中华风湿病学杂志
中華風濕病學雜誌
중화풍습병학잡지
CHINESE JOURNAL OF RHEUMATOLOGY
2012年
2期
124-128
,共5页
刘宇宏%王莎莎%沈凌汛%徐玉兰%陈正望
劉宇宏%王莎莎%瀋凌汛%徐玉蘭%陳正望
류우굉%왕사사%침릉신%서옥란%진정망
白细胞介素32%细胞增殖%细胞凋亡%shRNA%成纤维样滑膜细胞
白細胞介素32%細胞增殖%細胞凋亡%shRNA%成纖維樣滑膜細胞
백세포개소32%세포증식%세포조망%shRNA%성섬유양활막세포
Intedeukin-32%Cell proliferation%Apoptosis%ShRNA%Fibroblast-like synovioeytes
目的 研究靶向白细胞介素(IL)-32γ的shRNA对类风湿关节炎(RA)成纤维样滑膜细胞(FLS)增殖与凋亡的影响及其机制.方法 设计合成靶向IL-32γ的短发夹RNA (EASY-shRNA-IL-32γ),用脂质体法导入RA-FLS细胞中；反转录-聚合酶链反应(RT-PCR)和蛋白印迹法检测IL-32γ、cyclin D1及p-Akt的表达；四甲基偶氮唑蓝(MTT)法检测细胞增殖；流式细胞术检测细胞周期；TUNEL法检测细胞凋亡.采用t检验进行统计分析.结果 ①针对序列1、2和3的EASY-shRNA-IL-32γ均能有效抑制FLS的IL-32γ的表达(P＜0.01),最大抑制率分别为75.6％、66.2％和64.1％.②实验组在接种后3d[A值:(0.23±0.03)对(0.35±0.03)和(0.36±0.04),均P＜0.05]和5 d[A值:(0.27±0.03)对(0.52±0.05)和(0.53±0.04),均P＜0.01]的细胞数量明显少于对照质粒组和未转染组.③实验组处于G1期的细胞比例为[(88±6)％],较对照质粒组[(69±5)％]和未转染组[(68±4)％]显著为高(均P＜0.05)；处于S+G2期的细胞比例为[(13.6±3.0)％],较对照质粒组[(30.2±4.1)％]和未转染组[(32.1±4.3)％]显著为低(均P＜0.01).④实验组凋亡率为[(20.50±3.21)％],较对照质粒组[(9.20±0.32)％]和未转染组[(8.60±0.22)％]显著为高(均P＜0.01).⑤实验组cyclin D1和p-Akt的表达[分别为(0.36±0.04)和(0.31 ±0.03)]明显低于对照质粒组[分别为(0.59±0.08)和(0.53±0.06)]和未转染组[分别为(0.61±0.07)和(0.52±0.06),均P＜0.01].结论 靶向IL-32γ的shRNA抑制RA-FLS增殖并促进凋亡.其机制可能部分与下调cyclin D1和p-Akt的表达,从而延缓RA-FLS通过整个细胞周期和抑制其存活有关.
目的 研究靶嚮白細胞介素(IL)-32γ的shRNA對類風濕關節炎(RA)成纖維樣滑膜細胞(FLS)增殖與凋亡的影響及其機製.方法 設計閤成靶嚮IL-32γ的短髮夾RNA (EASY-shRNA-IL-32γ),用脂質體法導入RA-FLS細胞中；反轉錄-聚閤酶鏈反應(RT-PCR)和蛋白印跡法檢測IL-32γ、cyclin D1及p-Akt的錶達；四甲基偶氮唑藍(MTT)法檢測細胞增殖；流式細胞術檢測細胞週期；TUNEL法檢測細胞凋亡.採用t檢驗進行統計分析.結果 ①針對序列1、2和3的EASY-shRNA-IL-32γ均能有效抑製FLS的IL-32γ的錶達(P＜0.01),最大抑製率分彆為75.6％、66.2％和64.1％.②實驗組在接種後3d[A值:(0.23±0.03)對(0.35±0.03)和(0.36±0.04),均P＜0.05]和5 d[A值:(0.27±0.03)對(0.52±0.05)和(0.53±0.04),均P＜0.01]的細胞數量明顯少于對照質粒組和未轉染組.③實驗組處于G1期的細胞比例為[(88±6)％],較對照質粒組[(69±5)％]和未轉染組[(68±4)％]顯著為高(均P＜0.05)；處于S+G2期的細胞比例為[(13.6±3.0)％],較對照質粒組[(30.2±4.1)％]和未轉染組[(32.1±4.3)％]顯著為低(均P＜0.01).④實驗組凋亡率為[(20.50±3.21)％],較對照質粒組[(9.20±0.32)％]和未轉染組[(8.60±0.22)％]顯著為高(均P＜0.01).⑤實驗組cyclin D1和p-Akt的錶達[分彆為(0.36±0.04)和(0.31 ±0.03)]明顯低于對照質粒組[分彆為(0.59±0.08)和(0.53±0.06)]和未轉染組[分彆為(0.61±0.07)和(0.52±0.06),均P＜0.01].結論 靶嚮IL-32γ的shRNA抑製RA-FLS增殖併促進凋亡.其機製可能部分與下調cyclin D1和p-Akt的錶達,從而延緩RA-FLS通過整箇細胞週期和抑製其存活有關.
목적 연구파향백세포개소(IL)-32γ적shRNA대류풍습관절염(RA)성섬유양활막세포(FLS)증식여조망적영향급기궤제.방법 설계합성파향IL-32γ적단발협RNA (EASY-shRNA-IL-32γ),용지질체법도입RA-FLS세포중；반전록-취합매련반응(RT-PCR)화단백인적법검측IL-32γ、cyclin D1급p-Akt적표체；사갑기우담서람(MTT)법검측세포증식；류식세포술검측세포주기；TUNEL법검측세포조망.채용t검험진행통계분석.결과 ①침대서렬1、2화3적EASY-shRNA-IL-32γ균능유효억제FLS적IL-32γ적표체(P＜0.01),최대억제솔분별위75.6％、66.2％화64.1％.②실험조재접충후3d[A치:(0.23±0.03)대(0.35±0.03)화(0.36±0.04),균P＜0.05]화5 d[A치:(0.27±0.03)대(0.52±0.05)화(0.53±0.04),균P＜0.01]적세포수량명현소우대조질립조화미전염조.③실험조처우G1기적세포비례위[(88±6)％],교대조질립조[(69±5)％]화미전염조[(68±4)％]현저위고(균P＜0.05)；처우S+G2기적세포비례위[(13.6±3.0)％],교대조질립조[(30.2±4.1)％]화미전염조[(32.1±4.3)％]현저위저(균P＜0.01).④실험조조망솔위[(20.50±3.21)％],교대조질립조[(9.20±0.32)％]화미전염조[(8.60±0.22)％]현저위고(균P＜0.01).⑤실험조cyclin D1화p-Akt적표체[분별위(0.36±0.04)화(0.31 ±0.03)]명현저우대조질립조[분별위(0.59±0.08)화(0.53±0.06)]화미전염조[분별위(0.61±0.07)화(0.52±0.06),균P＜0.01].결론 파향IL-32γ적shRNA억제RA-FLS증식병촉진조망.기궤제가능부분여하조cyclin D1화p-Akt적표체,종이연완RA-FLS통과정개세포주기화억제기존활유관.
Objective To investigate the effects and mechanisms of IL-32γ-shRNA-mediated gene silencing on the proliferation and apoptosis of fibroblast-like synoviocytes in patients with rheumatoid arthritis (RA).Methods A eukaryotic expression plasmid of shRNA targeting IL-32γ was transfected into fibroblastlike synoviocytes by liposome in patients with rheumatoid arthritis.RT-PCR was used to determine the expression level of IL-32γ.Western blotting was used to detect the levels of cyclin D1 and p-Akt.The proliferation of RA-FLS was examined by MTT.Cell cycles were analyzed by flow-cytometry.The apoptosis of cells were measured by TUNNEL.Comparisons between groups were tested by t test.Results ① The expression of IL-32γwas significantly inhibited by shRNA-IL-32γ-expressing plamid PGCsi 3.0 targeting sequence 1,2 and 3,and the inhibition rate had reached 75.6％,66.2％ and 64.1％,respectively.② The absorbance value of proliferation of RA-FLS in EASY-shRNA-IL-32γ group was significantly lower than that in the shRNA-control group and normal group at day 3 [(0.23±0.03) vs (0.35±0.03) and (0.36±0.04),P＜0.05] and 5 [(0.27±0.03) vs (0.52±0.05) and (0.53±0.04),P＜0.01 ] after transfection.③ The rate of RA-FLS at phase G1 in the EASY-shRNA-IL-32γ group was significantly higher than that in the shRNA-control group and normal group respectively [(88±6)％ vs (69±5)％ and (68±4)％,P＜0.05],while those at phase S+G2 in the EASY-shRNA-IL-32γgroup was significantly lower than that in the shRNA-control group and normal group [ ( 13.6±3.0)％ vs (30.2±4.1)％ and (32.1±4.3)％,P＜0.01].④The rate of RA-FLS apoptosis in the EASY-shRNA-IL-32γ group was significantly higher than that in the shRNA-control group and normal group [(20.50±3.21 )％ vs (9.20±0.32)％ and (8.60±0.22)％,P＜0.01].⑤ The expression of cyclin D1(0.36±0.04) and p-Akt(0.31±0.03) in the EASY-shRNA-IL-32γ group was significantly lower than that in the shRNA-control group [ (0.59±0.08) and (0.53±0.06)] and normal group [(0.61±0.07) and (0.52±0.06),P＜0.01].Conclusion EASYshRNA-IL-32γ can inhibit RA-FLS proliferation by down-regulating the expression of cyclin D1 and induce RA-FLS apoptosis by down-regulating the expression of p-Akt.
