多西环素抑制视网膜母细胞瘤血管生成拟态的体外研究及组织学观察

vitro Inhibitory effect of doxycycline on vasculogenic mimicry in retinoblastoma cell line in

目的研究多西环素对人视网膜母细胞瘤（RB）细胞体外血管生成拟态（VM）和细胞增生的抑制效应。方法建立人RB细胞株三维培养系统，缺氧诱导后观察RB细胞能否形成血管网状结构及其特点；用5～20mg／L不同浓度多西环素处理RB细胞，过碘酸雪夫氏（PAS）染色观察对RB细胞体外血管生成拟态的抑制效应；噻唑蓝（MTT）比色法检测多西环素对RB细胞增生活性的变化；逆转录聚合酶链式反应（RT-PCR）技术检测不同条件下基质金属蛋白酶2（MMP-2）、基质金属蛋白酶9（MMP-9）mRNA的相对表达量；收集12例RB石蜡包埋样本进行CD34／PAS免疫组织化学双重染色，观察RB中的血管生成拟态。结果RB细胞在三维培养系统形成管网状的拟态血管；5～20mg／L多西环素能抑制RB细胞体外管状结构数量。MTT检测显示多西环素加药组3、5、7d的吸光度A[旧称光密度（OD）]值与对照组比较差异均有统计学意义（t=15．320，P〈0．01），且不同浓度的多西环素组之间细胞的增生抑制率差异亦有统计学意义（F=9．442，11．729，12．120；P〈0．05），其浓度与细胞的增生呈负相关（r=-0．924，P〈0．01），氯化钻缺氧组MMP-2、MMP-9的mRNA表达量明显增强，各浓度多西环素加药组显著低于氯化钴缺氧组（t=16．469，P〈0．01）。在12例RB组织标本中可见CD34阴性PAS阳性的瘤细胞围成管道样结构，部分管腔中可见红细胞。结论多西环素能够抑制RB体外血管生成拟态形成和细胞的增生，其机制可能是通过抑制MMP-2、MMP-9mRNA的表达而实现。

Objective To investigate the effect of doxycycline on the proliferation and vasculogenic mimicry in retinobiastoma （RB） cell line in vitro. Methods RB cell line were tested for their ability to form perfusable tubular networks in 3-D culture with doxycycline in the concentrations ranging from 5 to 20 rag/L, and COC12 was used as chemical hypoxia-inducing reagent to mimic tumor hypoxic microenvironment. The effect of doxycycline on proliferation were detected by MTT assay in vitro, and the effect on tube formation of RB cells were detected by tube-like structure formation assay and PAS staining. The mRNA levels of MMP-2 and MMP-9 at different hypoxic culture and different doxycycline concentrations were detected by semi-quantitative reverse transcription polymerase chain reaction （RT-PCR）. Results The micrograph showed that RB cells linked each other to form cavity and network tructure in 3-D culture, the number of tubules in doxycycline group were significantly lower than which in the control group in the concentrations ranging from 5 to 20 mg/L （P〈0. 001 ）. OD of doxycycline group was significantly lower than which in the control group （t=15. 320, P〈0.01） , The proliferation of RB cells had a negative correlation with the concentration of doxycycline （r =-0. 924, P〈0.01）. The levels of MMP-2 and MMP-9 mRNA of RB cells under hypoxia were significantly higher than which in the control group （t = 16. 469,P〈0.01）. As the concentration of doxycycline increased, the expression of MMP-2 and MMP-9 decreased. The result of double staining also showed that VM, formed by CD34-negative and PAS-positive tumor cells, existed in 12 simples of retinoblastoma. Conclusion RB cells have the capacity of self- metamorphosing and vasculorizing in 3-D culture. Doxycycline can inhibit their proliferation and vasculogenic mimicry formation in vitro by down-regulating the expression of MMP-2 and MMP-9 .