中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2012年
1期
45-49
,共5页
贾秀红%范文文%李建厂%朱淑霞%唐慎华
賈秀紅%範文文%李建廠%硃淑霞%唐慎華
가수홍%범문문%리건엄%주숙하%당신화
RNA,小分子干扰%K562细胞%基因,同源盒%细胞增殖%凋亡
RNA,小分子榦擾%K562細胞%基因,同源盒%細胞增殖%凋亡
RNA,소분자간우%K562세포%기인,동원합%세포증식%조망
RNA,small interfering%K562 cells%Genes,homeobox%Cell proliferation%Apoptosis
目的 通过构建靶向同源盒A10(HOXA10)的真核表达载体,探讨利用RNA干扰沉默HOXA10基因对人慢性髓系白血病细胞株K562增殖和凋亡的影响.方法 根据筛选的针对HOXA10的特异性有效小干扰RNA(siRNA)序列设计合成短发夹RNA(shRNA)寡核苷酸链,构建pGPHI-GFP-Neo-HOXA10真核表达载体并测序,应用阳离子脂质体转染K562细胞.实验分为细胞对照组(仅加等量细胞及培养基),阴性对照组(脂质体转染阴性对照质粒)、实验组(脂质体转染pGPHI-GFP-Neo-HOXA10).转染载体24 h后利用RT-PCR检测各组HOXA10 mRNA表达；转染载体24、48、72 h后应用MTT法检测各组细胞增殖并计算细胞抑制率；转染载体48 h后应用流式细胞术检测各组细胞凋亡.结果 成功构建pGPHI-GFP-Neo-HOXA10载体并转染K562细胞.与细胞对照组和阴性对照组比较,实验组转染载体能有效降低HOXA10 mRNA的表达水平[(38.86±4.49)％比(88.52±9.24)％、(86.75±7.38)％,P＜0.05],而阴性对照组与细胞对照组比较差异则无统计学意义(P＞0.05).与阴性对照组比较,实验组载体作用于K562细胞24、48、72 h后,细胞增殖能力均明显下降,细胞抑制率明显升高[(39.92±0.74)％比(7.98±5.52)％;(55.62±1.18)％比(8.27±3.45)％;(66.30±1.26)％比(8.63±3.58)％;均P＜0.05].实验组细胞凋亡率较细胞对照组、阴性对照组也显著升高[(22.29±1.67)％比(9.82±0.69)％、(10.14±0.96)％,P＜0.05],而阴性对照组与细胞对照组比较差异则没有统计学意义(P＞0.05).结论 构建的真核表达载体pGPHI-GFP-Neo-HOXA10可有效沉默K562细胞中HOXA10基因的表达,能明显抑制K562细胞增殖并诱导其凋亡.
目的 通過構建靶嚮同源盒A10(HOXA10)的真覈錶達載體,探討利用RNA榦擾沉默HOXA10基因對人慢性髓繫白血病細胞株K562增殖和凋亡的影響.方法 根據篩選的針對HOXA10的特異性有效小榦擾RNA(siRNA)序列設計閤成短髮夾RNA(shRNA)寡覈苷痠鏈,構建pGPHI-GFP-Neo-HOXA10真覈錶達載體併測序,應用暘離子脂質體轉染K562細胞.實驗分為細胞對照組(僅加等量細胞及培養基),陰性對照組(脂質體轉染陰性對照質粒)、實驗組(脂質體轉染pGPHI-GFP-Neo-HOXA10).轉染載體24 h後利用RT-PCR檢測各組HOXA10 mRNA錶達；轉染載體24、48、72 h後應用MTT法檢測各組細胞增殖併計算細胞抑製率；轉染載體48 h後應用流式細胞術檢測各組細胞凋亡.結果 成功構建pGPHI-GFP-Neo-HOXA10載體併轉染K562細胞.與細胞對照組和陰性對照組比較,實驗組轉染載體能有效降低HOXA10 mRNA的錶達水平[(38.86±4.49)％比(88.52±9.24)％、(86.75±7.38)％,P＜0.05],而陰性對照組與細胞對照組比較差異則無統計學意義(P＞0.05).與陰性對照組比較,實驗組載體作用于K562細胞24、48、72 h後,細胞增殖能力均明顯下降,細胞抑製率明顯升高[(39.92±0.74)％比(7.98±5.52)％;(55.62±1.18)％比(8.27±3.45)％;(66.30±1.26)％比(8.63±3.58)％;均P＜0.05].實驗組細胞凋亡率較細胞對照組、陰性對照組也顯著升高[(22.29±1.67)％比(9.82±0.69)％、(10.14±0.96)％,P＜0.05],而陰性對照組與細胞對照組比較差異則沒有統計學意義(P＞0.05).結論 構建的真覈錶達載體pGPHI-GFP-Neo-HOXA10可有效沉默K562細胞中HOXA10基因的錶達,能明顯抑製K562細胞增殖併誘導其凋亡.
목적 통과구건파향동원합A10(HOXA10)적진핵표체재체,탐토이용RNA간우침묵HOXA10기인대인만성수계백혈병세포주K562증식화조망적영향.방법 근거사선적침대HOXA10적특이성유효소간우RNA(siRNA)서렬설계합성단발협RNA(shRNA)과핵감산련,구건pGPHI-GFP-Neo-HOXA10진핵표체재체병측서,응용양리자지질체전염K562세포.실험분위세포대조조(부가등량세포급배양기),음성대조조(지질체전염음성대조질립)、실험조(지질체전염pGPHI-GFP-Neo-HOXA10).전염재체24 h후이용RT-PCR검측각조HOXA10 mRNA표체；전염재체24、48、72 h후응용MTT법검측각조세포증식병계산세포억제솔；전염재체48 h후응용류식세포술검측각조세포조망.결과 성공구건pGPHI-GFP-Neo-HOXA10재체병전염K562세포.여세포대조조화음성대조조비교,실험조전염재체능유효강저HOXA10 mRNA적표체수평[(38.86±4.49)％비(88.52±9.24)％、(86.75±7.38)％,P＜0.05],이음성대조조여세포대조조비교차이칙무통계학의의(P＞0.05).여음성대조조비교,실험조재체작용우K562세포24、48、72 h후,세포증식능력균명현하강,세포억제솔명현승고[(39.92±0.74)％비(7.98±5.52)％;(55.62±1.18)％비(8.27±3.45)％;(66.30±1.26)％비(8.63±3.58)％;균P＜0.05].실험조세포조망솔교세포대조조、음성대조조야현저승고[(22.29±1.67)％비(9.82±0.69)％、(10.14±0.96)％,P＜0.05],이음성대조조여세포대조조비교차이칙몰유통계학의의(P＞0.05).결론 구건적진핵표체재체pGPHI-GFP-Neo-HOXA10가유효침묵K562세포중HOXA10기인적표체,능명현억제K562세포증식병유도기조망.
Objective To investigate the impact of RNA interference silencing HOXA10 gene on proliferation and apoptosis of K562 cell strain of human chronic myeloid leukemia (CML) by construction of eukaryotic expression vector targeting HOXA10.Methods Based on short hairpin RNA(shRNA) oligo that was designed and compounded by the selected specific small interference RNA (siRNA) targeting HOXA 10,the eukaryotic expression vector of pGPHI-GFP-Neo-HOXA10 was successfully constructed and sequenced,and the cationic liposome was then used to transfect K562 cells.There were cell control group (equivalent cells and culture medium only),negative control group (negative control plasmid transfected by liposome)and experimental group (pGPHI- GFP- Neo- HOXA10 transfected by liposome).The HOXA10 mRNA expression was detected by RT-PCR at 24 h after transfection,cell proliferation by MTT at 24,48 and 72 h for cell inhibition ratio,and the apoptosis by flow cytometry at 48 h in all the groups.Results The vector pGPHI-GFP-Neo-HOXA10 was successfully constructed and used to transfect K562 cells.The transfection vector in experimental group could effectively decrease the expression level of HOXA10 mRNA [ (38.86±4.49)％ vs (88.52±9.24)％,(86.75±7.38)％,P＜0.05] as compared with that in cell control and negative control group,with no difference between negative control and cell control group (P＞0.05).Compared with negative control group,the experimental group had a significant decrease in cell proliferation capacity but an increase in cell inhibition ratio at 24,48 and 72 h after transfection [ (39.92±0.74)％ vs (7.98±5.52)％;(55.62±1.18)％ vs (8.27±3.45)％; (66.30±1.26)％ vs (8.63±3.58)％; all P＜0.05].Moreover,the apoptosis rate was significantly increased in experimental group as compared with that in control and negative control groups [ (22.29± 1.67)％ vs (9.82±0.69)％,(10.14±0.96)％,P＜0.05],however,no difference was found between negative control and cell control group (P＞0.05).Conclusion Since eukaryotic expression vector pGPHI-GFP-Neo-HOXA10 can effectively silence the expression of HOXA10 in K562 cells,pGPHI-GFP-Neo-HOXA10 may significantly inhibit proliferation of K562 cell and induce its apoptosis.
