摘要
目的观察人细胞毒性T淋巴细胞相关抗原4-免疫球蛋白融合蛋白(hCTLA4-Ig)基因修饰的人骨髓间充质干细胞(hMSCs)作为种子细胞异种移植到F344大鼠体内是否成骨,探索获得骨组织工程异基因种子细胞的一种方法。方法应用含有目的基因hCTLA4-Ig和增强绿色荧光蛋白(EGFP)的逆转录病毒感染第1代hMSCs,应用G418筛选出抗性细胞群(hMSCs-CTLA4);用逆转录-聚合酶链反应(RT-PCR)和免疫细胞化学方法分别检测hMSCs-CTLA4中hCTLA4-IgmRNA和蛋白质表达。用荧光激活细胞分选仪(FACS)检测hMSCs-CTLA4中表达hCTLA4-Ig蛋白的阳性率。将hMSCs-CTLA4作为种子细胞在体外构建组织工程骨并移植到F344大鼠皮下,用X线片、组织形态学、免疫组织化学方法分别观察其体内成骨状况和hCTLA4-Ig表达情况。结果分离的hMSCsCD105表达阳性,CD34表达阴性。hMSCs-CTLA4的细胞形态呈梭形,与hMSCs比较无明显改变;倒置荧光显微镜观察见绝大多数细胞呈绿色,强阳性表达EGFP。RT-PCR、免疫细胞化学结果分别证实hMSCs-CTLA4表达hCTLA4-IgmRNA和蛋白。FACS检测结果显示:hMSCs-CTLA4表达hCTLA4-Ig蛋白的阳性率为78.4%。本实验构建的组织工程骨,每克脱钙骨基质(DBM)上吸附、生长的细胞约(1～1.5)×106个。DBM/hMSCs-CTLA4组植入F344大鼠皮下术后2～12周均可检测到hCTLA4-Ig阳性表达细胞,8～12周出现人源性新生骨组织;而单纯DBM组与DBM/hMSCs组表现为DBM逐渐被吸收,代之以纤维结缔组织,没有新生骨组织出现。结论以hMSCs-CTLA4作为种子细胞构建的组织工程骨异种移植到F344大鼠皮下可以成骨。hCTLA4-Ig基因修饰的人骨髓间充质干细胞(hMSCs)可能作为骨组织工程异基因种子细胞的来源。
Objective To observe osteogenesis after human cytotoxic T lymphocyte associated antigen 4-Ig gene （hCTLA4-Ig） is transfccted into human bone marrow derived mesenchymal stem cells （hMSCs） and explore the method for obtaining tissue-engineered allogeneic seed cells. Methods The first generation of hMSCs were infected with retrovirus containing hCTLA4-Ig and EGFP. Then, hMSCs- CTLA4 was isolated from bone marrow of the iliac crest of voluntary normal adults by density gradient centrifugation of Ficoll （1. 073 g/ml）. Culture-expanded hMSCs were transfected by a retroviral vector containing the gene of hCTLA4-Ig and screened by G418 （0.5 mg/ml）, hCTLA4-Ig mRNA and protein expressions of hMSCs-CTLA4 were observed by RT-PCR and immunocytochemistry respectively. In addition, the positive expression rate of hCTLA4-Ig in hMSCs-CTLA4 was investigated by fluorescence activated cell sorter （FACS）. The tissue engineered bone was reconstructed by using hMSCs-CTLA4 as seed cells and transplanted into subcutaneous part of F 3 4 4 of rats. Thereafter , expression of hCTLA 4 - Ig and osteogenesis of hMSCs-CTLA4 were observed by means of X-ray photography, morphology and immunohistochemistry. Results hMSCs-CTLA4 was fusiform shaped, with insignificant change compared with hMSCs. The inverted fluorescence microscope showed that majority of cells were green in color with strongly positive expression of EGFP. RT-PCR and immunocytochemical resulted both proved that hMSCs- CTLA4 expressed hCTLA4-Ig mRNA and protein. Results of FACS detection showed that positive rate of hMSCs-CTLA4 expressing hCTLA4-Ig protein was 78. 4%. Every grain of decalcified bone matrix （DBM） constructed by ourselves absorbed cells for about （1-1.5） × 10^6. In group DBM/hMSCs-CT- LA4, after hMSCs-CTLA4 was transplanted into subcutaneous part of F344, positive expression cells of hCTLA4-Ig could be detected at 2-12 weeks and neonatal bone tissue emerged at 8-12 weeks. While in groups of only DBM and DBM/hMSCs, DBM was gradually resorb
出处
《中华创伤杂志》
CAS
CSCD
北大核心
2005年第11期838-844,共7页
Chinese Journal of Trauma
基金
国家自然科学青年基金资助项目(30300090)
国家重点实验室开放课题基金资助项目
