Objective To establish axenic cultivation of Pneumocystis carinii (P.c). Methods The organisms of P.c were isolated from the bronchoalveolar lavage fluid (BALF) of the rats with Pneumocystis carinii pneumonia (PCP) and cultured in a medium which was based on IMDM (GIBCO) supplemented with S-adenosyl-L-methionine, putrescine, N-acetyl glucosamine, putrescine, L-cysteine and L-glutamine, and newborn calf serum. The organisms cultured in the system were identified by observing the morphology of cysts in smears stained with Gomori's methenamine silver nitrate stain (GMS). Ultrastructure of the cysts/trophozoites was examined by transmission electron microscopy. The sequences of mitochondria] large ribosomal DNA subunit of the cutured organisms were compared with the Pneumocysti carinii f.sp. ratti variant isolate (GenBank No U20173) and Pneumocystis carinii f.sp.hominis (GenBank No M58605). Results Five isolates of P.carinii received from BALF of 8 rats with PCP were cultured axenically and continuously in the system. The cultured organisms could be stored in frozen condition and used to reinitiate culture, and were amplified by 19-22 times within 72 h. The morphology, ultrastructure and gene sequencing of the cultured organisms confirmed that the isolated organisms were P.carinii. Conclusion Five continuously and axenicly cultured isolates of P.carinii have been received.

Objective To construct a recombinant adenovirus encoding ?-galactosidase gene(?-gal),which may be used to treat the lactose intolerance caused by lactase deficiency,with modified AdEasy system.Methods The sequence fragment of ?-gal was cloned into the shuttle plasmid pAdTrack-CMV,and the homo-logous recombination was completated with backbone plasmid pAdEasy1 in the E.coli BJ5183 to construct the recombinant adenoviral plasmid.The adenoviruses were packaged and amplified in the HEK293 cells mediated by liposome.The viral titre was measured by limiting dilution assay and the transfection efficiency was observed by X-gal histochemical staining.Results The recombinant adenovirus with ?-gal was constructed successfully and the viral titre was(2.5~4.0)?1011EFU/ml.More than 70% HEK293 cells were transfected when the MOI was 10.Moreover,more than 65% cells were blue after X-gal histochemical staining.Conclusion Recombinant adenovirus carrying ?-gal with high titre and efficient transfection is constructed successfully by AdEasy system,which supplies valuable experimental basis for the gene therapy to the lactose intolerance caused by lactase deficiency.

Group A β-hemolytic streptococcus (GAS),namely Streptococcus pyogenes,is one of the most im-portant human pathogen.GAS can cause skin and mucous membrane superficial infectious diseases,life -threatening invasive disease,toxin -mediated diseases and immune -related diseases.Antibiotic is an effective mean to control GAS infection.The β-lactam antibiotics remain the first -choice treatment for GAS infection and the macrolides are often recommended as a replacement therapy for β-lactam antibiotics allergic patients or a means to blocking GAS exotoxin product.But with the widespread use of macrolides autibiotics,macrolide -resistant GAS spread in the world. This paper will elaborate the situation of macrolide -resistant clones.

Objective To investigate the relationships among cerebellar mutism (CM), relapsed medulloblastoma (MB) and the primary tumor location.MethodsA retrospective analysis was conducted in 114 children over 3 years old with MB from November 2011 to April 2015.ResultsThe median onset age was 84.7 months (36.4 to 184.7 months) in 114 children with MB (77 boys and 37 girls), of whom there were 48 cases of recurrence. There were twenty two cases of CM and the overall incidence of CM was 19.3% (22/114). The incidence of CM was 19.7% (13/66) in non-recurrent cases and 18.8% (9/48) in recur-rent cases, and there was no signiifcant difference between two groups (P=0.899). The incidence of CM was 17.6% (9/51) in cas-es with primary tumor in the fourth ventricle, 7.1% (1/14) in cases with primary tumor in the cerebellar vermis, 21.4% (3/14) in cases with primary tumor in both fourth ventricle and cerebellar vermis, 45.5% (5/11) in cases with primary tumor in fourth ven-tricle and other parts of the brain, and 50.0% (4/8) in cases with primary tumor in cerebellar vermis and other parts of the brain. No CM incidence was observed in cases with primary tumor in central nerve system except for the fourth ventricle and cerebellar vermis. The incidence of CM between the cases with fourth ventricle/cerebellar vermis involvement and those without fourth ventricle/ cerebellar vermis involvement had signiifcant difference (P=0.039). ConclusionsThere is no relationship between CM and relapsed MB. Children with MB whose primary tumor is located in the fourth ventricle and/or the cerebellar vermis is susceptible to CM.

Objective: To make a study of density and affinity of IL-6R in human leukemic cell lines, and discuss the affection of high affinity IL-6R to the targeted treatment of leukemia with IL-6-PE40 fusion protein. Methods: Radial binding assay with scatchard plot and FACS were used to analysis the density and affinity of IL-6R and protein expression of IL-6Rα and β subunits in totally 8 representative human leukemic cell lines. Results: Myelocytie, monocytic and erythrocytic leukemic cell lines U937, HL-60, KG1 and TF1 express high affinity IL-6R, whose average density per cell is 2 502,2 874, 2 319 and 9 329 respectively, however no 125I-IL-6 binding was detected on chronic myelocytic leukemic cell line K562 and lymphoblastic leukemic cell lines such as Raji, CEM and HUT28. These results correlate with those of FACS highly. Conclusion:These observations suggest that acute nonlymphoblastic leukemic cells may be more suitable for targeted treatment with IL-6-PFA0 fusion protein.

Objective To investigate the clinical characteristics of children with medulloblastoma (MB). Methods The correlations amongst MB histopathological subtype,age at diagnosis,gender,primary tumor locations, relapsed tumor and relapsed tumor locations were analyzed retrospectively in 83 children who were diagnosed as MB by histopathology subtypes from February 2012 to April 2015 in Beijing Shijitan Hospital Affiliated to Capital Medical Uni-versity.The data was conducted by using SPSS 22.0 statistical software.Results Among the 83 cases (53 boys and 30 girls),there were 14 patients younger than 3 years old (9 boys and 5 girls)and 69 patients (44 boys and 25 girls)ol-der than 3 years old,including 28 relapsed (19 boys and 9 girls)and 55 non -relapsed cases (34 boys and 21 girls). The median age was 80.2 (13.1 -184.7)months at diagnosis.Of these 83 cases,48.2% (40 /83 cases)was classic medulloblastoma (CMB)(2 cases less than 3 years old),24.1 % (20 /83 cases)was desmoplastic /nodular medullo-blastoma (DMB)(6 cases less than 3 years old),12.1 % (10 /83 cases)was large cell/anaplastic medulloblastoma (LC /AMB)(1 case less than 3 years old),3.6% (3 /83 cases)was extensive nodular medulloblastoma (MBEN)(1 case less than 3 years old),and 12.1 % (10 /83 cases)(3 cases less than 3 years old)was mixed subtype.The rela-tionships between age at diagnosis and histopathological subtype,gender and primary tumor location were all statistically significant (χ2 =0.014,0.013,all P <0.05).Conclusions The incidence of boys with MB is higher than girls.CMB is the main histopathologic subtype in children over 3 years old.The primary tumor location involving the cerebellar vermis or cerebellar vermis and the fourth ventricle is higher in girls with MB.The primary tumor location involving the fourth ventricle,the fourth ventricle and other parts of the central nervous system,Cerebellar vermis and other parts of the central nervous system or other parts of the central nervous system is higher in boys with MB.

Objective To investigate the pathogen-related genes of atmospheric polluting disease so as to clarify the biology mechanism and provide the scientific basis.Methods By using the technique of dot blot hybridization,we analyzed genes’differential expression with cloning by exposure to ≥75 μg/m3 PM2.5 in heating season and < 75 μg/m3 PM2.5 in un-heating season in WI-38 human embryo lung cells.The levels of cytokines TNF-α,IL-2, IL-6 and IL-8 were determined by radio immunity assay. Results After 24h of treatment, compared with control group,more than 100μg/mL PM2.5 significantly increased TNF-α,IL-6 and IL-8 levels,and decreased IL-2 in WI-38 human embryo lung cells (P < 0.05 ).The clear stripe was found in 350 bp in 48 gene samples with segment with differential expression of genes exposed to different concentrations of PM2.5 in WI-38 human embryo lung cells.Through the dot blot hybridization,black brown spots were found in 41 samples in Tester cDNA hybridization,and no similar spots were found in all of the same samples in Driver cDNA hybridization. Conclusion PM2.5 exposure may induce the inflammatory damage of WI-38 human embryo lung cells.Obvious genetic damage was observed in those cells exposed to ≥75 μg/m3 PM2.5 in heating season.

Objective:To evaluate the efficacy of remimazolam-propofol-sufentanil for anesthesia in patients undergoing painless gastroscopy.Methods:Eighty American Society of Anesthesiologists physical statusⅠor Ⅱ patients, aged 20-59 yr, weighing 44-69 kg, scheduled for elective painless gastroscopy, were divided into 2 groups ( n=40 each) using a random number table method: remimazolam-propofol-sufentanil group (group RPS) and propofol-sufentanil group (group PS). The patients in group RPS received successive intravenous injection of sufentanil 0.1 μg/kg, remimazolam 0.15 mg/kg and propofol (at a rate of 4 mg/s). The patients in group PS received intravenous injection of sufentanil 0.1 μg/kg and propofol (at a rate of 4 mg/s). When Observer′ s Assessment of Alertness/Sedation Scale score was 0, gastroscopy was performed.The consumption of propofol, time of anesthesia, time for gastroscopy, emergence time and discharge time were recorded.The number of intraoperative assisted respiration cases, body movement and occurrence of adverse reactions at the time of discharge were observed. Results:Compared with group PS, the consumption of propofol was significantly decreased, and the time of anesthesia, emergence time and discharge time were shortened in group RPS ( P<0.05). There was no significant difference in the time for gastroscopy, the number of intraoperative assisted respiration cases, body movement and the occurrence of adverse reactions at discharge time between the 2 groups ( P>0.05). Conclusion:Remimazolam-propofol-sufentanil produces better efficacy for anesthesia than propofol-sufentanil in patients undergoing painless gastroscopy.

Objective:Identification of some biochemical and physical properties for a new recombinant B7-2-PE40KDEL exotoxin fusion protein.Methods:12%SDS-PAGE separating and gel imaging analyzing,peptide mass fingerprinting,Western blot and MTT assasying were used respectively for identification of the protein.Results:Molecular weight of the recombinant B7-2-PE40KDEL was 72 628,5% of the difference to its theoretical value 69 561.The result of Western blot indicated that the purified recombinant B7-2-PE40KDEL could specifically bind with mAb anti-human B7-2 and the antibody against PEA,while the negative control did not.The recombinant B7-2-PE40KDEL digested with trypsin and then detected by MOLTI-TOF-MS.It was shown that the detected 15 peptides lied in the extracellular part of B7-2 and the truncated Pseudomonas extoxin PE40KDEL.Searching in the peptident data bank of Expasy website,we did not find any known proteins which was accordant with the above terms.The cytotoxic activity of the recombinant toxin with MTT method showed that the B7-2-PE40KDEL selectively killed Jurkat cell line which expressesed CD28 receptor well and had no killing effect on the Raji cell line unexpressing CD28 receptor.Conclusion:Recombinant B7-2-PE40KDEL exotoxin fusion protein we construct proves to be a new one with targeted killing bioactivity to B7:CD28 system.

AIM: To obtain the high expression of recombinant human stem cell factor - thrombopoitin (SCF-TPO) fusion gene and predict its structure property. METHODS: Tour primers were designed according to known sequence of TPO and SCF to amplify the functional amino acid domain of TPO and SCF by RT- PCR, respectively from fetus hepatocytes. The expression plasmid pET32a/SCF- TPO was constructed by VOE gene fusion technique and expressed in BL21(DE3)plysS. The fusion protein property, such as second structure, flexibility, and hydrophilicity were predicted by DS Gene and Protscale software. RESULTS: The expression vector, pET32a/SCF - TPO was constructed and the high expression of the SCF/TPO fusion protein was obtained, with the expression amount of up to 40% of the total cellular protein. DS Genel .5 and Protscale predict no new antigenicity in fusion protein, and the second structure and ioelectric point have no changes except four amino acids change in first structure. There are high flexibility and low hydrophilieity in the linker peptide. CONCLUSION: High expression of SCF- TPO fusion protein has been obtained and protein prediction shows that the fusion protein design is reasonable, which lay foundation for further study of biological fundation of SCF - TPO fusion protein.