Objective To establish a rapid SNP( single-nucleotide polymorphism) genetic identification method for the frozen samples, such as frozen embryos and sperm of inbred mice.Methods In this study, the frozen embryos and sperm of inbred mice were provided by Shanghai Lab.Animal Research Center.Whole genome amplification and PCR-LDR genotyping system were used to get the rich DNA sample.Forty-five SNP were genotyped by multiple polymerase chain re-action and ligase detection reaction( PCR-LDR) .Results The electrophoresis results showed that the whole genome am-plification technique could highly increase the total DNA of frozen embryos.PCR-LDR typing method was suitable for the mouse genome typing of 45 SNPs.Ten strains of inbred frozen embryos and sperms of C57BL/6, BALB/c, FVB/NJ mice were genotyping identified, and their SNP loci data obtained by PCR-LDR were as the same as those of database.The num-ber of frozen mouse embryos was proportional to the number of SNPs detected, and when the embryo number reached more than 12, the detection rate of SNP was 100%.Conclusions This method can be used to the genetic quality identification, and rapidly identify the inbreed frozen mouse embryos and sperms.

Objective To investigate the role of p38MAPK in the differentiation of murine osteoblasts, and to observe the expressions of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG). Methods The calvarial osteoblasts of newborn BALB/c mice were cultured in MEM medium containing 10% NCS. Raloxifene (10~(-7) mol/L) and 17β-estradiol (10~(-8) mol/L) were added respectively when cells reached 70%-80% confluence combined with or without 5 μmol/L SB202190, an inhibitor of p38MAPK. The osteoblasts alkaline phosphatase activity assays were performed 72 hours later using PNPP method, and mRNA levels of alkaliphosphatase (ALP), OPG and RANKL were determined by RT-PCR. Results 17β-estradiol and raloxifene increased ALP activity and ALP mRNA level in osteoblasts in vitro which were blocked by p38MAPK inhibitor.The mRNA levels of RANKL and OPG were up-regulated by 17β-estradiol and raloxifene while the ratio of OPG/RANKL kept constant. SB202190 (5 μmol/L) inhibited the highly expressed RANKL and OPG in osteoblasts, and obviously decreased the ratio of OPG/RANKL. Conclusions p38MAPK inhibition blocked the differentiation of osteoblasts and decreased the up-regulated OPG and RANKL expressions in osteoblasts significantly (P<0.05).

Objective To observe the effect of Zhibo Dihuang Pill(ZDP) on leptin-induced idiopathic central precocious puberty(ICPP) in mice.Methods Eighteen BALB/c female mice aged 18 months were randomized into 6 groups: normal group,model group,high-and low-dose ZDP groups(at the dose of 101.4 and 50.7 g?kg-1?d-1 respectively),and megestrol acetate(3.9 mg?kg-1?d-1)group and gonadotropin releasing hormone analogues(GnRH-A) group(at the first dose of 1573.5 ?g/kg,the second and the third dose of 1180 ?g/kg,once every other day).Except the normal group,the mice in other groups received intraperitoneal injection of leptin 20mg/kg to establish ICPP model.After treatment,the vaginal opening,body weight,uterus weight and ovaries weight as well as the serum levels of luteinizing hormone(LH),estrogen(E2) were detected.Meanwhile,the uterus index and ovaries index were also examined.Results High-dose ZDP had an inhibition on the advance of vaginal opening induced by leptin after modeling for 4~6 days(P0.05).Conclusion The therapeutic effect of ZDP for ICPP is probably related with the inhibition of the advance of hypothalamic-pituitary-gonad axis activation.

Objective:To establish a liquid chromatography-mass spectrometry(LC-MS) method for determining the concentration of amlodipine besylate in human plasma and to evaluate the bioequivalence of 2 kinds of amlodipine besylate tablets.Methods: Twenty healthy male volunteers were enrolled into a single crossover study.A single dose of the suspension equivalent to 10 mg amlodipine besylate or a reference preparation was given in a crossover way.The plasma concentrations of amlodipine besylate were determined by LC-MS method in the volunteers at different time points;the pharmacokinetic parameters and relative bioavailability were calculated and the bioequivalence of the 2 preparations were evaluated.Results: The pharmacokinetic parameters for experimental and the reference preparations were: C_max(6.21?1.88) vs(6.03?1.08) ng/ml;AUC_0-120(250.68?52.61) vs(246.14?52.11) ng h/ml;T_max(6.0?2.3) vs(6.1? 2.5) h;t_1/2(40.45?6.68) vs(43.74?9.05) h,respectively.The linear range of the present method was 0.1-20.0 ng/ml;the lowest detectable concentration of amlodipine besylate was 0.1 ng/ml.There was no significant difference in pharmacokinetic parameters between the 2 tablets.Conclusion: The present method is simple to use,fast,and accurate.The 2 preparations of amlodipine besylate are bioequivalent.

Flagellum is a slender and wavy protein-attached filament on the surface of certain bacterial cells. It not only plays an important role in the movement and pathogenic ability of bacteria, but also participates in a variety of host immune regulation. Flagellin is a structural protein that forms the main part of flagellar filaments and can be recognized by TLR5 and other receptors in the host cell to induce the body′s immune response. At present, flagellin is widely used in the research of new immune adjuvants due to its immune activation, and its inflammation inhibitory effect also has good prospects against immune pathological damage. In this review, we summarized and analyzed the recent progress on the basic structure and function of flagellin, the host recognition mechanism, and its role in regulating the host immune system.

OBJECTIVETo compare the differences between computer aided identification and ultra-thin layer slicing in measuring the lesions of femoral head necrosis,and to confirm the accuracy and practicability of computer aided method.

METHODSFrom June 2012 to December 2013, the X-ray and MRI of 24 patients (24 hips on unilateral) were reviewed, who had avascular necrosis of the femoral head (ANFA) at late stage (stage III and IV) according to the ARCO international staging system. There were 15 males and 9 females, with an average age of (65.1 ± 8.8) years old, ranged 33 to 74 years old. Based on the software system with seeds point identification, the ragional adaptive search method with computer aid was used to calculate the volume of necrotic lesion in femoral on MRI. Then the pathological slices of those intraoperative femoral heads were made to measure the gross volume of necrotic lesion in femoral head,and the values were compared with the data in the computer.

RESULTSFor 24 hips, by the calculation of computer, the necrotic volume was (20.00 ± 3.04) cm (ranged, 18.72 to 21.29 cm³). Under the pathological section, the necrotic volume of the femoral head was (19.89 ± 3.17) cm³ (ranged, 18.55 to 21.23 cm³). In computer and pathology two kinds of measurement, the two entire femoral head volume had no significant difference using these two measurements (t = -1.227, P = 0.232).

CONCLUSIONComputer aided identification for necrotic area of femoral head adaptive can demonstrate the morphology of femoral head necrosis accurately and reliably, which will help surgeon better understand the morphology and orientation in femoral head.

Adult ; Aged ; Diagnosis, Computer-Assisted ; Female ; Femur Head Necrosis ; pathology ; Humans ; Magnetic Resonance Imaging ; Male ; Middle Aged

In this paper, Fourier transform infrared spectroscopy fingerprint analysis of Marsdenia tenacissima samples was used to develop a reliable method of tracing the geographical origins. Forty-eight samples from four provinces of China were analyzed by FTIR. We analyzed and characterized the fingerprints in both the full spectrum peaks and characteristic peaks, then the principal component analysis and the cluster analysis were carried out. The results of fingerprint analysis, correlation analysis, principal component analysis and cluster analysis can identify the geographic origins correctly, which verified and supplemented each other; the identification results and the actual location showed a high degree of consistency, namely the lower the space distance, the greater the similarity of different samples. These results revealed the obvious superiority and practical value in comparison to the more tedious and time-consuming wet chemistry method normally used. Using appropriate metrology methods can trace the geographical source correctly. The M. tenacissima materials from the region of Maguan should be considered as genuine medicinal materials taking into account the good quality.
China ; Cluster Analysis ; Drugs, Chinese Herbal ; analysis ; classification ; standards ; Geography ; Marsdenia ; chemistry ; classification ; Medicine, Chinese Traditional ; Principal Component Analysis ; Quality Control ; Reproducibility of Results ; Spectroscopy, Fourier Transform Infrared ; methods

Objective To establish a high throughput general multiple competitive polymerase chain reaction ( cPCR) detecting method of copy number variations ( CNVs) for the population of chromosome 1 substitution strains from wild mice.Method The selected 14 loci, including 11 CNVs on chromosome 1 and internal control loci on other three chromosmes (Chr 7, Chr 19 and Chr X), were detected based on the universal fluorescent primer multiple competitive pol-ymerase chain reaction.All specific cloned plasmids were constructed as competitors.Results Altogether 11 CNVs were designed in one panel, and the copy of Chr X accurately reflects the gender.Conclusions A rapid and high-throughput fluorescent multiplex cPCR assay is established which can be used for detection of copy number variations on chromosome 1 in mice.

Hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase(HCT) is an key enzyme involved in the biosynthesis of chlorogenic acid in Lonicera japonica. In this study, eight putative HCT genes were cloned with RACE (rapid amplification of cDNA ends) technology based on the analysis of transcriptome in L. japonica. Among them, one was suggested as HCT gene (LjHCT) in L. japonica through analysis of sequence similarity, physical and chemical properties, and domain conservation of the proptein. LjHCT gene containing 1 275 bp encodes a protein with the molecular weight of 47 kDa. These results will provide foundation for exploring the function of LjHCT in Lonicera japonica.

Objeetive To explore the mechanism of drug resistance of New Delhi metallo-β-lactamase-1 (NDM-1) producing Enterobacteriaceae,and to investigate the characteristics of blaNDM-1 carrying plasmid and its gene environment.Methods A total of 48 strains of carbapenem-resistant Enterobacteriaceae were successively collected from six general hospitals in south China during August 2011 and January 2013.Escherichia coli J53 was used for plasmid conjugation.Modified Hodge test was performed,and PCR method was used for the detection of carbarpenase-related genes.The relative molecular mass of the blaNDM-1 carrying plasmid was determined using pulsed field gel electrophoresis (PFGE)assay,and enzyme digestion was performed to investigate the homology and incompatibility group of the plasmid.Clinical feature of blaNDM-1 producing Enterobacteriaceae infection was also investigated.Results Among 48 strains of carbapenem-resistant Enterobacteriaceae,43 were positive in modified Hodge tests.blaVIM,blaGIM and blaSPM genes were negative in all strains,while blaNDM-1 was positive in 19 strains including 3 strains of Escherichia coli,5 strains of Klebsiella pneumoniae,6 strains of Enterobacter cloacae,3 strains of Citrobacterfreundii,1 strain of Klebsiella oxytoca and 1 strain of Providencia rettgeri.All the 19 strains were resistant to imipenem,cefotaxime,ceftazidime,cefepime,aztreonam and piperacillin/tazobactam,47.3％ strains were resistant to ciprofloxacin and levofloxacin,but 68.4％ strains were sensitive to amikacin.Conjugation experiment showed that,blaNDM-1 carrying plasmids in 13 strains were transmitted to the Escherichia coli J53.The conjugants were resistant to imipenem,ceftazidime,cefotaxime and piperacillin/tazobactam,but were sensitive to amikacin,ciprofloxacin and levofloxacin.All genes in conjugant J-FR90 (Providencia rettgeri) were negative,while the remaining 12 conjugants carried blaNDM-1,blaSHV and aac-(6')-Ib genes.PFGE showed that,the sizes of all blaNDM-1 carrying plasmids were about 50 kb,and more than 80％ of their macrorestriction maps were similar.The plasmid belonged to incompatibility group IncX3,and exhibited 100％ passage stability after 500 generations of propagation.Among 19 patients infected with NDM-1 producing Enterobacteriaceae,6 died and 13 survived.Conclusions NDM-1 producing Enterobacteriaceae is emerging in south China,and blaNDM-1 is transmitted to Enterobacteriaceae through IncX3.Patients infected with NDM-1 producing Enterobacteriaceae usually have good prognosis.