Background Thyroid associated ophthalmopathy (TAO) is an autoimmune disease.Current research on the pathogenesis focuses on common autoantigen.Insulin-like growth factor-1 receptor (IGF-1R) is necessary for the function of IGF-1,also IGF-1 plays an important role in signaling pathway of thyroid stimulating hormone receptor (TSHR).Objective This study was to investigate the effects of IGF-1 on the proliferation,expression of IGF-1R and TSHR on cultured orbital fibroblasts (OFs) derived from TAO.Methods Human orbital tissue was obtained from 17 TAO patients who received orbital adipectomy and 4 normal controls who received cosmetic surgery in West China Hospital from March 2016 to June 2016.OFs were cultured by explant culture with DMEM/F12 containing 5％ fetal bovine serum and identified by immunochemistry.The OFs were treated with different concentrations of IGF-1.IGF-1 at different concentrations (0,50,100,125 μg/L) was added into the medium,respectively,and the proliferation of the cells (absorbancy) was detected by MTS.The percentages of IGF-1R and TSHR expressions in the cells were assayed by flow cytometry.Results Cultured cells appeared to be spindle-like in shape and grew well with abundant cytoplasm.The characteristics of the cells derived from TAO patients were consistant with normal ones.The cells showed the positive response for vimentin and absent respose for desmin,S-100,myoglobin and cytokeratin.The proliferative values of OFs were gradually elevated with the increase of IGF-I dose in both TAO group and normal group (Fgroup =219.639,P＜0.001;F ion =17.752,P＜0.001) with the optimal effects in 100 μg/L IGF-1.The expression levels of IGF-1R in the OFs were (0.009 1 ±0.008 7)％,(0.095 3±0.023 3) ％,(0.083 7±0.022 7) ％ and (0.070 9 ± 0.024 1) ％ in the TAO group,and those in the normal group were (0.0023± 0.0006)％,(0.0093±0.0012)％,(0.0073±0.0015)％ and (0.0083±0.0012)％ after treatment of 50,100,125 μg/L IGF-1.The expression levels of IGF-1 R were significantly higher after treatment of 50,100 and 125 μtg/L IGF-1 than those treatment of 0 μg/L IGF-1 in both TAO group and normal group,and the expression levels of IGF-1R in the OFs were significantly increased in the TAO group compared with the normal group (all at P＜0.05).No statistical difference was seen in the TSHR expression between the TAO group and normal group after treatment of 0,50,100 and 125 μg/L IGF-1 (Fgroup =0.133,P ＞ 0.05;F ion =0.004,P ＞ 0.05).Conclusions IGF-1 can promote the proliferation of OFs and up-regulate the expression of IGF-1R in OFs.However,IGF-1 dose not play a regulating effect on the expression of TSHR in OFs.

Objective To investigate the effect of the revision of Chinese Versions of Reading the Mind in the Eyes Test(RME) on the diagnosis of mild cognitive impairment (MCI) and mild Alzheimer's dementia (AD).Methods We compared performance on the cognitive test battery between 96 patients with MCI and 55 patients with AD and 95 control individuals.Neuropsychological tests, encompassing RME-T, Mini Mental State Examination (MMSE) and Auditory Verbal Learning Test (AVLT), were conducted on 96 patients with MCI, 55 patients with AD and 95 healthy control individuals.Completion rate of RME-T in the normal group and the total scores and each item score of RME-T were analyzed, and the feature between RME-T and RME-S (RME-short) was compared.Results The total score of RME-T was (23.7±4.7) in normal group, (19.9 ± 4.9) in MCI group,and (19.9±7.5) in AD group.There were significant differences in the total score of RME-T among the three groups (t=13.9, P＜0.01).The differences in the total score of RME-T between normal group and AD group, between normal group and MCI group were also significant (both P＜0.01).RME-S was composed of 20 items after rejecting 16 items of RME T whose project completion rate was lower than 50％ in the normal group and in which there was no significant difference among normal, MCI and AD groups.The correlations between the total score of RME-T and 3 item scores of RME-T were not significant (P＞0.05).However, the total score of RME-S was highly correlated with each item score of RME-S (P＜0.01).The total score of RME-T and RME S were highly correlated with each total score of the cognitive test battery (P＜0.01).With the normal and MCI groups, normal and AD groups as the subjects, the receiver operating characteristic (ROC) curve analysis found that AUC(RME-S) was＞ AUC(RME-T) in normal and AD groups (P＜0.01), whereas, AUC(RME-S) was＜AUC(RME-T) in normal and MCI groups (P＞0.05).Conclusions RME has good reliability and validity, and RME-S is better than RME-T in internal consistency, which is more suitable for identifying mild cognitive impairment and Alzheimer's disease in Chinese population, and it is worthy of further popularization and application.

Objective To establish the Logistic regression model by reporting and data system version 2 (PI-RADS v2)and prostate specific antigen (PSA),and to evaluate the diagnostic efficiency in transition zone prostate cancer (PCa).Methods MRI and PSA data of 33 patients with PCa and 54 patients with non-PCa confirmed by pathology were analyzed retrospectively.The PI-RADS v2 was used to evaluate the risk of 2 groups (from low to high as 1 to 5 points).Total PSA (t-PSA),free to total PSA ratio (f-PSA/t-PSA),PSA density (PSAD) and PI-RADS v2 scores were compared between 2 groups.The Logistic regression models were established with parameters which were significantly different between 2 groups.The Logistic regression was divide into three protocols:PI-RADS v2-+ t-PSA (A),PI-RADS v2 + f-PSA/t-PSA (B),PI-RADS v2+PSAD (C).The ROC curves were constructed by the new parameters Logit (P) and PI-RADS v2 scores for assessing the diagnostic efficiency.Results The t-PSA,f-PSA/t-PSA,PSAD and PI-RADS v2 scores had significant differences between the 2 groups (all P＜0.01).Predictive multivariate model of A,B,C was established as Logit (P)=-8.682+1.507 PI-RADS v2+0.234 t-PSA (x2=65.993,P＜0.01),Logit(P)=-5.425+1.906 PI-RADS v2 13.921 f-PSA/t-PSA (x2 =65.993,P＜0.01),Logit(P)=-7.534+1.045 PI-RADS v2+13.318 PSAD (x2 =74.036,P＜0.01),their area underthe curve (0.945,0.919,0.960) were all higher than that of PI-RADS v2 score (0.861,all P ＜0.01).The protocol C had the best diagnostic efficiency,and the sensitivity and specificity were 87.88 ％ and 92.59 ％.The sensitivity and specificity of PI-RADS v2 score were 87.88％ and 77.78％.Conclusion The diagnostic efficiency of the Logistic regression model which includes the PI-RADS v2 score and PSA are superior to the PI-RADS v2 score alone for transition zone PCa,which can provide a reliable basis for patients whether need biopsy or not.

Objective To compare the display effect of contrast enhanced MR angiography (CE-MRA) and CTA in evaluating blood supply of early stage cervical cancer.Methods CE-MRA and CTA before the operation of 30 patients with pathologically confirmed early stage cervical cancer were analyzed retrospectively.The origins and continuity of bilateral uterine arteries and the conspicuity of bilateral decending branch of uterine arteries between CE-MRA and CTA were evaluated.Results Fifty uterine arteries could classify as origins clearly visible and vessels clearly visible without discontinuity,5 uterine arteries as origins clearly visible but vessels unclearly visible on both CEMRA and CTA.Both CE-MRA and CTA were conformed well in evaluating the origin and continuity of uterine arteries (Kappa=0.80,P＜0.05).The conspicuity of decending branches for CE-MRA was 55.00％ (33/60),which was lower than that of CTA (73.33％ [44/60]),which had statistically significant difference between CE-MRA and CTA (x2 26.22,P＜0.01).Conclusion For early stage cervical cancer,CE-MRA and CTA can both provide a good performance in evaluating the origin and continuity of uterine arteries,CTA reveals advantage over CE MRA in detecting conspicuity of decending branch of uterine arteries.

Objective To explore the anti-inflammatory and antipruritic effects of Chushizhiyang ointment in a mouse model. Methods A total of 40 male 8-week-old BALB/c mice were included in this study, and randomly and equally divided into 4 groups. A mouse model of atopic dermatitis (AD)was established in three groups of mice by repeated application of 2,4-dinitroflurobenzene (DNFB)to shaved abdominal skin for sensitization and to shaved dorsal skin for stimulation. After establishment of the AD model, the three groups were topically treated with sodium chloride physiological solution (model group), hydrocortisone cream (hydrocortisone group)and Chushizhiyang ointment (Chushizhiyang group)respectively for 14 consecutive days. The remaining group receiving no sensitization or treatment served as the normal control group. All the mice were sacrificed 12 hours after the final treatment, and the dorsal skin of mice was resected followed by the determination of skin thickness and weight as well as hematoxylin-eosin(HE)staining and toluidine blue staining for the counting of leukocytes and mast cells respectively. Moreover, enzyme-linked immunosorbent assay(ELISA)was performed to measure the levels of interferon-gamma(IFN-γ), tumor necrosis factor alpha(TNF-α), interleukin 4(IL-4)and IL-5 in dorsal skin lesions. In addition, a local skin itching model was induced by histamine phosphate in Hartley guinea pigs, which was used to explore the effect of Chushizhiyang ointment on itch thresholds. Results Compared with the model group, both the Chushizhiyang group and hydrocortisone group showed reduced thickness and weight of dorsal skin in mice (all P < 0.01), numbers of infiltrating lymphocytes and mast cells (all P < 0.01)and levels of IFN-γ, TNF-α, IL-4 and IL-5 in skin lesions (P < 0.05 or 0.01)on day 15 after the start of treatment. The thickness and weight of dorsal skin in mice were significantly decreased in the hydrocortisone group (P <0.01), but experienced no significant changes in the Chushizhiyang group compared with the normal control group. Additionally, Chushizhiyang ointment could significantly increase itch thresholds in guinea pigs induced by histamine phosphate(P < 0.01). Conclusions Chushizhiyang ointment can significantly inhibit DNFB-induced AD in mice, likely by restoring the balance between Th1 and Th2 type cytokines. Moreover, Chushizhiyang ointment could markedly relieve itching induced by histamine phosphate in guinea pigs.

Objective To investigate the relationship between pelvic asymmetry and Crowe classification of unilateral developmental dysplasia of the hip in adults. Methods According to the inclusion criteria, 100 cases of unilateral DDH in adults were collected, including 78 females and 22 males, with an average age of 56.3 years old (range, 21-79 years). In terms of Crowe classification, there were 30 cases of Crowe typeⅠ, 24 cases of Crowe typeⅡ, 23 cases of Crowe typeⅢ, and 23 cases of CroweⅣ. The normal?side and affected?side pelvic vertical height (the distance from the highest point of the ischial tuberosity to the iliac crest peak) were measured via the radiogram of anteroposterior pelvis. Furthermore, we compared the affected?side pelvic vertical height with the normal?side and analyzed the relationship between Crowe classification and the difference in pelvic verti?cal height. Results The average pelvic vertical height was 23.38±1.46 cm in the normal?side and 22.41±1.38 cm in the affected?side. The average D?value of pelvic vertical height between two sides is 0.98 ± 0.76 cm. From Crowe typeⅠto Ⅳ, the average heights of the normal?side were 22.89±1.09 cm, 23.23±2.07 cm, 23.75±1.16 cm, 23.79±1.22 cm, while those of the affected?side were 22.70 ± 0.98 cm, 22.41 ± 1.98 cm, 22.47 ± 1.12 cm and 21.92 ± 1.32 cm. The average D?values in each group were 0.19 ± 0.37 cm, 0.82±0.43 cm, 1.28±0.32 cm, 1.87±0.59 cm. The differences between the normal?side and the affected?side in each type were statistically significant. The average D?value increased significantly with Crowe type increased, and there was a positive correla?tion between the D?value of bilateral pelvic vertical height and the degree of hip dislocation. Conclusion The pelvic asymmetry does exist in adult patients with unilateral developmental dysplasia of the hip. Moreover, the asymmetry has positive correlation with the degree of hip dislocation.

Objective To explore the feasibility of intravoxel incoherent motion DWI (IVIM-DWI) in differential diagnosis of hepatocellular carcinoma (HCC) and focal nodular hyperplasia (FNH).Methods A total of 407 patients with clinically-suspected HCC or FNH underwent conventional and dynamic enhanced MRI and IVIM-DWI,60 patients (40 cases of HCC,20 cases of FNH) were enrolled.Parameters of ADC,slow apparent diffusion coefficient (D),fast apparent diffusion coefficient (D*) and fraction of fast apparent diffusion coefficient (f) were obtained by monoexponential model and biexponential model respectively.Results The values of ADC,D,D* and f in FNH group were (1.60±-0.25) × 10-3mm2/s,(1.12±0.17)×10-3mm2/s,(44.89±18.23)× 10-3 mm2/s and (34.80 ± 9.68)％,and those in HCC group were (1.32 ± 0.21) × 10-3 mm2/s,(0.82±-0.21) × 10-3mm2/s,(49.82±20.11) × 10 3mm2/s and (28.72±13.84) ％,respectively.Significant inter-group differences were observed in ADC and D (both P＜0.001),however,there were no significant differences in D* and f (both P＞0.05).The areas under the ROC curve of D were 0.90,and taking D=0.96 × 10-3 mm2/s as cut-off value,the sensitivity and specificity of D in diagnosis of HCC were 84.44％ and 90.02％.Conclusion IVIM-DWI is useful to distinguish FNH from HCC,and the D value in biexponential model has the best diagnostic efficacy for differentiations.

Objective To study the effect of Buyang Huanwu Decoction (BHD) on adhesion molecules in vascular endothelial cells (VECs) of blood stasis rats. Methods Expression of ICAM-1, VCAM-1, PECAM-1, iNOS in vascular endothelial cells of blood stasis rats receiving different doses of BHD was observed by immunohistochemical method and RT-PCR.Results The expression of ICAM-1, VCAM-1, PECAM-1, iNOS was increased in model group, while was decreased by BHD. There was an increase tendency on the expression of molecules and significant dose-effect relationship along with the decrease of the BHD doses. Conclusion The increased expression of adhesion molecules in vascular endothelial cells of blood stasis rats could be reduced by BHD in a dose dependent manner.

AIM: To study the effects of xijiao dihuang decoction, a Chinese medicine, on the expression of adhesion molecules in vascular endothelial cells (VECs) of adrenine and hypothermia-treated rats. METHODS: The expression of VCAM-1, ICAM-1, PECAM-1, iNOS and their corresponding mRNA in VECs was assayed by immunohistochemistry analysis and RT-PCR, respectively. RESULTS: Immunohistochemistry analysis showed the expression of VCAM-1, ICAM-1, PECAM-1 and iNOS are higher in adrenine and hypothermia-treated rats than that in controls, xijiao dihuang decoction decreased the expression of VCAM-1, ICAM-1, PECAM-1 and iNOS in a dose-dependent manner in adrenine and hypothermia-treated rats. CONCLUSION: Xijiao dihuang decoction can down-regulate the expression of adhesion molecules in vascular endothelial cells of adrenine and hypothermia-treated rats.

Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.
Base Sequence ; Cloning, Molecular ; Genetic Vectors ; genetics ; Mutagenesis, Site-Directed ; methods ; Nucleic Acid Amplification Techniques ; Plasmids ; Polymerase Chain Reaction