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BACKGROUND:Transgenosis of basic fibroblast growth factor (bFGF) gene has been successfully performed into the muscle satellite cells of rat extraocular muscles in the previous study of the research group, proving that bFGF could express in the myoblasts of extraocular muscles, also promote cell proliferation and differentiation.OBJECTIVE: To further investigate the methods for regulating the expression of the bFGF in myoblasts following transfection. METHODS: Target gene bFGF was connected with inducing expression vector pcDNA4/T0/myc-His?A, followed by masculine clone sequencing identified by colony PCR and enzyme digestion, EcoR I and Hind III restriction enzyme digestion, as well as Xho I single enzyme verification. C2C12 myoblasts antibiotics sensitivity was screened and finally defined. By use of lipofection transfection technology, cell lines where C2C12 stably expressed pcDNA6/TR were estabolishd and then identified by Western blot. The pcDNA4/TO/myc-His?A-bFGF was transfected into pcDNA6/TR- C2C12 cells. The bFGF expression and secretion in C2C12 cells following tetracycline-induced pcDNA4/TO/myc-His?A-bFGF transfection were determined by immunofluorescence and Western blot, the controls were established.RESULTS AND CONCLUSION: ① The conjunction between the bFGF and inducing expression vector pcDNA4/TO/myc-His?A was proved successfully by sequencing comparison, double digestion and single digestion. ②The minimal lethal concentration of blasticidin to C2C12 cells was 10 mg/L, while that of zeocin was 750 mg/L. ③ The pcDNA6/TR-C2C12 cell lines were established correctly. ④ The myoblasts treated by tetracycline and transfected with pcDNA4/TO/myc-His?A-bFGF were positive for gene expression, those untreated exhibited a negativity; bFGF protein could be produced in myoblasts treated by tetracycline and transfected with pcDNA4/TO/myc-His?A-bFGF, the production reached a peak at 24 hours, while those untreated can not produce bFGF protein. Results suggest that the bFGF expression in the myoblasts can be controlled by tetracycline inhibition and regulatory systems.
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BACKGROUND:Taurine is an important non-enzymatic system antioxidant in the lens.The mechanism of its anti-oxidative effect is mainly to protect lens from oxidative injury by anti-lipid peroxidation.OBJECTIVE:This study was to observe the effect of exogenous taurine on lens epithefial cell apoptosis-induced by H2O2 in vitro.DESIGN:A randomized controlled animal experiment.SETTING:Department of Ophthalmology,Affiliated Hospital of Qingdao University Medical College.MATERIALS:Seventy-five adult New Zealand standard tabbits,of either gender,weighing 1.5-2.5 kg,were provided by Qingdao Laboratory Animal Center.Reagent kit for in situ detecting cell apoptosis(Sigma Company,USA),taurine and H2O2(Shanghai Guangda Chemical Reagent Factory,China)were included in this study.METHODS:This study was performed in the Department of Ophthalmology and Central Laboratory.Affiliated Hospital of Qingdao University Medical College from Mav 2005 to June 2007.Rabbit lenses were harvested and randomly divided into 3 groups:control group,in which,clear lenses were incubated in non-serum and non-phenolsulfonphthalein MEM medium which was renewed every 24 hours,H2O2 group,in which,clear lenses were incubated in non-serum and non-phenolsulfonphthalein MEM medium containing 1 mmol/L H2O2 with addition of 62 μL H202(30 g/L)every 6 hours,and H2O2+taurine group,in which,clear lenses were incubated in non-serum and non-phenolsulfonphthalein MEM medium containing 1 mmol/L H2O2 and 10 g/L taurine.which was renewed every 6 hours.The protocol was conducted in accordance with ethical guidelines for the use and care of animals.MAIN OUTCOME MEASURES:Observation of lens opacity 6.12,24.48 and 72 hours after culture;Lens epithelial cell apoptosis determined by DNA in situ end labeling and DNA fragment analysis.RESULTS:Lens opacity:The lens opacity in the H2O2 group was aggregrated gradually along with the time of oxidative injury.The lens opacity in the H2O2 group was severer than that in the H2O2+taurine group.Lens epithelial cell apoptosis:There were no apoptotic cells in the control group within 72 hours.The number of apoptotic cells in the H2O2 group was increased gradually with the prolonged time of oxidative injury.Till the 72nd hour,the cells were all tamed into apoptotic cells.A few aopototic celIs were found in the H2O2+taurine group since hour 24,and then were more and more,and the number of apoptotic cells accounted for about 30%at hour 72.The apoptotic rate in the H202+taurine group was significantly lower than that in the H2O group at each time point(q=8.6845,P＜0.01).There was no significant difference in apoptotic rate between the H202+taurine group and the control group (P＞0.05). Findings of DNA fragmentation assay:The DNA"ladder"was found in the H2O2 group at hours 24,36,48 and 72,while no DNA"ladder"but only normal electrophoresis straps were present in the other two groups 24 hours after culture.CONCLUSION:Taurine can inhibit the oxidative injury-induced apoptosis of rabbit lens epithelial cells,and alleviate lens opacity.
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BACKGROUND:Previous experiments reported that basic fibroblast growth factor(bFGF) can change the shape of corneal endothelial cells,accelerate cell division and chemotaxis,and repair corneal endothelia cells injury in vivo and in vitro. OBJECTIVE:To investigate the effect of bFGF on cat corneal endothelial cells proliferation. DESIGN,TIME AND SETTING:The cell morphological observation was performed at the Central Laboratory of the Affiliated Hospital of Medical College of Qingdao University between January 2005 and December 2006. MATERIALS:Domestic cats were purchased from Experimental Animal Center of the Affiliated Hospital of Medical College of Qingdao University. METHODS:The corneal endothelial cells of cats were primarily cultured,and neurone specific enolase(NSE) immunocytochemistry staining was performed. After cell passage,bFGF(1,10,100 ?g/L) was added,continuously hatched for 1-5 days. MAIN OUTCOME MEASURES:MTT method was used to determine the proliferation of corneal endothelial cells;meantime,inverted phase contrast microscope and transmission electron microscope were used to observe the ultrastructure of cells. RESULTS:At days 1,3 and 5 after bFGF was added,the absorbance value in 490 nm was significantly higher than that of control group,especially in 10 ?g/L group. CONCLUSION:The bFGF can promote proliferation of cat corneal endothelial cells with effective concentration of 10 ?g/L .
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BACKGROUND: Experiments have demonstrated that exogenous basic fibroblast growth factor (bFGF) promotes the growth and survival of skeletal muscle satellite cells, and endogenous bFGF also has obvious effect on muscular repair.But whether bFGF gene transfection has the same effect on extraocular muscle satellite cells is unclear.OBJECTIVE: The goal of this study was to investigate the effect of bFGF gene transfection on the bio-behavior of rat extraocular muscle satellite cells.DESIGN: Single sample observation.SETTING: Department of Ophthalmology, Qingdao University Medical College.MATERIALS: Primary rat extraocular muscle satellite cells were cultured (purity ＞ 90%). Human PEGFP-N3-bFGF eukaryotic expression vector was successfully constructed (reported in other papers).METHODS: This study was carried out in the Central Laboratory of Qingdao University Medical College between September 2005 and December 2006. Experimental intervention and grouping: During the experiment, 3 groups were divided: Experimental group, in which, recombinant PEGFP-N3-bFGF was used for transfection; Control group A, in which, empty-load PEGFP-N3 was used for transfection; Control group B: in which, only F 10 medium (0.1 volume fraction of fetal bovine serum)was used for transfection. Recombinant PEGFP-N3-bFGF plasmid was used to transfect rat extraocular muscle satellite cells cultured in vitro by liposome-mediated transgenic technology. Experimental evaluation: bFGF expression was observed by immubohistochemical method; Transfection efficiency was detected with fluorescence microscope; The protein secretion of bFGF of satellite cells in each group was detected by SBC-ELISA method on the 1st, 3rd, 5th, 9th, 11th, 13th, 15th, 21st and 28th days of culture; The proliferation of rat extraocular muscle satellite cells in each group was detected by methyl thiazolyl tetrazolium(MTT) method; Creatine kinase (CK) activity of cells in each group was determined according to the A value of some standard whose concentration was known.MAIN OUTCOME MEASURES: ① Transfection efficiency of bFGF gene to rat extraocular muscle satellite cells cultured in vitro and bFGF gene expression after transfection; ②bFGF protein secretion after transfection, and effect of bFGF gene transfection on the growth and proliferation of extraocular muscle satellite cells.RESULTS: ① Transfection efficiency of bFGF gene to rat extraocular muscle satellite cells cultured in vitrowas (42.8±1.2)%. ② Immunohistochemical detection showed positive reaction. ②Transfected cells could secrete bFGF protein,which was the highest on the 5th day (662.935 ng/L). ④ The proliferation activity of transfected cells was obviously enhanced, and its A value was significantly higher than that of control group at the same time point (P ＜ 0.05). ⑤CK value was higher than that of the control group from the 5th day after transfection to the end (P ＜ 0.01).CONCLUSION: bFGF gene transfering into rat extraocular muscle satellite cells can make extraocular muscle satellite cells to secrete bFGF, promote cell proliferation, survival and differentiation.
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BACKGROUND: Oxidation injury can lead to the apoptosis of lens epithelial cells. Some researches have found that body supplement of vitamin C is beneficial to inhibit oxidation injury by increasing the level of vitamin C in aqueous humor.OBJECTIVE: To observe the inhibitive effect of vitamin C on H2O2 induced apoptosis of lens epithelial cell.DESIGN: Randomized controlled trial taking animal's lens as subject.SETTING: Department of Ophthalmology, Medical College of Qingdao University.MATERIALS: 120 adult New Zealand rabbits of both genders, weighing 3-5 kg, were selected. Vitamin C and 300 g/L H2O2 were purchased from Shanghai Guangda Chemical Reagent Factory.METHODS: The experiment was carried out in the Central Laboratory, the Affiliated Hospital of Medical College of Qingdao University from January 2002 to January 2004. ①Culture of lens: 240 lenses were isolat ed from 120 rabbits after being killed. Among them, 192 clear lenses were selected and divided randomly to three groups with 64 lenses in each group: control group, H2O2 group and H2O2+vitamin C group. Lenses in the control group were incubated in non-serum and non- phenolsulfonphthalein MEM medium, those in the H2O2 group incubated in non serum and non- phenolsulfonphthalein MEM medium containing 1 mmol/L H2O2 (62 μL of 30 g/L H2O2 was added into the medium every 6 hours to keep H2O2 level maintain 1 mmol/L in the medium), and those in the H2O2+vitamin C group incubated in non-serum and non- phenolsulfonphthalein MEM medium containing 1 mmol/L H2O2 and 1 mmol/L vitamin C. Under the same culture condition, the lenses in each group were detected at hours 6, 12, 18, 24, 36, 48, 72 and 108 after culture, respectively. ②Measurement of the opacity of lens: Under the white background, two black lines 0.5 mm in width, which were mutually vertical with an interval of 10 mm. The lenses were placed above the crossing to measure the opacity. ③Detecting the apoptosis of lens epithelial cells:The apoptosis of lens epithelial cells was examined using TUNEL method and DNA fragmentation assay under light microscope to calculate the apoptotic rate of epithelial cells.MAIN OUTCOME MEASURES: ①Opacity of the lenses in each group;②apoptotic rate of lens epithelial cells in each group.RESULTS: ①After 108 hours of culture, the opacity of the lenses in the H2O2+vitamin C group was obviously lower than that in the H2O2 group. ②The apoptotic rates in the control group were lower that those in the H2O2group at different time point and those in the H2O2+vitamin C groupat hours 24, 48 and 72 after culture(P＜0.05-0.01). The apoptotic rates in the H2O2+vitamin C group were significantly lower than those in the H2O2group at different time points after culture (P＜0.01). ③The findings of DNA fragment analysis showed that the DNA "ladder" was found in the H2O2 group during 24-72 hours, while no DNA "ladder" but only normal electrophoresis strap were present in the other two groups 24 hours after culture.CONCLUSION: Lens can take refuge from the oxidative injury of H2O2with the addition of vitamin C. As a result, the apoptosis rate of lens epithelial cells and further, cataract formation can be restrained.
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<p><b>INTRODUCTION</b>To determine the differentiation of human limbal epithelial cells in tissue culture.</p><p><b>MATERIALS AND METHODS</b>Epithelial cells from the human limbus (n = 29) were isolated and cultured in supplemental hormonal epithelial medium (SHEM) in the presence of mitomycin C-treated 3T3 feeder layer. Confluent cells were airlifted to form multiple layers. The expression of cytokeratin 3 (K3), cytokeratin 12 (K12), involucrin, connexin 43 (Cx43), proliferation cell nuclear antigen (PCNA) and p63 was studied in normal and airlifted cells by immunohistochemistry. Expression levels of K3 and K12 mRNA were examined by real-time polymerase chain reaction (PCR).</p><p><b>RESULTS</b>The colony-forming efficiency of primary cultured (P0) cells was about 19.35 +/- 6.46% (mean +/- SD, n = 7). Real-time PCR analysis showed that the transcription level of K3 and K12 in cultured cells was lower than in freshly isolated limbal cells or cells from central cornea (P <0.01). Few cells were positive for K3 in P0 or P1 cells [(1.99 +/- 1.27)% (n = 7, P0) and (3.96 +/- 1.35)% (n = 4, P1), P = 0.046]. More cells at all levels were found to stain positive for PCNA and p63 as compared to K3, K12 and involucrin. After air-lifting, cell sheets of 3 to 5 epithelial cell layers formed. Involucrin showed positive staining in suprabasal layers of the cell sheets while connexin 43 was only observed in the basal layer. Staining of K3 remained sparse.</p><p><b>CONCLUSIONS</b>Human limbal cells isolated from cadaveric tissues were able to proliferate in vitro and exhibited a phenotype with characteristics similar to that of the limbal stem or progenitor cells.</p>
Subject(s)
Humans , Cell Differentiation , Coculture Techniques , Connexin 43 , Metabolism , Cornea , Cell Biology , Epithelial Cells , Immunohistochemistry , Keratins , Metabolism , Limbus Corneae , Cell Biology , Protein Precursors , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells , Cell Biology , Tissue Culture TechniquesABSTRACT
<p><b>OBJECTIVES</b>To observe the expression of betaig-h3 in normal cornea and keratoconus and to elucidate the role of extracellular matrix in keratoconus.</p><p><b>METHODS</b>In situ hybridization was used to detect the expression of betaig-h3 in the cornea. The cDNA library was screened with human betaig-h3 cDNA probe to locate betaig-h3 mRNA in cells.</p><p><b>RESULTS</b>Expression of betaig-h3 was found mainly in the stroma of the normal cornea and keratoconus, but decrease depending on the degree of keratopathy. In some serious cases, no expression signal was detected. The strongest expression was seen at the border of the normal region and keratoconus.</p><p><b>CONCLUSIONS</b>betaig-h3, the structural component of the extracellular matrix, can affect cell adhensiveness in the development of corneal fibrous interstitial organization. During the development of keratoconus, decreasing levels of betaig-h3 cause the diminution of corneal steadiness, which is related to formation of keratoconus.</p>