中华耳鼻咽喉头颈外科杂志
中華耳鼻嚥喉頭頸外科雜誌
중화이비인후두경외과잡지
CHINESE JOURNAL OF OTORHINOLARYNGOLOGY HEAD AND NECK SURGERY
2013年
5期
405-411
,共7页
张春鸿%方潇碧%李文峰%施清圆%吴丽萍%陈晓云%黄振校%武鹏%王珍珍
張春鴻%方瀟碧%李文峰%施清圓%吳麗萍%陳曉雲%黃振校%武鵬%王珍珍
장춘홍%방소벽%리문봉%시청원%오려평%진효운%황진교%무붕%왕진진
鼻咽肿瘤%细胞系,肿瘤%原癌基因蛋白质c-bcl-2%辐射耐受性%微RNAs
鼻嚥腫瘤%細胞繫,腫瘤%原癌基因蛋白質c-bcl-2%輻射耐受性%微RNAs
비인종류%세포계,종류%원암기인단백질c-bcl-2%복사내수성%미RNAs
Nasopharyngeal neoplasms%Cell live,tumor%Proto-oncogene proteins c-bcl-2%Radiation tolerance%microRNAs
目的 探讨微小RNA(microRNA,rniR)-15a/16-1重组慢病毒对人鼻咽癌CNE-2Z细胞生物特性的影响,并探讨其可能机制.方法 重组慢病毒和空载体慢病毒转染CNE-2Z细胞后筛选绿色荧光蛋白阳性细胞,实时定量PCR (RT-qPCR)检测miR-15a、miR-16-1表达水平；将实验分为对照组、转染组、放射线照射组、转染联合放射线照射组,四甲基偶氮唑蓝法分析各组细胞增殖情况；流式细胞术检测各组细胞凋亡情况；克隆形成实验分析对照组和转染组对放射线敏感性的变化；RT-qPCR、Western blot法检测bcl-2 mRNA及其蛋白的表达水平；分光光度法检测Caspase-2、Caspase-3的活化程度.结果 转染组miR-15a、miR-16-1相对表达量((x)±s,下同)分别为524.80±40.79、466.11±40.96,同对照组比较,差异有统计学意义(t=494.611,t=386.840,P=0.000)；转染联合放射线照射组细胞增殖抑制最明显(F =424.255,P=0.000).对照组、转染组、放射线照射组及转染联合放射线照射组的细胞凋亡率分别为(2.2±1.4)％、(9.6±0.8)％、(2.9±1.1)％、(18.6±0.7)％,差异有统计学意义(F=158.519,P=0.000).转染组同等剂量下(2、4、6、8 Gy)存活分数、平均致死剂量(mean lethal dose,Do)、准阈剂量(quasi-threshold dose,Dq)均低于对照组.二者放射增强比(sensitization enhancement ratios,SER) (Do)、SER(Dq)分别为1.473、1.581.转染组、放射线照射组、转染联合放射线照射组bcl-2 mRNA表达相对量组间比较差异无统计学意义(P＞0.05).转染联合放射线照射组bcl-2蛋白表达下降最明显,转染组次之；对照组、放射线照射组、转染组、转染联合放射线照射组的Caspase-2活化程度分别为0.12 ±0.01、0.24±0.04、0.35±0.02、0.44±0.04(F=115.500,P=0.000),Caspase-3的活化程度分别为0.13±0.01、0.27±0.01、0.43±0.02、0.83±0.06(F =439.921,P=0.000).结论 重组慢病毒LV-miR15a/16-1可提高CNE-2Z细胞中miR-15a、miR-16-1的表达水平,抑制CNE-2Z细胞的增殖；促进CNE-2Z细胞的凋亡,增强CNE-2Z细胞的放射线敏感性.其可能通过抑制CNE-2Z细胞中bcl-2蛋白的表达,促进Caspase-2、Caspase-3的活化以实现对鼻咽癌细胞CNE-2Z生物学特性的调控.
目的 探討微小RNA(microRNA,rniR)-15a/16-1重組慢病毒對人鼻嚥癌CNE-2Z細胞生物特性的影響,併探討其可能機製.方法 重組慢病毒和空載體慢病毒轉染CNE-2Z細胞後篩選綠色熒光蛋白暘性細胞,實時定量PCR (RT-qPCR)檢測miR-15a、miR-16-1錶達水平；將實驗分為對照組、轉染組、放射線照射組、轉染聯閤放射線照射組,四甲基偶氮唑藍法分析各組細胞增殖情況；流式細胞術檢測各組細胞凋亡情況；剋隆形成實驗分析對照組和轉染組對放射線敏感性的變化；RT-qPCR、Western blot法檢測bcl-2 mRNA及其蛋白的錶達水平；分光光度法檢測Caspase-2、Caspase-3的活化程度.結果 轉染組miR-15a、miR-16-1相對錶達量((x)±s,下同)分彆為524.80±40.79、466.11±40.96,同對照組比較,差異有統計學意義(t=494.611,t=386.840,P=0.000)；轉染聯閤放射線照射組細胞增殖抑製最明顯(F =424.255,P=0.000).對照組、轉染組、放射線照射組及轉染聯閤放射線照射組的細胞凋亡率分彆為(2.2±1.4)％、(9.6±0.8)％、(2.9±1.1)％、(18.6±0.7)％,差異有統計學意義(F=158.519,P=0.000).轉染組同等劑量下(2、4、6、8 Gy)存活分數、平均緻死劑量(mean lethal dose,Do)、準閾劑量(quasi-threshold dose,Dq)均低于對照組.二者放射增彊比(sensitization enhancement ratios,SER) (Do)、SER(Dq)分彆為1.473、1.581.轉染組、放射線照射組、轉染聯閤放射線照射組bcl-2 mRNA錶達相對量組間比較差異無統計學意義(P＞0.05).轉染聯閤放射線照射組bcl-2蛋白錶達下降最明顯,轉染組次之；對照組、放射線照射組、轉染組、轉染聯閤放射線照射組的Caspase-2活化程度分彆為0.12 ±0.01、0.24±0.04、0.35±0.02、0.44±0.04(F=115.500,P=0.000),Caspase-3的活化程度分彆為0.13±0.01、0.27±0.01、0.43±0.02、0.83±0.06(F =439.921,P=0.000).結論 重組慢病毒LV-miR15a/16-1可提高CNE-2Z細胞中miR-15a、miR-16-1的錶達水平,抑製CNE-2Z細胞的增殖；促進CNE-2Z細胞的凋亡,增彊CNE-2Z細胞的放射線敏感性.其可能通過抑製CNE-2Z細胞中bcl-2蛋白的錶達,促進Caspase-2、Caspase-3的活化以實現對鼻嚥癌細胞CNE-2Z生物學特性的調控.
목적 탐토미소RNA(microRNA,rniR)-15a/16-1중조만병독대인비인암CNE-2Z세포생물특성적영향,병탐토기가능궤제.방법 중조만병독화공재체만병독전염CNE-2Z세포후사선록색형광단백양성세포,실시정량PCR (RT-qPCR)검측miR-15a、miR-16-1표체수평；장실험분위대조조、전염조、방사선조사조、전염연합방사선조사조,사갑기우담서람법분석각조세포증식정황；류식세포술검측각조세포조망정황；극륭형성실험분석대조조화전염조대방사선민감성적변화；RT-qPCR、Western blot법검측bcl-2 mRNA급기단백적표체수평；분광광도법검측Caspase-2、Caspase-3적활화정도.결과 전염조miR-15a、miR-16-1상대표체량((x)±s,하동)분별위524.80±40.79、466.11±40.96,동대조조비교,차이유통계학의의(t=494.611,t=386.840,P=0.000)；전염연합방사선조사조세포증식억제최명현(F =424.255,P=0.000).대조조、전염조、방사선조사조급전염연합방사선조사조적세포조망솔분별위(2.2±1.4)％、(9.6±0.8)％、(2.9±1.1)％、(18.6±0.7)％,차이유통계학의의(F=158.519,P=0.000).전염조동등제량하(2、4、6、8 Gy)존활분수、평균치사제량(mean lethal dose,Do)、준역제량(quasi-threshold dose,Dq)균저우대조조.이자방사증강비(sensitization enhancement ratios,SER) (Do)、SER(Dq)분별위1.473、1.581.전염조、방사선조사조、전염연합방사선조사조bcl-2 mRNA표체상대량조간비교차이무통계학의의(P＞0.05).전염연합방사선조사조bcl-2단백표체하강최명현,전염조차지；대조조、방사선조사조、전염조、전염연합방사선조사조적Caspase-2활화정도분별위0.12 ±0.01、0.24±0.04、0.35±0.02、0.44±0.04(F=115.500,P=0.000),Caspase-3적활화정도분별위0.13±0.01、0.27±0.01、0.43±0.02、0.83±0.06(F =439.921,P=0.000).결론 중조만병독LV-miR15a/16-1가제고CNE-2Z세포중miR-15a、miR-16-1적표체수평,억제CNE-2Z세포적증식；촉진CNE-2Z세포적조망,증강CNE-2Z세포적방사선민감성.기가능통과억제CNE-2Z세포중bcl-2단백적표체,촉진Caspase-2、Caspase-3적활화이실현대비인암세포CNE-2Z생물학특성적조공.
Objective To study the influence of recombinant lentiviral vector encoding miR-15a/16-1 on biological features of human nasopharyngeal carcinoma CNE-2Z cells and underlying mechanisms.Methods GFP-positive CNE-2Z cells transfected with recombinant lentiviral vector were selected.The experiment was divided into control group,transfected group,radiotherapy group,transfected-radiotherapy group.Cell proliferation was analyzed by MTT.Apoptosis was detected by flow cytometry.Radiotherapy sensitivity of the cells in control group and transfected group was evaluated by colony forming experiment.The expressions of miR-15a,miR-16-1 and bcl-2 mRNA were detected by real-time quantitative polymerase chain reaction (RT-qPCR).The expression of bcl-2 protein was detected by Western blot.The activation of Caspase-2 and Caspase-3 was evaluated by spectrophotometry.Results Relative expression quantities of miR-15a and miR-16-1 in infected group were 524.80 ±40.79 (t =494.611,P =0.000) and 466.11 ±40.96 (t =386.8,P =0.000),rcspcctively.Thc prolifcration of the ccells in transfected-radiotherapy group was the most obvious,followed by the cells in radiotherapy group and transfected group (F =424.3,P =0.000).The apoptosis rates of control group,transfected group,radiotherapy group and transfectedradiotherapy group were (2.2 ± 1.4)％,(9.6 ± 0.8)％,(2.9 ± 1.1)％,and (18.6 ± 0.7)％respectively(F =158.5,P =0.000).Clonogenic assay showed that the values of SF2,Do (1.473) and Dq (1.581) in transfected group were lower than those in control group.The relative expression levels of bcl-2 mRNA in transfected group,radiotherapy group,and transfected-radiotherapy group had no significant difference (P ＞ 0.05).Decrease in the expression of bcl-2 protein in transfeeted-radiotherapy group was most significantly,followed by that in transfected group.The percentages of activated Caspase-2 in control group,radiotherapy group,transfected group and transfected-radiotherapy group were 0.12 ± 0.01,0.24 ±0.04,0.35 ± 0.02,and 0.44 ± 0.04,respectively (F =115.500,P =0.000).The percentages of activatedCaspase-3 in the groups were 0.13 ±0.01,0.27 ± 0.01,0.43 ±0.02,and 0.83 ± 0.06,respectively (F =439.921,P =0.000).Conclusions Recombinant lentiviral vector LV-miR15a/16-1 could improve the expression of miR-15a and miR-16-1 in CNE-2Z cells,inhibit the proliferation of CNE-2Z cells,promote apoptosis and enhance the sensitivity of the cells to radiotherapy probably by inhibiting bcl-2 expression,activating Caspase-2 and Caspase-3.
