3.0 T核磁共振在大鼠体内示踪环氧化酶-2基因沉默的干细胞

In vivo tracking human bone marrow -derived mesenchymal stem cells after genetically modified

目的利用临床3.0 T核磁共振成像（MRI）对通过尾静脉注射Molday ION Rhodamine-B^TM（MIRB）标记慢病毒介导环氧化酶-2（COX-2）基因沉默的人骨髓间充质干细胞（hBMSCs）的大鼠活体示踪。 方法利用慢病毒介导在hBMCSs中沉默COX-2基因，通过反转录-聚合酶链反应（RT-PCR）和Western blot检测基因表达。将转基因hBMSCs进行新型氧化铁颗粒MIRB标记，测定最佳标记浓度，通过细胞计数试剂盒（CCK-8）测定标记后细胞增殖能力，分组检测细胞成骨、成脂能力。将MIRB标记、慢病毒介导COX-2基因沉默的hBMSCs，单纯hBMSCs与磷酸盐缓冲液（PBS）分为3组，分别通过尾静脉注射的方法注射入大鼠体内。分别于注射后1 h、7 d、14 d通过临床3.0 T MRI检测大鼠脑、肝脏，检测后处死大鼠，取脑、肝脏组织石蜡切片，通过荧光显微镜观察组织内荧光，普鲁士蓝染色观察组织内铁粒子位置。 结果通过RT-PCR和Western blot证实COX-2在hBMSCs中成功敲除，与对照组比较，MIRB浓度为10 μg Fe/ml时标记率可达到90%，当浓度上升到20 μg Fe/ml时标记率可达到98%，浓度为50 μg Fe/ml时标记率与20 μg Fe/ml时比较差异无统计学意义，MIRB浓度〈50 μg Fe/ml时对转基因干细胞可成功标记并不影响其分化、增殖能力。在大鼠体内注射7 d后，通过MRI梯度回波T2加权在大鼠肝脏组织内发现低信号逃避区域，并随时间延长逐渐加强，注射14 d后低信号区域趋于稳定，通过组织切片在大鼠肝脏血管、血窦、肝小叶间和被膜中发现铁粒子的存在。 结论MIRB对转基因干细胞具有生物安全、高效标记、多重方法检测的优点。MIRB标记的慢病毒介导COX-2基因沉默的hBMSCs在注射入大鼠体14 d内，可以通过3.0 T MRI检测并且追踪。

Objective To evaluate in vivo magnetic resonance imaging （MRI） for tracking the magnetically labeled human bone marrow- derived mesenchymal stem cells （hBMSCs） which sliencing of cyclooxygenase- 2 （COX -2） transplanted into nude rat through tail vein injection. Methods In the present study, we knockdown COX -2 in hBMSCs by lentivirus infection, detected the expression of COX -2 by reverse transcriptase- polymerase chain reaction （RT- PCR） and Western blotting. Then la- beled with new superparamagnetic iron oxide （SPIO） -Molday ION Rhodamine -BTM（ MIRB）, Cell via- bility, proliferation and differentiation capacity were assessed in vitro using appropriate functional assays. Labeled lenti - shCOX2 hBMSCs, hBMSCs, phosphate buffered saline （PBS） were divide into three groups injected into tail vein of nude rat models, respectively. All nude rats underwent GRE T2 ＊ - weighted MRI 1 h, 7 and 14 days post - injection. After MRI examination, the animals were sacrificed, the brain and liver were prepared for fluorescence observation and prussian blue staining. Results This study confirmed that we down -regulated COX -2 at mRNA and protein level in hBMSCs cells by lentivirus infection suc- cessfully. MIRB positive ceils were over 90% for cells labeled with MIRB （10 μg Fe/ml）and more than 98% for cells labeled with MIRB （20 -50 μg Fe/ml）. The resuhe shows that 〈50 μg Fe/ml MIRB labe- ling did not affect hBMSC viability or the ability to differentiate into either bone or fat. At the 7 clay, the hypointense signal void areas of livers was observed on GRE T2 - weighted MR images and intensified and increased over time. At the 14 day after cell transplantation, iron particles were located in liver blood vessel, sinusoids, interlobular septum and capsule. Conclusion The MIRB labeled lenti - shCOX2 hBMSCs transplanted into nude rat through tail vein can be detected and monitored in vivo with a 3.0 T clinical MRI for up to 14 days after cell transplantation.