Objective: To investigate the regulatory effects of two kinds of SV40 poly(A) signals on the upstream gene expression in three cell lines, so as to provide theoretical evidence for selection of poly(A) of vectors. Methods: A dual luciferase reporter vector Dual-Luc was constructed, and two SV40 poly(A) signal sequences were inserted in the downstream of the R-Luc gene separately. Then the two diverse dual luciferase reporter gene vectors Dual-Luc2 and Dual-Luc3 were transfected into 293, L-02, and HeLa cells. The relative quantities of the target gene (F-Luc) to control gene (R-Luc) were measured by Dual-Glo™ Luciferase Assay System and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Results: The two Dual-Luciferase vectors (Dual-Luc2 and Dual-Luc3) were successfully constructed. Dual-Glo™ Luciferase Assay showed that the mean F-Luc/R-Luc values were 3.25±0.43 and 3.03±0.14 in Dual-Luc2 and Dual-Luc3 transfected 293 cells(P>0.05), 6.16±0.39 and 3.83±0.39 in L-02 cells(P<0.05), and 1.21±0.10 and 0.66±0.02 in HeLa cells(P<0.05), respectively. The results of RT-PCR were consistent with those of luciferase assay. Conclusion: The two kinds of SV40 poly(A) signals have different regulatory effects on the upstream gene expression in different cell lines. SV40 poly(A) signals regulate the upstream gene expression at the transcriptional stage.

Child ; Cystitis ; Eosinophils ; Humans ; Male

Bronchiolitis Obliterans ; Child ; Humans

Objective:To establish a human monocyte cell line U937 stably expressing CD200.Methods:The eukaryotic expression vector pcDNA3-CD200 containing human CD200 cDNA and vector pcDNA3 were transferred into human monocyte U937 cell line by electrotransfer.These two cell lines were selected by G418, and the selected cell lines were subcultured. U937 cell line was enclosed as control group.The expression of CD200 mRNA and protein was detected by RT-PCR and flow cytometry.Results:After G418 selection,U937 cell line in control group was died, and the cell lines transferring pcDNA3 and recombined pcDNA3-CD200 were subcultured over 30 generations.The CD200 mRNA expression in pcDNA3-CD200 group was confirmed with RT-PCR.The CD200 expression in U937-pcDNA3-CD200,U937-pcDNA3 and U937 were 77.20%,3.20% and 2.10%,compared with other two groups (F=133 996.40,P0.05).Conclusion:Our study provides foundation for mechanism of action for CD200 in future.The human monocyte series U937 cell line that expresses CD200 protein stably is established successfully by gene transfection method.

BACKGROUND:It is wel known that long-term aerobic exercise aleviates renal dysfunction in spontaneously hypertensive rats. However, the underlying molecular mechanism remains unclear.
OBJECTIVE:To investigate the effect of long-term aerobic exercise on endogenous formation of hydrogensulfide in the kidney of spontaneously hypertensive rats.
METHODS:Rat models of long-term aerobic exercise were established and randomly assigned to four groups: Wistar-Kyoto (WKY) rat static group, WKY rat exercise group, spontaneously hypertensive rat static group and spontaneously hypertensive rat exercise group. Moderate-intensity exercise on treadmil was given for 12 weeks. At 24 hours after model establishment, weight was weighted. Blood pressure was detected in the caudal artery. Blood and urine were colected for measuring biochemical indicators related to kidney functions. The degree of glomerular sclerosis was observed. Hydrogen sulfide production activity was detected in the kidney. RT-PCR was used to detect mRNA expression levels of hydrogen sulfide production-related enzymes. Simultaneously, oxidative stress of the kidney was observed.
RESULTS AND CONCLUSION:(1) Long-term aerobic exercise obviously reduced body mass, systolic blood pressure and diastolic blood pressure, increased glomerular filtration rate and renal blood flow, decreased serum creatinine and urea nitrogen levels, urinary albumin levels, significantly reduced glomerular sclerosis score, increased hydrogen sulfide content in plasma and the rate of hydrogen sulfide formation in renal tissue, up-regulated cystathionine γ-lyase expression, obviously diminished malondialdehyde content in serum and kidney, and remarkably increasedthereduced glutathione and oxidized glutathione ratio in spontaneously hypertensive rats. (2) Results indicated that long-term aerobic exercise could increase the generation of endogenous hydrogen sulfide in kidney, lessen oxidative stress in the kidney, and amelioraterenal dysfunction in spontaneously hypertensive rats.

Objective To study the effects of sodium arsenite on the mRNA expression of GTP cyclohydrolase (GTPCH) and 6-pyruvoyl-tetrahydropterin synthase (PTPS) in Chang liver cells. Methods Human Chang liver cells were cultured with sodium arsenite at doses of 0, 50, 200 and 400 ?mol/L for 12 hours. Cell viability was tested by MTT assay, and the expression of mRNA of GTPCH and PTPS was detected by RT-PCR. Results Cell viability in 50 ?mol/L group was almost the same as that in control, while the viability in the other two groups were significantly lower than that in control. All of the mRNA expression levels in the three groups were significantly lower than that in control with dose-dependent manner. Conclusion The down-regulated expression of GTPCH and PTPS, the main enzymes of the synthesis of BH4, induced by sodium arsenite exposure may be related with arsenic related skin depigmentation.

Objective To investigate the association of gene polymorphism of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) with ulcerative colitis (UC) in Chinese patients. Methods The A+49G transition polymorphism at position 49 (exon 1) and C-318T transition polymorphism at position -318 in promoter of the CTLA-4 gene were determined by polymerase chain reaction with sequence specific primers (PCR-SSP) method in 82 Chinese patients with UC and 204 healthy controls of Han nationality. Results No significant differences in the distribution of genotype and allele frequencies were observed between CTLA-4 C-318T and A+49G gene polymorphisms in UC patients and normal controls. How- ever , comparing with healthy subjects,the frequency of haplotype 2,3 (C -318 -G 49 /T -318 -A 49 ) in UC patients was significantly reduced (26% vs 41%, P

Objective To study the change of the structural and biological properties of the cardiovascular tissue after cell extracting. Methods The native and treated pulmonary cusp and pulmonary artery wall were evaluated by HE stain, ETVG stain, immunochemistry stain and scan electron microscopy. The contents of the main macro-molecules were measured. The content and position of calcium was investigated by subcutaneous implantation in a rat model. Results The acellularization procedure resulted in an almost complete removal of the original cells while the construction of the matrix remained. The contents of colleagen and elastin increased while soluble GAG reduced. The content of calcium in acellular tissue by xenogenic implantation was less obviously than that in fresh one. Conclusion A suitable acellularization procedure can remove all cells and most soluble proteins, and matrix remains nearly intact, so as to alleviate obviously calcification via xenogenic implantation in vivo.

Objective To investigate the effect of stress induced by fear on germ cells apoptosis in rat's testis. Methods 36 SD rats were randomly assigned to acute stress group, subacute stress group, chronic stress group and control group, respectively. The model of the stress rat induced by fear was reproduced. The levels of caspase-3 mRNA and bax /bcl-2 mRNA expression in rats' testis were determined by fluorescent quantitative RT-PCR. The apoptosis of spermatogonic cell, primary spermatocyte, secondary spermatocyte and spermatid was identified by TUNEL. Results Caspase-3 mRNA, compared with that in control group, was expressed highly in the subacute stress group (5.34?1.13 vs 3.67?0.34, P0.05). The ratio of bax /bcl-2 mRNA, compared with that in the control group, was increased significantly in the subacute stress group (2.68?0.86 vs 1.60?0.42, P0.05). Compared with that in control group (1.50?0.58), the apoptotic index of germ cells was higher in the subacute stress group (10.50?1.29, P0.05). Conclusion Stress induced by fear plays an important role in inducing germ cells apoptosis, and caspase-3, bax and bcl-2 were involved in this process.

Objective To investigate the characteristics and possibility of using an image analyzer-aided method to count axotomized retinal ganglion cells (RGCs). Methods The left optic nerves of 18 rats were transected intraorbitally and a piece of gelform soaked in 5% fluorogold was applied to the ocular stump to retrogradely label the surviving RGCs. All animals were executed 2, 7 or 14 days after the operation (n=6 for each time point), respectively. The left retinae were removed, post-fixed and whole-mounted on the slides. The numbers of labeled RGCs were counted using both the conventional sampling method and image analysis, and compared statistically between the two methods. Results The number of surviving RGCs decreased sharply [(12 0663?9 089), (59 285?17 071) and (17 802?19 84) cells/mm 2 for image analyzer-aided method, and (118 237?7 898), (57 648?14 533) and (18 070?1 461) cells/mm 2 for conventional sampling method] when the survival time increased from 2 to 7 and 14 days. No significant difference was detected between the two groups at any corresponding time points. Conclusion The image analyzer-aided method is convenient, objective and reproducible, which can be used in the studies where counting RGCs is needed.