AIM: To explore the effect of complement on the cerebral ischemia/reperfusion injury in rat and the protection by sCR1-SCR15-18. METHODS: 75 male SD rats were randomly divided into three groups: sham operation group (SO, n=15), middle cerebral artery occlusion and reperfusion (MCAO) without treatment group (I/R, n=30); MCAO treated with sCR1-SCR15-18 group (sCR1-SCR15-18, n=30). After the MCAO for 2 h, then reperfusion for 24 h, the scores of neural behavioral functional deficits were determined. Infarction area was measured by TTC staining. Activity of MPO in cerebral cortex was detected. C3b deposition and pathological change were observed by immunohistochemial staining and HE staining, respectively. RESULTS: After reperfusion for 24 h, the neurological deficits score, infarction area and activity of MPO in sCR1-SCR15-18 group were decreased compared to I/R group. In sCR1-SCR15-18 group, C3b deposition in ischemic area was decreased and pathological injury was improved compared to I/R group. CONCLUSION: Complement plays a role in cerebral ischemia-reperfusion injury and sCR1-SCR15-18 exerts a protective effect by inhibiting the excessive activation of complement.

Objective To construct and express a small complement receptor type 1 (CR1) deriv-ative which contained two functional domains. Methods Total RNA was isolated from the peripheral blood mononuclear cell. The functional fragment Ⅰ of CR1 was amplified using the RT-PCR. The functional frag-ment Ⅱ was amplified with the plasmid of pET-32a-CR1-SCR15-18 as template which had been already con-structed in our laboratory. Then the chimeric gene that contained the two fragments was constructed with spli-cing overlap extention PCR. The chimeric gene was then inserted into the plasmid of pET-32a (+) and transformed into E. coli Rosetta(DE3). The inserted gene was verified by enzyme digestion and DNA se-quencing. After induced by IPTG, the expressed protein was analyzed by SDS-PAGE and Western blot. Then the fusion protein was purified by Ni-NTA affinity column and renatured through dialysis. The comple-ment inhibition activity was determined by CH50 method. Results The chimeric gene was successfully cloned into pET-32a(+). The result of SDS-PAGE and Western blot confirmed the expressed protein and showed that the molecule mass(Mr) of the expressed protein was 63×103. The purity of the recombinant protein was up to 92% after Ni-NTA column affinity chromatography. The bioactivity assay showed the fusion protein had a concentration-dependent complement inhibition activity within the concentration range of 0-200 μg/ml. Conclusion The two functional domains contained small CR1 derivative was successfully construc-ted and expressed in E. coli Rosetta. The fusion protein had a relative high bioactivity, providing a basis for further function experiment in vivo.

Objective To express the short consensus repeat 15-18 (SCR15-18) domain of human complement receptor 1 (CR1) in Pichia pastoris as a secreted protein in order to found a base for large-scale industry fermentation. Methods The gene fragment of CR1-SCR15-18 was amplified by PCR from plasmid pET32a-sCR1-SCR15-18. The obtained sequence was subcloned into Pichia pastoris secretory expression vector pPIC9. After identification the recombinant plasmid pPIC9-CR1-SCR15-18 was electrotransported into the yeast. Positive clones were identified using colony PCR. Positive recombinants were fermented in shake flaskes and induced with methanol. The recombinant proteins were identified with SDS-PAGE and Western blot analysis. After the recombinant protein was purified by Ni-NTA agarose metal chelate affinity chromatography, inhibiting complement hemolysis testing was used to detect the biological activity. Results Recombinant expression plasmid pPIC9-CR1-SCR15-18 was successfully constructed. SDS-PAGE and Western blot analysis revealed that the target gene was successfully expressed in the yeast and the recombinant protein was successful secreted in the culture supernatant. After purification, the protein inhibited complement hemolysis in vitro. Conclusion CR1-SCR15-18 is successful expressed in Pichia Pastoris with high activity of inhibiting complement hemolysis.

Objective To observe the interventional effect of Qingqihuatan Decoction on airway inflammation and inflammatory reaction of lung tissue in asthmatic mouse.Methods Forty-five BALB/c mice were randomly divided into the normal control group(CON),asthmatic model group(MOD),dexamethasone group(TRE),Qingqihuatan Decoction group(X1) and Qingqihuatan Decoction combined dexamethasone group(X2).The asthmatic mouse model was established by the sensitization and inhalation of OVA and aluminium hydroxide gel.The bronchoalveolar lavage fluid (BALF) was performed the cell counts and eosinophil counts,and the pathological changes of lung tissue were observed.Results Compared with CON group,BALF cell count and eosinophil count in the model group were increased obviously,which in the treatment group were significantly decreased.The effect of the X1 group and X2 group had statistical difference between on 5 d and 15 d.(P＜0.05 or P＜0.01);the treatment groups could reduce the pathogenical infiltration,lung bullae production and airway epithelial thickening of mouse lung tissue,especially the inflammation damage in X2 group was mild.Conclusion Qingqihuatan Decoction can relieve the airway inflammation and improves the lung tissue inflammatory response in model mouse.

Objective To investigate the expressions of KLF2 mRNA and KLF4 mRNA in the acute lung injury (ALl) rats induced by lipopolysaccharide (LPS),and to analyze the correlation between KLF2,KLF4 and ALI.Methods A total of 100 SD rats were randomly divided into 2 groups:normal control group and LPS treated group,then the latter group was randomly further divided into 3 subgroups as per the serum and lung tissue samples taken separately at 2,4 and 24h after modeling.The ALI model was made by injecting 5mg/kg LPS into tail vein.The pathological changes of lung tissue were observed in each group,and the expressions of KLF2,KLF4 mRNA in serum and lung tissue were detected by RT-PCR.The data of laboratory findings were analyzed with SPSS 17.0 software for statistical analysis.Results The histopathological changes showed the most obvious damage of lung tissue occurred at 4 hours after modeling.The expressions of KLF2 mRNA and KLF4 mRNA in the lung tissue and serum of control group were significantly higher compared to LPS treated subgroups (P ＜0.01).The expression of KLF2 mRNA in LPS treated subgroup at 2 hours was lower than that in LPS subgroups at 4 hours and 24 hours (P ＜ 0.01),while the expression of KLF4 mRNA in LPS treated subgroup at 4 hours was lower than that in LPS treated subgroups at 2 hours and 24 hours (P ＜ 0.01).Conclusions The expression of KLF2 mRNA was occurred earlier than the pathological changes in acute lung injury,while the expression of KLF4 was emerged synchronously,and both KLF2 and KLF4 could be used as candidates of predictive and diagnostics molecular markers of ALI.

Objective To analyze the epidemiological characteristics of Klebsiella pneumoniae car-bapenemase(KPC)-producing Escherichia coli(E. coli)strains isolated in Hangzhou,China. Methods A total of 25 KPC-producing Escherichia coli strains were collected from four hospitals in Hangzhou from July 2012 to January 2014. Antibiotic susceptibility of the isolates to 22 common antimicrobial agents was deter-mined by using Kirby-Bauer(K-B)disk diffusion method. PCR analysis and gene sequencing were used for bla KPC gene screening. The modified Hodge test was performed to detect the production of carbapenemase. Pulsed-field gel electrophoresis(PFGE)and multi-locus sequence typing(MLST)were used for homology analysis. Results All of the 25 clinical isolates were confirmed to be KPC-producing E. coli strains,harbo-ring the blaKPC-2 gene. These KPC-producing isolates showed high drug resistance rates and were resistant to almost all β-lactam antibiotics. PFGE typing classified the 25 isolates into three main homologous clone groups,including clone group A(4 isolates),clone group B(5 isolates)and clone group C(2 isolates), and some single clones(14 isolates). MLST typing classified the isolates into eight ST types,including ST131(14 isolates),ST167(3 isolates),ST2003(3 isolates),ST410(1 isolate),ST457(1 isolate), ST1463(1 isolate),STnew1(1 isolate)and STnew2(1 isolate). The typing results of PFGE and MLST were consistent with each other. Conclusion The prevalent KPC-producing E. coli strains in Hangzhou, China were ST131 type,which were resistant to multiple antibiotics and had been detected in several hospi-tals. The epidemic of KPC-producing E. coli strain often occurred at some special wards,such as Intensive Care Unit(ICU)and emergency ICU.

ObjectiveTo observe the effect of exercise training on chronic obstructive pulmonary disease (COPD) stable patients in community. MethodsTraditional Chinese exercise prescription was given to 20 COPD patients. They were assessed with 6-minute walk distance (6MWD), the Borg scale and the St. George's Respiratory Questionnaire (SGRQ) before and after the training. ResultsThe Borg scales dropped from (4.45±2.04) to (3.15±2.13) （P＜0.05）. 6MWD increased from (370.32±74.48) m to (403.75±76.15) m （P＜0.05）. SGRQ scores also showed statistical significant difference （P＜0.05）. ConclusionExercise training can improve the tolerance and decrease dyspnea in COPD patients in stable stage. It also can improve the quality of life.

Objective To explore the clinical characteristics and gene mutation in a patient clinically diagnosed as dopa-responsive dystonia (DRD) without family history.Methods The clinical characteristics of a patient clinically diagnosed as DRD without family history were collected and molecular and bioinformatic analyses were performed.Results The patient demonstrated as type A tyrosine hydroxylase deficiency and a compound heterozygous mutation of tyrosine hydroxylase (TH) gene was found,including a known nonsense mutation,c.457C＞T and a novel missense mutation,c.734G＞T that was probably pathologically predicted by bioinformatic analysis.Conclusion c.734G＞T may be a novel pathological mutation of TH gene.

Objective:To summarizes the diet management of one child with GSD I and severe hyperlipidemia.Methods:Key points of diet management include: making an individual diet plan, correcting parents' dietary misunderstanding, adjusting dietary and keeping following up regularly and keeping a food diary.Results:Following up for 11 months, the children basically formed a stable diet pattern, the blood glucose level was basically maintained between 4~6 mmol/L, the indicators of hyperlipidemia, hyperlactic acid and liver function were significantly improved compared with the previous period, and the height increase was guaranteed, while the weight gain was effectively controlled.Conclusions:It shows that individualized dietary guidance has a significant effect on the maintenance of blood glucose level, improvement of growth and development status and metabolic control in children with GSD I.

Objective:Charcot-Marie-Tooth disease (CMT) comprises a group of clinically and genetically heterogeneous inherited neuropathies with an estimated prevalence of 1 in 2500. This study aimed to analyze the clinical and mutational characteristics of Chinese CMT patients with MFN2, BSCL2 and LRSAM1 variants.Methods:In this study, genetic analysis was performed in 206 Chinese patients at Chinese PLA General Hospital from December 2012 to March 2020 with clinical diagnosis of CMT, and reported variants of MFN2, BSCL2 and LRSAM1 related to CMT2.Results:We reported ten MFN2 mutations in ten unrelated patients (7 male, 3 female), two of whom had positive family history. Three novel mutations were detected including c.475-2A>G (splicing); c.687dupA (p.E230Rfs*16) and c.558dupT (p.S186fs). We reported three BSCL2 mutations of four unrelated patients, including c.461C>G (p.S154W), c.461C>T(p.S154L), and novel variants of c.1309G>C (p.A437P) and c.845C>T (p.A282V). Furthermore, two novel variants of LRSAM1, including c.1930G>T (p.G644C) and c.1178T>A (p.L393Q) were detected in two unrelated patients.Conclusion:Mutational spectrum of MFN2-, BSCL2-and LRSAM1-related CMT disease is expanded with the identification of novel variants in Chinese patients.