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 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 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 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 evoke cerebral potentials by stimulating nociceptive fibers with contact heat evoked potentials stimulator (CHEPS)and estimate the nerve conduction velocities of peripheral nerve fibers mediating these responses.Methods Subjects were set in supine position.A heat-foil technology with a rapid rising speed at 70 ℃/s was used to elicit pain and contact heat evoked potentials(CHEP).Contact heat was delivered via one circular thermode (diameter 27 mm,area 573 mm~2).Thermal stimuli were sent at two intensity levels (49.5 ℃ and 54.5 ℃) to three body sites:thenar eminence,the dorsum of hand and proximal volar forarm.Contact heat evoked potentials were recorded from Cz and Pz.A systemic effect between stimulus intensities and pain rating were observed,the main components of this evoked potential were observed.Nerve conduction velocity was calculated from latency difference of CHEP and center to center distance of distal and proximal stimulus arrays.Results The pain intensity rating was 3.2?0.3 and 4.4?0.5 when thenar eminence was stimulated at the temperature of 49.5 ℃ and 54.5 ℃ respectively;the rating was 6.3?0.8 and 7.2?0.5 when the dorsum of hand and proximal volar forarm were stimulated at the temperature of 54.5 ℃ respectively.Three components,Cz/N550,Cz/P750 and Pz/P1000,were found in the evoked potentials.Nerve conduction velocities of the fibers were (12.9?7.5) and (1.7?0.4) m/s respectively,which were corresponding to those of A8 fiber and C fiber.Conclusions CHEPs can be elicited reliably and stably.Velocities of peripheral nerve fibers demonstrate that A8 fiber and C fiber mediate the response.

Objective To establish the method of contact heat evoked potential(CHEP)and to explore the value of this evoked potential in pain testing of patients with cerebral infarction.Methods A total of 100 healthy volunteers and 30 patients were examined.The healthy volunteers were divided into 3 groups according to the length of their arms:(Group A:56.0～65.0 cm ;Group B :65.5～74.0 cm ;Group C :74.5～83.0 cm).A recently de- veloped heat-foil technique with a rapid temperature rising rate at 70℃/s was used to elicit pain and contact heat e- voked potentials.Contact heat was delivered via one circular thermode(diameter 27 mm,area 573 mm~2)and set at two intensity levels(49.5℃and 54.5℃)to three body sites:the thenar eminence,the dorsum of hand and proximal volar forearm.The subjects were asked to rate the pain with numerical rating scale after each stimulus and CHEP was recorded from Cz and Pz.The association between stimulus intensities and pain rating was explored,the main compo- nents of the evuked potential were watched.CHEP,sensory conduction velocity(SCV)and somatosensory evoked potentials(SEP)were performed in patients with hemi-anesthesia caused by cerebral infarction.Results The pain intensity ratings were 3.2?0.3 and 4.4?0.5 at thenar eminence,5.0?0.7 and 6.3?0.8 at the dorsum of hand and 5.3?0.6 and 7.2?0.5 at the proximal volar forearm when the temperature of 49.5℃and 54.5℃was applied, respectively;Three components,Cz/N550,Cz/P750 and Pz/P1000,were identified in the evoked potentials.Cz/ N550 and Cz/P750 appeared when the dorsum of hand and proximal volar forearm were stimulated.In contrast,Pz/ P1000 could be identified when nociceptors of thenar eminence and proximal volar fbrearm were excited.In the pa- tients with cerebral infarction,CHEP disappeared or became abnormal on one side,while SCV and SEP were normal on that side.Conclusion It was suggested that CHEP could be elicited reliably in the controls.CHEP is helpful in the assessment of analgesia in patients with cerebral infarction.

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.

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.

Recurrent laryngeal nerve injury is one of the serious complications after thyroidectomy.Unilateral injury causes hoarseness,while bilateral injury causes difficulty in breathing or even life-threatening glottis obstruction.Analyzing the root cause of the thyroid injury,firstly it is the anatomical factors of recurrent laryngeal nerve,namely the close and complex relationship between the recurrent laryngeal nerve and the inferior thyroid artery,the existence of branches of recurrent laryngeal nerve and its variation,and the presence of non recurrent laryngeal nerve and Zuckerkandl nodules.Those are all made the recurrent laryngeal nerve easy to be damaged.Secondly it is because of the vulnerability of the recurrent laryngeal nerve itself.Last improper using of energy operation instrument will cause heat injury on nerves.Below counter measures can be implemented to prevent recurrent laryngeal nerve injury.Dissect to show recurrent laryngeal nerve or make it ‘ visualization’ during thyroidectomy.Elaborately anatomize recurrent laryngeal nerve to appropriate degree.Be familiar with the property of energy operation instrument and thus safely use them to reduce the heat injury of recurrent laryngeal nerve.Reasonably use the intraoperative nerve monitoring in the surgery,which assist to reduce the risk of injury of recurrent laryngeal nerve.