In comparison with their in vivo counterparts, the in vitro produced mammalian embryos had markedly lower rates of morula/blastocyst development and pregnancy after transfer to the recipients. Things became even worse in the cloned embryos. This necessitates improvement of the embryo culture system. Co-culture of embryos with different types of somatic cells was found beneficial for embryo development in vitro and many studies have been conducted in this area in recent years. In this paper, recent developments and the authors' own work in studies of co-culture of early mammalian embryos with somatic cells were reviewed, with emphasis on the effects of cell type, stage of estrous cycle and number of passages of somatic cells and supplement of serum on embryo development, and the mechanisms by which co-culture promote embryo development. The recent developments are summarized as follows: 1. Somatic cells of both homogeneous and heterogeneous origins can be used for co-culture of mammalian embryos, with similar developmental rates. 2. Supplementation of animal serum at appropriate concentrations improved the somatic cell growth and consequently the development of embryos in co-culture. 3. The estrous cycle stages of oviduct epithelial cells used for co-culture had no effect on the development of embryos. 4. Over-passaging of somatic cells reduced their efficiency in promoting development of the co-cultured embryos. In conclusion, studies have shown that co-culture overcame the block of embryo development in vitro and improved embryo quality with increased rates of implantation and pregnancy, but many problems remain to be solved on its influencing factors and mechanisms of action.
Animals ; Coculture Techniques ; methods ; Embryo Culture Techniques ; methods ; Embryo, Mammalian ; physiology ; Humans

The effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation (COH) implantation dysfunction were studied.The COH implantation dysfunction model in mice was established by intraperitoneal injection of 7.5 IU pregnant mare's serum gonadotrophin (PMSG),followed by 7.5 IU human chorionic gonadotrophin (HCG) 48 h later.Then the female mice were mated with male at a ratio of 2:l in the same cage at 6:00 p.m.The female mice from normal group were injected intraperitoneally with normal saline and mated at the corresponding time.Day 1 of pregnancy was recorded by examining its vaginal smears at 8:00 a.m.of the next day.Fifty successfully pregnant mice were equally randomly divided into 5 groups:normal control pregnant group (NC),COH implantation dysfunction model group (COH),low dosage of Bu-Shen-An-Tai recipe group (LOW),middle dosage of Bu-Shen-An-Tai recipe group (MID) and high dosage of Bu-Shen-An-Tai recipe group (HIGH).Then from day 1,the mice in different groups were respectively intragastrically given corresponding treatments at 9:00 a.m.for 5 consecutive days.The concentrations of 17β-estradiol (E2) and progesterone (P4) were determined by radioimmunoassay (RIA).The ultrastructural changes of ovarian tissues were observed by transmission electron microscope (TEM).The histopathological changes of ovarian tissues were observed by HE staining.The number of atretic follicles and pregnant corpus luteum were also recorded.TUNEL was applied to measure apoptotic cells of ovarian tissues.Western blotting was used to detect the protein expression of apoptosis-related factors like Bax,Bcl-2 and cleaved-caspase-3 in ovarian tissue of mice.The results showed that ovarian weight,the concentrations of E2 and P4,the number of atretic follicles and pregnant corpus luteum,as well as the apoptosis of granulosa cells were significantly increased in the COH group.The ultrastructures of ovarian tissues in the COH group showed that chromatin in granulosa cells was increased,agglutinated,aggregated or crescent-shaped.The focal cavitation and the typical apoptotic bodies could be seen in granulosa cells in the late stage of apoptosis.After the treatment with different doses of Bu-Shen-An-Tai recipe,the ultrastructural changes of ovarian granulosa cells apoptosis were dramatically improved and even disappeared under TEM.Visible mitochondria and mitochondrial cristae were increased and vacuoles were significantly reduced.The lipid dropltes were shown in a circluar or oval shape.The protein expression levels of Bax and cleaved-caspase-3 were decreased,and the expression of Bcl-2 protein was increased after treatment.It was concluded that Bu-Shen-An-Tai recipe can inhibit the apoptosis of ovarian granulosa cells,probably by up-regulating the protein expression of Bcl-2 and down-regulating Bax and cleaved-caspase-3,which contributes to the formation and maintenance of ovarian corpus luteum.It's helpful to promote the embryonic implantation,to reduce embryo loss and ultimately to improve the success rate of pregnancy.

OBJECTIVETo understand the genotypic mapping of Mycobacterium leprae identified in China and to compare with those from other countries to select suitable alleles for epidemiological investigation in the transmission chain of leprosy.

METHODSVarious number of tandem repeat(VNTR) in genomic DNA of Mycobacterium leprae was used in the present genotyping study. 33 skin biopsies from Wenshan prefecture,Yunnan province and 17 from other parts of China were studied. DNA extracted from skin biopsies of leprosy patients was subjected to PCR followed by agarose gel analysis and DNA sequencing to determine the number of repeats.

RESULTSLoci GGT-5,12-5,21-3 and 23-3 were as highly homogenous as 100%; The homogeneity of loci AC-8, 18-8, 27-5 and rpoT were 97%, 94%, 97% and 85% respectively. Loci GTA-9, AC-9 and 6-7 showed significant allelic diversity in isolates and the diversity of GTA-9 in Mycobacterium leprae isolated from China was also different from those identified other countries. We had subjected loci GTA-9 and the ten loci to phylogenetic tree analysis respectively.

CONCLUSIONThe present study revealed that the genotype of Mycobacterium leprae identified from China was close to the strains from the Philippines and India although a few loci were somehow differentiate. Locus 12-5 manifested as only 3 copies in China whereas 4-5 copies predominating in other countries. 12-5 locus might serve as a useful marker to diffrentiate Chinese strains from those in other countries. However, further study on the diversity of GTA-9 was needed in China. The molecular typing of Mycobacterium leprae from different geographic areas might be useful in studying the transmission of leprosy.

Alleles ; China ; epidemiology ; DNA, Bacterial ; Genotype ; Humans ; Leprosy ; epidemiology ; transmission ; Molecular Epidemiology ; Mycobacterium leprae ; genetics ; isolation & purification ; Polymerase Chain Reaction ; Skin ; microbiology

OBJECTIVETo analyze the changes of carotid-femoral and carotid-radial pulse wave velocity in patients with essential hypertension, and effects of age and levels blood pressure levels on these changes.

METHODSAutomatic pulse wave velocity (PWV) measuring system was applied to examine carotid-femoral PWV (CFPWV) and carotid-radial PWV (CRPWV) as the parameters reflecting central elastic large arterial and peripheral muscular medium-sized arterial elasticity respectively. 517 hypertensive patients aged 17-82 years (272 males and 245 females, mean age 52.0 +/- 13.0 years) and 118 healthy subjects aged 19-82 years (52 males and 66 females, mean age 54.2 +/- 13.8 years) were recruited in the study.

RESULTSCFPWV increased with age in both groups (P < 0.001), whereas CRPWV did not change significantly. CFPWV and CRPWV both increased with blood pressure. A stepwise multiple regression analysis demonstrated that age and systolic blood pressure were positively related to CFPWV independently, while diastolic blood pressure was significantly associated with CRPWV.

CONCLUSIONSAge and components of blood pressure exert different effects on elasticity of central elastic large artery and peripheral muscular medium-sized artery. Examining the change of large arterial elasticity has important clinical value in hypertensive patients.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Blood Flow Velocity ; Blood Pressure ; Carotid Arteries ; physiopathology ; Case-Control Studies ; Elasticity ; Female ; Femoral Artery ; physiopathology ; Heart Rate ; Humans ; Hypertension ; physiopathology ; Male ; Middle Aged ; Pulse ; Radial Artery ; physiopathology ; Young Adult

OBJECTIVETo study the role of contrast-enhanced ultrasound (CEUS) in the management of splenic trauma

METHODSForty patients with splenic trauma underwent CEUS examination and then different management were provided according to the examination results and the general status of patients: conservative treatment (group I, n = 19); CEUS-guided injective therapy (group II, n = 6); and surgery (group III, n = 15).

RESULTSEighteen patients were cured in group I and one patient experienced rehaemorrhagia. The haemostatic effect of CEUS-guided injective therapy was obvious in all six patients in group II. Among them, one patient experienced arteriovenous fistula, which was resolved after one week of injective therapy. Fifteen patients in Group III underwent surgery and were cured.

CONCLUSIONCEUS can provide reliable information for therapy mode selection in patients with splenic trauma and can be used to guide injective therapy.

Contrast Media ; Humans ; Spleen ; diagnostic imaging ; injuries ; Ultrasonography ; Wounds, Nonpenetrating ; diagnostic imaging ; therapy

OBJECTIVETo investigate the clinical features of invasive pulmonary fungal infections (IPFIs) after biliary atresia (BA) surgery and related risk factors.

METHODSA retrospective analysis was performed for the clinical data of 49 children with IPFIs after BA surgery, including clinical features, lung imaging findings, and pathogenic features. The risk factors for IPFIs after BA surgery were also analyzed.

RESULTSThe most common pathogens of IPFIs after BA surgery was Candida albicans (17 strains, 45%), followed by Candida tropicalis (7 strains, 18%), Aspergillus (6 strains, 16%), Candida krusei (3 strains, 8%), Candida glabrata (3 strains, 8%), and Candida parapsilosis (2 strains, 5%). Major clinical manifestations included pyrexia, cough, and shortness of breath, as well as dyspnea in severe cases; the incidence rate of shortness of breath reached 78%, and 35% of all children had no obvious rale. The multivariate logistic regression analysis showed that age at the time of surgery, time of glucocorticoid application, cumulative time of the application of broad-spectrum antibiotics, and recurrent cholangitis were major risk factors for IPFIs after BA surgery.

CONCLUSIONSThe three most common pathogens of IPFIs after BA surgery are Candida albicans, Candida tropicalis, and Aspergillus. It is important to perform surgery as early as possible, avoid recurrent cholangitis, and shorten the course of the treatment with broad-spectrum antibiotics and glucocorticoids for decreasing the risk of IPFIs.

OBJECTIVE: To investigate the effect of placement of peripherally inserted central catheter (PICC) via the upper versus lower extremity veins in neonates through a Meta analysis. METHODS: CNKI, Wanfang Data, VIP Data, CBMdisc, PubMed, Web of Knowledge, Embase, Medline, Cochrane Library and Google Scholar were searched for control studies on the effect of PICC placement via the upper versus lower extremity veins in neonates. RevMan 5.3 was used to perform a Meta analysis of the studies which met the inclusion criteria. RESULTS: A total of 18 studies were included, among which there were 8 randomized controlled trials and 10 cohort studies, with 4 890 subjects in total. Compared with those undergoing PICC placement via the upper extremity veins, the neonates undergoing PICC placement via the lower extremity veins had significantly lower incidence rates of complications (RR=0.83, 95%CI: 0.75-0.92, P<0.05), catheter-related infections (RR=0.77, 95%CI: 0.60-0.99, P<0.05), catheter malposition (RR=0.28, 95%CI: 0.18-0.42, P<0.05), extravasation of the infusate (RR=0.52, 95%CI: 0.40-0.70, P<0.05), and unplanned extubation (RR=0.82, 95%CI: 0.69-0.98, P<0.05). They also had a significantly higher first-attempt success rate of puncture (RR=1.17, 95%CI: 1.05-1.30, P<0.05) and a significantly shorter PICC indwelling time (MD=-0.93, 95%CI: -1.26-0.60, P<0.05). CONCLUSIONS The above evidence shows that PICC placement via the lower extremity veins has a better effect than PICC placement via the upper extremity veins in neonates.
Catheterization, Central Venous ; Catheterization, Peripheral ; Cohort Studies ; Humans ; Infant, Newborn ; Lower Extremity ; Retrospective Studies

Systematical studies are lacking on the influencing factors and mechanisms of the heparin enhanced sperm capacitation, although many studies have shown that heparin enhanced sperm capacitation. The effect of heparin concentration and exposure time, incubation temperature and co-culture with oviductal epithelial cells or cumulus cells on goat sperm capacitation were investigated in this study. The motility, membrane and acrosome integrity and capacitated percentage of goat spermatozoa were assessed after different heparin treatments, and rates of fertilization and embryo cleavage were compared after in vitro insemination of oocytes with spermatozoa capacitated by different heparin treatments. The major results are summarized as follows: 1) When spermatozoa were capacitated with heparin at 5, 10, 25, 50 and 100 microg/mL for 45 min, 50 and 100 microg/mL heparin treatments produced the highest capacitated percentages of 55% and 56%, respectively, but the percentage of spermatozoa with intact acrosomes in the 100 microg/mL heparin treatment decreased significantly (P < 0.05) in comparison with that in the control group, indicating that the optimal heparin concentration for goat sperm capacitation would be 50 microg/mL. 2) Capacitated percentage of spermatozoa increased with extension of treatment time when goat sperm were treated with 50 microg/mL heparin for 0, 10, 20, 30, 45, 60 or 120 min. Although heparin treatments for 45 to 120 min did not differ significantly (P > 0.05) in capacitated sperm percentages, sperm motility and membrane integrity decreased significantly when treated with heparin for 120 min. This suggested that the optimal exposure time of heparin at 50 microg/mL for goat sperm capacitation would be 45 to 60 min. 3) Significantly higher capacitated percentages of spermatozoa were obtained when goat sperm were treated at 42 and 38.5 degrees C than at 15 and 37 degrees C, but sperm motility and acrosome integrity were significantly lower when spermatozoa were treated at 42 degrees C than they were treated at other temperatures. Temperature of 38.5 degrees C would, therefore, be the optimal temperature for goat sperm capacitation. 4) The capacitated percentage of spermatozoa was significantly higher when goat sperm were co-cultured with oviductal epithelial cells than when treated with heparin alone or co-cultured with cumulus cells, but sperm motility and membrane and acrosome integrity did not differ significantly among the three treatments. Rates of fertilization (91.3%) and cleavage (72.2%) were significantly higher in the oviductal epithelial cell co-culture group than those in the heparin alone group. This indicated that co-culture with oviductal epithelial cells significantly enhanced goat sperm capacitation by heparin treatment.
Acrosome Reaction ; drug effects ; physiology ; Animals ; Coculture Techniques ; Epithelial Cells ; cytology ; Fallopian Tubes ; cytology ; Female ; Fertilization in Vitro ; Goats ; Heparin ; pharmacology ; Male ; Sperm Capacitation ; drug effects ; physiology ; Sperm Motility ; Spermatozoa ; cytology ; physiology

To investigate the seasonal influenza split vaccine's immune protective effectiveness against the homologous and heterogonous subtypes of influenza A virus challenge and the relationship between the protective effectiveness and hemagglutination inhibition (HI) antibody titer in mice. Two components of H1N1 and H3N2 in Chinese 2008-2009 seasonal influenza spilt vaccine, were derived from vaccine strain A/Brisbane/59/2007 (H1N1)-like virus and A/Brisbane/10/2007 (H3N2)-like virus respectively, and were used to immune BALB/c mice. Firstly, different doses of the vaccines were used to immunize mice and the HA immunization dosage that can induce the HI antibody titer of 40 in mice was identified; Secondly, H1N1 vaccine immunized mice were challenged with different doses of influenza virus mouse adaptation strains of A/Brisbane/59/2007 (H1N1)-like virus (MA) (referred to as A1 virus, well matched-strain in the homologous subtype) and A/Purto Rico/8/34 (H1N1) (referred to as PR8 virus, poor matched-strain in the homologous subtype) respectively, and H3N2 vaccine immunized mice were challenged with H1N1 influenza virus of A1 strain (Heterogonous subtype), body weight changes and survival rates were observed to explore the immune protective effectiveness of influenza split vaccine against the homologous and heterogonous subtypes of influenza A virus in mice. Results indicated that HI antibody titers were elevated as the HA protein immunization dosages increased from 0.15 microg, 0.5 microg, 1.5 microg, 5 microg to 15 microg in mice, and 1.5 microg HA of the seasonal influenza split vaccine could induced HI antibody titer of 40 in mice; 3LD50, 10LD50, 30LD50, 100LD50, 300LD50,1000LD50 and 3000LD50 of influenza virus strain A1 were used to challenge the H1N1 immunization mice, 1.5 microg HA of H1N1 vaccine could 100% protect mice against challenge with 1000LD50 of matched and homologous subtype of influenza virus strains A1, mice immunized with 15 microg HA of H1N1 vaccine even could 100% protect mice against challenge with 3000LD50 of influenza virus strains A1; but mice immunized with both the 1.5 microg and 15 microg HA of H1N1 vaccine were all sacrificed when challenged with 3LD50 of the mismatched and homologous subtype of influenza virus strain PR8, and mice immunized with the high dosage of 15 microg HA of H3N2 vaccine also were all sacrificed when challenged with 3LD50 of the heterogonous subtype of influenza virus strain A1. These results suggest that 1.5 microg HA of seasonal influenza split vaccine could induced HI antibody titer of 40 after one dose in mice, this dosage of HA can effectively protect mice against matched homologous subtype of influenza virus strain, but hardly to protect mice against mismatched homologous or heterogonous subtype of influenza virus strain. These results provide materials for the establishment of influenza vaccine evaluation system based on seasonal influenza vaccine.
Animals ; Antibodies, Viral ; blood ; Cells, Cultured ; Chick Embryo ; Dogs ; Female ; Influenza A Virus, H1N1 Subtype ; immunology ; Influenza Vaccines ; immunology ; Mice ; Mice, Inbred BALB C ; Orthomyxoviridae Infections ; prevention & control ; Vaccination

To efficiently express nucleoprotein (NP) of influenza A virus A/Jingke/30/95 (H3N2) in E. coli for further immunogenicity study, three forms of NP gene, NP(His) (NP fused with 6 x His tag), NPwt (wild type NP, non-fused NP with native codon) and NP(O) (codon optimized, non-fused NP) were cloned by the technologies of restriction enzyme digestion, PCR, codon optimization and gene synthesis. Three recombinant plasmids were subsequently constructed based on the prokaryotic vector pET-30a, respectively. The comparative studies with these plasmids were carried out on the gene expression efficiency, induction temperature and time, purification process and immune reactivity. It was confirmed by restriction enzyme digestion and sequencing analysis that the three NP genes were inserted into the expression plasmid pET-30a correctly. SDS-PAGE showed that all three forms of NP gene could be efficiently ex pressed in E. coli, among which NP(O) was expressed with the highest expression level. The lower temperature fermentation (T=25 degrees C) and longer time induction (t=10 h) were necessary for high-level expression of protein in soluble form. The purity of tag-free NP was up to 90% through the two-step purification process with anion-exchange and gel filtration chromatography. It was indicated by Western blot that purified NP reacted well with the serum from mice immunized with PR8 virus. These results suggest that the codon-optimized influenza A virus NP gene can be efficiently expressed in E. coli and the expressed NP protein with specific immune reactivity could be purified from the supernatant of bacterial lysate.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; RNA-Binding Proteins ; chemistry ; genetics ; isolation & purification ; metabolism ; Solubility ; Viral Core Proteins ; chemistry ; genetics ; isolation & purification ; metabolism