FIA检测AGEs和CAM整体模型实验方法的建立及其防治糖尿病血管并发症中药筛选
|
摘要
第一部分 FIA检测AGEs方法的建立和应用
一 FIA检测AGEs方法的建立
目的 建立糖基化终未产物(advanced glycation end products，AGEs)的一种检测方法。方法 利用高效液相色谱仪(high performance liquid chromatography,HPLC)构建流动注射分析系统，检测体外制备的AGEs，并与普通荧光法等进行比较。荧光检测的激发波长和发射波长(Ex/Em)为370/440nm和335/385nm。结果 Ex/Em为370/440nm时，在原反应液的0.1-0.0001浓度范围内仍有良好的线性，Y=67979.98x+4.38，r=0.99999；重复性CV为1.11％；日内、日间CV分别为0.94％和2.51％；平均回收率98.5％；与其它检测AGEs的方法相比，其相互之间测定的数据均有较好的相关性，结果趋势基本一致。Ex/Em 335/385nm与370/440nm结果一致。结论 流动注射分析(Flow injection assay,FIA)是较可靠的检测AGEs水平的新方法。
二 体外蛋白质非酶糖基化的建立
目的 建立可用于药物筛选的体外蛋白质非酶糖基化模型。方法 1、葡萄糖与牛血清白蛋白组成反应系统。37℃孵育1、2周后与NBT反应，用流动注射法于530nm处测定早期糖化蛋白(果糖胺)；2、甘油醛与牛血清白蛋白组成反应系统。37℃孵育2周后用流动注射法于Ex/Em：370/440，335/385nm处测定糖基化终产物；3、3-脱氧葡萄糖醛酮与牛血清白蛋白组成反应系统。37℃孵育1周后用流动注射法于Ex/Em：370/440，335/385nm处测定糖基化终产物。结果 1、反应1和2周后，反应系统内葡萄糖浓度与果糖胺的吸收度值呈显著正相关，分别为：
Part 1
Establishment of FIA employed as an analyzing approach for AGEs
1 Establishment of an analyzing approach for advanced glycation end products formed in vitro by flow injection assay
ABSTRACT Objective To establish an analyzing approach for advanced glycation end products formed in vitro. Methods Flow injection system was assembled using HPLC to detect AGEs formed in vitro. Other analyzing approaches were employed as controls. Fluorescence of each sample was determined at excitation/emission wavelengths of 370/440 nm as well as 335/385 nm. Results There was a good liner between AGEs concentration (diluted to 0.1-0.0001) and peak area (obtained at 440nm with excitation at 370nm). The equation was y=67978.98x+4.38, 1=0.99999. The coefficient of variance of reproducibility, within-day and between-day assayed by flow injection assay for AGEs was 1.11%, 0.94% and 2.51% respectively. The average recovery rate was 98.5%. There were correlations between flow injection assay and other measures both in data and the results. The data, as well as the results, detected by flow injection was correlated with those detected by other measures. The same results were observed at 335/385nm. Conclusion AEGs
detected by flow injection assay shows excellent accuracy and reproducibility. It is a novel method for monitoring AGEs formed in vitro.
一 FIA检测AGEs方法的建立
目的 建立糖基化终未产物(advanced glycation end products，AGEs)的一种检测方法。方法 利用高效液相色谱仪(high performance liquid chromatography,HPLC)构建流动注射分析系统，检测体外制备的AGEs，并与普通荧光法等进行比较。荧光检测的激发波长和发射波长(Ex/Em)为370/440nm和335/385nm。结果 Ex/Em为370/440nm时，在原反应液的0.1-0.0001浓度范围内仍有良好的线性，Y=67979.98x+4.38，r=0.99999；重复性CV为1.11％；日内、日间CV分别为0.94％和2.51％；平均回收率98.5％；与其它检测AGEs的方法相比，其相互之间测定的数据均有较好的相关性，结果趋势基本一致。Ex/Em 335/385nm与370/440nm结果一致。结论 流动注射分析(Flow injection assay,FIA)是较可靠的检测AGEs水平的新方法。
二 体外蛋白质非酶糖基化的建立
目的 建立可用于药物筛选的体外蛋白质非酶糖基化模型。方法 1、葡萄糖与牛血清白蛋白组成反应系统。37℃孵育1、2周后与NBT反应，用流动注射法于530nm处测定早期糖化蛋白(果糖胺)；2、甘油醛与牛血清白蛋白组成反应系统。37℃孵育2周后用流动注射法于Ex/Em：370/440，335/385nm处测定糖基化终产物；3、3-脱氧葡萄糖醛酮与牛血清白蛋白组成反应系统。37℃孵育1周后用流动注射法于Ex/Em：370/440，335/385nm处测定糖基化终产物。结果 1、反应1和2周后，反应系统内葡萄糖浓度与果糖胺的吸收度值呈显著正相关，分别为：
Part 1
Establishment of FIA employed as an analyzing approach for AGEs
1 Establishment of an analyzing approach for advanced glycation end products formed in vitro by flow injection assay
ABSTRACT Objective To establish an analyzing approach for advanced glycation end products formed in vitro. Methods Flow injection system was assembled using HPLC to detect AGEs formed in vitro. Other analyzing approaches were employed as controls. Fluorescence of each sample was determined at excitation/emission wavelengths of 370/440 nm as well as 335/385 nm. Results There was a good liner between AGEs concentration (diluted to 0.1-0.0001) and peak area (obtained at 440nm with excitation at 370nm). The equation was y=67978.98x+4.38, 1=0.99999. The coefficient of variance of reproducibility, within-day and between-day assayed by flow injection assay for AGEs was 1.11%, 0.94% and 2.51% respectively. The average recovery rate was 98.5%. There were correlations between flow injection assay and other measures both in data and the results. The data, as well as the results, detected by flow injection was correlated with those detected by other measures. The same results were observed at 335/385nm. Conclusion AEGs
detected by flow injection assay shows excellent accuracy and reproducibility. It is a novel method for monitoring AGEs formed in vitro.
引文
01 Glaser BM, D Amare, Michels RG et al, Demonstration of vasoproliferative activity from mammalian retina. J. Cell Biol 1980; 84:298．
02 Glaser BM, Campochiaro PA, Davis JL, Sato M. Retinal pigment epithelial cell release an inhibitor of neovascularization. Arch Ophthalmol 1985; 103: 1870-1875．
03 Aiello LP, Wong JS. Role of vascular endothelial growth factor in diabetic vascular complications. Kidney Int Suppl 2000 Sep. 77:S113-9．
04 Okamota T, Yamogishis, Inagaki Y, Amanos, Koga K, Aba R, Takenki M, Ohno s, Yoshimura A, Makitaz. Angiogenesis induced by advanced glycation end products and its prevention by cerivastatin. FASEB J, 2002 Dec 16(14) , 1928-30, Epub 2002 Oct 04．
02 Glaser BM, Campochiaro PA, Davis JL, Sato M. Retinal pigment epithelial cell release an inhibitor of neovascularization. Arch Ophthalmol 1985; 103: 1870-1875．
03 Aiello LP, Wong JS. Role of vascular endothelial growth factor in diabetic vascular complications. Kidney Int Suppl 2000 Sep. 77:S113-9．
04 Okamota T, Yamogishis, Inagaki Y, Amanos, Koga K, Aba R, Takenki M, Ohno s, Yoshimura A, Makitaz. Angiogenesis induced by advanced glycation end products and its prevention by cerivastatin. FASEB J, 2002 Dec 16(14) , 1928-30, Epub 2002 Oct 04．