Background How to harvest the purified corneal endothelial seed cells is very important for the corneal tissue engineering technology. Herein,to establish a good culture method and effective identification method of corneal endothelial cells ( CECs) is the key. Objective Present study wag to establish the cultivating and identifying approach of the rabbit CECs and detect the biological characteristics of passaged cells. Methods Rabbits CECs were isolated from Descemet's membrane peeled off completely in 30 New Zealand white rabbits and then digested with 0. 25% trypsin-0. 02% EDTA and primarily cultured in CECs medium containing 15% fetal bovine serum. The growth situate and cellular morphology of rabbit CECs were observed under the inverted phase-contrast microscope following the alizarin red staining. Rabbit CECs were identified by cellular morphology as well as in gene and protein level, including the detection of expressions of collagen type IV α2(COL4A2) ,vascular endothelial growth factor receptor 2 ( FLK1 ) , Na+-K+ ATPase alpha 1subunit ( ATP1 A1 ) , aquaporin 1( AQP1 ) , voltage-dependent anion channels ( VDACs) by reverse transcription-polymerase chain reaction ( RT-PCR ). The expression and distribution of neurone specific enolase ( NSE) , Na+-K+ ATPase, zonula occludens-1 ( ZO-1 ) were also detected by immunocytochemistry under the fluorescence microscope. The proliferation activity of the passage cells was dynamically observed by MTT assay,and Na+-K+ ATPase activity of different generations cells was detected by ATPase kit. Results Majority of the primarily cultured cells adhered in 24 hours, infused in 2-3 days with the hexagon shape in appearance. The morphology of the cells was very varied with passage. Alizarin red staining showed a well- defined and well-lined cellular morphology similar to the corneal cells in vivo. Target genes of C0L4A2, FLK1, ATPIAI ,AQPI and VDACs were positively expressed in the cells. However,the expression of CK12 was absent in the cells. NSE, Na+-K+ ATPase, ZO-1 positive cells were respectively observed under the laser confocal scanning microscopy. MTT results showed a gradually low growth curve with the passage. Quantitative results also revealed that the Na+-K+ ATPase activity was gradually declined in different generations of cells ( F = 77. 174, P = 0. 000 ). Conclusion The enzyme digestion is a better approach of isolating and culturing comeal endothelial cells. Cultured cells can be identified by cells staining, gene expression and protein level. Earlier generation of CECs are ideal seed cells for cornea tissue engineering.

AIM: To study the effect of the subretinal fluid (SRF) on proliferation of retinal pigment epithelium (RPE) cells and retinal glial (RG) cells and associated activation and translocation of protein kinase C (PKC) as well as the application of PKC inhibitor.MTEHODS: RPE and RG cells were disintegrated to obtain PKC activity of cytoplasm and cellular membrane after being treated by the subretinal fluid (SRF) from the different stages of PVR patients (grade B and C) or being treated with PKC specific activator [phorbol-12-myris-tate-13-acetate (PMA)] or normal vitreous or DMEM culture medium. PKC activity in cytoplasm and cellular membrane was measured using radioactive isotope 32P labeling in a specific reaction of phosphorylation on PKC substrate. In addition, the PKC inhibitor, dequalinium chloride, was used to pretreat the RPE and RG cells before the cells exposed to SRF or PMA or normal vitreous. 3H-TdR (tritiated thymidine) was used to measure the levels of proliferation of RPE and RG cells with or without the activation and translocation.RESULTS: SRF and PMA promoted the proliferation of RPE and RG cells. SRF and PMA activated PKC in the cytoplasm of RPE and RG cells and the activated cytoplasm PKC translocated to the cellular membrane of RPE or RG cells. The cell proliferation or PKC activation or translocation were not equally active in RPE as in RG cells. However, PKC inhibitor which attenuated the cell proliferation did not show significant difference on inhibition of RPE and RG cell proliferation. (P ＞0.05).CONCLUSION: SRF can lead to the activation and translocation of PKC in RPE and RG cells, which promote the proliferation of RPE and RG cells. Dequalinium chloride can inhibit PKC activation and translocation hence slow down the cells proliferation.

Objective To investigate the role of intraeellular Ca2+ and MERTK in the phagocytosis of human retinal pigment epithelial (RPE) cells, and reveal the relationship between MERTK and intraeellular Ca2+. Methods The euhured RPE cells were incubated with rod outer segments (ROS) at 37℃, the phagoeytosis was terminated at different incubation time points. The concentration of intraeellular Ca2+ was assayed by Fluro-3/AM loading methods combined with fluorescence microscope and CCD system, and the mRNA level of MERTK gene was measured by reverse transcription polymerase chain reaction (RT-PCR). Treating the RPE cells with stimulator (A23187)or inhibitor(verapamile)of intraeellular Ca2+ to observe the changes of MERTK gene expression. Results ROS adhered to hRPE cells at the 15th minute, and the ingestion saturated at the 24th hour. The concentration of intracellular Ca2+ increased at the 15th minute, and kept the high level in 24 hours. The level of MERTK mRNA increased at the 5th minute, and kept the high level duration the whole incubation. When RPE cells were treated by A23187, the expression of MERTK increased in a dose-dependent manner. After RPE cells was pretreated by A23187, the expression level of MERTK was higher in the proceeding incubation groups than which in the control group except at the 3rd hour. When RPE cells were treated by verapamil, the expression level of MERTK decreased in a dose-dependent manner. After RPE cells were pretreated by verapamil , the expression level of MERTK was lower in all the proceeding incubation groups than which in the control group (P<0.05). Conclusion MERTK gene and Ca2+ play an important role in sustaining RPE cells phagoeytizing ROS. As an up-stream regulator, the receptor tyrosine kinase MERTK keeps RPE cells phagocytizing ROS by starting the intracellular Ca2+.

[Objective] To compare the axial length (AL) measured with IOLMaster and contact A-Scan in eyes with macular edema (ME) and to investigate the correlation between measurement difference and foveal thickness.[Methods] Sixty-seven ME eyes of 42 patients (ME group) and 40 healthy eyes of 30participants (control group) were enrolled in this study.Foveal thickness was measured with 3D optical coherence topography (OCT)-1000.The AL was prospectively measured by IOLMaster and contact A-scan,The correlation between measurement difference and foveal thickness was analyzed by Pearson correlation analysis.[Results] Mean foveal thickness of ME eyes was (377.85± 119.84) μm.Mean AL by IOLMaster and contact A-scan were (22.95±0.97) mm and (22.82±1.04) mm in ME group,and (23.21±1.08) mm and (23.17 ± 1.15) mm in the control group respectively.The difference between two groups was statistically significant (t=-3.102,P=0.003).There was no correlation between measurement difference and foveal thickness in ME group (r=-0.097; P =0.447).[Conclusion]s There is a difference of AL measurements using contact A-scan and IOLMaster in ME eyes.However,there was no correlation between measurement difference and the foveal thickness.

Background Retinal neovascularization is associated with various disorders.Studying the pathogenesis of retinal neovascularization is of important significance.Oxygen-induced retinopathy(OIR) mouse model is a common animal model for the study of retinal neovascular diseases.However, conventional modeling methods usually cause high animal mortality and low rate of success.Objective This study aimed to establish a modified method of mouse OIR model.Methods Eighty 1-week-old SPF C57BL/6J mice were randomly divided into normal control group and OIR group with 40 mice for each.The newborn mice of the normal control group were kept in a normal air environment with their breast-feeding mothers, but the mice of postnatal 2 days (P2) in the OIR group were raised with two litters per cage until P7.The P7 mice exposed to oxygen tank containing 80％ oxygen together with one or another mother mouse alternately daily for 5 days and then returned to the normal air environment.The success rate of modeling,mortality rate of maternal mice and survival rate of immature mice were evaluated.The mixed solution of fluorescein isothiocyanate (FITC) and PBS with 4％ paraformaldehyde was infused into the hearts of P12, P14,P17 and P21 mice and the eyeballs were obtained after the mice were sacrificed for histopathological examination of retinas and preparation of retinal flatmounts.The number of vascular endothelial cells extending inner limiting membrane was counted and the distribution of retinal vessels was evaluated.The use and care of the animals complied with the Statement of ARVO.Results The survival rate of the neonatal mice was 100％ both in the normal control group and the OIR group,and the survival rate of maternal mice was 85.7％ in the OIR group.Retinal new vessels were found in the mice of the OIR group,with the success rate of modeling 100％.The retinal vessels distributed from optical disc toward periphery in P14 mice in the normal group.However,in the OIR group,non-perfusion area at the posterior pole was seen in P12 mice,new blood vessels at the periphery were found in P14 mice, neovascularization at the junction area between vascular area and non-perfusion area as well as leakages were exhibited in P17 mice,and less non-perfusion area and new vessels were seen in P21 mice.Retinal inner limiting membrane was smooth in the mice of the normal group, and the vascular endothelial cell nucleus extending inner limiting membrane were seen in P12 mice and peaked in P17 mice.The vascular endothelial cell nucleus were (11.44±2.01), (31.24±1.50) and (9.23-±1.12)/slide in P14, P17 and P21 mice in the OIR group,which were significantly more than (0.27±0.14) , (0.30±0.11) and (0.32±0.16)/slide in P14, P17 and P21 mice in the normal group (t=47.90,61.30,40.70,all at P＜0.05).Conclusions The method of OIR modeling is modified by alternating maternal mice,exposing to 80％ oxygen-nitrogen mixture gas and cohabitating immature mice.Modified modeling method is simple with the low death rate of maternal mice and stable OIR phenotype.

Objective To explore the effects of cryopreservation on biological characteristics of cultured limbal stem cells.Design Experimental study.Participants Twenty New Zealand white rabbits.Methods Twenty New Zealand white rabbits(40 eyes) were preparated of peripheral cornea with 2 mm and 2 mm circular shallow conjunctival limbal graft.Rabbit limbal stem cells(Rb-LSCs) used for cell-suspention culture were isolated form New Zealand white rabbit limbal tissues.The limbal stem cells,having formated monolayer cultured limbal stem cells,were cryopreservated one month,then thawing.By immunofluorescence staining,flow cytometry,inverted microscope,MTT assay,we compared the subculture of limbal stem cells cryopreservation before and after the structure and properties.Main Outcome Measurements Limbal stem cell activity and biological characteristics.Results Compared with freshing cells,the cells after cryopreservation in morphology were poor adherence late,small nuclear cytoplasm ratio,irregular shape.Im munofluorescence staining to detect the low-temperature frozen cells ABCG2 monoclonal antibody 81.7% positive rate decreased to 46.9%,the difference was statistically significant(P

Many malfunctions may happen in the operation of plasma sterilization machine.The basic principle and running procedure of plasma sterilization system under normal condition are introduced,and the causes of malfunctions are mainly analyzed so that users can discover and obviate them immediately.

Objective To enhance the quality and efficiency of the outpatient cashier service.Methods A comprehensive outpatient cashier management system based on RFID was developed and put into use.Results The system could be used in outpatient cashier department for attendance checking,schedule management,bonus distribution,personnel management,data statistics and report printing.Conclusion The comprehensive outpatient cashier management system can improve the efficiency of the outpatient department.

ObjectiveTo evaluate the therapeutic efficacy of bleeding cupping with different acupoints and frequencies in treating acne vulgaris.MethodSixty-four patients were divided by using stratified randomization method into Dazhui (GV14) group of 32 cases (16 cases in the subgroup of once every week, and the other 16 cases in the subgroup of twice every week) and Geshu(BL17) group of 32 cases (16 cases in the subgroup of once every week, and the rest 16 cases in the subgroup of twice every week). Dazhui group received bleeding cupping once every week or twice every week, while Geshu group also received bleeding cuppingonce every week or twice every week. The acne symptoms scores and total score were observed before and after intervention to evaluate the therapeutic efficacy.ResultThe symptoms scores including oily condition, skin lesion nature, counts, color, and swelling pain were decreased after intervention in the two groups (P<0.01). The improvement of oily condition in Geshugroup was more significant than that in Dazhuigroup (P<0.05). The symptoms scores including oily condition, skin lesion nature, counts, color, and swelling pain were decreased after intervention in all the subgroups of different frequencies (P<0.01). The improvements of skin lesion nature and swelling pain were more significant in the subgroups of twice a week than that in the subgroups of once a week (P<0.05).ConclusionBleeding cupping with different acupoints and frequencies all can produce satisfactory efficacies; bleeding cupping at Geshucan produce a more significant efficacy than that at Dazhui;treatment at a frequency of twice a week can produce a more significant effect than once a week in improving skin lesion nature and swelling pain.

Through analyzing the data of ectopic thyroid glands, to know this kind of congenital lesion deeply. In all the cases, parathyroid gland types were more than aberrant thyroid types; the majority cases were single ectopic thyroid; most of ectopic thyroid were present in the neck and mouth; local mass was the most common symptom; adenoma was often associated with ectopic thyroid and there was a high proportion of combined malignant; most cases could not be correctly diagnosed.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Child, Preschool ; Choristoma ; pathology ; Female ; Humans ; Infant ; Male ; Middle Aged ; Thyroid Gland ; pathology ; Young Adult