中华糖尿病杂志
中華糖尿病雜誌
중화당뇨병잡지
CHINES JOURNAL OF DLABETES MELLITUS
2011年
2期
161-166
,共6页
过氧化物酶增殖激活受体-δ%腺病毒%脂代谢%胰岛细胞
過氧化物酶增殖激活受體-δ%腺病毒%脂代謝%胰島細胞
과양화물매증식격활수체-δ%선병독%지대사%이도세포
Peroxisone proliferators-octivated%Adenovirus vector%Lipid metabolism%INS-1 cells
目的 构建小发夹状RNA(shRNA)腺病毒沉默过氧化物酶增殖激活受体-δ(PPAR-δ)表达,探讨PPAR-δ在大鼠胰岛细胞瘤细胞(INS-1)脂代谢中的作用和地位.方法 用限制性内切酶Sal Ⅰ和HindⅢ将shRNA质粒pGenesil-1载体中的PPAR-δ-shRNA片段连同U6启动子一起切下,连接至线性化的穿梭质粒pAdtrack-CMV上,将pAdtrack-CMV与骨架质粒pAdeasy在腺病毒载体电转感受态细菌(BJ5183)中重组得到PPAR-δ-shRNA腺病毒重组质粒.限制性内切酶Pac Ⅰ线性化重组质粒后用脂质体转染试剂Lipofectamine 2000转染人胚肾细胞(HEK293),包装得到含PPAR-δ-shRNA的病毒重组子,病毒重组子在HEK293细胞中扩增后,将收集的病毒液感染INS-1细胞、RT-PCR和Western blot检测PPAR-δ蛋白的表达.RT-PCR检测该病毒对INS-1细胞脂代谢相关基因酰基辅酶A氧化酶(ACO)、肉毒碱棕榈酸转移酶1(CPT1)、脂肪酸转运蛋白1(FATP1)、长链酰基辅酶A脱氢酶(LCAD)表达的影响,并检测INS-1细胞内甘油三酯含量的变化.采用SPSS 12.0软件进行统计学分析,组间差异采用方差分析.结果 成功构建了含有大鼠PPAR-δ-shRNA基因的重组腺病毒载体,病毒滴度为1×1010PFU/ml.重组腺病毒感染后INS-1细胞PPAR-δ mRNA和蛋白表达明显下降.与空病毒组相比,PPAR-δ-shRNA腺病毒使ACO的表达下降40%(分别为0.72±0.05,0.44±0.07,P＜0.05),CPT1表达下降27%(分别为0.66±0.08,0.48±0.02,P＜0.05),使FATP1的表达下降55%(分别为0.65±0.07,0.30±0.02,P＜0.05),使LCAD的表达下降32%(分别为0.66±0.12,0.45±0.10,P＜0.05).与空病毒组相比,PPAR-δ-shRNA腺病毒使INS-1细胞内甘油三酯含量上升65%(分别为5.27±0.19,8.68±0.34,P＜0.05).结论 成功构建了PPAR-δ-shRNA腺病毒,该腺病毒能够抑制INS-1细胞的脂肪酸氧化,促进细胞内脂质沉积.
目的 構建小髮夾狀RNA(shRNA)腺病毒沉默過氧化物酶增殖激活受體-δ(PPAR-δ)錶達,探討PPAR-δ在大鼠胰島細胞瘤細胞(INS-1)脂代謝中的作用和地位.方法 用限製性內切酶Sal Ⅰ和HindⅢ將shRNA質粒pGenesil-1載體中的PPAR-δ-shRNA片段連同U6啟動子一起切下,連接至線性化的穿梭質粒pAdtrack-CMV上,將pAdtrack-CMV與骨架質粒pAdeasy在腺病毒載體電轉感受態細菌(BJ5183)中重組得到PPAR-δ-shRNA腺病毒重組質粒.限製性內切酶Pac Ⅰ線性化重組質粒後用脂質體轉染試劑Lipofectamine 2000轉染人胚腎細胞(HEK293),包裝得到含PPAR-δ-shRNA的病毒重組子,病毒重組子在HEK293細胞中擴增後,將收集的病毒液感染INS-1細胞、RT-PCR和Western blot檢測PPAR-δ蛋白的錶達.RT-PCR檢測該病毒對INS-1細胞脂代謝相關基因酰基輔酶A氧化酶(ACO)、肉毒堿棕櫚痠轉移酶1(CPT1)、脂肪痠轉運蛋白1(FATP1)、長鏈酰基輔酶A脫氫酶(LCAD)錶達的影響,併檢測INS-1細胞內甘油三酯含量的變化.採用SPSS 12.0軟件進行統計學分析,組間差異採用方差分析.結果 成功構建瞭含有大鼠PPAR-δ-shRNA基因的重組腺病毒載體,病毒滴度為1×1010PFU/ml.重組腺病毒感染後INS-1細胞PPAR-δ mRNA和蛋白錶達明顯下降.與空病毒組相比,PPAR-δ-shRNA腺病毒使ACO的錶達下降40%(分彆為0.72±0.05,0.44±0.07,P＜0.05),CPT1錶達下降27%(分彆為0.66±0.08,0.48±0.02,P＜0.05),使FATP1的錶達下降55%(分彆為0.65±0.07,0.30±0.02,P＜0.05),使LCAD的錶達下降32%(分彆為0.66±0.12,0.45±0.10,P＜0.05).與空病毒組相比,PPAR-δ-shRNA腺病毒使INS-1細胞內甘油三酯含量上升65%(分彆為5.27±0.19,8.68±0.34,P＜0.05).結論 成功構建瞭PPAR-δ-shRNA腺病毒,該腺病毒能夠抑製INS-1細胞的脂肪痠氧化,促進細胞內脂質沉積.
목적 구건소발협상RNA(shRNA)선병독침묵과양화물매증식격활수체-δ(PPAR-δ)표체,탐토PPAR-δ재대서이도세포류세포(INS-1)지대사중적작용화지위.방법 용한제성내절매Sal Ⅰ화HindⅢ장shRNA질립pGenesil-1재체중적PPAR-δ-shRNA편단련동U6계동자일기절하,련접지선성화적천사질립pAdtrack-CMV상,장pAdtrack-CMV여골가질립pAdeasy재선병독재체전전감수태세균(BJ5183)중중조득도PPAR-δ-shRNA선병독중조질립.한제성내절매Pac Ⅰ선성화중조질립후용지질체전염시제Lipofectamine 2000전염인배신세포(HEK293),포장득도함PPAR-δ-shRNA적병독중조자,병독중조자재HEK293세포중확증후,장수집적병독액감염INS-1세포、RT-PCR화Western blot검측PPAR-δ단백적표체.RT-PCR검측해병독대INS-1세포지대사상관기인선기보매A양화매(ACO)、육독감종려산전이매1(CPT1)、지방산전운단백1(FATP1)、장련선기보매A탈경매(LCAD)표체적영향,병검측INS-1세포내감유삼지함량적변화.채용SPSS 12.0연건진행통계학분석,조간차이채용방차분석.결과 성공구건료함유대서PPAR-δ-shRNA기인적중조선병독재체,병독적도위1×1010PFU/ml.중조선병독감염후INS-1세포PPAR-δ mRNA화단백표체명현하강.여공병독조상비,PPAR-δ-shRNA선병독사ACO적표체하강40%(분별위0.72±0.05,0.44±0.07,P＜0.05),CPT1표체하강27%(분별위0.66±0.08,0.48±0.02,P＜0.05),사FATP1적표체하강55%(분별위0.65±0.07,0.30±0.02,P＜0.05),사LCAD적표체하강32%(분별위0.66±0.12,0.45±0.10,P＜0.05).여공병독조상비,PPAR-δ-shRNA선병독사INS-1세포내감유삼지함량상승65%(분별위5.27±0.19,8.68±0.34,P＜0.05).결론 성공구건료PPAR-δ-shRNA선병독,해선병독능구억제INS-1세포적지방산양화,촉진세포내지질침적.
Objective To construct the recombinant adenovirus vector containing peroxisome proliferators-activated receptor-δ-shRNA,and observe its effect on lipid metabolism of INS-1 cells. Methods The pGenesil-1 plasmid was digested with Sal Ⅰ and Hind Ⅲ to obtain PPAR-δ-shRNA and U6 promoter,which were then ligated to the pAdtrack-CMV vector. The plasmid of pAdtrack-PPAR-δ-shRNA was linearized with Pme Ⅰ ,and then the fragment was reclaimed and transformed into BJ5183 bacteria which contained pAdeasy. After having been screened,the extracted plasmid of positive bacteria linearized with Pac Ⅰ was transfected into HEK293 cells with lipofectamine 2000. The harvested virus was amplified in other HEK293 cells and infected INS-1 cells. After infection,the expression of PPAR-δ was proved by the RT-PCR and Western blot analysis. Then the mRNA level of lipid metabolic related proteins including ACO,CPT1,FATP1 and LACD were determined by RT-PCR,and the intracellular content of TG was measured by biochemical methods. The data of each culture condition were analyzed using SPSS 12. 0 statistical software.Results The PPAR-δ-shRNA adenovirus was constructed successfully and the titer was 1 × 1010 PFU/ml.The mRNA and protein level of PPAR-δ were both down-regulated by PPAR-δ-shRNA adenovirus. Compared with the null-adenovirus groups,mRNA level of ACO,CPT1,FATP1,LACD of the PPAR-δ-shRNA adenovirus groups decreased by 40. 0% ( 0. 72 ± 0. 05,0. 44 ± 0. 07,P ＜ 0. 05 ),27.0% ( 0. 66 ± 0. 08,0.48 ±0.02,P ＜0.05) ,55.0% (0.65 ±0.07,0.30 ±0.02,P ＜0.05) and 32.0% (0.66 ±0. 12,0. 45 ±0. 10,P ＜0. 05 ),respectively. By contrast,the intracellular content of TG increased by 65.0% (5.27 ±0. 19,8.68 ± 0. 34,P ＜ 0. 05 ) of the PPAR-δ-shRNA adenovirus group. Conclusion The PPAR-δ-shRNA was constructed successfully,which can depress the fatty acid oxidation and promote the lpid deposition of INS-1 cells.
