中国血液流变学杂志
中國血液流變學雜誌
중국혈액류변학잡지
CHINESE JOURNAL OF HEMORHEOLOGY
2013年
3期
420-423,427
,共5页
李申篪%任园园%杨百霞%许玉杰
李申篪%任園園%楊百霞%許玉傑
리신지%임완완%양백하%허옥걸
SPATA5L1%真核表达载体%KB细胞%肿瘤复发转移
SPATA5L1%真覈錶達載體%KB細胞%腫瘤複髮轉移
SPATA5L1%진핵표체재체%KB세포%종류복발전이
SPATA5L1%eukaryotic expression vector%KB cell%recurrence and metastasis
目的：通过构建过表达质粒并筛选稳定细胞株，研究SPATA5L1基因对KB细胞增殖和迁移的影响。方法 PCR扩增SPATA5L1基因全长，用载体pEX-1构建重组质粒，测序成功后转染人口腔癌KB细胞，并设空载体作对照，经过G418压力筛选后获得单克隆细胞株，Real-time PCR、Western blot验证SPATA5L1基因的表达，光镜观察SPATA5L1对KB细胞形态的影响，CCK-8法检测细胞增殖情况，划痕实验观察细胞迁移能力。结果测序结果显示质粒构建成功，经过G418压力筛选所得到的稳定表达细胞株，在转录水平和蛋白水平均检测到目的基因的高表达，且细胞由正常状态下的多边形变为梭形，细胞增殖能力和体外迁移能力明显提高（P＜0.05）。结论成功构建pEX-1-SPATA5L1表达载体并获得稳定细胞株，过表达SPATA5L1蛋白能使KB细胞形态变长，增殖和迁移加快。
目的：通過構建過錶達質粒併篩選穩定細胞株，研究SPATA5L1基因對KB細胞增殖和遷移的影響。方法 PCR擴增SPATA5L1基因全長，用載體pEX-1構建重組質粒，測序成功後轉染人口腔癌KB細胞，併設空載體作對照，經過G418壓力篩選後穫得單剋隆細胞株，Real-time PCR、Western blot驗證SPATA5L1基因的錶達，光鏡觀察SPATA5L1對KB細胞形態的影響，CCK-8法檢測細胞增殖情況，劃痕實驗觀察細胞遷移能力。結果測序結果顯示質粒構建成功，經過G418壓力篩選所得到的穩定錶達細胞株，在轉錄水平和蛋白水平均檢測到目的基因的高錶達，且細胞由正常狀態下的多邊形變為梭形，細胞增殖能力和體外遷移能力明顯提高（P＜0.05）。結論成功構建pEX-1-SPATA5L1錶達載體併穫得穩定細胞株，過錶達SPATA5L1蛋白能使KB細胞形態變長，增殖和遷移加快。
목적：통과구건과표체질립병사선은정세포주，연구SPATA5L1기인대KB세포증식화천이적영향。방법 PCR확증SPATA5L1기인전장，용재체pEX-1구건중조질립，측서성공후전염인구강암KB세포，병설공재체작대조，경과G418압력사선후획득단극륭세포주，Real-time PCR、Western blot험증SPATA5L1기인적표체，광경관찰SPATA5L1대KB세포형태적영향，CCK-8법검측세포증식정황，화흔실험관찰세포천이능력。결과측서결과현시질립구건성공，경과G418압력사선소득도적은정표체세포주，재전록수평화단백수평균검측도목적기인적고표체，차세포유정상상태하적다변형변위사형，세포증식능력화체외천이능력명현제고（P＜0.05）。결론성공구건pEX-1-SPATA5L1표체재체병획득은정세포주，과표체SPATA5L1단백능사KB세포형태변장，증식화천이가쾌。
Objective To construct the recombinant eukaryotic vector expressing SPATA5L1 cDNA,and explore the effect of SPATA5L1 over-expression on the proliferation and migration of human oral cancer cell line KB.Methods The fragment of SPATA5L1 gene was ampliifed by polymerase chain reaction(PCR).An eukaryotic vector expressing SPATA5L1(pEX-1-SPATA5L1) was constructed and transfected into KB cells which were then selected in culture medium containing G418 to acquire the monoclonal cell lines.The transcription and expression of SPATA5L1 gene were detected by Western blotting.The effect on the proliferation and migration of KB cells were evaluated by CCK-8 assay and wound healing assay respectively.Results The plasmid vector with target SPATA5L1 gene was designed correctly.The monoclonal cell line which showed spindle shape was obtained after screened with G418.The protein level of SPATA5L1 in the pEX-1-SPATA5L1 transfected cells was signiifcantly higher than controls.CCK-8 assay showed that the proliferation of pEX-1-SPATA5L1-KB cells was much faster than the controls.Wound healing assay revealed that up-regulating SPATA5L1 in KB cells could promote the wound healing.Conclusion Up-regulating SPATA5L1 gene could stimulate the proliferation of KB cells and promote the migration of KB cells.
