Although it has many important functions in physiology,glutamine is not used widely in clinic.Low dissolution and unstabitity in water are the main disadvantages of glutamine.L-alanyl-L-glutamine dipeptide is the product of high dissolution and stability in water and can be metabolised and utilized rapidly in the body.The metabolism and clinical use of the dipeptide are reviewed here.

[Objective] To investigate the difference of bone Absorption function of the rat osteoclast(OC) in different cultural methods, and the difference in the levels of mRNA expression of bone Absorption key gene, ATPase a3 gene, and provide the basis for further investigation on the OC in vitro. [Methods] with mechanical separation method, mature osteoclast was separated from inner wall of 24 hours newborn rat long bones medullary cavity; and induction culture methods, bone marrow cell was induced to osteoclast like cell(OLC) by using 1,25(OH)2D3.The morphological and functional change of osteoclast was observed and the difference in the level of ATPase a3 mRNA expression was determined.[ Result]OC and OLC are multinucleated giant cells, which can be stained positive by potartrate resistant acid phosphatase(TRAP), and can form bone Absorption lacuna. The number of OLC in induction method is more than in mechanical separation method, but bone Absorption lacuna is smaller and shallower than the early stage of induction. The late stage of OCL is extremely similar to OC. There is no significant difference of ATPase a3 mRNA expression from the cells between mechanical separation 8 hour and induction culture 6 days, but either of them is significantly less than inducing 8 days. [Conclusion]Induction method can produce a large number of OLC which is superior to mechanical separation method, but its bone resorption function is weaker in the earlier stage, because bone resorption function is associated with the number of nucleus. The late stage of OCL is extremely similar to OC of mechanical separation method. And it can be used in any experiments.

Objective This study was to investigate the expression of human interferon ?(huIFN ?) gene in colon cancer cells. MethodsThe whole length cDNA of huIFN ? was subcloned into the expression vector pcDNA 3 after sequence measurement. Lipofectamine was used to transfer huIFN ? gene into three colon cancer cell lines(LOVO?SW620?HCT116BG) .The integration and expression of huIFN ? were identified by PCR and RT PCR respectively, the activity of IFN ? expressed in different cell lines was measured by ELISA. Results PCR and RT PCR showed successful integration and sustained expression of huIFN ? in 3 colon cancer cell lines. Conclusion The huIFN ? can be effectively transduced into human colon cancer cells, and sustained expression was obtained.

ObjectiveTo evaluate the effect of combined cytosine deaminase(CD) gene therapy and chemical reagents on colon cancer cell line.MethodsLovo cells transducted with CD gene in a recombinant adenovirus AdCMVCD vector were treated with 5 fluorocytosine(5 FC) and ciplatin(CDDP) or mitomycine C(MMC). Cytotoxic effect was assayed with MTT method. ResultsLow dose of CDDP or MMC reduces the IC 50 of 5 FC significantly on Lovo cells transducted with CD gene and enhances the bystander effect of AdCMVCD/5 FC system.ConclusionCombined therapy with CD/5 FC system and chemical reagents may have a potential application in the treatment of colon cancer.

Objective:To investigate the effects of IL-12p35 small interference RNA on immune functions of Dendritic cells in rats.Methods:DCs were generated by culturing bone marrow progenitor cells of rats with GM-CSF, IL-4 and LPS in vitro. The IL-12p35 siRNA was synthesized and transfected into DCs. The expressions of CD80 and MHCⅡwere examined by flow cytometry. Reverse transcriptase PCR analysis was used to detect IL-12p35 mRNA transcription and ELISA was used to detect IL-12 and IL-10 protein levels, respectively. The antigen presenting by DCs was evaluated by mixed lymphocyte responses.Results:IL-12p35 siRNA silenced DCs expressed lower IL-12, higher IL-10,and exhibited weak activity in stimulating the proliferation of allogenic T cells, but no changes on CD80 and MHCⅡexpressions.Conclusion:IL-12p35 siRNA interference exerts no effects on maturation of DC, but negative effect on immume function of DCs.

Objective To analyze and summarize the risk factors of failed internal fixation for intertrochanteric fracture.Methods From April 2008 to April 2011,267 patients with intertrochanteric fractures in 4 hospitals were treated with internal fixation.The relationship between the failure of internal failure and possible factors as age,gender,hypertension,diabetes,the abuse of alcohol and tobacco,use of glucocorticoid,the degree of osteoporosis and fractures type were studied.According to the surgical risk assessment table,the patients were divided into low-risk,mid-risk,and high-risk group.The rate of internal fixation failure was compared in the 3 groups.Results We found 42 cases which showed radiographic failures.The internal fixation failure directly related with advanced age,diabetes,severe osteoporosis,unstable type fracture,but not gender,hypertension,the abuse of alcohol and tobacco,use of glucocorticoid.Risk factors of internal fixation failure included diabetes,osteoporosis degree,and fracture stability.Failed intertrochanteric fracture fixation mainly occurred in the mid-risk and high-risk groups.Conclusion Severe osteoporosis,unstable fracture,diabetes are risk factors of failure of intertrochanteric fracture fixation.These factors will affect the quality of surgery.For the patient with intertrochanteric fractures in the low-risk groups,internal fixation should be the first choice for treatment.For the patients in the mid-risk and high-risk group,internal fixation should be applied cautiously.For the aged patients in high-risk groups,hip arthroplasty is a wise option.

Objective:To explore the effects of miR-27a on the phenotype and cytokine secretion in LPS-stimulated dendritic cells.Methods:Murine bone marrow-derived dendritic cells were transfected with miR-27a mimics and negative control mimics,and then stimulated by LPS for 24 hours.Dendritic cells exposed to LPS were collected for analysis of the DC immunophenotype by flow cy-tometry and supernatants were collected to determine cytokine lever.Moreover,the capability of stimulating allogeneic CD4 T+cell prolif -eration was measured by MLR ( mixed lymphocyte reaction) .Results: The levels of MHCⅡ, CD80, and CD86 were significantly increased in LPS-stimulated dendritic cells when compared with imDC ( P<0.001).Transfection with miR-27a mimics resulted in sig-nificantly lower expression levels in levels of MHCⅡ,CD80,and CD86 (P<0.001).For cytokine secretion,transfection with miR-27a mimics enhanced IL-10 production (P<0.01) and reduced the production of IL-12 (P<0.05).For MLR,transfection with miR-27a mimics suppressed allogeneic CD4+T cell proliferation.Conclusion: MiR-27a may play critical roles in regulating the maturation process and cytokine secretion in LPS-stimulated dendritic cells.

Objective To investigate the anti-tumor effect of interferon-?(IFN-?) gene therapy on colon cancer. Methods BALB/c mice were inoculated subcutaneously with CT26 (murine colon carcinoma cell line) cells to prepare an in vivo tumor model. Eight days after tumor inoculation,the tumor-bearing mice were divided into 3 groups and injected intra-tumorally with one of the following preparations: pcDNA3-IFN-?/Lipofectamine,pcDNA3 /Lipofectamine,and PBS respectively. The expression of IFN-?,the immunity function of mice,the histological changes of the tumor,the tumor volume in tumor-bearing mice were tested after gene therapy. ResultsThe level of IFN-? in the serum and the CTL activity increased significantly( P

Objective:To construct a mutant D314A of Escherichia coli cytosine deaminase (EC-CD, substitution of an alanine (A) for the aspartic acid (D) at position 314 of cytosine deaminase) and investigate its antitumor effect. Methods: Eukaryotic expression plasmid containing EC-CD gene (pcDNA3.1-CD~(wt)) was constructed, and the mutant pcDNA3.1-CD~(D314A) plasmid, with aspartic acid (D) at position 314 of EC-CD gene substituted by alanine (A) (EC-CD~(D314A)), was established by site-directed mutation. EC-CD~(wt) and EC-CD~(D314A) were transfected into human colon cancer cell line LoVo via Lipofectamine~(tm) 2000, and positive LoVo-CD~(wt) and LoVo-CD~(D314A) cells stably expressing corresponding genes were selected by G418. The cytotoxicity and bystander effects of EC-CD and EC-CD~(D314A) genes on LoVo cells were e-valuated by MTT assay. Results: The mutant D314A was confirmed by sequence analysis. EC-CD and EC-CD~(D314A) mRNA were expressed after transfected into LoVo cells. The IC_(50) of Lovo-CD~(D314A) cells was (85.13±0.60) mmol/L, which was significantly lower than that of LoVo-CD~(wt) cells ([689.76±0.45] μmol/L, P=0.000). Bystander effect assay showed that, when at the ratio of 30%, the survival rates of LoVo-CD~(wt) cells and Lovo-CD~(D314A) cells were (48.5±0.49)% and (17.3±0.40) % (P = 0.000), respectively. Conclusion: Mutatant EC-CD gene (EC-CD~(D314A)) has a significantly in-creased antitumor effect on LoVo cells compared with wild type EG-CD gene, and it may become a new candidate gene for tumor gene therapy.

[ ABSTRACT] AIM:To explore the mechanisms of fluctuant high blood glucose-induced apoptosis of hepatocytes. METHODS:SD rats were randomly divided into normal control group ( N) , stable high blood glucose group ( S) , fluctu-ant high blood glucose group ( F) and insulin group ( I) .Diabetic rats were induced by intraperitoneal injection of strepto-zotocin (65 mg/kg) , and the fluctuant high blood glucose animal model was induced by intraperitoneal injection of ordinary insulin and glucose at different time points every day.The blood glucose fluctuation patterns of the animals in F group with-in 12 weeks were similar every day and no significant difference of the HbA1c concentration was observed compared with S group, indicating that the fluctuant hyperglycemia was successfully established in F group.The activity of superoxide dis-mutase ( SOD) and glutathione peroxidase ( GSH-Px) , and the content of malondialdehyde ( MDA) and nitric oxide ( NO) in the homogenate of the liver tissues were detected by colorimetry.The mRNA and protein levels of JNK, p-JNK, Bax and Bcl-2 were examined by RT-PCR and Western blot.RESULTS:After 12 weeks, the increases in the intakes of food and water, the urine output, and the abnormal liver function were observed in S group, I group and F group.Compared with N group, the MDA level was increased, the content of NO and the activity of SOD and GSH-Px were decreased, and up-regu-lation of JNK mRNA and p-JNK and Bax proteins, and down-regulation of Bcl-2 were also found in S group, I group and F group.The above effects were more obviously showed in F group.CONCLUSION:Oxidative stress activates JNK-MAPK signaling pathway, which is involved in fluctuant high glucose-induced apoptosis of hepatocytes.