AdLacZ基因修饰山羊耳软骨细胞复合F127构建组织工程化软骨

Construction of tissue engineered cartilage by using AdLacZ gene modified goat auricular chondrocytes and Pluronic F127 scaffold

目的:应用AdLacZ基因修饰体外培养的山羊耳软骨细胞,并与Pluronic F127支架材料复合,观察裸鼠皮下软骨形成的情况,为进一步应用软骨分化因子基因修饰重建软骨的研究提供参数。方法:实验于2004-11/2006-12在上海交通大学医学院附属第九人民医院完成。①实验材料:取健康雄性山羊,体质量26～30kg。6周龄BALB/c裸鼠24只,体质量20g。体外培养山羊耳软骨细胞,转染AdLacZ基因,检测基因转染效率。②实验方法:将基因修饰的细胞与可吸收生物材料PluronicF127混合形成细胞-生物材料复合物,并注射到6只裸鼠皮下,每只在其背部皮下分别接种2个点,分别在术后2,4周取材,每个时间点有标本6例。另取裸鼠18只,同样设计,分别以未转基因的软骨细胞-材料复合物、单独注射的软骨细胞、或单独注射的F127支架作为对照组,每组6只。③实验评估:标本行苏木精-伊红染色、阿利新蓝染色,并对4周标本行X-gal染色,Ⅱ型胶原免疫组织化学检测。结果:纳入BALB/c裸鼠24只,均进入结果分析。①50pfu/细胞可以获得80%的转染效率。②2周时细胞-材料复合物形成了不成熟的软骨样组织,苏木精-伊红染色显示外周呈纤维样组织包裹,中部大部分为不成熟的软骨细胞;4周实验组中央部位出现少量稍成熟的软骨,阿利新蓝染色显示该部位为深蓝色的巢状软骨,部分细胞有Ⅱ型胶原阳性表达。冰冻切片X-gal染色提示细胞来自于植入的软骨-材料复合物。对照组未转基因的软骨细胞-材料复合物,其组织学及Ⅱ型胶原免疫组织化学表现与实验组没有明显差别,而X-gal染色为阴性。单独注射软骨细胞或单独注射F127支架,在4周内则均未见到软骨形成。结论:腺病毒载体可向软骨细胞高效转移目的基因,F127支架材料是软骨再生较为理想的支架材料。

AIM： To observe the chondrogenesis after AdLacZ gene modified goat auricular chondrocytes were combined with Pluronic F127 scaffold and injected into nude mice subcutaneously, so as to provide preliminary parameters for future chondrogenic differentiation factor gene therapy in cartilage reconstruction. METHODS： The experiment was carded out in Shanghai Ninth People＇s Hospital affiliated to Shanghai Jiao Tong University Medical School from November 2004 to December 2006. ①Health male goat of 26-30 kg and twenty-four 6 weeks old BALB/c nude mice of 20 g were selected. Goat auricular chondrocytes were cultured in vitro and transfected with AdLacZ over-expression viruses, and gene transfer efficiency was tested. ②Those gene-modified chondrocytes were then combined with biodegradable Pluronic F127 scaffold and injected at 2 points in 6 nude mice subcutaneously. Samples were collected 2 and 4 weeks postoperatively with 6 cases at each time point. Another 18 nude mice were selected and subjected to the previous process, and the non-transfected chondrocytes with scaffold, cells along or scaffold along were regarded as control group with 6 animals in each group. ③All samples were given H-E staining, and alcian blue staining, and the 4-week samples were given X-gal staining, and collagen Ⅱ immunohistochemistry staining. RESULTS： Twenty-four nude mice were involved in the result analysis. An MOI of 50 pfu/cell produced optimal effects in transfer efficiency over 80%. At week 2, immature cartilage-like tissue formed at AdLacZ treated chondrocytes injection sites, and H-E staining showed that the outside was fiberlike encapsulation, while the middle part was composed of immature chondrocytes. At week 4, a little of comparatively mature cartilage was observed in the middle of the sample; alcian blue staining appealed there was deep blue nidulant cartilage, and type Ⅱ collagen positive staining was detected in a few cells. X-gal staining suggested that the cells were derived from the implanted cartilage and scaffold composite. Non-transfected chondrocytes plus F127 group showed similar HE and type Ⅱ collagen expression as the AdLacZ transfected experimental group, but X-gal staining was negative. While no cartilage formation was found in cells along or F127 alone samples within 4 weeks. CONCLUSION： Adenovirus gene transfection achieved comparatively high transfer efficiency on primary cultured chondrocytes. Pluronic F127 scaffold is a suitable scaffold material for tissue engineered cartilage regeneration.