加减大黄蛰虫丸抑制视网膜色素上皮细胞增殖、移行及其机制研究

An experimental study on the proliferation and migration of retinal pigment epithelial cells inhibited by Adjusted Dahuang Zhechong Pill and its mechanism

目的观察加减大黄蛰虫丸对人视网膜色素上皮(RPE)细胞(CRL-2302)增殖、移行以及表达血管内皮生长因子(VEGF)和基质金属蛋白酶2(MMP-2)的影响,了解其干预RPE细胞参与脉络膜新生血管(CNV)形成的途径及初步机制。方法MTS比色法观察加减大黄蛰虫丸对VEGF诱导的CRL-2302细胞增殖的影响;Transwell小室检测加减大黄蛰虫丸对CRL-2302细胞移行的影响;WesternBlot和实时定量逆转录聚合酶链反应(RT-PCR)的方法,分别检测加减大黄蛰虫丸对CRL-2302细胞表达VEGF、基质金属蛋白酶2(MMP-2)蛋白和mRNA的影响。结果MTS法显示,0.05、0.1和0.2g/ml浓度的加减大黄蛰虫丸对VEGF诱导的CRL-2302细胞增殖具有抑制作用;Transwell小室实验显示,加减大黄蛰虫丸浓度为0、12.5、25和50mg/ml时CRL-2302细胞移行数分别为198.33±19.86、121.67±14.05、62.33±11.24和18.67±4.51;50、100mg/ml浓度的加减大黄蛰虫丸具有抑制CRL-2302细胞VEGF、MMP-2蛋白和mRNA表达的作用,200mg/ml浓度的加减大黄蛰虫丸具有抑制CRL-2302细胞VEGF、MMP-2蛋白和MMP-2mRNA表达的作用。结论加减大黄蛰虫丸可能通过抑制CRL-2302细胞增殖、移行的途径抑制其参与CNV形成,其机理可能与抑制CRL-2302细胞VEGF、MMP-2的表达有关。

OBJECTIVE To observe the effect of Adjusted Dahuang Zhechong Pill on proliferation, migration, and expression of vascular endothelial growth factor （VEGF） and matrix metalloproteinase 2 （MMP-2） of human retinal pigment epithelial cell line （CRL-2302） cells in vitro in order to understand the action pathway and mechanism of Adjusted Dahuang Zhechong Pill interfering on choroidal noevascularization （CNV）. METHODS MTS method was used to access the effect of Adjusted Dahuang Zhechong Pill on the proliferation of CRL-2302 cells induced by VEGF. Transwell insert was used to access the effect of Adjusted Dahuang Zheehong Pill on the migration of CRL-2302 cells. Western Blot and real-time quantita- tive reverse transcription polymerase chain reaction （RT-PCR） were used to detect Adjusted Dahuang-Zheehong Pill on the expression of VEGF and matrix metalloproteinase 2 （MMP-2） protein and mRNA of CRL-2302 cells. RESULTS The proliferation of CRL-2302 cells induced by VEGF was inhibited by Adjusted Dahuang Zhechong Pill at concentrations of 0.05, 0.1, and 0.2 mg/ml. When the concentration of Adjusted Dahuang-Zhechong Pill was 0, 12.5, 25, and 50 mg/ml,cells number that migrated to under the Transwell membrane was 198.33±19.86, 121.67±14.05, 62.33±11.24, and 18.67±4.51 respectively. At con-centrations of 50 and 100 mg/ml, Adjusted Dahuang Zhechong Pill could inhibit the expression of VEGF and MMP-2 protein and mRNA of CRL- 2302 cells. At concentrations of 200 mg/ml, Ad-justed Dahuang Zhechong Pill could inhibit the protein expression of MMP-2 and VEGF and mRNA expression of MMP-2 of CRL-2302 cells. CONLUSlONS Adjusted Dahuang Zhechong Pill could inhibit CNV concerned with CRL-2302 cells through the path of suppression of proliferation, migration of CRL- 2302 ceils. The inhibition of the expression of VEGF and MMP-2 protein and mRNA of CRL-2302 cells may be one of the important mechanisms.