Objective To investigate the role of Catbindin-D28k in the kidney on calcium metabolism.Methods VDR/CaBP-D28k double knockout(KO)mice was made.Body weight,diet intake and serum,urinary parameters and length,density of the long bones,histological staining of the tibia of WT,CaBP-D28(-/-),VDR(-/-)and VDR(-/-)/CaBP-D28k(-/-)mice were determined on regular and high Ca-Lac diet.Results On a regular diet,the double KO mice were growth-retarded more and smaller than VDR KO mice.Compared with VDR KO mice,the double KO mice had higher urinary calcium excretion and rachitic skeletal phenotype,which were manifested with higher serum parathyroid hormone levels,lower bone mineral density,and more distorted growth plate with mole osteoid formation in the trabecular region.On high calcium and high lactose diet,blood-ionized calcium levels were normal in both VDR KO and the double KO mice.However,in contrast to VDR KO mice,the skeletal abnormalities were not completely corrected in the double KO mice.Conclusion These results directly demonstrate that CaBP-D28k plays a critical role in maintaining calcium homeostasis and skeletal mineralization and suggest that its caleemic role can be mostly compensated by CaBP-D9k.

Objective To construct the prokaryotic expression vector bearing fusion gene NT4-ADNF-9 and lay foundation for further study on genetic therapy of neuraseusory deafness. Methods By means of asymmetrical prince/ template, double stranded eDNA of activity dependent neurotrophic factor-9 (ADNF-9) was obtained, which included restriction enzymes sites on the two extremities. ADNF-9 eDNA was ligated to the signal and leader peptides of nenrotrophin 4 (NT4), and the fusion gene was named NT4-ADNF-9. Then it was suheluned into prokaryotic expression vector pBV220, and called pBV220/ NT4-ADNF-9. Results Evidences of DNA sequence analysis and restrtction enzymes digestion showed that we recombined ADNF-9 eDNA to the 3'terminal of the signal and leader peptides of NT4, and the fusion gene was subcluned into pBV220 successfully. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of dorsal root ganglia (DRG) of embryonic day-8 cbicken neurons as compared to the control. Conclusion Prokaryotic expression vector pBV220/NT4-ADNF-9 can be constructed successfully and the bioactivtty is satisfactory.

Objective To express and purify Japanese encephalitis virus ( JEV) EDⅢprotein and evaluate the possibility of using it as a candidate antigen in JEV diagnostic kit .Methods PCR primers spe-cific for the gene encoding JEV EDⅢprotein were designed and used to amplify the gene fragment by RT-PCR.The cloned gene fragment was then inserted into pET-30a (+) to construct the recombinant expression plasmid.The transformed E.coli BL21 carrying expression plasmid were induced by IPTG to express JEV EDⅢ protein.The expressed JEV EDⅢprotein and a control antigen of tick-borne encephalitis virus protein were deposited in small spots to set up ELISA microarray .The serum samples from patients with Japanese encephalitis and healthy people were detected by Array-ELISA.The results obtained by Array-ELISA were compared with those by using indirect immunofluorescence assay .Results The gene fragment encoding JEV EDⅢprotein was successfully cloned and expressed in E.coli BL21.The recombinant protein could be used in Array-ELISA assay for the detection of serum samples from patients with Japanese encephalitis and healthy subjects .The results were consistent with those by using indirect immunofluorescence assay.Conclusion The recombinant JEV EDⅢprotein can be used as a candidate antigen for the diagnosis of JEV infection .

Objective To express and purify the recombinant nucleoprotein fragments of hemor-rhagic fever with renal syndrome ( HFRS) virus and to evaluate their diagnostic efficacy by using array-ELISA technology .Methods The target genes encoding nucleoprotein fragments of HFRS virus were amplified by PCR, and then inserted into prokaryotic expression vectors to construct the recombinant plasmids of pET -32a (+)/Pn and pET-32a(+)/Pc.The plasmids were transformed into E.coli BL21 ( DE3) to induce the ex-pression of nucleoprotein fragments by IPTG and the expressed products were purified by affinity chromatog -raphy using Ni-NTA agarose.The specificity and sensitivity of the recombinant antigens were evaluated by the assay of array-ELISA using commercial colloidal gold assay kit as a comparison .Results The recombi-nant nucleoprotein fragments of HFRS virus were correctly expressed in E.coli and highly purified by affinity chromatography .Array-ELISA showed that 13 of 16 suspected serum samples were positive by using the His-Pn protein as diagnostic antigen , consistency with the commercial colloidal gold assay kit reaching 94%. Conclusion The recombinant His-Pn protein expressed in E.coli cells could be used for specific serodiag-nosis of HFRS virus as its high antigenicity and sensitivity .The array-ELISA is an effective assay for the de-tection of virus at protein level .

Objective To explore the relationship between gastric juice pH and hospital-acquired pneumonia ( HAP) , the gastric bacterial colonization and etiology of HAP in neurologic intensive care unit patients by monitoring gastric juice pH value.Methods From October 2014 to May 2015, consecutive seventy-two tube feeding patients admitted in the Department of Neurology Intensive Care Unit in Xijing Hospital were enrolled in this research.The type and concentration of pathogens from gastric contents were collected, while samples from upper respiratory tract and pharynx were detected dynamically at the same time.Results (1)The group with new onset HAP was higher in gastric juice pH (6.4(5.4,6.4) vs 5.4 (2.5, 6.4), Z=-2.37, P=0.01); (2) The isolation rate of colonized bacteria in gastric cavity was associated with the pH of gastric juice , achieving 60.8% ( 42/69 ) in HAP group; ( 3 ) When the gastric juice pH was >4, the isolation rate of Gram-negative bacilli in gastric cavity obviously increased (63.6%(28/44) vs 35.7%(10/28),χ2 =5.323, P=0.021); (4)The same pathogens were found in stomach-pharynx-upper respiratory tract in 7 cases ( 17.5%) of the total 40 HAP patients.Conclusion Increased gastric juice pH was associated with gastric colonization , especially Gram-negative bacilli , and may lead to a higher incidence of new onset HAP in patients on enteral feeding.

Objective To explore the effect of vitamin D receptor (VDR) in intestinal tumor development and the relationship between VDR and β-catenin signaling pathway. Methods The interaction of vitamin D receptor and β-catenin were detected by co-immunoprecipitation assay after human colonic carcinoma cells SW480 were treated with vitamin D in vitro for 4 hours. The expression of E-cadherin protein was detected by Western blot after treated for 24 hours. To compare APCmin/+VDR-/- and APCmin/+ mice in vivo, the expression of VDR,β-catenin and BrdU proteins in intestinal tumor were determined by immunohistochemistry. The expression of β-catenin protein in tumor and adjacency intestinal was further determined by Western blot. Results After SW480 cells were treated with vitamin D, vitamin D receptor and β-catenin protein showed binding, the expression of E-cadherin protein further increased (Gray value the control group 145.57±4.21,Gray value of the experimental group 109.35±3.56,t＝32.63,P＜0.05). Immunostaining and Western blot detection（Gray value 166.47±2.36） showed a marked increase of β-catenin level(Gray value 140.51±2.57) in APCmin/+VDR-/- tumor compared to APCmin/+ tumor(145.41±3.62,182.35±3.24,t＝2.65,4.36,P＜0.05). Conclusions The role of vitamin D suppressing intestinal tumor may be achieved through VDR affectingβ-catenin signaling pathway.

Objective To develop perfluorocarbon lipid particles and investigate their basic properties,and target them to human hepatocellular carcinoma cells in vitro by hepatoma monocolonal antibody HAb18 with avidin-biotin interaction.Methods Rotary evaporation and high pressure homogen were used to prepare perfluorocarbon lipid particles, and the appearance and distribution of them were investigated by microscope and electron microscope, the concentration and the size and electric potential were detected.The biotinylated monoclonal antibody HAbl8 was prepared, then the biotinylated degree of the antibody was determined.The biotinylated perfluoroearbon lipid particles labelled with NBD were prepared and targeted to human hepatocellular carcinoma cells in vitro with avidin-biotin interaction.Results These perfluorocarbon lipid nanoparticles were uniform and stable,and the mean diameter of them was 171.9 nm.Hepatocellular carcinoma cells were surrounded by the biotinylated particles labelled with NBD.Conclusions A steady perfluoroearbon lipid particles were prepared and the biotinylated particles can be targeted to hepatocellular carcinoma cells with avidin-biotin interaction.

BACKGROUND: How to effectively and rapidly induce the osteogenic differentiation of human umbilical cord mesenchymal stem cells is the focus of the current stem cell research. Increasing evidence has demonstrated some growth factors, such as bone morphogenetic protein-2, have important effects on the transdifferentiation of umbilical cord mesenchymal stem cels into osteoblasts in vitro. However, widespread use of growth factors is limited because of high cost. Insulin is widely used in the cell culture and induction, but there is no report about the effect of insulin on the osteogenic differentiation of human umbilical cord mesenchymal stem cells. OBJECTIVE:To observe the effect of insulin on osteogenic differentiation of human umbilical cord mesenchymal stem cels and to explore the feasibility of human umbilical cord mesenchymal stem cell transplantation in the treatment of diabetic delayed fracture healing. METHODS:The passage 3 human umbilical cord mesenchymal stem cells were inoculated in two flasks, denoted as experimental group and control group. The insulin (10-7mmol/L) was added to the experimental group but not to the control group. The proliferative capacity of human umbilical cord mesenchymal stem cels was evaluated by cell count kit-8 and alkaline phosphatase activity. The osteogenic differentiation capacity of human umbilical cord mesenchymal stem cells was evaluated by measuring the protein and mRNA expressions of type I colagen as well as osteocalcin mRNA level. RESULTS AND CONCLUSION: After 1-2 weeks of induction, compared with the control group, insulin could significantly increase the number of human umbilical cord mesenchymal stem cells in the experimental group, the activity of alkaline phosphatase and expressions of type I collagen osteocalcin mRNA (P< 0.05). These data indicate that insulin can promote the proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells.

Objective To investigate the prevalence rate of heart valve calcification and its relativity with pathological changes and clinical pathogenic factors in elder patients at autopsy Methods Pathology data at autopsy of 1047 patients with age from 60 to 106 years in Beijing Hospital from November 1954 to March 2016 were collected.Cases of heart valve calcification verified at autopsy were retrospectively reviewed.The prevalence of heart valve calcification and its relativity with age,clinical disease and coronary atherosclerosis was investigated.Results Among 1047 autopsies,aortic valve calcification(AVC)was found in 15.2 ％ (n=159),mitral valve calcification(MVC)in 9.6 ％ (n=101),both AVC and MVC calcification in 5.4 ％ (n =57)and heart valve calcification in 19.4 ％ (n =203).The prevalence of heart valve calcification was 6.4％ (15/234)at age of 60-69,12.8％(37/289)at age of 70-79,22.5％(70/311)at age of 80-89 and 38.0％ (81/213) at age of 90-106 years,respectively(tendency x2 =82.523,P＜0.01).Calcification prevalence was significantly increased when complicated with coronary artery stenosis,hypertension,coronary artery disease (CAD),diabetes and chronic kidney disease (CKD).Multivariate regression analysis showed that age and CAD were independently risk factors for heart valve calcification(OR=1.066,95％ CI:1.048-1.086,P＜ 0.01;OR =2.238,95％ CI:1.396-3.589,P＜0.01,respectively),while hypertension,diabetes and CKD were not independent risk factors(OR =1.223,95％ CI:0.859-1.741,P＞ 0.05;OR =1.053,95％ CI:0.700-1.586,P ＞0.05;OR =0.924,95％ CI:0.610-1.399,P＞ 0.05,respectively).As compared with patients without heart valves calcification,patients with heart valve calcification had more increased risk for coronary atherosclerosis(OR =2.983,95a％CI:1.868-4.765,P＜0.01).Conclusions Prevalence of heart valve calcification is increased in elder patients with increasing age.Prevalence of heart valve calcification is higher in CAD patients than in non-CAD patients.And heart valve calcification is sigmficantly associated with coronary atherosclerosis.

Objective To investigate the correlation between apparent diffusion coefficient (ADC)of diffusion weighted imaging (DWI)and prognostic factors in rectal cancer.Methods 5 5 patients with rectal cancer were confirmed pathologically.Conventional pelvic MRI and DWI examination were performed,and the mean ADC values of tumor were measured preoperatively.The patients were divided into two groups with or without lymph node metastasis,and were also divided into four groups with negative,weakly positive,positive or strongly positive expression of EGFR in rectal cancer.The ADC values were calculated in each group,and the correlation of ADC values with the lymph node status and EGFR expression classification were analyzed.Results In 5 5 patients with colorectal cancer,there were 13 lesions with lymph node metastasis and the positive expression rate of EGFR was 67.2%.There were no significant difference in mean ADC value between the groups with and without lymph node metastasis (P=0.342).The number of lesions with negative,weakly positive,positive and strongly positive EGFR expression were 18,15,12 and 10.The difference in the mean ADC values among negative,weakly positive,positive and strongly positive expression groups of prognostic factor EGFR was not significantly different (P=0.412).There were also no correlations in the prognostic factors mentioned above (r=0.183 and -0.324,all P>0.05).Conclusion The ADC value can not be used to predict the prognosis and to provide more valu-able information for individualized therapy in patients with rectal carcinoma,which needs further studiy in the future.