pLXSN—BDNF体外转染神经干细胞及其外源基因的表达  

Measuring the expression of exogenous BDNF of NSCs after infection of supernatant from recombinant retrovirus pLXSN-BDNF

作　　者：周雪美[1] 原慧萍[1] 吴东来[2] 王雅[1] 王卓[1] 

机构地区：[1]哈尔滨医科大学附属第二医院眼科,150086 [2]中国农业科学院哈尔滨兽医研究所

出　　处：《中华微生物学和免疫学杂志》2009年第9期786-791,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目（30471844）;黑龙江省教育厅资助项目（10551187）

摘　　要：目的建立体外稳定表达脑源性神经生长因子（BDNF）的神经干细胞（NSCs）。方法采用有限稀释法纯化体外培养的NSCs。用重组和空白逆转录病毒感染NSCs，分别采用荧光定量PCR法和ELISA法检测3组（pLXSN．BDNF病毒感染组、pLXSN病毒感染组、空白对照组）NSCs中外源基因的表达水平。结果荧光定量PCR法测定pLXSN-BDNF组BDNF原始模板拷贝数为（19．57±0．65）×10^3 copies／μl，与其余两组比较差异有统计学意义（P〈0．05）；ELISA法测定pLXSN-BDNF组NSCs细胞上清中BDNF含量最高，与其余两组比较差异有统计学意义（P〈0．05）。结论用携带BDNF的重组逆转录病毒能成功地将外源基因导入NSCs中，为NSCs眼内移植研究，也为基因治疗视神经损伤的量化研究提供实验依据。Objective To develop neural stem cells（NSCs） which can stably express exogenous brain-derived neurotrophic factor（BDNF） in vitro. Methods NSCs from the subependymal zone of embryonic day 14.5 （E14.5） rat brain were purified by limiting dilution assay and then infected with supernatant of recombinant retrovirus pLXSN-BDNF and retrovirus pLXSN. The original copy numbers of exogenous gene templates from three groups NSCs（ pLXSN-BDNF viral infection group, pLXSN viral infection group, control group） were detected by fluorescent quantitative PCR（FQ-PCR）. ELISA assay was used for determining the protein contents of BDNF of supernatant from three groups NSCs for six days continually after seeded in 24- well plates in the same cell density. Results NSCs were purified successfully by limiting dilution assay. The original copy numbers of exogenous BDNF gene templates from pLXSN-BDNF viral infection group by FQ-PCR were （19.57 ±0.65） ×10^3 copies/μl, higher than those of another two groups（P 〈0.05）. The protein contents of BDNF of supernatant from NSCs of pLXSN-BDNF viral infection group was highest among three groups and compared with another two groups had statistical significance （ P 〈 0.05 ） . Conclusion The purified NSCs can be transduced exogenous BDNF successfully with supernatant of recombinant retrovirus pLXSN-BDNF which provide experimental evidences and laying foundations for further research of retinal transplantation and quantization investigation of gene therapy for optic nerve injury.

关 键 词：神经于细胞 纯化 重组逆转录病毒 荧光定量PCR法 ELISA法 

分 类 号：R775[医药卫生—眼科]