摘要
目的探讨SPIO标记3.0T磁共振成像辅助示踪技术在TRPV1基因修饰BMSCs治疗脑缺血再灌注损伤大鼠的相关影响。方法提取并培养鼠源骨髓间充质干细胞(BMSCs),利用流式细胞仪进行鉴定。通过Cas-9技术构建TRPV1的过表达载体,利用慢病毒转染BMSCs,根据病毒转染情况将BMSCs分成3组,即空白对照组、阴性对照组和过表达载体组。利用RT-PCR和Western blot法检测TRPV1过表达载体转染后对BMSCs的促神经分化作用。利用超顺磁性氧化铁(SPIO)试剂盒标记BMSCs染色,构建缺血再灌注损伤SD大鼠模型。利用3.0T磁共振成像SPIO标记BMSCs的归巢检测。结果1)BMSCs的表面蛋白CD44和CD29的表达为60.2%和58.3%;CD34和CD45表达为3.4%和2.6%;2)慢病毒可以将TRPV4过表达载体质粒转染至BMSCs细胞内,且效率较高,为90%以上;3)体外细胞实验结果显示,过表达载体组TRPV4,Peripherin和NSE的mRNA相对表达量和蛋白表达量要明显高于空白对照组和阴性对照组(P<0.05);4)BMSCs细胞可以与超顺磁性氧化铁(SPIO)相结合,从而可以作为示踪分子,检测后续BMSCs细胞尾静脉注射后BMSCs的示踪行为;5)相较于BMSCs组,将TRPV1基因过表达载体转染BMSCs后,可以更好的保护脑组织。结论TRPV1过表达载体可以促进BMSCs向神经细胞分化,且对缺血再灌注损伤SD大鼠具有一定程度的保护作用。
Objective To investigate the effect of SPIO labeled 3.0T MR assisted tracing technique in the treatment of cerebral ischemia-reperfusion injury in rats with TRPV1 gene modified BMSCs.Methods BMSCs were extracted and cultured and identified by flow cytometry.The overexpression vector of TRPV1 was constructed by cas-9 technology,and BMSCs were transfected by lentivirus.BMSCs were divided into three groups according to virus transfection,namely blank group,negative control group and overexpression vector group.RT-PCR and Western blot were used to detect the effect of TRPV1 on the neural differentiation of BMSCs.Using superparamagnetic iron oxide(SPIO)kit to label BMSCs,the SD rat model of ischemia-reperfusion injury was established.3.0T MRI SPIO was used to detect the homing of BMSCs.Results 1)the expressions of CD44 and CD29 were 60.2%and 58.3%in BMSCs,and 3.4%and 2.6%in CD34 and CD45;2)Lentivirus can transfect TRPV4 over expression vector plasmid into BMSCs cells,and the efficiency was higher,more than 90%;3)The results of cell experiments in vitro showed that the relative mRNA expression and protein expression of TRPV4,peripherin and NSE in the overexpression vector group were significantly higher than those in the blank control group and the negative control group(P<0.05);4)BMSCs can be combined with superparamagnetic iron oxide(SPIO),which can be used as a tracer molecule to detect the tracing behavior of BMSCs after tail vein injection;5)Compared with BMSCs group,TRPV1 gene overexpression vector can protect brain tissue better.Conclusion TRPV1 overexpression vector can promote the differentiation of BMSCs into neural cells,and has a certain degree of protective effect on SD rats with ischemia-reperfusion injury.
作者
张海霞
刘峰
ZHANG Haixia;LIU Fen(Department of Imaging, Shangdong Province coal Taishan Sanatorium, Taian 271000, P.R,China;Institute of Radiology, Shandong Academy of Medical Sciences, Jinan 250062, P.R.China;Department of Imaging, Xintai People's Hsopital Taishan Medical College, Taian 271200, P.R.China)
出处
《医学影像学杂志》
2021年第5期873-877,共5页
Journal of Medical Imaging
