摘要
目的构建表达外源性胶质细胞源性神经营养因子(GDNF)的转基因大鼠骨髓基质干细胞(BMSCs),拟应用于脊髓损伤的基因治疗研究。方法提取新生大鼠肾组织mRNA,用逆转录PCR法扩增GDNF全长cDNA,构建其真核表达载体PEG-GFP-GDNF,酶切及测序鉴定;然后转染原代培养的大鼠BMSCs,荧光免疫细胞化学及细胞形态学检测GDNF的表达。结果扩增到全长687bp序列准确的GDNF编码片段,并在GDNF转基因原代大鼠BMSCs中检测到重组GDNF的表达。结论体外可构建成功表达GDNF的转基因BMSCs,有望应用于脊髓损伤等中枢神经系统疾病的治疗研究。
Objective To construct genetically modified rat bone marrow stem cells （BMSCs） expressing exogenetic glial cell-line derived neurotrophic factor （GDNF） which can be used in gene therapy of spinal cord injury （SCI） . Methods The total mRNA was extracted from newborn rat kidney by one-step method. GDNFeDNA was acquired by RT-PCR. The vector of PEG-GFP-GDNF was constructed and identified by double enzyme digestion. Its sequence was analyzed. The transgenetic expression results were identified with immunofluoreseenee detection and immunoeytoehemieal stain. Results 687 bp eDNA of GDNF was obtained and the sequence preciseness was identified by sequence analysis. GDNF was expressed in the transgenetie BMSCs. Conclusion Since recombinant genetic engineered BMSCs are capable of expressing GDNF, they may be further researched on for transgenetie treatment of trauma and disease of central nervous system.
出处
《中华创伤骨科杂志》
CAS
CSCD
2005年第9期848-851,共4页
Chinese Journal of Orthopaedic Trauma
