Résumé
Background Gene transfection is an effective therapeutic avenue to target many kinds of eye diseases.Non-viral vectors with high transfection efficiency,long-term expression,low toxicity and high expression levels are pivotal in gene therapy of corneal disease.Objective This study was to evaluate and compare the safety and efficiency between EntransterTM and liposome vectors for transfer of CD25 siRNA in rat cornea.Methods Eighty male SPF SD rats were randomly divided into EntransterTM-CD25 siRNA group,liposome-CD25 siRNA group,simple CD25 siRNA group and normal saline solution (NSS) group with the right eye as experimental eyes.Corneal epithelia of the rats were completely removed after ocular surficial anesthesia,and 50 μl EntransterTM-CD25 siRNA,liposome-CD25 siRNA,CD25 siRNA solution and NSS were topical administered in the eyes respectively.Ocular response and green fluorescence number on the corneas were examined under the slit lamp assisted microscope 12 hours,24 hours,3 days and 7 days after use of the drugs.The rats were sacrificed and the corneas were obtained,and corneal histopathological examination was performed by using hematoxylin eosin stain.The gene transferred efficiency in the corneas was evaluated by fluorescence technology,and the safety of EntransterTM and liposome carriers was assessed using TUNEL stain.The expression and location of CD11b in the corneas were detected by immunofluorescence technology.The use and care of the experimental animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Committee.Results The quantity and intensity of fluorescence staining in the corneas were significantly increased in the EntransterTMCD25 siRNA group in comparison with the liposome-CD25 siRNA group,and the corneal fluorescence appeared earlier in the simple CD25 siRNA group,but it disappeared in 24 hours after transfection.Corneal histopathological examination revealed that the corneal edema and inflammatory cell infiltration in corneal epithelium after gene transfection were more dominant in the liposome-CD25 siRNA group than those in the EntransterTM-CD25 siRNA group,simple CD25 siRNA group and NSS group,and no abnormality was seen in the stroma and endothelium.The number of inflammatory cells was more in the liposome-CD25 siRNA group than that in the EntransterTM-CD25 siRNA group,simple CD25 siRNA group and NSS group (all at P =0.00).The number of apoptosis cells was significantly more in the liposome-CD25 siRNA group than that in the EntransterTM-CD25 siRNA group,simple CD25 siRNA group and NSS group in 12 hours and 3 days after transfection (all at P =0.00).Immunofluorescence assay showed the expression of CD11b primarily located in the corneal epithelial and stromal layers.The expression of CD11b was gradually enhanced over time in the liposome-CD25 siRNA group and peaked in 24 hours after transfection.However,the expression was absent in the EntransterTM-CD25 siRNA group,simple CD25 siRNA group and NSS group.Conclusions EntransterTM nanometer material-mediated transfection of CD25 siRNA in corneas of normal SD rats appears to have high transfection efficiency,low toxicity and slight irritating response to corneas,and EntransterTM vector is currently available for the gene therapy of corneal disease.
Résumé
Background Laser assisted subepithelial keratomileusis (LASEK) is one of surgical procedures for refractive correction.Dilute alcohol that is used for the removal of epithelium during LASEK induces the apoptosis of corneal epithelial cells.Researches showed that thymosin β4 (Tβ4) can arrest apoptosis, but whether Tβ4 plays inhibitory effect on ethanol-induced damage of corneal epithelial cells is still unelucidated.Objective The aim of this study was to investigate the protective effects of Tβ4 on ethanol-induced rabbit corneal epithelial cell damage in vitro.Methods The corneal tissue of deendothelium was obtained from 10 New Zealand white rabbits.Corneal epithelial cells were cultured in vitro by using explant culture method.Cultured cells were identified by detecting the expression of keratin 12 and connexin 43 with reverse transcription PCR (RT-PCR).The cells of second generation were collected and divided into 4 groups.The cells were regularly cultured in the normal control group, and Tβ4 was added in the culture medium at the final concentration of 1 μg/ml in the Tβ4 treated group.Ethanol-induced rabbit corneal epithelial cell damage models were established by adding PBS containing 20％ alcohol in medium for 20 seconds in the model group,and Tβ4 was added in the medium of model cells at the final concentration of 1 μg/ml in the model+Tβ4 group.The survival rate of the cells was detected by MTT assay, and the apoptosis rate of the cells was examined by TUNEL method.The relative expression levels of cyclin D1 mRNA and cyclin-depensent protein kanase 4 (CDK4) mRNA in the cells were detected by real-time flurescenee quantitative PCR.The content of bcl-2 protein in the cells was detected by ELISA assay.Spectrophotometry was employed to measure the activity of caspase-3.The study complied with the Regulation for the Adminstration of Affairs Concerning Experimental Animals by State Science and Technology Commission.Results The mean cell survival rate was (52.1 ± 14.07) ％ in the model group,which was significantly reduced in comparison with 100％ of the normal control group and (77.7± 19.60) ％ of the model+Tβ4 group (P=0.001 ,P=0.035).TUNEL staining revealed more positive cells in the model group and the model+Tβ4 group,and the percentage of TUNEL positive cells was (30.0±6.7)％ in the model+Tβ4 group, showing an evident decline in comparison with (42.4±4.0)％ of the model group (P=0.01).The relative expression levels of cyclin D1 mRNA and CDK4 mRNA were 0.93±0.17 and 0.88±0.09 in the model+Tβ4 group, which were significantly higher than 0.68±0.05 and 0.54±0.07 in the model group (P=0.027,0.002).ELISA assay exhibited that bcl-2 content in the cells was considerably lower,and caspase-3 activity was significantly higher in the model group than those in the model+Tβ4 group (P =0.030,0.021).Conclusions Tβ4 plays a protective effect on rabbit corneal epithelial cells from apoptosis by lowing the caspase 3 activity and increasing bcl-2 content in ethanoldamaged rabbit corneal epithelial cells.In addition, Tβ4 promotes the regrowth of corneal epithelial cells by upregulating the expression of cyclin D1 and CDK4.
Résumé
Background Oxidative stress is a pathophysiological process of retina,so it is very important to explore a protective way against retinal oxidative stress.Studies determined that extract of ginkgo biloba (EGb) has antioxidant,anti-apoptosis,anti-thrombosis and anti-inflammatory effects,however,the effect of EGb on human Müller cells in oxidative stress is still below understood.Objective This study was to investigate the protection of EGb against oxidative stress of human retinal Müller cells induced by As2O3 in vitro.Methods Human retinal Müllercell line was cultured in DMEM/F12 with 10％ fetal bovine serum (FBS),2 μmol/L glutamine and 1％ antibiotics.As2O3 solution at the final concentration 5 mg/L was added in the medium for 24 hours to establish oxidative models,and then the EGb with the final concentrations of 5,10 and 20 mg/L was used to cell models for 24 hours,respectively.Cell viability was detected by MTT assay,and reactive oxygen species (ROS) levels in the cells were detected with CM-H2DCFDA fluorescent probe.The relative expression levels of caspase-3 mRNA in cytoplasm and cell nuclei were assayed by reverse-transcription PCR (RT-PCR),and the expressions of Nrf2 protein were quantitatively detected by Western blot.Results Müller cells adhered well 24 hours after cultured.At 6-7 days after culture,Müller cell body was large with abundant cytoplasm and mosaic-like arrangement.However,floating cells were seen after As2 O3 treatment.Cell viability (absorbance) was significantly different among the normal culture group,As2 O3-treated group,As2 O3 + 5 mg/L EGb group,As2 O3 + 10 mg/L EGb group and As2 O3 + 20 mg/L EGb group,with the strongest viability in the normal culture group and the weakest viability in the As2 O3-treated groups (F=163.57,P =0.00).The fluorescence intensity of ROS was the weakest in the normal culture group and the strongest in the As2 O3-treated group and was gradually weakened with the increase of EGb doses,showing a remarkable difference among the groups (F =4 013.61,P =0.00).The relative expression level of caspase-3 mRNA in the cells was gradually reduced with the increase of EGb doses,with a statistically significant difference among the groups (F =2 199.72,P =0.00).In addition,no considerable difference was seen in the expression level of Nrf2 protein (grey scale) in cytoplasm among the groups (F=15.42,P=0.40);while in the nuclei,the expression levels of Nrf2 protein were 100.01 ±0.04,46.59±0.63,54.51 ±0.62,59.93 ±0.17 and 67.60±0.24 in the normal culture group,As2 O3-treated group and As2O3+5 mg/L EGb group,As2O3+10 mg/L EGb group,As2O3+20 mg/L EGb group respectively,with a significant difference among them (F=7 271.72,P=0.00).Conclusions EGb can protect human retinal Müller cells against As2O3-induced damage in a dose-dependant manner by antioxidant and antiapoptotic effects in vitro,and the activities occur primarily in cell nucleus.
Résumé
Objective To investigate the mechanism of inhibitor of calpain on hyperglycemia-induced apoptosis in cultured rat cardiomyocyte.Methods Cardiomyocytes were randomly divided into three groups (control,high glucose,and ALLN).MTT assay was used to detect the viability of cultured cardiomyocytes.Laser confocal microscopy was used to observe the mitochondrial permeable transition and membrane potential.The change of Caspase-3 activity in cardiomyocytes was detected by western blot.Results MTT assay showed that,after 72 h of hyperglycemia,the viability of cardiomyocytes was significantly declined (55％ ± 11％),and the viability in the ALLN pretreatment group was (70％ ± 15％) (P ＜0.05).After hyperglycemia,the mitochondrial permeable transition of cardiomyocyte was increased (30％ ± 15％ vs 60％ ± 11％,P ＜0.05),and membrane potential was declined.Hyperglycemia could increase the expression of cleaved capsase-3,while with pretreatment of ALLN the expression of cleaved caspase-3 was downregulation(0.42 ± 0.11 vs 0.21 ±0.12,P ＜0.05).Conclusions The calpain inhibitor can protect cardiomyocytes from apoptosis under the high glucose condition.
Résumé
Objective To compare the pharmacological actions of Liandai Tablet(LT) and its main active components on apoptosis of gastric cancer cells and DNA damage. Methods Effects of Liandai Tablet(LT) and its main active components,berberine and indirubin,on growth and apoptosis of gastric cancer cell strain MGC_803 were explored and their effects on DNA damage were also studied. Results LT serum in high and low dosages and berberine could inhibit the growth of MGC_803 as compared with the control group,and typical morphological features of apoptosis were found in the MGC_803 by methyl green pyronin stain assay.But indirubin at various concentrations showed no obvious inhibitory effects. Agarose gel electrophoresis assay revealed that the MGC_803 cell DNA was split into large fragments when treated with berberine. Conclusion LT serum exerts a similar inhibitory effect on the growth and apoptosis of gastric cancer cells as compared with berberine.The effects of LT at various serum concentrations on MGC_803 DNA was less than that of berberine,and indirubin at the given concentration had no this effect.
Résumé
Objective To investigate the effects of all-trans-retinoic acid(atRA) on proliferation and apoptosis of smooth muscle cell in human saphenous vein organ culture.Methods Thirty segments(1cm) of human saphenous vein were divided into 3 groups: HSV group,atRA group and control group.The human saphenous vein segments in atRA group and control group were maintained in organ culture for 14 days with 100?M atRA and equivalent concentrations of pure ethanol respectively.All 30 segments human saphenous vein were harvested and the level of proliferating cell nuclear antigen(PCNA) was detected immunohistochemically.The smooth muscle cell proliferation was observed pathologically.Results Cultured saphenous vein segments developed significant neointimal formation and marked smooth muscle cell proliferation(P