Background Aberrant proliferation of residual lens epithelial cells (LECs) is one of main causes of posterior capsular opacification (PCO).Researches indicated that Wnt3a signaling pathway promote proliferation of epithelial cells,but its effect on LECs is still unclear. Objective The present study was to investigate the effects of Wnt3a on proliferation of human LECs and its mechanism and to provide a new gene target in the prevention and treatment of PCO. Methods Human LECs line (SRA01/04 cells ) was cultured and then incubated to 6-well plate at the density of 4×105/well.A human Wnt3a cDNA expressing vector targeted human LECs was constructed to increase the Wnt3a expression in SRA01/04 cells,and pcDNA3-HA expression vector was used as the control group.The expression of Wnt3a was identified by Western blot assay after transfected.The growth and proliferation of SRA01/04 cells were detected by MTT and flow cytometry (FCM).The expressions of β-catenin,cyclin D1 and c-myc in the cells were detected by Western blot assay.β-Catenin expression was localized using immunofluorescence assay,and the expression and localization of proliferating cell nuclear antigen ( PCNA ) were analyzed by immunocytochemistry for the exploration of the active mechanism of Wnt3a to proliferation of LECs.Results Human Wnt3a cDNA expression vector was designed successfully and transiently transfected to SRA01/04 cells,and Wnt3a/SRA01/04 cells and pcDNA3-HA/SRA01/04 cells were obtained.The expression of Wnt3a was verified in the Wnt3a transfected group compared with the control group.MTT indicated that the cell proliferating rate was significantly different between the Wnt3a transfected group and the control group ( Fgroup =15.235,P =0.005 ;Ftime =369.677,P =0.000),and that in various time points after transfected was significantly different (t =20.843,P=0.001 ;t =26.214,P＜0.001 ;t=25.177,P=0.001 ;t =35.516,P＜0.001 ;t =615.056,P＜0.001 ).The proportion of SRA01/04 cells in G1 phase was 51.74％ in the Wnt3a cDNA transfected group,with a significantly decrease in comparison with 79.44％ of the control group.However,the proportion of SRA01/04 cells in S phase in the Wnt3a cDNA transfected group was higher than that of the control group (36.23％ versus 12.34％ ).The positive expression rate of PCNA protein in SRA01/04 was (47.00％ ±7.58％ ) in the Wnt3a cDNA transfected group and ( 16.00％ ±3.61％ ) in the control group with a significant difference between them (t =8.256,P＜0.01 ).After 48 hours of transfection of the Wnt3a cDNA,the expression amount of β-catenin proteins was higher and the immunofluorescence was stronger in cell nucleus,and the expressions of cyclin D1 and c-myc proteins were elevated in Wnt3a/SRA01/04 cells. Conclusions The overexpression of Wnt3a activates the Wnt/β-catenin signaling pathway and downregulates the expression of a subset of target genes,including cyclin D1 and c-myc,which plays an important role in promoting the proliferation of human LECs.

Animals ; Electromagnetic Fields ; Embryo, Mammalian ; metabolism ; Female ; Mice ; Pregnancy ; Proto-Oncogene Proteins c-fos ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

AIM: To measure the anatomical character of nasolacrimal dust by spiral CT 3D reconstruction in children.METHODS: The length of nasolacrimal dust and the angles between nasolacrimal dust and vertical plane,horizontal plane and coronal plane in 27 children(54 eyes) and 15 adults(30 eyes) were surveyed using spiral CT 3D reconstruction. While the length of nasolacrimal dust in 7 dead children(14 eyes) body were surveyed as comparison by anatomical method. The RESULTS:were analyzed by software SPSS 13.0 statistically. RESULTS: The length of nasolacrimal dust was 10.06±0.29mm in children and 11.51±1.54mm in adults by spiral CT 3D reconstruction,as 9.95±0.31mm in dead children body by anatomical method,with significant statistical difference between that of children and adults. The angles between nasolacrimal dust and vertical plane,horizontal plane and coronal plane in children were 7.96°±1.62°,73.24°±6.75°,and 12.31°±2.03° respectively,while the corresponding angels in adults were 8.08°±0.63°,72.69°±3.85° and 12.09°±1.21°. The difference between them had no statistical meaning. CONCLUSION: The anatomical data of children nasolacrimal dust obtained from spiral CT 3D reconstruction have important guidance to the therapy of nasolacrimal dust diseases in children.

OBJECTIVETo investigate the relations between anti-myelin basic protein antibody (anti-MBP) variation and myelinoclasis in the brain stem following brain trauma.

METHODSIn rat models of brain trauma, MBP content and anti-MBP titer in the blood were measured using enzyme-linked immunosorbent assay (ELISA) at different time points after brain trauma, and the degree of myelinoclasis in the brain stem slices was assessed with osmic acid staining.

RESULTSEarly after brain trauma, MBP content in the blood increased followed by significant reduction 10 days later. Four days after the trauma, anti-MBP titer was markedly increased, accompanied by obvious exacerbation of myelinoclasis in the brain stem, both reaching the highest levels on day 10, at the point of which anti-MBP titer increased by 4 folds and the number of myelinoclasis by 10 folds compared with the control group. Anti-MBP titer and brain stem myelinolysis both lowered 30 days later. Correlation analysis showed an intimate positive correlation between anti-MBP titer and the degree of myelinoclasis.

CONCLUSIONAfter brain trauma, MBP is released as a specific antigen into the blood to stimulate the immune system for anti-MBP production, and the antibody is intimately related to the brain stem myelinoclasis.

Animals ; Antibodies ; metabolism ; Brain Injuries ; complications ; Brain Stem ; immunology ; pathology ; Demyelinating Autoimmune Diseases, CNS ; etiology ; immunology ; Female ; Male ; Myelin Basic Protein ; Nerve Tissue Proteins ; blood ; immunology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Transcription Factors ; blood ; immunology

AIMTo investigate the effects of extremely low magnetic field on acute and chronic arthritis of rats.

METHODSThe acute arthritis animal models were built by the right thenar hypodermic injection of carrageenan. Then they were exposed to magnetic field for 6 hours and 8 hours, respectively. The chronic arthritis animal models were built by the right thenar hypodermic injection of complete freund's adjuvant. They were exposed to magnetic field for 7 days and 6 hours each day after the secondary affection appeared 15 days later. The swelling and inflammatory cytokine were observed in both the acute and chronic arthritis models.

RESULTSThe ankle and thenar swellings decreased in both the acute and chronic arthritis models after different time exposure. The cytokine of serum and articular lixivium did not change in 6 hours exposed groups of acute arthritis models. The serum IL-6 and articular lixivium IL-6, TNF-alpha decreased compared with sham groups. Though the rest indexes were unchanged, they had the tendency of decrease.

CONCLUSIONThe extremely low magnetic field has the effects of restraining acute and chronic arthritis of rats, and can also partially restrain the cytokines when the rats were exposed for 8 hours. The mechanisms need to be further investigated.

Animals ; Arthritis, Experimental ; metabolism ; pathology ; Disease Models, Animal ; Edema ; prevention & control ; Freund's Adjuvant ; adverse effects ; Interleukin-6 ; metabolism ; Magnetic Fields ; Male ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVESThe mutations of growth hormone receptor (GHR) gene results in growth hormone insensitivity (Laron syndrome) or partial growth hormone insensitivity. This study aimed to understand the relation between mutations of GHR gene and short stature with non-growth-hormone deficiency, and the clinical feature of the patients with the GHR gene mutations.

METHODS(1) Forty-seven patients with non-growth-hormone deficiency and short stature were enrolled in this study, 33 were male and 14 female. The age of the patients were at a range of 2 - 16 years. (2) The mutations of GHR gene were identified by PCR-SSCP and DNA sequencing. (3) The characteristics of the GHR mutation was assumed by screening for the same mutations in patients' family members and the control samples.

RESULTS(1) Four GHR mutations were identified in 5 patients with non-growth-hormone deficiency: H56R, G148E, IVS6-30, -31CA > TG and IVS8 + 10G > C. These mutations were located within the extracellular domain of GHR and not reported before. Five patients were the heterozygous of H56R, G148E, IVS6-30, -31CA > TG and IVS8 + 10G > C. The detection rate of mutant heterozygous individual accounted for 10.6% (5/47). The mutations were considered non-polymorphism by the GHR gene analysis in patients' family members and control samples. (2) Comparison of the amino acid sequence of different species and the position of the mutations H56R and G148E in the GHR protein structure suggested impact of the mutations on the protein function. (3) A polymorphism site was identified in exon 6 of GHR gene: G168G (GGA > GGG). The allelic frequency of G168G had no difference between the patients with non-growth-hormone deficiency and control samples but had significant difference between Chinese and Caucasian. It seems that the G168G was a polymorphism and has no relationship with the height stature. However, there was the allele diversity in different races.

CONCLUSIONThe mutations of GHR gene were detected in the patients with non-growth-hormone deficiency. Special attention should be paid clinically to its potential pathogenesis for short stature.

Adolescent ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Case-Control Studies ; Child ; Child, Preschool ; DNA Mutational Analysis ; European Continental Ancestry Group ; genetics ; Female ; Growth Disorders ; genetics ; Humans ; Male ; Molecular Sequence Data ; Mutation ; Polymorphism, Genetic ; Receptors, Somatotropin ; genetics

OBJECTIVETo investigate the mutation in mitochondrial DNA displacement-loop (mtDNA D-loop) region in oncocytoma and its relationship with tumorigenesis and tumor development.

METHODSThe mtDNA D-Loop region of 20 thyroid or renal oncocytomas and the adjacent normal tissues were amplified by PCR, and then sequenced. Five human fetal renal tissues were collected as matched controls.

RESULTSAmong the 20 oncocytomas, 21 mutations which focused on hypervariable region I (HVI) were found in 7 tumor tissues and 1 normal tissue with the mutation rates of 35% and 5%, respectively. At the same time, 191 polymorphisms were found in the 20 cases.

CONCLUSIONmtDNA D-loop region, especially HV I, is the mutational hotspot of oncocytomas, which may be closely related with mtDNA duplicating rate and the function of mitochondria.

Adenoma, Oxyphilic ; genetics ; Base Sequence ; DNA Mutational Analysis ; DNA, Mitochondrial ; genetics ; Female ; Humans ; Kidney Neoplasms ; genetics ; Male ; Middle Aged ; Mitochondria ; genetics ; Mutation ; Polymorphism, Genetic ; Thyroid Neoplasms ; genetics

Objective To observe the inhibitory effect of CDglyTK double suicide gene on the growth of C6 glioma in vivo. Methods Thirty 4- to 6-week-old male Balb/c nude mice with subcutaneous injection of C6 glioma cells were randomized into two groups for injections with 5x105 CFU of CDglyTK (treatment group) or 50 μL PBS (control group) for 5 consecutive days. RT-PCR was performed to examine the mRNA expression of the CDglyTK fusion gene in the tumor cells. All the mice received intraperitoneal injections of 5-FC (500 mg/kg) and GCV (60 mg/kg) for 7 days, and the tumor volume, weight and mice survival time were observed. The effect of CDglyTK gene transfer on the apoptosis of the C6 glioma cells was evaluated using flow cytometry. Results RT-PCR demonstrated effective expression of the fusion gene in C6 glioma cells. In the fourth week of tumor cell inoculation, the tumor in the control group grew to the volume of 20 mmx30 mm with occasional death of the mice, and in week 6, all the control mice died. In the treatment group, the tumors showed no obvious growth with a volume of around 5 mm, and some of tumors even disappeared; no death of the mice occurred at the end of the third month. By gross observation, the tumors in the control mice were large and red with rich blood supply, but those in the treatment group showed reduced volume. On day 21 following tumor cell inoculation, the weight of the tumors was significantly greater in the control group than in the treatment group (2.51±0.58 vs 0.35±0.26 g, P<0.05), and the growth inhibition rate was 86.1% in the latter group. Flow cytometry showed significantly higher apoptotic rate of the tumor cells in the treatment group than in the control group (34.41%±5.2% vs 2.92%±1.3%, P<0.05), and electron microscope demonstrated the formation of apoptotic bodies in the tumor cells in the former group. Conclusion Significant antitumor effects can be obtained with the CKglyTK fusion gene in combination with the two prodrugs.

OBJECTIVETo investigate the effect of simulated microgravity on erythroid differentiation of K562 cells and explore the possible mechanism.

METHODSThe fourth generation rotating cell culture system was used to generate the simulated microgravity environment. Benzidine staining was used to evaluate the cell inhibition rate, and real-time quantitative PCR (qRT-PCR) was used to detect GATA-1, GATA-2, Ets-1, F-actin, β-Tubulin and vimentin mRNA expressions. The changes of cytoskeleton were observed by fluorescence microscopy, and Western blotting was employed to assay F-actin, β-tubulin and vimentin protein expression levels.

RESULTSBenzidine staining showed that simulated microgravity inhibited erythroid differentiation of K562 cells. K562 cells treated with Hemin presented with increased mRNA expression of GATA-1 and reduced GATA-2 and Ets-1 mRNA expressions. Simulated microgravity treatment of the cells resulted in down-regulated GATA-1, F-actin, β-tubulin and vimentin mRNA expressions and up-regulated mRNA expressions of GATA-2 and Ets-1, and reduced F-actin, β-tubulin and vimentin protein expressions. Exposure to simulated microgravity caused decreased fluorescence intensities of cytoskeletal filament F-actin, β-tubulin and vimentin in the cells.

CONCLUSIONSimulated microgravity inhibits erythroid differentiation of K562 cells possibly by causing cytoskeleton damages to result in down-regulation of GATA-1 and up-regulation of GATA-2 and Ets-1 expressions.

Actins ; metabolism ; Cell Differentiation ; Down-Regulation ; GATA1 Transcription Factor ; metabolism ; GATA2 Transcription Factor ; metabolism ; Hemin ; pharmacology ; Humans ; K562 Cells ; Proto-Oncogene Protein c-ets-1 ; metabolism ; Tubulin ; metabolism ; Up-Regulation ; Vimentin ; metabolism ; Weightlessness Simulation

OBJECTIVETo study the role of carbamyl phosphate I (CPS-I)and ornithine transcarbamoylase (OCT) levels in cirrhosis patients with and without hepatic encephalopathy, and to analyze the correlations between CPS-Iand OCT with the development of hepatic encephalopathy.

METHODSCPS-I, OCT, plasma ammonia and liver function of 95 cirrhosis patients with hepatic encephalopathy and 25 cirrhosis patients without hepatic encephalopathy in our hospital from January 2008 to December 2009 were analyzed. 60 healthy controls were recruited in the control group. The differences of serum CPS-I, OCT levels among the cirrhosis patients with and without hepatic encephalopathy and the healthy controls were analyzed; the correlations of CPS-I, OCT levels with plasma ammonia and total protein in cirrhosis patients,and the correlations of CPS-I, OCT levels with Child-Pugh classification of cirrhosis symptom severity in cirrhosis were analyzed. the clinical characteristics between patients who had HE and no HE with chi-square tests were compared. Comparisons of CPS-I, OCT levels across patients based on the Child-Pugh classification were performed with One-Way ANOVA and Student-Newman-Keuls, correlation of CPS-I, OCT with other indicators were performed with Pearson correlation analysis.

RESULTSSerum CPS-I and OCT levels in cirrhosis patients with hepatic encephalopathy were (143.3+/-48.5) U/L, (297.0+/-102.6) is multiplied by 10 U/L, which were lower than that in cirrhosis patients without hepatic encephalopathy (180.3+/-51.5) U/L, (351.8+/-109.0) is multiplied by 10 U/L (t = 2.53, t = 2.78, P < 0.01). Compared with healthy controls, serum CPS-I and OCT levels in cirrhosis patients with and without hepatic encephalopathy were all lower (t = 3.21, t = 4.16, t = 2.12, t = 3.15, P < 0.05). CPS-I was correlated with OCT, (r = 0.946, P < 0.05); CPS-I and OCT were negatively correlated with ALT and AST (r = -0.284, r = -0.239, r = -0.303, r = -0.322, P < 0.05). Additionally, CPS-I and OCT levels were negatively correlated with the Child-Pugh classification in Cirrhosis (F = 10.13, F = 20.28, P < 0.01).

CONCLUSIONThe serum CPS-I and COT levels were important factors affecting plasma ammonia in patients with cirrhosis and played an important role in the development of hepatic encephalopathy.

Adult ; Ammonia ; blood ; Carbamoyl-Phosphate Synthase (Ammonia) ; metabolism ; Case-Control Studies ; Female ; Hepatic Encephalopathy ; blood ; enzymology ; Humans ; Male ; Middle Aged ; Ornithine Carbamoyltransferase ; metabolism