大鼠骨髓间充质干细胞的钆-荧光双标记及MRI体外示踪的研究

Gadolinium and fluorescent bi-functionally labeling and in vitro MRI of rat bone marrow mesenchymal stem cells

目的应用多聚胺载体，对大鼠骨髓间充质干细胞（MSCs）进行钆喷替酸葡甲胺（Gd-DTPA）及荧光双标记，探讨MSCsMR磁性标记及体外示踪的可行性。方法以聚乙烯亚胺-罗丹明复合物（JetPEI—FluoR）为载体，制备Gd—DTPA双标记示踪剂，培养分离sD大鼠骨髓MSCs，以此示踪剂体外标记间MSCs。对标记后细胞行生物学性状检测及电镜、荧光镜观察。应用1．5TMR仪，对标记的干细胞进行sET1WI及T2WI及混合（mixed）回波序列的T1测量，标记细胞正常传代后进行MR检查，观察标记的持久性。标记细胞、未标记细胞的T1WI信号强度、T1之间的比较使用t检验，台盼蓝拒染率采用两因素重复资料方差分析，不同浓度示踪下细胞吸光度比较采用单因素方差分析。结果双标记示踪剂标记5×10^5个MSCs，标记成功了4．25×10^5个，荧光镜下标记率为85％，电镜下钆（Gd）颗粒主要位于胞质内高尔基体周围。双标记示踪剂孵育3、6、12、24h内标记细胞的台盼蓝拒染率分别为（96．55±2．90）％、（94．17±2．56）％、（97．16±3．12）％、（94．23±2．67）％，相应的未标记细胞的拒染率分别为（95．86±2．67）％、（92．04±2．21）％、（93．38±3．64）％、（92．12±2．53）％，24h内标记细胞与未标记细胞拒染率差异无统学意义（F=4．523，P〉0．05）。细胞增殖实验中，在2．5、5．0、10．0、20．0、30．0、40．0μl不同浓度示踪剂时，标记细胞的吸光度分别为（0．1884±0．0151）、（0．1878±0．0190）、（0．1741±0．0160）、（0．1135±0．0215）、（0．1079±0．0145）、（0．0811±0．0079），未标记细胞为（0．1940±0．0116），Gd—DTPA30．0μl以下标记细胞与未标记细胞吸光度差异无统计学意义（q’=0．2225～0．9458，P〉0．05）。标记后细胞凋亡指数为5．08％，对照组未标记细胞为3．86％。未标记细胞T1WI平均信号强度及T1分别为240．3±24．7、（2457±56）ms，而标记细胞为336．2±20．7、（1102±64）ms，两者间差异有统计学意义（t值分别为12．656、17．889，P值均〈0．01），T1WI可监测到最低密度为5×10^3个标记细胞。标记细胞正常传代后，MRI体外可持续显示至第4代标记细胞。结论应用多聚胺载体对大鼠间充质干细胞进行Gd—DTPA及荧光双标记，安全有效，MRI能示踪体外双标记的干细胞。

Objective To determine the feasibility of magnetically labeling and tracking mesenchymal stem cells （MSCs） in vitro by using a gadolinium and fluorescent bi-functionally transfection agent of polyethylenimine. Methods A gadolinium bifunctional transfection reagent complex was obtained after the linear polyethylenimine derivative （JetPEI-FluoR） was incubated with Gd-DTPA. Mesenchymal stem cells isolated from the bone marrows of SD rats were cultured and expanded. The mesenchymal stem cells were incubated with the bi-functional labeling agents. After labeling, the MSCs were examined with fluoroscope and electron microscope and the biological characters were detected including trypan blue exclusion test, MTT, and apoptosis detection. On a 1.5 T MR system, the labeled MSCs were examined with spin echo T1WI and T2WI and T1 measurement with mixed sequence. After labeling, the cells were cultured and undergone routine passage. Prior MR examinations were repeated for each passage of labeled cells. All data was statistically prolessed with SPSS for Windows. Results Of 5 ×10^5 MSCs incubated with the bi-functional agents,4. 25 × 10^5 MSCs were successfully labeled, the percentage of labeled MSCs was 85% fluoroscopically. The high density electron particles of gadolinium observed electron microscopically existed around cellular apparatuses, especially around Golgi apparatus. In trypan blue exclusion test, the exclusion rate of labeled MSCs with incubation duration of 3,6,12,24 h was （96.55 ± 2.90） % , （94.17 ± 2. 56） % , （97.16 ± 3. 12） % and （94. 23 ± 2. 67 ） % , respectively. The corresponding exclusion rate of unlabeled MSCs was （95.86 ±2.67）%, （92.04 ±2.21）%, （93.38 ±3.64）% and （92. 12 ±2.53）%, respectively. There was no statistical difference of trypan blue exclusion rate between labeled cells and control unlabeled cells within 24 hours of incubation （ F = 4. 523, P 〉 0.05 ）. In the proliferation test, the optical absorption value of labeled MSC with 2. 5, 5.0, 10. 0, 20. 0, 30. 0 and 40. 0 μl bi-functional labeling agent was （0. 1884 ±0. 0151） , （0. 1878 ±0. 0190） , （0. 1741 ±0. 0160）, （0. 1135 ±0. 0215）, （0. 1079 ± 0. 0145 ） and （0. 0811 ± 0. 0079）, respectively. The corresponding optical absorption value of unlabeled MSCs was （0. 1940 ±0. 0116）. The optical absorption value of labeled cells was not affected in case of less than 30. 0 μl of Gd-DTPA （ q＇ = 0. 2225-0. 9458, P 〉 0. 05 ）. The apoptosis index for labeled cells and unlabeled cells were 5.08% and 3. 86%, respectively. On T1WI, the signal intensity and Tt relaxation time of unlabeled cells and labeled cells were 240. 3 ± 24. 7 and （ 2457 ± 56） ms, 336. 2 ± 20. 7 and （1102 ± 64）ms, respectively, and there were significant statistical difference （t = 12. 656, 17. 889, P 〈 0. 01 ）. The minimal amount of cells which was detectable for T1WI was 5 ×10^3. After routine passage, the gadolinium in the cells gradually decreased and could be tracked by MRI until the fifth passage. Conclusions The gadolinium and fluorescent bi-functionally labeling rat bone marrow mesenchymal stem cell by using the transfection agent of polyethylenimine is feasible, efficient and safe, The labeled ceils could be tracked in vitro on MR imaging.