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@#【Objective】To form a new PTMAc- PEG- PTMAc triblock（PPP）copolymerhydrogel and to evaluate its sustained released performance and antiproliferative effects as an ocular drug carrier of Pirfenidone（PFD）【Methods】One type triblockcopolymers PPP hydrogel was chosen. The morphology of the material was evaluated by scanning electron microscopy（SEM）. The swelling properties of the PPP hydrogel was analyzed in PBS solution（37 ℃ ，pH 7.4）. The in vitro drug release of the pirfenidone loaded hydrogels were evaluated with non-dialysis method and the curves of drug release were drawed. We evaluated the adhesion，survival of human Tenon′s capsule fibroblasts（HTF）around hydrogels. The cell cytotoxicity of hydrogels and antiproliferative effects were evaluated through CCK-8 assay.【Results】The hydrogel has stable gelation conditions. The swelling rate decreased by increasing hydrogel concentration.The SEM images indicated the fibrous and porous structure. We also observed that the encapsulated pirfenidone were sustained released from hydrogels with an initial burst release at early stage（within 4 d）and then the release rate were declined for all hydrogels during the following 14 d. The PPP hydrogel can inhibit cell adhesion. The cell viability in hydrogels at four time point（24，48，72 and 96 h）were 85.7% ，93.0% ，82.0% ，81.6%. The inhibition rate of drug loaded hydrogel with two drug concentration（1 mg/mL or 2 mg/mL）are 25.8%，21.8%，55.4%，25.6%；44.6%，35.9%，55.5%，31.4%. While that of drug solution are 28.9% ，29.7% ，7.8% ，7.7%. The suppressive effects of the PFD loaded hydrogels on HTF proliferation followed a dose-dependent fashion and time-dependent fashion.【Conclusions】Such biocompatible copolymers hydrogel can be effectively used as an drug sustain released system. It can induce significant inhibition of HTF proliferation. With equal amount of drug，the inhibition effect of drug loaded gel was longer than that of drug solution.
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Background The rat model of N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell apoptosis is often used to study retinal degeneration.But the changes in the gene expression patterm in retinal degeneration in rats have not been reported.Objective This study was undertaken to investigate regulation of gene expression in the retina of MNU-induced retinal degeneration in rats by performing microarray analysis of retinal RNA.Methods Fifty 6-week-old SD rats were numbered and randomized into the normal group and the model group.The retinal degeneration model was established by a single hypodermic injection of 40 mg/kg of MNU,and the rats in the normal group received equivalent volume of physiological saline in the same way.The rats were sacrificed 12 hours or 24 hours after injection.Retinal sections from the right eyes were prepared for the measurement of the retinal thickness by histopathological examination,and retinas from the left eyes were used to confirm the differential gene expression as detected by microarray (normal group and 12 hours model group).Genes exhibiting changes in expression by ≥2.0 folds were further confirmed using real-time PCR.Results The whole thickness of the retina declined in the rats from the 24 hours model group compared to the normal group and 12 hours model group (t =9.926,P=0.002;t=2.736,P=0.028).The thickness of the outer nuclear layer was (26.58±2.90) μm in the 24 hours model group,showing a significant decrease in comparison with (38.11 ± 1.01) μm in the normal group and (35.07t3.03) μm in the 12 hours model group (t=6.028,P=0.009;t=6.839,P=0.006).However,there was no significant difference in retinal thickness between the normal group and the 12 hours model group (whole thickness:t=1.541,P=0.324;outer nuclear layer thickness:t=2.040,P=0.134).Microarray analysis of the rat genes showed that out of 17 000 genes,142 genes involved in biological process and 94 genes involved in molecular functions were differentially expressed,where most of them participate in the mitogen activated protein kinase signaling pathway,Tolllike receptor signaling pathway and apoptosis pathway.Real-time PCR analysis demonstrated that the expression of CCL2,IL-1b,CCL3,c-fos,c-myc,p53 and MMP3 were consistently up-regulated,conforming with the results from microarray analysis.Conclusions The changes in gene expression pattern appear in the early stage of MNUinduced retinal degeneration.These microarray results provided clues to understanding the molecular pathways underlying photoreceptor degeneration and indicating the directions for future studies.
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BackgroundOur previous study determined that tetrandrine (Tet) has an inhibitory effect on the proliferation of human Tenon capsule fibroblasts ( TCFs ) in vitro,but its mechanism is poorly understood.ObjectiveThis study was to investigate the effect mechanism of Tet on human TCFs.MethodsHuman TCFs were isolated and cultured from scleral tissue of donor using explant technique.The cells were identified by vimentin antibody staining and morphology.The third generation of cells were seeded in the culture plate at the density of 1 × 105 cells/ml.Twenty-four hours after inoculation,the Tet of 1 × 10-5 mol/L was added in the well of culture plate,and the cells cultured only in 1640 medium served as the control group.The apoptosis of the cells was assessed by TUNEL,and the expressions of bax,bel-2,transforming growth factor-β2 (TGF-β2 ) in TCFs were detected using immunochemistry.Results The cultured cells showed the features of the fibroblasts in shape with the positive response for vimentin.A number of TUNEL positive cells were seen in Tet group and no TUNEL positive response was found in control group.The expression levels (A value) of bax,bcl-2 and TGF-~ protein in TCFs were 0.577 ± 0.009,0.430±0.012 and 0.341 ±0.017 in Tet group,and those in control group were 0.320±0.015,0.819±0.021 and 0.624±0.014 respectively,showing statistically significant differences between two groups( t =33.277,-35.356,-28.093,P＜0.01 ).Conclusions Tet suppresses the proliferation of human TCFs through up-regulating the expression of bax and down-regulating the expressions of bcl-2 and TGF-β2 in vitro.
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<p><b>BACKGROUND</b>Accumulating evidence indicates that both innate and adaptive mechanisms are responsible for the postnatal development of the mammalian visual cortex. Most of the studies, including gene expression analysis, were performed on the visual cortex during the critical period; few efforts were made to elucidate the molecular changes in the visual cortex during much earlier postnatal stages. The current study aimed to gain a general insight into the molecular mechanisms in the developmental process of the rat visual cortex using microarray to display the gene expression profiles of the visual cortex on postnatal days.</p><p><b>METHODS</b>All age-matched Sprague-Dawley rats in various groups including postnatal day 0 (P0, n = 20), day 10 (P10, n = 15), day 20 (P20, n = 15) and day 45 (P45, n = 10) were sacrificed respectively. Fresh visual cortex from the binocular area (Area 17) was dissected for extraction of total RNA for microarray analyses. Taking advantage of annotation information from the gene ontology and pathway database, the gene expression profiles were systematically and globally analyzed.</p><p><b>RESULTS</b>Of the 31 042 gene sequences represented on the rat expression microarray, more than 4000 of the transcripts significantly altered at days 45, 20 or 10 compared to day 0. The most obvious alteration of gene expression occurred in the first ten days of the postnatal period and the genomic activities of the visual cortex maintained a high level from birth to day 45. Compared to the gene expression at birth, there were 2630 changed transcripts that shared in three postnatal periods. The up-regulated genes in most signaling pathways were more than those of the down-regulated genes.</p><p><b>CONCLUSIONS</b>Analyzing gene expression patterns, we provide a detailed insight into the molecular organization of the developing visual cortex in the earlier postnatal rat. The most obvious alteration of gene expression in visual cortex occurred in the first ten days. Our data were a basis to identify new relevant candidate genes that control visual cortex development.</p>
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Animals , Rats , Gene Expression Profiling , Gene Expression Regulation, Developmental , Genetics , Physiology , Oligonucleotide Array Sequence Analysis , Visual Cortex , MetabolismABSTRACT
<p><b>BACKGROUND</b>Equatorial lens epithelial cells proliferate and differentiate into fiber cells throughout life, while central lens epithelial cells proliferate little and do not form fiber cells. This study aimed to investigate the differences in gene expression between the central and the peripheral epithelial cells of the bovine lens.</p><p><b>METHODS</b>Lens epithelia were dissected into central (<or= 11.5 mm diameter, cLEC) and peripheral regions (pLEC). The differences in gene expression and protein accumulation between these two regions were assayed by microarray analysis and two-dimensional electrophoresis (2-DE) combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Differently expressed proteins were validated by immunoanalyses.</p><p><b>RESULTS</b>By microarray analysis, 67 transcripts were at least two-fold lower and 269 at least two-fold higher in pLEC compared with that in cLEC. Thirty-four protein spots, including 20 in cLEC and 14 in pLEC, were identified by two dimensional electrophoresis and mass spectrometry. Of these 34 protein products, 28 were represented by probe sets on the microarray. Nine transcripts changed in the same direction and four transcripts in the opposite direction to their protein products. Immunoanalyses revealed that three (mitogen-activated protein kinase 1 (MAPK1), nidogen (NID), small nuclear ribonucleoprotein N (SNRPN)) out of four transcripts with opposite change between 2-DE and microarray assay showed the same changes as the results of 2-DE gel analyses. The genes differently expressed between cLEC and pLEC mainly include those related to the MAPK, transforming growth factor beta (TGFbeta) signaling and glycolysis pathways.</p><p><b>CONCLUSION</b>The results suggested that there were distinctly different genome activities, including a specific group of pathways, between central and peripheral lens epithelial cells.</p>
Subject(s)
Animals , Cattle , Physiology , Electrophoresis, Gel, Two-Dimensional , Epithelium , Metabolism , Gene Expression Profiling , Gene Expression Regulation , In Vitro Techniques , Lens, Crystalline , Metabolism , Oligonucleotide Array Sequence Analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
AIM:To find new biomarkers in the sera of retinoblastoma (Rb) patients with surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI TOF MS) and protein chip technique.METHODS:SELDI TOF MS, IMAC30 and CM10 protein chips were used to analyze the protein profiles from sera of 18 patients with Rb and 17 age matched controls. The protein profiling was analyzed statistically by Ciphergen protein chip software 3.0.2. The test was applied to compare the protein peak intensity. Fisher's exact test was used to compare the predominance of differential protein peaks appeared in patients.RESULTS:With IMAC30 protein chips, there were 26 proteins which appeared different in sera of patients with Rb compared to normal children. Among them, 21 proteins, I.e. 7746, 7014, 11713, 3049, 7084, 7299, 5888, 2544, 12575, 5489, 9658, 9575, 9929, 10161, 8955, 1886, 10617, 6209, 2411, 7374, 6614m/z were up regulated and 5 proteins, I.e. 8382, 7923, 7972, 8590, 66576m/z, were down regulated(P<0.01). Using the 7014 protein peak for statistical analysis, we could differentiate the patients with Rb from the healthy children with a sensitivity of 94.4% and a specificity of 82.4%. By CM10 protein chips, 4 proteins, including 3 up regulated proteins(5888, 6097, 7798 and 1 down regulated protein (8590m/z), were detected in Rb patients (P<0.01). The sensitivity and specificity were 83.3% and 70.6% respectively when 7798m/z protein peak was selected for statistical analysis.CONCLUSION:There are a few candidates as Rb biomarkers in the sera of Rb patients. SELDI TOF MS protein chip technology could be a potential method in the clinical screening test of Rb.