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.

ObjectiveTo investigate the expression of hypoxia inducible factor 1 (HIF-1α) in rats' retinae during the embryonic and earlier postnatal period.MethodsThe retinal expression patterns of HIF-1α protein and mRNA of embryonic day 12 (E12), E16, E20, and postnatal day 1(P1) and P5 rats were determined by immunohistochemical staining and reverse transcription polymerase chain reaction (RT-PCR).ResultsHIF-1α protein was detected in the neural epithelial layer and the pigment epithelial layer at all those 5 timepoints, with higher expression in the ganglion cell layer and the inner plexiform layer, and seems limited to the ganglion cell layer when retina became more mature. Embryonic rat retina had higher expression of HIF-1α protein and mRNA than postnatal retina, the difference was significant (P<0. 01). ConclusionThe expression of HIF-1α in rats' retinae differs from embryonic to earlier postnatal stages.

Anthracosis ; complications ; Cerebrovascular Disorders ; complications ; Cohort Studies ; Humans ; Male ; Middle Aged ; Retrospective Studies

Background Pigmentary glaucoma and pseudoexfoliation glaucoma are characterized by pigment dispersion in trabecular meshwork,and the dipersitional pigment probably contributes to the resistance of aqueous outflow pathway,irreversible damage of the trabecular meshwork and the remodeling abnormality of extracellular matrix.Researches determined that contents of transforming growth factor-β (TGF-β) in the aqueous humor are increased in glaucomatous eyes.However,the effects of TGF-β and fibronectin (FN) in the trabecular meshwork cells (TMCs) acted by iris pigmentary particles still are not elucidated.Objective This study was to investigate the effects of iris pigment particles on TGF-β1 and FN expression in bovine TMCs (BTMCs) cultured in vitro.Methods BTMCs were cultured in vitro by explant culture method and identified by morphological evaluation.The third generation of BTMCs were divided into normal control group and pigment group,and 100 μ1 PBS and 100 μl iris pigment suspension (final concentration of 1 × 107 particles/ml) were added into the medium for 24 hours,respectively.The expressions of TGF-β1 mRNA,FN mRNA and their proteins in the BTMCs were assayed by real-time fluorescence quantitative PCR and ELISA,respectively.Results Cultured cells grew well and showed the fusiform,polygon and dendritic like in shape,with pigmented and round nuclei.The relative expression levels of TGF-β1 mRNA and FN mRNA in the cells were 2.98±0.27 and 0.36±0.10 in the iris pigment group,which were significantly higher than 1.00±0.00 and 1.00±0.00 in the normal control group (t =12.68,10.60,both at P =0.00).The concentrations of TGF-β1 protein and FN protein in the cell suspension were (156.60±9.74)ng/L and (59.29±15.79)mg/ml in the iris pigment group,showing significant differences in comparison with (65.46 ± 14.24) ng/L and (102.10 ± 12.14)mg/mlin the normal control group (t=9.15,P=0.00;t=3.72,P=0.02).Conclusions The expression of TGF-β1 is up-regulated and that of FN is down-regulated in BTMCs cultured by iris pigment,inferring that TGF-β1 and FN participate in the pathogenesis and development of pigmentary and pseudoexfoliation glaucoma.

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.

Objective To observe the curative effects of octreotide on gastrointestinal cancer and its influenee on the serum level of IGF-1.Methods 33 patients diagnosed as advanced gastrointestinal cancer were randomized into 2 groups.15 cases in therapy group received octreotide 400μg/d subcutaneous injection or intravenous injection,other patients were taken as control.Curative effects of octreotide and serum level of IGF-1 on different time were observed before and after therapy.Results 4 cases in therapy group kept stable condition during treatment,all 18 cases in control group deteriorated gradually.The median survival time of octreotide therapy was 6.9 months,longer than that of control group(2.3 months)(P＜0.05),the feeling of well being in therapy group was reported a remarkable improvement.Serum level of IGF-1 decreased obviously after octreotide injection(P＜0.05),but no significant difference in control group.Conclusion Octreotide can prolong the survival duration and improve living quality of patients with advanced gastrointestinal tumors.The mechanism of antitumor is probably through suppression of IGF-1.

Objective To investigate the effect of heat treatment on the proliferation of, melanin synthesis and tyrosinase activity in cultured normal human melanocytes. Methods Normal human foreskin tissue was obtained by sterile circumcision and melanocytes were harvested by using methods for epidermal cell culture. Basic fibroblast growth factor (bFGF) was utilized as the primary mitogen to establish the culture system of normal human epidermal melanocytes. Masson-Fontana staining was proformed to identify melanocytes.Third-passage melanocytes were treated with hyperthermia at various temperatures (39 ℃, 41 ℃, 42 ℃, 43 ℃ and 45℃) for 1 hour a day for consecutive 3 days followed by the measurement of cell viability with MTT assay. The hyperthermia at optimized temperature was used to treat fourth-passage melanocytes for 1 hour a day for consecutive 3 days; subsequently, the tyrosinase activity were detected with L-Dopa as the substrate, and melanin content was determined in heat-treated and untreated (control) melanocytes. Results The hyperthermia at 42 ℃ exhibited the strongest promotive effect on the proliferation of melanocytes among these 5 hyperthermia conditions. After treatment with hyperthermia at 42 ℃ for 1 hour a day for consecutive 3 days, melanocytes showed an increment in tyrosinase activity by 36.4% and melanin synthesis by 78% compared with the untreated melanocytes (both P＜0.05). Conclusions Heat treatment can enhance the proliferation of cultured human melanocytes, promote their melanin synthesis and tyrosinase activity.

Background Erythropoietin (EPO) was proved to be express in hematopoietic tissue and nervous system and play the effects of stimulating blood cell production and protecting nervous tissue.Researches showed that EPO is expressed in the embryon brain of animal.However,whether EPO exist in nervous-derived retina and its action on retina with the development is concerned. Objective This research was to investigate the expression of EPO during the embryonic development period of rat retina and explore the role of EPO in retina development process.Methods Clean Wistar rats with pregnancy for 12 days,16 days and 20 days were collected,and the embryonic 12-day rats (E12 d,5 rats),embryonic 16-day rats (E16 d,5 rats) and embryonic 20-day rats ( E20 d,5 rats) were obtained by caesarean operation,and 5 12-month W istar rats were used as controls.The rats were sacrificed by cervical dislocation and the retinal sections were prepared in the different-embryo-phase (12 d,16 d,20d) and growth phase.The expression of EPO protein and mRNA in rat retina was detected by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR),respectively.The feed and use of the animals followed the Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results EPO was positively expressed in the cytoplasm and nuclei in the neuroepithelial layer and pigment epithelium of every-embryo-phase rats but only in retinal ganglion cell layer in 12-month-old rats.The gray scale values of EPO expression in retina were 105.55±10.35,99.35± 8.71,83.27± 7.84and 30.30± 3.80 in E12 d rats,E16 d rats,E20 d rats and 12-month-old rats respectively with a statistically significant difference (F=76.13,P＜0.01 ).RT-PCR revealed that the relative values of EPO mRNA expression in retina were 0.876±0.10,0.861 ±0.09 and 0.256±0.03 in E16 d rats,E20 d rats and 12-month-old rats respectively,presenting a elevated value in embryonic rats compared with adult rats ( P =0.00).Gel imaging deletion showed that the A value of EPO amplification products was highest in E16 d rats and lowest in adult rats.Conclusions The expression of EPO appears a high to low fashion during the embryonic development of Wistar rats,which is closely associated with the developing procedure of retina.

Aim To explore the effects of tanshinone IIA on cell proliferation via G protein-coupled estrogen receptor inductive and regulative pathway in typical es-trogen receptor and G protein-coupled estrogen receptor positive T47D breast cancer cells. Methods The pro-liferation rate of T47 D cells influenced by tanshinone IIA was analyzed by MTT assay. G protein-coupled es-trogen receptor agonist G1 and GPER antagonist G15 were employed as tools. GPER SiRNA was applied to build GPER gene silence T47D cells. GPER expres-sion influenced by tanshinone IIA was measured by Western blot. Results The proliferation rates of T47D cells treated with 1 × 10 -5 mol·L-1 - 1 × 10 -7 mol· L-1 of tanshinone IIA were decreased significantly. Such effects could be attenuated by G1 or enhanced by G15 . Growth of GPER SiRNA transfected T47 D cells were significantly inhibited by 1 × 10 -5 mol·L-1 - 1 × 10 -7 mol·L-1 of tanshinone IIA treating. Result of Western blot showed that tanshinone IIA at 1 × 10 -5 mol· L-1 and 1 × 10 -6 mol · L-1 could induce de-crease of GPER protein expression in T47D cells. Conclusions Tanshinone IIA shows inhibitory effects on proliferation rate of T47 D breast cancer cells via GPER pathway. Tanshinone IIA could perform regula-tive function on GPER expression level in target cells.

BACKGROUND: Hypoxia inducible factor-1 α(HIF-1 α) is not only related to physiological reaction of hypoxia,but also takes part in normal embryonic development.OBJECTIVE: To study the alteration of HIF- 1 α in retina during rat development.DESIGN,TIME AND SETTING: Randomized contrast animal study,which was performed in the Shandong Provincial Molecular Virus Key Laboratory,Medical College of Qingdao University between January and September 2007.MATERIALS: Adult Wistar rats,nulliparity,clean grade,and weighing 200 250 g were used in this study.METHODS: Male and female rats were caged as the ratio of 1 : 1.Embryos were obtained at 12-day,16-day,and 20-day pregnancy.Eyeballs were obtained from newborn rats by anesthesia at 1-day,5-day,10-day,and 12-month birth.Retina was separated and made into paraffin section.MAIN OUTCOME MEASURES: Expressions of HIF 1 α protein and HIF-1 α mRNA in retina were measured by immunohistochemistry and semiquantitative reverse transcription-polymerase chain reaction at various dine points of embryonic development.RESULTS: HIF-1 α positively expressed at stratum neuroepitheliale retinae and purpurogenous membrane in the embryonic phase.Additionally,HIF-1 α still positively expressed at stratum neuroepitheliale retinae and purpurogenous membrane,especially in ganglionic cells and inner plexiform layer,in early development.With the gradual development,the positive expression was mainly located at stratum ganglionare retinae.HIF-1 α protein and mRNA expressions were the highest in the embryonic phase,lower in the development,and the lowest in the adult period.There were significantly differences among these three phases (P ＜ 0.01).CONCLUSION: HIF-1 α decreases gradually in retina and its expression is mainly located at stratum ganglionare retinae.