AIM:To investigate the correlation of serum miR-146a with nuclear factor-κB(NF-κB)and vascular endothelial growth factor(VEGF)in diabetic retinopathy patients.

METHODS: A total of 100 patients with T2DM treated in our hospital from July 2016 to December 2017 were assigned into T2DM patients with DR(DR group, n=32)and T2DM patients without DR(T2DM group, n=68). Thirty healthy volunteers were selected as control group. Real-time PCR was used to examine the expression of miR-146a. Enzyme linked immunosorbent assay was used to detect the levels of NF-κB and VEGF. The correlation between miR-146a and NF-κB and VEGF was analyzed.

RESULTS: Compared with the control group, HbA1c in T2DM group and DR group increased(t=6.822, 5.709; P<0.001), FBG increased(t=8.889, 7.923; P<0.001), 2hPBG increased(t=6.646, 5.514; P<0.001). Compared with T2DM group, the duration of diabetes in DR group was longer(t=2.431, P=0.017). Compared with the control group, serum miR-146a in T2DM and group DR significantly decreased(t=3.967, 7.169; P<0.001), and the DR group was lower than that in the T2DM group(t=4.444, P<0.001). Compared with the control group, the serum NF-κB in the T2DM and DR group increased significantly(t=6.063, 14.851; P<0.001), VEGF increased significantly(t=7.613, 12.943; P<0.001), NF-κB and VEGF in DR group were larger than those in T2DM group(t=11.406, 7.560; P<0.001). Pearson analysis showed that miR-146a was negatively correlated with NF-κB and VEGF(r=-0.503, -0.574; P<0.05).

CONCLUSION: The serum miR-146a in DR patients significantly decreased, the NF-κB and VEGF significantly increased. MiR-146a may be involved in the pathogenesis of DR by mediating inflammatory reaction and vascular proliferation.

The cytoplasm of E. coli is a reducing environment where cysteines do not engage in disulfide bonds. Any disulfide bonds that do appear are rapidly reduced through the action of disulfide reducing enzymes such as thioredoxin and glutaredoxin. To study the influence of E. coli cytoplasm on the solubility of recombinant proteins produced in it, bovine fibroblast growth factor (BbFGF), with single disulfide bond, and anti-HBsAg single-chain Fv (HBscFv), with two disulfide bonds, were selected as the pattern molecules of simple protein and complex protein, respectively. pJN98-BbFGF, a BbFGF expressing plasmid based on the vector pET3c, was constructed and transformed into normal host BL21(DE3) and a reductase deficient strain, E. coli Origami(DE3). At the same time, pQE-HBscFv, a HBscFv expressing plasmid was constructed and transformed into M15 [pREP4] and Origami(DE3). The recombinant BbFGF and HBscFv were produced in 2 types of bacteria and their solubilities and bioactivities were determined, respectively. It was found that the majority of BbFGF had formed inclusion body in the cytoplasm of BL21 (DE3) and all of them turned into soluble protein in Origami(DE3). It was also found the productivity of BbFGF in Origami (DE3) was 5% - 10% of the total protein and the value was 15% - 23% in BL21(DE3). BbFGFs produced in 2 recombinant bacteria were purified by cation exchange and heparin affinity chromatography. MTT assay revealed that the bioactivity of BbFGF purified from Origami(DE3) was higher than its counterpart from BL21(DE3). The ED50 of BbFGFs from different bacteria was 1.6ng/mL and 2.2ng/mL, respectively. As far as HBscFvs, both of them formed inclusion body in the cytoplasm of M15 [pQE-HBscFv] and Origami [pQE-HBscFv]. The inclusion body was solubilized in 6mol/L GuHCl, purified with a His-Trap column and then refolded by dialysis step-by-step against buffers containing downtrend concentration of GuHCl. Indirect ELISA was applied to determine the HBsAg binding activity of HBscFvs. It was found there was no obvious difference between the bioactivity of refolded HBscFvs produced from 2 recombinant bacteria. On the other hand, the supernatant of Origami [pQE-HBscFv] lysate displayed weak bioactivity and its counterpart from M15 [pQE-HBscFv] displayed without any bioactivity. The soluble HBsFv in the cytoplasm of Origami [pQE-HBscFv] was purified by cation exchange and immobilized metal affinity chromatography (IMAC) and the yield was 1 - 2mg/L. Those results suggested that modification of the redox environment of E. coli cytoplasm greatly improved the solubility of recombinant disulfide-bonded proteins produced in it. In the next step, we had like to co-express of molecular chaperones or refoldase to raise the yield of soluble recombinant proteins, as well as optimizing the culture condition of the "oxidizing" E. coli.
Animals ; Antibodies ; genetics ; immunology ; metabolism ; Cattle ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; enzymology ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; Fibroblast Growth Factors ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hepatitis B Surface Antigens ; immunology ; Inclusion Bodies ; chemistry ; metabolism ; Oxidoreductases ; genetics ; Plasmids ; genetics ; Protein Engineering ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Solubility

Objective To investigate and analyze the clinical efficacy of cefoperazone and Shubatan in the treatment of severe infection in emergency department. Methods 100 cases of severe infection were randomly divided into control group and experimental group, with 50 patients in each group. The control group received routine treatment, and the experimental group was treated with cefoperazone and Shubatan on this basis. Pay close attention to the related vital signs change of the experimental group and the control group, the relevant clinical indicators of the two groups of patients were compared and analyzed. Results After the corresponding treatment, the bacteria removal rate of the experimental group was 90.00%, significantly higher thant 60.00 % of the control group (P<0.05). The effective rate of the experimental group was 90.00%, which was significantly higher than that of the control group (74.00%), which was statistically significant (P<0.05). There were no obvious adverse reactions in the two groups. After treatment, the total score of quality of life in the experimental group was (75.2±11.2) points, and the total score of QOL in the control group was (64.2±9.3) points, which was statistically significant (P<0.05). Conclusion The clinical efficacy of cefoperazone and Shubatan in the treatment of emergency severe infection is satisfactory. It can significantly improve the clinical symptoms and improve the quality of life in patients with severe acute respiratory infection.