Objective To investigate the effect of intravitreal injection of fibrillin-2(FBN2)recombinant protein on FBN2-deficient retinopathy.Methods Thirty-two SPF-grade C57BL/6J mice were randomly divided into 4 groups:nor-mal control group,negative control group,FBN2 knockdown group,and FBN2 recombinant protein group,with 8 mice in each group.The right eyes were taken as the experimental eyes.Mice in the normal control group did not receive any inter-vention,mice in the negative control group were intravitreally injected with 3 μL empty vector(1 mg·L-1),and mice in the FBN2 knockdown group and FBN2 recombinant protein group were intravitreally injected with 3 μL adeno-associated vi-rus(1 mg·L-1).After 4 weeks,mice in the FBN2 recombinant protein group were intravitreally injected with 3 μL FBN2 recombinant protein(1 mg·L-1).Then,electroretinogram(ERG)and optical coherence tomography(OCT)were used to measure the amplitude of Rod-b and Max-a waves and the changes in the retinal structure.Real-time quantitative poly-merase chain reaction(RT-PCR)and Western blot were used to detect changes in FBN2,microfibril-associated glycopro-tein 2(MAGP-2),collagen I(COL1)mRNA and protein expression in the mouse retina.Results The ERG findings showed that compared with the negative control group and normal control group,the amplitude of Rod-b and Max-a waves in the retina of mice in the FBN2 knockdown group and FBN2 recombinant protein group decreased(all P＜0.05);com-pared with the FBN2 knockdown group,the amplitude of Rod-b and Max-a waves in the retina of mice in the FBN2 recom-binant protein group significantly increased(both P＜0.05).The OCT findings showed that compared with the FBN2 knock-down group,the structure of the retinal pigment epithelium and the light reflex in the FBN2 recombinant protein group be-came more regular.The RT-PCR detection results showed that compared with the FBN2 knockdown group,the expression of FBN2 mRNA in the retinal tissue of mice in the FBN2 recombinant protein group significantly increased,while the ex-pression of COL1 and MAGP-2 mRNA significantly decreased(all P＜0.05).Western blot assay results showed that com-pared with the FBN2 knockdown group,the expression of FBN2 protein in the retinal tissue of mice in the FBN2 recombi-nant protein group increased significantly,while the expression of COL1 and MAGP-2 proteins decreased significantly(all P＜0.05).Conclusion Intravitreal injection of FBN2 recombinant protein can compensate for the endogenous deficiency of FBN2 in mice with FBN2-deficient retinopathy and achieve therapeutic effects by regulating COL1 and MAGP-2 expres-sion.

Objective:To investigate the expression of latent transforming growth factor-β-binding protein (LTBP), transforming growth factor-β (TGF-β), cyclin-dependent kinase 2 (CDK2) and cyclin D2 (CCND2) in fibrillin-2 ( FBN2) interfering induced mouse retinopathy. Methods:Twenty-seven 8-week-old C57BL/6J mice were randomly divided into normal control group, empty vector group and FBN2 interference group according to the random number table method, with 9 mice in each group.The normal control group was not treated.The empty vector group and FBN2 interference group were intravitreally injected with 3 μl empty vector and 3 μl adeno-associated virus (AAV) carrying the sh-FBN2 interference plasmid in the right eye, respectively.The structural and functional changes of the retina were detected at 4 weeks after injection by optical coherence tomography (OCT) and full-field electroretinography (ERG).The expression and distribution of FBN2 protein in the retina were detected by immunofluorescence staining.The mRNA and protein expression levels of FBN2, LTBP-1, TGF-β2, CDK2 and CCND2 in mouse retina were detected by real-time fluorescence quantitative PCR and Western blot.All experiments complied with the ARVO statement.The research scheme was approved by the Experimental Animal Ethics Committee of Shandong University of Traditional Chinese Medicine (No.2019036).Results:Four weeks after injection, the results of OCT examination showed that compared with normal control and empty vector groups, the retinal pigment cortex of the FBN2 interference group was irregular with high density reflection areas.Full-field ERG results showed that compared with normal control and empty vector groups, the amplitude of Rod-a, Rod-b, Max-a and Max-b waveforms in FBN2 interference group decreased, and the differences were statistically significant (all at P<0.05).The results of immunofluorescence staining showed that FBN2 was expressed in the whole retina, and the fluorescence intensity of FBN2 was weaker in FBN2 interference group than that in normal control and empty vector groups.The fluorescence intensity of FBN2 in normal control group, empty vector group and FBN2 interference group was 16.21±2.21, 15.57±3.63 and 5.32±1.06, respectively, with a statistically significant overall difference ( F=66.03, P<0.05).The fluorescence intensity of FBN2 protein in FBN2 interference group was significantly lower than that in empty carrier group and normal control group (both at P<0.05).Compared with normal control and empty vector groups, the relative expression levels of LTBP-1 and TGF-β2 mRNA and protein were significantly increased in FBN2 interference group, while the relative expression levels of FBN2, CDK2 and CCND2 mRNA and protein were significantly decreased, and the differences were statistically significant (all at P<0.05). Conclusions:The increase of LTBP-1 and TGF-β2 and the decrease of G1/S phase related proteins CDK2 and CCND2 are involved in the development of FBN2-deficient retinopathy.

Objective To investigate the mechanism of electroacupuncture in relieving the apoptosis of retinal cells in experimental myopia based on mitochondrial dynamics.Methods In the study,140 two-week-old tricolor guinea pigs were divided into the normal control(NC)group,lens-induced myopia(LIM)group,LIM+sham acupoint(LIM+SHAM)group,and LIM+electroacupuncture intervention(LIM+EA)group,with 35 guinea pigs in each group.Guinea pigs in the NC group were fed normally,and those in the LIM group,LIM+SHAM group and LIM+EA group wore a-6.0D lens on the right eye to induce myopia.Guinea pigs in the LIM+EA group were treated at Hegu and Taiyang acupoints,while those in the LIM+SHAM group were treated at sham acupoints.After 2-and 4-week myopia induction,the diopter and axial length of the guinea pigs were measured,and the messenger ribonucleic acid(mRNA)and protein levels of dynam in-related pro-tein 1(DRP1),optic atrophy 1(OPA1),Bcl-2 associated X protein(BAX)and B cell lymphoma-2(BCL-2)in the retinas of the guinea pigs in each group were detected by quantitative PCR and Western blot,respectively.Meanwhile,HE staining was taken to observe the morphological changes in the retina of the guinea pigs in each group,and TUNEL staining was used to detect apoptosis.Results After 2-and 4-week myopic induction,compared with the NC group,the differences in diopter and axial length between the right and left eyes of guinea pigs in the LIM and the LIM+SHAM groups significantly increased(all P＜0.001).Compared with the LIM group,the differences in diopter and axial length between both eyes of guinea pigs in the LIM+EA group significantly decreased(all P＜0.01).At 4 weeks after myopic induction,HE staining re-sults showed that the retinas of the guinea pigs in the NC group were evenly arranged,and the morphology of inner and out-er nuclear layer cells was normal.Compared with the NC group,the retinas of the guinea pigs in the LIM and the LIM+SHAM groups were significantly thinner and disorderly arranged.Compared with the LIM group,the retinal thickness of guinea pigs in the LIM+EA group slightly increased,and the arrangement was relatively regular.The TUNEL staining re-sults showed that compared with the NC group,the green fluorescence of the guinea pig retina in the LIM group and LIM+SHAM group was significantly enhanced;compared with the LIM group,the green fluorescence of the guinea pig retina in the LIM+EA group was significantly weaken.After 2-and 4-week myopic induction,the mRNA and protein expression lev-els of DRP1 and BAX in the LIM and the LIM+SHAM groups were significantly higher than those in the NC group(all P＜0.05),while the mRNA and protein expression levels of OPA1 and BCL-2 were significantly lower(all P＜0.05).The mR-NA and protein expression levels of DRP1 and BAX in the LIM+EA group were significantly lower than those in the LIM group(all P＜0.05),while the mRNA and protein expression levels of OPA1 and BCL-2 were significantly higher(all P＜0.05).Conclusion Experimental myopia can enhance retinal mitochondrial fission.Electroacupuncture intervention can alleviate retinal cell apoptosis and improve the morphological structure of the retina by inhibiting retinal mitochondrial fis-sion and increasing mitochondrial fusion,thereby delaying myopia development.

CDIO is a new education model composed of Conceive, Design, Implement and Operate modules. In order to meet the needs of social development, this paper discusses the construction of the training system of applied innovative talents in ophthalmology of integrated traditional Chinese and western medicine guided by CDIO concept. Combing with practice, this paper puts forward teaching reform strategies from such five aspects as teaching purpose, curriculum system, construction of the teaching team, evaluation system, and practice effect, providing a new thought for the cultivation of applied innovative talents in ophthalmology.

Objective To investigate the regulatory role of rno-miR-30b-5p in the expressions of interleukin-10 (IL-10) and toll-like receptor 4 (TLR4) in uveitis.Methods Both IL-10 and TLR4 gene 3'UTR lucfferase vectors and relevant binding site mutant vectors were constructed.Further,both rno-miR-30b-5p mimics and reporter gene vector were co-transferred into 293 T cells to validate the fluorescent alterations of the reporter gene expression to detect the interactions between rno-miR-30b-5p and the related target genes.Moreover,an experimental autoimmune uveitis (EAU) model was induced with IRBP peptide emulsion in rats,and both lymph node and spleen were isolated on day 12 after EAU induction.In order to measure rno-miR-30b-5p levels and IL-1 0,TLR4 expressions in spleen and lymph node,quantitative PCR and ELISA techniques were applied.Results The results of double lucfferase reporter gene expression analysis showed rno-miR-30b-5p mimic apparently down-regulated the fluorescence intensity of both IL-10 and TLR4 in wild type cells.After the mutation of the target site,the fluorescence intensity of the mutant vector was significantly reduced,accompanied by a significantly statistical difference (all P ＜ 0.01).Moreover,animal results revealed the expressions of rno-miR-30b-5p were apparently decreased,whereas IL-10 and TLR4 were markedly increased in both lymph node and spleen (all P ＜ 0.05).Conclusion Target identification shows that rno-miR-30b-5p can obviously regulate the expressions of 3'UTR gene with either IL-10 or TLR4 gene fragment,though its regulation might not be through the predicted site.The down-regulated expression of rno-miR-30b-5p in both spleen and lymph node in EAU rats result in the up-regulated expressions of both IL-10 and TLR4,further influence the development of uveitis.This study paves a way for the modulation of microRNA on the occurrence and development of uveitis,and will provide a new insight on treating uveitis.

Autophagy is of highly conserved self degradation under physiological and pathological conditions.Recent studies has been shown that autophagy play the important role in the development of ocular diseases.This article reviews the role of autophagy in the development of ocular diseases,and provides new ideals for clinical treatment of ocular diseases.

Objective To study the immunomodulatory effects of Longdan Xiegan Tang on related inflammatory cytokines in rots with experimental autoimmune uveitis (EAU).Methods Lewis rats were randomly divided into control group (6 rats),EAU group (18 rats) and LXT group (18 rats).Rats in EAU and LXT groups were immunized with interphotoreceptor retinoid-binding protein (IRBP) emulsion.The expressions of IFN-γ,IL-17,IL-10 and TNF-α were investigated by quantitative real-time PCR and ELISA,respectively.Results In blood of LXT group,the mRNA level of IFN-γ on day 8 was higher than that in control group (P =0.000),but obviously lower than that in EAU group (P =0.000);The mRNA level of IL-17 was peaked on day 12,but was lower than EAU group (P=0.000);The mRNA level of TNF-α on day 12 was higher than that in control group (P =0.000),but obviously lower than that in EAU group (P =0.000);The mRNA level of IL-10 on day 16 was higher than that in EAU group (P =0.042).In lymph node and spleen of LXT group,the mRNA levels of IFN-γ,IL-17 and TNF-α on day 12 were lower than those in EAU group (all P=0.000);The mRNA level of IL-10 was peaked on day 12,but was lower than EAU group (P =0.000).In serum of LXT group,the mRNA levels of IFN--γ,IL-17 and TNF-α on day 12 and day 16 were lower than those in EAU group;The level of IL-10 in EAU and LXT group on day 12 were higher than that in control group,peaked on day 16,but the LXT group was the highest.Conclusion Longdan Xiegan Tang can reduce the expressions of proinflammatory cytokines including IFN-γ,IL-17 and TNF-α.Meanwhile,it can also promote the production of IL-10 and further accelerate the recovery of EAU,indicating that Longdan Xiegan Tang can play a significant role in treating uveitis.

Background The excessive growth of human Tenon fibroblasts (HTFs) is a primary cause of failure of anti-glaucomatous filtering surgery.To seek a drug of anti-fibrosis is of an important significance for improving the successful rate of anti-glaucomatous filtration surgery.Objective The goal of this study was to investigate the effect of paclitaxel on proliferation of HTFs in vitro.Methods Human Tenon tissue was obtained during the anti-glaucomatous filtering surgery.HTFs were cultivated using explant method and 3-6 generations of cells were used in the experiment.The cells were identified by immunochemistry using keratin,vimentin,fibronectin and S-100.Different concentrations (0,1 ×10-s,1 × 10-7,1 × 10-6 mol/L) of paclitaxel were added into the medium,and then the cell indexes (CI) in the various groups were detected by real-time cell electronic sensing (RT-CES) 24 hours after affection of paclitaxel.Apoptosis of the cells was examined using DAPI staining,and the proportion of the cells in different cycles were assayed by flow cytorneter 12 hours after addition of paclitaxel.The expressions of matrix metalloproteinase-1 (MMP-1) protein and mRNA were detected by ELISA and real-time PCR,respectively.Results The cells migrated from the cultivated tissue within 7 days with the fibrocyte-like shape.The cells showed the positive response for vimentin and absent response for keratin,fibronectin and S-100.The CI values were 1.093 ±0.191,0.665 ± 0.093 and 0.473 ± 0.117 in the 1 × 10-8,1 × 10-7 and 1 × 10-6 mol/L paclitaxel groups,showing significant rise in comparison with the 1.514 ±0.283 of the 0 mol/L paclitaxel group (all at P =0.000).The cell nuclei were normal in the 0 mol/L paclitaxel group,however,blue-fluorescent particles and apoptotic bodied were found in the cell nuclei after affection of paclitaxel.The proportion of G2/M phase of cells were (9.20±0.80) ％,(12.37±0.45)％ and (13.80±0.35)％ in the 1×10-8 mol/L,1×10-7 mol/L and 1×10-6 mol/L paclitaxel groups,which were higher than the (7.17±0.50) ％ in the 0 mol/L paclitaxel group (P=0.005,0.000,0.000).In addition,the relative expressing level of M MP-1 mRNA (MMP-1 mRNA/GAPDH mRNA) and the expression level of MMP-1 protein in the HTFs were significantly lower in the 1 ×10-8 mol/L,1 × 10-7 mol/L and 1 × 10-6 mol/L paclitaxel groups than those in the 0 mol/L group (all at P＜0.05).Conclusions Paclitaxel at the concentrations of 1 × 10-8 mol/L-1 × 10-6 mol/L inhibits the proliferation of HTFs in vitro by arresting the cellular mitosis and inducing cell apoptosis.These effects probably associated with down-regulation of MMP-1 expression in the HTFs.

Background Plasma membrane calcium ATPase 3 (PMCA3) participates in the regulation of Ca2+ level in lens epithelial cells (LECs),which may be associated with the pathogenesis of cataract.It has been proved that ultraviolet B (UVB) is one of causing-factors of cataract.However,the effect of UVB on the expression of PMCA3 in LECs is unclear.Objective This study was to investigate the effects of UVB irradiation on the expression of PMCA3 in human LECs B-3.Methods HLE B-3 cells were cultured and passaged.The cells were exposed to 0,5,10 and 20 mJ · s/cm2 UVB for 0,20,40,80 s,respectively and further cultured for 24,48 and 72 hours.MTT assay was used to detect the cell proliferation rate.JC-1 staining was used to detect the changes of mitochondrial membrane potential (△ψm) in the cells.The intracellular reactive oxygen species (ROS) level was detected by DCFH-DA staining,and cell apoptosis was evaluated using annexin V-FITC/PI staining.In addition,the intracellular calcium ion (Ca2+) concentration in the cells was assayed with Fluo-3/AM staining.The expression levels of PMCA3 mRNA and PMCA protein in the HLE B-3 cells were detected by real-time PCR and Western blot,respectively.Results The survival rates of the cells were significantly reduced with the increase of irradiated intensity of UVB and the lapse of time (Fgroup =72.411,P =0.000 ; Ftime =36.588,P =0.000),and the survival rates of the cells in the 10 mJ · s/cm2 and 20 mJ · s/cm2 UVB for 24 hours were (75.3 ± 2.2) ％ and (48.7 ±4.5) ％,respectively,which were significantly lower than those in the 0 mJ · s/cm2 UVB group ([100.0±0.0] ％) (P=0.001,0.000).The survival rates of the cells in the 5,10,20 mJ · s/cm2 UVB for 48 hours were (84.9± 1.2) ％,(69.3±17.4)％ and (32.8±4.5)％,showing significant declines in comparison with the 0 mJ · s/cm2 UVB group ([100.0±0.0] ％) (P =0.047,0.000,0.000).In 72 hours following 5,10,20 mJ · s/cm2 UVB irradiation,the survival rates of the cells were (55.1 ± 3.0) ％,(42.1 ± 1.9) ％ and (26.1 ±4.7) ％,respectively,with significant differences in comparison with the 0 mJ · s/cm2 UVB group ([100.0 ± 0.0] ％) (P =0.000,0.000,0.000).JC-1staining exhibited the intracellular red fluorescence in the normal cells group.However,in the 5 mJ · s/cm2 UVB group,weak green fluorescence was seen,and the green fluorescence was enhanced in the 10 mJ · s/cm2 and 20 mJ · s/cm2 UVB groups.After irradiated by 5,10 and 20 mJ · s/cm2 UVB,the ROS levels in the cells increased from 0.4％ to 35.8％,51.9％ and 76.7％,respectively.The apoptosis and necrosis rate of the cells was 2.0％ in the 0 mJ · s/cm2 UVB and 4.2％,7.6％,15.1％ in the 5,10,20 mJ · s/cm2 UVB groups,respectively.The Ca2+ level raised by (1.2±0.1) and (1.3±0.1) folds in the 10 and 20 mJ · s/cm2 UVB groups more than that in the 0 mJ · s/cm2 group (P =0.039,0.004).The expression levels of PMCA3 mRNA in the cells were significantly reduced (P=0.001,0.004,0.000),and the expression levels of the PMCA protein were declined in the 5,10 and 20 mJ · s/cm2 UVB groups compared with the 0 mJ · s/cm2 UVB group (P=0.000,0.000,0.001).Conclusions UVB irradiation causes cataract probably through downregulating the expression of PMCA3 in human LECs and inducing apoptosis of LECs in a dose-and time-dependent manner.

To foster interdisciplinary talents with the highly fusion of clinical skills and the capacity for scientific research, a preliminary exploration of teaching for the cultivation of scientific innovation ability was carried out for clinical postgraduate students. In the cultivation of clinical post-graduate students' innovation consciousness and innovative spirit, we focused on the transformation of attaching more importance to the clinical skills than to the capacity for scientific research to establish the foundation of the competitive compound talents. Then in the early stage of medical project writing, basic knowledge learning and exchange, we stressed clinical postgraduate students' solid grasp of basic knowledge of medical science to consolidate the way of enhancing the scientific research ability. Furthermore , under the guidance of the second research supervisors allocated by the department , we strengthened the clinical postgraduate students' writing of scientific research project bid and pro-fessional paper to promote the organic combination of the clinical practice and scientific research innovation and enhance their scientific research ability.