The Effect Of TGF-beta 1 Intervention On HTFs Autophagic Positive Cells In TGF-beta 1

Objective: through the establishment of glaucoma filtration surgery sex cells in vitro culture model,to green light Ann drug-containing serum intervention,HTF cells induced autophagy,prove that autophagy sex's death is a green light for the important mechanism of the HTF cell proliferation inhibition,for the green light,mechanism of action research to explore new territory,and provides the experimental and theoretical basis.Methods:(1)according to the method of random 20 Sprague wrote-Dawley(SD)male rats were divided into four groups,divided into the green light,10 times group 5times,the green light,green light 2.5 times group and blank group,the green light,take 10 times of serum for high dose group,5 times in serum for dose group,2.5 times from the serum of low dose group.(2)take the Tenon's capsule group,cut into 1x1x1mm3 size,after rinsing in DMEM medium temperature and training of 15% FBS,the cells of 70%-80% of trypsin digestion to extend,after cell climbing and fixed,cell morphology,observed under inverted microscope immunofluorescence parallel waveform protein identification.(3),then take 3-6 cell experiment is divided into normal control group,model group,low dose group,middle dose group,high dose group,by joining CCK 8 read enzyme standard instrument of 450 nm absorbance(A)value,the green light to represent different concentrations Ann drug-containing serum influence on HTFs proliferation ability,in order to determine the drug-containing serum on the HTFs optimal mass concentration.(4)the cell model of GFS was constructed after TGF-beta 1 induced HTFs into myofibroblasts.(5)Add green light Ann drug-containing serum,the experiment group(blank)divided into two groups,the TGF-beta 1 group(model group),TGF-beta 1 + drug-containing serum group(treatment group),serum medicated group(control group),use Cyto-ID autophagy detection kits,observed under Cytation5 around the nucleus and cytoplasm of autophagosome and autophagy-lysosome green fluorescence,namely autophagy positive cells,expressed as a number of autophagy positive cell percentage of HTF cell autophagy level,at the same time detection average fluorescence intensity of autophagy positive cells.Results :(1)to complete the preparation of the serum containing serum of qingguang'an,and to choose the dosage of the drug serum in the 5 times group as the intervention concentration of the experiment.(2)successful training of Tnon,s cystic fibroblasts(HTFs)and identification.(3)TGF-beta 1 promotes the proliferation of HTF cells,becomes myofibroblasts,and successfully simulates the postoperative cells of GFS.(4)Autophagy positive cell identification,24 h,the blank group,control group little green fluorescent dye in the cell,model group and treatment group green fluorescent dye rare inside the cell,compared with the blank group,control group,model group and treatment group no obvious enhanced green fluorescent dye in the cell,compared with model group,treatment group green fluorescent dye has no obvious increase in the cell;After 48 h,green fluorescent dye in cells almost no blank group,control group green fluorescent dye rare inside the cell,the model group green fluorescent staining part visible inside the cell,the treatment group the strongest green fluorescent dye in the cell,compared with the blank group,control group,model group and treatment group obviously enhanced green fluorescent dye in the cell;Compared with the model group,the green fluorescence staining was enhanced in the treatment group.72 h,the blank group green fluorescent staining part visible inside the cell,the control group in the cell enhanced green fluorescent dye,model group and treatment group green fluorescent dye in cells significantly enhanced,compared with the blank group,control group,model group and treatment group green fluorescent dye in the cell significantly enhanced,model group and treatment group green fluorescent staining were similar in the cell;(5)Autophagy level was detected,and the results showed that the amount of autophagy in each group was positively correlated with the time dependence of the drug.(6)The average fluorescence intensity test of autophagic positive cells showed that the average fluorescence intensity of the autophagy positive cells in each group was positively correlated with the time dependent of the drug.Conclusion: green light Ann drug-containing serum can promote TGF-beta 1intervened HTF cell autophagy strength increase,led to the deaths of cell autophagy,thus inhibiting cell proliferation,reducing subconjunctival scar area,viral postoperative scarring resistant to glaucoma,the prevention of postoperative intraocular pressure,improve the success rate of filtration surgery.