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摘要

目的观察羟苯磺酸钙（CaD）对斑马鱼胚胎血管新生的抑制作用及其机制。方法选取斑马鱼胚胎180个，随机分为空白组、高糖组（130 mmol/L 葡萄糖）、阳性药+高糖组、高糖+CaD 30 μg/mL 组、高糖+CaD 60 μg/mL 组、高糖+CaD 90 μg/mL 组，其中高糖均为130 mmol/L 葡萄糖，阳性药物为浓度500 ng/mL 内皮细胞生长因子受体酪氨酸激酶抑制剂（VRI）2 mL，CaD 各浓度组给予相应浓度的CaD 2 mL。通过参考致死率实验选用3个给药剂量浓度，分别为1/10 最大非致死浓度（MNLD）（30 μg/mL）、1/5 MNLD（60 μg/mL）、3/10 MNLD（90 μg/mL）作为毒性指标观察，给予药物暴露液2 mL，连续给药72 h，每24 小时更换1次。在体式显微镜下观察并统计斑马鱼胚胎体节间血管指数及镜下荧光点数，荧光定量PCR 检测处理后胚胎血管内皮生长因子（VEGF）、一氧化氮合酶（NOS）表达，采用ELISA法检测VEGF 和NOS含量。结果高糖组的斑马鱼胚胎体节间血管指数、镜下荧光点数及VEGF、NOS mRNA 蛋白表达水平高于空白组，差异有统计学意义（P＜0.05）；阳性药+高糖组及高糖+CaD 90 μg/mL、60 μg/mL 组斑鱼胚胎的体节间血管指数及镜下荧光点数均显著低于高糖组（P＜0.01）。对CaD 给药组进行组间比较，发现随着CaD 给药剂量的增加，斑马鱼胚胎体节间血管指数降低，差异有统计学意义（P＜0.05）。与高糖组相比，药物处理后各组斑马鱼胚胎的荧光点数呈下降趋势，差异有统计学意义（P＜0.05）。阳性药+高糖组及高糖+CaD 90 μg/mL、60 μg/mL 组VEGF、NOS的mRNA表达水平低于高糖组（P＜0.05）。高糖+CaD 30 μg/mL 组VEGF、NOS的mRNA表达水平与高糖组比较，差异无统计学意义（P＞0.05）。阳性药+高糖组及高糖+CaD 90 μg/mL、60 μg/mL组的VEGF、NOS 蛋白表达水平低于高糖组（P＜0.05）。高糖+CaD 30 μg/mL 组的VEGF、NOS 蛋白表达水平与高糖组比较，差异无统计学意义（P＞0.05）。结论 CaD 具有抑制高糖环境斑马鱼胚胎血管新生的作用，与抑制VEGF/i-NOS 有关。

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	唐志宇
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	吴金峡

关键词 ：羟苯磺酸钙, 高糖, 血管新生, 胚胎毒性, 斑马鱼

Abstract：Objective To observe the inhibitory effect of calcium dobesilate (CaD) on zebrafish embryos angiogenesis and its mechanism.Methods A total of 180 zebrafish embryos were selected and randomly divided into the blank group,the high glucose group (130 mmol/L glucose),the positive drug+high glucose group,the high glucose+CaD(30 μg/mL) group,the high glucose+CaD (60 μg/mL) group,and the high glucose+CaD (90 μg/mL) group.Among them,the high glucose concentration was 130 mmol/L,and the positive drug was 2 mL endothelial cell growth factor receptor tyrosine kinase inhibitor (VRI) at a concentration of 500 ng/mL.CaD in 2 mL was provided in each CaD group at different concentrations.Three doses of 1/10 maximum non-lethal dose (MNLD,30 μg/mL),1/5 MNLD (60 μg/mL),and 3/10 MNLD (90 μg/mL) were selected as the toxicity indicators with reference to lethality experiment.Drug exposure solution in 2 mL was given for continuous 72 h,which was changed every 24 h.The zebrafish embryonic intersegment blood vessel indexes and the number of fluorescence points were observed and calculated under stereoscopic microscope.Fluorescence quantitative polymerase chain reaction was used to detect the expression of vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS) in embryos after intervention.The contents of VEGF and NOS were determined by enzyme-linked immuno sorbent assay (ELISA).Results The zebrafish embryonic intersegment blood vessel index,the number of microscopic fluorescence points and the expression levels of VEGF and NOS mRNA in the high glucose group were higher than those in the blank group,and the differences were statistically significant (P＜0.05).The zebrafish embryonic intersegment blood vessel indexes and the number of fluorescence points in the positive drug+high glucose group,the high glucose+CaD (60 μg/mL) group,and the high glucose+CaD (90 μg/mL) group were significantly lower than those in the high glucose group (P＜0.01).The CAD administration groups were compared,the blood vessel indexes of zebrafish embryonic intersegment decreased when the CaD administration dose gradually increased,and the differences were significant (P＜0.05).Compared with the high glucose group,the number of fluorescene points of zebrafish embryos in each group showed a downward trend after drug treatment,and the difference was statistically significant (P＜0.05).The mRNA expression levels of VEGF and NOS in the positive drug+high glucose group,the high glucose+CaD (60 μg/mL)group,and the high glucose+CaD (90 μg/mL) group were lower than those in the high glucose group (P＜0.05).There were no significant differences in the VEGF and NOS mRNA expression between the high glucose+CAD (30 μg/mL)group and high glucose group (P＞0.05).The expression levels of VEGF and NOS in the positive drug+high glucose group,the high glucose+CaD (60 μg/mL) group,and the high glucose+CaD (90 μg/mL) group were lower than those in the high glucose group (P＜0.05).There was no significant difference in the expression of NOS and VEGF between the high glucose+CAD (30 μg/mL) group and high glucose group (P＞0.05).Conclusion CaD can inhibit the angiogenesis of zebrafish embryos in high glucose environment,which is related to the inhibition of VEGF/iNOS.

Key words： Calcium dobesilate High glucose Angiogenesis Embryotoxicity Zebrafish

基金资助:重庆市永川区科技计划项目（自然科学基金）（Ycstc，2016nc5005）

作者简介: 唐志宇，男，医学博士，副主任中医师，硕士研究生导师，主要从事中西医结合肿瘤及呼吸系统疾病临床工作

引用本文:

唐志宇;邓杰强;王猛;左玲;吴金峡.

基于斑马鱼模型羟苯磺酸钙抑制高糖环境血管生成及机制研究

[J]. 中国当代医药, 2021, 28(4): 4-7.
TANG Zhi-yu；DENG Jie-qiang；WANG Meng；ZUO Ling；WU Jin-xia. Inhibition of angiogenesis in high glucose environment by calcium dobesilate based on zebrafish model and its mechanism study. 中国当代医药, 2021, 28(4): 4-7.

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http://dangdaiyiyao.com/CN/ 或 http://dangdaiyiyao.com/CN/Y2021/V28/I4/4

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