中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2012年
35期
2509-2512
,共4页
陶绍能%何晓东%沈佐君%董林
陶紹能%何曉東%瀋佐君%董林
도소능%하효동%침좌군%동림
甲氨蝶呤%对映体%抗药性,肿瘤%肿瘤侵袭
甲氨蝶呤%對映體%抗藥性,腫瘤%腫瘤侵襲
갑안접령%대영체%항약성,종류%종류침습
Methotrexate%Enantiomer%Drug resistance,neoplasm%Neoplasm invasion
目的 探讨甲氨蝶呤(MTX)对映体耐药A549细胞的体外运动能力、浸润侵袭能力差异,从而说明MTX对映体在肿瘤细胞耐药后的侵袭转移过程中的差异.方法 采用浓度递增结合低剂量持续诱导,获得耐受15 μmol/L MTX对映体的细胞系,分别命名为L-(+)-MTX/A549、D-(-)-MTX/A549；用MTT法测定其耐药指数；通过平板划痕实验反映D-(-)-MTX/A549和L-(+)-MTX/A549细胞移动能力；采用双层软琼脂克隆形成实验检测D-(-)-MTX/A549组、L-(+)-MTX/A549组细胞的克隆形成率和集落形成的大小；应用transwell小室实验检测3组细胞体外侵袭能力.结果 L-(+)-MTX/A549、D-(-)-MTX/A549耐药指数分别为(6.1±1.0)和(20.3±1.8).平板划痕实验72 h后L-(+)-MTX/A549细胞进入划痕区的数量少于D-(-)-MTX/A549细胞；软琼脂实验发现D-(-)-MTX/A549组、L-(+)-MTX/A549组以及亲本细胞的集落数分别为(50±7)个克隆、(44±6)个克隆、(52±7)个克隆以及集落形成率分别为(1.68％±0.23％)、(1.49％±0.18％)、(1.73％±0.23％),3组之间差异无统计学意义(P＞0.05),但D-(-)-MTX/A549组细胞的集落形状大小明显大于L-(+)-MTX/A549细胞；transwell小室检测D-(-)-MTX/A549、L-(+)-MTX/A549以及亲本细胞的体外浸润侵袭穿过人工基底膜Matrigel的细胞数分别为(267 ±30)、(106±16)、(134±16)；3组实验的数据均显示D-(-)-MTX/A549组和L-(+)-MTX/A549组以及亲本细胞组差异有统计学意义(P＜0.05),而L-(+)-MTX/A549组与亲本细胞组差异无统计学意义(P＞0.05).结论 D-(-)-MTX诱导的非小细胞肺癌A549耐药细胞在体外运动、浸润侵袭力均大于L-(+)-MTX耐药细胞,从而说明MTX对映体耐药细胞后细胞的转移力有差异.
目的 探討甲氨蝶呤(MTX)對映體耐藥A549細胞的體外運動能力、浸潤侵襲能力差異,從而說明MTX對映體在腫瘤細胞耐藥後的侵襲轉移過程中的差異.方法 採用濃度遞增結閤低劑量持續誘導,穫得耐受15 μmol/L MTX對映體的細胞繫,分彆命名為L-(+)-MTX/A549、D-(-)-MTX/A549；用MTT法測定其耐藥指數；通過平闆劃痕實驗反映D-(-)-MTX/A549和L-(+)-MTX/A549細胞移動能力；採用雙層軟瓊脂剋隆形成實驗檢測D-(-)-MTX/A549組、L-(+)-MTX/A549組細胞的剋隆形成率和集落形成的大小；應用transwell小室實驗檢測3組細胞體外侵襲能力.結果 L-(+)-MTX/A549、D-(-)-MTX/A549耐藥指數分彆為(6.1±1.0)和(20.3±1.8).平闆劃痕實驗72 h後L-(+)-MTX/A549細胞進入劃痕區的數量少于D-(-)-MTX/A549細胞；軟瓊脂實驗髮現D-(-)-MTX/A549組、L-(+)-MTX/A549組以及親本細胞的集落數分彆為(50±7)箇剋隆、(44±6)箇剋隆、(52±7)箇剋隆以及集落形成率分彆為(1.68％±0.23％)、(1.49％±0.18％)、(1.73％±0.23％),3組之間差異無統計學意義(P＞0.05),但D-(-)-MTX/A549組細胞的集落形狀大小明顯大于L-(+)-MTX/A549細胞；transwell小室檢測D-(-)-MTX/A549、L-(+)-MTX/A549以及親本細胞的體外浸潤侵襲穿過人工基底膜Matrigel的細胞數分彆為(267 ±30)、(106±16)、(134±16)；3組實驗的數據均顯示D-(-)-MTX/A549組和L-(+)-MTX/A549組以及親本細胞組差異有統計學意義(P＜0.05),而L-(+)-MTX/A549組與親本細胞組差異無統計學意義(P＞0.05).結論 D-(-)-MTX誘導的非小細胞肺癌A549耐藥細胞在體外運動、浸潤侵襲力均大于L-(+)-MTX耐藥細胞,從而說明MTX對映體耐藥細胞後細胞的轉移力有差異.
목적 탐토갑안접령(MTX)대영체내약A549세포적체외운동능력、침윤침습능력차이,종이설명MTX대영체재종류세포내약후적침습전이과정중적차이.방법 채용농도체증결합저제량지속유도,획득내수15 μmol/L MTX대영체적세포계,분별명명위L-(+)-MTX/A549、D-(-)-MTX/A549；용MTT법측정기내약지수；통과평판화흔실험반영D-(-)-MTX/A549화L-(+)-MTX/A549세포이동능력；채용쌍층연경지극륭형성실험검측D-(-)-MTX/A549조、L-(+)-MTX/A549조세포적극륭형성솔화집락형성적대소；응용transwell소실실험검측3조세포체외침습능력.결과 L-(+)-MTX/A549、D-(-)-MTX/A549내약지수분별위(6.1±1.0)화(20.3±1.8).평판화흔실험72 h후L-(+)-MTX/A549세포진입화흔구적수량소우D-(-)-MTX/A549세포；연경지실험발현D-(-)-MTX/A549조、L-(+)-MTX/A549조이급친본세포적집락수분별위(50±7)개극륭、(44±6)개극륭、(52±7)개극륭이급집락형성솔분별위(1.68％±0.23％)、(1.49％±0.18％)、(1.73％±0.23％),3조지간차이무통계학의의(P＞0.05),단D-(-)-MTX/A549조세포적집락형상대소명현대우L-(+)-MTX/A549세포；transwell소실검측D-(-)-MTX/A549、L-(+)-MTX/A549이급친본세포적체외침윤침습천과인공기저막Matrigel적세포수분별위(267 ±30)、(106±16)、(134±16)；3조실험적수거균현시D-(-)-MTX/A549조화L-(+)-MTX/A549조이급친본세포조차이유통계학의의(P＜0.05),이L-(+)-MTX/A549조여친본세포조차이무통계학의의(P＞0.05).결론 D-(-)-MTX유도적비소세포폐암A549내약세포재체외운동、침윤침습력균대우L-(+)-MTX내약세포,종이설명MTX대영체내약세포후세포적전이력유차이.
Objective To study the differential in vitro motor and invasion capacities of methotrexate (MTX) enantiomer-resistant tumor cells.Methods The incremental concentrations and successive low-dose induction were employed to acquire the cell series resistant to 15 μmol/L MTX enantiomer,namely L-( + )-MTX/A549 and D-(-)-MTX/A549.Their drug-resistant indices were determined by MTT assay and their migration capacities by wound healing assay.Double soft-agar clone formation was used to detect the colony efficiency and size.And Transwell was employed to detect the in vitro movement and invasion capacity of three cell types.Results The resistance indices of L-( + )-MTX/A549 and D-(-)-MTX/A549 were (6.1 ± 1.0)and (20.3 ± 1.8) respectively.At 72 hours after wound healing assay,the number of L-( + )-MTX/A549 entering scratch zone was fewer than that of D-(-)-MTX/A549 ;The numbers of colony formation in D-(-)-MTX/A549,L-( + )-MTX/A549 and parental cells were (50 ±7),(44 ±6),(52 ±7)and the rates of colony formation(1.68％ ±0.23％ ),(1.49％ ±0.18％ ),(1.73％ ± 0.23％ )respectively.And there was no significant significance among three groups (P ＞ 0.05 ).But the size of D-(-)-MTX/A549 was larger than that of L-( + )-MTX/A549.Transwell detected infiltration and invasion through artificial basement membrane Matrigel.The numbers of D-(-)-MTX/A549,L-( + )-MTX/A549 and parent cells were (267 ± 30),(106 ± 16) and (134 ± 16)respectively.The data were significant between D-(-)-MTX/A549 cells and L-( + )-MTX/A549 or parent cells (P ＜0.05) but not significant between L-( + )-MTX/A549 and parent cells (P ＞0.05 ).Conclusion The D-(-)-MTX-induced NSCLC A549 cells have greater motor and invasion capacities than those of L-( + )-MTX-induced ones.It suggests that MTX enantiomer has different capacities of tumor invasion and metastasis after acquiring resistance.
