小鼠活体内hMSCs干细胞生物发光示踪监测

In vivo monitoring of hMSCs stem cell by bioluminescent tracer in mice

目的:利用慢病毒载体转染和生物发光技术实时监测小鼠活体内人骨髓间充质干细胞(hMSCs)。方法:构建萤火虫荧光素酶(LUC)慢病毒载体,转染hMSCs后注射到小鼠皮下,(IVIS)生物发光成像系统连续检测体外hMSCs和小鼠注射部位的发光强度。结果:1.慢病毒载体对hMSCs的转染率为(29.6±1.5)%,转染后20代的hMSCs仍稳定表达LUC;2.慢病毒载体对hMSCs的生长和增值没有影响;3.小鼠活体内生物发光强度与注射细胞数量成正比(R2=0.9826);4.小鼠活体内生物发光时间持续6d。结论:可利用慢病毒载体转染LUC对小鼠活体内移植的hMSCs进行生物发光示踪监测。

Background：Bone marrow-derived mesenchymal stem cells （MSCs） have been proved in vitro to inducible differentiate into many kinds of cells of mesenchymal tissues such as osteocytes,chondrocytes, adipocytes,myocytes et al,especially myocardium cells. Recently it was shown that the wall thickness and heart function were improved after MSCs implantation in animal experiments and large-scale clinical trials. But other studies didn’t show that grafted stem cells can bring benefits to heart function in patients with cardiac disease. These therapeutic differences may be rise at least because of lack of visible method to trace in vivo grafted stem cells bio-behaviors such as survive,proliferation,distribution and differentiation. In recent decade an in vivo cellular tracking techniques by using luciferase marker so called BLU have been developed for long-term study of stem cells homing and migration. Bioluminescent imaging （ BLU） technology is a technology to integrate exogenous report luciferase genes into host cells,and express luciferase. This enzyme will give out the visible light when it reacts with its substrate fluorescein under the condition of existing O2 and ATP,to track living cells in real time. In BLU technology the vector for gene transfection is considered a principal because it very closely links to gene transfection efficiency and hence cells lumination. Objective：By using reconstructed-lentivirus vector transfection and bioluminescent imaging techniques to monitor in vivo survival and proliferation and migration of grafted human mesenchymal stem cells （hMSCs） in mice. Methods：Firefly-Luciferase （LUC） -Lentivirus vector were constructed and co-cultured with hMSCs in vitro. Then transfected hMSCs were subcutaneously injected into mice. Either in vitro and in vivo biophotonic imaging of hMSCs were detected by using Xenogen Visible System. Results：1. When multiplicity of infection （MOI） was 100,transfection efficiency of LUC-lentivirus vector could come to （29. 6 ±1. 5）%. Transfected- hMSCs had expressed LUC after 20 successive generation. Indicating this reconstructed lentivirus-LUC-vector has the characteristics of high transfection sensitivity to hMSCs,and high stability for exogenous reporter gene integration and its expression. 2. No morphological and proliferation effect on hMSCs were observed after co- culture with constructed lentivirus vector for 20 generation. This demonstrating that neither lentivirus vector nor exogenous reporter gene has cytotoxicity to targeting cells. 3. Detected in vivo bioluminescence intensity was positively correlated with grafted mass of transfected-LUC hMSCs （ R2 = 0. 9826 ）. 4. Since cellular transplantation,in vivo bioluminescence signal bounded injection location were detectable and the signal intensity were linear faded out until to 6th day. Means the survival,homing,proliferation and migration of Firefly-Luciferase gene transfected hMSCs can be quantitatively in vivo monitored in real time. Conclusion： Reconstructed descytotoxicityly lentivirus vector is an effective and safety tool for Firefly-Luciferase gene integrating into hMSCs. Thus by using bioluminescent tracer techniques the phenotype and potential involving homing,proliferation and migration of LUC-transfectied hMSCs can be quantitatively in vivo real-timely monitored.