Green fluorescent protein as marker in chondrocytes overexpressing human insulin-like growth factor-1 for repair of articular cartilage defects in rabbits

Objective:To label the primary articular chondrocytes overexpressing human insulin-like growth factor (hIGF-1) with green fluorescent protein (GFP) for repair of articular cartilage defects in rabbits. Methods: GFP cDNA was inserted into pcDNA3. 1-hIGF-1 to label the expression vector. The recombinant 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 hIGF-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 II collagen. Results: The expression of hIGF-1 and GFP was confirmed in transfected chondrocytes by in situ hybridization, immunocytochemical analysis and fluorescence microscope observation. Green articular chondrocytes overexpressing hIGF-1 could expand and maintain their chondrogenic phenotypes for more than 4 weeks. After the transfection of IGF-1, the proliferation of chondrocytes was enhanced and the chondrocytes could effectively maintain the expression of typeⅡcollagen. Conclusions: The hIGF-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 hIGF-1. The labeled articular chondrocytes overexpressing hIGF-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

To label human insulin-like growth factor-Ⅰ (hIGF-Ⅰ) eukaryotic expression vector with green fluorescent protein (GFP) for the repair of articular cartilage defects. Methods: GFP cDNA was inserted into pcDNA3.1-hIGF-Ⅰ to construct the co-expression vector with two multiple cloning sites mammalian expression vector under two cytomegalovirus promoters/enhancers respectively. Recombinant pcGI was transfected into NIH 3T3 cells with the help of lipofectamine. Results : Enzyme digestion and agarose gel electrophoresis analysis revealed that pcGI vector contained correct GFP and hIGF-Ⅰ eDNA. Expression of hIGF-Ⅰ and GFP was confirmed in transfected NIH 3T3 cells by immunocytochemical analysis and fluorescence microscopy. Conclusions : hIGF-Ⅰ eukaryotic expression vector has been successfully labeled with GFP.     

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…     

转肝细胞生长因子基因肝细胞模型的建立

目的：利用脂质体介导法在体外建立转肝细胞生长因子（HGF）基因的人肝细胞模型。方法：建立HGF真核细胞表达载体，利用脂质体介导法在体外将HGF基因转染入人肝细胞，利用荧光显微镜观察、免疫组织化学、原位杂交方法检测HGF真核细胞表达载体的转录和表达情况。结果：以阳离子脂质体LipofectAMINE为载体将HGF基因转染人肝细胞后，经400mg/L的G418筛选后可形成抗性克隆；Neo基因原位杂交结果显示转染基因的细胞有阳性表达；荧光显微镜下观察到有绿色荧光；免疫组织化学证实转染HGF基因的肝细胞有HGF蛋白的表达。结论：HGF基因可被成功转染入人肝细胞并能有效表达，这可能为肝病的基因治疗提供一种新途径。     

重组人BMP-7真核表达质粒的构建及转染软骨细胞后的表达

[目的]构建重组人骨形成蛋白-7（rhBMP-7）基因真核表达质粒，转染兔关节软骨细胞，探讨外源性人rhBMP-7基因在转染软骨细胞中的表达情况及对细胞生物学性状的影响。[方法]采用PCR技术扩增rhBMP-7基因，将其插入真核表达载体pcDNA3．1中，体外分离培养兔关节软骨细胞，然后用构建的rhBMP-7质粒转染软骨细胞，经G418筛选、免疫组化染色和逆转录PCR检测其表达，同时检测表达产物对维持软骨细胞表型的作用。[结果]经过PCR及酶切鉴定证实获得了BMP-7真核表达质粒pcDNA3．1-rhBMP-7，通过免疫组化染色和逆转录PCR鉴定证实rhBMP-7在转染后的软骨细胞中得到了表达，其表达产物促进软骨细胞表型的维持。[结论]外源性rhBMP-7基因转染软骨细胞可以获得高效表达，并具有一定的维持软骨细胞表型的作用，为软骨组织工程的研究提供了改良的种子细胞。     

转染重组人骨形态发生蛋白-7基因对骨髓基质细胞生物学行为的影响

目的 观察重组人骨形态发生蛋白-7(rhBMP-7)基因转染骨髓基质细胞(BMSCs)后的稳定表达及表达产物对BMSCs生物学行为的影响。方法利用脂质体介导法将rhBMP．7基因转染BMSCs，以G418筛选出阳性克隆并扩大培养，用免疫组织化学链霉抗生物素蛋白-生物素-过氧化物酶复合物(SABC)法检测转基因细胞的稳定表达；转染48h后，用转基因细胞的培养液上清刺激正常培养的BMSCs，利用^3H标记的胞腺嘧啶氧核苷(^3H-TdR)掺入法、Na2^35SO4掺入法以及氯胺T法检测基因表达产物对BMSCs增殖、合成蛋白多糖以及胶原的影响。结果 转基因细胞4周时仍能表达外源性基因；基因表达产物能明显促进BMSCs的增殖以及蛋白多糖和胶原的合成。结论 rhBMP-7基因转染BMSCs后可获得稳定表达，且基因表达产物能促进BMSCs增殖。诱导其向软骨细胞分化并增强其生物学活性。     

TGF-beta-1 gene transfer in joint cartilage cells. Stimulating effect in extracellular matrix synthesis

TGF beta-1 has been shown to upregulate matrix synthesis in articular chondrocytes. TGF beta-gene transfer to chondrocytes has the potential to increase the local production of this key component within regenerating cartilage after trauma and could support the repair process in articular cartilage lesions. Primary rabbit articular chondrocytes were cultured and retrovirally transfected with the experimental TGF beta-1 and the lacZ marker gene for control purposes. After radioactive labeling of new synthesized matrix proteins results were compared with normal primary chondrocytes. After TGF beta-1 gene transfer the endogenous growth factor concentration was doubled compared to normal chondrocytes and decreased in the lacZ control group. The proteoglycan synthesis in TGF beta-1 transfected chondrocytes showed a 96% increase compared to the basal production of normal chondrocytes. The LacZ transfected group revealed the opposite effect by a 44% decrease. The collagen synthesis of TGF beta-1 transfected chondrocytes was 304% compared to normal chondrocytes, predominantly type II collagen. The lacZ group collagen production was reduced by 35%. We conclude that TGF beta-1 gene transfer overcomes the decreasing effect observed by transfection with the LacZ marker gene and increases matrix synthesis in articular chondrocytes. Genetically altered chondrocytes might improve the repair of cartilage lesions by stimulating matrix synthesis and supporting the expression of the hyaline phenotype.     

Fas Ligand gene transfer enhances the survival of tissue-engineered chondrocyte allografts in mini-pigs

BACKGROUND AND PURPOSE: Since the Fas/Fas Ligand (FasL) interaction has been recognized as an apoptotic pathway, it eliminates the activated T cells and promotes the survival of grafts. In this study, the effect of FasL transfection of pig chondrocytes on allogeneic transplantation was examined in vitro and in vivo. METHODS: Chondrocytes were isolated from articular and aural cartilages of anesthetized Guizhou Xiang (Gz) pig. The cells were transfected with G418 selected virus, packed from PA317 cells with a constructed plasmid using pig FasL (pGCEN-FasL). The apoptotic effect of FasL transfection was examined on Jurkat cells and activated recipient Gz T cells. The FasL expression was assessed by Western blot and flow cytometry. FasL+chondrocytes-Pluronic F-127 complex was injected into the right abdomen of recipient Gz pig. Histology and morphology of the engineered tissue were examined after 2 and 5 weeks of transplantation. RESULTS: The FasL expression was confirmed in pGCEN-FasL transfected chondrocytes. The expression of FasL of chondrocytes from Gz pig was analyzed by FACS. The apoptosis of Jurkat cells and activated recipient Gz T cells was increased by co-culture with FasL(+) chondrocytes (53.41% and 30.38% (E/T=10:1), in contrast of 32.27% and 13.16% with the control chondrocytes, respectively, P<0.01). FasL(+) chondrocytes-Pluronic F-127 implant expressed FasL and Type II collagen at the 5th week and survived until the 8th week. INTERPRETATION: The result indicates that the expression of FasL by chondrocytes is capable of inducing apoptosis of activated T cells. This suggests a potential role for allogeneic transplantation with chondrocytes.     

人胎儿表皮干细胞的体外分离培养及基因转染

目的：探讨人胎儿表皮干细胞体外分离培养的方法以及作为体外基因转染靶细胞的可行性。方法：利用Ⅳ型胶原快速贴附法分离人胎儿表皮干细胞，以人胎儿成纤维细胞条件培养液配制表皮干细胞培养基，通过角蛋白19（K19）和整合素β1免疫组化染色、细胞周期分析及克隆形成率测定，对培养细胞进行鉴定。采用脂质体介导法，以含血管内皮细胞生长因子165（VEFG165）基因片段的真核表达载体pcDNA3.1(pcDNA3.1/VEGF165)转染培养细胞；采用病毒载体介导法，以含报告基因绿色荧光蛋白（GFP）的重组腺相关病毒载体（raav/GFP)转染培养细胞。应用免疫组化染色及荧光显微镜观察检测转染效果。结果：人胎儿表皮干细胞呈明显克隆性生长、克隆形成率高，G1期细胞比例明显高于普通基底层角质细胞，K19和整合素β1免疫组化染色呈强阳性。pcDNA3.1/VEGF165转染的表皮干细胞VEGF165免疫组化染色阳性，raav/GFP转染的表皮干细胞呈现强荧光。结论：利用Ⅳ型胶原快速贴附法及人胎儿成纤维细胞条件培养基，可初步实现人胎儿表皮干细胞的分离培养。以质体为介导或以腺相关病毒为载体进行人胎儿表皮干细胞的体外基因转染是可行的。     

增强绿色荧光蛋白标记比较腺病毒、腺相关病毒体外转染兔软骨细胞的效果

目的 通过增强绿色荧光蛋白(eGFP)标记,比较腺病毒(Ad)、腺相关病毒(AAV)对体外培养软骨细胞的转染效果.方法 体外培养正常3月龄新西兰兔关节软骨细胞,以rAd5-eGFP、rAAV2-eGFP分别转染原代关节软骨细胞,计算最佳传染复数.之后以最佳传染复数的病毒重组体转染软骨细胞,分别应用流式细胞仪检测绘制转染率的时间一反应曲线,倒置荧光显微镜下观察转染后细胞的大体形态、绿色荧光的表达情况并应用荧光定量PCR检测转染前后软骨细胞Ⅱ型胶原mRNA表达水平的变化.结果 rAd5-eGFP、rAAV2-eGFP的最佳传染复数分别为1×103 vp/cell和1×105 vg/cell.分别应用最佳传染复数体外转染原代培养的软骨细胞后,rAd5-eGFP在转染后1～2 d荧光表达即可达到高峰,但之后迅速衰减,在转染后第28天基本检测不到荧光表达.rAAV2-eGFP在转染第7天荧光表达达高峰,随之缓慢衰减,在转染后第56天仍可检测到绿色荧光表达.荧光定量PCR检测显示,rAd5-eGFP在转染软骨细胞后,软骨细胞合成Ⅱ型胶原mRNA的水平明显下降,而rAAV2-eGFP则影响不显著.结论 Ad转染体外培养的关节软骨细胞后,目的基因的表达迅速,但维持时间短,对软骨细胞表型的影响较明显;AAV转染软骨细胞后,目的基因的表达缓慢,但维持时间长,且对软骨细胞表型的影响较小.AAV更适于作为软骨细胞的体外转染载体.     

PcDNA3-hBMP3基因转染关节软骨细胞及其稳定表达

目的 探讨外源性基因ｈＢＭＰ３在软骨细胞获得稳定表达的可能性。方法 将和ＢＭＰ３的ｃＤＮＡ构建于真核表达载体ＰｃＤＮＡ３,形成重组真核表达载体ＰｃＤＮＡ３－ｈＢＭＰ３,转染培养状态下兔关节软骨细胞。利用重组ＤＮＡ和基因克隆技术构建重组真核表达载体ＰｃＤＮＡ３－ｈＢＭＰ３;利用细胞２和细胞基因转染技术体外转染ｈＢＭＰ３基因至关节软骨细胞;     

人IL-1受体拮抗蛋白重组逆转录病毒载体的构建、鉴定及在骨关节炎软骨细胞中的表达

目的 构建含人IL-1受体拮抗蛋白(IL-1 receptor antagonist, IL-1Ra)逆转录病毒表达载体(PLXRN-IL-1Ra),体外转染人骨关节炎(osteoarthritis, OA)软骨细胞,研究其相关特性.方法 利用细菌内同源重组技术快速构建PLXRN-IL-1Ra逆转录病毒重组质粒,经测序及酶切鉴定正确后转染PT67细胞,包装成为重组PLXRN-IL-1Ra逆转录病毒,并使用小鼠肾成纤维细胞系NIH/3T3对病毒进行滴度测定.实验分为3组未转染组(A组)、PLXRN空质粒转染组(B组)、PLXRN-IL-1Ra转染组(C组),病毒感染人OA软骨细胞后,RT-PCR检测细胞内IL-1Ra基因的转录和表达;ELISA法检测细胞培养上清液中人IL-1Ra蛋白表达.结果 酶切鉴定及基因测序证实重组逆转录病毒质粒中含有人IL-1Ra cDNA,测定包装的病毒滴度为3 × 104 CFU/mL.原代软骨细胞体外培养呈多角形或梭形,甲苯胺蓝染色见细胞内有紫色异染颗粒.RT-PCR结果显示在C组出现311 bp人IL-1Ra mRNA片段,A、B组未见人IL-1Ra mRNA的表达带,GAPDH在各组均有表达.ELISA检测发现C组细胞上清有一定量的人IL-1Ra表达,蛋白浓度为(60.47±15.13)ng/L,A组和B组均无人IL-1Ra表达.结论 构建的IL-1Ra逆转录病毒表达载体成功地感染人OA软骨细胞,并在体外获得稳定表达,为将表达人IL-1Ra基因的人OA软骨细胞用于OA基因治疗提供了实验依据.     

Cathepsin K基因抑制后对软骨细胞功能的影响研究

目的研究应用RNA干扰技术早期抑制人CathepsinK（CTSK）基因的表达对延缓软骨细胞的去分化过程及维持软骨细胞表型的影响。方法pGCsilencer TMH1／Nco／GFP／CTsKRNAi质粒体外转染人软骨细胞,并用G418筛选3周．再通过RT—PCR检测CTSK、Ⅱ型胶原和AggrecancDNA转录的表达,Western-Blot、免疫荧光检测转染后软骨细胞的CTSK、Ⅱ型胶原和Aggrecan在蛋白水平的表达。结果装入质粒载体转染第1代的软骨细胞,在体外通过细胞形态观察可发现,抑制CTSK基因后3周软骨细胞仍大多数保持多角形,而对照组则向成纤维样细胞形态变化。RT—PCR结果显示,在mRNA水平抑制了CTSK的表达,而不影响对软骨细胞Ⅱ型胶原和Aggrecan的mRNA表达。免疫荧光和Wesfem—blot的结果证实了在早期抑制了软骨细胞CTSK基因表达并维持3周左右。软骨细胞的特异性基质Ⅱ型胶原和Aggrecan在蛋白水平明显增加。结论早期抑制了软骨细胞CTSK基因的表达,可使软骨细胞去分化过程中其软骨特异性基质Ⅱ型胶原和Aggrecan增加,说明可以维持软骨细胞的表型。     

猪TGF-β1基因重组慢病毒载体的构建及其在BMSCs中的表达

目的构建猪TGF-β1重组慢病毒表达载体,并转染BMSCs,为构建组织工程骨软骨提供TGF-β1修饰的BMSCs,作为持续、高效的种子细胞。方法将已获取的目的基因TGF-β1cDNA包装至慢病毒载体中,通过PCR及基因测序对阳性克隆进行鉴定,并测定病毒滴度。取2月龄巴马香猪(体重约15 kg)骨髓制备BMSCs,取第2～3代用于实验。用TGF-β1重组慢病毒载体以感染复数(multiplicity of infection,MOI)为10、50、70、100、150分别转染BMSCs,通过激光共聚焦显微镜观察,并以Western blot检测不同MOI值的转染效果,确定最佳MOI值。用TGF-β1重组慢病毒载体以最佳MOI值感染BMSCs作为实验组,以空载体转染的BMSCs(空载体组)及未转染的BMSCs(空白组)作为对照,通过RT-PCR、免疫细胞化学染色、ELISA等方法检测TGF-β1基因及蛋白在BMSCs中的表达情况,并检测Ⅱ型胶原表达情况。结果经PCR及基因测序鉴定TGF-β1重组慢病毒表达载体构建成功,并成功转染BMSCs,激光共聚焦显微镜下可观察到强绿色荧光;Western blot示MOI为70时转染效果最佳;RT-PCR示实验组TGF-β1基因的表达量明显高于空载体组及空白组,差异有统计学意义(P<0.05);免疫细胞化学染色示实验组TGF-β1蛋白及Ⅱ型胶原呈阳性表达,而空载体组及空白组呈弱阳性或阴性表达;ELISA示实验组TGF-β1蛋白至转染后21 d仍有较高表达。结论 TGF-β1重组慢病毒表达载体可成功转染BMSCs,TGF-β1蛋白可长期、稳定表达,促使BMSCs向成软骨细胞方向分化。     

转导人端粒酶逆转录酶基因致人骨髓间充质干细胞永生化并向软骨细胞诱导分化的研究

目的 建立永生化人骨髓间充质干细胞系并向软骨细胞诱导分化,以供软骨组织工程基础研究及临床应用.方法 原代培养人骨髓间充质干细胞(hMSC),用含有人端粒酶逆转录酶(hTERT)基因的逆转录病毒转染hMSC,G418筛选得到阳性克隆,体外连续培养,检测端粒酶的表达及活性.TGF-β1和地塞米松对转化后的hMSC-hTERT细胞诱导,使其向软骨细胞分化,并用原位杂交和免疫组化检测II型胶原.结果 外源性hTERT在转染细胞中稳定表达并传至第50代,永生化的hMSC细胞经TGF-β1和地塞米松诱导在体外分化为软骨细胞.结论 外源性hTERT基因可以有效地在体外使hMSC永生化,永生化的hMSC细胞经诱导可在体外分化为软骨细胞,从而作为软骨组织工程研究的细胞来源.     

重组腺病毒介导外源性SOX9在正常关节软骨细胞中诱导软骨基质合成的体外表达

背景：关节软骨损害是临床一种常见的损伤,软骨形成转录因子SOX9是一种调控Ⅱ型胶原合成的关键因子。 目的：观察以表达外源性SOX9的腺病毒体外成功感染关节软骨细胞后对Ⅱ型胶原和蛋白聚糖（Aggrecan）合成的影响,探讨软骨损伤后修复软骨缺损的基因治疗方法。 方法：体外构建腺病毒载体AdSOX9和AdGFP,成功感染培养的人关节软骨细胞,分别以逆转录聚合酶链式反应（RT-PCR）技术检测了病毒感染前后SOX9、II型胶原和蛋白聚糖基因mRNA的表达,同时以免疫组化技术检测了病毒感染前后关节软骨细胞中Ⅱ型胶原的表达。 结果：应用AdEasy腺病毒构建专利技术体外成功构建了能高效表达外源性SOX9的腺病毒AdSOX9和只表达绿色荧光蛋白GFP的腺病毒AdGFP;以腺病毒AdSOX9和AdGFP体外成功感染人关节软骨细胞后48h,未感染对照组和AdGFP感染组,均检测到了Ⅱ型胶原和蛋白聚糖的表达;而AdSOX9感染组的细胞中,检测到了SOX9基因mRNA的表达明显增高,与未感染对照组和AdGFP感染组相比有显著性差异（P〈0．05）,同时,Ⅱ型胶原和蛋白聚糖的表达也较未感染对照组和AdGFP感染组明显增高,差异显著（P〈0．05）。 结论：以外源性SOX9为目的基因的腺病毒介导基因治疗方法,在促进关节软骨细胞Ⅱ型胶原和蛋白聚糖合成方面得出了初步满意的结果,SOX9可能是关节软骨缺损基因治疗研究领域一个新的理想靶点,值得继续深入研究。     

Type II collagen synthesis in the articular cartilage of a rabbit model of osteoarthritis: expression of type II collagen C-propeptide and mRNA especially during early-stage osteoarthritis

Background The aim of this study was to observe time course changes in type II collagen synthesis in various regions of articular cartilage affected with osteoarthritis (OA) by examining the expression of type II collagen C-propeptide (pCOL II-C) and mRNA in a rabbit OA model. Methods Osteoarthritis was experimentally induced by partial lateral meniscectomy in the knees of Japanese white rabbits. The cartilage of the animals was then examined histologically over time. The degenerative area of articular cartilage was divided into three areas, according to the degree of degeneration. The ability to synthesize type II collagen was estimated by the immunohistological staining of pCOL II-C and the in situ hybridization of mRNA in type II collagen. Results The positive rate of pCOL II-C immunostaining in chondrocytes was highest in the central-degenerative region 1 week after surgery, and the highest rate in the para-degenerative region was observed 2 and 4 weeks after surgery. The percentage of pCOL II-C positive cells increased as the histological degeneration score increased to moderate degeneration and then decreased with further progression of the severity of cartilage degeneration. Examination by in situ hybridization revealed that the regions marked by strong pCOL II-C mRNA expression were similar to those indicated by the immunohistology results. Conclusions These results suggest that the type II collagen-synthesizing potential of chondrocytes is highest in moderately degenerated areas of OA articular cartilage. Cartilage repair continues to be seen even as OA advances, although the reaction varies depending on the stage of OA.     

Fibroblast growth factor-18 is a trophic factor for mature chondrocytes and their progenitors

OBJECTIVE: The aim of this study was to examine the effects of recombinant human Fgf18 on chondrocyte proliferation and matrix production in vivo and in vitro. In addition, the expressions of Fgf18 and Fgf receptors (Fgfr) in adult human articular cartilage were examined. METHODS: Adenovirus-mediated transfer of Fgf18 into murine pinnae and addition of FGF18 to primary cultures of adult articular chondrocytes were used to assess the effects of FGF18 on chondrocytes. In situ hybridization was used to examine the expression of Fgf18 and Fgfr s in adult human articular cartilage. RESULTS: Expression of Fgf18 by adenovirus-mediated gene transfer in murine pinnae resulted in a significant increase in chondrocyte number. Chondrocytes were identified by staining with toluidine blue and a monoclonal antibody directed against type II collagen. Fgf18, Fgfr 2-(IIIc), Fgfr 3-(IIIc), and Fgfr 4 mRNAs were detected within these cells by in situ hybridization. The nuclei of the chondrocytes stained with antibodies to PCNA and FGF receptor (FGFR) 2. Addition of FGF18 to the culture media of primary articular chondrocytes increased the proliferation of these cells and increased their production of extracellular matrix. To assess the receptor selectivity of FGF18, BaF3 cells stably expressing the genes for the major splice variants of Fgfr1-3 were used. Proliferation of cells expressing Fgfr 3-(IIIc) or Fgfr 2-(IIIc) was increased by incubation with FGF18. Using FGFR-Fc fusion proteins and BaF3 cells expressing Fgfr 3-(IIIc), only FGFR 3-(IIIc)-Fc, FGFR 2-(IIIc)-Fc or FGFR 4-Fc reduced FGF18-mediated cell proliferation. Expression of Fgf18, Fgfr 3-(IIIc) and Fgfr 2-(IIIc) mRNAs was localized to chondrocytes of human articular cartilage by in situ hybridization. CONCLUSION: These data demonstrate that Fgf18 can act as a trophic factor for elastic chondrocytes and their progenitors in vivo and articular chondrocytes cultured in vitro. Expression of Fgf18 and the genes for two of its receptors in chondrocytes suggests that Fgf18 may play an autocrine role in the biology of normal articular cartilage.     

绿色荧光蛋白体外转染与体内示踪成骨细胞的研究

目的观察经腺病毒介导的绿色荧光蛋白(greenfluorescentprotein,GFP)转染的成骨细胞的体外表达及体内示踪情况,探讨GFP作为组织工程种子细胞示踪剂的可行性。方法以腺病毒为载体,293A细胞为包装细胞,介导GFP转染成人骨髓源成骨细胞,与未转染的同期细胞作对照,在相差显微镜和荧光显微镜下观察,流式细胞术检测GFP表达效率;分别检测转染后两组细胞的碱性磷酸酶(ALP)活性与骨钙素(OCN)的含量;并将GFP转染8d后的成骨细胞植入裸鼠股部肌袋内,术后4、8周取材进行荧光显微镜、HE染色组织学和免疫组织化学染色观察。结果GFP转染的骨髓源成骨细胞表达绿色荧光的阳性率达75%以上,转染8d后的成骨细胞表面抗原标志CD29、CD44高效表达,而CD34不表达;转染后4、8d细胞内ALP活性与OCN含量与未转染组比较差异无显著性意义(P>005)。GFP转染8d后的成骨细胞植入裸鼠体内4、8周均可表达明显的绿色荧光,并具有成骨细胞的形态特征,ALP免疫组化染色呈黄褐色。结论GFP能在体外转染、裸鼠体内示踪成骨细胞,是一种可用于组织工程研究的理想的活细胞示踪剂。     

PTEN编码蛋白对TGF-β1刺激大鼠肾成纤维细胞增殖和ColⅣ与FN分泌的抑制作用

目的：观察张力蛋白同源物基因（PTEN）编码蛋白对转化生长因子-β1（TGF-β1）刺激所致大鼠肾成纤维细胞增殖和Ⅳ型胶原（ColⅣ）、纤连蛋白（FN）分泌的影响。方法：用重组PTEN腺病毒转染体外培养大鼠肾成纤维细胞,倒置荧光显微镜检测绿色荧光蛋白表达,RT-PCR检测PTEN mRNA表达。转染36h后TGF-β1体外刺激,TGF-β1刺激24h后MTT法检测细胞增殖活力,免疫细胞化学法检测PCNA表达,ELISA法检测细胞上清中ColⅣ、FN水平。结果：PTEN腺病毒转染36h后可见明显绿色荧光蛋白表达,PTEN mRNA表达明显增多（P〈0.01）。PTEN腺病毒转染后给予TGF-β1刺激组较单纯TGF-β1刺激组平均光密度值（OD）显著降低（P〈0.01）,PCNA表达显著减少（P〈0.01）,且ColⅣ、FN的分泌水平明显下降（P〈0.05）。结论：PTEN基因编码蛋白能抑制TGF-β1刺激所致大鼠肾成纤维细胞增殖、PCNA表达及ColⅣ、FN分泌,可能具有抑制残肾纤维化、延缓残肾毁损速度的作用。