全长型脾酪氨酸激酶基因转染对喉癌细胞恶性生物学行为的抑制作用
전장형비락안산격매기인전염대후암세포악성생물학행위적억제작용
Inhibitory effect of full-length spleen tyrosine kinase on invasion and metastasis of human laryngeal squamous cells
  • 万方数据
    中华耳鼻咽喉头颈外科杂志 중화이비인후두경외과잡지
    CHINESE JOURNAL OF OTORHINOLARYNGOLOGY HEAD AND NECK SURGERY
    2014年 11期 943-949 ,共7页

    李志海%蔡志毅%陶宝鸿%金巧智

    리지해%채지의%도보홍%금교지

    喉肿瘤%蛋白酪氨酸激酶类%转染%细胞系,肿瘤 喉腫瘤%蛋白酪氨痠激酶類%轉染%細胞繫,腫瘤
    후종류%단백락안산격매류%전염%세포계,종류
    Laryngeal neoplasms%Protein-tyrosine kinases%Transfection%Cell line,tumor
    目的 探讨全长型脾酪氨酸激酶[full length spleen tyrosine kinase,Syk(L)]基因转染对喉癌细胞恶性生物学行为的抑制作用.方法 构建Syk(L)基因真核表达载体pIRES2-EGFP-Syk(L),测序验证正确后,用脂质体介导法及G418筛选法,分别建立转染pIRES2-EGFP-Syk (L)载体和pIRES2-EGFP空白载体的喉癌细胞系Hep-2-Syk (L)及Hep-2-neo细胞.实时荧光定量聚合酶链反应(quantitative real time fluorescence polymerase chain reaction,Q-RT-PCR)及蛋白印迹法(Western Blot)分析转染细胞Hep-2-Syk (L)、Hep-2-neo及未转染细胞Hep-2的Syk (L) mRNA及蛋白表达水平;采用CCK-8实验、Transwell实验、裸鼠成瘤实验分别检测转染细胞及未转染细胞的体外增殖能力、侵袭能力、体内成瘤能力.采用SPSS 18.0统计软件中的方差分析进行组间样本均数的比较.结果 成功构建了Syk(L)基因真核表达载体pIRES2-EGFP-SyK (L);成功建立分别稳定转染pIRES2-EGFP-Syk(L)载体、pIRES2-EGFP空白载体的Hep-2-Syk(L)、Hep-2-neo细胞系;Q-RT-PCR结果显示Hep-2-Syk(L)细胞Syk (L) mRNA表达水平明显高于Hep-2-neo及Hep-2细胞(相对表达量2-△△CT分别为28.395±0.067、3.891±0.021及1.005 ±0.012),差异有统计学意义(F=104.02,P＜0.01);Western Blot结果提示Hep-2-Syk(L)细胞Syk(L)相对蛋白含量为(0.821 ±0.047),明显高于Hep-2-neo细胞的(0.558 ±0.031)和Hep-2细胞的(0.468±0.031),差异有统计学意义(F=112.32,P＜0.01);CCK-8实验72 h Hep-2-Syk(L)细胞的平均吸光度值为1.390±0.067,低于Hep-2-neo细胞的1.830±0.067和Hep-2细胞的1.920±0.040,差异有统计学意义(F=107.64,P＜0.01);Transwell实验中,Hep-2-Syk(L)组每个视野的平均侵袭细胞数目为(176.04±22.32)个,较Hep-2-neo组的(301.02±21.45)个、Hep-2组的(336.04±26.01)个明显减少,差异有统计学意义(F=123.46,P＜0.01);裸鼠成瘤实验中,Hep-2-Syk(L)细胞组体内移植瘤生长受到抑制,平均体积为(250.77±34.83) mm3,低于Hep-2-neo组的肿瘤平均体积(750.77±40.83) mm3及Hep-2组的肿瘤平均体积(770.77±30.83) mm3差异有统计学意义(F=165.78,P＜0.01).结论 喉癌细胞中Syk(L)低表达与细胞的恶性生物学行为相关,通过基因转染技术提高Syk(L)表达可以抑制喉癌细胞的恶性生物学行为,Syk(L)可能成为喉鳞癌基因治疗良好的干预靶点.
    목적 탐토전장형비락안산격매[full length spleen tyrosine kinase,Syk(L)]기인전염대후암세포악성생물학행위적억제작용.방법 구건Syk(L)기인진핵표체재체pIRES2-EGFP-Syk(L),측서험증정학후,용지질체개도법급G418사선법,분별건립전염pIRES2-EGFP-Syk (L)재체화pIRES2-EGFP공백재체적후암세포계Hep-2-Syk (L)급Hep-2-neo세포.실시형광정량취합매련반응(quantitative real time fluorescence polymerase chain reaction,Q-RT-PCR)급단백인적법(Western Blot)분석전염세포Hep-2-Syk (L)、Hep-2-neo급미전염세포Hep-2적Syk (L) mRNA급단백표체수평;채용CCK-8실험、Transwell실험、라서성류실험분별검측전염세포급미전염세포적체외증식능력、침습능력、체내성류능력.채용SPSS 18.0통계연건중적방차분석진행조간양본균수적비교.결과 성공구건료Syk(L)기인진핵표체재체pIRES2-EGFP-SyK (L);성공건립분별은정전염pIRES2-EGFP-Syk(L)재체、pIRES2-EGFP공백재체적Hep-2-Syk(L)、Hep-2-neo세포계;Q-RT-PCR결과현시Hep-2-Syk(L)세포Syk (L) mRNA표체수평명현고우Hep-2-neo급Hep-2세포(상대표체량2-△△CT분별위28.395±0.067、3.891±0.021급1.005 ±0.012),차이유통계학의의(F=104.02,P＜0.01);Western Blot결과제시Hep-2-Syk(L)세포Syk(L)상대단백함량위(0.821 ±0.047),명현고우Hep-2-neo세포적(0.558 ±0.031)화Hep-2세포적(0.468±0.031),차이유통계학의의(F=112.32,P＜0.01);CCK-8실험72 h Hep-2-Syk(L)세포적평균흡광도치위1.390±0.067,저우Hep-2-neo세포적1.830±0.067화Hep-2세포적1.920±0.040,차이유통계학의의(F=107.64,P＜0.01);Transwell실험중,Hep-2-Syk(L)조매개시야적평균침습세포수목위(176.04±22.32)개,교Hep-2-neo조적(301.02±21.45)개、Hep-2조적(336.04±26.01)개명현감소,차이유통계학의의(F=123.46,P＜0.01);라서성류실험중,Hep-2-Syk(L)세포조체내이식류생장수도억제,평균체적위(250.77±34.83) mm3,저우Hep-2-neo조적종류평균체적(750.77±40.83) mm3급Hep-2조적종류평균체적(770.77±30.83) mm3차이유통계학의의(F=165.78,P＜0.01).결론 후암세포중Syk(L)저표체여세포적악성생물학행위상관,통과기인전염기술제고Syk(L)표체가이억제후암세포적악성생물학행위,Syk(L)가능성위후린암기인치료량호적간예파점.
    Objective To study the effect of gene transfection of full length spleen tyrosine kinase (Syk (L)) on the biological behavior of malignant cancer cells.Methods Eukaryotic expression vector pIRES2-EGFP-Syk (L) was constrauted and sequenc.Laryngeal carcinoma cell line Hep-2 were transfected with pIRES2-EGFP-Syk (L) vectors or blank vectors.The expressions of mRNA and protein were examined by real time fluorescence quantitative polymerase chain reaction (Q-RT-PCR) and Western blot analysis.CCK-8 method was used for evaluating cell proliferation,Transwell for cell invasion capacity in vitro,and tumor formation in nude mice for in vivo tumorigenicity.Results pIRES2-EGFP-Syk (L) vectors were successfully construct and transfected to Hep-2 cells.Q-PCR showed that mRNA expression level in Hep-2 cells transfected with Sky (L) (28.395 ± 0.067) was higher than those in Hep-2-neo cells transfected with blank vectors (3.891 ± 0.021) and Hep-2 cells with no transfection (1.005 ± 0.012),with statistically significant difference (F =104.02,P ＜ 0.01).Western blot showed that protein expression level of transfected-Sky (L) cells (0.821 ± 0.047) was significantly higher than those of Hep-2-neo cells (0.558 ±0.031) and Hep-2 cells (0.468 ± 0.031),and the difference was statistically significant (F =112.32,P ＜ 0.01) ; CCK-8 assay showed OD value (1.390 ± 0.067) of transfected-Sky (L) cells was lower than those of Hep-2-neo cells (1.830 ± 0.067) and Hep-2 cells (1.920 ± 0.040),and the difference was statistically significant (F =107.64,P ＜ 0.01).Transwe11 assay showed average cell number per field of transfected-Sky (L) cells (176.04 ± 22.32) was higher than those of Hep-2-neo cells (301.02 ± 21.45) and Hep-2 cells (336.04 ± 26.01) with statistically significant difference (F =123.46,P ＜0.01).The volume (250.77 ± 34.83) mm3 tumor formed from transfected-Sky (L) cells in nude mice,was less than those from Hep-2-neo cells (750.77 ± 40.83) mm3 and Hep-2 cells (770.77 ±30.83) mm3,with statistically significant difference (F =165.78,P ＜0.01).Conclusion Downregulation of Syk in Hep-2 cells is associated with the malignant biological behaviors of the cells.Syk (L)may be a potential target in gene therapy for laryngeal squamous cell carcinoma.
    원문보기 원문다운로드 사이트로이동
저자의 최근 논문