In order to verify that bypass-activated complement can enable polymorphonuclear neutrophils (PMN) to lose their bactericidal capacity,3 experiments were performed as follows:(DA monolayer culture of human PMN was added with zymosan-activated human serum (ZAHS).then the superoxide ion (O2-,the specific granules,and the intracellular bactericidal activity of the cultured PMNs were signigicantly decreased and reached the lowest point in the 6th hour after the adding of ZAHS.0.05 ml of ZAHS showed the maximal effects on the PMNs.(2)Mice were injected intravenously with 0.5 ml of ZAHS and were killed 6 hours later.All the 3 parameters mentioned above dropped markedly in the PMNs isolated from the blood and the lungs of the mice.Pathologically,the lungs of the rats showed acute interstitial inflammation,focal edema,hemorrhage and atelectasis.and subcellular damages on the pulmonary blood-barrier.(3)ZAHS,after neutralization in vitro with antiserum against human C3 and C5,lost most of its harmful effects mentioned above.The findings suggest that the harmful effects of ZAHS originate from the fragments of C3 and Cs and they are most effective when its dosage is suitable and there must be a sufficient length of time to react before the phagocytosis of PMNs.The possible mechanism of the origination of the harmful effects of ZAHS is as follows:Before PMNs undertake phagocytosis,they releas the bactericidal and inflamnagenic (O2-and specific granules extra-cellularly.So the intracellular bactericidal activity of PMNs is decreased.The accumulation of PMNs in the pulmonary tissues can damage the PMNs themselves and the adjacent pulmonary blood-barrier,which will leads to the occurrence of infection and multiple organ failure.

Objective To study the roles of protein kinase C (PKC) in the signal transduction pathway of LPS induced nuclear transcription factor (NF) ?B activation of alveolar macrophages(AMs). Methods NF ?B level in nuclear protein extraction of AMs was detected by sandwich ELISA. Intranuclear translocation of NF ?B was observed by immunocytochemical staining. Results LPS could induce NF ?B activation of AMs in time and dose dependent manners. Immunohistochemical staining revealed intranuclear translocation following LPS induction. Specific calphostin C (Cal C) and pyrrolidine dithiocarbamate (PDTC) could inhibit LPS induced NF ?B activation. Conclusion PKC, as a up stream messenger, is involved in the signal transduction pathway of LPS induced NF ?B activation.

Objective To clone and express the gene encoding the binding domain of human soluble complement receptor type 1 (sCR1). Methods sCR1-SCR15-18 cDNA was amplified using RT-PCR from human monocytes of peripheral blood and sequenced using vector pMD-18T. Recombinant pET32-sCR1-SCR15-18 was constructed using prokaryotic expression vector pET32 and transformed into bacterium BL21. IPTG was used to induce gene expression and the obtained expression product was identified by immunoblotting. Results The gene segment that specifically encodes sCR1 was synthesized, the sequence of which was consistent with that of sCR1-SCR15-18 cDNA as registered at GenBank. A prokaryotic expression recombinant pET32-sCR1-SCR15-18 was constructed. The amount of target protein accounted for 40% of the total bacterial proteins and inclusion bodies were present in the bacteria. Immunoblotting showed a single positive band at the site of 43?10~(3). Conclusion The gene encoding sCR1-SCR15-18 was cloned from human monocytes and efficiently expressed in E.coli.

Objective To prepare highly expressed, purified and refolded SCR15-18 of human soluble complement receptor type 1 (sCR1-SCR15-18) protein. Methods The expression of recombinant pET32-sCR1-SCR15-18 in E.coli.. BL21 was induced by IPTG of different concentrations for different time period under different temperatures and the bacteria were split by sonication. The sCR1-SCR15-18 protein was purified by Ni2+-NTA resin affinity chromatography. The purified protein was refolded under different conditions. Then the bioactivity of the protein was analyzed. Results The sCR1-SCR15-18 protein of high expression, purity and bioactivity was attained. Conclusion The parameters of expression, purification and refolding of sCR1-SCR15-18 protein were optimized, which may pave a way for further studies.

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.

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 analyze and summarize the clinical characteristics of patients with spontaneous hemor -rhage of hepatic adenoma in glycogen storage disease type Ⅰa.Methods Reporting 1 case in our hospital and making a summary about general situation , category, etiology, diagnosis and treatment of the hemorrhage of hepatic adenoma with glycogen storage disease type Ⅰa through checking literatures .Results The patient was a 27 year old male who had been diagnosed as glycogen storage disease for 14 years, as well as was first found hepatic adeno-ma at the age of 17 .He once was diagnosed as intra-adenoma bleeding with persistent abdominal pain and dizziness and was underwent selective hepatic artery embolization at the age of 22.Hepatic adenoma in glycogen storage dis-ease typeⅠa generally appeared at the age of puberty .One common complication of this disease was hemorrhage of hepatic adenoma , which can be found by ultrasonography and CT .Clinical management includs observation , selec-tive hepatic artery embolization , radiofrequency ablation , surgical resection and liver transplantation .Conclusions Glycogen storage disease type Ⅰa is an autosomal recessive genetic disease with hepatic adenoma as a common complication of GSD Ⅰa, serious liver adenoma's hemorrhage can be life threatening , the radiological examination can be helpful to detect hepatic adenoma .Then appropriate intervention can improve the life quality and prognosis .

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.

AIM: To investigate the influences of bypass-activated complement and TNF-? on intercellular adhesion molecule 1(ICAM-1) expression in endothelial cells, and polymorphonuclear neutrophil-endothelial cell (PMN-EC) adhesion. METHODS: In vitro, zymosan-activated human serum(ZAHS) and/or TNF-? directly stimulated the HUVECS monolayers. PMN-EC adhesion percentage was measured, and modified whole-cell ELISA was used for measurement of ICAM-1. RESULTS: ZAHS alone resulted in no significant change in the degree of PMN adhesion and the level of ICAM-1. Activation of HUVECS with TNF-? followed by ZAHS stimulation resulted in greater increase in PMN-EC adhesion and ICAM-1 expression, as compared with the activation with TNF-? alone. CONCLUSION: Bypass-activated complement can promote ICAM-1 expression and neutrophil -endothelium adhesion induced by TNF-?, and so it is effective for promoting inflammation.

Objective:To observe the risk factors of acute renal injury(AKI) after coronary artery bypass grafting(CABG) and the influence of blood pressure on AKI.Methods:980 patients in CABG of Cardiology Department of TEDA International Cardiovascular Hospital were diagnosed with AKI according to the AKIN standard, with 706 males and 274 females, averaged(61.9±8.0)years old. Patients were divided into two groups according to whether AKI occurred: AKI group(86 cases) and non AKI group(894 cases). The baseline clinical data, operation related data were compared between the two groups. At the same time, according to the preoperative mean systolic blood pressure(SBP) level, LSP[mean systolic blood pressure<120 mmHg(1 mmHg=0.133 kPa), 374 cases], MSP(mean systolic blood pressure 120-140 mmHg, 481 cases) and HSP(mean systolic blood pressure≥140 mmHg, 125 cases) were classified as covariates, and the influencing factors of dependent variable AKI were analyzed by multivariate logistic regression.Results:The prevalence of AKI was 8.7%(86/980). Compared with non-AKI group, preoperative SBP[(129.8±13.8)mmHg vs.(124.4±13.3)mmHg, P=0.000], mean arterial pressure[(91.9±8.8)mmHg vs.(88.8±9.1)mmHg, P=0.004], and mean pulse pressure[(56.9±10.7)mmHg vs.(53.2±9.8)mmHg, P=0.001]were increased significantly. After adjusted for other risk factors, preoperative SBP elevation, hypertension history, cardiopulmonary bypass(CPB), use of intra-aortic-balloon-pump(IABP), secondary thoracotomy, preoperative diuresis, intraoperative blood transfusion and baseline low glomerular filtration rate(eGFR) were independent risk factors for AKI after CABG. Compared with LSP group, the relative risk of AKI after CABG in HSP group was 2.743(95% CI: 1.595-4.715). In patients with hypertension history, AKI in HSP group was significantly higher than that in LSP group(18.4% vs. 8.1%, P=0.001). However, the preoperative blood pressure level of patients who denied the history of hypertension had no effect on AKI. Conclusion:Preoperative SBP is a risk factor for AKI after CABG. The incidence of AKI after CABG can be significantly reduced by controlling SBP below 140 mmHg in patients with hypertension.