Objective To develop a diagnostic test based on indirect immunofluorescence assay(IFA) to detect special antibodies in the serum of SARS patients, thus to provide a reference material for confirmation of the clinical diagnosis of SARS. Methods SARS coronavirus GZ01 and BJ01 strains isolated in our laboratory were used to infect Vero E6 cells. When CPE reached 25%, cells were trypsinized and transferred to 10 well slides in a quantity of 40?l with a cell density of 2?10 7 /ml. After 4 hour incubation at 37℃,the slides were fixed with acetone, and IFA was used to detect antibodies in serum samples, which were obtained from 154 SARS patients and 14 non SARS patients with respiratory disease, as well as 100 healthy volunteers. Results IFA method for detecting antibodies of SARS coronavirus was developed. Sera from one hundred and forty two out of 154 clinically diagnosed patients were IFA positive, with a positive rate of 92 3%. Sera from 14 non SARS patients with respiratory disease and 100 healthy persons were all IFA negative. Conclusion The IFA method we developed was sensitive and specific in detecting SARS antibodies in serum, and was a reliable test for laboratory diagnosis of SARS coronavirus.

Prognostic factors of differentiated thyroid carcinomas ( DTC ) were analyzed and the related literatures were systematically reviewed in order to justify the diagnostic and therapeutic modalities for improving the patient′s survival.150 patients ( female,n =113 ; male,n =37 ) with histopathologically diagnosed DTC,including papillary thyroid carcinoma ( n =131,87.3％ ) and follicular thyroid carcinoma ( n =19,12.7％ ),were postoperatively followed up and their clinical data were retrospectively reviewed.Patients were followed up for 4.15-31 years wherein 140 patients( 93.3％ ) survived but with relapse in 30 patients( 20.0％ ),and 10 patients( 6.7％ ) died.Surgical procedures consisted of near-total or subtotal thyroidectomy ( n =83,55.3％ ),partial thyroidectomy ( n =64,42.7％ ),and total thyroidectomy ( n =3,2.0％ ).Out of the patients receiving lymph node dissection ( n =63 ),45 patients( 71.4％ ) had detectable lymph node metastasis.Age of onset,tumor size at initial visit,and early metastasis showed the statistically significant difference between mortality group and survival group (P＜ 0.05 ),as well as between relapse group and relapse-free group( P＜0.05 ).Age of onset,tumor size at initial visit,and early metastasis are prognostic factors for DTC.

Objective To compare the efficacy of Graves disease animal models induced by thyroid stimulating hormone receptor (TSHR) plasmid DNA (pcDNA3.1-TSHR) and by TSHR A subunit recombinant adenovirus(Ad-TSHR289),and to investigate the influence of duration for preparing animal model induced by Ad-TSHR289 on Graves hyperthyroidism and its related indices.Methods The plasmid group and the adenovirus group were set up respectively.The plasmid group:21 female BALB/c mice were randomly divided into model group (n =12) and control group (n =9).The model group were injected intradermally with pcDNA3.1-TSHR 50 μg,once every 3 weeks,totally 3 times.Then 4 weeks after the last immunization,the mice were euthanized to obtain blood for testing TSHR antibody (TRAb),total T4,and thyroid tissue for histological examination.The controls were injected with the same dose of pcDNA3.1 in the same way.The adenovirus group:52 female BALB/c mice were divided into 10-week model group (n =8),14-week model group (n =10) and 18-week model group (n =8),and the respective controls (n =8,n =10,n =8) were set up.All model groups were injected intramuscularly with Ad-TSHR289,three times at three weekly intervals.Then the mice were euthanized at 4,8 and 12 weeks to test TRAb,total T4 level and to observe the change of thyroid histology.The controls were treated with the same dose of Ad-lacz in the same way.Another 8 mice were scheduled to test the dynamic variation of TRAb before and after the 3 times immunization.Results In the plasmid model group,only two of 12 mice developed weak antibody responses against TSHR,and no elevated total T4 level and no hyperplasia changes of thyroid were observed.In the 10-week model group,all mice had high level TRAb [(807.65 ± 136.33)U/L,Six-eighths mice had hyperthyroidism exhibited hyperplasia changes.In the 14-week model group,the TRAb level [(650.12 ± 192.88) U/L]and the incidence of hyperthyroidism (3/10) were lower than those in 10-week group.Histologically,the degree of thyroid hyperplasia lightened to a small extent,but its positive rate did not decline.In the 18-week model group,only 2 of 8 mice displayed slightly elevated TRAb level,and no mice showed increased total T4 level.Additionally,thyroid tissues of 2 mice were mildly abnormal.Compared with the model groups at different time,the change of antibody levels of the mice for TRAb dynamic observation exhibited the similar trend.Conclusions Being good at repeatability and high incidence of hyperthyroidism,the animal model of Graves disease induced by Ad-TSHR289 is still an ideal research tool presently.The duration of model ean be maintained 18 weeks,and 10 weeks is the best period to snstain characteristic of Graves disease.

Objective To investigate the feasibility of inducing neonatal immunotolerance against Graves'disease by gene TSH receptor (TSHR) 289 and its possible mechanism.Methods Neonatal (0-24 h) female BALB/c mice were divided into intraperitoneal injection group,intramuscular injection group,model group,and normal control group.The intraperitoneal group and the intramuscular group were further divided into low-dosage,middle-dosage,high-dosage tolerance groups,and the coresponding control groups.The tolerance groups and the controls were intraperitoneally or intramuscularly pretreated with low-dosage( 1×106 particles),middle-dosage( 1 × 108particles),high-dosage( 1 × 1010 particles)of Ad-TSHR 289 or Ad-lacz respectively.6 to 7 weeks later,the normal control group received intramuscular injection with Ad-lacz; the other groups were immunized with Ad-TSHR289,three times at 3 weeks interval.10 days after the first immunization,serum TRAb was detected.4 weeks after the last immunization,serum TRAb,TT4,splenic CD4 + CD25 + Foxp3/CD4 + were tested,and the thyroid tissues were examinated histologically.Results Ten days after the first immunization,no antibody response against TSHR was detected in the two high-dose tolerance groups,but the TRAb titer in respective controls was significantly higher( P＜0.05 ).4 weeks after the last injection,in high-dose tolerance groups,only 1/10 of mice immunized by intraperitoneal or intramuscular injection elicited anti-TSHR antibody,and no mice immunized intraperitoneally had elevated serum TT4.Two of ten mice challenged intramuscularly showed slightly increased TT4 levels,but the respective controls displayed a strong antibody response( P＜0.01 ) and elevated TT4 level ( P＜0.05 ).The similar percentages of high TT4 and thyroid hyperplasia were found in all groups.Additionally,the frequencies of CD4+CD25 +Foxp3/CD4+in two high-dose tolerance groups were significantly increased as compared to those in controls( P＜0.05 ).The incidence of Graves' disease in the other groups by intraperitoneal or intranuscular injections was not statistically different from those in the corresponding control groups and the model group.Conclusions The immune tolerance against Graves'disease is induced in neonatal mice by either intraperitoneal or intramuscular pathway with specific antigen of TSHR 289,carried by adenovirus vector,and then inhibits Graves' disease in adults. Stimulation with the high-dosage antigen is liable to induce immune unresponsiveness.CD4 + CD25 + Foxp3 +T cells may play an important role in the induction and maintenance of tolerance.

Objective To investigate the effect of mice gender on the TSH receptor antibody(TRAb)titers, the levels of TT4,and the degree of thyroid hyperplasia by establishing an animal model of Graves′ disease in male and female BALB/c mice. Methods Male and female BALB/c mice were immunized with recombinant adenovirus expressing TSHRA subunit(Ad-TSHR289)to induce Graves′ disease. Animals were injected 3 times at intervals of 3 weeks. All mice were sacrificed 4 weeks after the last injection to obtain blood for measurement of TSHR antibody titers and TT4evels, and thyroid glands for histological examination. Results TRAb positive rates were 100% both in female or male mice. No significant difference was observed in titers of TRAb between them. The incidence of hyperthyroidism in female mice was higher than that in male mice, being 75.0% and 41.7% respectively. There was statistical difference in levels of TT4between females and males(P＜0.01). Mice with high TT4exihibited marked thyroid hyperplasia. Conclusion Despite TSHR antibodies were similar between female and male mice, the incidence and degree of hyperthyroidism showed sex bias in Graves′s animal model. The results indicated that it was easier to induce model in females than in males by immunizing BALB/c mice with Ad-TSHR289.

Objective To establish the model of distraction osteogenesis for rabbit femur bone defect, and to observe the effect of icariin on regenerate ossification after distraction osteogenesis, thus to find a method for promoting regenerate ossification after distraction osteogenesis. Methods After the rabbit model of bone defect had been established successfully, the rabbits were equipped with distraction device. And then the 24 modeled rab bits were randomly divided into 2 groups. The experimental group was injected with icariin extract of Herba Epimedii into the interspace of bone distraction, and the control group was given local injection of recombinant human bone morphogenetic protein-2 (rhBMP-2, 100μg/kg) . On week 0, 1, 4 and 6 after the resting period, X-ray photography was carried out in both groups. On week 6 after distraction osteogenesis, the bone specimens of distraction osteogenesis region in both groups were observed by micro-computerized tomography ( CT) for the comparison of bone mass, and bone mineral content and mineral density of the newly-formed bone. Results The results of the features of imageology, and the statistical data of the bone mineral content and density showed that osteogenesis speed and osteogenic quality of the experimental group were superior to those of the control group. Conclusion The rabbit model of distraction osteogenesis for femur bone defect has been established preliminarily, and icariin can promote the speed and quality of regenerate ossification after distraction osteogenesis.

Objective To identify the infection and the replication of Tick-borne encephalitis virus(TBEV) in human neuroblastoma cells.Methods After being inffected with TBEV,the cell culture supernatant of human neuroblastoma cell line SK-N-SH was collected and assayed at different time points.Byusing real-time RT-PCR and plaque assay to measure the titer of virus in the supernatant,the replication andproliferation of TBEV in human neuroblastoma cell was identified.Meanwhile,the morphological change of SK-N-SH after TBEV infection was also visualized by observation under microscope and immunmquorescenceassay.Results Real-time RT-PCR and plaque assay both demonstrated that TBEV could replicate effectively in SK-N-SH cells,the peak titer could reach 2.92× 107 PFU/ml on 3 days post-inoculation.And significant morphological change occured on infected SK-N-SH cells after 2 days post inoculation.By immunofluorescence assay,the virus particles could be detected and visualized.Conclusion TBEV can replicate andproliferate effcctively and cause significant cell morphological changes in human neuroblastoma cell SK-N-SH,which demonstrated that SK-N-SH could be a suitable cell model for TBEV culture.

According to the training requirement of the biological-psycho-social medical model to the clinical medical students,in order to promote the comprehensive improvement of medical students' professional knowledge,hands-on ability and human qualities,we design the course of introduction to clinical medicine.Through six major functional modules such as basic professional quality,clinical diagnosis basic technology,basic skills related to the operation,the new progress in clinical medicine and technology,clinical nursing and medical relationship,and medical information management,we build up the core content of integrated course of introduction to clinical medical.In this way,the clinical and related basic knowledge and skills are integrated,the clinical course is closely connected with the basic curriculum,the medical and the humanities exchanges.Through this design,the foundation is laid out for the collaborative efforts of the organ-system of integrated curriculum reform.

Objective To investigate the correlation between the improvement of insulin resistance(IR)and T lymphocyte subsets in patients with type 2 diabetes mellitus(T2DM)after intensive insulin therapy.Methods A total of 160 patients with T2DM who were treated in the endocrinology department of our hospital from February 2020 to March 2023 were selected.They were divided into simple T2DM group(n=80)and T2DM combined with family history of diabetes mellitus(FHD)group(n=80)based on whether they had FHD.The changes in blood glucose,pancreatic islet cell function,T lymphocyte subsets,and inflammatory cytokine levels were compared before and after treatment between the two groups.Pearson correlation analysis was used to investigate the correlation between insulin resistance index(HOMA-IR)and T lymphocyte subsets,and multiple linear regression analysis was used to explore the influencing factors for IR in T2DM patients.Results After treatment,the FPG,2 hPG,and coefficient of variation(CV)of blood glucose were lower than before treatment in the T2DM group(P<0.05).In the FHD group,the FPG,2 hPG,bedtime blood glucose,and CV were lower after treatment than before treatment(P<0.05).After treatment,the FPG,2 hPG,and CV were lower in T2DM group than in FHD group(P<0.05).After treatment,FC-P,2 hC-P,HOMA-β and IL-10 were higher(P<0.05),while HOMA-IR,IL-21,IL-1β,CD3+,CD4+,CD8+,CD4+/CD8+were lower than before treatment in both groups(P<0.05).After treatment,FC-P,2 hC-P,HOMA-β and IL-10 were higher,while HOMA-IR,IL-21,IL-1β,CD3+,CD4+,CD8+,CD4+/CD8+were lower in T2DM group than in FHD group(P<0.05).Pearson correlation analysis showed that HOMA-IR was positively correlated with CD3+,CD4+,CD8+,CD4+/CD8+(P<0.05).Multiple linear regression analysis shows that BMI and CD8+are influencing factors for IR in T2DM patients.Conclusions Intensive insulin therapy can improve the islets β cell function inpatients with T2DM and IR.There is a correlation between IR and the increase of CD8+T lymphocyte level in patients.

Objective To construct a Nomogram prediction model based on body position exer-cise training for patients with chronic constipation.Methods A total of 327 patients with chronic constipation from March 2021 to March 2023 were divided into qualified group(n=279)and unqual-ified group(n=48)according to their performance in body position exercises.General clinical mate-rials of the patients were collected,and univariate and multivariate Logistic regression analyses were performed to identify risk factors for unqualified body position exercises in patients with chronic con-stipation;the R software was used to construct a Nomogram model for predicting unqualified body po-sition exercises in patients with chronic constipation,and the receiver operating characteristic(ROC)curve and calibration curve were applied to verify the discrimination and consistency of the Nomogram model.Results There were no significant differences in gender,education level,history of smok-ing,history of alcohol abuse,history of hypertension,history of abdominal surgery,history of colonoscopy,and correct medication administration ratio between the two groups(P＞0.05);the un-qualified group had higher age,longer duration of chronic constipation,and a higher proportion of di-abetes history compared to the qualified group(P＜0.05);age,duration of chronic constipation,and diabetes history were independent risk factors for unqualified body position exercises in patients with chronic constipation(P＜0.05).The constructed Nomogram prediction model demonstrated excellent discrimination(area under the curve was0.923,95％CI,0.888 to 0.949,with sensitivity and specificity of 87.50％and 81.00％respectively)and consistency(H-L goodness-of-fit test x2=2.246,P=0.973).The clinical value of this Nomogram model in predicting unqualified body posi-tion exercises for patients with chronic constipation was high when the high-risk threshold probability ranged from 0.06 to 0.80.Conclusion Age,duration of chronic constipation,and diabetes history are independent risk factors for unqualified body position exercises in patients with chronic constipa-tion,and the Nomogram model constructed based on these multi-factor results has high discrimination and consistency in predicting unqualified body position exercises for patients with chronic constipation.