抑制NRP2表达对淋巴管内皮细胞小管成型的影响

Effects of inhibiting the neuropilins-2 expression on the tubular formation of lymphatic tube endothelial cells

目的探讨抑制神经纤毛蛋白质2(NRP2)基因表达对人淋巴管内皮细胞(LECs)体外形成小管能力的影响。方法从人新鲜包皮中分离纯化LECs,并进行鉴定。实验设NRP2-RNAi/LECs组、mock/LECs组和hECs组,三组分别转染pGensil-NRP2(携带NRP2 siRNA的真核表达载体)、pGensil-1(空载体)及正常LECs细胞。采用RT-PCR和Western blotting检测NRP2 mRNA和蛋白的表达;绘制细胞生长曲线以观察各组细胞生长的差异。对细胞进行三维胶培养,计数小管样结构的数量,并测量小管外径、内径和管壁厚度。结果与hECs组和mock/LECs组比较,NRP2-RNAi/LECs组NRP2表达水平明显下调(P<0.05),而前两组间无显著差异(P>0.05)。生长曲线显示三组细胞的增殖能力无显著差异,均于接种后3～5d进入对数生长期,6～7d进入停滞期。三维胶培养形成的管样结构在NRP2-RNAi/LECs组很少见,其管样结构的数量及小管外径、内径、管壁厚度都明显低于mock/LECs组和hECs组(P<0.05),而后两组间无显著差异(P>0.05)。结论抑制NRP2的表达可抑制LECs在体外形成小管的能力,并可能抑制肿瘤淋巴管的生成。

Objective To explore the role and influence of inhibiting the neuropilins-2 （NRP2） expression on the tubular formation of human lymphatic tube endothelial cells （LECs） in vitro. Methods Human LECs were isolated and purified from freshly excised human foreskins,and they were identified. The NRP2-RNAi/LECs group and mock/LECs group were respectively transfected with pGensil-NRP2 （a plasmid combined with NRP2 siRNA） and pGensil-1 （empty plasmid）,and the hECs group consisting of normal LECs. RT-PCR and Western blotting were applied to detect the mRNA and protein expressions of NRP2. The difference in cell proliferation was reflected in cell growth curves. LECs were cultured with three-dimensional cultivation method,and the quantity of tubular structures,external and internal diameters of tubes,and thickness of tube wall were calculated. Results The NRP2 expression significantly declined in NRP2-RNAi/LECs group compared with that of both hECs group and mock/LECs group （P0.05）,while no significant difference was found between the two latter groups （P0.05）. The growth curves showed no difference in proliferation among the three groups,the cells achieved the exponential phase within 3-5 days and a lag phase within 6-7 days. Lymphatic tubular structure was seldom observed,but only budding was sometimes found in the NRP2-RNAi/LECs group,in which the number of tubal structure,external and internal diameters of tubes,and thickness of tube wall were less and lower than that in the mock/LECs group and hECs group （P0.05）,while no significant difference on those parameters was found between the two latter groups （P0.05）. Conclusion Down-regulation of NRP2 expression may suppress the lymphatic tube formation in vitro,thereafter inhibit the formation of lymphatic vessels in tumor.