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 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 explore the relationship between lymphovascular invasion and non-sentinel lymph node (NSLN) metastasis in early-stage invasive breast cancer with positive sentinel lymph node (SLN) and its significance.Methods:The clinicopathological data of 79 patients with stage cT 1-2N 0M 0 invasive breast cancer who had positive SLN by biopsy and underwent axillary lymph node dissection (ALND) from January 2015 to February 2021 in the Central Hospital of Wuhan were retrospectively analyzed. The correlation between patients' clinicopathological characteristics and NSLN metastasis was analyzed. Results:Among 79 patients, 58 patients (73.4%) underwent total mastectomy, 61 patients (77.2%) were Luminal type, 38 patients (48.1%) had lymphovascular invasion, 64 patients (81.0%) had 1-2 positive SLN, and 42 patients (53.2%) with NSLN metastasis were found after ALND. Univariate analysis showed that the proportions of patients with lymphovascular invasion diagnosed by immunohistochemistry [86.8% (33/38) vs. 51.2% (21/41)], Ki-67 positive index>30% [60.5% (23/38) vs. 36.6% (15/41)], positive human epidermal growth factor receptor 2 [36.8% (14/38) vs. 14.6% (6/41)], and elevated lymph node pathological staging [57.9% (22/38) vs. 31.7% (13/41)] in the lymphovascular invasion group were higher than those in the non-lymphovascular invasion group (all P < 0.05). Multivariate logistic regression analysis showed that lymphovascular invasion was an independent risk factor for NSLN metastasis ( OR = 2.935, 95% CI 1.081-7.970, P = 0.035). Conclusions:Lymphovascular invasion is an independent risk factor for NSLN metastasis in SLN-positive stage cT 1-2N 0M 0 invasive breast cancer. It may help to guide the decision-making of local axillary treatment, so as to avoid over or under treatment.

This study explores the effects of enhancing the definitive hematopoiesis(DH)signals during the differentiation of human embryonic stem cells(hESCs)into hematopoietic stem/progenitor cells on the generation efficiency and effector function of natural killer(NK)cells generated from hESCs(also known as hESC-NK cells).The hESC(H1)were transformed into embryoid bodies(Ebs)by centrifugation,and during the induction of K562,were used to analyze the efficiency of hematopoietic differentiation,the efficiency of NK cell generation from hESC,the in vitro effector functions,and the expression of effector function related surface receptors.Compared to the control group,the DH group had a significant increase in the number of arterial hematopoietic endothelial cells(CD34+DLL4+)and a significant decrease in primitive hematopoietic related cells(CD34-CD43+)on day 8 of hematopoietic differentiation(P<0.05).On day 28 of NK cell differentiation,the DH group demonstrated a significant increase in the number of NK cells(CD45+CD56+),while a slight increase in the expression of effector function-related molecules such as IFN-γ,Granzyme B,Perforin and CD107a without statistical significance.Furthermore,the activation receptors CD16a and CD69 were significantly increased,NKP46 was significantly decreased,the inhibitory receptor NKG2A was significantly increased,while CD96 was significantly decreased on hESC-NK cells of DH groups(P<0.05).Conclusively,enhancing the signals for definitive hematopoiesis during hESC differentiation into hematopoietic stem/progenitor cells significantly improves the yield of NK cells and the expression of CD16a without affecting their in vitro effector functions.Our study provides a new approach to improving the efficiency of hESC-NK cell or iPSC-NK cell generation.