重组慢病毒GV303 Rat IL-33体外转染原代大鼠骨髓间充质干细胞的研究

Establishment of rat bone mesenchymal stem cells expressing IL-33

目的:探索构建稳定表达IL-33的原代大鼠骨髓间充质干细胞(BMSC)细胞系的方法。方法:体外分离、培养、鉴定大鼠骨髓间充质干细胞,并进行ELISA实验检测转染前IL-33的表达情况。构建GV303 Rat IL-33慢病毒载体,并进行慢病毒的包装。3d后荧光显微镜下观察GFP的表达,分别使用Western blot、ELISA检测转染后的BMSC中IL-33的表达。结果:大鼠BMSC被成功分离并传代,流式细胞术鉴定BMSC可得到CD90、CD29为阳性,CD11b、CD45、CD34为阴性,与之前的文献报道相符。并且BMSC被成功诱导分化为骨细胞、软骨细胞、脂肪细胞。对转染前的BMSC行ELISA实验检测IL-33的表达,结果为阴性。病毒转染3d后于荧光显微镜下可观察到GFP的表达;ELISA实验检测转染后BMSC中IL-33的表达,结果为阳性;Western blot亦可以检测到IL-33的表达。结论:应用GV303慢病毒载体可以建立稳定表达IL-33的大鼠BMSC原代细胞系。

AIM： To explore a way to establish a rat bone mesenchymal stem cell（BMSC）line expressing Interleukin（IL）-33. METHODS： BMSCs were isolated from 6-week-old Wistar Rat. GV303 virus were used as a IL-33 gene carrier to tansfect isolated BMSCs.Three days later, transfected BMSCs were observed under a fluorescence microscope. The expression level of IL-33 of transfected BMSCs was detected by Western blot and ELISA. RESULTS： The BMSCs were successfully isolated, since flow cytometry results showed that rat BMSCs CD90 and CD29 positive, CD45,CD34 and CD11b negtive. Furthermore, BMSCs were able to be differentiated to osteoblasts, adipocytes and chondrocytes respectively. Green fluorescence of GFP BMSCs was observed under the fluorescence microscope 3d after virus transfecting. Expression of IL-33 was detected by Western blot and ELISA in the transfected rat BMSCs. CONCLUSION： A rat BMSC cell line expressing IL-33 was established by GV303 virus transfection.