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 evaluate the performance of emergency test and the applicability under complex field conditions of biochemical modules of field point-of-care test ( POCT ) system ( type A ) .Methods The precision and anti-interference ability of albumin ( ALB ) , total bilirubin ( TBIL ) , alanine transaminase ( ALT ) , aspartate transaminase (AST),blood area nitrogen(BUN),creatinine(CREA),uric acid(UA),lactate dohydrogenase(LDH),creatine kinase ( CK) ,and glucose( GLU) detected by field POCT system( A) were analyzed according to standards formulated by National Committee for Clinical Laboratory Standards(NCCLS).Field POCT system(A) and Coulter Beckman AU2700 automatic biochemical analyzer were used to detect the serum of 22 clinical cases respectively.After simulating the field environment by adjusting the temperature and humidity, we compared the results of mixed serum under different environment conditions. Results The coefficients of variation in total precision of ALB,TBIL,ALT,AST,BUN,CREA,UA,LDH,CK,and GLU detected by field POCT system(A) were 3.34%,6.54%,6.01%,4.80%,3.95%,5.59%,3.33%,6.19%,7.40%,and 4.56%(LevelⅠ);and 3.08%,4.47%,4.02%,4.31%,3.76%,4.22%,2.93%,5.25%,6.39%,and 4.35%(LevelⅡ) respectively.When triglycerides( TG) level was at 21 mmol/L, the interference rate was below 10%.When bilirubin level was at 120 μmol/L, the interference rate of ALT,AST and CREA was -33.33%,-22.99%,20.00%(LevelⅠ), and -22.13%,-14.55%,and 8.70%(LevelⅡ),respectively.When its level was at 240 μmol/L, the interference rate of UA was -16.67%and -24.69%, respectively at two levels;if hemoglobin( Hb) was at 170 mg/dl, the interference rate of TBIL and LDH was 20.00%,and 99.26%(LevelⅠ),and 15.38%,and 40.79%(LevelⅡ),respectively;if it was at 340 mg/dl, the interference rate of ALT and AST was 9.84% and 13.79%(LevelⅠ), and 12.30%,and 12.27%(LevelⅡ),respectively;if it was at 510 mg/dl, the interference rate of CREA,UA and Ck in LevelⅠwas 26.67%, 16.67%,and 11.74%.The R2 of linear regression between field POCT system( A) and AU2700 automatic biochemical analyzer were 0.961,0.995,0.989,0.995,0.990,0.989,0.989,0.963,0.978,and 0.993, respectively.The POCT system could not work at 35℃ or higher temperature, and there was no difference in the results of detection between temperatures of 10-30℃or RH of 70%-90% and normal temperature and humidity(20℃,RH 50%) (P>0.05). However, the result of ALT and CK at high temperature and humidity was significantly higher than at normal temperature and humidity(P<0.001,and P=0.011, respectively).Conclusion The biochemical module of field POCT system(A) has a good correlation with the common large biochemical analyzer, and its precision meets the requirement of laboratory detection, but jaundice and hemolytsis can interfere in several tests to varying degrees.The POCT system can basically ensure accurate detection under field conditions of temperature and humidity, but should not work under extreme environments.

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.

Objective Deletion detection and annotation of 18 lines from the population of specific chromosome 1 substitution strains ( PCSSs) derived from Chinese wild mice based on whole genome re-sequencing data. Methods Whole genome re?sequencing of the 18 lines were performed on the Illumina Hiseq platform. SpeedSeq software was used to detect the deletion after read alignment. Further annotation was obtained using SnpEff software. Results 13803 dele?tions were identified among the 18 lines, the length of deletion was ranged from 51bp to 70 kb, among them nearly 50%were less than 500 bp. Through functional annotation,we found most of the variants were located in intronic (50. 361%) and intergenic (28. 745%) regions. However, we also identified 31 protein coding genes harboring loss?of?function dele?tions. Among them, 3 genes were associated with human diseases, 7 genes were participated in 11 KEGG pathways. Conclusion The chromosome 1 of PCSSs harbors abundant deletion mutations which can be used as genetic markers in genetic studies.