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@#【Objective】To evaluate the effect of preoperative conjunctival sac irrigation with Anerdian Ⅲ disinfectant and its effect on the function of eye surface and tear film. 【Methods】 This clinical study was involved of thirty cases （30 eyes） undergoing phacoemulsification. Conjunctiva sac irrigation with Anerdian disinfectant （type III ，5g/L） was performed before the phacoemulsification procedure. Secretions of conjunctiva sac were examined by bacterial culture pre-and post-irrigation. Schirmer test，non-invasive first tear break-up time（NITBUT-F），non-invasive average tear break-up time（NITBUT-A），tear meniscus height（TMH），and goblet cell density（GCD）were also evaluated day 1 before and day 7 ，day 30 after operation.【Results】3 cases were positive in bacterial culture pre-irrigation ，while all were negative post-irrigation. There was no significantly change between Schirmer test and TMH from day 1 before ，day 7 and day 30 after operation . NITBUT-F，NITBUT-A，and GCD were significantly different between day 1 before and day 7 after operation（P<0.05），and shown no significantly difference between day 1 before and day 30 after operation.【Conclusions】 Our results suggested that conjunctiva sac irrigation with Anerdian disinfectant was efficient，while it might be related to the decrease of GCD and dysfunction of tear film. However，GCD would gradually improve as pre-operation and function of tear film could restore to normal.
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@#【Objective】To detect the ability and the efficiency of recombinant PTD-HSP27 transport across RGC-5 cells and measure the role of recombinant PTD- HSP27 against oxidative stress damage induced by cobalt chloride on RGC- 5 cells，and explored its potential mechanism tentatively. 【Methods】 RGC- 5 cells were incubated with PTD- HSP27 labeled with FITC，followed by the observation of fluorescence using fluorescence microscope. We also lysed the cells by radio immunoprecipitation assay and measure the transport efficiency of PTD- HSP27- FITC through ultraviolet spectrophotometer subsequently. We established the RGC- 5 cell damage model. Cellular experiments were divided into three groups：normal control group，cobalt chloride damage group，PTD-HSP27 + cobalt chloride treatment group. Those three groups of cells were experimented using Annexin V- FITC/PI staining kit：to detect apoptotic cell ratio；Western Blotting：to detect the expression level of apoptosis related protein，including Bcl-2，Bax and Caspase-3.【Results】The fluorescence of PTD- HSP27- FITC was visualized inside the RGC- 5 cells using the fluorescence microscope，while the transport efficiency of PTD-HSP27-FITC was（47.29 ± 2.33）%. Cobalt chloride inhibited the survival vitality of RGC- 5 cells in a concentration- dependent manner. The comparison among normal control group，cobalt chloride damage group and PTD-HSP27+CoCl2 treatment group showed：PTD-HSP27 could effectively suppressed early apoptosis induced by cobalt chloride（P<0.01）. Western Blotting results showed that PTD-HSP27 could effectively enhance the expression ratio of Bcl-2/Bax，and suppressed the activation of Caspase- 3（P<0.01）.【Conclusions】Recombinant protein PTD- HSP27 could protect RGC- 5 cells against oxidative stress injury induced by cobalt chloride. Iit mainly regulated the expression ratio of Bcl-2/Bax，suppressed early apoptosis and improved cell viability in RGC-5 cells.
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?AlM: To evaluate the clinical effects and security of posterior chamber implantable Collamer lens ( lCL ) implantation in patients with extreme highly myopia. ?METHODS:ln this study, 18 patients ( 32 eyes ) with extreme highly myopic patients who had undergone posterior chamber lCLs implantation from July 2010 to July 2013 were evaluated. Diopter -10. 5 ~ 19. 0D, and astigmia -0. 5 ~4. 5DC. Changes in intraocular pressure ( lOP ) , refraction, visual acuity and corneal endothelium, anterior chamber depth, iris, high arch, lens were noted at 1d, 1wk, 1, 3mo and 1a after surgery respectively, and follow-up was of 1a. ? RESULTS: Before surgery, the uncorrected visual acuity (UCVA) were 0. 01~0. 05, and the best spectacle-corrected visual acuity ( BSCVA) were 0. 4 ~ 1. 0. One month after surgery, the UCVA were 0. 5~1. 2. The mean vault were 547±222 μm (95%CI 442~672μm) and 528±268μm (95%CI 354 ~635μm) for 1mo and 1a, respectively (P = 0. 81), and there was no significant difference. Anterior subcapsular opacities in 1 eye, mild and transient increase in lOP in 3 eyes, and chronic pigment dispersion in 2 eyes were observed. There was no serious complication. ?CONCLUSlON: Posterior chamber phakic intraocular lens implantation is an effective and safe method for correcting patients with extreme highly myopia.
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Progressive loss of retinal ganglion cells (RGCs) and their axons is the main pathogenesis of glaucoma. The cause of glaucoma is not fully understood, but the neurodegeneration of glaucoma involves many mechanisms such as oxidative stress, glutamate toxicity and ischemia/reperfusion insult. In order to target these mechanisms, multiple neuroprotective interventions have been investigated to prevent the death of RGCs. Of note are some tonic herbs from the traditional Chinese medicine (TCM) pharmacopeia that have shown neuroprotective effects in glaucoma. TCM differs from Western medicine in that TCM exhibits complicated bioactive components, triggering many signaling pathways and extensive actions on vital organs. Modern scientific approaches have demonstrated some of their underlying mechanisms. In this review, we used Lycium barbarum and Ginkgo biloba as examples to elaborate the characteristics of TCM and their potential applications in neuroprotection in glaucoma.
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Humans , Clinical Trials as Topic , Drugs, Chinese Herbal , Therapeutic Uses , Ginkgo biloba , Glaucoma , Drug Therapy , Medicine, Chinese Traditional , Neuroprotective Agents , Therapeutic Uses , Retinal Ganglion Cells , PathologyABSTRACT
Background The construction of tissue-engineered corneal endothelium needs the functional seeding cells,so how to culture a large amount of functional corneal endothelial cells (CECs) is an urgent problem to be solved.Objective The aim of this study was to evaluate the role of aqueous humor on bovine CECs in vitro.Methods Aqueous humor of 1.2 ml was collected from the anterior chamber of bovine and sterilized,and the liquid supernatant was obtained.The bovine CECs were isolated from bovine cornea and then cultured in low glucose Dulbecco Modified Eagle Medium with 10％ fetal bovine serum (FBS) in vitro.Aqueous humor was added into the medium with the final concentration of 2.5％,5.0％,l0.0％,15.0％ and 20.0％,respectively,and no aqueous humor was added in the control group.Cell counting kit-8 (CCK-8) assay was used to detect the absorbency value of CECs for the evaluation of cell proliferation.Progression of the cell cycle was analyzed by flow cytometry (FCM).After confluence of the cells was reached,1 ml plastic spear tip was used to scratch the cell single layer,and the cells were incubated consequently in medium with 10％ FBS and with or without aqueous humor for 24 hours.Healing area of the cell single layer was measured.The cells were incubated at a density of 6 × 105 cells/ml and cultured using medium with or without 10.0％ aqueous human for 5 days,and the number of the cells was analyzed by DAPI fluorescence technique.Results Under the phase-contrast microscopy,the confluent CECs showed a slabstone-like and hexagonal appearance.CCK-8 assay revealed that the absorbance values of CECs was significantly different among the various culture groups (F=4.051,P =0.007),and the absorbance value in different concentrations of aqueous human culture groups was significantly higher than that in the control group (P ＜ 0.01).FCM showed that the percentage of the cells in S-G2 phases was (34.80-±3.13)％ in the 10.0％ aqueous humors group and (23.06±1.13)％ in the control group,showing a significant difference (t =-5.729,P=0.005).The scratch test showed that the healing area of the cell signal layer was (0.116±0.019) mm2 in the 10.0％ aqueous humors group and (0.358 ±0.049) mm2 in the control group,showing a significant difference (t =13.842,P =0.000).The density of cells in the 10.0％ aqueous humor group was (1439± 1 10)/field,which was more than (1162±45)/field in the control group (t =-11.020,P=0.000).Conclusions Aqueous humor at the concentration of 10.0％ promote the growth and proliferation of bovine CECs.The result suggests that 10.0％ aqueous humor can be used as a promoting agent during the culture of CECs.
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Background Induced pluripotent stem cells (iPSCs)can differentiate into various types of somatic cells without causing ethical controversy and immune rejection in clinical activity,which is similar to differentiation ability of embryonic stem cells.So,iPSCs may be used as seed cells for tissue engineering corneal endothelial reconstruction.Objective The present study was to survey the morphologic change of iPSCs after coculture with corneal endothelium cells(CECs) under the atomic force microscopy(AFM).Methods Rabbit CECs and human MMC-iPSCs were isolated and cultured respectively.The iPSCs were identified with the marker by immunochemistry.iPSCs passaged for 7 days were then cultured with 60％ confluent CECs to establish the co-culture model.The surface morphology and cellular membrane ultrastructure of differentiated iPSCs after induced by CECs were examined by AFM combination with inverted microscope,and compared with CECs and undifferentiated iPSCs.Results Thelengthand width were(66.93±10.48)μm and (44.85 ± 8.14) μm in CECs,(12.51±1.40)μm and (10.93 ±1.69) μm in uninduced iPSCs,and(36.12±10.29) μm and(31.53±9.65)μm in CECs-induced iPSCs.Both the length and width values of CECs-induced iPSCs were statistically bigger than those uninduced iPSCs,with significant differences between them (P＜0.05),but no significant difference was seen in the width valne of CECs-induced iPSCs in comparison with CECs(P＞0.05).The convex structure of CECs cytomembrane surface showed the digitation in shape with the size and height(2.11 ± 1.03) μm and (115.68±92.08) nm respectively,and the concave structure of cytomembrane surface of CECs was fenestrae-like depression and the size was (1.49 ± 0.65) μm.The numerical valuc of mean square root roughness (Rq)and average roughness (Ra)of cytomembrane surface of CECs were(39.20±7.82)nm and (30.37±5.32)nm respectively.The convex surface of cytomembrane of iPSCs was granular-like in shape with size and height(0.39±0.22)μm and(13.11±9.18)nm respectively.The concave surface of cytomembrane of iPSCs was worm-eaten-like concave with the size(0.34±0.18)μm.The numerical value of Rq and Ra of geometrical parameters of cytomembrane surface of iPSCs were (26.60 ± 4.93)nm and (9.97 ± 3.78) nm respectively.The convex surface of cytomembrane of induced iPSCs was digital-like in shape with the size and height (1.91±0.76) μm and(106.55±77.27) nm respectively.The concave surface of cytomembrane of induced iPSCs was fenestrae-like depression and the size of concave was(1.6l±1.25) μm.The numerical value of Rq and Ra on surface of cytomembrane of induced iPSCs was (57.33± 12.80) nm and (43.63± 11.17) nm respectively.The numerical values of the size and height of convex,the size of concave,Rq and Ra on surface of cytomembrane in induced iPSCs were statistically bigger than in iPSCs(P＜0.05)and were not significant differences in comparison with CECs (P＞0.05).Conclusions Morphology of iPSCs translate toward the CECs after induce for 7 days under the AFM.This outcome lays the foundation for further study on iPSCs.
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Background Clinical studies indicated that the pathogenesis of most corneal dystrophy is associated with the mutation of the transforming growth factor beta-induced (TGFBI) gene.However,the molecular mechanism of mutated TGFBI gene in corneal dystrophy is unclear. Objective The present study was to investigate the expression of the TGFBI gene in human corneal tissue and cells in vitro.MethodsHuman corneal epithelial cells and keratocytes were cultured and passaged,and donor corneal tissue was obtained for the section preparation.RT-PCR was used to detect the expression of TGFBI mRNA in human corneal tissue and cells.Immunofluorescence was used to test the expression of the TGFBI protein in the human corneal tissue,and immunohistochemistry was used to test the expression of the TGFBI protein in human corneal epithelial cells and corneal stromal cells.ResultsRT-PCR analysis showed that TGFBI mRNA could be detected as a 1274 bp band in human corneal tissue and corneal stromal cells,but no TGFBI mRNA was observed in corneal epithelial cells.Immunofluorescence assay revealed that corneal stromal cells were positive ly expressed for the TGFBI protein,but the corneal epithelial cells did not express the TGFBI protein.Immunohistochemistry indicated that the expression of TGFBI was detected the red fluoressence in the cytoplasm of corneal stromal cells;however,no positive response was found in corneal epithelial cells.ConclusionsThe expression of the TGFBI gene occurs in human corneal stromal cells but not in the corneal epithelial cells.This result might be of helpful for studying the function and role of TGFBI gene in pathogenesis of corneal dystrophy.
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Background The human transforming growth factor beta-induced gene (TGFBI) is the first determined pathogenic gene to corneal dystrophy.But the molecular genetic mechanism is completely unknown.The study of concerning role of TGFBI is very important for us understand the physiological function of cornea,and the pathogenesis of corneal dystrophy.Objective The vector of human transforming growth factor beta-induced gene (TGFBI) in eukaryotic expression was constructed and transfected into the human corneal epithelial cells in order to explore its influence on the growth of human corneal epithelial cells.Methods Total RNA was extracted from normal donor cornea tissue and cDNA was obtained by reverse transcription.TGFBI cDNA was synthesized by reverse transcription-PCR and cloned into pCMV-N-HA vector and identified by sequencing with PCR and EcoRV,XhoI double restriction endonuclease.The cells were grouped into recombinant pCMV-N-HA-TGFBI plasmid group,pCMVN-HA plasmid group,non-transfected group and pGFP-C2 transfected group.The recombinant pCMV-N-HA-TGFBI plasmid was transfected to human corneal epithelial cells and identified by observing the expression of enhanced green fluorescence protein(EGFP) in the cells.The TGFBI mRNA and proteins were harvested from the cells for real-time PCR analysis and Western blot assay respectively in 58 hours after transfection.The growth of the transfected cells was assessed by Cell Counting Kit-8.The expressions of matrix metalloproteinase(MMP) and tissue inhibitors of matrix metalloproteinase (TIMP) proteins and their mRNA in transfected cells were detected using SYBR fluorescence realtime PCR analysis and Western blot assay.Results The sequencing result of pCMV-N-HA-TGFBI positive clone plasmid showed that amplified TGFBI eDNA inserted into the vector at the correct sequence.EGFP was expressed in transfected cells in 48 hours after transfer of pGFP-C2 with the transfer efficacy 70％.The expression intensity of TGFBI mRNA was significantly higher in recombinant pCMV-N-HA-TGFBI plasmid group compared with pCMV-N-HA plasmid group and non-transfected group,and TGFBI protein was expressed in recombinant pCMV-N-HA-TGFBI plasmid group.No significant difference was found in the A450value among recombinant pCMV-N-HA-TGFBI plasmid group,pCMV-N-HA plasmid group and non-transfected group ( F=3.34,P＞0.05 ).The mRNA level of MMP1,MMP3in the transfected cells was significant elevated but that of TIMP1 was declined in the recombinant pCMV-N-HA-TGFBI plasmid group compared with pCMV-N-HA plasmid group and non-transfected group (all P ＜ 0.05 ).Meanwhile,the expressions of MMP1,MMP3 and TIMP1 proteins appeared the same tendency( all P＜0.05).Conclusions Eukaryotic expression vector harboring human TGFBI eDNA can be successfully constructed and efficiently overexpressed in human corneal epithelial cells.TGFBI gene is involved in the physical and pathological conditions of human corneal epithelial cells by regulating the activity of MMP1,MMP3 and TIMP1.The results offer a new approach for the study of the role of TGFBI in pathogenesis of corneal transparency.
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<p><b>OBJECTIVE</b>To investigate the role of oxidative stress in the pathogenesis of retinal injury induced by hyperoxia.</p><p><b>METHODS</b>Sixty immature Sprague-Dawley (SD) rats born at a gestational age of 21 days, were randomly exposed to room air (air group, n=30) or 95% oxygen (hyperoxia group, n=30) immediately after birth. Plasma 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha) levels were determined by ELISA. The ultrastructures of the retina were observed under a transmission electron microscope.</p><p><b>RESULTS</b>The plasma 8-iso-PGF2alpha contents of the air group were 19.09 +/-5.57, 18.24+/-5.91 and 17.00 +/- 5.58 pg/mL on the 3rd, 7th and 14th days after birth, respectively (F=1.024, P> 0.05). The plasma 8-iso-PGF2 contents in the hyperoxia group on the 3rd (28.33 +/- 5.59 pg/mL), the 7th day (51.20 +/- 15.01 pg/mL) and 14th day (84.54 +/- 14.85 pg/mL) after birth were significantly higher than those of the air group (t=2.863, P< 0.05; t=5.073, P< 0.01; t=11.006, P< 0.01). Moreover, the plasma 8-iso-PGF2 contents in the hyperoxia group increased with the prolonged hyperoxia exposure (F=150.7, P < 0.01). The ultrastructures of retina in the air group were normal. Hyperoxia exposure resulted in abnormalities of the ultrastructures of retina, manifesting as the membrane discs rarefied, twisted and disrupted and mitochondrial swelling.</p><p><b>CONCLUSIONS</b>Oxidative stress can results in retinal injury in immature rats. An increased plasma level of 8-iso-PGF2alpha is related to the injury degree of retina.</p>