CDMP1转基因细胞片修复兔甲状软骨缺损的实验研究

发布时间：2018-05-27 19:35

  本文选题：细胞片 + CDMP1　； 参考：《辽宁医学院》2014年硕士论文

【摘要】：目的 构建软骨源性形态发生蛋白-1（cartilage derived morphogeneticprotein-1,CDMP1）转基因的细胞片，并对其生物活性进行鉴定，运用细胞膜片技术（cell sheet technology，CST）与组织工程技术相结合的方法探讨新型无支架组织工程细胞片在兔甲状软骨缺损中的修复效果，以期为软骨重建及再生医学提供一个新的思路和方法。 方法 取1月龄左右的新西兰白兔，无菌条件下分离兔双侧股骨，优化方案分离纯化BMSCs，体外扩增培养传代。取第4代兔BMSCs分别给予以下三种处理：（A）转染Ad-CMV-hCDMP1-IRES-eGFP组；（B）转染Ad-CMV-eGFP组；（C）BMSCs组，行MTT检测细胞增殖活性，连续培养14d后接种至温度敏感性培养皿制备细胞片，行Real time PCR、Western blot及组织学方法检测转基因细胞片中糖胺聚糖（Glycosaminoglycan,GAG）、CDMP1及Π型胶原的表达。将上述3组组织工程细胞片移植入兔甲状软骨缺损处，，常规喂养，于术后4周和8周无痛处死，行大体和组织切片观察。 结果 体外培养的BMSCs增殖活跃，腺病毒介导的hCDMP1转基因并未对其增殖活性产生影响，采用温度敏感性培养皿体外成功收获了完整的细胞片结构，且通过CST成功构建了多层三维立体组织工程细胞片，有一定韧性和可操作性。Real time PCR和Western blot可检测到CDMP1和Π型胶原的表达。HE染色显示A组细胞片基质丰富，阿利新蓝染色呈均一蓝色异染，Π型胶原表达阳性，B、C组细胞片未见明显特异性蓝染颗粒，Π型胶原表达阴性，A组与B、C组比较有统计学差异（P0.05）。体外实验结果显示，A组软骨缺损修复区有类软骨样细胞生长，质韧，表面光滑， GAG和Π型胶原表达阳性，而B组和C组基本为纤维结缔组织和肌肉组织填充，表面凹陷粗糙，GAG和Π型胶原表达阴性。 结论 1、体外成功构建CDMP1转基因细胞片。 2、细胞片对修复兔甲状软骨的缺损有促进作用。
[Abstract]:Purpose The chondrogenic morphogenetic protein-1 derived cartilage derived CDMP1 was constructed and its biological activity was identified. In order to provide a new idea and method for cartilage reconstruction and regenerative medicine, the method of cell sheet technology combined with tissue engineering technique was used to study the repair effect of new scaffolding tissue engineering cell piece in rabbit thyroid cartilage defect. Method BMSCs were isolated from New Zealand white rabbits at the age of about 1 month under aseptic condition. BMSCs were isolated and purified by optimization method and cultured in vitro. The fourth generation of rabbit BMSCs was divided into three groups: Ad-CMV-hCDMP1-IRES-eGFP group was transfected with the following three treatments: Ad-CMV-hCDMP1-IRES-eGFP group (BMSCs) were transfected into Ad-CMV-eGFP group. The proliferative activity of Ad-CMV-eGFP group was measured by MTT. After 14 days of continuous culture, the cells were cultured in a temperature-sensitive dish to prepare the cells. The expression of glycosaminoglycan (Gago) CDMP1 and collagen 蟺 in transgenic cells was detected by Real time PCR Western blot and histological method. The above three groups of tissue engineering cells were transplanted into the defect of thyroid cartilage in rabbits. The rabbits were given routine feeding and were killed painlessly at 4 and 8 weeks after operation. The gross and histological sections were observed. Result The proliferation of BMSCs in vitro was active, and the hCDMP1 gene mediated by adenovirus had no effect on its proliferative activity. The whole cell piece structure was successfully harvested in vitro by using temperature-sensitive culture dish. The multilayer three-dimensional tissue engineering cell pieces were successfully constructed by CST. The results showed that the cells in group A were rich in matrix. Real time PCR and Western blot could detect the expression of CDMP1 and 鈪

本文编号：1943486