1.8.PCR was performed using the human Gpx-1 gene,and we obtained the needed target gene fragments.Conclusion The alkaline lysis extraction method is reliable for obtaining high quantities of DNA from blood clot suited for PCR amplification.

Objective We used the DNA methylation microarrays to investigate the differential methylation genes and loci sites in Kashin-Beck disease (KBD),to study the relationship between DNA methylation and KBD pathogenesis.Methods Totally 12 KBD adults and 12 healthy adults were selected and peripheral blood samples were collected and DNA was extracted.Illumina 450K bead-chip was applied to detect methylation status in KBD and healthy controls.Aberrant hyper-methylated sites were filtrated according to the P value after correction and methylation differences,together with GenomeStudio soft.Screened genes were validated using bisulfite sequencing polymerase chain reaction (BSP) technology.Results A total of 484 948 loci sites were analyzed and compared,93 differential methylated loci were found by comparing KBD and normal people,including 34 hypermethylated sites and 59 hypomethylated sites.There were 50 genes corresponding to the loci,43 genes not reported in literature.According to gene ontology analysis,the genes were involved in the immune response,antigen processing,phosphate and phosphoric acid metabolism and phosphorylation and the process of metal ions in combination.However,in the verification test using BSP method,there was no significant difference in methylation rate in human leukocyte antigen (HLA)-DRB1 between the case and the control group (48％ vs 70％,x2 =3.688,P ＞ 0.05).Conclusions The high and low differentially methylated sites in peripheral blood DNA of KBD patients are significantly different from those of the health control.HLA-DRB1 locus is not significantly different between the BSP verification test and methylation chip.

Objective To study basic thyroid stimulating hormone (bTSH) levels impact on outcomes of in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) in Qinghai.Methods Totally 282 cases with IVF cycles and 93 cases with ICSI cycles were studied prospectively,according to bTSH level,patients were divided into four groups.Reproduction rate,clinical pregnancy rate,miscarriage rate and live birth rate were studied among four groups.Results (1) In 375 cases with IVF/ICSI cycles,bTSH was positively correlated with abortion rate (r=0.42,P=0.04),but live birth rate and growing rate showed negative correlations with bTSH (r=-0.42,-0.28; P=0.04,0.03).bTSH and the number of eggs,the number of fertilized eggs,the number of embryos,biochemical pregnancy rate,and clinical pregnancy rate were no significant correlation (all P＞0.05).(2) Among women at group of ≤1.7,＞1.7 and ≤2.5,＞2.5 and ≤3.5,＞3.5 mU/L,the implantation rates were 28.7％,27.3％,37.7％ and 19.2％,live birth rates were 80.9％,75.0％,82.7％,and 59.8％,abortion rates were 19.0％,15.0％,16.7％,40.1％; they all showed significant difference (all P＜0.05).Abortion rate in women with high bTSH level was higher than that of women with lower bTSH level,however implantation rate,live birth rate in women with high bTSH level were lower.Conclusion When bTSH level is ＞3.5 mU/L,the abortion rate were increased,but live birth rate,rate of implantation were decreased.

Objective To develop an effective process for isolating and purifying haptoglobin ( Hp) from Cohn fractionⅣby a new ion exchange chromatography and to preliminarily identify and analyze the product of each purification step . Methods The fraction was first diluted and impurities were adsorbed with Rivanol .Then, the supernatant was treated with 50%ammonium sulfate.Finally, the precipitate was redissolved , and Hp was purified further with Q Sepharose Fast Flow chromatography .Native-PAGE was used to measure the activity of the haptoglobin-bound hemoglobin , while SDS-PAGE analysis and immunoblot were used for identification of the target protein .Results After pretreatment , some of the impuri-ties were removed from the Cohn fraction Ⅳ, and the target protein was enriched .In our case, the target protein was Hp and Hp2-2 was the main phenotype in the human plasma fraction Ⅳ.Target protein band and high purity were identified by SDS-PAGE.Immunoblot analysis further proved that this method could successfully isolate the target protein Hp , and the activity of 2.8 U/ml was measured by Native-PAGE method.Conclusion Haptoglobin is successfully isolated from human Cohn fractionⅣwith this method.The purification process is simple and suitable for scale-up production with a good prospect.

Objective To investigate the rate of termination of pregnancy (TOP) in gravidas with prenatally diagnosed fetal malformation and to analyze the influences of medical and non-medical factors on decision making.Methods This was a prospective cohort study. Gravidas who took part in a multidisciplinary consultation due to fetal malformation and finished a questionnaire after consulting from September 12, 2012 to May 2, 2013 were recruited. Exclusion criteria were chromosomal disorders and isolated abnormal ultrasound soft markers. The questionnaire survey was conducted to understand the patient's backgrounds and to collect their feedbacks on the consultation. Decisions of the gravidas on TOP were followed up by phone in 2014 and 2016. If a gravida chose to continue her pregnancy, her baby's outcome was also recorded.T test,Chi-square test or Fisher's exact test, or rank-sum tests (Mann-Whitney or Wilcoxon) or Logistic regression was used for statistical analysis.Results (1) Altogether 229 gravidas were recruited and 10 of them were lost to follow-up, so 219 cases were finally analyzed. Among the 219 cases, 35.6% (78/219) chose to terminate their pregnancies. (2) Neonatal prognosis was predicted based on the type and severity of the disease and was divided into four levels including good prognosis (122 cases, 55.7%), medium prognosis (20 cases, 9.1%), poor prognosis (17 cases, 7.8%) and unsure prognosis (60 cases, 27.4%). (3) Gravidas who chose to terminate their pregnancies were younger than their counterparts choosing to continue to term (average age: 27.8±4.1 vs 29.0±3.9,t=2.257,P<0.05). Gravidas who went to the consultation before the 24th gestational week carried double risk of TOP than those after the 24th gestational week [termination rate: 52.5% (31/59) vs 29.4% (47/160),χ2=10.089,P<0.01). (4) Gravidas with fetal growth restriction (FGR) were at triple risk of TOP than those without (OR=2.850, 95%CI: 1.323-6.140) after adjusting for maternal age, gestational age at consultation and prognostic evaluation. Comparing with the good prognosis group, in which the rate of TOP was 19%, the unsure (OR=2.354, 95%CI: 1.108-5.004), medium (OR=16.188, 95%CI: 4.732-55.372) and poor (OR=14.515, 95%CI: 3.61-58.359) prognosis groups had higher risk of TOP. (5) There were 63 women informed us their reasons for TOP (multiple choices), among which 57 (90.5%) were due to unsure neonatal outcomes, and 10 (15.9%) were due to emotional factors. (6) Maternal satisfaction with neonatal prognosis was 2 to 5 points (medium score, ten-point system) lower in gravidas choosing to TOP than in those choosing to continue pregnancy regardless of good, unsure, or medium neonatal prognosis. No significant difference in maternal satisfaction was found among gravidas with poor neonatal prognosis.Conclusions The rate of TOP in gravidas with prenatally diagnosed fetal malformation remains high in China. Factors that can negatively influence the rate of TOP are consultation after the 24th gestational week, better perceived neonatal prognosis and higher maternal satisfaction with neonatal prognosis. Uncertainty of the neonatal prognosis is the leading cause of maternal dissatisfaction.

Objective To investigate effect of early enteral nutrition supplemented with probiotics on gastrointestinal motility disturbance and nutritional status in patients with severe craniocerebral trauma.Methods Forty patients with severe craniocerebral trauma were randomized into study group (18patients) and control group (22 patients).Patients of both groups received enteral nutrition via nasogastric tube at 24-72 hours after admission,but the patients of study group were also supplemented with probiotics simultaneously.Rate of abdominal distention,vomiting,gastro-oesophageal reflux,gastric retention,constipation and diarrhea were recorded during the whole study.Time to first defecation and time to targeted nutritional goals were also recorded.Prealbumin and transferrin in serum were detected at days 0,4,7,and 15 after the beginning of enteral nutrition.Length of ICU stay was compared between groups.Results There were no significant differences of the two groups in terms of rate of abdominal distention,vomiting,gastric retention and diarrhea.However,less gastro-oesophageal reflux or constipation patients were observed in study group,as compared with control group (P ＜ 0.05).Time to first defecation and time to targeted nutritional goals were shorter in study group,as compared with control group (P ＜ 0.05).Levels of prealbumin and transferrin had no significant differences between the two groups at days 0,4,and 7,but study group showed both were higher than control group at day 15 (P＜0.05).Moreover,length of ICU stay showed no significant difference between the two groups.Conclusions Compared with simple enteral nutrition,early enteral nutrition with probiotics improves gastrointestinal motility,facilitates the delivery of enteral nutrition,and further ameliorates nutritional status in patients with severe craniocerebral trauma.

Objective To assess the association between NOD2 gene single nucleotide polymorphisms (SNPs) and leprosy in Chinese Yi population.Methods Whole blood samples were obtained from 300 patients with leprosy and 300 healthy human controls of Yi nationality in Sichuan province.Genomic DNA was extracted,and a SNaPshot assay was performed to determine the genotypes of four single nucleotide polymorphisms (SNPs) of the NOD2 gene,including rs9302752,rs7194886,rs8057341 and rs3135499.Chi-square test was conducted to compare allele frequency,and Hardy-Weinberg equilibrium was tested.Results The genotype distribution of all the four SNPs was consistent with Hardy-Weinberg equilibrium (all P ＞ 0.05).Significant differences were observed between the patients with leprosy and healthy controls in both genotype distribution and allele frequency of the SNP rs3135499 (both P ＜ 0.01),but not in those of the other three SNPs (all P ＞ 0.05).Conclusion The SNP rs3135499 of the NOD2 gene may be associated with the development of leprosy in Chinese Yi population.

Objective To establish an auto-control system for high-throughput and multi-channel DNA synthesis which can simultaneously and quickly synthesize up to 96 different oligonucleotides in a 96-well microtiter format.Methods The PLC and its extended modules is used as the main-control unit, which executes the DNA automatic synthesis process according to the synthesis sequences and steps set by the user,and the manual injecting reagent etc.And the configuration software and VC6.0 were used for programming the man-machine interface sofeware to set synthesis parameters, position calibration,flux calibration data etc, and communicated with PLC.Results The synthesis application of about 150 000 DNA chains has proved that the synthesis cycle time for 96 couplings was 4 min,the average coupling efficiency was 99%across the entire 96-well plate,the monomer reagent usage was reduced by 50 percent,and the synthesis configuration was more flexible.Conclusion A reliable and simple auto-control system is provided for parallel synthesis of 96-channel oligonucleotide chains,which can meet the demands of high-throughput and multi-channel DNA synthesis.

Objective To investigate the changes in the expression of matrix metalloproteinase-1 (MMP-1),matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of metalloproteinase-3 (TIMP-3) in the articular cartilage of patients with Kashin-Beck disease (KBD) and the role of these proteins in pathogenesis of KBD.Methods The postoperative cartilage samples were collected from patients with KBD,osteoarthritis and patients with non-bone disease (control).The expression of MMP-1,MMP-3 and TIMP-3 in the cartilage was detected by immuohistochemistry,and the positive chondrocytes were counted in different layers of the articular cartilage under microscope.Results The positive rates of MMP-1 in the upper [(67.00 ± 1.69)％] and deeper [(22.07 ± 29.66)％] layers of articular cartilage from patients with KBD,and in the deeper layer of articular cartilage from patients with osteoarthritis [(70.52 ± 37.84)％] were significantly higher than those in the control group [(51.73 ± 36.74)％,(3.75 ± 6.85)％,all P ＜ 0.05].The positive rates of MMP-3 in the deeper layer of articular cartilage from patients with KBD [(28.84 ± 31.13)％] and in the middle and deeper layers of articular cartilage from patients with osteoarthritis [(55.69 ± 35.00)％,(45.96 ± 35.38％)] were significantly higher than those in normal cartilage [(34.09 ± 28.54)％,(14.46 ± 18.32)％,all P ＜ 0.05].The positive rates of TIMP-3 in the middle layer of articular cartilage from patients with KBD [(21.25 ± 25.23)％] and in the middle and deeper layers of articular cartilage from patients with osteoarthritis [(20.40 ± 22.19)％,(18.10 ± 22.58)％] were significantly lower than those in normal cartilage [(36.74 ± 26.61)％,(7.81 ± 20.58)％,all P ＜ 0.05].Conclusion MMP-1,MMP-3 and TIMP-3 play important roles in the articular cartilage damage of KBD.

Super hydrophobic interface modified with silver nanoparticles was fabricated for the detection of pesticide residues.By using a chemical reduction method,silver nanoparticles were deposited on the substrate surfaces with different microscopic pore structures.Two kinds of composite substrates,including regular stainless steel mesh and cellulose polyester film,were used.The pre-treatment of the substrate with fluoridated reagents was used to form a super hydrophobic interface,which made the target molecules on the surface concentrate effectively.The surface with the cellulose polyester substrate was used to detect Rhodamine 6G (R 6G) effectively with surface enhanced Raman scattering (SERS) technique.The results showed that the detection hmit was 10-16 mol/L.In addition,the surfaces based on the stainless steel mesh and cellulose polyester substrate were used to detect trichlorfon pesticide with detection limits of 1 × 10-15 mol/L and 1 × 10-16 mol/L,respectively.