Objective To investigate the effect of hypoxia on expressions of erythropoietin(EPO)mRNA and protein in retinal M?ller cells cultured in vitro. Methods Retina tissues from the new-born Wistar rats were dissected into cell suspension after digested by pancreatin.M?ller cells were separated and purified by mechanical concussion and blowing and striking method.The expression of EPO mRNA and protein under the condition of hypoxia was detected by semi-quantitative reverse transcriptase(RT)-polymerase chain reaction(PCR)and immunocytochemical method. Results Retinal M?ller cells were cultured successfully,95% of which were positively stained by glial fibrillary acidic protein(GFAP).Positively stained EPO protein was located in the cytoplasm and protuberance.The expression of EPO mRNA and protein was faint in the normal retinal M?ller cells,but increased obviously and time-dependently after hypoxia. Conclusion Expression of EPO mRNA and protein increases in M?ller cells after hypoxia,which may be one of the protective factors for the nerves in anoxic retinopathy.

BACKGROUND: Basic fibroblast growth factor (bFGF), a kind of polypeptide growth factor possessing multifunctional biological activities,can protect neurons and promote the growth of nerves. It has been corfirmed that bFGF has therapeutic effects on retina ischemia/reperfusion injury (RIRI).OBJECTIVE: To establish RIRI model and analyze the effects of bFGF on cellular apoptosis of retina and the expression of regulatory gene protein.DESIGN: Randomized grouping and validating trial.SETTING: Department of Ophthalmology, the Affiliated Hospital of Medical College of Qingdao University.MATERIALS: The experiment was conducted at the Research Laboratory of Pathology, Department of Ophthalmology, Medical College of Qingdao University, from April 2002 to December 2003. Twenty-eight healthy Wistar rats were enrolled in this experiment. Four rats were randomly chosen for normal control group, the left eyes of the other 24 rats were set as normal saline control group, and the right eyes were set as bFGF group.METHODS: Normal saline control group and bFGF group adopted the rat RIRI models established by transiently elevating intraocular pressure. Normal saline of 12 μL was injected into the vitreous cavity of the left eyes of the rats in normal control group. 12 μL bFGF was injected into the vitreous cavity of the right eyes of the rats in bFGF group, 4 rats once. No administration was given in normal control group. The expression of apoptotic cells was detected and apoptosis indexes were calculated with the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) method and immunohistochemical staining method at the 1st, 6th,12th, 24th,48th and 72nd hours after reperfusion and ischemia for 1 hour.MAIN OUTCOME MEASURES: ① The detection results of apoptotic cells in situ of retina tissuesat different time points after reperfusion. ②The expression of Fas and caspases-2 in retina tissues at different time points after reperfusion.RESULTS ① Comparison of apoptosis indexes of retina tissues at different time points after ischemia reperfusion: There were no apoptotic cells in the retina tissues of the rats in normal control group. As compared with those in normal saline control group, apoptosis indexes in bFGF group were significantly decreased at ischemia 1 hour and reperfusion 1, 6, 12, 24, 48and 72 hours, especially at the 12th, 24th and 48th hours after reperfusion (t =5.362-5.595, P ＜ 0.05). ② The change of Fas expression at different time points after ischemia reperfusion: There was hardly any Fas expression in normal control group. As compared with that in normal saline control group, Fas expression in bFGF group was significantlydecreased at ischemia 1 hour and reperfusion 1, 6, 12, 24, 48 and 72 hours, especially at the 6th, 12th and 24th hours after reperfusion (t=3.954-9.327, P ＜ 0.05). ③The changes of caspase-2 expression at different time points after ischemia reperfusion: There was no caspase-2 expression in normal control group.Compared with that in normal saline control group, the number of caspase2 positive cells in bFGF group was significantly decreased at the 6th,12th,24th, 48th and 72nd hours after ischemia for 1 hour and reperfusion (t=4.125-15.641, P ＜ 0.05).CONCLUSION: bFGF can significantly inhibit the expression of apoptosis gene Fas and caspase-2 in the ischemia and reperfusion of retina, thus reducing cellular apoptosis of ganglion cells and exerting therapeutic effects on the ischemia and reperfusion of retina.

Aim This study aimed to assess the protection of recombinant human erythropoietin (rhEPO) in light-induced injuries in human retinal pigment epithelial(RPE)cells by researching the inhibition of rhEPO for apoptosis in human RPE cells by light-induced injuries.Methods Cultured human RPE cells were exposed to light of 8 w (2 000?500) lux for 12hours,then the culture were stopped at 24 hours after 12hours light stimulation. The effect of inhibiting apoptosis of rhEPO was detected by AnnexinV-flunorescein isothiocyanate/Propidium iodium labeling and flow cytometry. The enzyme linked immunosorbant assay(ELISA)and immunocytochemical staining were used to assess the expressions of caspase-3 and Bcl-2 treated by different doses of rhEPO in light-induced injury on human RPE cells and research the protective mechanism of rhEPO by adding AG490(the special inhibitor of Jak2).Results There was a obviously increased effects on inhibiting apoptosis in every rhEPO group, which was the most conspicuous in 40 IU?ml-1 rhEPO group,and the value was (4.93?1.45)?ml-1. The decrease of expression of caspase-3 was most obvious in 40 IU?ml-1 rhEPO group, and the value was (0.125?0.029) ?g?L-1. The increase of expression of Bcl-2 was the most obvious in 40 IU?ml-1 rhEPO group and the value was 168.21?3.87. But these effects on inhibiting apoptosis in rhEPO group were restrained by adding AG490, the value of apoptosis was (11.29?2.11)?ml-1 and the density of caspase-3 increased to (0.362?0.042) ?g?L-1,the expression of Bcl-2 dropped.Conclusion It is suggested that rhEPO can inhibit the apoptosis of human RPE cells in the light-induced injuries and inhibit the expression of caspase-3 and up-regulate the expression of Bcl-2, so rhEPO can protect the light-induced injuries for human RPE cells. Its protective mechanism is accomplished principally by the pathway of combining EPO with EPOR ,then the combination activates Jak2.

To study the possible etiopathology of senile cataract,the changes in the activity of lens epithelial enzyme in senile cataract and the effects of oxidative damage on the enzymatic activity of the cultured bovine lens epithelial cells. Methods 1. The activities of succinate dehydrogenase ( SDH)、 Lactate dehydrogenase ( LDH)、 Glucose - 6 - phosphate dehydrogenase (G60D) and Adenosine triphosphatase (ATPase) collected from senile cataract and normal lens epithelial cells were observed by enzyme histochemistry methods. 2. An experimental oxidative damage model of cultured bovine lens epithelial cells in vitro was established. Then the activities of SDH、 LDH and G6PD were observed after oxidative damage and then,after VitC treatment. Results 1. The activities of SDH、 LDH、 G6PD and ATPase of senile cataract lens epithelial cells were apparently lower than those of normal transparent lens.2. After oxidative damage, the activities of SDH、 LDH and G6PD of cultured bovine lens epithelial cells dramatically decreased. Vitamin C partly raised the enzyme activities, rather than bring them to normal.Conclusion Activities of enzyme related with energetic metabolism are decreased following oxidative damage, which may be a possible cause of senile cataract.

Objective To describe the clinical characteristics,and to analyze the AGXT gene mutation in three siblings with primary hyperoxaluria type I (PHI).Methods AGXT gene mutation was analyzed by direct sequencing analysis in this family,and the minor allele status was also tested.One hundred unrelated healthy subjects were also analyzed as controls.Results Three mutations in AGXT were identified in each of three patients including two novel heterozygous missense mutations and one previously reported variant.One mutation was a methionine to leucine substitution at position 49 (p.M49L,c.145A ＞ C) in exon 1,one was an asparagine to isoleucine transition at codon 72 (p.N72I,c.215A ＞ T) in exon 2,and another was a heterozygous nonsense mutation at codon 333 (p.R333*).Both p.M49L and p.R333* occured in cis configuration with the minor allele IVS1 +74 bp.Conclusions Two novel mutations are identified probably in association with PHI,however their pathogenicity and potential molecular mechanisms should be explored by further investigations.This is the first investigation on mutant gene analysis of PHI in China.

OBJECTIVETo investigate the relationship between P16 gene (the tumor suppressor gene) and the bcl-2 gene (the apoptosis inhibitor gene) and the incidence and development of malignant eyelid tumors.

METHODSThe streptavidin-biotin-peroxidase complex immunohistochemistry method was used to study the expression of P16 gene and the bcl-2 gene in 96 cases of malignant eyelid tumors.

RESULTSAmong the 96 cases, there were 40 basal cell carcinomas (BCCs), 33 squamous carcinomas and 23 sebaceous carcinoma, with P16 protein positive (nuclear staining) rates 70%, 54.6% and 56.5%, respectively. The P16 positive rate was negatively correlated with the degree of tumor histological differentiation, and the rate difference between the high differentiated carcinomas was significant (P < 0.05). Positive Bcl-2 protein expression was detected in the cytoplasm. All 40 BCC cases were Bcl-2 positive, and nearly all of the tumor cells showed positive cytoplasmic expression, while in the 33 squamous cell carcinoma cases only one showed positive focal reaction, and the staining in the other 32 cases was relatively faint. None of the 23 sebaceous carcinomas expressed Bcl-2.

CONCLUSIONSThe expression of the P16 protein was related to the occurrence and degree of differentiation of malignant eyelid tumors. The overexpression of the Bcl-2 protein suggests that suppression of apoptosis might play a role in the tumorigenesis of BCC.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Basal Cell ; chemistry ; Carcinoma, Squamous Cell ; chemistry ; Cell Differentiation ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Eyelid Neoplasms ; chemistry ; pathology ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Proto-Oncogene Proteins c-bcl-2 ; analysis

OBJECTIVETo investigate the expression of cluster of differentiation 44 variant 6 (CD(44V6)) and proliferating cell nuclear antigen (PCNA) in ocular squamous cell carcinomas.

METHODSStreptavidin-biotin complex (SABC) immunohistochemistry was used to explore the expression of CD(44V6) and PCNA in 35 cases of ocular squamous cell carcinomas, 20 cases of papillomas, and 11 cases of normal eyelid tissue.

RESULTSThe CD(44V6) positive rate was 62.9% (22/35) in ocular squamous cell carcinomas, 15.0% (3/20) in papillomas, but not detectable in the 11 cases of normal eyelid tissue. The positive expression rates of CD(44V6) in ocular squamous cell carcinomas were significantly higher than in benign tumors (chi(2) = 11.57, P < 0.01) or control tissue (P = 0.001), and the positive expression rates of CD(44V6) in metastasis were significantly higher than without metastasis (P = 0.049). PCNA labeling indexes (PI) in tumors with CD(44V6) expression were significantly higher than those without (t = 20.21, P < 0.01).

CONCLUSIONSOverexpression of CD(44V6) is correlated with the progress and metastasis of ocular squamous cell carcinomas. CD(44V6) protein positive staining is associated with high PI. CD(44V6) and PCNA are useful for evaluating prognosis.

Carcinoma, Squamous Cell ; chemistry ; pathology ; Eye Neoplasms ; chemistry ; pathology ; Glycoproteins ; analysis ; Humans ; Hyaluronan Receptors ; analysis ; Immunohistochemistry ; Lymphatic Metastasis ; Proliferating Cell Nuclear Antigen ; analysis ; Skin ; chemistry

Objective To investigate the effects of simulated microgravity by rotary cell culture system (RCCS) on expression profiles of miRNA in mouse fibroblasts L929 cell line.Methods L929 cells were cultured in vitro and divided into simulated microgravity (SMG) group and normal gravity (NG) group according to the simple random method.Samples of two groups were collected on 7th day of culture and the total RNAs were extracted,labeled,and hybridized in sequence.Feature Extraction Software was used to collect the array images and get raw data,which were analyzed by Genespring Software.Differentially expressed miRNAs were identified and then validated by qRT-PCR.Target genes of differentially expressed miRNAs were predicted by the databases of Targetscan and microRNAorg,and the intersections of databases were identified as potential regulatory target genes.Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis were applied to determine the roles of these target genes.Relevant biological functions and/or signaling pathways of the regulated genes especially related with wound healing process were categorized based on their enrichments.Then integration predictions of the miRNA and mRNA expression profiles had been proposed to refine the functional miRNA-mRNA relationships.The miRNA-mRNA functional network and miRNA-mRNA control network were constructed.Results Four miRNA genes were up-regulated significantly including mmumiR-669j,-122-5p,-30a-3p,-6516-3p,among which mmu-miR-669j was up-regulated at 52.84 folds with the greatest significance (P ＜ 0.05).Seventeen miRNA genes were down-regulated significantly including mmu-miR-21a-3p,-miR-28a-5p,-218-5p,-210-3p,-miR-19a-3p,-miR-31-3p,and-miR-19b-3p,among which mmu-miR-28a-5p was down-regulated at 15.47 folds with the greatest significance (P ＜ 0.05).The qRT-PCR showed a high concordance with the microarray results (P ＜ 0.05).Target gene prediction and functional enrichment analysis suggested that a variety of biological processes and signaling pathways involved in wound repair were significantly enriched (P ＜ 0.05).Function network and regulation network of miRNA-mRNA covered all the differentially expressed miRNAs,which suggested that miR-21 a-3p and predicted target gene Smad3 might play an important role in wound healing under microgravity.Conclusions Simulated microgravity by RCCS can significantly affect the expression of stress-related miRNAs in mouse fibroblasts L929.The miRNA target gene prediction and functional enrichment analysis based on gene chip technology may provide theoretical basis for illustrating the mechanism and management of weightlessness stress injury.

Objective To analyze the risk factors for persistent corneal epithelial defects (PCED) after pars plana vitrectomy (PPV) in patients with proliferative diabetic retinopathy (PDR).Methods A total of 201 PDR patients (201 eyes) who received PPV were enrolled in this retrospective study.There were 86 males (86 eyes) and 115 females (115 eyes).The patients aged from 30 to 81 years,with the mean age of (57.94±9.65) years.Among them,159 patients were ≥50 years of age,and 42 patients were ＜50 years of age.There were 36 patients with HbA1c ＜7.0％,165 patients with HbA1c ≥7.0％.There were 93 right eyes and 108 left eyes.There were 93 right eyes and 108 left eyes.The diabetic retinopathy stages were as follows:stage Ⅳ in 24 eyes,stage Ⅴ in 78 eyes and stage Ⅵ in 99 eyes.The operation time was ranged from 1 to 4 hours,with an average of 2 hours.Among the 201 eyes,corneal epidermis were scraped on 25 eyes;70 eyes were combined with cataract surgery;a laser photocoagulation count ＜ 1000 points was performed in 78 eyes,and ＞ 1000 points were performed in 123 eyes.Sixty-one eyes involved intravitreal silicone oil tamponade,18 eyes involved intravitreal tamponade with C3F8,and 122 eyes were not involved with intraocular tamponade.Postoperative persistent intraocular hypertension was defined as an intraocular pressure (IOP) ≥21 mmHg (1 mmHg=0.133 kPa) after PPV with necessary treatment using IOP-lowering medications for ≥ 2 weeks.The diagnostic criteria for corneal epithelial defects were taken from the Expert Consensus on Clinical Diagnosis and Treatment of Corneal Epithelial Defect in China (2016).The corneal epithelial defect was diagnosed as PCED if it was treated with common methods such as a lacrimal substitute or corneal contact lens,but showed no improvement and no signs of healing for ≥2 weeks.The incidence of PCED after eye surgery was recorded and its related risk factors were analyzed.A multivariate logistic regression was used to analyze the risk factors for PCED,which were expressed as a odds ratio (OR) and a 95％ confidence interval (CI).Results Of 201 eyes,16 eyes (7.96％) presented with PCED after surgery and 185 eyes (92.04％) with no PCED.There was no significant difference in the age,sex,and eyes between the patients with or without PCED (x2=6.548,0.927,0.044;P=0.011,0.336,0.833).A multivariate logistic regression showed that intraoperative epithelial debridement (OR=1 3.239,95％CI 2.999-58.442,P=0.001),intraoperative treatment in combination with cataract surgery (OR=7.448,95％CI 1.975-28.091,P=0.003),intravitreal tamponade with C3F8 (OR=11.344,95％CI 2.169-59.324,P=0.004),and postoperative persistent intraocular hypertension (OR=10.462,95％CI 2.464-44.414,P=0.001) were risk factors for PCED after PPV.Conclusion Intraoperative epithelial debridement,intraoperative treatment in combination with cataract surgery,intravitreal tamponade with C3F8,and postoperative persistent intraocular hypertension are risk factors for PCED in patients with PDR after PPV.

Objective To analyze the relationship between planning factors of intensity-modulated radiation therapy (IMRT) and gamma index and investigate the effect of each parameter upon the γ passing rate of IMRT.Methods Gamma analysis was performed using 3％/3 mm acceptance criteria for 457 IMRT beams with different planning factors.During multi-factor ANOVA analysis of planning factors and gamma passing rate,the control variables primarily included the minimum segment area,minimum number of monitor unit (MU),number of segment,segment conversation,and the spatial resolution in the measured dose distribution.Results The percentage of pixels with passingγsignificantly differed under different minimum segment area,segment conversation and the spatial resolution in the measured dose distribution (all P＜ 0.05).No significant correlation was observed between the passing rate and the minimum number of MU and the number of segment (P＞ 0.05).Conclusions According to the actual situation of the equipment,the minimum segment area should be determined during IMRT planning.Direct machine parameter optimization should be performed.Appropriate resolution of the plane dose images can be chosen according to the minimum detector interval of dose matrix device,