角蛋白在血管生成中的作用实验研究

AN EXPERIMENTAL STUDY ON THE ROLE OF KERATIN IN ANGIOGENESIS IN VITRO

目的研究角蛋白17(keratin17,K-17)对血管内皮细胞迁移、增殖和管样结构形成的影响,了解K-17在血管生成过程中的作用。方法将人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)于含10%FBS的DMEM培养液培养过夜,采用脂质体转染法向HUVEC中分别加入K-17-小分子干扰RNA(small interfer-ingRNA,siRNA)-转染试剂(Lipofectamine2000)混合液(实验组)、siRNA-转染试剂混合液(阴性对照组),使siRNA终浓度为50nmol/L;对照组加相同体积仅含载体(脂质体Lipofectamine2000)的培养基,采用RT-PCR和Westernblot法检测K-17-siRNA沉默效果。于培养后36h采用细胞计数法检测细胞增殖能力;培养后30h,分别采用24孔板Millicell小室检测细胞迁移能力以及胶原纤维凝胶实验法检测细胞分化成管能力。另取未经siRNA处理的HUVEC分别在含10%FBS的培养基(A组)、含2%FBS的培养基(B组)和含2%FBS及10ng/mLbFGF的培养基(C组)中培养24h,检测HUVECK-17的表达。结果实验组K-17-siRNA作用于HUVEC30h后,RT-PCR检测示细胞内K-17mRNA的表达为0.09±0.01,较阴性对照组0.35±0.07和对照组0.34±0.06均降低了约74%(P<0.01);Westernblot检测示实验组K-17-siRNA作用于HUVEC30h后,细胞内K-17蛋白表达为0.19±0.01,较阴性对照组0.52±0.01和对照组0.55±0.02分别降低了63%和65%(P<0.01);阴性对照组和对照组间比较差异均无统计学意义(P>0.05)。K-17-siRNA作用HUVEC36h后,实验组细胞增殖能力与阴性对照组和对照组比较差异均无统计学意义(P>0.05)。HUVEC转染K-17-siRNA30h后,实验组HUVEC24h穿过Millicell小室上室膜进入下室的细胞数为(3719.0±319.0)个,明显低于阴性对照组(7356.3±795.7)个和对照组(7437.5±212.0)个,差异有统计学意义(P<0.01),阴性对照组和对照组间差异无统计学意义(P>0.05)。实验组、阴性对照组和对照组HUVEC24h分化形成的管数分别为(1.1±0.5)、(3.6±0.5)和(3.2±0.6)管/视野,实验组与阴性对照组、对照组间比较差异均有统计学意义(P<0.01),阴性对照组和对照组间比较差异无统计学意义(P>0.05)。A、B、C组HUVECK-17的表达分别为0.25±0.02、0.08±0.01和0.72±0.03,3组间比较差异均有统计学意义(P<0.01)。结论K-17对HUVEC增殖无影响,但增强其迁移能力,有利于血管生成。

Objective To investigate the effect of keratin 17 （K-17） on the migration, proliferation and tube formation of human umbilical vein endothelial cell （HUVEC）, and to realize the role of K-17 in angiogenesis. Methods After HUVEC were cultured in DMEM medium supplemented with 10% FBS overnight, K-17-siRNA-mixture （experimental group） and Ncontrol-siRNA-mixture （negative control group） were added into HUVEC, respectively, by Lipofectamine 2000 transfection assay, and the final concentration of the siRNA was 50 nmol/L. Lipofectamine 2000 alone was used as the control. After the cells were cultured for 36 hours, the cell proliferation ability was detected by cell counting. After 30-hour culture, the cell＇s abilities of migration and differentiation to tube were detected by 24-well Millicell units and the collagen gel assay, respectively. In addition, non-siRNA-treated HUVEC were cultured for 24 hours in DMEM medium supplemented with 10% FBS （group A）, 2%FBS （group B） and 2%FBS＋10 ng/mL bFGF （group C）, respectively, and then the expression of K-17 in HUVEC was detected by RT-PCR and Western blot. Results After the treatment with K-17-siRNA for 36 hours, HUVEC exhibited no significant difference in the proliferation, compared with both control and negative control groups （P 〉 0.05）. After transfected with K-17-siRNA for 30 hours, the number of HUVEC in the experimental group which migrated from the upper chamber to the lower chamber of Millicell wells within 24 hours （3719.0 ± 319.0） was smaller than both control （7 437.5 ± 212.0） and negative control （7 356.3 ± 795.7） groups, with significant difference （P 〈 0.01）. However, there was no significant difference between the control group and the negative control group （P 〉 0.05）. After HUVEC were transfected with K-17-siRNA for 30 hours, the number of tubes in the experimental group, the negative control group and the control group in 24 hours was （1.1±0.5）, （3.6± 0.5） and （3.2±0.6） per field, respectively. The experimental group was significantly different from both control and negative control groups （P 〈 0.01）, and there was no significant difference between the negative control group and the control group （P 〉 0.05）. The expression of K-17 protein in HUVEC in groups A, B and C was 0.25±0.02, 0.08 ± 0.01 and 0.72 ± 0.03, respectively. There was significant difference among these three groups （P 〈 0.01）. Conclusion K-17 has no impact on cell proliferation, but may augment endothelial cell migration, which may facilitate angiogenesis.