Huang Dong-Yu, Zhang Zhi-Jian, Chen Bai-Ling, Wu Xiu-Li, Wang Ning, Zhang Yan-Ding
Department of Neurology, The First Hospital Affiliated to Fujian Medical University, Fuzhou 350005, China.
Sheng Wu Gong Cheng Xue Bao. 2007 Mar;23(2):235-40. doi: 10.1016/s1872-2075(07)60020-x.
Recently, mesenchymal stem cells (MSCs) have been one of the target cells of gene engineering. To construct the lentiviral (LV) vectors carrying the brain-derived neurotrophic factor (Bdnf) gene, the rat mesenchymal stem cells (rMSCs) were infected and finally the Bdnf gene-modified rMSCs was obtained. The CDS region of the rat Bdnf gene was obtained with reverse transcriptase-polymerase chain reaction (RT-PCR), and the transfer plasmid (PNL-BDNF-IRES2-EGFP) of the LV vector was constructed. The three plasmids of LV vector: PNL-BDNF-IRES2-EGFP, HELPER, and VSVG were cotransfected to 293T cells to produce the LV vectors, which enabled the coexpression of the Bdnf gene and the enhanced green fluorescent protein (Egfp) gene. rMSCs were separated from the bone marrow of 2-month-old F344 rats, cultured in vitro, and identified. rMSCs were infected by the LV vectors that were produced already and were identified with fluorescent microscope, RT-PCR, immunocytochemical staining, and western blot. The result of sequencing showed that the sequence of the cloned Bdnf gene was consistent with that reported in the GenBank. The PNL-BDNF-IRES2-EGFP plasmid that was identified showed the correct sequence. After the 3 plasmids of LV vectors were cotransfected to the 293T cells, considerable green fluorescence in 293T cells was observed under the fluorescent microscope; the supernatant was collected and concentrated using ultracentrifugation, and the titer of the replication-defective LV vector particles measured was found to be 6.7 x 10(7) TU/mL. After the constructed LV vectors infected the rMSCs, the results obtained using RT-PCR, immunocytochemical staining, and western blot showed that the expression of BDNF in the Bdnf-rMSCs group (experimental group, EG) was significantly higher than that in the PNL-IRES2-EGFP-rMSCs group (mock group, MG) and the rMSCs group (control group, CG) at both mRNA and protein levels. LV vectors carrying the Bdnf gene were constructed successfully. The Bdnf gene-modified rMSCs could express BDNF to a higher degree. This greatly facilitates the next step in the study, such as the long period of therapeutic observation of cerebral ischemia with Bdnf gene-modified rMSCs.
近年来，间充质干细胞（MSCs）一直是基因工程的靶细胞之一。为构建携带脑源性神经营养因子（Bdnf）基因的慢病毒（LV）载体，对大鼠间充质干细胞（rMSCs）进行感染，最终获得Bdnf基因修饰的rMSCs。通过逆转录聚合酶链反应（RT-PCR）获得大鼠Bdnf基因的编码区（CDS），并构建LV载体的转移质粒（PNL-BDNF-IRES2-EGFP）。将LV载体的三种质粒PNL-BDNF-IRES2-EGFP、HELPER和VSVG共转染至293T细胞以产生LV载体，该载体可使Bdnf基因和增强型绿色荧光蛋白（Egfp）基因共表达。从2月龄F344大鼠的骨髓中分离rMSCs，进行体外培养和鉴定。用已产生的LV载体感染rMSCs，并通过荧光显微镜、RT-PCR、免疫细胞化学染色和蛋白质印迹法进行鉴定。测序结果表明，克隆的Bdnf基因序列与GenBank中报道的序列一致。鉴定后的PNL-BDNF-IRES2-EGFP质粒序列正确。将LV载体的三种质粒共转染至293T细胞后，在荧光显微镜下观察到293T细胞中有大量绿色荧光；收集上清液并通过超速离心进行浓缩，测得复制缺陷型LV载体颗粒的滴度为6.7×10⁷ TU/mL。构建的LV载体感染rMSCs后，RT-PCR、免疫细胞化学染色和蛋白质印迹法的结果表明，Bdnf-rMSCs组（实验组，EG）中BDNF在mRNA和蛋白质水平的表达均显著高于PNL-IRES2-EGFP-rMSCs组（空载体组，MG）和rMSCs组（对照组，CG）。成功构建了携带Bdnf基因的LV载体。Bdnf基因修饰的rMSCs能够更高程度地表达BDNF。这极大地促进了下一步研究，如用Bdnf基因修饰的rMSCs对脑缺血进行长期治疗观察。
