[Effects of direct antiviral agent on the frequency of peripheral blood mononuclear cells and their activating factors sCD14s and CD163 in patients with chronic hepatitis C].

Objective: To explore the effects of direct antiviral agent (DAAs) on the frequency of peripheral blood mononuclear cells and their activating factors sCD14s and CD163 in patients with chronic hepatitis C. Methods: Data of 15 treatment-naive chronic hepatitis C patients and 10 healthy controls were collected. Patients with chronic hepatitis C were treated with DAAs for 12 weeks. Blood samples were collected at 0, 4 and 12 weeks respectively, and blood samples of healthy controls were used as controls. Flow cytometry was used to detect the frequency of classical CD14(++)CD16(-) mononuclear cells and pro-inflammatory CD14(+)CD16(+) mononuclear cells in peripheral blood. Serum sCD14s and sCD163 were detected by enzyme-linked immunosorbent assay. The comparison between the two groups was performed by t-test. The comparison between multiple groups was performed by analysis of variance, and further pairwise comparison was performed by LSD-t test. Results: Prior DAAs treatment, peripheral blood CD14(+)CD16(+) mononuclear cell frequency (18.49% ± 1.54% vs. 10.65% ± 0.83%), serum sCD14s [(64 407.38 ± 5778.49) pg/ml vs. (28 370.76 ± 2 357.68 ) pg/ml] and sCD163 [(22 853.80 ± 4 137.61) pg/ml vs. (2 934.41 ± 223.31) pg/ml] were all higher than healthy controls (P < 0.05), while the frequency of CD14(++)CD16(-) mononuclear cells in peripheral blood was lower than healthy controls (59.14%±0.54% vs. 72.75%±1.31%, P < 0.01). During DAAs treatment, CD14(+)CD16(+) mononuclear cells frequency, serum sCD14 and sCD163 were all decreased significantly. After 12 weeks of treatment, CD14(+)CD16(+) mononuclear cells had decreased to nearly normal level (12.42% ± 1.60% vs. 10.65% ± 0.83%, P > 0.05), and serum sCD14 and scd163 were still higher than those of healthy controls [sCD14: (44 390.06 ± 3 330.17) pg / ml vs. (28 370.76 ± 2 357.68) pg/ml, Scd163: (11 494.79 ± 1 836.97) pg / ml vs. (2 934.41 ± 223.31) pg / ml, P < 0.01], while the frequency of CD14(++)CD16(-)mononuclear cells had gradually increased during the course of treatment and neared healthy control level after 12 weeks of treatment. There was no statistically significant difference between the two groups (71.54) % ± 2.99% vs. 72.75% ± 1.31%, P > 0.05). Conclusion: DAAs therapy can reduce the activation of peripheral blood mononuclear cells in patients with chronic hepatitis C.

[Risk factors for early death in acute myocardial infarction patients complicating with ventricular septal rupture].

Objective: To assess the clinical characteristics and identify the risk factors in the acute myocardial infarction (AMI) patients complicating with ventricular septal rupture (VSR). Methods: A retrospective study was performed on 96 AMI patients complicating with VSR, who were hospitalized in the Second Xiangya Hospital of Central South University, Hunan Provincial Peoples' Hospital, the First Affiliated Hospital of University of South China, the Second Affiliated hospital of University of south China, Xiangtan Central Hospital from December 2007 to May 2017. There were 46 females and the age was (66.2±10.7) years (from 43 to 90 years). Patients were divided into in-hospital survival group (n=64) and in-hospital death group (n=32). The 96 patients were also divided into the early death group (survived ≤2 weeks after admission, n=50) and non-early death group (survived>2 weeks after admission, n=46). Multivariate logistic regression was used to analyze the independent risk factors of the early death. Results: Location of VSR was available in 71 patients, VSR was located at the apical or anterior septum near the apical region in 64.0% (32/50) patients with the anterior AMI, VSR was located at the posterior wall and basal inferior segment in 57.1% (12/21) patients with non-anterior AMI. Compared to the in-hospital survival group, patients in the in-hospital death group were older ((69.6±11.3) years vs. (64.6±10.1) years, P=0.031), incidence of non-ventricular aneurysm (71.9% (23/32) vs. 37.5% (24/64), P=0.001) and anterior AMI (84.4%(27/32) vs. 62.5%(40/64), P=0.028) was significantly higher in the in-hospital death group than in the in-hospital survival group. The comparison between the early death group and non-early death group showed that older age, female, no history of angina or myocardial infarction, Killip grade>Ⅲ, and non-ventricular aneurysm were related to increased risk of the early mortality in this patient cohort. Logistic regression analysis revealed that female (OR=5.109,95%CI 1.19-22.00, P=0.012), no history of angina or myocardial infarction (OR=23.34, 95%CI 3.44-158.37, P=0.001), Killip grade>Ⅲ(OR=5.35, 95%CI 1.26-22.66, P=0.019) and non-ventricular aneurysm (OR=6.30,95%CI 1.67-23.73, P=0.005) were independent risk factors for early death in this patient cohort. Conclusion: The risk factors of in-hospital death include older age, non-ventricular aneurysm and anterior AMI. Female, no history of angina or myocardial infarction, Killip grade>Ⅲ and non-ventricular aneurysm are independent risk factors for the early death of AMI patients complicating VSR.

[Expression of extracellular domain of VEGF receptor KDR with recombinant AAV].

New vessel formation plays a key role in tumor growth and transforming, and the Vascular endothelial growth factor (VEGF) is the most important factor inducing tumor vessel formation. The cDNA of extracellular domain of VEGF receptor KDR was cloned from primary cultured HUVEC by RT-PCR and subcloned into AAV vector pSNAV. Recombinant AAV was obtained from BHK cells transformed with pSNAV/KDR after adding helper virus. The recombinant AAV expressing soluble KDR that can bind to VEGF. In vivo experiment demonstrated that the recombinant virus can inhibit the new vessel formation of melanoma in C57 mice model.

[Cloning of VEGF receptor KDR and its expression in insect cells].

The cDNA fragment of the first 3 loops of VEGF receptor, KDR, was cloned by PCR and inserted into a baculovirus expression plasmid pFASTBACI. The competent E. coli DH10BAC cell, which contain another plasmid with baculovirus genome in it, was transformed with pFASTBACI-KDRn3. Homologous recombination in the prokaryotic cells resulted in a recombinant plasmid containing KDRn3 in baculovirus genome. Transfection of the insect cell SF-9 with above plasmid generated a recombinant baculorvirus contain target gene fragment. SDS-PAGE and Western blot analysis of the supernatant of the infected SF-9 cell showed that KDRn3 was secreted in the medium. The recombinant protein was verified with Western blot and tested for their binding activity with VEGF. Its anti-angiogenic activity was assayed on chorionic allantoic membrane(CAM) of fertilized egg. The results showed that the recombinant protein could inhibit new vessel formation on CAM of fertilized eggs.

Expression of human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop cDNA in Pichia pastoris, purification of the expressed product and detection of its biological activity.

OBJECTIVE: To express human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop cDNA in Pichia. pastroris, and to purify the expressed product and detect its biological activity. METHODS: By inserting human Flt-1 (1-3 loop) cDNA coding 316 amino acid residues into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and the sequences of alpha secreting signal peptides, a recombinant expression plasmid pPIC9K/Flt-1 (1-3) was constructed and transformed to yeast host strain GS115, then His+ Muts phenotype transformant was screened out and cultured in flasks, and Flt-1 (1-3) was expressed under the induction of 1% methanol. RESULTS: SDS-PAGE showed that after being induced with 1% methanol for 4d, the expressed product existed in supernatant in the form of soluble molecule and contained 60% of total protein expressed. Western blot showed good antigenicity and specificity of expressed product. After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, the purity of the expressed product reached above 90%. Biological assay proved that the expressed product could bind to hVEGF165 and inhibit the proliferation of HUVEC stimulated by hVEGF165. CONCLUSION: Human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop was successfully expressed. The study lays a foundation for further application of the expressed product in the treatment of vasoformation related diseases, such as tumor and diabetic retinopathy.

[Expression and characterization of human vascular endothelial growth factor receptor Flt-1 extracellular domain in Pichia pastoris].

By inserting VEGF receptor Flt-1(1-3 loop) cDNA into Pichia pastoris expression vector pPIC9K containing AOX1 promotor and the sequences of alpha secreting signal peptides, the expression plasmid pPIC9K/Flt-1(1-3) was constructed and transformed into GS115. The multi-copy insert transformants were selected and cultivated in flasks. After 4 days of 1% methanol induction, the expressed Flt-1(1-3) accumulated up to 30% of total proteins in supernatant. The expressed Flt-1(1-3) was further proved with good antigenicity and high specificity by ELISA and Western blot. They can bind to VEGF and inhibit HUVEC proliferation stimulated by VEGF.