Objective:To construct a recombinant adeno-associated virus vector rAAV2-PD-L1 and to investigate the biological efficiency of rAAV2-PD-L1-transfected vascular endothelial cells in co-stimulating secretion of cytokines by T cells.Methods: Mouse PD-L1 cDNA was amplified by RT-PCR from total RNA of mouse liver tissues and was cloned into shuttle vector pSNAV1;the products were then transferred into BHK21 cells by lipofectamine and rAAV2-PD-L1 was screened out.Mouse vascular endothelial cell line 2F-2B was infected with rAAV2-PD-L1 and were co-cultured with activated mouse T cells,and the IFN? content was identified by ELISA in the supernatant.Results: The sequences of PD-L1 cDNA and pSNAV-PD-L1 were confirmed to be correct.The recombinant rAAV2-PD-L1 was verified by PCR and SDS-PAGE analysis.The virus physical titer was 4?10~(12) virus genome/ml and the protein concentration was 0.355 mg/ml.There was a high expression of PD-L1 in mouse vascular endothelial cells infected with rAAV2-PD-L1.The content of IFN? in the culture supernatant was significantly decreased 48 hours after co-culture.Conclusion: The recombinant rAAV2-PD-L1 can infect vascular endothelial cells and inhibit secretion of IFN? by activated T cells through costimulation.

By reviewing experiment teaching,the primal problems and solutions of practical training in preventive medicine specialty are explored in order to provide some beneficial reference and help for experimental teaching.

Objective To analyze the accuracy,repeatability and feasibility of Ultrafast Doppler for renal artery ultrasonography.Methods One hundred and one cases were selected that were suspected to have renal artery disease and were successful of conventional and Ultrafast Doppler for renal artery ultrasonography,and 101 cases were grouped by age (≤40 years old,41-60 years old,≥61 years old),body mass index (BMI) (normal 18.5-23.9 kg/m2,overweight 24.0-27.9 kg/m2,obesity ≥28.0kg/m2) and whether there were the presence of renal artery stenosis (no significant renal artery stenosis and renal artery stenosis ＞60％).Each case was respectively examined by conventional and Ultrafast Doppler for renal artery ultrasonography in a random order.The consistency of Doppler parameters was tested.The duration of each Doppler study was compared and the feasibility of Ultrafast Doppler for renal artery ultrasonography was explored.The Doppler parameters included:renal artery peak systolic velocity (PSV),resistance index (RI),renal segmental artery acceleration time (T) and time consuming (△T).The concordence and △T of two Doppler method were compared.Results ① Ultrafast Doppler had good reproducibility,intraclass correlation coefficient (ICC) values were ＞ 0.6.② For renal artery ultrasonography,the successful number of cases examined by Ultrafast Doppler were more than those examined by conventional Doppler,but the difference was not statistically significant (P ＞0.05).③The Doppler parameters from all subjects and different groups showed a strong positive correlation between the two Doppler studies (P ＜0.05).④ UltraFast Doppler required a shorter time than conventional Doppler (P ＜0.05).⑤The △T of cases with different ages and with or without renal artery stenosis showed no statistically significant (P ＞0.05),however,their △T were increased with body mass index increasing (P＜0.05).Conclusions Ultrafast Doppler for renal artery ultrasonography has a high success rate,a good repeatability and consistency,and a shorter time consuming and simple operation than conventional Doppler.

B-Raf kinase (BRAF) gene is a driver mutation,and is an effective target in the treatment of non-small cell lung cancer (NSCLC).Studies have shown that BRAF inhibitors are effective for treatment of NSCLC with BRAF mutant.It is important to understand the clinicopathologic features and the research progress of BRAF inhibitors for the individual treatment of NSCLC.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs)and anti-angio-genic drugs have individually demonstrated clinical benefit in the treatment of patients with advanced non-small cell lung cancer (NSCLC).Recent studies demonstrate that the combination of anti-EGFR and anti-angiogene-sis can more significantly enhance clinical benefit,and even can remit EGFR-TKIs resistance in the treatment of advanced NSCLC.According to the different kinds of anti-angiogenesis drugs,recent clinical studies mainly include the combination of anti-vascular endothelial growth factor monoclonal antibody bevacizumab plus EGFR-TKIs and multi-targeted receptor anti-angiogenic tyrosine kinase inhibitor plus EGFR-TKIs,and the for-mer results show a more significant improvement in terms of safety and efficacy in the treatment of advanced NSCLC.Therefore,the combination of bevacizumab plus EGFR-TKIs can be used as a new treatment standard in the treatment of some patients with NSCLC.

Objective To evaluate the performance of virtual touch tissue quantification imaging (VTQ) for differential diagnosing in uterine hysteromyoma and adenomyoma. Methods Blood flow parameters and SWV (shear wave velocities) from 42 regular hysteromyomas,14 abnormal hysteromyomas and 25 uterine adenomyomas were compared and analyzed through color Doppler. Results The SWV of regular hysteromyomas was on average of (2.72 ± 0.31) m/s;that of abnormal hysteromyomas (1.88 ± 0.25) m/s;while the SWV of uterine adenomyomas was on average of 1.91 ± 0.27 m/s. The mean SWV in regular hysteromyomas group was significantly higher than that in abnormal hysteromyomas group and uterine adenomyomas group (P < 0.05). There was no significant difference of SWV between abnormal hysteromyomas group and uterine adenomyomas group (P > 0.05).The resistance index and SWV of uterine hysteromyomas and adenomyomas had positive correlation (r = 0.753,0.544,0.506,P < 0.05). Conclusion VTQ can quantify the stiffiness of uterine hysteromyoma and adenomyoma.

ObjectiveTo investigate the expression and localization of midkine (MK) in human hepatocellular carcinoma. MethodsIn situ hybridization and immunohistochemical analysis for MK were performed on samples of both tumor tissues and paratumor tissues from HCC and benign liver tumors. ResultsThe distribution and localization of the MK transcripts′ signals determined by in situ hybridization were similar to those obtained by immunohistochemical analysis. Most HCC tissues showed enhanced positive reaction within cytoplasm to both MK probe and MK immunostaining. There was no significant difference in clinicopathological parameters between MK negative and positive cases of HCC. ConclusionsHuman hepatocellular carcinoma overexpresses MK at the mRNA and protein level.

Objective To explore the clinical value of high-frequency color Doppler ultrasound in the diagnosis of isolated calf muscle vein thrombosis (ICMVT). Methods Sonographic features of 175 patients with isolated calf muscular venous thrombosis were analyzed retrospectively and outcome of anticoagulant therapy in 1, 3, and 6 months was followed up. Diagnosis was established with high-frequency color Doppler ultrasound examination. Results One hundred and seventy-ifve patients presenting with 190 calf muscle vein thrombosis were included. One hundred and iffty-eight cases with 173 calf muscle vein thrombosis were diagnosed by high-frequency color Doppler ultrasound, 7 cases of misdiagnosis, missed diagnosis in 10 cases. The accuracy rate was 91.1%(173/190). Seven cases were misdiagnosed with 1 euroifbromatosis, 1 mixed hemangioma, 5 gastrocnemius hematoma. After diagnosis of ICMVT, all patients prescribed thrombolysis and anticoagulation therapy. High-frequency color Doppler ultrasound for 1, 3, 6 months after treatments revealed partial or complete recanalization without calf deep vein thrombosis. Typical sonographic features included:calf muscle venous lumen dilation, tortuous anechoic lumen or hypoechoic iflling, with tubular or branched shape in the longitudinal view and oval or round shape in the transversal view. Conclusion High-frequency color Doppler ultrasound is an accurate and reliable method in the diagnosis of the isolated calf muscular venous thrombosis.

Objective To explore the role of type Ⅶ collagen (COL7A1) gene in the pathogenesis of pretibial dominant dystrophic epidermolysis bullosa (DDEB-Pt).Methods Peripheral blood samples were obtained from a sporadic Chinese patient of Han nationality with DDEB-Pt,his parents and 100 healthy human controls.A modified salting-out method was used to extract genomic DNA from the blood samples,and PCR was performed to amplify 118 exons of the COL7A1 gene followed by DNA sequencing.Results A G→A mutation was identified at position 6109 (G6109A) in exon 78 of the COL7A1 gene in this patient,which caused a change from GCT to ACT at codon 2037 in the triple helix region,and resulted in the substitution of glycine (Gly) by arginine (Arg) (p.Gly2037Arg).Conclusion A novel glycine substitution mutation was identified in the COL7A1 gene in the patient with DDEB-Pt,which may be a pathogenic mutation.

OBJECTIVE:To evaluate the bioavailability of two nimesulide preparations.METHODS:A total of 20 healthy male volunteers were enrolled in a randomized crossover study in which the subjects were randomly assigned to receive single dose of 200 mg nimesulide orally disintegrating tablets(test)or nimesulide tablets(reference).The plasma concentrations of nimesulide were determined by RP-HPLC,and the pharmacokinetic parameters and bioavailability were calculated with DAS program.RESULTS:The main pharmacokinetic parameters of nimesulide test and reference preparations were as follow:AUC0～24:(54.67?18.25)vs.(56.15?15.54)?g?h?mL-1;AUC0～∞:(56.38?18.03)vs.(57.63?15.26)?g?h?mL-1;Cmax:(7.61?2.72)vs.(7.50?2.19)?g?mL-1;tmax:(3.83?1.39)and(3.80?1.28)h.The relative bioavailability of nimesulide or-ally disintegrating tablets as against nimesulide tablet was(98.7?22.9)%.CONCLUSION:Nimesulide test and reference preparations were bioequivalent.