Objective To establish relative quantification in real time reverse transcription polymerase chain reaction to measure cytokine expression.Methods TNF? and GAPDH were used as target gene and internal reference respectively. Two gene fragment were cloned, vectors were purified and quantificated. Standard curves were established using a series dilution of quantificated plasmids to measure the amplification efficiency of TNF? and GAPDH. By changing reaction conditions, the amplification efficiency of two gene were nearly 100%. Ct value of TNF? and GAPDH were measured in 14 samples stimuteneously.Results The method can detected as low as 10 3 copies with the linear range was 10 3～10 9 copies, the intra assay and interassay variation was 1% and 8% respectively. TNF? increased 8 6～9 8 fold with the average 9 2 relative to untreated control group analyzed by the 2 -??Ct methods. Conclusions The method we established has fine sensitivity and reproducibility and the data analysis was simple and reliable and can be apply to any genes.

Vesicoureteral reflux,a most common congenital anomaly of the kidney and urinary tract,is associated with the malformation of ureterovesical junction. It does not cause any specific symptoms or signs un-less it is part of a syndrome or complicated by urinary tract infection. The exact cause is not clear,and genes or environmental factors may result in vesicoureteral reflux. The prevalence of siblings and offspring of reflux pa-tients are higher than normal control groups,so the genetic screening is necessary. This article will review the ge-netics of vesicoureteral reflux and possible interactions.

Objective:To study adenovirus mediated transfer of TGF ?1 in treatment of mice autoimmune myocarditis. Methods: Experimental autoimmune myocarditis was induced in BALB/c mice using purified myosin from porcine heart. TGF ?1 was transferred into mice by intravenous injection of adenovirus encoding TGF ?1. Plasma level of myosin autoantibody was detected by indirect ELISA method and plasma level of troponin I was assayed using fluorescence immune assay method. The protective effects of TGF ?1 were evaluated from 3 aspects: (1) the degree of infiltrative inflammatory cells in the heart; (2) the plasma level of myosin autoantibody; (3) the plasma level of troponin I. Results: TGF ?1 successfully ameliorated the myocarditis. Plasma level of troponin I and myosin autoantibody decreased significantly( P

BACKGROUND:Most of the traumatic brain injury and stroke survivors have foot drop and varus deformity, that need to be treated with ankle-foot orthosis. OBJECTIVE:To investigate the advantages and effect of early application of front-ankle-foot orthosis on the walking function of the patients with hemiplegia after stroke. METHODS:The clinical data of 54 patients with hemiplegia after stroke treated in the Changshu No. 2 People’s Hospital from June 2008 to October 2009 were retrospectively analyzed. The patients treated with front-ankle-foot orthosis were the front group (n=28), and the patients treated with rear-ankle-foot orthosis were the rear group (n=26). Al the patients received 10 m maximum walking speed test, and the recovery of walking function of the patients in two groups were observed daily. RESUTLS AND CONCLUSION:There were no patients exited and dead in the observation period. The activities of daily living Barthel index in the front-ankle-foot orthoses group (60.0±12.9) was significantly higher than that in the rear-ankle-foot orthoses group (59.1±10.9), and there was no significant difference in the activities of daily living Barthel index between two groups (P>0.05). For the 10 m maximum walking speed test, the walking speed of (39.6±11.6) m/min in the front-ankle-foot orthosis group was significantly faster than that of (33.0±12.4) m/min in the rear-ankle-foot orthosis group (P<0.05). Front-ankle-foot orthosis is more appropriate for patients with hemiplegia after stroke to improve the walking speed.

Objective:To clone the rat cardiac myosin ? heavy chain cDNA fragment encoding aa736 960 and construct its recombinant retrovirus vector Methods:The 681 bp target gene was amplified from heart tissue of young rats with RT PCR Fusion gene of hIL 2/myosin was constructed by splicing with preserved region of hIL 2 cDNA using ligation methods and subsequently the plasmid pLNC hIL 2 myosin was constructed NIH3T3 cells and PA317 cells were transfected with plasmid pLNC hIL 2 myosin using Lipofectamine After screening with medium containing G418, the positive clone was chosen and was detected using RT PCR, immunohistochemistry, immune electron microscope and dot blot Results:The determination of nucleotide sequence showed that the nucleotide and amino acid sequence of the gene cloned was the same as the reported sequence, and its open reading frame was correct RT PCR analysis indicated that mRNA of the fused gene was present in the positive clone Immunohistochemistry, immune electron microscope and dot blot showed that the fused gene IL 2 myosin was successfully expressed Conclusion:The fused gene of rat cardiac ? heavy chain fragment and the preserved region of human IL 2 was constructed and expressed successfully

Objective To set up a HPLC method for the determination of the contents of notoginsenoside R1,ginsenoside Rg1 and Rb1 in Kangliu pills.Methods It was performed on Kromasil C18(4.6 mm?150 mm,5 ?m),and the mobile phase was acetonitrile-0.05% phosphoric acid with gradient elution system.The flow rate was 1.0 mL/min,the detective wavelength was 203 nm,injection volume was 10 ?L and the column temperature was 30 ℃.Results The liner range of notoginsenoside R1 was 82.8～621 ?g(r=0.999 9) and the average recovery was 100.057%(RSD=0.399 6%).The liner range of ginsenoside Rg1 was 109.8～ 823.5 ?g(r =0.999 6) and the average recovery was 99.248%(RSD=1.004 6%).The liner range of ginsenoside Rb1 was 112.6～844.5 ?g(r=0.999 9) and the average recovery was 99.812%(RSD=1.474 4%).Conclusion The method is simple,accurate and reproducible,and suitable for the content determination of notoginsenoside R1,ginsenoside Rg1 and Rb1 in Kangliu pills.

Objective:To construct adenovirus containing murine MHC class II transactivator (CIITA) mutant gene and to observe expression of this gene mediated by adenovirus as well as function of the expression product in vitro.Methods:The type IV CIITAcDNA gene was cloned from peritoneal macrophage of BALB/c mouse by conventional way of molecular biology, and then it was cloned into expression vector pIRES. CIITA mutant gene was constructed by the way of overlap extension by PCR (OE-PCR), and then it was also cloned into expression vector pIRES. By using of pAdEasy-1 system we acquired the deficient recombination adenovirus Ad-CIITAm, containing CIITA mutant gene, which had the ability of infection and the no-loaded control adenovirus Ad-GFP.Then the two adenoviruses strains were processed for a great deal amplification,purification and titer determining. The Ad-CIITAm and Ad-GFP were infected into HeLa cells and Raji cells.Inducible or constitutive expression of HLA-DR molecule and the following changes were observed by the use of flow cytometry.Results:The murine CIITA gene was cloned successfully and the recombinantant adenovirus Ad-CIITAm containing murine CIITA mutant gene was also constructed successfully. It was proved by flow cytometry that expression of HLA-DR molecules on the surface of HeLa cells and Raji cells infected by Ad-CIITAm were all decreased remarkably in comparison with those of control cells infected by Ad-GFP.Conclusion:This experiment proves that expressed murine CIITA mutant mediated by adenovirus could inhibit the expression of MHC II molecules efficiently.

AIM: To construct a recombinant adenovirus vector which regulated expression of mouse transforming growth factor beta (TGF-?1). METHODS: Total RNA was extracted from Balb/c mice spleen pre-treated with Con A to clone TGF-?1. pTRE-shuttle vector was used as mediator to ligate TGF-?1 gene and the backbone of the replication-incompetent adenoviral vector. The constructed recombinant adenovirus contains tetracycline-responsive element which can regulate the expression of inserted genes. Identifying the desired recombinant adenovirus, it was packaged in HEK 293 cells. Supernatant of high titer adenovirus was collected to detect the TGF-?1 gene expression by ELISA kit. RESULTS: The constructed recombinant adenovirus had been identified by restriction endonucleases cutting, sequencing and PCR. In the supernatant of infected cells, it expressed the mice TGF-?1 in high concentration to 91.16 ng/L. CONCLUSION: The mice TGF-?1 recombinant adenovirus has been built which make a basis for further research and use in gene therapy.

Objective:To produce a fusion protein(ICOS-Ig)between extracellular portion of human ICOS and Fc portion of mouse IgG 2a and study on its biological activity.Methods:cDNA encoding the extracellular domain of human ICOS was prepared by RT-PCR from the RNA of the stimulated human peripherial blood mononuclear cells. The Fc portion of mouse IgG2a was cloned by PCR from the vector that contains the sequence-encoding Fc portion of mouse IgG2a.Two resulting amplified PCR products were ligated into a secrection mammalian expression vector, pSecTag2/Hygro A. The recombined vector was transfected into CHO cells by lipofectamine2000.Results:RT-PCR demonstrated the integration and mRNA synthesis of fusion gene. ELISA and Western blot analysis showed the expression of ICOS-Ig and its molecular weight was between 43-66 kD, its concentration was 5-25 ?g/ml. FACS analysis assured that ICOS-Ig had ligand specific binding activity. Addition of ICOS-Ig to MLR resulted in inhibition of T-cell proliferation and IL-2,IFN-? secretion.Conclusion:The fused gene ICOS-Ig was constructed and expressed successfully. It had the bioactivity of inhibition of T cell proliferation and cytokine secretion.

Taking Coxsackie B3 virus cDNA labelled by Biotin as detecting probes and 25 kinds of standard enteroviruses as detected viusr, a spot nucleic acid hybridization method has been established to detect enterovirus RNA gene. The method has simple operation and its sensitivity is 50-80 TCID50 spot. Reagent is safe and stable, and may be stored for long period. pCBHⅢ/35.51 probe used has wider hibridization spectrum to enteroviruses; pCBⅢ/29 only reacts with RNA of Coxsackie B3 virus in strict condition. Consequently, the hybridization character which these probes possess is suitable to determining enlerovirus in clinical samples.