CAJ | 학술논문

目的观察RNA干扰沉默PPAγ基因对小鼠骨髓基质干细胞成脂与成骨分化潜能的影响.方法针对小鼠PPARγ基因,构建短发夹RNA(shRNA)真核表达载体并转染小鼠骨髓基质干细胞,逆转录-聚合酶链反应(RT-PCR)和免疫印迹沉淀(Wetem-blot)观察PPARγ的基因沉默效果.分别在成脂及成骨诱导培养基条件下,诱导培养14 d,采用油红O(Oil Red O)染色及茜素红染色鉴定分化结果.并与空载体转染组和未转染组比较.结果测序证实成功构建PPARγ的shRNA真核表达载体pSilencer-shPPARγ,转染小鼠骨髓基质干细胞后,PPARγ基因表达在转录水平和翻译水平都显著受到抑制,与两对照组相比,抑制率分别为92%和90%.成脂及成骨诱导分化后,shPPARγ转染组小鼠骨髓基质干细胞的成骨分化率为56.46%±1.92%,明显高于pSilencer-neo空载体转染组和未转染组(P<0.01);成脂分化率为19.24%±0.98%,显著低于pSilencer-shPPARγ-neo空载体转染组和未转染组(P<0.01).结论 RNA干扰沉默PPARγ基因后,可增强小鼠骨髓基质干细胞的成骨分化潜能,抑制其成脂分化潜能.
목적 관찰RNA간우침묵PPAγ기인대소서골수기질간세포성지여성골분화잠능적영향.방법 침대소서PPARγ기인,구건단발협RNA(shRNA)진핵표체재체병전염소서골수기질간세포,역전록-취합매련반응(RT-PCR)화면역인적침정(Wetem-blot)관찰PPARγ적기인침묵효과.분별재성지급성골유도배양기조건하,유도배양14 d,채용유홍O(Oil Red O)염색급천소홍염색감정분화결과.병여공재체전염조화미전염조비교.결과 측서증실성공구건PPARγ적shRNA진핵표체재체pSilencer-shPPARγ,전염소서골수기질간세포후,PPARγ기인표체재전록수평화번역수평도현저수도억제,여량대조조상비,억제솔분별위92%화90%.성지급성골유도분화후,shPPARγ전염조소서골수기질간세포적성골분화솔위56.46%±1.92%,명현고우pSilencer-neo공재체전염조화미전염조(P<0.01);성지분화솔위19.24%±0.98%,현저저우pSilencer-shPPARγ-neo공재체전염조화미전염조(P<0.01).결론 RNA간우침묵PPARγ기인후,가증강소서골수기질간세포적성골분화잠능,억제기성지분화잠능.
Objective The purpose of the study was to investigate the effects of RNA interference targeting PPARγ gene on adipogenic and osteogenic potential of mouse bone marrow stem cells. Methods The short hairpin RNA (shRNA) eukaryotic expression vector targeting PPARγ gene, named pSilencer-PPARγ was constructed and transfected into cultured bone marrow stem cells (BMSCs) via liposome reagent. The inhibition effects on PPARγ gene were determined by semi-quantitative reverse transcriptiou-polymerase chain reaction (RT-PCR) and Western blot analysis. BMSCs were harvested from the femur and tibia of the mouse, then separated, purified, proliferated and induced with osteogenic and adipogenic induction medium for 14 days, respectively. Differentiated adipocytes and osteoblasts were identificated by Oil Red O staining and Alizarin Red staining, respectively. Results The successful construction of pSileucer-PPARγ plasmid was identified with sequencing. After the shRNA expression vector was transfected into the BMSCs, the ex-pression of PPARγ gene was decreased 92% in the level of gene transcription and 90% in the translation repectively.The osteogenetic differentiation rate is 56.46%±1.92% in the BMMSCs transfected with psi-lencer-PPARγ plasmid, which was significantly higher than those in the negative control group transfected with pSilencer-neo and in the nontransfected group. Meanwhile, the adipogenic differentiation rate is 19.24% ±0.98%, which was significantly lower than those in the negative control group transfected with pSilencer-neo and in the non transfected group. Conclusion RNA interference targeting PPARγ gene of mouse bone marrow stem cells could increase the adipogenic potential and decrease the osteogenic potential.