CAJ | 학술논문

目的观察桥梁分子1-S(ITSN1-S)的SH3结构域对恶性胶质瘤细胞LN229增殖和运动能力的影响,并探讨其相关分子机制.方法将ITSN1-S的全长和去除SH3结构域的ITSN1-S(即EH1-EH2-CC片段)分别连接人双酶切后线性化的PCDH-CMV-MCS-EF1-Puro载体中作为实验组,用不作处理的PCDH-CMV-MCS-EF1-Puro载体作为对照组,通过慢病毒转染上述3个重组质粒进入LN229细胞,筛选稳定表达的细胞克隆；Western blot检测目的蛋白的表达；噻唑蓝(MTT)实验检测连续培养6d各组胶质瘤细胞的增殖；软琼脂克隆形成实验检测连续培养2周的各组胶质瘤细胞克隆形成能力；划痕试验检测各组胶质瘤细胞在0、3、6、9、12、24 h的非定向运动能力.结果 MTT实验和软琼脂克隆形成实验均证明ITSN1-S全长组细胞的增殖能力比EH1-EH2-CC片段组和空载体对照组显著增强(P＜0.05),但EH1-EH2-CC片段组和空载体对照组两组比较差异无统计学意义(P＞0.05).第6天时ITSN1-S全长组、EH1-EH2-CC片段组和空载体对照组细胞增殖率分别为(523.60±32.12)％、(409.54±31.33)％和(353.89±13.98)％；第14天时ITSN1-S全长组克隆形成率达(46.49±2.34)％,而EH1-EH2-CC片段组和空载体对照组克隆形成率分别为(23.00±1.66)％和(17.68±3.47)％.划痕试验12 h时ITSN1-S全长组细胞的运动速度比EH1-EH2-CC片段组和空载体对照组明显提高(P＜0.05),但EH1-EH2-CC片段组和空载体对照组两组比较差异无统计学意义(P＞0.05).24 h时ITSN1-S全长组运动距离为(0.39±0.02) mm,EH1-EH2-CC片段组和空载体对照组分别为(0.30±0.02) mm和(0.29±0.01)mm.结论 ITSN1-S参与调节胶质瘤细胞的增殖和运动,ITSN1-S中SH3结构域是影响胶质瘤细胞增殖与运动能力的主要功能结构域.
목적 관찰교량분자1-S(ITSN1-S)적SH3결구역대악성효질류세포LN229증식화운동능력적영향,병탐토기상관분자궤제.방법 장ITSN1-S적전장화거제SH3결구역적ITSN1-S(즉EH1-EH2-CC편단)분별련접인쌍매절후선성화적PCDH-CMV-MCS-EF1-Puro재체중작위실험조,용불작처리적PCDH-CMV-MCS-EF1-Puro재체작위대조조,통과만병독전염상술3개중조질립진입LN229세포,사선은정표체적세포극륭；Western blot검측목적단백적표체；새서람(MTT)실험검측련속배양6d각조효질류세포적증식；연경지극륭형성실험검측련속배양2주적각조효질류세포극륭형성능력；화흔시험검측각조효질류세포재0、3、6、9、12、24 h적비정향운동능력.결과 MTT실험화연경지극륭형성실험균증명ITSN1-S전장조세포적증식능력비EH1-EH2-CC편단조화공재체대조조현저증강(P＜0.05),단EH1-EH2-CC편단조화공재체대조조량조비교차이무통계학의의(P＞0.05).제6천시ITSN1-S전장조、EH1-EH2-CC편단조화공재체대조조세포증식솔분별위(523.60±32.12)％、(409.54±31.33)％화(353.89±13.98)％；제14천시ITSN1-S전장조극륭형성솔체(46.49±2.34)％,이EH1-EH2-CC편단조화공재체대조조극륭형성솔분별위(23.00±1.66)％화(17.68±3.47)％.화흔시험12 h시ITSN1-S전장조세포적운동속도비EH1-EH2-CC편단조화공재체대조조명현제고(P＜0.05),단EH1-EH2-CC편단조화공재체대조조량조비교차이무통계학의의(P＞0.05).24 h시ITSN1-S전장조운동거리위(0.39±0.02) mm,EH1-EH2-CC편단조화공재체대조조분별위(0.30±0.02) mm화(0.29±0.01)mm.결론 ITSN1-S삼여조절효질류세포적증식화운동,ITSN1-S중SH3결구역시영향효질류세포증식여운동능력적주요공능결구역.
Objective To investigate the effects of Src homology 3 domain (SH3) of intersectin 1-S (ITSN1-S) on proliferation and migration of glioma cell line LN229 and the possible molecular mechanisms.Methods ITSN1-S and fragmental EH1-EH2-CC domain of ITSN1-S were spliced into the linear PCDH-CMV-MCS-EF1-Puro vector which was excised by incision enzyme digestion,and PCDH-CMV-MCS-EF1-Puro vector without treatment served as control group.Each of the three recombinant plasmids was transfected into the LN229 cells by lentivirus,and the stably expressed cell clones were screened.Western blotting was applied to detect the expression of each protein.MTT assay was performed to detect proliferation of LN229 cells persistently cultured for 6 days.Soft agar assay was performed to detect the colony formation ability of glioma cells persistently cultured for 2 weeks.Wound-healing assay was performed to detect the migration of LN229 cells at 0,3,6,9,12,24 h.Results MTT assay and soft agar assay showed that ITSN1-S total-length group proliferated more rapidly than EH1-EH2-CC fragment group and vector control group (P ＜ 0.05),but there was no significant difference between EH1-EH2-CC fragment group and vector control group (P ＞ 0.05).The proliferation rate at 6th day in ITSN1-S total-length group,EH1-EH2-CC fragment group and vector control group was (523.60 ± 32.12)％,(409.54 ± 31.33)％and (353.89 ± 13.98) ％,respectively.The clone formation rate at 14th day in ITSN1-S total-length group,EH1-EH2-CC fragment group and vector control group was (46.49 ± 2.34)％,(23.00 ±1.66) ％ and (17.68 ± 3.47)％,respectively.Wound-healing assay revealed that the cells in ITSN1-S total-length group migrated faster than in EH1-EH2-CC fragment group and vector control group after 12 h (P ＜0.05),but there was no significant difference between EH1-EH2-CC fragment group and vector control group (P ＞0.05).The migration distance at 24th h in ITSN1-S total-length group,EH1-EH2-CC fragment group and vector control group was (0.388 ± 0.023) mm,(0.307 ± 0.021) mm and (0.287 ±0.011) mm,respectively.Conclusion ITSN1-S is involved in regulating the proliferation and migration of glioma cells.The SH3 domains were the crucial functional domain of ITSN1-S to regulate the proliferation and migration of glioma cells.