中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2009年
3期
254-259
,共6页
黄渝侃%张明昌%王勇%范可顺%姜冬玲%张光红%周龚莉
黃渝侃%張明昌%王勇%範可順%薑鼕玲%張光紅%週龔莉
황투간%장명창%왕용%범가순%강동령%장광홍%주공리
RNA,小分子干扰%周期素依赖激酶抑制剂p27%质粒%内皮,角膜%细胞增殖
RNA,小分子榦擾%週期素依賴激酶抑製劑p27%質粒%內皮,角膜%細胞增殖
RNA,소분자간우%주기소의뢰격매억제제p27%질립%내피,각막%세포증식
RNA,small interfering%Cyclin-dependent kinase inhibitor p27%Plasmids%Endothelium,corneal%Cell proliferation
目的 探讨针对p27Kip1基因的小发夹RNA(shRNA)表达质粒对牛角膜内皮细胞(bCEC)增殖能力的影响.方法 实验研究.设计有发夹状结构的3条p27Kip1-shRNA对应模板DNA序列,并构建无关序列HK-shRNA作为阴性对照.构建并鉴定重组质粒Pgenesil-P1、Pgenesil-P2、Pgenesil-P3及Pgenesil-HK.以脂质体法将以上4种质粒分别转染bCEC,并设立空白组.稳定转染后采用RT-PCR和Western免疫印迹法榆测bCEC内p27Kip1的mRNA及其蛋白水平,筛选出抑制效果最好的阳性siRNA质粒.用四甲基偶氮唑盐比色法(MTT)检测该质粒组、Pgenesil-HK组及空白组的细胞生长情况.流式细胞术检测各组细胞周期的分布.以上数据结果均采用单因素方差分析.结果酶切及测序证实4个重组质粒均构建成功.与空白组比较,Pgenesil-P1、Pgenesil-P2、Pgenesil-P3各组mRNA的抑制效率分别为32.71%、67.76%及80.28%(F=453.102,P=0.000),蛋白表达水平降低为29.27%、64.73%及76.13%(F=75.385,P=0.000),以Pgenesil-P3抑制效果最明显.空白组与Pgenesil-HK组比较蛋白水平(P=0.356)和mRNA水平(P=0.246)均差异无统计学意义.与空白对照和阴性siRNA相比,Pgenesil-P3组bCEC增殖能力提高,G1期细胞比例下降(F=134.224,P=0.000),s期比例增加(F=334.957,P=0.000).结论针对p27Kip1的shRNA可有效降低p27Kip1基因及其蛋白的表达水平,并可提高bCEC增殖能力.RNA干扰介导的p27Kip1基因沉默可能是促进角膜内皮细胞再生的有效手段.(中华眼科杂志,2009,45:254-259)
目的 探討針對p27Kip1基因的小髮夾RNA(shRNA)錶達質粒對牛角膜內皮細胞(bCEC)增殖能力的影響.方法 實驗研究.設計有髮夾狀結構的3條p27Kip1-shRNA對應模闆DNA序列,併構建無關序列HK-shRNA作為陰性對照.構建併鑒定重組質粒Pgenesil-P1、Pgenesil-P2、Pgenesil-P3及Pgenesil-HK.以脂質體法將以上4種質粒分彆轉染bCEC,併設立空白組.穩定轉染後採用RT-PCR和Western免疫印跡法榆測bCEC內p27Kip1的mRNA及其蛋白水平,篩選齣抑製效果最好的暘性siRNA質粒.用四甲基偶氮唑鹽比色法(MTT)檢測該質粒組、Pgenesil-HK組及空白組的細胞生長情況.流式細胞術檢測各組細胞週期的分佈.以上數據結果均採用單因素方差分析.結果酶切及測序證實4箇重組質粒均構建成功.與空白組比較,Pgenesil-P1、Pgenesil-P2、Pgenesil-P3各組mRNA的抑製效率分彆為32.71%、67.76%及80.28%(F=453.102,P=0.000),蛋白錶達水平降低為29.27%、64.73%及76.13%(F=75.385,P=0.000),以Pgenesil-P3抑製效果最明顯.空白組與Pgenesil-HK組比較蛋白水平(P=0.356)和mRNA水平(P=0.246)均差異無統計學意義.與空白對照和陰性siRNA相比,Pgenesil-P3組bCEC增殖能力提高,G1期細胞比例下降(F=134.224,P=0.000),s期比例增加(F=334.957,P=0.000).結論針對p27Kip1的shRNA可有效降低p27Kip1基因及其蛋白的錶達水平,併可提高bCEC增殖能力.RNA榦擾介導的p27Kip1基因沉默可能是促進角膜內皮細胞再生的有效手段.(中華眼科雜誌,2009,45:254-259)
목적 탐토침대p27Kip1기인적소발협RNA(shRNA)표체질립대우각막내피세포(bCEC)증식능력적영향.방법 실험연구.설계유발협상결구적3조p27Kip1-shRNA대응모판DNA서렬,병구건무관서렬HK-shRNA작위음성대조.구건병감정중조질립Pgenesil-P1、Pgenesil-P2、Pgenesil-P3급Pgenesil-HK.이지질체법장이상4충질립분별전염bCEC,병설립공백조.은정전염후채용RT-PCR화Western면역인적법유측bCEC내p27Kip1적mRNA급기단백수평,사선출억제효과최호적양성siRNA질립.용사갑기우담서염비색법(MTT)검측해질립조、Pgenesil-HK조급공백조적세포생장정황.류식세포술검측각조세포주기적분포.이상수거결과균채용단인소방차분석.결과매절급측서증실4개중조질립균구건성공.여공백조비교,Pgenesil-P1、Pgenesil-P2、Pgenesil-P3각조mRNA적억제효솔분별위32.71%、67.76%급80.28%(F=453.102,P=0.000),단백표체수평강저위29.27%、64.73%급76.13%(F=75.385,P=0.000),이Pgenesil-P3억제효과최명현.공백조여Pgenesil-HK조비교단백수평(P=0.356)화mRNA수평(P=0.246)균차이무통계학의의.여공백대조화음성siRNA상비,Pgenesil-P3조bCEC증식능력제고,G1기세포비례하강(F=134.224,P=0.000),s기비례증가(F=334.957,P=0.000).결론침대p27Kip1적shRNA가유효강저p27Kip1기인급기단백적표체수평,병가제고bCEC증식능력.RNA간우개도적p27Kip1기인침묵가능시촉진각막내피세포재생적유효수단.(중화안과잡지,2009,45:254-259)
Objective To clarify the proliferation of bovine corneal endothelial cells(bCEC)by interference with the recombinant plasmid of short hairpin RNA(shRNA)against p27Kip1,a kind of cyclin dependent kinase inhibitor(CKI).MethodsIt was an experimental study.Three p27Kip1-shRNA template DNA sequences containing small hairpin structure were designed and synthesized as experimental groups.Plasmid expressing irrelevant shRNA with a random combination was used as negative shRNA.The products were inserted into the Pgensil-1 plasmid and the recombinant plasmid of Pgenesil-P1,Pgenesil-P2,Pgenesil-P3 and Pgenesil-HK were constructed.The recombinant plasmids were transfected into bCEC cells with lissome and a blank group.The expression of mRNA and protein of p27 Kip1 was detected by RT-PCR and Western blot after stable transfection,and the plasmid with the best inhibitory effect was selected.The growth of the experimental group.Pgenesil-HK group and blank group were assessed by MTT.The influence of shRNA-p27Kip1 on bCEC cell cycle was deteceted by flow cytometry(FCM).All statistical analyses were performed using one-way ANOVA.Results Restrictive enzyme digestion and sequence analysis showed that four recombinant plamids were constructed successfully and the aim sequence was obtained.The expression of p27Kip1 mRNA and p27Kip1 protein of Pgenesil-P1 group.Pgenesil-P2 group and Pgenesil-P3 group were all lower than that in the control group,including blank group and negative siRNA group.The inhibitive rate of mRNA reached 32.71%,67.76% and 80.28%(F=453.102,P=0.000 in each group)and the inhibitive rate of protein reached 29.27%,64.73% and 76.13%(F=75.385,P=0.000 in each group)compared with the blank group.As the lowest expression among the three positive shRNA group,Pgenesil P3 was selected for the next steps.There was no significant difference between blank group and negative Pgenesil-HK of the expression of p27Kip1 protein(P=0.356)and the express of p27Kip1 mRNA(P=0.246).Compared with the control group and the blank group,the growth of the bCEC transfected by Pgenesil-P3 was significantly promoted with increased cell percent of S-phrase(F=334.957,P=0.000)and decreased cell percent of G1-phrase(F=134.22,4,P=0.000).Conclusions shRNA-p27Kipl can down-regulate the expression of bCEC effectively and increase the growth of bCEC.shRNA-p27 Kip1 RNA interference may be an effective method to promote the proliferation of CEC.(Chin J Ophthalmol,2009,45:254-259)
