels differed from those in age-matched healthy children and to examine the relationship between these levels and heights. IGF-1 and IGFBP-3 levels did not differ from those in normal children. IGF-1 and IGFBP-3 were significantly correlated, increased with pubertal stage in all children and with age in non-dialysis patients. IGF-1, but not IGFBP-3, correlated with age in dialysis patients. There was no correlation between IGF-1 or IGFBP-3 levels (corrected for age) and height standard deviation score (SDS) in either non-dialysis or dialysis patients. Study group 2 comprised 19 children (7 on dialysis) who were studied prospectively for 1–2 years to examine the relationship between IGF-1 and IGFBP-3 levels, growth rates and nutritional parameters. Mean values of IGF-1 and IGFBP-3 (corrected for age) did not change over 1-year periods, while height SDS fell by –0.38 ±0.21 SD/year in dialysis patients and by –0.11 ±0.29 SD/year in non-dialysis patients. No significant correlations were found between IGF-1 or IGFBP-3 levels and growth rates or nutritional parameters. Thus growth retardation in children with CRF is not related to circulating levels of IGF-1 or IGFBP-3.     

Repair of full-thickness cartilage defects using liposomal transforming growth factor-β1

 Transforming growth factor-β1 (TGFβ1) is a well- known, potent growth factor implicated in both in vitro and in vivo chondrogenesis. Liposomes have been employed as a drug delivery system to promote the efficient use of drugs. The objective of this study was to demonstrate that a single injection of liposomal TGFβ1 has an accelerating effect on the repair of an articular cartilage defect. Full-thickness articular cartilage defects were prepared on the patellar grooves of the femurs in knee joints of Japanese white rabbits. One week after surgery, various reagents including liposomal TGFβ1, free TGFβ1, and phosphate-buffered saline were injected into the operated knee joints. At 3 weeks after surgery the specimens obtained from the lesions were evaluated histologically, and the glycosaminoglycan content was quantified. Histological examination revealed that the defects were filled with thicker fibrous cartilage and showed more intense metachromatic staining in the liposomal TGFβ1 group than in the other groups. The glycosaminoglycan content of the repair tissue was also significantly higher in the liposomal TGFβ1 group than in the other groups. This study indicated that the intraarticular injection of liposomal TGFβ1 could accelerate the early-stage repair of full-thickness articular cartilage defects. Received: April 18, 2002/Accepted: September 4, 2002 Acknowledgments. We express our deep appreciation to Professor Yutaka Yabe for providing the opportunity to perform this study. Offprint requests to: T. Abe, Department of Orthopaedic Surgery, Saiseikai-Utsunomiya Hospital, 911-1 Takebayashi-cho, Utsunomiya 321-0964, Japan     

人骨形态发生蛋白对体外培养胎儿关节软骨块的影响

为了解人骨形态发生蛋白（ｈＢＭＰ）对软骨细胞的影响,作者观察了ｈＢＭＰ对体外培养关节软骨块中软骨细胞表达骱钙素（ＢＧＰ）。ｈＢＭＰ的影响及对软骨基质中钙沉积的影响。结果显示：ｈＢＭＰ可诱导软骨块中的关节软骨的细胞表达ＢＧＰ、ｈＢＭＰ并可促进软骨基质中的钝沉积。作者认为,ｈＢＭＰ有诱导软骨块中软骨细胞进一步分化为成骨细胞样细胞的作用,并推测这可能是软骨内化骨过程中,成骨细胞的另一来源的。     

EGF和IGF对体外培养兔关节软骨细胞的影响

目的　了解表皮生长因子 (EGF)和胰岛素样生长因子 (IGF)对体外培养兔关节软骨细胞存活数目和分裂增殖百分数的影响。方法　体外培养兔关节软骨细胞传至第 2代 ,分别以EGF、IGF ,以及二者不同的浓度组合作用于软骨细胞。通过酶联免疫检测仪 (MTT)测定软骨细胞存活数 ,流式细胞仪测定软骨细胞分裂增殖百分数。结果　不同浓度EGF对兔关节软骨细胞存活和分裂增殖均有促进作用 ,作用浓度强度次序为 :10ng/ml>0 1ng/ml>10 0ng/ml。不同浓度IGF对兔关节软骨细胞的存活和分裂增殖均有较强促进作用 ,浓度为 5 0ng/ml时 ,刺激效果最显著。EGF与IGF联合使用时 ,刺激效果优于任何一种因子单独使用 ,而以EGF 10ng/ml和IGF 5 0ng/ml为最佳浓度组合。 结论　EGF和IGF都可促进兔关节软骨细胞存活和分裂增殖 ,但以协同作用效果最佳。     

Effect of insulin-like growth factor-1 and hyaluronic acid in experimentally produced osteochondral defects in rats

Background:The common purpose of almost all methods used to treat the osteochondral injuries is to produce a normal cartilage matrix. However current methods are not sufficient to provide a normal cartilage matrix. For that reason, researchers have studied to increase the effectiveness of this methods using chondrogenic and chondroprotective molecules in recent experimental studies. Insulin-like growth factor-1 (IGF-1) and hyaluronic acid (HA) are two important agents used in this field. This study compared the effects of IGF-1 and HA in an experimental osteochondral defect in rat femora.Results:According to the total Mankin scale, there was a statistically significant difference between IGF-1 and HA groups and between IGF-1 and control groups. There was also a significant statistical difference between HA and control groups.Conclusion:It was shown histopathologically that IGF-1 is an effective molecule for osteochondral lesions. Although it is weaker than IGF-1, HA also strengthened the repair tissue.     

Recombinant insulin-like growth factor-1 as a therapy for IGF-1 deficiency in renal failure

Renal disease in children disrupts the growth hormone (GH) and insulin-like growth factor (IGF) axis and causes growth failure. Although GH therapy stimulates growth in these children, their short stature is likely due to a form of IGF-1 deficiency (IGFD) rather than GH deficiency. Recent experimental data have caused us to reconsider the importance of IGF-1 and IGFD to human growth. Pharmacology studies in rodents, as well as studies in patients with no functional GH receptors and primary IGFD, have shown that IGF-1 is an effective growth-promoting therapy. Gene knockout studies in mice have shown that IGF-1, rather than GH, is the major hormone controlling growth. In addition, both pharmacological and genetic studies have shown that there are effects of GH and IGF-1 that require their combined presence. In children with primary IGFD, where there is no GH signaling, recombinant human (rh)IGF-1 produces a large growth response, while in children who are GH and IGF-1 deficient, treatment with rhGH is the most-appropriate therapy. Children with short stature due to renal failure are GH sufficient and have some GH receptor signaling capacity, so that rhIGF-1, or rhIGF-1 plus rhGH, are logical therapeutic options and merit clinical testing.This work was presented in part at the IPNA Seventh Symposium on Growth and Development in Children with Chronic Kidney Disease: The Molecular Basis of Skeletal Growth, 1–3 April 2004, Heidelberg, Germany