中华小儿外科杂志
中華小兒外科雜誌
중화소인외과잡지
CHINESE JOURNAL OF PEDIATRIC SURGERY
2011年
4期
285-289
,共5页
陈鑫%董蒨%鹿洪亭%郝希伟%邢士超
陳鑫%董蒨%鹿洪亭%郝希偉%邢士超
진흠%동천%록홍정%학희위%형사초
受体,趋化因子%RNA干扰%神经母细胞瘤
受體,趨化因子%RNA榦擾%神經母細胞瘤
수체,추화인자%RNA간우%신경모세포류
Receptors,chemokine%RNA interference%Neuroblastoma
目的 构建趋化因子受体4(CXCR4)小分子干扰RNA(siRNA)表达载体,研究其对体外神经母细胞瘤侵袭能力的影响.方法 选择CXCR4高表达的神经母细胞瘤SH-SY5Y细胞系,设计合成人CXCR4基因不同靶点的能编码siRNA的3条双链DNA序列,克隆到真核表达载体pSilencerTMneo中构建siRNA表达载体,体外脂质体介导转染SH-SY5Y细胞,用半定量RT-PCR分析CXCR4基因mRNA的变化,用免疫组织化学和Western blot分析CXCR4蛋白表达,Transwell小室检测细胞的侵袭能力.结果 成功构建了CXCR4-siRNA表达载体,转染后半定量RT-PCR检测神经母细胞瘤细胞CXCR4 mRNA丰度分别为siR1转染组0.32±0.09、siR2转染组0.35±0.13和siR3转染组0.33±0.11,相对于对照组0.58±0.13表达下降(P＜0.05);转染后免疫组化检测神经母细胞瘤细胞CXCR4的蛋白表达分别为siR1转染组75.98±4.81、siR2转染组75.52±3.95和siR3转染组76.35±6.51,相对于对照组92.196±3.89表达下降(P＜0.01);转染后Western b1ot检测神经母细胞瘤细胞CXCR4的蛋白表达分别为siR1转染组0.1103±0.0023、siR2转染组0.1203±0.015和siR3转染组0.1308±0.0018,相对于对照组0.4832±0.0012表达下降(P＜0.01);且转染后神经母细胞瘤细胞侵袭能力较对照组53.11±3.72降低(P＜0.05),siR1转染组为25.48±2.81、siR2转染组为30.89±2.77、siR3转染组为18.83±1.79.结论 CXCR4-siRNA表达载体通过降低CXCR4基因的蛋白表达能显著抑制神经母细胞瘤细胞的体外侵袭能力,有望为神经母细胞瘤的基因治疗开辟新途径.
目的 構建趨化因子受體4(CXCR4)小分子榦擾RNA(siRNA)錶達載體,研究其對體外神經母細胞瘤侵襲能力的影響.方法 選擇CXCR4高錶達的神經母細胞瘤SH-SY5Y細胞繫,設計閤成人CXCR4基因不同靶點的能編碼siRNA的3條雙鏈DNA序列,剋隆到真覈錶達載體pSilencerTMneo中構建siRNA錶達載體,體外脂質體介導轉染SH-SY5Y細胞,用半定量RT-PCR分析CXCR4基因mRNA的變化,用免疫組織化學和Western blot分析CXCR4蛋白錶達,Transwell小室檢測細胞的侵襲能力.結果 成功構建瞭CXCR4-siRNA錶達載體,轉染後半定量RT-PCR檢測神經母細胞瘤細胞CXCR4 mRNA豐度分彆為siR1轉染組0.32±0.09、siR2轉染組0.35±0.13和siR3轉染組0.33±0.11,相對于對照組0.58±0.13錶達下降(P＜0.05);轉染後免疫組化檢測神經母細胞瘤細胞CXCR4的蛋白錶達分彆為siR1轉染組75.98±4.81、siR2轉染組75.52±3.95和siR3轉染組76.35±6.51,相對于對照組92.196±3.89錶達下降(P＜0.01);轉染後Western b1ot檢測神經母細胞瘤細胞CXCR4的蛋白錶達分彆為siR1轉染組0.1103±0.0023、siR2轉染組0.1203±0.015和siR3轉染組0.1308±0.0018,相對于對照組0.4832±0.0012錶達下降(P＜0.01);且轉染後神經母細胞瘤細胞侵襲能力較對照組53.11±3.72降低(P＜0.05),siR1轉染組為25.48±2.81、siR2轉染組為30.89±2.77、siR3轉染組為18.83±1.79.結論 CXCR4-siRNA錶達載體通過降低CXCR4基因的蛋白錶達能顯著抑製神經母細胞瘤細胞的體外侵襲能力,有望為神經母細胞瘤的基因治療開闢新途徑.
목적 구건추화인자수체4(CXCR4)소분자간우RNA(siRNA)표체재체,연구기대체외신경모세포류침습능력적영향.방법 선택CXCR4고표체적신경모세포류SH-SY5Y세포계,설계합성인CXCR4기인불동파점적능편마siRNA적3조쌍련DNA서렬,극륭도진핵표체재체pSilencerTMneo중구건siRNA표체재체,체외지질체개도전염SH-SY5Y세포,용반정량RT-PCR분석CXCR4기인mRNA적변화,용면역조직화학화Western blot분석CXCR4단백표체,Transwell소실검측세포적침습능력.결과 성공구건료CXCR4-siRNA표체재체,전염후반정량RT-PCR검측신경모세포류세포CXCR4 mRNA봉도분별위siR1전염조0.32±0.09、siR2전염조0.35±0.13화siR3전염조0.33±0.11,상대우대조조0.58±0.13표체하강(P＜0.05);전염후면역조화검측신경모세포류세포CXCR4적단백표체분별위siR1전염조75.98±4.81、siR2전염조75.52±3.95화siR3전염조76.35±6.51,상대우대조조92.196±3.89표체하강(P＜0.01);전염후Western b1ot검측신경모세포류세포CXCR4적단백표체분별위siR1전염조0.1103±0.0023、siR2전염조0.1203±0.015화siR3전염조0.1308±0.0018,상대우대조조0.4832±0.0012표체하강(P＜0.01);차전염후신경모세포류세포침습능력교대조조53.11±3.72강저(P＜0.05),siR1전염조위25.48±2.81、siR2전염조위30.89±2.77、siR3전염조위18.83±1.79.결론 CXCR4-siRNA표체재체통과강저CXCR4기인적단백표체능현저억제신경모세포류세포적체외침습능력,유망위신경모세포류적기인치료개벽신도경.
Objective To explore the effect of silencing chemokine receptor type 4 (CXCR4) by siRNA on the invasion capability of neuroblastoma cell line SH-SY5Y in vitro. Methods Three siRNAs targeting CXCR4 were chemically synthesized and transfected into SH-SY5Y cells. The transfection efficiency was observed under fluorescence microscope. CXCR4 expression at mRNA and protein levels were detected by semi-quantitative RT-PCR and Western blotting. The invasion capability of the cells was evaluated by Boyden Chamber in vitro. Results Compared with control groups, after the SH-SY5Y cells being transfeeted with the three CXCR4 targeting siRNAs, CXCR4 mRNA in transfected cells significantly decreased (0. 32 ± 0. 09, 0. 35 ± 0. 13 and 0. 33 ± 0. 11 vs 0. 58 ± 0. 13, P＜0. 05 ), CXCR4 protein detected by immunohistochemistry was decreased (75. 98 ± 4. 81, 75. 52 ± 3. 95and 76. 35 ± 6. 51 vs 92. 196 ± 3. 89, P＜0. 01 ), CXCR4 protein detected by Western blotting was also decreased (0. 1103 ± 0. 0023, 0. 1203 ± 0. 0015 and 0. 1308 ± 0. 0018 vs 0. 4832 ± 0. 0012, P＜0. 01 ).The invasion capability of the SH-SY5Y cells was decreased 48 hours after the cells were transfected (25.48±2.81, 30.89±2.77 and 18.83± 1.79 vs 53. 11 ±3.72, P＜0.05). Conclusions Silencing CXCR4 by siRNA decreases the invasion capability of SH-SY5Y cells.
