两种复合阳离子聚合物/pDNA的基因活化基质转染骨髓间充质干细胞的效果比较

Comparison of Efficacy of Two Cationic Polymer/pDNA Nanoparticle-embedded Gene-activated Matrixes in Transfection of Bone Mesenchymal Stem Cells

目的比较2种复合阳离子聚合物/pDNA的基因活化基质转染骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)的效果。方法以壳聚糖/胶原复合支架为基质材料,以壳聚糖/pDNA复合物或PEI/pDNA复合物为基因传递系统,合成了2种不同的基因活化基质(gene-activated matrix,GAM)。以含有裸DNA的GAM作为对照,将BMSCs种植于各组GAM中共培养7d,采用MTT法通过检测细胞的吸光度值测定细胞的增殖能力,检测各种GAM的细胞相容性;各组GAM采用共培养转染和直接转染2种方式体外转染BMSCs,通过检测萤光素酶表达水平比较各种GAM体外转染BMSCs的能力。结果与对照组相比,含有壳聚糖/pDNA复合物的GAM并未影响BMSCs的增殖(P>0.05),具有良好的细胞相容性;而与含有PEI/pDNA复合物的GAM共培养的BMSCs增殖能力明显低于对照组(P<0.05),对BMSCs具有一定的细胞毒性。采用共培养转染方式,壳聚糖/pDNA与PEI/pDNA复合物组均获得比对照组高的转染结果(1.28×105,1.62×105 vs.2.14×104 RLU/mg protein),但两复合物组相比差异无统计学意义(P>0.05);采用直接转染方式,壳聚糖/pDNA组的萤光素酶表达水平与对照组相比差异无统计学意义(4.51×104 vs.3.23×104 RLU/mg protein,P>0.05),而PEI/pDNA组的表达水平(2.50×105 RLU/mg protein)高于对照组(P<0.05)。结论含有壳聚糖/pDNA复合物的GAM,体外共培养转染BMSCs具有较好的生物相容性及一定的转染能力,具有用于体内基因治疗的潜力。

Objective To compare the efficacy of two cationic polymer/pDNA nanoparticle-embedded gene-activated matrixes（GAM）in the transfection of bone mesenchymal stem cells（BMSCs）.Methods Two different GAMs were synthesized with chitosan/collagen composite scaffold as matrix materials and chitosan/pDNA complexes or PEI/pDNA complexes as gene delivery systems.Naked DNA embedded in the GAM served as control.BMSCs were co-cultured with different GAMs for 7 days.Then MTT method was used to measure the viability and proliferation of cells as well as the cytocompatibility of GAMs.The luciferase expression level was detected to compare the transfection efficiency of the two GAMs in BMSCs which were co-cultured with GAMs or directly transfected with GAMs in vitro.Results The proliferation of BMSCs in the chitosan/ pDNA-embedded GAM group was similar to that in the control group（P0.05）,suggesting the good cytocompatibility of the chitosan/pDNA-embedded GAM.The proliferative ability of BMSCs co-cultured with the PEI/pDNA-embedded GAM was significantly lower than that in control group（P0.05）,suggesting that the PEI/pDNA-embedded GAM has certain cytotoxicity on BMSCs.The co-cultured transfection experiment revealed that the chitosan/pDNA and PEI/pDNA groups both obtained higher transfection results than control group（1.28×105,1.62×105 vs.2.14×104 RLU/mg protein）.There was no statistical difference in the transfection efficiency between the chitosan/pDNA and PEI/pDNA groups（P0.05）.The direct transfection experiment showed that the luciferase expression levels in the chitosan/pDNA group were not significantly different from those in the control group（4.51×104 vs.3.23×104 RLU/mg protein,P0.05）,while the expression level in PEI/pDNA group（2.50 ×105 RLU/mg protein）was much higher than that in the control group.Conclusion The chitosan/pDNA-embedded GAM has good cytocompatibility and transfection ability in co-cultured BMSCs.It holds promises for application in in vivo gene therapy.