腺病毒介导神经生长因子在绿色荧光蛋白基因标记的间充质干细胞中表达及意义

Adenovirus-mediated nerve growth factor expression in mesenchymal stem cells labeled with green fluorescent protein gene

目的为提高间充质干细胞(MSCs)表达神经生长因子(NGF)的能力,同时使MSCs 被绿色荧光蛋白(GFP)稳定标记,探讨通过病毒载体将NGF和GFP基因高效转染到MSCs的方法。方法取2个月龄100～150 g Wistar大鼠,全骨髓法分离培养纯化大鼠MSCs,利用携带人 NGFβ基因的复制缺陷性重组腺病毒(Ad-hNGFβ)和携带GFP基因的复制缺陷性逆转录病毒(Rt- GFP)转染MSCs,荧光显微镜观察GFP阳性的MSCs,免疫细胞化学和Western blot检测hNGFβ的表达。结果 Rt-GFP转染后,80％～90％MSCs可激发出明亮的绿色荧光,且荧光不随培养时间延长和传代次数增加而衰减。Ad-hNGFβ的转染MSCs中hNGFβ阳性率(90．17±2．14)显著高于阴性对照组(2．17±0．75,P<0．01)和空白对照组(1．83±0．98,P<0．01),转染后MSCs中NGFβ表达量(188．67±8．71)显著高于阴性对照组(25．67±4．08,P<0．01)和空白对照组(27．50± 3．33,P<0．01)。结论 Ad-hNGFβ可以高效转染MSCs,实现NGF在MSCs中高效表达,Rt-GFP 可以使MSCs获得长效标记。

Objective To enhance the expression of nerve growth factor （NGF） in mesenchymal stem cells （MSCs） and explore the ways of NGF and green fluorescent protein （GFP） gene highly transfected to MSCs by virus vectors. Methods In 2-month 100-150 g Wistar rats, MSCs were isolated and-purified by using complete bone marrow method. MSCs were transfected with replication-defieient recombinant adenovirus vector （Ad-hNGFβ） and replication-deficient recombinant retrovirus vector （Rt-GFP） respectively. The GFP positive MSCs were observed under fluorescent microscopy and the expression of hNGFβ detected by using immunocytochemistry and Western blot. Results After the MSCs were transfected with Rt-GFP in rats, green fluorescence was triggered in 80% to 90 % MSCs. in Ad-hNGFβ transfected group, the hNGFβ positive rate was （90.17 ± 2.14）%, significantly higher than in negative control group （2.17 ± 0.75） % and blank control group [ （ 1.83 ± 0.98）%, P 〈 0.01 ]. The gray scales of NGFβ in transfected group （188.67 ± 8.71 ） were also significantly higher than those in negative control group （25.67±4.08） and blank control group （27.50±3.33, P〈0.01）.Conclusion Ad-hNGFβ can efficiently transfect MSCs and make NGF mass expression in MSCs. Rt-GFP can make MSCs be labeled in long-term.