中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2011年
11期
804-809
,共6页
王赞宏%李莉%彭芝兰%段振玲
王讚宏%李莉%彭芝蘭%段振玲
왕찬굉%리리%팽지란%단진령
Beclin1基因%自噬%细胞凋亡%宫颈肿瘤%HeLa细胞
Beclin1基因%自噬%細胞凋亡%宮頸腫瘤%HeLa細胞
Beclin1기인%자서%세포조망%궁경종류%HeLa세포
Beclin1 gene%Autophagy%Apoptosis%Uterine cervical neoplasms%Cell line,HeLa
目的 研究自噬基因Beclin1在宫颈癌细胞株HeLa中过表达对HeLa细胞体内外生长的影响.方法 构建自噬基因Beclin1的真核表达载体pcDNA3.1(+)-Beclin1,转染HeLa细胞[pcDNA3.1(+)-Beclin1组],同时设空载体转染组[pcDNA3.1(+)组]和空白对照组.采用荧光定量PCR和Western blot法检测3组HeLa细胞中Beclin1 mRNA和蛋白的表达变化,采用四甲基偶氮唑蓝(MTT)法检测3组HeLa细胞生长的变化,采用流式细胞术检测3组HeLa细胞的凋亡情况,采用单丹磺酰尸胺染色检测3组HeLa细胞的自噬情况.将3组HeLa细胞分别接种于裸鼠皮下,观察体内成瘤性和生长情况.结果 pcDNA3.1(+)-Beclin1组、pcDNA3.1(+)组和空白对照组HeLa细胞中Beclin1 mRNA的表达水平分别为994.718 ±468.764、0.570±0.121和0.736±0.251.pcDNA3.1 (+ )-Beclin1组HeLa细胞中Beclin1 mRNA的表达水平较pcDNA3.1(+)组和空白对照组明显增高(P＜0.05),而pcDNA3.1(+)组与空白对照组间的差异无统计学意义(P＞0.05)；3组HeLa细胞中Beclin1蛋白的表达情况与mRNA相似.pcDNA3.1(+)-Beclin1组的自噬细胞率为10.9％,明显高于空白对照组和pcDNA3.1(+)组(分别为2.5％和3.1％,P＜0.05).pcDNA3.1(+)-Beclin1组HeLa细胞的凋亡率为(28.2±2.3)％,明显高于pcDNA3.1(+)组和空白对照组[分别为(14.6±4.6)％和(11.2±3.0)％,P＜0.05].Beclin1在HeLa细胞中过表达可抑制肿瘤细胞在体外的生长,抑制率为58.7％；并可使HeLa细胞在裸鼠体内成瘤时间延长,瘤体体积和重量明显小于pcDNA3.1 (+)组和HeLa组(P＜0.05),抑瘤率为52.2％.结论 自噬基因Beclin1过表达可抑制HeLa细胞在体内外的增殖,促进细胞的自噬与凋亡,为宫颈癌基因治疗提供了新的选择途径.
目的 研究自噬基因Beclin1在宮頸癌細胞株HeLa中過錶達對HeLa細胞體內外生長的影響.方法 構建自噬基因Beclin1的真覈錶達載體pcDNA3.1(+)-Beclin1,轉染HeLa細胞[pcDNA3.1(+)-Beclin1組],同時設空載體轉染組[pcDNA3.1(+)組]和空白對照組.採用熒光定量PCR和Western blot法檢測3組HeLa細胞中Beclin1 mRNA和蛋白的錶達變化,採用四甲基偶氮唑藍(MTT)法檢測3組HeLa細胞生長的變化,採用流式細胞術檢測3組HeLa細胞的凋亡情況,採用單丹磺酰尸胺染色檢測3組HeLa細胞的自噬情況.將3組HeLa細胞分彆接種于裸鼠皮下,觀察體內成瘤性和生長情況.結果 pcDNA3.1(+)-Beclin1組、pcDNA3.1(+)組和空白對照組HeLa細胞中Beclin1 mRNA的錶達水平分彆為994.718 ±468.764、0.570±0.121和0.736±0.251.pcDNA3.1 (+ )-Beclin1組HeLa細胞中Beclin1 mRNA的錶達水平較pcDNA3.1(+)組和空白對照組明顯增高(P＜0.05),而pcDNA3.1(+)組與空白對照組間的差異無統計學意義(P＞0.05)；3組HeLa細胞中Beclin1蛋白的錶達情況與mRNA相似.pcDNA3.1(+)-Beclin1組的自噬細胞率為10.9％,明顯高于空白對照組和pcDNA3.1(+)組(分彆為2.5％和3.1％,P＜0.05).pcDNA3.1(+)-Beclin1組HeLa細胞的凋亡率為(28.2±2.3)％,明顯高于pcDNA3.1(+)組和空白對照組[分彆為(14.6±4.6)％和(11.2±3.0)％,P＜0.05].Beclin1在HeLa細胞中過錶達可抑製腫瘤細胞在體外的生長,抑製率為58.7％；併可使HeLa細胞在裸鼠體內成瘤時間延長,瘤體體積和重量明顯小于pcDNA3.1 (+)組和HeLa組(P＜0.05),抑瘤率為52.2％.結論 自噬基因Beclin1過錶達可抑製HeLa細胞在體內外的增殖,促進細胞的自噬與凋亡,為宮頸癌基因治療提供瞭新的選擇途徑.
목적 연구자서기인Beclin1재궁경암세포주HeLa중과표체대HeLa세포체내외생장적영향.방법 구건자서기인Beclin1적진핵표체재체pcDNA3.1(+)-Beclin1,전염HeLa세포[pcDNA3.1(+)-Beclin1조],동시설공재체전염조[pcDNA3.1(+)조]화공백대조조.채용형광정량PCR화Western blot법검측3조HeLa세포중Beclin1 mRNA화단백적표체변화,채용사갑기우담서람(MTT)법검측3조HeLa세포생장적변화,채용류식세포술검측3조HeLa세포적조망정황,채용단단광선시알염색검측3조HeLa세포적자서정황.장3조HeLa세포분별접충우라서피하,관찰체내성류성화생장정황.결과 pcDNA3.1(+)-Beclin1조、pcDNA3.1(+)조화공백대조조HeLa세포중Beclin1 mRNA적표체수평분별위994.718 ±468.764、0.570±0.121화0.736±0.251.pcDNA3.1 (+ )-Beclin1조HeLa세포중Beclin1 mRNA적표체수평교pcDNA3.1(+)조화공백대조조명현증고(P＜0.05),이pcDNA3.1(+)조여공백대조조간적차이무통계학의의(P＞0.05)；3조HeLa세포중Beclin1단백적표체정황여mRNA상사.pcDNA3.1(+)-Beclin1조적자서세포솔위10.9％,명현고우공백대조조화pcDNA3.1(+)조(분별위2.5％화3.1％,P＜0.05).pcDNA3.1(+)-Beclin1조HeLa세포적조망솔위(28.2±2.3)％,명현고우pcDNA3.1(+)조화공백대조조[분별위(14.6±4.6)％화(11.2±3.0)％,P＜0.05].Beclin1재HeLa세포중과표체가억제종류세포재체외적생장,억제솔위58.7％；병가사HeLa세포재라서체내성류시간연장,류체체적화중량명현소우pcDNA3.1 (+)조화HeLa조(P＜0.05),억류솔위52.2％.결론 자서기인Beclin1과표체가억제HeLa세포재체내외적증식,촉진세포적자서여조망,위궁경암기인치료제공료신적선택도경.
Objective To investigate the effects of autophagy gene Beclin 1 on growth of cervical cancer HeLa cells in vitro and vivo.Methods The eukaryotic expression vector of Beclinl was constructed and transfected via lipofectamine into HeLa cells.The experimental cells were classified into 3 groups:pcDNA3.1 ( + )-Beclinl group,pcDNA3.1 ( + ) group and HeLa group.Real fime-ploymerase chain reaction and Western blot were used for detecting expression of Beclin1 mRNA and protein in the transfected cells.Flow cytometry (FCM) was employed to observe the effect of transfection on the apeptosis of HeLacells,and proliferation was analyzed by MTT assay.The formation of autophagic vacuoles was measured by MDC staining.HeLa cells transfected with plasmid PcDNA3.1 ( + )-Beclin1 and PcDNA3.1 ( + ) were inoculated subcutaneously in nude mice.The carcinogenic and growth activities of cancer cells in vivo were observed.Results Eukaryotic expression vector peDNA3.1 ( + )-Beclin1 was constructed successfully.It significantly improved the expression of Beclin1 mRNA and protein in HeLa cells.The proliferation of HeLa cells was inhibited,and the inhibition rate was 58.7％.FCM investigation showed that the apoptotic rate was (28.22 ± 2.34) ％ of pcDNA3.1 ( + )-Beclin1 group,significantly higher than the ( 14.6 ± 4.6) ％ in the pcDNA3.1 ( + ) group and ( 11.2 ± 3.0) ％ in the HeLa group ( P ＜ 0.05 ).The monodansylcadaverin (MDC) staining showed significantly more autophagic vacuoles in the pcDNA3.1 ( + )-Beclin1 group ( 10.9％ ) than that in the pcDNA 3.1 ( + ) group (3.1％ ) and HeLa group (2.5％) ( P ＜ 0.05 ).After transfected with vector pcDNA3.1 ( + )-Beclin1,the carcinogenic activity of HeLa cells was decreased in nude mice,and the inhibition rate of tumor growth was 52.2％.Conclusions Autophagy gene Beclin 1 overexpression can inhibit the proliferation and growth of HeLa cells in vitro and vivo,while promote autophagy and apoptosis of HeLa cells.So it might be one of new gene therapy strategies for cervical carcinoma.
