酪氨酸激酶受体B介导的卵巢上皮性癌OVCAR3细胞失巢凋亡抑制与其侵袭的关系

Anoikis-suppression and invasion induced by tyrosine kinase receptor B in OVCAR3 ovarian cancer cells

目的探讨失巢凋亡抑制因子酪氨酸激酶受体B（TrkB）介导的卵巢上皮性癌（卵巢癌）OVCAR3细胞的失巢凋亡抑制与其侵袭的关系。方法采用RT—PCR和实时定量PCR技术检测4种细胞包括3种不同病理类型的卵巢癌细胞系OVCAR3、SKOV3（两者均为卵巢浆液性乳头状囊腺癌细胞系）和ES2（卵巢透明细胞癌细胞系）细胞及神经母细胞瘤细胞系SK—N—DZ细胞中，以及经不同方式[贴壁培养和立体培养（培养后形成团簇细胞）]培养的OVCAR3细胞中TrkBmRNA的表达。转染TrkB小分子干扰RNA（siRNA）后，流式细胞仪检测OVCAR3细胞的凋亡率，以转染无意义的（scrambled）siRNA者为阴性对照；体内、体外侵袭实验检测不同方式培养的OVCAR3细胞的侵袭能力。结果（1）OVCAR3细胞中TrkBmRNA表达水平为0．0240±0．0017，明显高于SKOV3细胞的0．0030±0．0006、ES2细胞的0．0027±0．0009及SK—N-DZ细胞的0．0087±0．0003（P〈0．01）；OVCAR3团簇细胞中TrkBmRNA表达水平为0．04374-0．0021，明显高于贴壁细胞的0．0240±0．0017（P〈0．01）。（2）转染TrkBsiRNA后，OVCAR3细胞的凋亡率为（46．3±5．9）％，明显高于阴性对照的（9．0±0．8）％（P〈0．01）。（3）体外侵袭实验显示，OVCAR3团簇细胞的穿膜细胞数为（71．8-4-0．8）个，明显多于贴壁细胞的（47．7±0．8）个（P〈0．01）；体内侵袭实验显示，转染TrkBsiRNA后，OVCAR3细胞在裸鼠体内形成的肿瘤体积为（6．0-4-1．4）mm3，明显小于阴性对照的（16．3±4．7）mm。（P〈0．01）。结论TrkB可能通过诱导卵巢癌OVCAR3细胞的失巢凋亡抑制而赋予其高侵袭能力。

Objective To study the relationship between tyrosine kinase receptor B （TrkB） expression and anoikis-suppression and invasion in OVCAR3 ovarian cancer cells. Methods The expression of TrkB mRNA in OVCAR3 ovarian cancer cells under two culture conditions ： adhesive cells and cell-spheroids were evaluated by RT-PCR and real-time PCIL The relationship between TrkB expression and anoikis-suppression of OVCAR3 ovarian cancer cells was examined by RNA interference （ RNAi ） technic, anchorage independent culture and fluorescence-activated cell sorting analysis. The difference in invasion and metastatic ability of OVCAR3 cells under two culture conditions and with or without TrkB silenced by small interfering RNA （siRNA） was investigated by matrigel invasion assay and in vivo studies. Results The expression of TrkB mRNA was highest in OVCAR3 ovarian cancer cells, 0.0240 ±0. 0017, compared with the other three cell lines, 0. 0030 ± 0. 0006, 0. 0027 ± 0. 0009 and 0. 0087 ± 0. 0003 respectively, and the expression in OVCAR3 muhicellular spheroids was significantly higher than that in cells under monolayer adhesive culture, 0. 0437 ± 0. 0021 versus 0. 0240 ± 0. 0017 （ P 〈 0.01 ） . TrkB mediated anoikis- suppression in OVCAR3 ovarian cancer cells. OVCAR3 multieellular spheroids had a higher invasion ability than OVCAR3 cells under monolayer adhesive culture, and the penetrating ceils of the two groups were 71.8 ± 0. 8 and 47.7 ± 0. 8 respectively （ P 〈 O. 01 ）. The metastatic ability of OVCAR3 cells was attenuated when TrkB was silenced, and the volume of the tumors developed by OVCAR3 adhesive cells and OVCAR3 adhesive cells with TrkB silenced were （ 16. 3 ± 4.7）mm3 and （6.0 ± 1.4）mm3 respectively （P 〈0. 01 ）. Conduslon As an anoikis-suppressor, TrkB may increase the invasion and metastasis of OVCAR3 ovarian cancer cells.