Objective To use tetraploid embryo complementation combined with gene transfer to produce genetically modified embryonic stem cells (EsCs) clones. Methods In this study, EGFP was introduced into ESCs by electroporation, and transfected positive cells were selected by G418 resistance. The tetraploid embryos were obtained from diploid blastomere electrofusion which preformed at 2-cell stage. Afterwards, 19-21 EGFP-ESCs were inserted into each tetraploid blastocyst cavity by piezo drilled microinjection,then the injected blastocysts were transferred into the uterus of pseudo-pregnancy at 2.5-day or the oviduct of 0.5-day female mice. Results The transfected ESCs maintained normal karyotype even after long-term passage (2n=40). The rate of fusion was 95.07%, and the developmental rate of tetraploid blastocyst was 95%.Totally 410 injected blastocysts were obtained. Unfortunately, we have not got any vital offsprings, except 151 implantation sites (pseudo-pregnancy 2.5 days:29.41%;the oviduct of half one day:64.37%). Furthermore, scattered EGFP expressions in transgenic fetus were observed under invert fluorescent microscope. Conclusion The transfected ESCs were observed in transgenic fetus, and the implantation rate in oviduct was higher than that in uterine.

Objective To investigate the characteristics of quality of life (QoL) and self-awareness in girls with idiopathic central precocious puberty (ICPP) and the correlation between QoL and the self-awareness.Methods The inventory of subjective life quality (ISLQ) and Children's self-concept scale (PHCSS) were employed to evaluate the level of QoL and self-awareness in 24 ICPP girls with ICPP (ICPP group) and 24 normal control girls (control group),and the correlation analysis between QoL and the self-awareness in the ICPP group was made.Results The score of overall (70.85±14.11 vs 80.21±6.07) and emotion component satisfaction (62.61± 12.91 vs 73.74±5.02),dimensions satisfaction on school life (5.80± 1.44 vs 6.69±0.65),depression experience (6.55± 1.14 vs 7.29±0.45),anxiety experience (5.93± 1.35 vs 6.96±0.82) and physical emotion (6.33±1.17 vs 7.12±0.83) in ICPP group was respectively lower than control group (P＜0.05).There was no difference in cognitive component between control group and ICPP group,as well as for other dimension satisfaction.Moreover,the score in total self-awareness,physical appearance and anxiety in ICPP group was respectively lower than those in control group (P＜0.05).In addition,a positive correlation was found between the subjective QoL and the children self-concept in ICPP group (P＜0.01).Conclusion The girls with ICPP show a lower subjective QoL and self assessment than normal girls.It is important to take care of the psychological feature of the girls with ICPP to improve the self-assessment and overall QoL during the medication.

Objective To develop a short Chinese version of the center for epidemiological studies depression scale(CES-D).Method A stratified random nationwide sample with 30801 normal people and a mentally ill sample with 415 patients were tested.Items in the short version were chosen according to both the result of independent t-test in current study and the result of reliance and validity.Cut-off scores were offered to check up depressions.Result The internal consistency reliability of the 9-item Chinese version CES-D ranged from 0.85 to 0.88.The test-retest reliability was 0.49(P＜0.001).All item-total correlations were higher than 0.5.The correlation between sumscores of the original scale and sunscores of the Chinese short version ranged from 0.94 to 0.96 (P＜ 0.001) in all the samples.The sum scores of initial diagnosis depression sample were significantly higher than that of treated depression sample and common sample.A two-dimension structure was testified.The cut-off scores of the short version were 10 and 17.Conclusion The 9-item Chinese version of CES-D can be used in various samples with reliable and valid effect.

Objective To establish a key technological system for spider fibroin gene code tandem connection , vector construction , prokaryotic expression and purification using genetic engineering in order to achieve MaSp 1 heterologous ex-pression in Escherichia coli and its separation and purification .Methods Isocaudarner ligation method was used to connect synthetic spider fibroin gene monomer code in tandem , and a recombinant clone concatemer was obtained .The identified recombinant clones were connected with prokaryotic expression vector pET 28a(+), and then transformed into E.coli BL21 (DE3).After being induced by IPTG for 6 hours, the expression product was identified by SDS-PAGE and Western blot-ting.Engineering bacteria were fermented in high density , and the obtained protein was purified through ammonium sulfate fractionation.Results and Conclusion The expression plasmids of MaSp1concatemers were successfully constructed , and the induced expression genetic engineering MaSp 1 protein was of the expected relative molecular mass .In addition, the pu-rity of the purified protein was above 80%.This study has developed crucial technologies for mass production of genetic en-gineering spider silk proteins .

Objective To establish an ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for detecting α-hydroxybutyrate (α-HB) in serum.Methods Electrospray ionization negative ion and multiple reaction monitoring mode were used to detect serum α-HB.The linearity,low limits of quantification,precision,recovery and interference of UHPLC-MS/MS were evaluated.The reference interval of this method was established in 130 serum samples (62 males and 68 females) from Shanghai East Hospital.Dixon method was used to judge the outliers and K-S test was used to analyze the data normality.The standard curve was scored by linear regression analysis.Results The total run time was 4 min of UHPLC-MS/MS method for the determination of α-HB.It has a good linear relationship in the range of 0.5-40.0 mg/L(r=0.999 4);the low limit of quantification was 0.5 mg/L;the in-batch and inter-batch coefficient of variation precision were less than 4.1％ and 6.3％,respectively;the recovery ranged between 95.8％ and 103.8％.Hemolytic samples (about 5 g/L hemoglobin),lipemic samples (about 12 mmol/L triglyceride),icteric samples (about 150 μmol/L total bilirubin) had no significant interference to the detection.The reference range of the apparent healthy population was 1.46-6.48 mg/L.Conclusions A method for the determination of serum α-HB by UHPLC-MS/MS was established.The method was simple,rapid,and could be used for the detection of clinical samples.

Objective: To determine the pharmacokinetics of ephedrine hydrochloride in rats after intragastric administration of Shegan mixtures. Methods:Shegan mixtures (1. 0 ml/100 g) were administered to each rat by gavage. Blood samples were collected after the administration. Plasma concentration of ephedrine hydrochloride was determined by LC-MS/MS. The pharmacokinetic parame-ters of ephedrine hydrochloride were obtained using the pharmacokinetic software. Urine and fecal samples were collected in 24 hours after the administration using metabolic cage to determine the recovery of ephedrine hydrochloride. Results: The pharmacokinetic pa-rameters of ephedrine hydrochloride were as follows:Tmax of (1. 30 ± 0. 23)h,T1/2 of (21. 17 ± 1. 35)h, Cmax of (278. 86 ± 46. 41)ng ·ml-1,AUC0~∞ of (1221.98 ±412.64)ng·ml-1 and Vc/F of (1.70 ±0.15)L. Totally 85.66% ephedrine hydrochloride could be recovered from urine in 24 hours after the administration;however, it was not detected in the fecal samples. Conclusion: Most of e-phedrine hydrochloride is excreted through kidney in 24h,therefore, Shegan mixtures can't cause the accumulation of ephedrine hydro-chloride in rats.

The "quality-oriented education" is closely integrated with the problem-based learning(PBL) teaching mode. In the teaching of Technologies for Microbial Detection course, the PBL teaching mode is the carrier, the student is the host, and the brainstorm guides the students to conduct "dialectical thinking", so as to cultivate the students' "innovation", "social morality" and "self-learning ability". We choose the interactive platform of "WeChat" and "MosoInk WebSite" to make up for the deficiency of PBL teaching in the large class, and comprehensively assess students' knowledge and skills, humanistic quality, and value judgment.

Environmental endocrine disrupting chemicals are a kind of exogenous chemicals that generally exist in the environment, and can disturb the endocrine homeostasis and adversely affect reproductive, immune, neurological, and other functions after entering the body, among which the damage to the reproductive system is the most significant one. Studies have confirmed that the long-term exposure to environmental endocrine disrupting chemicals have irreversible and harmful effects on primordial germ cell growth, reproductive organ development, and reproductive endocrine regulation, and also have obvious correlations with the occurrence and development of various reproductive system tumors. This paper reviewed various reproductive toxicities induced by common environmental endocrine disrupting chemicals in the developmental and reproductive stages, and associated mechanisms involved in the occurrence and development of reproductive system tumors.

Objective:To investigate the effects of persistent isolated hypothyroxinemia in the first and second trimester of pregnancy on complications and adverse outcomes of pregnancy.Methods:A retrospective analysis was conducted in 784 pregnant women including 111 cases of persistent isolated hypothyroxinemia in the first and second trimester of pregnancy and 673 pregnant women with normal thyroid function as control group. All women were registered and delivered in the Department of Obstetrics of our hospital from April 2016 to April 2017. The complications and adverse outcomes of pregnancy in the two groups were analyzed.Results:Age, body weight before pregnancy, body mass index(BMI), 1 h plasma glucose and 2 h plasma glucose during oral glucose tolerance test in persistent isolated hypothyroxinemia group were higher than those in control group( P<0.05), with increased incidence of anemia during pregnancy( P<0.05). However, there were no significant differences in the incidences of gestational diabetes mellitus and gestational hypertension between the two groups( P>0.05). No significant statistical differences were found in macrosomia, stillbirth, neonatal malformation, postpartum hemorrhage, acute delivery, premature delivery, fetal intrauterine development delay, and small full-term infants between the two groups( P>0.05). Logistic regression analysis showed that age( OR=1.1, 95% CI 1.0-1.1, P=0.002) and pre-pregnancy body weight( OR=1.0, 95% CI 1.0-1.1, P=0.046) were risk factors for the occurrence of persistent isolated hypothyroxinemia in the first and second trimesters of pregnancy. Persistent isolated hypothyroxinemia in the first and second trimesters was associated with anemia during pregnancy( OR=1.9, 95% CI 1.1-3.2, P=0.024). Conclusions:Pregnant women who are older and heavier before pregnancy should pay more attention to their thyroid function. Pregnant women with persistent isolated hypothyroxinemia in the first and second trimesters should be concerned for anemia.

Objective: To establish a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the detection of serum oleic acid (OA), and preliminarily evaluate the role of OA in insulin resistance (IR) of type 2 diabetes (T2DM). Methods: OA-［ 13 C 5 ］ was used as isotope-labeled internal standard, and the ion pairs of OA and OA-［ 13 C 5 ］ were 281.3/281.3 and 286.3/286.3, respectively. The ultrapure water was used as mobile phase A and methanol: acetonitrile (1∶1, v/v) as mobile phase B in a ZORBAX SB-Aq C18 reversed phase column. Meanwhile, the gradient elution system with a flow rate of 0.3 mL/min was used. According to the CLSI guidelines (EP15-A3), the reliability of the established method was evaluated by detecting the performance indicators such as precision, trueness, linear range, stability and carrying contamination rate. Serum OA levels were detected by the established HPLC-MS/MS method in 109 patients with clinically diagnosed T2DM and 100 healthy controls. The insulin resistance index (HOMA-IR) was calculated to evaluate IR, and the relationship between OA and IR was further analyzed. Results: The established HPLC-MS/MS method for the detection of serum OA had good specificity and linearity in the range of 10-1 000 μmol/L (y=0.007 55x+0.004 83,r=0.997 7), and the low limit of quantification (LLOQ) was 10 μmol/L. It also had good precision, and the within-run coefficient of variation (CV) and total CV were not more than 1.62% and 1.73%, respectively, indicating that the method was suitable for the detection of serum OA. The serum OA levels in T2DM patients ［(425.58 ± 220.17) μmol/L］ were significantly higher than that in the healthy controls ［(113.20±58.00) μmol/L］, and serum OA levels were significantly correlated with HOMA-IR in T2DM patients and healthy controls. The area under the receiver operating characteristic (ROC) curve (AUC) of OA for the diagnosis of IR was 0.689. When the cut-off value identified by Youden index was 235.8 μmol/L, the sensitivity and specificity were 70.4% and 63%, respectively. When OA combined with fasting blood glucose (FBG) to diagnose IR, the AUC increased to 0.806, which was significantly higher than that of OA (P＜0.05). Conclusion A scientific and efficient HPLC-MS/MS method for the quantitative detection of serum OA is established successfully, which provides a reliable method for the dynamic monitoring of the changes of OA levels in the patients with metabolic diseases.