OBJECTIVE:To explore the effects of Qingguang'an(青光安)containing serum on the expression levels of autophagy related genes in the transforming growth factor beta 1(TGF-β1)-activated human Tenon's fibroblast...OBJECTIVE:To explore the effects of Qingguang'an(青光安)containing serum on the expression levels of autophagy related genes in the transforming growth factor beta 1(TGF-β1)-activated human Tenon's fibroblasts(HTFs).METHODS:(a)Primary HTFs were stimulated by TGF-β1 and underwent immunohistochemistry,which established a cell model after Glaucoma filtration surgery(GFS).(b)The cell models were divided into 4 group:normal group(normal cells),model group(+TGF-β1),treatment group(+TGF-β1+medicated serum),and positive control group(TGF-β1+rapamycin).Then,Qingguang'an medicated serum with optimum concentration was added to the corresponding group.The autophagy positive cells were identified by the Cyto-ID autophagy detection kits under fluorescent microscope and Cytation 5 multifunctional instrument for cell imaging.And the mean fluorescence intensity of autophagy positive cells was determined by flow cytometry.The expression levels of autophagy related genes—Beclin-1,autophagy related gene 5(ATG-5),and microtubule-associated protein 1 light chain 3(LC-3Ⅱ)were detected by quantitative reverse transcription-polymerase chain reaction and Western blot analysis.RESULTS:Compared with the normal group and the model group,the relative mRNA expression levels of autophagy-related genes(Beclin-1,ATG-5 and LC-3Ⅱ)in the experimental group were notably increased(P<0.05,P<0.01),and with the extension of treatment time,it had an increasing trend(48 h was more obvious),which showed a certain time dependency;the protein expression levels of autophagy-related genes(Beclin-1,ATG-5,and LC-3Ⅱ)were significantly increased in the experimental group(P<0.05,P<0.01).With the prolongation of treatment time,there was an increasing trend(48 h was relatively obvious),and it revealed a certain time dependency CONCLUSION:The Qingguang'an medicated serum could up-regulate autophagy related genes(Beclin1,ATG5,and LC3Ⅱ)in the TGF-β1-activated HTFs.展开更多
基金Supported by the National Natural Science Foundation of China(Inducing effect of Qingguang'an on Autophagy of Tenon's Capsule Fibroblasts after Glaucoma Filtration Surgery,No.81603665)And the Postdoctoral Science Foundation of China(Experimental Study on Autophagy of Tenon's Fibroblasts Induced by Qingguang'an after Glaucoma Filtration Surgery,No.2017M612565)。
文摘OBJECTIVE:To explore the effects of Qingguang'an(青光安)containing serum on the expression levels of autophagy related genes in the transforming growth factor beta 1(TGF-β1)-activated human Tenon's fibroblasts(HTFs).METHODS:(a)Primary HTFs were stimulated by TGF-β1 and underwent immunohistochemistry,which established a cell model after Glaucoma filtration surgery(GFS).(b)The cell models were divided into 4 group:normal group(normal cells),model group(+TGF-β1),treatment group(+TGF-β1+medicated serum),and positive control group(TGF-β1+rapamycin).Then,Qingguang'an medicated serum with optimum concentration was added to the corresponding group.The autophagy positive cells were identified by the Cyto-ID autophagy detection kits under fluorescent microscope and Cytation 5 multifunctional instrument for cell imaging.And the mean fluorescence intensity of autophagy positive cells was determined by flow cytometry.The expression levels of autophagy related genes—Beclin-1,autophagy related gene 5(ATG-5),and microtubule-associated protein 1 light chain 3(LC-3Ⅱ)were detected by quantitative reverse transcription-polymerase chain reaction and Western blot analysis.RESULTS:Compared with the normal group and the model group,the relative mRNA expression levels of autophagy-related genes(Beclin-1,ATG-5 and LC-3Ⅱ)in the experimental group were notably increased(P<0.05,P<0.01),and with the extension of treatment time,it had an increasing trend(48 h was more obvious),which showed a certain time dependency;the protein expression levels of autophagy-related genes(Beclin-1,ATG-5,and LC-3Ⅱ)were significantly increased in the experimental group(P<0.05,P<0.01).With the prolongation of treatment time,there was an increasing trend(48 h was relatively obvious),and it revealed a certain time dependency CONCLUSION:The Qingguang'an medicated serum could up-regulate autophagy related genes(Beclin1,ATG5,and LC3Ⅱ)in the TGF-β1-activated HTFs.
