基于TGF-β1/Sp-1建立抗干眼的中药高通量筛选体系及其在密蒙花中的应用

Establishment of a high-throughput screening system for Chinese medicine against dry eye based on TGF-β1 and its application in Buddleja officinalis

目的建立1种基于TGF-β1/Sp-1的抗干眼的中药高通量筛选体系,并旨在筛选出3种左右的密蒙花有效组分,检验该体系的实用性。方法(1)建立并评价筛选体系:首先获取质粒p4GCbox-ZsGreen1DR,将质粒转染大鼠泪腺上皮细胞(LGEC),使用G418筛选后得到sp-1的反应性报告细胞株LGEC-ZsGreen1DR;经过实验培养后,加入浓度为12.5 ng/ml的TGF-β1,借助荧光显微镜来检测每一个时相点大鼠LGEC中ZsGreen1-DR的表达;把细胞株(LGEC-ZsGreen1DR)冻存30天后,再经过复苏并传代培养30天,之后加入TGF-β1,观测每一个时相点的ZsGreen1-DR表达结果。(2)筛选出密蒙花抗干眼的有效组分:以不同溶剂分离联合大孔树脂分离的方法制备密蒙花中药组分库,建立密蒙花的中药组分库,共含有39种组分;利用24孔板测定荧光平均光密度,继而筛选密蒙花颗粒抗干眼症的有效组分。结果(1)当LGEC-ZsGreen1DR细胞刚刚加入TGF-β1时,只能在荧光显微镜(400×)下观察到少量带有弱荧光的细胞;当在4 h时,显微镜(100×)下就可见细胞所带的绿色荧光,并且能够看到多处发光点;荧光在12 h时达到了最高位,在24h时出现明显跌落。把细胞株(LGEC-ZsGreen1DR)冻存30天,再经复苏并传代培养30天后,也得到了类似结果。(2)筛选出的密蒙花抗干眼的有效组分为:2-2、2-2-3、2-2-4。结论构建的基于TGF-β1/Sp-1的高通量的中药筛选体系,具备比较好的实用性与稳定性,可为抗干眼的中药有效组分开发提供了实验基础。

Objective To establish a high-throughput screening system based on TGF-β1 for anti-dry eye Chinese medicine,and to screen out about three effective components of Buddleja officinalis to test the practicability of the system.Methods(1)Establish and evaluate the screening system:first obtain the plasmid p4GCbox-ZsGreen1DR,transfect the plasmid into rat lacrimal gland epithelial cells(LGEC),and obtain the Sp-1 reactive reporter cell line LGEC-ZsGreen1DR after G418 screening;add it after cultivation TGF-β1(12.5 ng/ml),through fluorescence microscope observation to detect the expression of ZsGreen1-DR in LGEC-ZsGreen1DR cells at different time points;after freezing the LGEC-ZsGreen1DR cell line for 1 month,recover and subculture for 1 month,Add TGF-β1 and observe the expression of ZsGreen1-DR at different time points.(2)Screen out the effective components of Buddleja officinalis:Use the separation of different solvents combined with macroporous resin separation to prepare the Buddleja officinalis traditional Chinese medicine component library,and establish the Buddleja officinalis traditional Chinese medicine component library,which contains 39 components in total Measure the average optical density of fluorescence using a 24-well plate,and then screen the effective components of Buddleja officinalis against dry eye.Results(1)When TGF-β1 was added to LGEC-ZsGreen1DR cells,only a few cells with weak fluorescence were observed under a 400-fold fluores-cence microscope;at 4 h,green fluorescence of the cells could be seen under a 100-fold microscope.Luminous point;it peaked at 12 h and decreased significantly at 24 h.Similar results were obtained after the LGEC-ZsGreen1DR cell line was frozen for 30 days,recovered and subcultured for 30 days.(2)The effective components of Buddleja officinalis screened out are:2-2,2-2-3,2-2-4.Conclusion We constructed A high-throughput screening system based on TGF-β1/SP-1 successfully.The system has good practicability and stability,which provides experimental basis for the development of effective components of traditional Chinese medicine against dry eye.