BACKGROUND: Recent studies demonstrate that erythropoietin (EPO) can protect retina from light injury, and is the mechanism related to the expression of Caspase-3 in the light-injured retinal pigment epithelium (RPE) cells. OBJECTIVE: To study the effects of EPO at different dosages on the expression of Caspase-3 in light-injured human RPE cells. DESIGN: Control observation.SETTING: Qingdao University Medical College. MATERIALS: Adult ARPE-19 cells (American Cell Culture Collection Company); DMEM/F12 mixed medium, fetal bovine serum and trypase (GIBCO Biotechnique Company); recombinant human EPO (rhEPO, Sigma Biotechnique Company); human Caspase-3 quantitative kit (Shanghai Xitang Biotechnique Co.,Ltd); Caspase-3 monoclonal antibody (American Santa Cruz Company); PV6001 immunohistochemistry kit and DAB color reagent kit (Zhongshan Biotechnology Company, Beijing).METHODS: The experiment was carried out in the Department of Pathophysiology at Qingdao University Medical College between May 2006 and January 2007. Human RPE-19 cell strain at passages 2-5 were harvested for light injury models, and the passage cells were divided into 7 groups randomly, with 4 apertures in each group:①normal control group: no light or EPO intervention;②light-injured model group: 12-hour illumination, no EPO intervention;③light-injury and EPO groups: 12-hour illumination with 10 000, 20 000 and 40 000 U/L EPO;④light-injury and 40 000 U/L EPO and AG490 group: 12-hour illumination with 40 000 U/L EPO and inhibitor of Jak2 enzyme 50 000 U/L;⑤light-injury and 40 000 U/L EPO and carbxyl-terminal modulator protein (CTMP) group: 12-hour illumination with 40 000 U/L EPO and specific inhibitor of protein kinase B enzyme 100 μmol/L.MAIN OUTCOME MEASURES: The enzyme linked immunosorbant assay (ELISA) and immunohistochemistry were used to assess the effects of rhEPO at the different doses on the expression of Caspase-3 in light-injured human RPE cells. RESULTS: Caspases-3 was not expressed in RPE cells of the normal control group and was positively expressed in the nucleus of RPE cells of the light-injured model group, showing a specific brown-yellow staining. Expression of Caspase-3 was gradually decreased in every rhEPO group with increase of EPO concentration, with the weakest expression in 40 000 U/L rhEPO group. The effects of EPO on Caspase-3 expression were strongly inhibited in light-injury+ 40 000 U/L EPO +AG490 group and the expression was positive in light-injury +40 000 U/L EPO+CTMP group, which was slightly weaker than light-injured model group. CONCLUSION: The rhEPO can reduce the expression of Caspase-3 in the light-injured human RPE cells, and one of the possible mechanisms is the inhibition of light-injured RPE cell apoptosis by the rhEPO.

Objective To investigate the possibility of affecting transgenic rescue for Wilson disease using the human ATP7B transgenic LEC rat.Methods The 7.1kb transgene constructed with human ATP7B cDNA and chicken ?-actin promoter was introduced into the fertilized oocytes of LEC rats, an animal model of Wilson disease, by microinjection. The expressions of human ATP7B protein in the transgenic rats were detected by Western blot. The plasma AST and ALT activities, and the total bilirubin levels in transgenic rats were measured continuously from 6 to 16 weeks using non-transgenic rats and LEA rat as controls. The pathological and histochemistry changes in the liver of the transgenic rats at 13 weeks were analyzed. Results The intact and correct product derived from human ATP7B was confirmed in the liver of transgenic rats. At the age around 12 weeks, the plasma AST and ALT activities, and the total bilirubin levels in transgenic rats were significantly decreased, while the inflammatory reation in the liver of transgenic rats was much mild as compared with that of non-transgenic rats, and the granules of stained copper were less in the hepatocytes of transgenic rats. By the age of 16 weeks, the transgenic rats were phenotypically normal, and the survival rate was 100%. These data showed that the LEC rats were successfully rescued from fulminant hepatitis after introducing of human ATP7B gene. Conclusion The hepatitis in Wilson disease is directly related to the toxicity of excessive accumulated copper, which attributed to the functional deficiency of the ATP7B. Gene transfer probably is the effective method for the therapy of Wilson disease.

Objective To investigate the possibility of affecting transgenic therapy for the hepatic cirrhosis and hepatoma in Wilson disease by human ATP7B cDNA. Methods The 7.1*!kb transgene consisting of human ATP7B cDNA and chiken ?-actin promoter was introduced into the LEC rats which is an animal model of Wilson disease by microinjection.The plasma AST and ALT activities in transgenic rats were measured continuously from weeks 17 to 30 using non-transgenic and LEA rats as controls.The histological and histochemistry changes of liver in the transgenic rats at 30 and 60 weeks of age were examined. Results The plasma AST and ALT activities in transgenic rats were kept at the relatvie lower levels from 17 to 60 weeks of age, as compared to the age-matched non-transgenic rats.By the age of 60 weeks,none of the transgenic males developed cholangiofibrosis or hepatoma,whereas all of the non-transgenic rasts had severe cholangiofibrosis at the age of 30 weeks and one male rat had hepatoma at 60 weeks.The transgenic rats were phenotypically normal,and the survival rate was 95.7%.In addition,the distribution and the numbers of the granules of stained copper in the hepatocytes of the transgenic rats did not show any significant difference between 30 and 60 weeks.Conclusion The human ATP7B successfully delayed the onset of hepatic cirrhosis,and suppressed the development of hepatoma in the LEC rats by gene transfer.The hepatic cirrhosis and hepatoma in Wilson disease may be not directly related to the copper accumulation.

Objective To analyze the clinical efficacy of compound 18 norgestrel combined with estrogen in treatment of dysfunctional uterine bleeding. Methods 78 cases of patients with dysfunctional uterine bleeding in Beijing Ji Shui Tan Hospital and Dongguan city people's hospital of guangdong Province were divided into control group (n=39)and observation group (n=39)by using randomized single-blind allocation. The control group was only given estrogen,the observation group was used compound 18 norgestrel combined with estrogen. The clinical efficacy of the two groups was compared. Results 31 cases were cured and 7 cases were valid of the study group,the total effective rate was 97.4%,significantly higher than the control group's total efficiency of 71.8%,the difference was statistically significant (P<0.05). Each index of observation group and the control group were improved after treatment,the difference was statistically significant (P<0.05 );After treatment,the menstrual cycle in observation group was (29.7 ±7.1)d,the menstrual period was (5.6 ±0.5)d,hemoglobin content was (135.5 ±21.7)g/L,endometrial thickness was (0.63 ±0.15)mm, significant changes than the control group,the difference was statistically significant (P<0.05 ). Conclusion Compound 18 levonorgestrel combined with estrogen has a better effect in patients with dysfunctional uterine bleeding and it can significantly improve patients'hemoglobin,the menstrual cycle and the uterus film thickness and other indicators which is worth promoting in clinical.

Objective To investigate the clinical features,treatment and prognosis of paraneoplastic pemphigus (PNP).Methods A retrospective study was performed on 11 patients with PNP hospitalized in the Department of Dermatology,Ruijin Hospital.Clinical characteristics of these patients were analyzed.Results Of the 11 patients,10 had oral or labial erosions or ulcers,and 6 had obstructive bronchiolitis.Computed tomography (CT)showed solitary internal tumors in all the patients after appearance of skin lesions,and 8 of them were diagnosed with Castleman's disease.All the patients had been treated with corticosteroids before operation,but achieved no obvious improvement.After 2-7 months of postoperative treatment with low-dose prednisone and thalidomide,both cutaneous and mucosal lesions healed with the relief of pulmonary symptoms in 5 patients.Conclusions Oral erosions or ulcers appear to be the most common initial manifestation of PNP with Castleman's disease as the most frequent accompanying tumor.Early detection and timely resection of tumors are keys to successful treatment of PNP,and postoperative treatment with glucocorticoids and thalidomide proves to be effective for PNP.

After the concept of symptoms in traditional Chinese medicine was defined, the classification of symp-toms in traditional Chinese medicine and the characteristics of clinical diagnosis and treatment in traditional Chinese medicine were described .It was suggested that the symptoms in traditional Chinese medicine should be classified into 3 kinds, namely symptoms and signs according to the 4 diagnostic methods, symptoms and signs according to the diagnostic devices, symptoms and signs according to the physical constitutions of patients in order to lay a foun-dation for improving the classification in clinical term system of traditional Chinese medicine.

Objective To investigate the effect of recombinant human erythropoietin (rhEPO) on the expression of glutamine synthetase (GS) of cultured rat retinal Müller cells in high glucose environment in vitro.Methods Mtüller cells were isolated from retinas of 10 Sprague-Dawley rats at postnatal day three to five by trypsin digestion,and were randomly divided into six groups,including normal control group,high glucose group,high glucose +5 U/ml rhEPO group,high glucose+ 10 U/ml rhEPO group,high glucose+ 20 U/ml rhEPO group,high glucose+40 U/ml rhEPO groups.After 48 hours,the apoptosis of retinal Müller cells were assayed by terminal transferase-mediated DNA end labelling assay,and the expression levels of GS protein were detected with semi-quantitative immunocytochemistry.Results Compared with the normal control group,the cell viability and GS protein were reduced while the cell death increased in Müller cells cultured in high glucose,the difference was statistically significant (t =27.4,P ＜ 0.01).Compared with the high glucose group,rhEPO treatment reduced the apoptotic Müller cells (t=857.2,2 374.6,2 473.2,2 537.7; P＜0.01),induced the expression of GS proteins (t=3.2,18.0,22.5,26.4; P＜0.05).Conclusions rhEPO can protect Müller cells from apoptosis under high glucose condition.The mechanism may be related to its function to up-regulate the GS protein expression,promote glutamic acid cycle,and reduce the excitotoxicity effects of high concentration of glutamate.

Insulin resistance and islet ? cell dysfunction are well-identified pathogenesis of type 2 diabetes. The generation of a great quantity of mitochondrial reactive oxygen species (ROS) impairs the function of mitochondria and induces oxidative stress during metabolic process of hyperglycaemia and hyperlipemia. Oxidative stress blocks insulin action pathway and induces insulin resistance; inhibit expression of insulin gene; decrease insulin secretion and impair ? cell function. In this paper, the mechanism of the ROS generation, insulin resistance and ? cell dysfunction induced by oxidative stress are reviewed.

Background Chrysin has many biological activities,and its anti-inflammatory effect has been confirmed.However,whether it can treat experimental autoimmune uveitis (EAU) is still not elucidated.Objective This study was to investigate the therapeutic effect of chrysin on EAU and explore the potential mechanism.Methods EAU animal models were established in 30 SPF C57BL/6J mice by the subcutaneous injection and ball pad injection of interphotoreceptor retinoid binding protein1-20 (IRBP1-20)/complete Freund adjuvant (CFA),and then the models were randomized into the model control group and chrysin-treated group.Chrysin solution dissolved by 10 μl dimethyl sulfoxide (DMSO)+140 μl PBS was administrated by gavaging in the mice with the dlose 25 mg/kg from 3 days before immunization through 21 days everyday in the chrysin group,and equal volume of solvent was used in the same way in the model control group.Retinas were examined by indirect ophthalmoscope once per day,and inflammation and pathological scores of retina were performed based on the criteria of Caspi on the 21st day after injection.The apoptosis of retinal cells was assayed by TUNEL staining,and the relative expressions of proinflammatory cytokines interferon-γ (IFN-γ),interleukin-17 A (IL-17A),IL-6 and tumor necrosis factor-α (TNF-α) and signal transducer and activator of transcription 1 (STAT1),STAT3,p-STAT1,p-STAT3 in mouse retinas were detected by Western blot.Results Compared with the model control group,the inflammation scores and pathological scores of retinal inflammation were significantly reduced in the chrysin group (inflammation scores:0.50± 0.45 vs 1.58±0.92,t=2.600,P=0.026;pathologic scores:0.58±0.38 vs 1.83±0.75,t=3.638,P=0.005).The infiltration of a large number of inflammatory cells,retinal vasculitis and granulomatous lesions were found in mouse retinas in the model control group,however,the morphology of mouse retinas in the chrysin group was normal based on hematoxylin-eosin staining.The number of apoptotic cells was remarkable lessened in the chrysin group compared with the model control group under the fluorescence microscope.Western blot assay resolved that significantly downregulation in the expressions of IFN-γ,IL-17A,IL-6 and TNF-α was seen in the chrysin group in comparison with the model control group (t =7.802,P =0.001;t =14.906,P =0.000;t =10.241,P =0.001;t =3.304,P =0.030),and the relative expression levels of STAT1,STAT3,p-STAT1 and p-STAT3 were considerably lower in the chrysin group than those in the model control group (t=8.965,P=0.001;t=8.358,P=0.001;t=4.864,P=0.031;t=4.730,P=0.009).ConclusionsChrysin or chrysin-like flavonoids ameliorate intraocular inflammatory symptoms in EAU mice by inhibiting the activity of STAT signal pathway.

By analyzed the problems of clinical medicine key disciplines in professional degree graduate education,such as pay more attention to the research rather than clinical work,pay more attention to using rather than cultivating,the new needs were identified in the focus adjustment of this education.The measurements and methodologies of focus adjustment were discussed to match the needs.