Objective To study the effective methods for extraction of clenbuterol from biological material. Methods Clenbuterol in blood samples was extracted with 5 different methods,the extracts from various extraction methods were determined by gas chromatography-mass spectrometry(GC-MS?雪. The efficiency of 5 extraction methods was compared with each other. Results The standard curve showed good linear relationship with a range from 0.01-10.0 mg/L clenbuterol hydrochloride(r=0.999 9?雪. The detection limit was 0.005 mg/L. After adding 1.0 ?g/ml clenbuterol hydrochloride into blood sample,the blood sample was immersed and extracted in 0.01 mol/L HCl and absolute alcohol.The extracts were extracted by chloroform at pH value of 11. The average recovery and RSD of this method were 96.5% and 5.21% respectively(n=6?雪. Conclusion The method showed easy operation and reliable results ,it was suitable for determination of clenbuterol in biological material without deviation.

Objective To observe the expression of apoptosis and invasion genes in different tissues of laryngeal lesions,and to detect the content of proliferation related protein and proliferation inhibition gene.Methods In our hospital,the clinical diagnosis of laryngeal carcinoma,precancerous lesions and vocal polyps in 88 cases of laryngeal carcinoma,surgical excision of lesion specimens cut from the amount of Pro apoptotic genes and promote invasion and proliferation related protein,gene the content of tissue proliferation inhibition gene detection.Results The laryngeal carcinoma group of Pro apoptotic genes nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) and large tumor suppressor gene 1 (LATS1) were all significantly lower than lesion group and polyp group (P ＜ 0.05).Laryngeal carcinoma invasion promoting gene tumour necrosis factor receptor associated factor 6 (TRAF6),and cellular FADD-like interleukin1βconverting enzyme inhibitory protein (c-FLIP) content were significantly higher than that of laryngeal pre cancerous lesions and vocal polyps (P ＜ 0.05).The proliferation related protein Cyclin-dependent kinase 6 (CDK6) and E2F1 in laryngeal carcinoma were significantly lower than that in the lesion and polyp group,and cyclin D1 (CCND1),Bmi-1,and Livin contents were significantly higher than that in the lesion group and the polyp group (P ＜ 0.05).The levels of differentiated embryo-chondrocyte expressed gene 1 (DEC-1),IκB kinase 16(IKK16),large tumor suppressor gene 1 (LAST-1),receptor-interacting proteins-1-(RIP-1),and c-myc promoter binding protein-1 (MBP-1) in the tissue of laryngeal carcinoma were significantly lower than those in the lesion group and polyp group (P ＜ 0.05).Conclusions The laryngeal carcinoma invasion promoting gene was higher than that in other tissues,inhibition of gene content was lower than that of other tissues proliferation related protein gene,apoptosis and proliferation,apoptosis and invasion of lesions in gene expression level,proliferation related protein and proliferation inhibition of gene content and laryngeal squamous cell carcinoma were related to laryngeal disease prevention significance of detection.

Objective To explore the role of human papillomavirus (HPV) in squamous cell carcinoma of the larynx.Methods A cohort of 152 laryngeal squamous cell carcinoma (LSCC) patients,40 premalignant lesions of the larynx,and 40 vocal fold polyp patients was evaluated for the expression of HPV16 E7 protein by immunohistochemistry (IHC).E7 protein expression was correlated with clinicopathological features and outcomes.Results The expression of E7 protein in vocal fold polyp was negative,and it's positive expression in precancerous lesions and laryngeaI carcinomas was 2.5％,and 32.9％,respectively.There were no other significant clinical or demographic differences between pl6-positive and-negative cases.There was no difference in overall survival (OS) between E7-positive and-negative patients with 5-year survival of 79％ and 66％ for p16 positive and negative patients,respectively (P =0.581).There was no statistically significant difference in failure-free survival (FFS) with 5-year FFS of 57.8％ and 42.3％ for p16 positive and negative patients,respectively (P =0.579).Conclusions E7 expression is infrequent in laryngeal carcinomas.There are no statistically significant correlations between E7 IHC status and OS or FFS.

Objective To investigate the effects of oestrogen-receptor(ER), progesterone-receptor(PR), vascular endothelial growth factor(VEGF) and kinase insert domaincontaining receptor(KDR) in uterine bleeding induced by intrauterine device(IUD). Methods Immunohistochemical method was used to detect the expressions of ER, PR, VEGF and KDR in endometrium from IUD bleeding group and normal control group, respectively. Microvessel density(MVD) was evaluated by using anti factor Ⅷ-related antigen (F8-RA) antibody to mark stromal microvessels. Results The expressions of ER, VEGF and KDR were significantly higher in IUD-induced endometrium bleeding group than those in control group(P0.05). MVD was significantly higher in IUD-induced endometrium bleeding than that in normal endometrium(P

Objective To summarize the treatment experience of refractory systemic juvenile idiopathic arthritis (JIA) by tocilizumab, and to explore the cost-effective treatment. Methods The clinical data of 6 pediatric patients with refractory systemic JIA treated by tocilizumab from 2014 to June 2016 were retrospectively analyzed in the aspects of course and effectiveness of tocilizumab, steroid reduction, adverse reaction, and growth. Results The median age of the six patients (3 males and 3 females) was 6 years, and the course of disease were from 16 to 63 months. All patients were treated by other immunosuppressive agents or biological agents in addition to steroid and traditional anti-rheumatic drug therapy. The courses of tocilizumab treatment were from 7 to 26 months and the median time was 9.5 months. All 6 patients responded to tocilizumab and achieved the clinical remission at different time. After the induced remission, the interval of the treatment intervention was increased from 2 weeks up to 4 weeks in 3 cases, and no disease activity was observed. Except one case, another 5 cases reduced and stopped the use of hormones at 5.8 months after tocilizumab treatment. After hormones was reduced and discontinued, the growth was improved. All 6 patients had no serious adverse reactions. Conclusions Tocilizumab is safe and effective for patients with refractory JIAs. The steroid can be reduced in short time to improve growth. After remission is induced, the interval of the treatment intervention could be prolonged.

Objective T o explore the relationship betw een the change rules of volatile organic com pounds (V O C s) in rat m uscle and postm ortem interval (PM I). Methods A total of 120 healthy rats w ere divided random ly into 12 groups (10 for each group). A fter the rats w ere sacrificed by cervical dislocation, the bodies w ere kept at (25±1)℃. R at m uscle sam ples w ere separately obtained at 12 PM I points, including 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 d. T he V O C s in rat m uscles w ere collected, detected and ana-lyzed by headspace solid-phase m icroextraction (H S-SPM E ) coupled to gas chrom atography-m ass spec-trom eter (G C-M S ). Results In total, 15 species of V O C s w ere identified, including 9 arom atic com-pounds, 3 sulfur com pounds, 2 aliphatic acids and 1 heterocyclic com pound. T he species of V O C s in-creased w ith PM I: no species w ere detected w ithin 1 day, 3 species w ere detected on day 2, 9 on day 3, 11 on day 4, 14 from day 5 to 7, and 15 from day 8 to 10. T otal peak area of 15 species of V O C s w as significantly correlated to PM I (adjusted R2=0.15-0.96): the regression function w as y=-17.05 x2+164.36 x-246.36 (adjusted R2=0.96) from day 2 to 5, and y=2.24 x+101.13 (adjusted R2=0.97) from day 6 to 10. Conclusion T he change rules of V O C s in rat m uscle are helpful for PM I estim ation.

Objective:To explore the effect of case-based learning (CBL) guided by ICF (International Classification of Functioning, Disability and Health) in rehabilitation practice teaching.Methods:Fifty-eight rehabilitation undergraduates who had practiced in the Department of Rehabilitation Medicine in The First Affiliated Hospital of Chongqing Medical University from February 2019 to December 2019 were randomized into observation group and control group. The observation group adopted ICF-guided CBL method for practice teaching, while the control group adopted traditional teaching methods. At the end of the internship, all the students were assessed in terms of their theoretical knowledge, case analysis and clinical practice ability. At the same time, questionnaires were used to conduct a satisfaction survey on the teaching method, and the results of the two groups were compared and analyzed by ttest and rank-sum test through SPSS 25.0. Results:The scores of the observation group were higher than those of the control group, with statistical significance ( P<0.05) . In the satisfaction survey, the observation group was superior to the traditional teaching method in terms of self-thinking ability and learning initiative, with statistical significance ( P<0.05). Conclusion:ICF-guided CBL method can strengthen students' independent thinking ability and learning initiative, and improve the students' clinical comprehensive ability of rehabilitation treatment.

BACKGROUND:The use of mesenchymal stem cels in the field of tissue engineering for osteoarticular injury repair is a very promising tool since these cels are readily expandable and able to differentiate into chondrocytes. Abundant evidence suggests that microRNAs play critical roles in chondrogenic differentiation of mesenchymal stem cels.
OBJECTIVE:To observe the chondrogenic effect of human bone marrow mesenchymal stem cels transfected with lentiviral vectors bearing miR-221-3p/222-3p inhibition, thereby provding new strategies for cartilage injury.
METHODS: miRNA microarray technology was applied to detect microRNAs expression profiles at three different stages of chondrogenic differentiation induction after transforming growth factor-β3 treatment and verified by real-time fluorescence quantitative PCR (RT-qPCR). Human bone marrow mesenchymal stem cels were infected with lentivirus bearing miR-221-3p/222-3p inhibition. After co-suppressing the expression of miR-221/222-3p, cel counting kit-8 was used to determine the cel proliferation, the differentiation of bone marrow mesenchymal stem cels towards chondrocytes was verified by type II colagen protein expression through immunohistochemistry and glycosaminoglycan accumulation was also elevated by sarranine O staining. RT-PCR was used to detect type II colagen and aggrecan mRNA expression at 21 days of chondrogenic induction.
RESULTS AND CONCLUSION: The expression of miR-221-3p/222-3p was inhibited after Lv-miR221-3p/222-3p inhibition co-transfected into bone marrow mesenchymal stem cels. microRNA microarray and RT-qPCR results showed that the expression of miR-221-3p/222-3p was declined significantly at the anaphase of chondrogenic differentiation. The expression levels of chondrogenic markers, Aggrecan and type II colagen were significantly increased in the miR-221-3p/222-3p inhibition group and cel proliferation was also inhibited significantly compared with non-transduced cels or transduced with the empty lentiviral vector group. miR-221-3p/222-3p knockdown in bone marrow mesenchymal stem cels could inhibit proliferation but promote chondrogenic differentiation of bone marrow mesenchymal stem cels.

Objective: Attention deficit hyperactivity disorder (ADHD) is a polygenic neurodevelopmental disorder with significant gender differences. The sexual dimorphism of ADHD may be associated with estrogen acting through estrogen receptors (ESR). This study investigates the impact of ESR gene polymorphism and its interactions with neurodevelopmental genes on ADHD susceptibility. Methods: The study compared genotyping data of single nucleotide polymorphisms in ESR1 and ESR2 in 1,035 ADHD cases and 962 controls. The gene-gene interactions between ESR genes and three neurodevelopmental genes (brain-derived neurotrophic factor [BDNF], synaptosomal-associated protein of 25 kDa gene [SNAP25], and cadherin-13 [CDH13]) in ADHD were investigated using generalized multifactor dimensionality reduction and verified by logistic regression analysis. Results: The G allele of rs960070/ESR2 (empirical p=0.0076) and the A allele of rs8017441/ESR2 (empirical p=0.0426) were found significantly higher in ADHD cases than in the controls but not in male or female subgroups. Though no difference was found in all subjects or females, the A allele of rs9340817/ESR1 (empirical p=0.0344) was found significantly higher in ADHD cases than controls in males. We also found genetic interaction models between ESR2 gene, neurodevelopmental genes and ADHD susceptibility in males (ESR2 rs960070/BDNF rs6265/BDNF rs2049046/SNAP25 rs362987/CDH13 rs6565113) and females (ESR2 rs960070/BDNF rs6265/BDNF rs2049046) separately, though it was negative in overall subjects. Conclusion The ESR gene polymorphism associates with ADHD among Chinese Han children, with interactions between ESR genes and neurodevelopmental genes potentially influencing the susceptibility of ADHD.

Objective To investigate the heterogeneity of chemosensitivity in colorectal cancer using an ATP-tumor chemosensitivity assay (ATP-TCA) and the feasibility of individual chemotherapy.Methods An ATP-TCA were used to determine the effect of 16 single or combined cytotoxic drugs in surgical specimens from 50 patients with colorectal cancer.Results There were considerable differences in chemosensitivity between individuals.The most active single drugs in the assay was identified as Navelbine, Hydroxycamptothecin, 5-Fluorouracil and Paclitaxel; 34.1%, 31.6%, 27.6% and 24.3% of specimens showed sensitivity to them, respectively.5-Fluorouracil+Mitomycin+Aytarabine was found to be the most effective combination, for 100% (11/11)specimens were sensitive to this regimen.5-Fluorouracil+Cisplatin+Adriamycin and Gemcitabine+Cisplatin were moderately active regimens.Conclusions There was the heterogeneity of the in vitro chemosensitivity in colorectal cancer.The use of the ATP-TCA provides a method of selecting appropriate anti-cancer drugs in colorectal cancer.