Objective:To establish a method for the determination of related substances in erythromycin ophthalmic ointment. Methods:The extraction method was established and optimized. An HPLC gradient elution method was used for the determination of related substances in erythromycin ophthalmic ointment. Results: One step extraction with PBS (pH 7.0) - methanol(1: 1) had promising effect. After the method validation, it was proved that the method could be used to determine the related substances in eryth-romycin ophthalmic ointment. Conclusion:The method established in the paper provides a better analytical extraction and determina-tion method for the quality control of erythromycin ophthalmic ointment.

Objective To observe the change of serum ischemia modified albumin (IMA) in continuous ambulatory peritoneal dialysis (CAPD) patients.Methods Seventy-four end stage renal disease patients undergoing CAPD more than 3 months (CAPD group) were divided into 2 groups according to clinical symptoms of cardiovascular diseases:CAPD symptoms group (29 cases) and CAPD asymptomatic group (45 cases).Seventy healthy subjects were selected as control group.The serum IMA level and abnormal rate of electrocardiogram and heart color ultrasonic were examined and compared.Results The serum IMA level in CAPD group was significantly higher than that in control group [(92.33 ± 17.17) kU/L vs.(69.63 ± 9.24)kU/L,P＜ 0.01].The serum IMA level in CAPD symptoms group was significantly higher than that in CAPD asymptomatic group [(109.37 ± 21.34) kU/L vs.(85.31 ± 8.58) kU/L,P ＜ 0.05].The elevatory rate of serum IMA level and abnormal rate of electrocardiogram and heart color ultrasonic in CAPD symptoms group were significantly higher than those in C APD asymptomatic group [37.9％ (11/29) vs.15.6％(7/45),62.1％ (18/29) vs.22.2％ (10/45),P ＜0.05].Conclusions The serum IMA level in CAPD patients is elevatory.Serum IMA level is significantly higher in CAPD patients with cardiovascular disease clinical symptoms,it can be used for diagnosis of myocardial damage in CAPD patients.

Objective To analyze the correlation between serum antinucleosome antibody (AnuA) and renal pathological characteristic,disease activity as well as some laboratory tests in patients with lupus nephritis (LN).Methods Serum AnuA levels were detected by enzyme-linked immunosorbant assay in 40 patients with LN (observation group) and 40 healthy people (control group).Renal biopsy was examined in all LN patients.The relationships between serum AnuA level and systemic lupus erythematosus disease activity index (SLEDAI),renal pathohistology,laboratory parameters were analyzed.Results The serum AnuA level in observation group before treatment was significantly higher than that in control group [ ( 110.23 ± 80.48) kU/L vs. ( 10.45 ± 8.20) kU/L,P ＜ 0.05 ].Four cases of renal biopsies were class Ⅱ,8 cases were class m,23 cases were class Ⅳ,and 5 cases were class V.Serum AnuA level had significant difference between each class by Kruskal-Wallis rank sum test (P ＜ 0.05),and serum AnuA level of class Ⅳ was the highest (P ＜ 0.05).Serum AnuA level had positive correlation with SLEDAI,urine protein quantitation and anti-double strands DNA antibody (r =0.462,0.521,0.394,P ＜0.05),negative correlation with complement C3 and C4 levels (r =-0.403,-0.489,P ＜ 0.05 ).Serum AnuA level after treatment [ (32.45 ± 18.31) kU/L] was significantly decreased than that before treatment [(110.23 ± 80.48) kU/L](P＜0.05).Conclusions Serum AnuA level is not only a good index of LN activity,but also reflect renal involvement.That serial measurement of serum AnuA level may provide better clinical strategies for the therapy.

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.

Objective To investigate the clinical outcome of patients who underwent whole embryo vitrification freezing and thawed frozen embryo transplantation, and to compare it with the patients at the same period in the fresh cycle to explore the value of vitrification technology in the whole embryo freezing combined with recovery transplantation. Methods The whole embryo freezing group included 40 untransplanted cases of in-vitro fertilization (IVF), while another 300 patients in the fresh cycle with in vitro fertilization and embryo transfer (IVF-ET) at the same period served as the fresh cycle group. The average number of transferred embryos, high-quality embryos, endometrial thickness, estradiol (E2) level, and clinical pregnancy rate were compared.Results The average number of transferred embryos, high-quality embryos, difference in endometrial thickness were not significant between the 2 groups (P>0.05); the E2 level of the fresh cylcle group was significantly higher than that of the whole embryo freezing group (P<0.05), and the clinical pregnancy rate in the fresh cylcle group was significantly lower than that of the whole embryo freezing group (P<0.05). Conclusion Vitrification technology is a simple, reliable, and efficient embryo freezing technology. For patients who meet the requirements, whole embryo vitrfication freezing and thawed frozen embryo transplantation is feasible. It is worth for further clinical research.

OBJECTIVE To determine pathogens and drug resistance of lower respiratory tract infection in patients receiving mechanical ventilation in ICU and to provide the reference for clinical prevention and treatment.(METHODS) The ages,underlying diseases,pathogens distribution and resistance rate of Gram-negative bacteria of 110 cases patients receiving mechanical ventilation and being complicated lower respiratory tract infection in ICU were investigated.RESULTS Totally 110 strains of pathogens were isolated by bacterial culturing.The ratio of the Gram-negative bacteria to total bacteria isolated was 72.7%(80 strains) and of the Gram-positive bacteria was(18.2%)(19 strains),of the fungi was 10%(11 strains),among them the Pseudomonas aeruginosa contributed the majority,and ESBLs accounted for 38.8%,MRSA contributed 31.6% in Staphylococcus.Multidrug-(resistance) of Gram-negative bacteria to antimicrobial agents was serious.CONCLUSIONS Gram-negative bacteria are the majority of the pathogens,isolated from patients with lower respiratory tract infection in ICU receiving mechanical ventilation.It is suggested that there be urgent need for surveillance of bacterial resistance and rational use of antimicrobial(agents) during clinical therapy.

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 obtain the disulfide stabilized Fv fragment (dsFv) against N-terminal fragment of human lip polysaccharide binding protein (NH-LBP) and to identify its biological vitality. Methods The disulfide stabilized Fv fragment antibody (dsFv) was obtained after the inclusion bodies of dsFvVH and dsFvVL had been refolded and purified. Then the characteristics of dsFv were determined in vitro by ELISA and by detecting the secretion of tumor necrosis factor alpha (TNF-?) in rats. Results There was 2.1 mg protein of dsFv obtained. dsFv had good combination with NH-LBP and could restrain inflammatory reaction caused by lip polysaccharide (LPS) in vivo. Conclusion It is feasible to get dsFv against NH-LBP by respective expression of VL and VH. The partial inhibition of the biological function of LBP by dsFv is a new way to restrain the over-inflammatory reaction in vivo.