中华口腔医学杂志
中華口腔醫學雜誌
중화구강의학잡지
Chinese Journal of Stomatology
2015年
8期
490-495
,共6页
杨月%王海丞%许舒宇%彭靖%江久汇%李翠英
楊月%王海丞%許舒宇%彭靖%江久彙%李翠英
양월%왕해승%허서우%팽정%강구회%리취영
癌,腺样囊性%基因敲除%短回文重复序列/常间回文重复序列丛集关联蛋白系统%基因外显子A
癌,腺樣囊性%基因敲除%短迴文重複序列/常間迴文重複序列叢集關聯蛋白繫統%基因外顯子A
암,선양낭성%기인고제%단회문중복서렬/상간회문중복서렬총집관련단백계통%기인외현자A
Carcinoma,adenoid cystic%Knockout%Clustered regularly interspaced short palindromic repeats/associated%Extra domain A
目的 利用成簇的规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)/常间回文重复序列丛集关联蛋白系统(associated proteins,Cas)编辑纤连蛋白(fibronectin)基因抑制其可变剪接片段基因外显子A(extra domain A,EDA),并观察对腺样囊性癌(adenoid cystic carcinoma,ACC)的促肿瘤作用.方法 根据纤连蛋白序列设计两段互补于EDA上游和一段与下游互补的引导RNA (single guide RNA,sgRNA,20 bp),分别连接至PX330质粒的U6启动子下游.质粒转染至ACC细胞系SACC-83,PCR扩增基因组并测序验证其定点敲除EDA结果及效率.质粒转染后的细胞进行稳定株的筛选及鉴定,将筛选后的稳定株作为EDA敲除实验组,SACC-83细胞为对照组,进行CCK-8细胞增殖和Transwell侵袭能力检测,每组实验重复3次.结果 sgRNA连接至PX330质粒U6启动子下游,成功构建了质粒敲除模型;SACC-83的基因组EDA外显子被敲除,敲除效率达70％以上,但纤连蛋白总量未发生明显变化.筛选出3株EDA敲除稳定株(A+C-2、A+C-6、B+C-10),并通过PCR鉴定证实其可靠性.划痕实验中实验组细胞运动速率[A+C-2 (21.67±1.87) μm/h;A+C-6(12.22±2.13) μm/h;B+C-10(20.00±2.56) μm/h]相对对照组[(27.78±3.20) μm/h]降低;增殖实验显示EDA敲除组细胞倍增时间增加[对照组SACC-83 (38.52±4.26)h,实验组A+C-2(62.05±5.80)h,A+C-6(46.32±6.35)h,B+C-10(40.7±3.88)h].结论 在sgRNA的引导下,CRISPR/Cas系统能简洁、高效地敲除细胞基因组中的EDA可变剪接外显子,EDA敲除对肿瘤细胞运动和侵袭有明显抑制作用.
目的 利用成簇的規律間隔的短迴文重複序列(clustered regularly interspaced short palindromic repeats,CRISPR)/常間迴文重複序列叢集關聯蛋白繫統(associated proteins,Cas)編輯纖連蛋白(fibronectin)基因抑製其可變剪接片段基因外顯子A(extra domain A,EDA),併觀察對腺樣囊性癌(adenoid cystic carcinoma,ACC)的促腫瘤作用.方法 根據纖連蛋白序列設計兩段互補于EDA上遊和一段與下遊互補的引導RNA (single guide RNA,sgRNA,20 bp),分彆連接至PX330質粒的U6啟動子下遊.質粒轉染至ACC細胞繫SACC-83,PCR擴增基因組併測序驗證其定點敲除EDA結果及效率.質粒轉染後的細胞進行穩定株的篩選及鑒定,將篩選後的穩定株作為EDA敲除實驗組,SACC-83細胞為對照組,進行CCK-8細胞增殖和Transwell侵襲能力檢測,每組實驗重複3次.結果 sgRNA連接至PX330質粒U6啟動子下遊,成功構建瞭質粒敲除模型;SACC-83的基因組EDA外顯子被敲除,敲除效率達70％以上,但纖連蛋白總量未髮生明顯變化.篩選齣3株EDA敲除穩定株(A+C-2、A+C-6、B+C-10),併通過PCR鑒定證實其可靠性.劃痕實驗中實驗組細胞運動速率[A+C-2 (21.67±1.87) μm/h;A+C-6(12.22±2.13) μm/h;B+C-10(20.00±2.56) μm/h]相對對照組[(27.78±3.20) μm/h]降低;增殖實驗顯示EDA敲除組細胞倍增時間增加[對照組SACC-83 (38.52±4.26)h,實驗組A+C-2(62.05±5.80)h,A+C-6(46.32±6.35)h,B+C-10(40.7±3.88)h].結論 在sgRNA的引導下,CRISPR/Cas繫統能簡潔、高效地敲除細胞基因組中的EDA可變剪接外顯子,EDA敲除對腫瘤細胞運動和侵襲有明顯抑製作用.
목적 이용성족적규률간격적단회문중복서렬(clustered regularly interspaced short palindromic repeats,CRISPR)/상간회문중복서렬총집관련단백계통(associated proteins,Cas)편집섬련단백(fibronectin)기인억제기가변전접편단기인외현자A(extra domain A,EDA),병관찰대선양낭성암(adenoid cystic carcinoma,ACC)적촉종류작용.방법 근거섬련단백서렬설계량단호보우EDA상유화일단여하유호보적인도RNA (single guide RNA,sgRNA,20 bp),분별련접지PX330질립적U6계동자하유.질립전염지ACC세포계SACC-83,PCR확증기인조병측서험증기정점고제EDA결과급효솔.질립전염후적세포진행은정주적사선급감정,장사선후적은정주작위EDA고제실험조,SACC-83세포위대조조,진행CCK-8세포증식화Transwell침습능력검측,매조실험중복3차.결과 sgRNA련접지PX330질립U6계동자하유,성공구건료질립고제모형;SACC-83적기인조EDA외현자피고제,고제효솔체70％이상,단섬련단백총량미발생명현변화.사선출3주EDA고제은정주(A+C-2、A+C-6、B+C-10),병통과PCR감정증실기가고성.화흔실험중실험조세포운동속솔[A+C-2 (21.67±1.87) μm/h;A+C-6(12.22±2.13) μm/h;B+C-10(20.00±2.56) μm/h]상대대조조[(27.78±3.20) μm/h]강저;증식실험현시EDA고제조세포배증시간증가[대조조SACC-83 (38.52±4.26)h,실험조A+C-2(62.05±5.80)h,A+C-6(46.32±6.35)h,B+C-10(40.7±3.88)h].결론 재sgRNA적인도하,CRISPR/Cas계통능간길、고효지고제세포기인조중적EDA가변전접외현자,EDA고제대종류세포운동화침습유명현억제작용.
Objective To investigate the effect of the fibronectin extra domain A on the aggressiveness of salivary adenoid cystic carcinoma (SACC) cells,via the clustered regularly interspaced short palindromic repeats (CRISPR)/associated proteins (Cas) system.Methods One sgRNA was designed to target the upstream of the genome sequences of extra domain A(EDA) exon and the downstream.Then the sgRNA was linked into plasmid PX-330 and transfected into SACC-83 cells.PCR and DNA sequence were used to testify the knockout cells,and the monoclones of EDA absent SACC cells were selected (A+C-2,A+C-6,B+C-10).CCK-8 cell proliferation and invasion was then tested in control group and the experimental group.Results The sgRNA was successfully linked into PX-330 plasmid.Part of adenoid cystic carcinoma cells' SACC-83 genomic EDA exon was knocked out,and the knockdown efficiency was above 70％,but the total amount of fibmnectin did not change significantly.Three monoclones of EDA absent SACC-83 cells were successfully selected with diminished migration and proliferation.Conclusions The CRISPR/Cas9 system was a simplified system with relatively high knockout efficiency and EDA knockout could inhibiting SACC cell's mobility and invasiveness.
