对比研究两种腺病毒载体对大鼠骨髓间充质干细胞的转染效率及机制

Study of infective efficiency of two adenovirus vectors to rBMSC and its mechanism

目的对比研究两种腺病毒载体Ad5／EGFP和AdF35／EGFP转染SD大鼠骨髓间充质干细胞（rBMSC）的效率及其机制。方法用密度梯度法从大鼠骨髓中分离出rBMSC，流式细胞仪检测其表面相关标志抗原（CI）90、CD29、CD44、CD34、CD11b和CIM5）以鉴定rBMSC。用不同梯度感染复数（MOI）的Ad5／EGFP和AdF35／EGFP分别转染rBMSC，MTT检测二者对rBMSC生长的影响。转染后第1～9天，荧光显微镜下观察转染情况，同时流式细胞仪检测转染效率及平均荧光强度，实时PCR检测rBMSC表面CAR及CIM6的表达水平。结果rBMSC表达CD90、CD29、CD44阳性，而CD34、CD11b、CIM5表达为阴性。MOI≤1600时，Ad5／EGFP对rBMSC的生长无明显抑制作用（t=3．12，P=0．052）；MOI≤1000时，AdF35／EGFP对rBMSC的生长无明显抑制作用（t=2．20，P=0．15））。荧光显微镜下，Ad5／EGFP转染rBMSC1d后即可见大量EGFP表达阳性的细胞，第2～3天阳性细胞数最多，随后减少，第9天仍可见少量的阳性细胞。AdF35／EGFP转染rBMSC后1～9dEGFP阳性细胞数始终很少。Ad5／EGFP的转染效率显著性高于AdF35／EGFP的转染效率（t=2．45，P=0．008）。经实时PCR检测，rBMSC高度表达CAR，而极低表达CIM6，二者之间差异有统计学意义（t=2．57，P〈0．01）。结论Ad5／EGFP对rBMSC的转染效率明显高于AdF35／EGFP，这可能与rBMSC表面高表达CAR而极低表达CD46有关。AdF35不合适作为目的基因转染rBMSC的载体，Ad5可作为目的基因有效转染rBMSC的载体，这为后续目的基因转染rBMSC治疗各种疾病的实验研究奠定了基础。

Objective To investigate the infective efficiency of AdS/EGFP versus Adl;35/EGFP to rat bone marrow mesenchymal stem cells （rBMSC） and understand its mechanism. Methods rBMSC were isolated from rat bone marrow by density gradient method. The expressions of CD90, CD29, CD44, CD34, CDllb and CD45 on the surface of rBMSC were measured by flow cytometry. The EGFP-carrying AdS and AdF35 were infected into rBMSC respectively. The effect of AdS/EGFP and AdF35/EGFP with different MOIs to rBMSC was tested by MTT. Infected rBMSC were observed by fluorescence microscopy. The infective efficiency and mean fluorescence intensity were determined by flow cytometry after a cellular infection for 1- 9 days. The expressions of CAR and CD46 on rBMSC were measured by real-time PCR. Results The expressions of CDg0, CD29 and CD44 on rBMSC were positive while those of CD34, CD11 b and CD45 were negative. When MOI ≤ 1600 and ≤ 1000, AdS/EGFP and AdF35/EGFP could not inhibit the growth of rBMSC respectively（t = 3.12, P = 0. 052; t = 2. 20, P = 0. 15）. RBMSC with EGFP expression could be observed under fluorescence microscope after a 1-day infection by AdS/EGFP. The number of observed ceils peaked after an infection for 2-3 days and then decreased gradually. And a few cells could be still observed for 9 days. Fewer cells could be observed after an infection by AdF35/EGFP for 1-9 days. The infective efficiency of AdS/EGFP was significantly higher than that of AdF35/EGFP （ t = 2.45, P = 0. 008 ）. There was a high expression of CAR on rBMSC. The expression of CD46 was quite low. There was significant difference between them （ t = 2.57, P 〈 0.01 ）. Conclusion The infective efficiency of AdS/EGFP is significantly higher than that of AdF35/EGFP. It may explain the expression patterns of CAR and CD46 on rBMSC. AdF35 is not a suitable gene-carrying vector to infect rBMSC . Yet AdS can be an efficient vector carrying target gene to infect rBMSC for curing diseases.