应用硝普钠促进经门静脉移植的骨髓间充质干细胞在肝内弥散分布

目的探讨硝普钠是否可促进经门静脉移植的骨髓间充质干细胞(MSCs)在肝内弥散分布。方法分离SD大鼠的骨髓MSCs,进行培养、传代,流式细胞仪鉴定MSCs表面标记,以Fe2O3-PLL标记供移植的MSCs。以彩色多普勒超声检查大鼠注射硝普钠后10～20 min时门静脉内径及血流速度变化,并计算其血流量。受者(SD大鼠)背部皮下注射四氯化碳,制成肝损伤模型,将模型随机分为实验组和对照组,实验组经门静脉注射以Fe2O3-PLL标记的MSCs悬液0.5 ml及含硝普钠的生理盐水(硝普纳的注入量为24 nmol/100g),对照组仅注射Fe2O3-PLL标记的MSCs悬液0.5 ml。分别于MSCs注射前及注射后3 h、1周、2周、4周行磁共振扫描,测定肝脏信噪比(SNR)。最后1次磁共振扫描后,处死全部受者,取各叶肝组织,行HE染色和普鲁士蓝染色,进行组织学观察。结果分离获得的MSCs经培养、传代,高表达CD29(99.88%)和CD90(99.84%),CD45阳性细胞极少(0.13%)。注射硝普钠后10～20 min,大鼠门静脉内径明显扩张(P<0.01),门静脉血流量明显增多(P<0.01)。移植后随着时间延长,两组肝脏SNR均逐渐增高,实验组移植后3 h、1周、2周时的肝脏SNR明显低于对照组(P<0.05),至移植后4周,两组肝脏SNR的差异无统计学意义;两组移植后3 h、1周、2周时的肝脏SNR均明显低于移植前(P<0.05),至移植后4周,肝脏SNR恢复至移植前水平。实验组肝组织普鲁士蓝染色阳性细胞多于对照组(P<0.01)。结论门静脉注入硝普钠后,其分支内径扩大,血流量增加,在经门静脉移植MSCs的同时注入硝普钠可促进MSCs向末梢分支与血窦弥散分布。

Sodium nitroprusside promoting intrahepatic diffusion and proliferation of bone marrow mesenchymal stem cells transplanted through portal vein

Objective To observe whether sodium nitroprusside (SNP) can promote intrahepatic diffusion and proliferation of bone marrow mesenchymal stem cells (MSCs) transplanted through portal vein. Methods Male SD rats aged 6 weeks and 7 weeks were chosen as cell transplantation donors and recipients respectively. MSCs of donors were isolated, cultured, passaged and identified by the detection of its membranous antigen using flow cytometer (FCM) and then labeled with Fe2O3-PLL. By using color Doppler imaging, the width of rat portal vein and the velocity of its blood flow were measured before and 10-20 min after intra-superior mesenteric vein injection of SNP, and the blood flow calculated finally. Recipient rats were subjected to cell transplantation after 4 subcutaneous injections of carbon tetrachloride (CCl4) which dissolved in olive oil by a ratio of 1:1. Recipient rats were randomly divided into experimental group (0. 5 ml Fe2O3-PLL-labled MSCs suspensions together with SNP at a dose of 24 nmol/100 g via portal veins, n = 5) and control group (0. 5 ml Fe2O3-PLL-labeled MSCs suspensions alone via portal veins, n = 5). Follow-up serial liver MRI was performed and signal-to-noice ratio (SNR) of liver on T2* WI was measured before and 3 h, 1 week, 2 weeks and 4 weeks after cell transplantation. All animal subjects were killed after the last MR scan, and histological examination of each liver lobe was carried out. Results After Prussian blue staining, blue particles were clearly displayed within almost every labeled cell, while they were not found in the un-labeled cells. For the MSCs passaged for 3 generations, the percentage of CD29-positive and CD90-positive cells reached 99. 88 % and 99. 84 % respectively, while the percentage of CD45-posive cells 0. 13 % only. The width of rat portal vein was expanded (P<0. 01) and the blood flow increased (P<0. 01) significantly 10-20 min after intra-superior mesenteric vein injection of SNP. MR imaging revealed that SNR of livers in both groups was increased gradually as time went on after the transplantation. The SNR of livers in experimental group was obviously lower than that in control group 3 h, 1 week and 2 weeks after transplantation (P<0. 05), while there was significant difference in SNR of livers between two groups 4 weeks after transplantation (P>0. 05). The SNR of livers in both groups was decreased significantly 3 h, 1 week and 2 weeks after injections of MSCs (P<0. 05) and gradually returned to that of rat livers before transplantation 4 weeks after transplantation. Histological examination proved that the average number of Prussian blue-stained cells per high-powered field of view in experimental group was significantly more than that in control group (P<0. 01). Conclusion Using SNP to expand the width of portal vein branches and increase the blood flow of portal vein can accelerate the diffusion of MSCs transplanted through portal vein into end vessels and sinusoids, resulting in a greater proliferation of transplanted MSCs in the host liver.