骨髓间充质干细胞介导大鼠肺内基因转移的可行性

Bone marrow mesenchymal stem cell mediated gene transfer into rat lung

目的评价骨髓间充质干细胞(MSCs)介导大鼠β-半乳糖苷酶(Lac-Z)基因肺内转移的可行性。方法对原代培养的Lewis大鼠MSCs进行Lac-Z表达载体(pSV-β-Gal)转染和DAPI荧光染色标记,再将MSCs(5×105细胞/每只大鼠)经颈静脉输入同基因背景受体Lewis鼠体内(n=10),48 h 和8周后处死大鼠(每时点5只大鼠),制作肺、脾、肝、肾、骨骼肌标本,荧光显微镜观察荧光标记的移植细胞,将肺组织与X-gal色素原一起孵育检测Lae-Z基因表达产物。结果静脉输入细胞后48 h及 8周,脾、肝、肾组织仅见少量DAPI标记的阳性细胞,骨骼肌组织未见阳性细胞,但肺组织内可见较多 DAPI标记的阳性细胞,主要分布于肺间质和肺泡腔内。经颈静脉输入转染了pSV-β-Gal的MSCs后48 h及8周,肺组织标本与X-gal色素原溶液一起孵育后,显微镜下可见大鼠肺内散布着许多深蓝色标记的MSCs,而脾、肾中则仅见少量深蓝色标记的MSCs,肝、骨骼肌未见深蓝色标记的MSCs。结论颈外静脉注射基因修饰的MSCs具有较好的肺内靶向性,且在受体鼠肺可较长时间表达外源基因。

Objective To determine the feasibility of achieving local transgenic expression in the rat lung using bone marrow mesenchymal stem cells （MSCs） transfected with Lac-Z gene. Methods Primary cultures of bone marrow MSCs from Lewis rats were transfected with the pSV-β-galactosidase control vector and labelled with a fluorescent, membrane impermeable dye DAPI. The transfected and labelled MSCs （5× 10^5 cells/animal） were injected into the jugular vein of syngenetic recipient rats. The animals were killed at 48 h and 8 wk after injection respectively. The lungs, spleens, livers, kidneys and skeletal muscle were then excised and examined under fluorescene microscope. The transgenic expression of Lac-Z gene was detected by incubating with the X-gal chromogen. Results Only a few DAPI labelled MSCs could be identified in the spleen, liver, kidney and skeletal muscle, whereas a large amount of DAPI labelled MSCs could be identified in the lung and most of them lodged in the lung parenchyma and air sac at 48h and 8wk after intravenous injection of transfected MSCs. After incubation with the X-gal chromogen, microscopic examination showed that a large number of MSCs with multiple intense blue staining were scattered throughout the lung. On the contrary only a few cells with blue staining could be identified in the spleen and kidney and no MSCs with blue staining could be seen in the liver pancreas and skeletal muscle. Conclusion Genetically modified MSCs injected into the jugular vein can target the lung effectively and achieve local transgenic expression for a long time.