绿色荧光蛋白基因转染示踪比格犬体内骨髓间充质干细胞分化

摘要

Objective To observe differentiation and transformation of bone mesenchymal stem cells (BMSCs)labeled with green fluorescent protein (GFP)technology in bone tissue engineering.Methods Adenoviral delivery of GFP genes (Ad-GFP)transfected to canine BMSCs.The morphology of BMSCs,alkaline phosphatase (ALP)and alizarin red staining were observed with the help of invert light microscope,and the expression of GFP was scrutinized by using fluorescence microscope.Once succeeding,the centrifuged BMSCs were collected and mixed with beta-tricalcium phosphate (β-TCP),then they were transplanted under tibial periosteum.The beagles using the above way to construct tissue engineering bone were marked group A,which only withβ-TCP transplated into tibial periosteum were marked group B, and which withβ-TCP subcutaneously placed were marked group C.In the postoperative 2 weeks,histology and immunohistochemistry results were studied and the expression of GFP was observed by laser scanning confocal microscope.Results After induced differentiation in vitro, observing BMSCs found that ALP staining and alizarin red mineralization nodules staining were positive.Histology observation revealed that compared with group B,abundant bone formed and neovascularized significantly in group A,and bone formation did not exist in group C. Immunohistochemistry staining showed that type Ⅰ collagen and platelet endothelial cell adhesion molecule-1 (CD31)were positive in group A, type Ⅰ collagen and CD31 were positive partly in group B rather than group C.The expression of typeⅠcollagen and CD31 were analyzed in group A and group B by the way of semi-quantitative immunohistochemical staining.The value of type Ⅰ collagen and CD31 had significant differences between group A and group B.The expression of GFP was observed by laser scanning confocal microscope in group A not in group B nor group C.Conclusion BMSCs is the important factor to accelerate the generation of tissue engineering bone instead of osteoblasts in surrounding.They could promote endothelial cell tube formation inside periosteum.%目的运用绿色荧光蛋白（GFP）标记技术，观察骨组织工程中种子细胞的变化和转归。方法使用重组腺病毒（Ad-GFP）将 GFP 基因转染比格犬骨髓间充质干细胞（BMSC），倒置相差显微镜下观察 BMSC 形态、碱性磷酸酶（ALP）和茜素红染色情况；荧光显微镜下观察细胞 GFP 表达状况。待细胞标记成功后将其离心，接种于β-磷酸三钙（β-TCP）上，回植于比格犬胫骨内侧骨膜囊内，构建组织工程骨记为 A 组；单纯β-TCP 植入对称胫骨内侧骨膜囊记为 B 组；单纯β-TCP 植入皮下记为 C 组。术后2周取材，分别行组织学观察、免疫组化检测，同时激光共聚焦显微镜下观察 GFP 表达情况。结果体外成骨诱导实验显示，ALP 染色以及茜素红染色阳性。组织学观察可见，A 组新生骨组织以及新生血管均较 B 组明显，C 组未见新生骨组织。免疫组化检测显示，A 组Ⅰ型胶原、血小板内皮细胞黏附分子 CD31染色阳性；B 组Ⅰ型胶原、CD31染色部分阳性；C 组未见阳性表达。免疫组化半定量检测显示，A、B 两组Ⅰ型胶原、CD31定量有统计学差异（P ＜0．05）。激光共聚焦显微镜观察结果显示，A 组新生骨组织内可见 GFP 表达，B、C 组未见 GFP 标记细胞。结论BMSC 是组织工程骨早期形成的重要来源，但周围成骨细胞并非新生骨组织主要来源；移植 BMSC 于骨膜下可促进新生血管形成。