基于TGF-β1的抗干眼症药物高通量筛选体系的建立

姚小磊; 彭清华

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基于TGF-β1的抗干眼症药物高通量筛选体系的建立

首发时间：2011-07-13

姚小磊 ¹
姚小磊,（1982-），男，医学博士、主治医师。研究方向：眼表疾病、眼底病。
彭清华 ²
彭清华

1、湖南中医药大学第一附属医院眼科重点学科
2、湖南中医药大学第一附属医院眼科重点学科，长沙410007

摘要：本文建立了基于TGF-β1的抗干眼症药物高通量筛选体系，为干眼症防治药物的研究提供一个理想的技术平台。首先建立了含有SV40弱启动子、4×GCbox基序、不稳定型墨绿色荧光蛋白ZsGreen1-DR基因片段的报告质粒p4GCbox-ZsGreen1DR。将其转染大鼠泪腺上皮细胞（LGEC），G418筛选后获得了Sp-1反应性报告细胞株LGEC-ZsGreen1DR，即基于TGF-β1的抗干眼症药物高通量筛选体系。TGF-β1诱导表达实验结果表明，该体系加入TGF-β1前及加入TGF-β1在0 h时，LGEC-ZsGreen1DR表达荧光的平均光密度为0.008±0.002；在4 h时LGEC-ZsGreen1DR荧光的平均光密度为0.164±0.044；在12 h达到高峰，平均光密度为0.495±0.024；在24h时细胞平均光密度明显下降，平均光密度为0.245±0.055。将LGEC-ZsGreen1DR细胞株冻存30天，复苏并传代培养1个月，加入TGF-β10 h时，LGEC-ZsGreen1DR表达荧光的平均光密度为0.007±0.001；4 h后LGEC-ZsGreen1DR的平均光密度为0.144±0.052；12 h时的平均光密度为0.387±0.084；24h时的平均光密度为0.188±0.034；说明LGEC-ZsGreen1DR细胞株经冻存、复苏、传代后仍能有效反映TGF-β1对Sp-1的诱导激活作用。

关键词：中医眼科学 TGF-β1 Sp-1 GCbox基序干眼症不稳定型墨绿色荧光蛋白

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Establishment of the High Throughput Screening System for Antagonistic Agents of Dry Eye Based on TGF-β1

Yao Xiaolei ¹
姚小磊,（1982-），男，医学博士、主治医师。研究方向：眼表疾病、眼底病。
peng qinghua ²
彭清华

1、Key discipline of ophthalmology，the first affiliated hospital of Hunan University of Traditional Chinese Medicine
2、Key discipline of ophthalmology，the first affiliated hospital of Hunan University of Traditional Chinese Medicine，Changsha Hunan China，410007

Abstract：IThrough the establishment of the high throughput screening system for antagonistic Herbs of dry eye based on TGF-β1. It provided an ideal technology platform of study for antagonistic herbs of dry dye. First, we established a vector which contained a weak promoter SV40, a oligodeoxynucleotide of GCbox motif and ZsGreen1DR motif. The plasmid p4GCbox-ZsGreen1DR was transfected into the rat lacrimal gland epithelial cells (LGEC). After selected with G418, reporter cell line which was based on TGF-β1 was gained and named LGEC-ZsGreen1DR. The expression of ZsGreen1DR induced by TGF-β1 was detected. Before and right after the addition of TGF-β1,the average optical density of fluorescence that LGEC-ZsGreen1DR expressed was 0.008±0.002 ; after 4 hours of the addition of TGF-β1 , the average optical density of fluorescence that LGEC-ZsGreen1DR expressed was 0.164±0.044;peak after 12 hours of the addition of TGF-β1, the average optical density of fluorescence that LGEC-ZsGreen1DR expressed was 0.495±0.024; At 24-hour time point, the average optical density of fluorescence that LGEC-ZsGreen1DR expressed reduced obviously and was 0.245±0.055. LGEC-ZsGreen1DR cell line which had been resuscitation culture and subculture for one month after 30 days of cryopreservation were stimulated by TGF-β1 to evaluate the stability of LGEC-ZsGreen1DR cell line. The average optical density of fluorescence that LGEC-ZsGreen1DR expressed was 0.007±0.001；The average optical density of fluorescence that LGEC-ZsGreen1DR expressed was 0.144±0.052 after 4 hours; The average optical density was 0.387±0.084 after 12 hours;The average optical density was 0.188±0.034 after 24 hours; These results indicate that LGEC-ZsGreen1DR cell line would still be effective after resuscitation culture、subculture and cryopreservation ,and TGF-β1 on the Sp-1 could induce activation.

Keywords： ophthalmology of Traditional Chinese Medicine TGF-β1 Sp-1 GCbox motif Dry eye ZsGreen1-DR

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姚小磊，彭清华. 基于TGF-β1的抗干眼症药物高通量筛选体系的建立[EB/OL]. 北京：中国科技论文在线 [2011-07-13]. https://www.paper.edu.cn/releasepaper/content/201107-199.

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论文编号	201107-199
论文题目	基于TGF-β1的抗干眼症药物高通量筛选体系的建立
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