Objective To investigate the effect of chronic ultra-low frequency electrical stimulation on mechanical characteristic of diaphragmatic muscle of emphysema rabbit.Methods The rabbit model of emphysema was made by inhaling papain of ultrasonic atomization once a week for 3 weeks in 24 adult Japanese rabbits,6 receiving 10 Hz chronic ultra-low frequency electrical stimulation,6 receiving 40 Hz,6 receiving 2.5 Hz and 40 Hz.Another 6 rabbits served as control.The twitch tension (Pt),titanic tension(Po),time to peak tension(TPT),half-relaxation time(1/2RT),fatigue index(FI) and fatigue recovery index(FRI) were measured in control group,emphysema group,emphysema+chronic ultra-low frequency electrical stimulation groups.Results As compared with the control rabbits, Pt and Po decreased,TPT and 1/2RT prolonged,FI and FRI increased in the only emphysema rabbits.Pt and Po were higher,TPR and 1/2Rt was shorter in the only emphysema rabbits than the rabbits in 40 Hz and(2.5+40) Hz groups(P0.05),more increase in FR and FRI(P

Objective To investigate the effect of chronic low-frequency electrical stimulation on the structural and metabolic changes of diaphragm of emphysema rabbits.Methods The rabbit model of emphysema was made by papain-inhaling of ultrasonic atomization once a week for 3 weeks in 30 adult Japanese rabbits,6 receiving 10 Hz chronic low frequency electrical stimulation,6 receiving 40 Hz,and 6 receiving(10+40)Hz.Other 6 emphysema rabbits and 6 normal rabbits served as control.An enzyme chemical method was used to observe the changes in the enzyme activities of sarcoplasmic reticulum Ca2+-ATPase,succinate dehydrogenase(SDH)and the lactate dehydrogenase(LDH)and changes in MHC in control group,emphysema group,and emphysema+chronic low frequency electrical stimulation group.Results Following chronic electrical stimulation using 40 Hz,10 Hz,and(10+40)Hz,the relative contents of MHC were increased in the diaphragmatic muscle of rabbits with emphysema.The percentage of MHC-Ⅰ was significantly increased in the 10 Hz group,while that of MHC-Ⅱa was significantly increased in the(10+40)Hz group.Following chronic electrical stimulation using 40 Hz and(10+40)Hz,diaphragmatic muscle SR Ca2+-ATPase activity was increased in rabbits with emphysema(P

ObjectiveTo observe the clinical effect of low molecular weight heparin calcium in the treatment of chronic obstructive pulmonary disease combined with pulmonary heart disease.MethodsA total of 64 patients with chronic obstructive pulmonary disease combined with pulmonary heart disease from December 2008 to December 2010 were enrolled in this study and divided into treatment group and control group by random digits table with 32 cases each.The control group was given routine treatment and the treatment group was given low molecular weight heparin calcium for 7 days at the basis of routine treatment.ResultsThe total effective rate in treatment group[ 90.6％ (29/32) ] was significantly higher than that in control group [ 68.8％ (22/32) ] (P ＜ 0.05 ).In treatment group,the levels of arterial carbon dioxide tension (PaCO2) and arterial oxygen tension (PaO2) after treatment [(54.64±9.63),(74.21 ± 11.76) mm Hg (1 mm Hg =0.133 kPa)] were significantly decreased compared with before treatment [(78.66 ± 11.22),(53.42 ± 8.84 ) mm Hg ] (P ＜ 0.01 ).In control group,the levels of PaCO2 and PaO2 after treatment [ (61.10 ±7.24),(65.07 ± 8.21 ) mm Hg] were significantly decreased compared with before treatment[ (79.52 ± 12.54),(51.35 ± 7.31 ) mm Hg ] (P ＜ 0.05 ).After treatment the levels of PaCO2 and PaO2 in treatment group were better than those in control group (P ＜ 0.05).ConclusionsThe low molecular weight heparin calcium in treatment of chronic obstructive pulmonary disease combined with pulmonary heart disease can effectively improve the clinical manifestation.It is worth the clinical promoted application.

Objective To clone and express recombinant outer membrane protein P6, determine its optimal expression conditions and to investigate the immunoprotective effects of the P6 protein on mice. Methods The P6 gene of nontypeable Haemophilus influertzae(NTHi) was amplified by PCR from the NTHi genome and cloned into expression vector pET-32a (+) to generate the pET-32a-P6 recombinants. They were confirmed by nuclease digestion and sequence analysis. The verified recombinant was transformed into E. coli BL21 (DE3). Its optimal expression conditions were determined such as engineering strains, the concentration of IPTG, inducing temperature, inducing time, different medium etc. The recombinant protein was purified by Q Sepharose~(TM) XL ion exchange and gel filtration chromatography. The protein was analyzed by SDS-PAGE, Western blot and sequencing. BALB/c mice were immunized with recombinant protein be-fore challenged by NTHi through intraperitoneal injection. Then the mortality rate of different group was com-pared. Results The recombinant P6 of NTHi was successfully constructed and expressed in E. coli at a rel-atively high level. The purity was up to 95% after purification. The relative molecular mass of the protein is 14 145. 848. The recombinant protein was confirmed to show specific reaction on the antiserum through Western blot. The animal experiments showed the mortality rates of immunization groups were significantly lower than that of the control group (P < 0.05). Conclusion The successful expression of the recombinant P6 will be very helpful for the further study on development of vaccine, its purified, immunological activity and antibody preparation.

OBJECTIVE:To investigate the dosage and consumption sum of opioid analgesic drug in Qingdao district. METH-ODS:The consumption data of drugs in 29 public hospitals at secondary or above level in Qingdao district were analyzed statistical-ly by ABC analytic methods and defined daily dose methods. RESULTS:In ABC analysis,5 kinds of class A drugs accounted for 20% of the total number of species,and the percentage of consumption sum was 77.38%;4 kinds of class B drugs accounted for 16.00% of all species numbers,and the percentage of consumption sum was 11.98%;other 16 kinds of drugs accounted for 64.00% of the total number of species,and the percentage of consumption sum was 10.64%. Oxycodone sustained-release tablets and Morphine sustained-release tablets with the highest DDDs consumed more health care costs,serial number ratio was 1.00,syn-chronization was good and conform to the actual needs of clinical work. CONCLUSIONS:The composition ratio of opioid analge-sic drug costs is consistent with the theoretical value,significant discrepancy between cost and DDDs does not appear.

ObjectiveTo detect the difference of cytokines and antibodies productions by immunologic system from mice and rabbits vaccinated with the M.RCAg-1 chimeric protein,expressed in E.coli,formulation with different adjuvants,including Freund's adjuvant and three clinically acceptable adjuvants,namely,Al(OH)3,Montanide ISA720 and Montanide ISA51.MethodsSix weeks female BALB/c mice were vaccinated with recombinant protein formulated with different adjuvants through intranasal.Serum were collected to detect specific antibodies of M.RCAg-1 and individual Epitope by ELISA ; natural parasite antigen was recognized by indirect immunofluorescence assay; mouse specific T lymphocyte activation was detected by enzyme-linked immunosorbent spot test (ELISPOT) ; Affinity assay between protein and immune IgG of rabbits with the biosensor,and the growth of Plasmodiumfalciparum in vitro to evaluate by growth inhibition assay(GIA).ResultsDifferent formulation can induce different levels of antibody titers,the effection of ISA51 adjuvant was most closely with Freund's adjuvant,and can induce a higher specific antibody of 11 epitopes within proteins,can effectively stimulate cellular immune response based on the IFN-γ,to avidity Montanide ISA51 adjuvant immune antibodies and M.RCAg-1 protein affinity than the other two adjuvants;and Montanide ISA720 adjuvants and Al (OH)3 adjuvant group in mice can't induce a significant IFN-γresponse(P＞0.05).On avidity assay,the Montanide ISA51 formulation group was better than the other two adjuvants; and Montanide ISA720 and Al (OH)3 adjuvant formulation group can't induce a significant IFN-γresponse in mice(P＞0.05) ; the inhibition rates were 60% and 100% in 3D7 and Dd2 Plasmodium falciparum at a concentration of 2 mg/ml IgG by Montanide ISA51 formulated protein,and IgG of Al( OH)3 formulation could not effectively inhibit the in vitro growth of Plasmodium falciparum( 10% ),while IgG of Montanide ISA720 formulation could not inhibit growth of parasite in vitro.ConclusionBy comparing three clinically acceptable adjuvants and Freund's adjuvant in BALB/c mice and New Zealand rabbit,Montanide ISA51 adjuvants can be acceptable formulated M.RCAg-1 protein induced humoral and cellular immune responses,can be used as one of the candidate adjuvants.

ObjectiveTo explore the effects of vector fusion peptides on the conformation and immune reactivity of recombinant polyepitope antigens,M.RCAg-1.MethodsWe subcloned polyepitope artificial antigen gene,M.RCAg-1,into prokaryotic expression vectors,pDS-ex,that contain different fusion tags at the N-terminus or no any tag by different restriction enzyme cutting site.Three recombinant proteins expressed by these vectors,named P312-1,P312-2,and P312-3,were purified and purity is greater than 95％.Then BALB/c mice were vaccinated with the three proteins formulated with Freund's adjuvant through intranasal.Serum were collected to detect specific antibodies of M.RCAg-1 and individual epitope by ELISA ; natural parasite antigen was recognized by indirect immunofluorescence assay; mouse specific T lymphocyte activation was detected by enzyme-linked immunosorbent spot test (ELISPOT) ; and the growth of Plasmodium falciparum in vitro to evaluate by growth inhibition assay(GIA),and analyze secondary and tertiary structures of recombinant proteins from different expression vectors by bioinformatics and circular dichroism technique.ResultsThe P312-1,P312-2 have almost the same amino acid sequence,and the three proteins have the same immunogenicity in animal models(P＞0.05),however,the different proteins elicited various T-cell responses,the rabbit antibody induced by these proteins showed diverse efficacy in malaria parasite growth inhibition assaysin vitro ( respectively,93.9％,14.7％,54.3％ ).The significant differences of secondary and tertiary structures were shown in recombinant proteins from different expression vectors,analyzed by bioinformatics and circular dichroism technique,which demonstrated the change of protein molecule spaces conformation,and the obviously change of some epitope locations.ConclusionThese results suggest that the expressed polyepitope artificial antigens originating from the different vector fusion peptides indeed affect the protein folding and,subsequently,the epitope exposure.Thus,these proteins are able to induce both distinct humoral and cellular immune responses in animal models,and they affect the efficacy of immune inhibition against the parasite,the enhancement or suppresses.

Objective To construct a stable infectious clone of S191 virus strain used for the pro-duction of live attenuated measles vaccine. Methods Full length cDNA of S191 strain and gene fragments encoding nucleocapsid(N),phosphoprotein(P)and RNA polymerase(L)were synthesis and respectively cloned into the vector pVAX1. The 293T cells were respectively transfected with the recombinant expression plasmids and co-cultured with Vero cells. The supernatants of cell culture were collected for identifying res-cued viruses. The indirect immunofluorescence assay was performed for virus identification. The rescued viruses at different passages in Vero cells and the sequences of the rescued viruses were analyzed. Results Restriction enzyme digestion and sequence analysis showed that the recombinant expression plasmids contai-ning the full length cDNA with an artificially engineered mutation at nucleotide 2101(C-A)and gene frag-ments encoding N,P and L proteins of S191 strain were constructed successfully. The N and P proteins were detected in Vero cells with immunofluorescence assay. A cytopathogenic effect on Vero cells was induced by rescued viruses. Conclusion The stable infectious clones of S191 virus used for the production of live at-tenuated measles vaccine were rescued successfully. An approach by using reverse genetics technique for S191 strain study was established which could be used for the development of new chimeric vaccines against measles virus.

Objective To investigate the clinical, biochemical and genetic features of a Chinese boy with holocarboxylase synthetase deficiency (HCSD). Methods The clinical and genetic data of a rare case of HCSD were retrospectively analyzed. Results After birth, the boy showed development delay. At 3 months old, the boy was started with rehabilitation. Tandem mass spectrum and gas chromatography analysis was carried in the 5th month after birth because of the recurrent upper respiratory tract infection and elevated level of C5-OH in the blood and decreased level of C0,and elevated level of 3-OH-propionic, pyruvic acid, methylcrotonylglycine in the urine were in accordance with the HCSD. Genetic analysis found compound heterozygous mutations of c.1648G>A and c.1544G>A in gene, of which the latter one is novel. After the treatment of biotin (20 mg/d) and L-Carnitine, the condition of this boy was gradually improved. Conclutions HCSD is characterized with slow onset and inconspicuous manifestations. The confirmed diagnosis can be built with MS/MS, GC/MS analysis and gene mutation analysis. The effect of early biotin treatment is satisfactory. In this study,we carried out clinical and genetic diagnosis,which lays a solid foundation for prenatal diagnosis and early treatment.

Objective To explore the clinical features, diagnosis, and treatment of congenital disorder of glycosylation type 1a (CDG-Ⅰa), a rare inherited metabolic disease. Methods The clinical data and the gene detection results of one case of CDG-Ia which was discovered because the case had encephalopathy and hepatopathy were retrospectively analyzed. The related literatures were reviewed. Results Male infant suffered with face and trunk rash, motor development retardation, malnutrition, cheek fat plump, low limbs muscle tone, and bilateral crater nipple at 3 months old. Abnormal liver function and mild renal impairment were found after examination. The development quotient was low. Head MRI showed that bilateral frontal and temporal sulcus widening, and cerebellar atrophy. Urinary organic acids, amino acids, carnitine, and biotin activities were normal. Gene sequencing revealed that there were two heterozygous mutations, c.430T>C (p.F144L) and c.713G>C (p.R238P), in the PMM2 gene. The diagnosis of CDG-Ⅰa was confirmed. Both of the infant's parents were healthy, and each of them carries a pathogenic mutation. The infant had an elder brother who had mental disorder and died for liver and kidney function damage and hydronephrosis at 8 months old. Conclusion CDG-Ⅰa is an autosomal recessive disease. For infants with unexplained multiple organ damage, especially combined with intelligent and motor development retardations, strabismus, nipple retraction, and cerebellar atrophy, the possibility of CDG-Ⅰa should be considered. Gene detection of PMM2 can help the diagnosis.