Objective To observe the occurrence of vascular calcification in maintenance hemedialysis (MHD) patients. Method Sixty-nine MHD patients were enrolled in this study. Vascular calcification was evaluated by plain X-my films of chest, upper and lower extremites. Analyzed the relationship of vascular calcification and cardiovascular disease. Results Vascular calcification on X-ray films presented in 38 cases (55%). Of the 23 cases with calcification on arteries of extremites, 14 cases were found to have both intimal calcification and medial calcification. Patients with vascular calcification were older. Ischemic heart disease and cardiac failure appeared more frequently in the patients with vascular calcification. Conclusions Both intimal calcification and medial calcification are frequently found in the MHD patients. Vascular calcification is probablly associated with cardiovascular disease in MHD patients.

Objective To explore the possible correlations under the interaction of multi-genes action at different stages and analyze the primary histophotomacrographic changes of hind buds tissue in congenital clubfoot and the pathodynamic developmental procedure.MethodsSeventy-seven female Wistar rats were administered with retinoic acid on the 10th day after pregnancy. And the hindlimb, buds and spinal cord were detected through transmission electron microscopic and molecular biological experiments.ResultsThere was clubfoot-like deformity in 61.8% of the experimental animals. Persistence of the embryonic position of the talus and tibia in fetuses was observed. Poor overlapping between talus and calcaneus was seen. Cell apoptosis at the anterior corner of spinal cord and hind buds were seen.ConclusionCongenital clubfoot deformities appear at early stage and exaggerate along with developmental procedure.

Objective To explore the effect of three-dimensional conformal radiation dose fractionation treatment of advanced non-small cell lung cancer(NSCLC).Methods 75 NSCLC patients were treated with hypofractionated 3DCRT( observation group),while 73 cases were treated with conventional fractionated radiotherapy (control group).The efficacy and adverse reactions were observed;Survival after treatment were followed up in 1,2,3 years.Lung function was detected before and after radiotherapy treatment,including FVC,FEV1 and CLCO.Results The total effective cases of control group were 47 cases patients ( 64.4％ ),observation group's was 60 cases ( 80.0％ ),total effective rate had statistically significant difference ( x2 =4.50,P ＜ 0.05 ).Survival of control group after treatment in 1,2,3year were 50.7％,24.7％,8.2％,the median survival was 13 months,observation group's were 73.3％,45.3％,20.0％,and 19 months.The survival of these two groups was statistically different (x2 =8.07,6,94,4.22,all P ＜ 0.05 ).The patients blood system side effects of observation group were significantly lower than the control group ( x2 =4.73,P ＜0.05 ) ;acute radiation pneumonia,esophagitis of these two groups had no significant difference in the incidence( P ＞ 0.05 ).Conclusion Hypofractionated 3DCRT treatment of advanced NSCLC had good effect,and its adverse reactions was low,and it was worthy of clinical application.

Colonic Diseases ; Hepatitis C, Chronic ; Humans ; Interferons ; Ischemia

Objective To investigate the association of serum leptin(LEP),adiponectin(ADPN)and leptin-to-adiponectin(L/A)ratio with cardio ankle vascular index(CAVI)in the patients maintaining hemodialysis(MHD).Methods Sixty MHD patients and thirty healthy volunteers were involved in the study.The levels of LEP and ADPN were determined by ELISA.Atherosclerosis was evaluated by the cardio ankle vascular index which was measured by using a VaSera VS-1000 machine.Results The levels of serum LEP and ADPN in hemodialysis patients were significantly higher than those in healthy controls([7.99 ± 1.21]μg/L vs.[4.18 ± 1.10]pg/L)and([15.12 ± 4.68]mg/L vs.[8.58 ± 2.23]mg/L),respectively,P ＜ 0.01].With the progression of atherosclerosis,the levels of serum LEP increased significantly,while the levels of serum ADPN decreased significantly.The L/A ratio was dramatically increased in patients with atherosclerosis.Multiple stepwise regression analysis showed that independent risk factors associated with CAVI include age,diabetes,levels of LEP and L/A ratio.Conclusion The increased serum LEP level and decreased serum ADPN level may be involved in the progression of atherosclerosis in MHD patients.The L/A ratio may be a powerful independentpredictor for CAVI in MHD patients.

Objective To detect the level of serum Klotho in chronic kidney disease (CKD) 3-5 stage patients and explore its relationship with the progress of renal function.Methods Twenty-nine patients with CKD 3-5 stage (CKD group) and 10 cases of age-matched non-CKD patients (control group) were enrolled in the study.The level of serum Klotho and fibroblast growth factor 23 (FGF23) were measured by enzyme-linked immunosorbent assay.Results The level of serum Klotho in CKD group was significantly lower than that in control group [(670.07 ± 146.22) ng/L vs.(1 125.50 ± 126.96) ng/L] (P ＜ 0.01).The level of serum FGF23 in CKD group was significantly higher than that in control group[(48.89 ± 44.28) ng/L vs.(10.48 ± 8.93) ng/L] (P ＜ 0.01).Pearson correlation analysis showed that the level of Klotho in CKD patients was positively correlated with estimated glomerular filtration rate (eGFR) and hemoglobin,and Klotho was negatively correlated with log urea nitrogen,log serum creatinine,log FGF23 and phosphate.Multiple regression analysis showed that log FGF23 and serum phosphorus were independent factors for serum Klotho level in patients with CKD.CKD patients were divided into rapid progression of renal function group (10 cases) and renal stability group (19 cases) according to annual rate of eGFR dechne.The patients in rapid progression of renal function group with lower serum Klotho [(580.27 ± 162.15) ng/L vs.(717.33 ± 115.22) ng/L],higher uric acid and more severe proteinuria [(448.00 ±65.21) mmol/L vs.(368.32 ±78.80) mmol/L,(1.99 ± 1.57) g/d vs.(0.60 ± 1.00) g/d] (P ＜0.05).Conclusions Serum Klotho level of CKD 3-5 stage patients decreases with the decline in renal function.Serum phosphate and FGF23 are the independent factors for serum Klotho.Serum Klotho may become the prediction index of renal function progress for patients with CKD.

Objective To reconstruct the proteinic sequence of human cathelicidin LL-37 to increase the bactericidal activity of LL-37 and to express the reconstructed LL-37 (rLL-37) in bacterium. Methods The two dimensional structure, three dimensional structure and chemical characteristic of LL-37 were analyzed by Soft Ware Anthepro 5.0 and SWISS-MODEL. Without the three dimensional changes of LL-37, some negative amino acids of human cathelicidin LL-37 were replaced by positive amino acids and the positive charge of LL-37 was increased. According to the proteinic sequence changes of rLL-37, the DNA sequence of rLL-37 was reconstructed by Touch-Down PCR and recombined with vector pET-28a (+), thus rLL-37 was expressed in E.coli. BL21 (DE3) by the induction of IPTG and was purified by chromatography. Results Glu~ 16 , Asp~ 26 , Glu~ 36 of LL-37 were replaced by Gln~ 16 , Asn~ 26 , Gln~ 36 and the static charge of LL-37 was increased from +5.8 to +9.0 at pH 7.4. The DNA sequence of rLL-37 was reconstructed and inserted into vector pET-28a (+), the rLL-37 was expressed in E.coli. BL21 (DE3) and purified by strong cation exchange supports Macro-Prep High S successfully. The rLL-37 was proved by the means of inhibitory zone to be able to kill Gram-negative bacteria and Gram-positive bacteria. Conclusion It is feasible to reconstruct human cathelicidin LL-37 and express the protein in bacteria by fusion, which make it possible to produce more rLL-37 and study its biological function deeply.

Objective To obtain and identify monoclonal antibody against human lipopolysaccharide binding protein (LBP). Methods Three clones of antibody with better activity against human LBP were isolated after human antibody library screening by human LBP and 5 rounds of enrichment. After plasmid extracting, geneⅢ cutting, self-recircling and electric transforming, soluble Fab was expressed in E.coli. XL1-blue by the induction of IPTG and its characteristics were determined. Results The positive antibody was concentrated to 8.16?104 folds and the highest activity of the three clones was 106 . Conclusion Phage antibody against human LBP has been obtained successfully, which lays a solid foundation for researching its function.

Objective To employ 2 approaches to construct and express reconstructed LL-37 (rLL-37) in procaryotic system, and to explore a better preparation method. Methods The first method: the rLL-37 was inserted into vector pET-28a (+), then was induced to express in E.coli. BL21 (DE3) and purified by chromatography; the second method: the rare codons in the rLL-37 gene sequence were substituted by the preferred codons of procaryotic cell, and a fragment of carrier protein molecule (CPM) was added to the N termination of the objective sequence to construct expression plasmid pET-30a(+)-CPM-rLL-37, then the rLL-37 was expressed in E.coli. BL21 Star(DE3) and purified by chromatography. The productive rates of the 2 methods were compared and the antimicrobial effects of obtained rLL-37 was studied. Results The first method: the DNA sequence of rLL-37 was obtained successively by Touch-Down PCR. The expression plasmid pET-30a(+)-CPM-rLL-37 was expressed with fusion protein in E.coli BL21 (DE3). The expression rate accounted for 20% of total bacterio-protein, then the expressed product was purified by using high positive ion exchange column Macro-Prep High S; The second method: a fragment of carrier protein molecule was designed that contained 28 amino-acid residue and its pHi was 2.7, net charge was-6.0 at pH 7.4. After the expression plasmid pET-30a(+)-CPM-rLL-37 was constructed successively, it was expressed in E.coli BL21 Star (DE3). The expressed fusion protein accounted for 35% of total bacterio-protein, then the expressed product was purified by using affinity binding chromatography with TALON resins successfully. The obtained 2 kinds of rLL-37 were able to kill both Gram-negative and-positive bacteria by the means of inhibitory zone. Conclusion It’s feasible to prepare efficiently rLL-37 in procaryotic system, which founds the basis for the further research on bactericidal activity of rLL-37.

Objective To introduce the mutated gene coding cysteine into the gene of dsFv VL of human antibody to N terminal fragment of lipopolysaccharide binding protein(LBP)and to express,purify the mutated dsFv VL in bacterium.Methods We reconstructed and sequenced the mutated gene of VL of human mAb Fab to LBP by Mega-primer PCR based on point mutagenesis method.Some codes of FWR1 of VL had been replaced by TGT in order to code cysteine.The DNA sequence of reconstructed VL was inserted into vector pET-28a(+),then VL was expressed by E.coli.BL21 star(DE3)and was purified by chromatography.Finally the activity of VL to bind NH-LBP was determined by ELISA.Results The results showed that the cysteine was introduced into the position 21 amino acid of VL to replace the threonine.The gene of VL was about 650 bp and relative molecular weight of VL was 28?103.VL could bind NH-LBP directly.Conclusion These have laid a foundation for producing the dsFv against NH-LBP.