OBJECTIVETo explore various factors affecting the clinical pregnancy outcomes of artificial insemination with donor sperm (AID).

METHODSWe retrospectively analyzed 15,744 cycles of AID in 6302 women and investigated the association of the clinical pregnancy outcomes of AID with the treatment protocols, the times of insemination per cycle, the age of the infertile women, the status of the oviduct, and the number of AID cycles.

RESULTSThe pregnancy rate of AID was higher in the chlomiphene-treated women than in those of the natural cycle group (P = 0.003) but showed no significant differences either between the chloramiphene and human menopause gonadotropin (HMG) or between the HMG and natural cycle groups (P > 0.05), and so was it in the women that had received AID twice per cycle before and after ovulation (26.3%) than in those that had undergone only once before (7.0%) or after ovulation (23.7%) (P < 0.05). However, the pregnancy rate was remarkably lower in the women aged 35-40 years (16.5%), especially in those over 40 years (1.2%), than in those under 35 years (26.0%) (P < 0.05). There was no significant difference in the success rate of AID between the women with oviductal adhesion and those without (27.4% vs. 28.1%, P > 0.05). The pregnancy rate of the first cycle of AID (27.6%) was markedly higher than those of the second (24.7%), third (23.9%), and fourth (23.1%) (P < 0.01), but with no significant differences among the latter three cycles (P > 0.05), while that of the fifth cycle (19.0%) was remarkably lower than those of the first four (P < 0.01).

CONCLUSIONThe age of the infertile women is an important factor affecting the success rate of AID. AID twice per cycle is better than once only. For those without oviductal factors, at least 4 cycles of AID are required before in vitro fertilization.

Adult ; Age Factors ; Female ; Fertilization in Vitro ; Humans ; Infertility, Female ; Insemination, Artificial ; Insemination, Artificial, Heterologous ; Ovulation ; Ovulation Induction ; Pregnancy ; Pregnancy Outcome ; Pregnancy Rate ; Retrospective Studies

Pseudorabies virus (PRV) is a swine herpesvirus of the Alphaherpesvirinae subfamily and a pathogen of swine resulting in devastating disease and economic losses worldwide. Cre/loxP site-specific system has the character of site specific, time specific, tissue specific and high efficiency in recombination, which makes this system universal in vivo and in vitro recombination of bacteria, fungus, plants, insects and mammals. A recombinant PRV which contain a loxP site in TK locus by using Cre/LoxP recombinant system was construsted. A pair of primers were synthesized according to the pEGFP-C1 sequence published on GenBank, and were used to amplify the EGFP gene expression cassette with two loxP sites flanking each side. This target gene was cloned into pSKLR, the resulting transfer vector pSKLR-GFP-loxP was then cotransfected into 293T cells with PRV SH strain genomic DNA. The recombinant virus rPRV1 was selected and purified in TK-143 cells by choosing fluorescent expressing plaques. Cre expression vector pOG231 was cotransfected into 293T cells with rPRV1 genomic DNA. The second recombinant virus rPRV2 was obtained, which contains only one loxP site in TK locus. Sequencing results of rPRV2 TK gene indicated that 34bp loxP site was inserted into rPRV2 genome and there were 270bp deletion in TK gene. PCR amplifying different generations of rPRV2 TK gene showed that the mutant was stable when passages in RK-13 cells. TCID_ 50 assay indicated that rPRV2 grows well on RK-13 cells. The LD_ 50 test results on BALB/C mice suggested that the virulence of rPRV2 was reduced. As a conclusion, the report gene GFP expression cassette was removed successfully from rPRV1 genome and only one LoxP site was leaved in rPRV2 genome by using Cre/LoxP recombinant system.

Objective To investigate the meaning of expression of apoptosis related molecules NFKBp65 and p53 and GPX1-mRNA in patients with Keshan disease(KSD).Methods Sixteen chronic Keshan Disease patients were enrolled in KSD group according to electrocardiogram,chest X ray film and clinical examinations on 15,September in 2009,and 23 healthy people were included in control group from physical examination taken in The Second Affiliated Hospital of Xi'an Jiaotong University.Fresh blood(5 ml)was collected from antecubital vein of all subjects in the fasting state.Total mRNA and protein of blood sample were isolated using Trizol.GPX Assay Kit was used to detect GPX enzyme activity,and GPX1-mRNA expression was determined by SYBR Real-Time PCR.Meanwhile,expression of apoptosis related molecules NFKBp65 and p53 were determined by Western blot.Results GPX enzyme activity decreased significantly in KSD group[(108.61±14.10)U]compared with control group[(122.78±11.89)U,t=2.874,P<0.05],GPX1-mRNA level of KSD group(0.553±0.299)notably KSD group(0.802±0.057)compared with control group[(1.065±0.355),t=6.829,P<0.01].p53 increased in KSD group(1.604±0.191)compared with control group[(1.137±0.186),t=3.033,P<0.05].Conclusiom Decreased GPX1-mRNA expression may result in lower GPX enzyme activity of patients with KSD.Thus oxidative damage increases and cadioeyte apoptosis is activated by activating apoptosis signal pathway.

OBJECTIVETo study the effects of Angelica sinensis Polysaccharides (ASP) on the hepatic drug metabolism enzymes activities in normal mice and those prednisolone (PSL)-induced liver injury.

METHODThe activities of phase II enzymes (GSH-related enzymes) and cytochrome P450 enzymes were measured by biochemical method.

RESULTASP increased the activities of glutathione S-transferase in liver microsomes and mitochondria. The cytochrome P450 content, NADPH-cytochrome c reductase, aminopyrine N-demethylase, and aniline hydroxylase activities in liver microsomes were also increased. PSL significantly increased serum ALT levels, and decreased the liver mitochondrial glutathione content. At the same time, other enzymes activities were all increased. When mice were treated with ASP 2.0 g.kg-1, the PSL-induced changes on cytochrome P450 enzymes, glutathione S-transferase, and GSH content were restored.

CONCLUSIONASP can modulate the activities of drug metabolism enzymes.

Aminopyrine N-Demethylase ; metabolism ; Angelica sinensis ; chemistry ; Aniline Hydroxylase ; metabolism ; Animals ; Chemical and Drug Induced Liver Injury ; enzymology ; etiology ; Cytochrome P-450 Enzyme System ; metabolism ; Glutathione Transferase ; metabolism ; Male ; Mice ; Microsomes, Liver ; enzymology ; Mitochondria, Liver ; enzymology ; NADPH-Ferrihemoprotein Reductase ; metabolism ; Plants, Medicinal ; chemistry ; Polysaccharides ; isolation & purification ; pharmacology ; Prednisolone

To investigate a baculovirus insect cell system for expressing an interferon alpha 2b (IFNa2b)/immunoglobulin G-4 (IgG4) Fc fusion protein, which has long-acting antiviral effects. Human IFNa2b and IgG4 Fc cDNAs were generated by molecular cloning and inserted into a baculovirus shuttle vector, which was then transposed into the DH10 Bac strain to form recombinant Bacmid-IFN/Fc. The Bacmid-IFN/Fc was transfected into High five insect cells, and expression of the IFN/Fc fusion protein was detected by Western blotting and its biological activity was assessed by the cytopathic effect inhibition method. The IFNa2b and IgG4 Fc cDNA fragments were successfully amplified by RT-PCR using human peripheral lymphocytes. After cloning into the baculovirus shuttle vector, pFastBac1, and transforming into DH10 Bac competent cells, screening identified positive clones carrying the recombinant Bacmid-IFN/Fc. A Bacmid-IFN/Fc clone was successfully transfected into the High five insect cells and packaged into the baculovirus for expression of the IFN/Fc fusion protein. Western blotting revealed that the fusion protein expression was specific, and yielded a protein of 45 kD in size. The in vitro antiviral activity of the IFN/Fc fusion protein was 580 IU/mL. A novel IFN/Fc fusion protein was successfully generated using a baculovirus insect cell system, which may prove useful for providing future experimental data for development of a new long-acting interferon to treat chronic viral hepatitis.
Animals ; Antiviral Agents ; metabolism ; Baculoviridae ; genetics ; Cell Line ; Cloning, Molecular ; Gene Expression ; Gene Fusion ; Genetic Vectors ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunoglobulin G ; biosynthesis ; genetics ; Insecta ; Interferon-alpha ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

Light intensity, gas temperature, soil temperature and gas exchange parameters were determined of three years old Panax notoginseng planted on different layers seedbed and different location (left, middle, right) of the same layer in greenhouse. Result show that diurnal variation of light intensity, gas temperature and soil temperature showed that upper layer > middle layer > lower layer; different locations of the same layer showed that light intensity of upper layer was not different among different locations; light intensity of middle and lower layer in right and left were the same, and significantly higher than those in the middle position; the gas temperature of each layer all with less different of each location; soil temperature of 12 cm depth is the lowest, and was gradually increased to the upper and lower surface; net photosynthetic efficiency of P. notoginseng showed that upper layer > middle layer > lower layer; there were significant correlation between soil temperature, stomatal conductance, intercellular CO2 concentration and photosynthetic rate were correlated with light intensity significantly; transpiration rates had notable correlation with light intensity and gas temperature. All above indicated that net photosynthesis rate of P. notoginseng was affected by light intensity directly, gas temperature and soil temperature indirectly. Inconclusion, stereoscopic cultivation of P. notoginseng was practicable in present study. The planting quality of P. notoginseng under stereoscopic cultivation could be improved by ameliorate the structure of seedbed to enhance the light intensity of middle and lower layer. Increase the thickness of the seedbed to decrease the temperature difference of soil. Further the management of ventilation facilities of greenhouse to control the gas temperature.
Carbon Dioxide ; metabolism ; Light ; Panax notoginseng ; growth & development ; metabolism ; Photosynthesis ; Soil ; Temperature

OBJECTIVEAs our previous comparative proteomics study on high and low metastasis human hepatocellular carcinoma (HCC) cell strains revealed that cytokeratin 19 (CK19) was related to higher metastasis potential, we further investigated the relationship between serum CK19 level in HCC patients and their clinico-pathologic characteristics.

METHODSSerum CK19 levels of 101 normal controls and 108 pathology-proven HCC patients were determined using radioimmunoassay, and the their correlation with clinico-pathologic parameters were studied.

RESULTSThe upper limit of one-side 98% confidence interval of normal serum CK19 level was 2.3 microg/L. Among 108 HCC patients, 24 (22.2%) had increased serum CK19 level, ranging from 2.4 to 45.5 microg/L. There were 12 patients (11.1%) with increased CK19 level but normal AFP level. The percentage of poor differentiated tumor was higher in CK19 increased cases (37.5%, 9/24) than in CK19 normal cases (20.2%, 17/84). Moreover, the presence of portal vein tumor emboli was significantly higher in CK19 increased cases (25.0%, 6/24) than in CK19 normal cases (6.0%, 5/84). (Chi-square = 7.403, P < 0.01) In addition, the percentage of TNM stage III/IV tumor was significantly higher in CK19 increased patients (54.2%, 13/24) than in CK19 normal cases. (chi-square = 13.300, P < 0.005)

CONCLUSIONSome HCC patients do have increased serum CK19 level, which could be related to portal vein tumor emboli, poor tumor differentiation and advanced tumor stages.

Adult ; Aged ; Biomarkers, Tumor ; blood ; Carcinoma, Hepatocellular ; blood ; pathology ; Female ; Humans ; Keratins ; blood ; Liver Neoplasms ; blood ; pathology ; Male ; Middle Aged ; Neoplasm Proteins ; blood ; Peptide Fragments ; blood ; genetics ; Proteome ; analysis

OBJECTIVETo investigate the effect of enhanced nutritional therapy on wound healing after endoscopic therapy in patients with liver cirrhosis and esophageal varices.

METHODSFifty patients with liver cirrhosis and esophageal varices were randomly divided into an enhanced nutritional therapy group (n = 25) and a control group (n = 25). The enhanced nutritional therapy group received one week of enhanced nutritional supplementation, including liver nutritional elements, prior to routine endoscopic therapy. The routine without any change to their diet. The rate of transformation and status of wound healing of esophageal varices were compared between the two groups.

RESULTSThe ratio of ulcers occurring at the injection site was lower in the enhanced nutrition group than in the control group (16/25 vs. 23/25; x2 = 5.711, P = 0.017). The enhanced nutrition group had only one case of minimal bleeding occurring during endoscopy as compared to the seven cases of bleeding in the control group (x2 = 5.357, P = 0.021). On average, the enhanced nutrition group required less sessions of endoscopic treatment to achieve eradication of esophageal varices than the control group (3.8 vs. 4.1; t = 2.069, P = 0.044).

CONCLUSIONPre-endoscopic enhanced nutritional therapy may benefit patients with liver cirrhosis and esophageal varices by promoting recovery of procedure-related local tissue injury and occlusion of varices.

Adult ; Endoscopy ; Esophageal and Gastric Varices ; etiology ; therapy ; Female ; Humans ; Liver Cirrhosis ; complications ; therapy ; Male ; Middle Aged ; Nutritional Support ; Wound Healing

Hairy root clones of Saussurea involucrata transformed with Agrobacterium rhizogenes strains R1601, R1000, and LBA9402 were established to investigate the flavonoid production. Opine synthesis and PCR analysis confirmed the integration of the T-DNA fragment of Ri plasmid from A. rhizogenes strain R1601 into the transformed root genome. The frequency of hairy root formation from root segments, which were pre-cultured 2 days in N6 solid medium without plant growth regulators, amounted to 100% following infection with R1601 strain of A. rhizogenes. The transformed roots were kept in hormone-free N6 liquid medium in the dark at 25 degrees C, 110r/min and routinely subcultured every 20 - 24 days. One hairy root clone, which grew vigorously with lateral branches, was periodically examined for the ability to produce flavonoid. The maximum of biomass and flavonoid yield achieved 66.7 g/L (fresh weight) and 102.3mg/g dry weight after incubation 20 days. The calli were induced from the hairy root culture in the presence of 0.5mg/L IBA and intact plantlets were regenerated from these calli. The regeneration plantlets from hairy roots, in which the flavonoid content were 53% in that of untransformed plants, weren't different in growth and morphology of the untransformed plantlets. Therefore plant regeneration from hairy roots may be also a means for producing transformed S. involucrata plants. Hairy root cultures of S. involucrata clearly showed higher flavonoid contents compared to the wild plant or the regeneration seedlings. As the wild S. involucrata grows only in special regions with peculiar climate, and cultivation of this species in a normal climate has been unsuccessful so far. The success in obtaining a method for high production of flavonoid might very well be one of the solutions for this problem in the future.
Culture Techniques ; Flavonoids ; biosynthesis ; Plant Roots ; growth & development ; Rhizobium ; physiology ; Saussurea ; growth & development

OBJECTIVETo study the dynamic effects of allogenic transplantations with the bladder acellular matrix grafts (BAMG) of rabbits.

METHODSHemi-cystectomies were performed in 25 rabbits, and the defects were repaired with BAMG about half bladder size. The rabbits underwent postoperative assessment of bladder function at 8 weeks, including cystometry, vesical volume, vesical compliance and cystography. The allografts were observed by light microscope and electron microscope at 1, 2, 4, 8, 12, 16 weeks after surgery.

RESULTSMacroscopic observation revealed that BAMG regenerated gradually. All urodynamic results of 8 weeks after surgery were not different statistically as compared with these of preoperation (P > 0.05). Cystography revealed that the morphous of bladder was recovered. Epithelialization and neovascularity occurred accompanied by infiltration of inflammatory cell at 1 week. Smooth muscle cell and stratified epithelium regenerated 2 weeks after grafting. Neural elements formed around smooth muscle bundles as early as 4 weeks. Each component regenerated on the frame of BAMG sequentially. After 16 weeks, it was difficult to delineate the junction between the host bladder and BAMG by histology.

CONCLUSIONAfter allogenic transplantation with rabbits' BAMG, the constitution and function of the allografts regenerate completely and gradually on the frame of BAMG.

Animals ; Extracellular Matrix ; transplantation ; Rabbits ; Regeneration ; Tissue Engineering ; Transplantation, Homologous ; Urinary Bladder ; cytology ; physiology ; transplantation