BACKGROUND:Intestinal acute graft-versus-host disease is one of the most aggressive complications after allogeneic hematopoietic stem cell transplantation with high lethality.How to improve intestinal inflammation and regulate autophagy by applying traditional Chinese medicine in order to treat intestinal acute graft-versus-host disease is a worthwhile research issue nowadays. OBJECTIVE:To investigate the mechanism of Huangqintang modulating intestinal flora for the treatment of intestinal acute graft-versus-host disease. METHODS:CB6F1 mice were irradiated with 60Co X radiation at a total dose of 8 Gy,and then single nucleated cell suspensions(bone marrow cells+splenocytes)from Balb/c H-2d mice were injected into the tail vein in order to prepare a model of intestinal acute graft-versus-host disease.These samples were randomly divided into the model group and the high-,moderate-,and low-dose Huangqintang groups.After modeling,the model,high-,moderate-,and low-dose groups received different doses of Huangqintang or an equal volume of saline by continuous gavage for 14 days.Clinical acute graft-versus-host disease grading,and survival time was recorded.Small intestinal tissues from each group were stained with hematoxylin and eosin for small intestinal mucosal pathology scoring.The intestinal flora of mice in each group was detected using 16S rDNA sequencing.Autophagy-related markers were detected using immunofluorescence,immunohistochemistry,and PCR. RESULTS AND CONCLUSION:(1)Compared with the model group,the survival time of mice was significantly prolonged(P<0.01);the clinical acute graft-versus-host disease scores were significantly reduced(P<0.01);the pathological grading scores of the small intestinal mucosa were significantly diminished(P<0.01);the levels of the small intestinal tissue inflammatory factors tumor necrosis factor-α,interleukin-1β,and interleukin-6,were significantly decreased(P<0.01);the structural integrity of the small intestinal mucosal epithelium was partially restored in mice after the intervention of moderate and high-dose Huangqintang.(2)The study of intestinal flora found that compared with the model group,the pro-inflammatory strain Enterococcus was significantly reduced(P<0.05),while beneficial bacteria such as Clostridium_innocuum and Rhodococcus,a pro-autophagy bacterium,were significantly elevated(P<0.05)in the moderate-dose Huangqintang group.(3)Compared with the model group,the autophagy markers were significantly elevated in the moderate-dose Huangqintang group(P<0.05);under transmission electron microscopy,the number of autophagic vacuoles of moderate-dose Huangqintang group increased significantly.(4)The results showed that Huangqintang significantly reduced the abundance of conditionally pathogenic bacteria and the level of inflammatory factors in small intestinal tissues,and increased the relative abundance of beneficial bacteria and promoted the expression of autophagy in the small intestinal mucosa,which resulted in a significant improvement of intestinal symptoms in mice with acute graft-versus-host disease.

OBJECTIVE: To explore the prevalence and characteristics of chromosomal abnormalities in abortuses during early pregnancy with single nucleotide polymorphism microarray (SNP-array). METHODS: For 520 abortuses, copy number variations (CNVs) in chorionic villi were analyzed with SNP-array. RESULTS: In 510 (98.1%) of the samples, the analysis was successful. Among these, 57.6% (294/510) of the samples were found to harbor clinically significant chromosomal abnormalities. 38.8% of the samples (198/510) had a normal result. 2.4% (12/510) of the samples harbored benign CNVs, and 1.2% (6/510) harbored variants of uncertain significance (VOUS). Aneuploidies, polyploidies, pathogenic CNVs and uniparental disomies (UPD) had accounted for 75.2% (221/294), 13.9% (41/294), 8.2% (24/294), and 2.7% (8/294) of the samples, respectively. 45,XO was the most common finding, which was followed by trisomy 16 and trisomy 22. 69,XXY was the most common polyploidy. CONCLUSION Chromosomal abnormalities are the main cause for early miscarriage, among which aneuploidies are most common. The prevalence of aneuploidies is significantly increased among women over 35. SNP-array analysis has the advantage of high success rate, high resolution and great accuracy, but the clinical significance of microdeletions/microduplications found by SNP-array can be difficult for interpretation.
Chorionic Villi ; Chromosome Aberrations ; Chromosome Disorders ; DNA Copy Number Variations ; Female ; Genetic Testing ; Humans ; Karyotyping ; Polymorphism, Single Nucleotide ; Pregnancy

Objective To evaluate the efficacy of non-invasive prenatal screening (NIPS) in the detection of fetal aneuploidies.Methods Cell free DNA was sequenced in 5 566 pregnant women to identify the fetal aneuploidies in the First Affiliated Hospital of Zhengzhou University from January 1st,2015 to March 15th,2016.Among them,5 230 (93.96％,5 230/5 566) were singleton pregnancies and 336 (6.04％,336/5 566) were twin pregnancies.In singleton pregnancies,1 809 (34.59％,1 809/5 230) were women with advanced maternal age,and 3 421 (65.41％,3 421/5 230) were young women.The positive results of NIPS were validated by karyotyping through invasive procedures and neonatal outcomes were followed up by telephone.Results Among the 5 566 women,69 (1.24％,69/5 566) got positive NIPS results,with 66 in singleton pregnancies and 3 in twin pregnancies.Two were monochorionic diamniotic twins and 1 was dichorionic twin pregnancy.The positive predictive value of NIPS for trisomy 21,18 and 13 were 100.0％,90.9％ and 100.0％,and was 55.6％ for sex chromosome aneuploidies.There was no false negative case found during the follow-up.In the advanced maternal age group and young women group,the prevalence rates of fetal chromosomal aneuploidies were 1.11％ (20/1 809) and 0.94％ (32/3 421),respectively.In the young women with soft markers in fetal ultrasound,the prevalence of fetal chromosomal aneuploidies was 1.44％ (7/487),and in serum high risk women,it was 0.94％ (7/747).In women with the serum screening risk with cut-off value,0.89％(9/1 016) had fetal aneuploidies,and the prevalence was 0.77％(9/1 171) in volunteers.There was no statistically significant difference among these groups (P=0.636).Conclusions There is no difference in the detection rate of fetal aneuploidies between high-risk women in serum screening and volunteers in NIPS.NIPS is more suitable as a first line screening test for women without fetal ultrasound abnormalities.It should be used carefully when there is ultrasound abnormalities.

OBJECTIVE:To establish a method for simultaneous determination of the contents of ligustroflavone,specnuezhe-nide,demethylwedelolactone and wedelolactone in Zibu ganshen pill. METHODS:Dual-wavelength HPLC was performed on the column of Elite C18 with mobile phase of acetonitrile-0.5%acetic acid(gradient elution)at flow rate of 0.9 ml/min,column temper-ature was 25 ℃,detection wavelengths were 224 nm(0-30 min) and 351 nm (30-50 min),and the injection volume was 20 μl. RESULTS:The linear range was 6.75-135.00μg/ml(r=0.999 5)for ligustroflavone,6.54-130.80μg/ml(r=0.999 8)for specnuezhe-nide,4.90-98.00μg/ml(r=0.999 4)for demethylwedelolactone and 6.42-128.40μg/ml(r=0.999 6)for wedelolactone;RSDs of pre-cision,stability and reproducibility tests were no more than 1.25%;average recoveries were 96.15%-99.96%(RSD<2%,n=6). CONCLUSIONS:The method is rapid,sensitive and accurate,and can be used for the contents determination of ligustroflavone, specnuezhenide,demethylwedelolactone and wedelolactone in Zibu ganshen pill.

Objective:To develop an HPLC-DAD-ELSD method for the determination of nitidine chloride, 5-ethoxychelerythrine, bergeninum and ardisiacrispin A in Shangtong tinctures ( STD) . Methods: A Hypersil C18 column was used as the chromatographic column, the flow rate was 0. 8 ml·min-1 . For nitidine chloride and 5-ethoxychelerythrine, the mobile phase A consisted of acetoni-trile,the mobile phase B consisted of 0. 1% formic acid-triethylamine (pH 4. 5),and the DAD detection wavelength was at 273 nm. For bergeninum and ardisiacrispin A, the mobile phase consisted of methanol-water(25∶75), the temperature of drift tube was set at 95℃, and the gas flow (N2) was set at 2. 5 SLPM·min-1. Results:There was a good linear relationship between the concentration and peak area for nitidine chloride and 5-ethoxychelerythrine within the range of 0. 021-0. 426 μg (r=0. 999 5) and 0. 075-1. 494 μg (r=0. 999 8), respectively. The average recovery was 99. 22%(RSD=0. 64%) and 98. 61%(RSD=0. 46%), respectively. There was a good linear relationship between the concentration and peak area for bergeninum and ardisiacrispin A within the range of 0. 215-4. 304 μg(r=0. 999 3) and 0. 286-5. 728 μg(r=0. 999 7), respectively. The average recovery was 99. 15%(RSD=0. 77%) and 99. 25%(RSD=0. 56%) accordingly. Conclusion:The method is accurate, sensitive and reproducible, and can be used in the de-termination of nitidine chloride, 5-ethoxychelerythrine, bergeninum and ardisiacrispin A in STD.

In this study, real-time PCR and immunohistochemistry were used to detect coxsakie and adenovirus receptor (CAR) expression. Both localization and quantity were evaluated in the uteri obtained at days post coitus (dpc) 2.5, 4.5, 6.5, 8.5. Outcome of PCR was assessed by 2(-ΔΔCt) method. Image Pro-Plus 6.0 software was used for quantifying mean density of CAR expression in immunohistochemical sections. We found relatively weak CAR expression in the mouse uteri during implantation window. PCR and immunohistochemistry revealed highest CAR expression was detected on dpc 2.5 followed by down-regulation of CAR at dpc 4.5 and 6.5 (with significant difference). At dpc 8.5, CAR expression was increased slightly again. It is concluded that during implantation, the expression of CAR mRNA and protein is declined, resulting in the impairment of tight junction between cavity epithelium cells. After implantation window closure, CAR appears again to maintain epithelium stability. CAR might play an important role during embryo implantation procedure.

In this study,real-time PCR and immunohistochemistry were used to detect coxsakie and adenovirus receptor (CAR) expression.Both localization and quantity were evaluated in the uteri obtained at days post coitus (dpc) 2.5,4.5,6.5,8.5.Outcome of PCR was assessed by 2-△△Ct method.Image Pro-Plus 6.0 software was used for quantifying mean density of CAR expression in immunohistochemical sections.We found relatively weak CAR expression in the mouse uteri during implantation window.PCR and immunohistochemistry revealed highest CAR expression was detected on dpc 2.5 followed by down-regulation of CAR at dpc 4.5 and 6.5 (with significant difference).At dpc 8.5,CAR expression was increased slightly again.It is concluded that during implantation,the expression of CAR mRNA and protein is declined,resulting in the impairment of tight junction between cavity epithelium cells.After implantation window closure,CAR appears again to maintain epithelium stability.CAR might play an important role during embryo implantation procedure.

To investigate the the possibility to utilize the cellular fatty acid (CFA) information as a method in Brucella typing, 90 Brucella strains were subjected to the study on CFAs, and all the experimental strains were inoculated on Brucella Agar plates for 48 hours. After that, cells were harvested, saponificated, methylated and extracted to provide fatty acids methylesters for gas chromatography analysis. Based on the CFAs data matrix, dendrogram of 90 experimental strains was generated by SPSS11.5 software package. As shown in the dendrogram, 90 Brucella strains could be divided into 5 clusters. The first cluster included some species of Brucella abortus,Brucella melitensis,Brucella suis, Brucella ovis; and some of the variant strains of Brucella abortus and Brucella melitensis and the typical strain of Brucella neotomae. The second cluster included typical strains of Brucella suis (1,2,3 and 5 types); vaccine strains of Brucella suis S2; vaccine strains of Brucella melitensis M28、Rev.1 and typical strain of Brucella ovis. The third cluster included some of Brucella melitensis; some of the variant strains of Brucella melitensis; some of Brucella abortus(3,6 types); Brucella canis and Brucella ovis. The fourth cluster was the typical strain of Brucella canis.and the fifth cluster included some of Brucella melitensis(1 type); some of Brucella abortus (1 type); some of the variant strains of Brucella melitensis and Brucella suis(1,3 type). It is apparent that CFAs information can be used in brucella typing. and Brucella suis and Brucella canis can be distinguished by the difference in the CFA contents of 3 fatty acids 19:0CYCLOω8c, 18:1ω7c and 16:0. The results of CFAs typing in Brucella species show that Brucella canis includes 2 biovars at least and the high homologization of Brucella abortus (3 type) and Brucella abortus(6 type) can be found.

ly decreased,which will increase during HAART.It may indicate CD4+ cells restoration was delayed during HAART compared with peripheral blood.

BACKGROUND: In recent years, nano-carriers have been regarded as the most promising technologies for breakthrough the bottleneck of gene transfer. Polyamidoamine dendrimer (PAMAM) is a kind of new nanometer material. PAMAM can transfer target gene to the cell with high efficiency and lower toxic both in vivo and in vitro.OBJECTIVE: To evaluate the antitumour effects of survivin antisense oligonucleotide (Survivin-asODN) carried by PAMAM on colorectal cancer transplanted subcutaneously in nude mice.DESIGN, TIME AND SETTING: An in vivo experiment regarding tumor gene therapy was performed from February to August in 2008 at the Laboratory of Bionanometer Engineering, Research Institute of Micro/nanometer Science & Technology of Shanghai Jiao Tong University and Central Laboratory of Zhujiang Hospital of Southern Medical University.MATERIALS: Human colorectal cancer cells SW620 were from Shanghai Cell Institute of Chinese Academy of Sciences.PAMAM dendrimer was offered by the Bionanometer Engineering Laboratory, Research Institute of Micro/nanometer Science & Technology, Shanghai Jiao Tong University. Lipofectamine ~(TM)2000 was purchased from Invitrogen, USA. Survivin-asODN was synthesized by Shanghai Bioengineering Company.METHODS: The PAMAM and cation liposome were respectively mixed with Survivin-asODN to generate the transfection complex carrying antisense gene. The shape of the complex was observed by transmission electron microscope, the particle size was determined by laser particle size analysator and the zeta potential was measured by an analytical tool. The encapsulating efficiency and release progress in vitro were determined by ultraviolet spectrophotometer in centrifuging method. Human colorectal cancer cells SW620 at logarithmic phase were inoculated into the abdominal region of 18 Blab/C nude mice subcutaneously to produce transplanted tumor models in colorectal cancer nude mice, which were randomly divided into 3 groups: liposome, PAMAM and blank control groups. They were injected respectively with Hposome-survivin-asODN complex,PAMAM-survivin-as ODN transfection complex and seroculture liquid. The volumes of tumor were surveyed in the 2 groups.Western blotting method was used to determine the survivin gene expression in the transplanted tumor tissue.MAIN OUTCOME MEASURES: Particle size, zeta potential, gene loading level, encapsulation efficiency, release rate of cationic liposome-survivin-asODN complex and PAMAM-survivin-asODN complex, as well as survivin expression rate and apoptosis rate after transfection, inhibition rate of the transplanted tumor growth, Survivin protein expression and activity in the transplanted tumor cells.RESULTS: The particle size of PAMAM-survivin-asODN complex was smaller (P < 0.01), but the zeta potential was greater (P < 0.05), compared with liposome-survivin-asODN. There was no significant difference between PAMAM and liposome groups in terms of gene loading rate and transfection efficiency. DNA release lasted for 14 days for PAMAM, but only 5 days for liposome.After colorectal cancer cell transfection, survivin protein expression was lower, but apoptosis rate was higher, in the PAMAM-survivin-asODN complex than in the liposome-survivin-asODN complex (P < 0.05).CONCLUSION: PAMAM facilitates delivery of Survivin-asODN into transplanted colorectal cancer cells SW620. As a result,survivin protein expression was decreased, and apoptosis rate was increased in vivo which inhibited transplanied tumour growth.