CAJ | 학술논문

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5～20 mg/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;5～20 mg/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、7 d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),氯化钴缺氧组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-9 mRNA的表达而实现.
목적 연구다서배소대인시망막모세포류(RB)세포체외혈관생성의태(VM)화세포증생적억제효응. 방법건립인RB세포주삼유배양계통,결양유도후관찰RB세포능부형성혈관망상결구급기특점;용5～20 mg/L불동농도다서배소처리RB세포,과전산설부씨(PAS)염색관찰대RB세포체외혈관생성의태적억제효응;새서람(MTT)비색법검측다서배소대RB세포증생활성적변화;역전록취합매련식반응(RT-PCR)기술검측불동조건하기질금속단백매2(MMP-2)、기질금속단백매9(MMP-9)mRNA적상대표체량;수집12례RB석사포매양본진행CD34/PAS면역조직화학쌍중염색,관찰RB중적혈관생성의태. 결과 RB세포재삼유배양계통형성관망상적의태혈관;5～20 mg/L다서배소능억제RB세포체외관상결구수량.MTT검측현시다서배소가약조3、5、7 d적흡광도A[구칭광밀도(OD)]치여대조조비교차이균유통계학의의(t=15.320,P<0.01),차불동농도적다서배소조지간세포적증생억제솔차이역유통계학의의(F=9.442,11.729,12.120;P<0.05),기농도여세포적증생정부상관(r=-0.924,P<0.01),록화고결양조MMP-2、MMP-9적mRNA표체량명현증강,각농도다서배소가약조현저저우록화고결양조(t=16.469,P<0.01).재12례RB조직표본중가견CD34음성PAS양성적류세포위성관도양결구,부분관강중가견홍세포. 결론 다서배소능구억제RB체외혈관생성의태형성화세포적증생,기궤제가능시통과억제MMP-2、MMP-9 mRNA적표체이실현.
Objective To investigate the effect of doxycycline on the proliferation and vasculogenic mimicry in retinoblastoma (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 mg/L, and CoCl2 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 doxyeycline 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. Doxyeycline can inhibit their proliferation and vasculogenic mimicry formation in vitro by down-regulating the expression of MMP-2 and MMP-9.