CAJ | 학술논문

目的：探索采用非病毒方法转染骨髓基质干细胞( bone mesenchymal stem cells，BMSC )膜片的可行性；antimiR-138转染的 BMSC 膜片促进同种冻干骨粉( FDB )成骨的可行性。方法采用 Vc 连续诱导法培养 BMSC 膜片，通过调整 Lipofectamine 2000-miRNAs 载体复合物的用量及转染步骤对 BMSC 膜片进行转染；采用荧光体式及显微观察、miRNA-PCR 方法比较各组的转染效率的差异；通过大体观察、HE 染色和扫描电镜观察 BMSC 膜片的形态。将 BMSC 膜片/ FDB 复合物移植到裸鼠皮下，术后4～8周，分别采用 HE 和 MT 染色对膜片的成骨能力进行评估。结果荧光体式及显微结果表明 antimiR-138可成功地转至 BMSC 膜片中，其中150 nM 组对内源性 miR-138的抑制率为85%；体内异位成骨实验表明 antimiR-138转染的 BMSC 膜片可显著促进并加速移植复合物的骨组织形成。结论(1)通过对以 Lipofectamine 2000为载体的转染复合物的用量及转染步骤进行优化，可以构建出具有较高转染效率，良好的细胞相容性以及结构完整的 antimiR-138修饰的 BMSC 膜片。(2) antimiR-138转染的 BMSC 膜片可显著促进并加速移植复合物的异位骨组织形成。
목적：탐색채용비병독방법전염골수기질간세포( bone mesenchymal stem cells，BMSC )막편적가행성；antimiR-138전염적 BMSC 막편촉진동충동간골분( FDB )성골적가행성。방법채용 Vc 련속유도법배양 BMSC 막편，통과조정 Lipofectamine 2000-miRNAs 재체복합물적용량급전염보취대 BMSC 막편진행전염；채용형광체식급현미관찰、miRNA-PCR 방법비교각조적전염효솔적차이；통과대체관찰、HE 염색화소묘전경관찰 BMSC 막편적형태。장 BMSC 막편/ FDB 복합물이식도라서피하，술후4～8주，분별채용 HE 화 MT 염색대막편적성골능력진행평고。결과형광체식급현미결과표명 antimiR-138가성공지전지 BMSC 막편중，기중150 nM 조대내원성 miR-138적억제솔위85%；체내이위성골실험표명 antimiR-138전염적 BMSC 막편가현저촉진병가속이식복합물적골조직형성。결론(1)통과대이 Lipofectamine 2000위재체적전염복합물적용량급전염보취진행우화，가이구건출구유교고전염효솔，량호적세포상용성이급결구완정적 antimiR-138수식적 BMSC 막편。(2) antimiR-138전염적 BMSC 막편가현저촉진병가속이식복합물적이위골조직형성。
Objective To verify the feasibility of transfecting the bone mesenchymal stem cells ( BMSC ) sheets with the non-viral way and to confirm the osteogenesis enhancing effects of antimiR-138 delivered BMSC sheet / freeze dried bone ( FDB ) complex.Methods A continuous vitamin C inducing method was used to culture the BMSC sheets, which were later transfected with different Lipofectamine 2000-miRNA lipoplexes. The transfection efficiencies of different formulations were assessed by the miRNA RT-PCR. H&E staining and SEM observation were performed to evaluate the harvested sheets. The ectopic bone regeneration ability of the BMSC sheet / FDB complexes were assessed in immunocompromised mice: H&E or Masson’s Trichrome staining were used to assess the osteogenesis of the BMSC sheet / FDB complexes in SCID mice 4 and 8 weeks after implantation.Results The BMSC sheets were fabricated by a vitamin C inducing method. They could be successfully transfected with antimiR-138 to achieve a transfection efifciency of nearly 85% by a Lipofactamine2000 based transfection formulation after proper adaption and optimization. The in vivo ectopic bone regeneration results conifrmed the robust enhancing effects of antimiR-138 transfection on the bone regeneration ability of BMSC sheets combined with freeze dried allograft bone. Conclusions ( 1 ) Satisfactory transfection efifciency of nearly 85%, good cytocompatibility and unimpaired cell sheet structure can be obtained by properly optimizing the Lipofactamine 2000 based transfection formulation. ( 2 ) The antimiR-138 transfection signiifcantly enhances the osteogenesis of the BMSC sheet/FDB complexes in vivo, showing great clinical signiifcance for bone repair and regeneration.