【作者】 姚小磊；

【导师】 彭清华；

【作者基本信息】 湖南中医药大学 ， 中西医结合临床， 2010， 博士

【摘要】 目的：建立基于TGF-β1与Bcl-2的抗干眼症中药高通量筛选体系,为干眼症防治药物的研究提供一个理想的技术平台。并利用所建立筛选体系对密蒙花小规模中药组分库进行阳性组分筛选,检验所建立筛选体系的实用性,初步考察密蒙花阳性中药组分的分布。方法：(1)1号筛选体系的构建和评价：首先采用PCR方法从质粒pGL2-control中扩增获得SV40弱启动子,并以其替换质粒pEGFP-N1中的强启动子CMVIE,获得pSV40-EGFP质粒。提取两份pSV40-EGFP质粒,其中一份以XhoⅠ、HindⅢ双酶切获得含有EGFP的质粒骨架,另一份用BglⅡ、HindⅢ双酶切获得含SV40启动子的质粒片段。用T4-DNA连接酶连接含有EGFP的质粒骨架、含SV40启动子的质粒片段及人工合成的4×GCbox基序(具有XhoⅠ、BglⅡ粘端),获得p4GCbox-EGFP质粒,并以pZsGreen1-DR质粒中ZsGreen1-DR片段替换p4GCbox-EGFP质粒中的EGFP片段,最终获取报告质粒p4GCbox-ZsGreen1DR。将p4GCbox-ZsGreen1DR报告质粒转染大鼠泪腺上皮细胞(LGEC),G418筛选后获得TGF-p1反应性报告细胞株LGEC- ZsGreen1DR。培养后加入TGF-β1(12.5ng/ml),通过荧光显微镜观察检测不同时相点LGEC-ZsGreenlDR细胞ZsGreenl-DR的表达。将LGEC-ZsGreenlDR细胞株冻存1个月后,复苏并传代培养1个月,加入TGF-β1,观察不同时相点ZsGreen1-DR的表达情况,据此评价TGF-β1反应性报告细胞株LGEC-ZsGreen1DR的稳定性。(2)2号筛选体系的构建和评价：XhoⅠ、BglⅡ双酶切已经建立的质粒p4GCbox-ZsGreen1DR,获得含有ZsGreen1DR的质粒大片段骨架。用T4-DNA连接酶连接含有含有ZsGreen1DR的质粒大片段骨架及人工合成的3×p53binding site (p53BS)基序(具有XhoⅠ、BglⅡ粘端),获得报告质粒p3p53BS-ZsGreen1DR。将p3p53BS-ZsGreen1DR报告质粒转染大鼠泪腺上皮细胞(LGEC), G418筛选后获得p53反应性报告细胞株LGEC-ZsGreen1DR。培养后加入p53(12.5ng/ml),通过荧光显微镜观察检测不同时相点LGEC-ZsGreen1DR细胞ZsGreen1-DR的表达。将LGEC-ZsGreen1DR细胞株冻存1个月后,复苏并传代培养1个月,加入p53,观察不同时相点ZsGreen1-DR的表达情况,据此评价p53反应性报告细胞株LGEC-ZsGreen1DR的稳定性。(3)密蒙花小规模中药组分库的试探性筛选：以不同溶剂分离联合大孔树脂分离的方法制备密蒙花小规模中药组分库,共含有39种组分,利用所建立的1号体系和2号体系进行筛选,利用24孔板进行荧光平均光密度进行组分筛选。结果：(1)质粒酶切及DNA测序鉴定结果表明,Sp-1反应性报告基因载体p4GCbox-ZsGreen1DR序列,以及p53反应性报告基因载体p3p53BS-ZsGreenlDR序列符合预期设想,报告质粒构建成功。(2)将报告质粒转染泪腺上皮细胞,经G418筛选后,获得反应性报告细胞株LGEC-ZsGreen1DR,提取LGEC-ZsGreen1DR细胞基因组DNA进行PCR鉴定,结果表明：1号体系细胞株LGEC-ZsGreen1DR中含有p4GCbox-ZsGreen1DR质粒,2号体系细胞株LGEC-ZsGreen1DR中含有p3p53BS-ZsGreen1DR质粒。反应性报告细胞株构建成功。(3) TGF-β1诱导表达实验结果表明,1号体系加入TGF-β1前及加入TGF-β1在0h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12h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-β10h时,LGEC-ZsGreenlDR表达荧光的平均光密度为0.007±0.001;4h后LGEC-ZsGreenlDR的平均光密度为0.144±0.052；12h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-ZsGreenlDR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。(4)p53诱导表达实验结果表明,2号体系加入P53前及加入P53在0h时,LGEC-ZsGreenlDR表达荧光的平均光密度为0.010±0.003；加入P53在4h时LGEC-ZsGreenlDR的平均光密度为0.134±0.023；在12h达到高峰,平均光密度为0.401±0.124；在24h时细胞平均光密度明显下降,平均光密度为0.149±0.045。将LGEC-ZsGreenlDR细胞株冻存30天,复苏并传代培养1个月,加入P53在0h时,LGEC-ZsGreenlDR表达荧光的平均光密度为0.007±0.001；加P53诱导4 h后LGEC-ZsGreenlDR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-ZsGreenlDR细胞株经冻存、复苏、传代后仍能有效反映P53的诱导激活作用。(5)经过溶剂分离和大孔树脂分离之后,实际得到39种中药组分,将所得到的各层次中药组分进行系统的相应编号,利用所建立的1号体系和2号体系进行筛选,各进行了4块24孔板的初筛和1块24孔板的复筛。1号体系筛选出的阳性中药组分编号为：2-2、2-2-3、2-2-4,复筛的平均光密度值分别为：0.213±0.034、0.315±0.079、0.222±0.034；2号体系筛选出的阳性中药组分编号为：2-2-3、2-2-4,复筛的平均光密度值分别为：0.174±0.035、0.158±0.042。结论：1.成功构建了以Sp-1顺式作用元件4×GCbox基序为增强子、以SV40为启动子,以ZsGreen1-DR为报告基因的真核表达载体p4GCbox-ZsGreen1DR。并将其转染LGEC细胞,经G418筛选后,获得了Sp-1反应性报告细胞株LGEC-ZsGreen1DR,即基于Sp-1的抗干眼症药物高通量筛选体系(1号筛选体系)。2.成功构建了以p53顺式作用元件3×p53BS基序为增强子、以SV40为启动子,以ZsGreen1-DR为报告基因的真核表达载体p3p53BS-ZsGreen1DR。并将其转染LGEC细胞,经G418筛选后,获得了p53反应性报告细胞株LGEC-ZsGreenlDR,即基于p53的抗干眼症药物高通量筛选体系(2号筛选体系)。3.经TGF-β1和p53诱导表达实验证实,LGEC-ZsGreen1DR报告细胞株可通过荧光强弱变化敏感地监测Sp-1和p53的活性改变,从而反映外源性物质对TGF-β1、bcl-2的效应,据此可进行抗干眼症药物的筛选。经冻存、复苏实验证实,1号、2号筛选体系具有可靠的稳定性。4.成功建立了密蒙花小规模中药组分库,所含组分39种,为后续研究提供了经验借鉴。利用所建立的1号、2号筛选体系和24孔板的初步筛选方法,在密蒙花小规模中药组分库中,筛选出能够促进或拟TGF-β1效应的中药阳性组分3种,组份编号为2-2、2-2-3、2-2-4；筛选出能够抑制p53效应的中药阳性组分2种,组份编号为2-2-3、2-2-4。证明筛选体系有良好的实用性。更多还原

【Abstract】 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.更多还原