桥梁分子1-S的SH3结构域对胶质瘤细胞增殖和运动能力的影响

Effect of Src homology 3 domain domains of intersectinl-S on proliferation and migration of gliomacells

目的观察桥梁分子1一S（ITSNl-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ITSNl-S的全长和去除SH3结构域的ITSNl一S（即EHl一EH2一CC片段）分别连接人双酶切后线性化的PCDH—CMV—MCS—EFl，Puro载体中作为实验组，用不作处理的PCDH—CMV．MCS．EFl一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；Westernblot检测目的蛋白的表达；噻唑蓝（M订）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、24h的非定向运动能力。结果MTF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EHl-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ITSNl．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（523．604-32．12）％、（409．54±31．33）％和（353．89±13．98）％；第14天时ITSNl-S全长组克隆形成率达（46．49±2．34）％，而EHl-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（17．684-3．47）％。划痕试验12h时ITSNl一S全长组细胞的运动速度比EHl-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）。24h时ITSN1-S全长组运动距离为（0．39±O．02）mill，EHl一EH2一CC片段组和空载体对照组分别为（0．304-0．02）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。

Objective To investigate the effects of Src homology 3 domain （SH3） of intersectin 1-S （ITSNI-S） on proliferation and migration of glioma cell line LN229 and the possible molecular mecha- nisms. 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 lentivilus, 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 col- ony 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 M3T 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-heallng assay revealed that the cells in ITSN1-S total-length group migrated faster than in EH1-EH2-CC fragtrfent 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 con-trnl group （ P 〉 0. 05 ）. The migration dislan~＇e at 24th h in ITSNI -S total-length group, EHI -EH2-CC frag- ment group and vector control group was （0. 388 ＋- 0. 023） ram, （0. 307 ± 0. 021） mm and （0. 287 - 0. 011 ） ram, respet＇lively. Conclusion [TSN1 -S is involved in regulating the proliferation and migration of glioma cells. The SH3 domains were the crucial fimctional domain of ITSN1-S to regulate the proliferation and migration of glioma cells.