Objective To construct recobinant pmRNA IRES-hKDR and lay a primary foundation for further study of gene therapy for tumors.Methods pmRNA IRES-hKDR was obtained from pDC520 hkDR and pmRNA IRES-Luc which was reserved by our laboratory through molecular biology technology and was transfected into Hepall-6 via lipsome after identification of PCR,restriction enzyme digesting and DNA ELISA and Western blot.Results The recombiant plasmid had pmRNA IRES-hKD gene which was expressed in Hepall-6//G418 in vitro and the expressed protein was secreted out of the cells could response with specific hKDR antibody,which was identified by Western blot.Conclusion An eukaryotic expression recombiant plasmid pmRNA IRES-hKDR can be constructed successfully which provides the possibility of further researches on its anti-tumor effect.

Objective To investigate the immune protection of dendritic cells(DCs) harboring kinase domain-containing receptor(KDR) fusion gene on mice carrying B16 melanoma.Methods The bone marrow precursor-derived dendritic cells(BMDCs) were induced from bone marrow progenitors of mice by GM-CSF.The KDR fusion gene mRNA was transfected into DCs in vitro.Mice were immunized with the same amount of DCs at 7-day interval and then each mouse was injected with 5?10~5 B16 cells.The mice without tumors were injected with B16 cells again 20 days later.Mice were randomly divided into 4 groups: group A(n=7),group B(n=8),group C(n=8) and group D(n=7).The mice were immunized with DCs one time in group A,2 times in group B,3 times in group C and as controls in group D.Results After one week,all mice in group D had tumors with average survival 15 days.All mice in group A,B and C had no tumors after 20 days later.After the second injection of B16 cells,2 mice in group A and 2 mice in group B had tumors.The mice in group C had no tumor.The average survival periods were calculated from first injection of B16 cells to the study end.The average survival period of group A was 50 days and that of group B was 72 days.Conclusions The dendritic cells vaccine harboring KDR fusion gene has immune protection against melanoma in mice.

Objective To study the inhibitory effect of fluorouracil combined with DDP on the growth of human osteosarcoma cell line MG-63,and to explore its influence on the expressions of transient receptor potential vanilloid 5 (TRPV5 )and transient receptor potential vanilloid 6 (TRPV6 )proteins.Methods The MG-63 cells were cultured by the density of 5 × 104 mL-1 .Fluorouracil group,DDP group,fluorouracil+ DDP group and control group containing 10% FBS were set up.The inhibitory rates of growth of MG-63 cells at different time were detected by CCK-8 assay.The apoptosis of MG-63 cells after treated with different drugs was determined by Hoechst staining Kit.The immunocytochemical staining was used to treatent to detect the expressions of TRPV5 and TRPV6 before and after treatment.Results Fluorouracil and DDP both inhibited the growth of MG-63 cells in a time-and dose- dependent manner.There were a lot of black particles in the MG-63 cells and the cells were smaller,aging or death when they were exposed to fluorouracil or DDP.Compared with 24 h group,the inhibitory rates of proliferation of MG-63 cells after treated with the sigle drug of fluorouracil or DDP for 48 and 72 h were increased significantly (P <0.05).Compared with control group,the apoptotic rates of MG-63 cells in fluorouracil group and DDP group 24,48,and 72 h after treatment were increased (P < 0.01)in a time-dependent manner. The expression levels of TRPV5 and TRPV6 in MG-63 cells 72 h after treatment of fluorouracil and DDP were decreased significantly compared with before treatment (P < 0.05 ). Conclusion Fluorouracil, DDP and fluorouracil combined with DDP could significantly inhibit the proliferation of MG-63 cells,induce the apoptosis, and decrease the expression levels of TRPV5 and TRPV6.

Objective:To prepare and characterize specific and discrepant mouse hybridoma antibodies on membrane of HL60 and HL60/ADR cell lines.Methods:BALB/c mice were immunized by subtractive immunization induced Cp(Cyclophosphamide).McAbs were prepared by hybridoma technique,screened and detected by FACS and LSCM.Results:51 candidates and discrepant antibodies were found,and one of them (5F6) was purified and identified.Conclusion:Combination of SI with discrepant screening method should facilitate the preparing and identifying discrepant McAbs for identifying antibodies that can distinguish the differences in proteins expressed in HL60 and HL60/ADR,which is a significative and potential method in the research and target therapy associated drug-resistance.

Aim To determine whether membrane cytokeratin 8(CK8 )and BCRP expression cooperatively contributed to multidrug resistance(MDR)in MCF-7/MX cells.Methods MCF-7/MX cells were transfected with specific anti CK8-siRNAs and anti BCRP-siRNAs via LipofectAMINE2000.The expression of CK8 and BCRP was determined using Western blot,and membrane staining was observed by laser confocal microscopy.Sensitivity to chemical drugs was examined by Sulforhodamine B method.Results The expression levels of cell surface CK8 and BCRP were obviously reduced by siRNAs,and inhibition of CK8 and BCRP expression could effectively restore the sensitivity to drugs and reverse MDR phenotype of MCF-7/MX cells.Conclusions CK8 together with BCRP may play significant roles in conferring the multifactorial MDR phenotype of MCF-7/MX cells,but may act independently via potentially different mechanisms.Combinational approaches that target multiple drug-resistance-related molecules/pathways in cancer cells may represent more efficacious strategies to overcome MDR.

Objective To investigate the clinical value of pulsed pulmonary function in the diagnosis of chronic obstructive pulmonary disease (COPD).Methods 32 patients with chronic obstructive pulmonary disease admitted to Haiyang People's Hospital from March 2016 to March 2017 were selected as observation group .Another 32 cases without COPD were selected as control group .All the patients received pulsed pulmonary function test .The relevant indicators of oscillation technology and pulmonary function test indicators were compared between the two groups.Results The resonance frequency , total respiratory impedance , central airway resistance and peripheral airway resistance in the observation group were (16.9 ±1.5)kPa · L-1· s-1,(0.46 ±0.03)kPa· L-1· s-1, (0.56 ±0.05)kPa · L-1· s-1 and (0.15 ±0.03)kPa · L-1· s-1,which were significantly higher than those in the control group [(9.5 ±0.3)kPa· L-1· s-1,(0.26 ±0.01)kPa · L-1· s-1,(0.24 ±0.01)kPa· L-1· s-1, (0.02 ±0.01)kPao· L-1· s-1] (t=30.595,40.000,39.691 and 26.000,all P<0.05).The FEV1％ in the observation group was (41.5 ±1.9)％,which was lower than (81.1 ±2.0)％ in the control group(t=47.229,P<0.05).The FEV1/FVC level in the observation group was (49.5 ±3.3),which was lower than (81.9 ±2.6) in the control group(t=21.678,P<0.05).Conclusion The results of pulsed pulmonary function are synchronous and consistent with routine pulmonary function test ,especially for elderly ,pediatric and critically ill patients .

To investigate the relationship between proliferative capacity of salivary gland cells in contiguous acini of parotid tumors and recurrent neoplasma, DNA contents of 30 fresh specimens of parotid were studied by using cytometry in tumors, normal and shallow or deep lobe acini of the masses. The results showed that the DI was 1.369, S % 16.95, PI 26.18 in malignant tumors;DI was 1.171, S % 12.41, PI 15.54 in recurrent pleomorphic adenoma; DI was 1.141, S % 12.74, PI 13.07 in pleomorphic adenoma, DI was 0.999, S % 5.10, PI 8.00 in normal acini. Analysis of variance showed there was a significant difference (P＜0.01). The average DNA contents of shallow on deep lobe of contiguous tumors was 1.08 in DI, 10. 65 in S %, 13.49 in PI in malignant tumor, 1.06 in DI, 8.96 in S % and 9.85 in PI in pleomorphic adenoma, which were all higher than in normal acini (P＞0.05). It was concluded that the levels of DI and S % of parotid tumor and its contiguous acini are related to degree of malignancy or recurrent condition of the tumors, suggesting contiguous acini of parotid tumors had the strong capacity of proliferation,which might play an important role in recurrent or malignant change of the parotid tumors.

To investigate the relationship between proliferative capacity of salivary gland cells in contiguous acini of parotid tumors and recurrent neoplasma, DNA contents of 30 fresh specimens of parotid were studied by using cytometry in tumors, normal and shallow or deep lobe acini of the masses. The results showed that the DI was 1.369, S % 16.95, PI 26.18 in malignant tumors;DI was 1.171, S % 12.41, PI 15.54 in recurrent pleomorphic adenoma; DI was 1.141, S % 12.74, PI 13.07 in pleomorphic adenoma, DI was 0.999, S % 5.10, PI 8.00 in normal acini. Analysis of variance showed there was a significant difference (P＜0.01). The average DNA contents of shallow on deep lobe of contiguous tumors was 1.08 in DI, 10. 65 in S %, 13.49 in PI in malignant tumor, 1.06 in DI, 8.96 in S % and 9.85 in PI in pleomorphic adenoma, which were all higher than in normal acini (P＞0.05). It was concluded that the levels of DI and S % of parotid tumor and its contiguous acini are related to degree of malignancy or recurrent condition of the tumors, suggesting contiguous acini of parotid tumors had the strong capacity of proliferation,which might play an important role in recurrent or malignant change of the parotid tumors.

In order to characterize the biological activity of fox (Vulpes vulpes) interferon gamma(VuIFN-gamma), We have isolated the cDNA encoding arctic fox (Alopex lagopus) VuIFN-gamma. This cDNA encodes a 23 amino acid signal peptide and a 144 amino acid mature protein, which shares 99.8% or 99.4% for nucleotide identity with silver fox and canine, respectively, and 100% for amino acid identity. Expression of recombinant mature arctic fox interferon gamma (mVuIFN-gamma) in bacterial system was confirmed by SDS-PAGE and Western blotting analysis. Recombinant VuIFN-gamma showed higher antiviral activity against vesicular stomatitis virus in cultured Vero and MDCK by inhibiting virus induced cytopathic effect, In view of the immunomodulatory and antiviral activities of VuIFN-gamma, it may provide a basis for further research on antiviral therapy of recombinant VuIFN-gamma in economic animal practice.
Animals ; Antiviral Agents ; pharmacology ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Foxes ; genetics ; Interferon-gamma ; genetics ; pharmacology ; Molecular Sequence Data ; Recombinant Fusion Proteins ; genetics ; pharmacology

Objective Based on the Kabat-Zinn's mindfulness decompression therapy and Teasdale's mindfulness cognitive therapy,and using the solution-focused approach as the frame structure,we establish comprehensive,standard mindfulness interventions scheme of domiciliary patients with chronic obstructive pulmonary disease,which can provide the basis for the development of the community and residential care.Methods Twenty-six experts were selected as the target of the study and two rounds of correspondence were used to establish the final mindfulness intervention plan of the domiciliary patients with chronic obstructive pulmonary disease.Meanwhile,the reliability and representativeness of the consultation are tested by using the coordination degree,positive coefficient and authoritative coefficient of expert opinions.Results The positive coefficient of the two round correspondence experts were 88.46%(23/26)and 100.00%(23/23),and the expert authority coefficient was 0.90 and 0.91.The variation coefficient of the indicators after two rounds of expert consultation was 0.04-0.14,the coordination coefficient of experts was 0.32(P＜0.01)and 0.59(P＜0.01)respectively.The final construct of domiciliary patients of chronic obstructive pulmonary disease with the intervention programme consists of 5 steps,25 entries,which was suitable for the positive reading intervention scheme.Conclusions The results show that experts enquiry for representative and high credibility,and the mindfulness intervention plan of domiciliary patients with chronic obstructive pulmonary disease is scientific and practical.