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.

Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Nursing Staff, Hospital ; psychology ; Surveys and Questionnaires

Protein post-translational modifications are important ways to regulate protein function and cell behavior, and oxidative stress directly influences protein post-translational modifications. P53 protein has various post-translational modifications, which can be quickly regulated under stress conditions to activate a series of downstream target genes and facilitate the p53 function diversity. The effects of oxidative stress on p53 post-translational modifications of phosphorylation, ubiquitination, sumoylation, acetylation and methylation are introduced in this paper.

Thyroglossal duct cyst is the most common congenital malformation of the neck. It is generally with non-specific symptoms. In our hospital, one case of laryngeal obstruction caused by giant thyroglossal duct cyst was cured, and this case was reported for reference.
Airway Obstruction ; Humans ; Laryngeal Diseases ; Thyroglossal Cyst

Background and purpose:Drug-resistance is the main obstacle in terms of efficacy of chemotherapy for leukemia, RNA interference(RNAi) strategy possesses the characteristics of specilization, high-efficiency and low-toxicity, and can effectively and specifically inhibit the overexpression of given gene. This study was designed to investigate the effect of small interfering RNA (siRNA) on expression of mdr1 gene and drug-resistance in multidrug-resistant human leukemia K562/ADM cell.Methods:Human multidrug-resistant leukemia cell line K562/ADM over-expressing mdr1 gene was used as the target cells, Two siRNAs (si-mdr1-1 and si-mdr1-2) targeted mdr1 gene were chemically synthesized and transfected into K562/ADM cells. Expression of mdr1 mRNA was determined by RT-PCR, P-glycoprotein (P-gp) expression was measured using flow cytometry (FCM), and the sensitivity of K562/ADM cells to adriamycin was assessed with a MTT colorimetric assay.Results:Two siRNAs (si-mdr1-1 and si-mdr1-2) specially designed in this study could markedly down-regulate the expression of mdr1 mRNA and its product P-gp in K562/ADM cells. After cells transfected with si-mdr1-1 or si-mdr1-2 for 24h and 48h, the inhibition of mdr1 mRNA expression in the cells for si-mdr1-1 was 55.5% and 22.5%; and for si-mdr1-2, 16.0% and 57.6%, respectively. Treated with siRNA for 72h, the expression intensity of P-gp in the two transfected cell lines decreased 74% and 85%, respectively. Both si-mdr1-1 and si-mdr1-2 significantly enhanced the sensitivity of K562/ADM cells to adriamycin and reversed their drug-resistance, the reversal efficiency was 2.52-folds and 1.96-folds, respectively.Conclusions:The siRNA could effectively and specifically silence the expression of mdr1 gene and overcome the drug-resistance mediated by P-gp in K562/ADM cells.

BACKGROUND:Selection of resin core materials may affect the overal strength of the fiber posts.
OBJECTIVE:To compare the overal flexural strength of five kinds of resin core materials combined with glass fiber posts.
METHODS:Fifty viva glass fiber posts were randomly divided into five groups respectively binding to five different resin materials for repair:group A, MEDENTAL dual curing resin cement+glass fiber post;Group B, Tina dual curing resin cement+glass fiber post;group C, Bisco BisCem+glass fiber post;group D, 3M nano composite resin curing light P60+glass fiber post;group E, PULPDENT dual curing resin cement+glass fiber post. The root canals were embedded with self-curing plastic, and fixed in the universal testing machine. The load in tooth length axis was added onto the core at a 135° angle with a loading speed of 1.0 mm/min, until the fracture. Then, the stress at fracture and the fracture mode were measured.
RESULTS AND CONCLUSION:The flexural strength was (83.248±7.857) N in group, (89.230±4.326) N in group B, (95.188±5.147) N in group C, (76.646±6.463) N in group D, and (83.064±3.964) N in group E. Except groups A and E, there were significant differences between every two groups (P<0.05). These findings indicate that Bisco BisCem resin cement binding to the fiber post can obtain a higher flexural strength.

Objective To investigate the correlation between uric acid level and macrovascular disease in type 2 diabetic patients.Methods Sixty type 2 diabetic patients with lower limb atherosclerosis of carotid artery were randomly selected in study group who hospitalized in the First Hospital of Zibo from Mar.to Feb.2012.Sixty type 2 diabetes mellitus(T2DM) without carotid and lower limb athemsclerosis were served as control group.The blood pressure,blood lipid,blood glucose and other biochemical indexes,including blood uric acid,serum insulin (FNS),fasting blood glucose (FPG),apolipoprotein a (LP (a)),apolipoprotein A1,B (APO-A1,APO-B),glycosylated hemoglobin (HbA1c),high density lipoprotein cholesterol (HDL-C),low density lipopmtein cholesterol (LDL-C),triacylglycerol (TG) and total cholesterol (TC) were measured and determined.Results There was no significant difference in terms of blood pressure,blood lipid levels,APO-A1,APO-B,HbA1C,FNS and FPG in study group patiems (P ＞ 0.05).The level LP(a) in study group was (0.4 ± 0.2) g/L,significantly higher than that in control group ((0.2 ± 0.2) g/L; t =3.842,P ＜ 0.01).The blood uric acid level in study group was (362.3 ± 112.8)mmol/L,significantly higher than that of the control group((284.8 ±68.6)mmol/L;t =3.188,P＜0.01).Conclusion Uric acid and LP(a) are involved in the oocurrence and development of athemsclemsis,which is close related to the development of type 2 diabetic macmangiopathy.Therefore,in the process of preventing type 2 diabetes with macroangiopathy,we should pay attention to uric acid and LP (a) of the patient beside effective control of blood glucose,blood pressure,blood lipid level.

Objective To investigate whether emodin enhances chemosensitivity of pancreatic cancer cells and the mechanism. Methods Normal human skin fibroblasts and pancreatic BXPC-3 cells were divided into control group, cisplatin (5 mg/L) treatment group, gemcitabine (0.5 μmol/L) treatment group, emodin (50 μmol/L)+ cisplatin (5 mg/L) co-treatment group and emodin (50 μmol/L) + gemcitabine (0.5 μmol/L) co-treatmentgroup. The cell viability was detected by MTT assay, the cell apoptosis rate by flow cytometry, the expression of the multidrug resistance-associated protein 1 ( MRP1 ), MRP2, breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) mRNA in different cell lines was determined by reverse transcription polymerase chain reaction.All data were analyzed using the t test. Results The cell viability and apoptosis rate of pancreatic BXPC-3 cells after treatment were 62.44% ± 3.42% and 27. 10% ± 4.24% in the cisplatin treatment group, and they were 30.53% ±0.05% and 66.33% ±9.37% in the emodin + cisplatin co-treatment group. There were significant differences in cell viability and apoptosis rate between the two groups (t = 13.20, 5. 35, P < 0.05 ). The cell viability and apoptosis rate of pancreatic BXPC-3 cells after treatment were 79.82% ±2.83% and 13.48% ± 1.65%in the gemcitabine treatment group, and they were 45.65% ± 2.46% and 62.74% ± 10. 18% in the emodin +gemcitabine co-treatment group. There were significant differences in cell viability and apoptosis rate between the two groups (t = 12.89, 8.28, P < 0. 05 ). There was no significant difference in the viability of normal human skin fibroblasts between the cisplatin treatment group and the emodin + cisplatin co-treatment group, and also between the gemcitabine treatment group and the emodin + gemcitabine co-treatment group (t = 2. 08, 0. 64, P >0.05 ). Expression of MRP1 mRNA was detected in pancreatic BXPC-3 cells, whereas the expression of MRP2,BCRP and P-gp mRNA was undetectable. The expression of MRP1 mRNA in pancreatic BXPC-3 cells was significantly down-regulated in the cisplatin treatment group, and cisplatin + emodin co-treatment had an additive effect on down-regulating the expression of MRP1 mRNA. Conclusion Emodin may enhance chemosensitivity of pancreatic cancer cells to cisplatin by down-regulating the expression of MRP1 mRNA.

Objective To establish a HPLC/MS/MS method for the determinati on of gentiopicroside in human urine.Methods The urine sample was treated by solid-phase extraction with internal standard of caffeine;The RESCEK C8 colum n(150mm?2.1mm,5 ?m) was used as the analytical column with a mobilep hase consisting of methanol-10mmol? L-1 NH4AC buffer(pH=6.5)-acetonit rile(50∶40∶10,V/V),the flow rate was 0.2 mL? min-1;A triple quadruple tandem mass spectrometer was used as the detector,Electrospray ioniza tion source was applied and operated in positiveion mode.Gentiopicroside and caffeine were detected by monitoring the ion transition of m/z 374.1→ 195.2 and m/z 195.2→ 138.2 respectively.Results The linear range was 30～ 9000 ng?mL-1(r=0.9980) for gentiopicroside in human urine.The recovery was 91.10% ～ 9 6.21 %,The absolute recovery was 100.52% ～ 103.83%,The within-day and between-day precisions were less than 10 %.Conclusion The method is proved to be sensitive,accurate,rapid,specific.