Establishment And Evaluation Of The High Throughput Screening System For Antagonistic Agents Of Dry Eye Based On TGF-β1 And Bcl-2

Objectives:Through the establishment of the high throughput screening system for antagonistic Herbs of dry eye based on TGF-(31 and Bcl-2,It provided an ideal technology platform of study for antagonistic herbs of dry dye.Using the established screening system, It selected the positive components on small-scale traditional Chinese medicine Buddleja components library, tested the practicality of screening system established, and preliminarily investigated the distribution component of positive traditional chinese medicine Buddleja.Methods:(1)Consreuction and evaluation of 1 screening system:First,A weak promoter SV40 was obtained by PCR from plasmid pGL2-control. After purified the promoter was used to replace the CMVIE promoter in the pEGFP-Nl plasmid and gained pSV40-EGFP plasmid. Two shares of pSV40-EGFP were ready, one share was digested by XhoⅠand HindⅢand a backbone with EGFP was obtained.The other was digested by BglⅡand HindⅢin order to get a fragment with SV40 promoter. The two fragments and a oligodeoxynucleotide of GCbox motif were linked together and generated p4GCbox-EGFP plasmid which contained the backbone with EGFP, the fragment with SV40 promoter and artificial synthetic 4 copies of GCbox motif which acted as the cis-acting element and possessed XhoⅠand BglⅡsites. After the EGFP fragment of the p4GCbox-EGFP plasmid was substituted with ZsGreenlDR in p ZsGreen1DR plasmid,reporter plasmid p4GCbox-ZsGreenlDR was finally constructed. The plasmid p4GCbox-ZsGreenlDR 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-ZsGreenlDR. TGF-(31(12.5 ng/ml) was added to LGEC-ZsGreen1DR cell line which was cultured in DMEM culture medium.The expression of ZsGreen1DR in LGEC-ZsGreen1DR cell line among different time points was detected through fluorescence microscope and flow cytometry. In order to evaluate the stability of the LGEC-ZsGreen1DR, the cell line had been resuscitation culture for one months after one months of cryopreservation. Then, TGF-β1 was added to the cells and the expression of ZsGreen1DR in different time points was detected.(2) Consreuction and evaluation of 2 screening system:p4GCbox-ZsGreen1DR which had obtained was digested by Xho I and Hind III and a backbone with ZsGreenlDR was obtained. The backbone with ZsGreenlDR and artificial synthetic 3×p53 binding site (p53BS) which acted as the cis-acting element and possessed Xho I and Bgl II sites were linked together by T4-DNA ligase and generated reporter p3p53BS-ZsGreenlDR plasmid. The reporter plasmid p3p53BS-ZsGreen1DR was transfected into the rat lacrimal gland epithelial cells (LGEC). After selected with G418, reporter cell line which was based on p53 was gained and named LGEC-ZsGreenlDR. TGF-β1(12.5 ng/ml) was added to LGEC-ZsGreenlDR cell line which was cultured in DMEM culture medium.The expression of ZsGreen1DR in LGEC-ZsGreen1DR cell line among different time points was detected through fluorescence microscope and flow cytometry. In order to evaluate the stability of the LGEC-ZsGreenlDR, the cell line had been resuscitation culture for one months after one months of cryopreservation. Then, p53 was added to the cells and the expression of ZsGreenlDR in different time points was detected.(3) Demonstration screening of Buddleja small-scale traditional Chinese medicine component library:The method of different solvents separation combined with macroporous resin is to gain Buddleja small-scale traditional Chinese medicine component library which had 39 components.Results:(1) Plasmid restriction enzyme digestion and DNA sequencing results showed that Sp-1 responsive reporter gene vector p4GCbox-ZsGreen1DR sequence and the p53 responsive reporter gene vector p3p53BS-ZsGreenlDR sequence were in line compared with expectations idea.The reporter of plasmid was constructed successfully. (2) The reporter plasmid was transfected into LGEC,after selected with G418, the responsive reporter cell line of LGEC-ZsGreenlDR was gained.The genome of LGEC-ZsGreenlDR cell line was extracted and was used to the PCR identification. The result of PCR indicates that 1 LGEC-ZsGreenlDR cell line system had p4GCbox-ZsGreenlDR plasmid and 2 LGEC-ZsGreen1DR cell line system had p3p53BS-ZsGreenlDR plasmid. The reporter responsive cell line was constructed successfully.(3)The expression of ZsGreenlDR 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-ZsGreenlDR expressed was 0.164±0.044;peak after 12 hours of the addition of TGF-β1, the average optical density of fluorescence that LGEC-ZsGreenlDR 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-ZsGreenlDR 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-ZsGreenlDR cell line would still be effective after resuscitation culture,subculture and cryopreservation,and TGF-β1 on the Sp-1 could induce activation.(4) The expression of ZsGreenlDR induced by p53 was detected, Before and right after the addition of p53,the average optical density of fluorescence that LGEC-ZsGreenlDR expressed was 0.010±0.003; after 4 hours of the addition of p53, the average optical density of fluorescence that LGEC-ZsGreen1DR expressed was 0.134±0.023;peak after 12 hours of the addition of p53, the average optical density of fluorescence that LGEC-ZsGreen1DR expressed was 0.401±0.124; At 24-hour time point, the average optical density of fluorescence that LGEC-ZsGreen1DR expressed reduced obviously and was 0.149±0.045.LGEC-ZsGreenlDR cell line which had been resuscitation culture and subculture for one month after 30 days of cryopreservation were stimulated by p53 to evaluate the stability of LGEC-ZsGreen1DR cell line. The average optical density of fluorescence that LGEC-ZsGreen1DR expressed was 0.007±0.002; The average optical density of fluorescence that LGEC-ZsGreen1DR expressed was 0.147±0.025 after 4 hours; The average optical density was 0.408±0.074 after 12 hours;The average optical density was 0.178±0.038 after 24 hours; These results indicate that LGEC-ZsGreen1DR cell line would still be effective after resuscitation culture,subculture and cryopreservation,and p53 could induce activation.(5) After solvent separation and macroporous resin, it was 39 kinds of Chinese medicine components which had actually gained, the medicine at all levels received had been given the corresponding number for system components, they were selected by the established system of 1 and 2 screening system, and used four different 24 well plates for screening and a 24-well plate of the re-screening.The numbers of those positive chinese medicine components that 1 screening system selected were 2-2.2-2-3.2-2-4, the average optical density values by re-screening were 0.213±0.034,0.315±0.079. 0.222±0.034; The numbers of those positive chinese medicine components that 1 screening system selected were 2-2-3,2-2-4, the average optical density values by re-screening were 0.174±0.035,0.158±0.042。Conclusions:1.The vector, p4GCbox-ZsGreenlDR Eukaryotic expression vector containing ZsGreenl-DRreporter gene was structed with Sp-1 cis-acting element 4×GCbox motif for the enhancer and SV40 as moter. The Eukaryotic expression vector p4GCbox-ZsGreenlDR was transfected into LGEC cells.After selected with G418,reporter SP-1 reactive cell line LGEC-ZsGreen1DR was gained. Further, high-throughput screening system (1 screening system) based on Sp-1 anti-dry-eye drug was gained. 2.The vector, p3p53BS-ZsGreen1DR Eukaryotic expression vector containing ZsGreen1-DRreporter gene was structed with p53 cis-acting element 3×p53BS motif for the enhancer and SV40 as moter. The Eukaryotic expression vector p3p53BS-ZsGreen1DR was transfected into LGEC cells.After selected with G418,reporter SP-1 reactive cell line LGEC-ZsGreen1DR was gained. Further, high-throughput screening system (2 screening system) based on p53 anti-dry-eye drug was gained.3. It was confirmed that LGEC-ZsGreen1DR report cell line could be sensitive to monitor of Sp-1 and p53 activity changes by fluorescence intensity by changes and reflect effects about exogenous substances on TGF-β1, bcl-2,which could be used to select for resistance to dry eye drug.It was confirmid that 1,2screening system possessed reliable stability after Cryopreservation and resuscitation experiments.4.It provided experience for the follow-up study that Buddleja small-scale chinese medine component library was successfully established, which contained 39 kinds of components. With the established 1,2, screening system and the 24-well plate initial screening method, it was three kins of positive components of chinese medicine which could promote or intend effect of TGF-β1 selected in Buddleja small-scale chinese medine component library,Component number of that were 2-2,2-2-3,2-2-4; It was 2 kinds of positive components of chinese medine which could inhibitory effect of p53 selected. Component number of that were 2-2-3,2-2-4.It was confirmed that screening system had good practicability.