Objective Getting the robust exogenous gene expression vector under the control of porcine insulin promoter, and to lay the foundation for pancreaticβ-cells specific transgene expressing pigs.Method Using porcine insu-lin promoter ( PIP, 1500 bp of the 5′UTR from the porcine INS gene including the first exon and the first intron) to con-struct expression vector, the HindIII restriction site which connected the sequences of PIP and EGFP was designed before ATG, named PIP-HindIII-EGFP.Considering that the different location of restriction site may affect the expression efficien-cy of the transgene, we optimized the expression vector.Firstly the HindIII restriction site was deleted to realize the seam-less connection of PIP and EGFP,the vector was named PIP-EGFP.Also we mutated the 3′intron splicing acceptor site( SA) of the first intron into HindIII restriction site, named as PIP-SA( M)-EGFP.Three different EGFP expression vectors were respectively transfected MIN-6 mouse pancreatic β-cells, pig ear fibroblasts and kidney cells.The transfected cells were cultured for 48 h and harvested for RT-PCR, flow cytometry and Western blot analysis, to analyze and compare the expres-sion efficiency of vectors.Results After transfection,green fluorescence was observed only in MIN-6 mouse pancreaticβ-cells.RT-PCR analysis and product sequencing showed that the three expression vectors did have different stability with in-tron splicing.The PIP-HindIII-EGFP construct and PIP-EGFP vector produced two kinds of mRNA with the first intron spliced and no spliced, indicating the instability of intron splicing.Mutation of the PIP splice site would cause the first in-tron not spliced, while flow cytometry and Western blot displayed that the mutation induced a most efficient expression of the downstream gene.Conclusions A robust and specific β-cells expression vector has been successfully generated by mutating the intron splicing acceptor site of the porcine insulin promoter.It provides the foundation for preparation of pigs with pancreaticβ-cells specifically expressing the transgene.

Objective To analyze the serotypes distribution characteristics of salmonella infection in infants below 2 years old in Dongguan.Methods Feces samples collected in children below 2 years old with diarrhea,caused by salmonella infection,were cultured for salmonella isolates,which were then identified for serotypes.The distribution characteristics of different serotypes of salmonella were analyzed.Results A total of 369 strains of salmonella were isolated,including 36 serotypes.The most common three serotypes were Salmonella typhimurium(S.ty),Salmonella stanley(S.st) and Salmonella enteritidis(S.en),which accounted for 68.7%.In all infants with diarrhea,there were 253 boys and 143 girls,and the male:female ratio was 1.77∶1.The average age of infants was (9.3±2.3) months.In male and female infants,the most serotype was S.ty,accounting for 62.3% and 53.3%,but the least serotypes in male and female infants were S.st(16.8%) and S.en(19.0%) respectively.S.ty was the most common serotype in different age groups.The least serotypes in low-age group(0-5.99 months) and middle-age group(6-11.99 months)were S.st(18.4%) and S.en(14.4%) respectively.In high-age group(12-24 months),the proportion of S.st and S.en were both 21.8%.The diversity of salmonella serotypes in male infants was higher than female infants,and with the increasing of age,the diversity was more obvious.Conclusion There could be unique serotype distribution characteristics of salmonella infection in infants with diarrhea,and the diversity of salmonella serotypes should be paid more attention.

Objective To analyze the short-term results of endovascular aortic repair (EVAR)for patients with acute type B aortic dissection and chronic renal insufficiency (CRI ).Methods Between February 2009 and December 2012,EVAR was performed in 30 patients with acute type B aortic dissection and CRI (CRI group).Consecutive 30 patients with acute type B aortic dissection whose renal function was normal during the same period was chosen as the control group (non-CRI group).All patients were within 14 days after onset,in which Marfan syndrome was excluded and diagnosis made by computed tomographic angiography (CTA) before the procedure.In 57 patients,EVAR was performed under looal anesthesia and associated procedures included insertion of a chimney stent in the left subclavian artery in 2 case and a bare metal stent in the renl artery in 2,In 3 patients,EVAR was done following right axillary artery-to-left axillary and left subclavian artery bypass with a Y-shaped graft under general anesthesia.Follow-up regimen included renal function and CTA at I month and 1 year postoperatively.Results Compared to the non-CRI group,patients in the CRI grup was significantly younger [ (44.7±13.2) years versus (53.7±16.2)years,P ＜0.05)and had a higher rate of perioperative complications (cerebrospinal ischemia,deterioration of renal dysfunction,and gastroenteral dysfunction) (16.7％ versus 3.3％,P ＜0.05 ),all of which resolved after surgical or medical treatment.One patient in CRI group was readmitted at 6 months for a redo EVAR to treat a new tear distal to the stent.At 1 month and I year postoperatively,no patients suffered from deterioration ofthe renal function,and their CTAs detected no apparent device deformation,alteration and endoleak,with remsrkable improvement in the blood supply of the aortic trie lumen and branches.Conchusion Satisfactory short-term results can be achieved with EVAR for patients with acute type B aortic dissection and CRI.At I month and 1 year postoperatively,no mortality or morbidity occumed such as endoleak,aortic rupture,neurologic and abdominal ischermia.

Objective To summarized the experience of simultaneous aortic operation and coroanry artery bypass graft (CABG).Methods Ninety seven patients who underwent combined aortic operation and CABG were reviewed from January 2009 to December 2011.All patients are divided into four groups according to etiology,63 aortic aneurysm,other 25 aortic dissection,7 coarctation of aorta or occlusion of main branch,and the other two aortic ulcer.Mean age of all patients is(57.6 ±9.5) years.The rate of preoperational diagnosis of coronary disease (CAD) were respectively 93.7％ (59/63),40.0％(10/25),100.0％,100.0％.The patients made the preoperative diagnosis of CAD were performed selective simultaneous CABG with aortic procedures.The others without diagnosis of CAD had to receive urgent CABG during the aortic procedures.Results The aortic procedures with simultaneous urgent CABG had signilicant higher mortality than with selective CABG,16.7％,3.8％,Fisher's exact test P =0.078.Conclusion Selective simultaneous CABG with aortic procedures is safely.In aortic surgery,patients with risk factors of CAD should undergo preoperative coronary artery angiography or spiral computed tomography.

[Objective]To establish a novel noninvasive fluorescent animal model for endometriosis in vitro and in vivo.[Methods]Adenovirus encoding enhancing green fluorescent protein(Ad-eGFP)was used to transfect endometrial glandular cells and stromal cells(cells transfection and injection,Method No.1),and fragments(tissues transfection and injection,Method No.2).Transfection efficiencies were compared between the two methods in vitro.Then GFP transfected glandular cells and stromal cells suspension were injected into nude mice subcutaneously(Method No.1),taking Method No.2 as a comparison.In vivo observation last for 25 days,and positive rates and duration times of fluorescent lesions were calculated.Histological examination was used to confirmed lesion formation.[Results]On the fifth day after injection,lesion positive rate of Method No.1 was 88.9%,which was statistically significantly higher than that of Method No.2(22.2%),P=0.015＜0.05.The fluorescent positive duration of Method No.1 and No.2 were 12 ± 8 days and 7±4 days.The structures of lesions were all identified as human original endometrium by histological examination,including HE staining and immunofluoresceney.[Conclusion]Noninvasive animal model of endometriosis can be built up by subcutaneously injection of Ad-EGFP transfected endometrial glandular cells and stromal cells suspension with higher positive rate and longer observation time

Objective To identify the risk factors for postoperative spinal cord injury in Stanford type A aortic dissection patients.Methods 210 Stanford type A aortic dissection(TAAD) patients underwent Sun's procedure in Beijing Aortic Disease Center during July 2014 to March 2015.14 patients had spinal cord injury after surgery.Clinical data and computed tomography angiography(CTA) imaging of aorta were retrospectively analyzed and multi-logistic regression analysis was performed to identify risk factors for spinal cord injury post operation.Results 14 out of 210(6.7％) patients had transient or permanent spinal cord injury after surgery.Univariate analysis showed only false lumen derived intercostal arteries at eighth thoracic vertebral level (T8) to first lumbar vertebral level (L1) was significantly associated with post-surgery spinal cord injury (P =0.000).Multi-logistic regression analysis showed that false lumen derived intercostal arteries (P =0.000) and age (P =0.016) were significantly associated with postoperative spinal cord injury.Conclusion Major intercostal arteries derived from false lumen and rapid thrombogenesis in false lumen are the major risk factors for postoperative spinal cord injury in Stanford type A aortic dissection patients.

Objective To identify the risk factors for acute kidney injury (AKI) in overweight patients who underwent surgery for the treatment of acute type A aortic dissection(TAAD).Methods A retrospective study including 108 consecutive overweight patients(body mass index(BMI) ≥24) between January 2010 and May 2013 in Beijing Anzhen Hospital was performed with Sun's procedure of total aortic arch replacement and frozen elephant trunk implantation.AKI was as defined according to Acute Kidney Injury Network (AKIN) criteria based on serum creatinine(sCr) or urine output.Results The mean age of the patients was(43.69 ± 9.66) years.During the postoperative period seventy-two patients(66.7％) developed AKI.The overall postoperative mortality rate was 7.4％,8.3％ in AKI group and 5.6％ in non-AKI group.There is no statistically significant difference between the two groups(P =0.32).A logistic regression analysis was performed to find out the independent risk factors for AKI:elevated preoperative sCr level and postoperative drainage volume.Renal replacement therapy(RRT) was performed in fifteen patients (13.9 ％).Conclusion A higher incidence of AKI (66.7 ％) in overweight patients following acute TAAD was identified.The logistic regression model found out elevated preoperative sCr level and 72 hour drainage volume as the two independent risk factors for AKI in overweight patients.More attention should be paid to prevent AKI in overweight patients following TAAD.

Objective This study was aimed to characterize the swine leukocyte antigen( SLA) class I genes of GGTA1 -/ - Wuzhishan minipigs and compare their similarity to human leukocyte antigen( HLA) . It has important implica?tions for understanding the cellular rejection in xenotransplantation. Methods Specimens of ear tissue from six founding GGTA1 -/ - Wuzhishan minipigs were collected, and the SLA class I genes (SLA?1, SLA?3, SLA?2) were amplified by RT?PCR. Purified products were cloned into pEASY?T1 vectors and sequenced, followed by BLAST alignment and using bioin? formatc analysis to characterize the SLA class I genes and compare with the similarity to HLA. Results A total of six al?leles were detected, among them alleles were previously reported (SLA?1?0703,SLA?2?1102, SLA?3?0401, SLA?3?0403), and the other were novel (SLA?1?0401wz01, SLA?2?11wz01). The homology between alleles of SLA class I genes in Wuzhishan minipigs and HLA was from 70?5% to 72?1%. The homology analysis of critical amino acid residues on HLA binding with human CD8 + molecules showed that SLA?1?0401wz01, SLA?1?0703, SLA?2?11wz01, SLA?2?1102 and SLA?3?0401 occurred mutant at amino acid positions 225 and 228 ( T→S,T→M) , whereas the other loci were highly conserved. There was a high homology at amino acid level between SLA?2?11wz01, SLA?2?1102 and HLA class I genes which are NK cell KIRs binding sites. Conclusions The amino acid sequences of SLA class I genes of GGTA1 -/ -Wuzhishan minipigs have a high homology to HLA. From the point of view of cell?mediated xenograft rejection, the amino acid sequences of SLA class I genes of GGTA1 -/ - Wuzhishan minipigs have a high homology to HLA, therefore, Wzhishan minipigs may become a good potential donor for pig?human xenotransplantation.

Objective To study the indication,feasibility and short-term efficacy of combined thoracoscopic and laparoscopic radical esophagectomy for the treatment of esophageal cancer.Methods Retrospective medical records analysis was conducted for 139 esophageal cancer patients who underwent combined thoracoscopic and laparoscopic esophagectomy in our department from December 2009 to August 2011.The tumors were located in upper esophagus in 16 cases,middle esophagus in 107 cases,and lower esophagus in 16 cases.The surgery started with the thoracoscopic mobilization of thoracic esophagus and lymph nodes dissection,which were followed by the laparoscopic stomach mobilization and gastroesophageal anastomosis in left neck.Postoperative pathological staging identified stage Ⅰ esophageal cancer in 25 cases ( stage Ⅰ a:13 cases,stage Ⅰ b:12 cases),stage Ⅱ esophageal cancer in 71 cases,stage Ⅲ esophageal cancer in 31 cases ( stage Ⅲ a:16 cases,stage Ⅲ b:15 cases) and stage Ⅳ esophageal cancer in 12 cases.Results Except for open conversions in 4 cases (2.9％),all surgical operations were completed smoothly.Postoperative anastomotic leak was found in 6 cases(4.3％ ),chylothorax in 1 case(0.7％ ),arrhythmia in 4 cases(2.9％ ),and dumping syndrome in 1 case( 0.7％ ).All of these complicated cases fully recovered after conservative treatments.Postoperative lung infection was found 11 cases (7.9％),3 of whom required tracheotomy and assisted ventilation and 1 case died as a result of the infection (mortality rate:0.7％ ).Ten cases(7.2％ ) presented with hoarseness postoperatively.Out of the 139 cases,130 cases were successfully followed up with durations ranged from 1 to 20 months,during of time the esophageal cancer spread to liver in 2 cases,celiac lymph nodes in 4 cases,lung in 2 cases,and bone in 1 case.Ten cases died,and all remaining cases remained alive during the follow up.The one-year survival rate was 88.9％ for these cases.Conclusion Combined thoracoscopic and laparoscopic radical esophagectomy is a technically safe and feasible treatment for esophageal cancer.The short-term efficacy results are satisfactory.This technique is indicated not only for early and middle stage esophageal cancer,but also for some of the advanced esophageal cancer cases.

Objective To investigate the role of fibroblast growth factor-23 (FGF23) in secondary hyperparathyroidism (SHPT). Methods (1)Serum FGF23 and intact parathyroid hormone (iPTH)from 38 maintenance hemodialysis (MHD) patients were measured by ELISA and chemiluminescence enzyme immunoassay respectively.(2) Parathyroid cells from six SHPT patients underwent parathyroidectomy with forearm autotrlansplantation were cultured for 24 h,then were induced by 0.1 mg/L FGF23.The supernatant was collected at 0.6,12,24 and 48 h respectively. The concentration of iPTH was measured by chemiluminescence enzyme immunoassay. (3)Protein expression of Klotho,FGFR1,FGFR3,GATA-3 and PCNA in parathyroid tissue from 33 SHPT eases and 3 healthy people were detected by immunohistochemistry SP and PV methods respectively. Positive cell rate and absorbance were calculated. Results (1) Serum FGF23 [(3901.85±2618.11) ng/L] was positively correlated with serum iPTH [(460.00±489.77) ng/L] in MHD patients. (2) 0.1 mg/L FGF23 suppressed iPTH secretion of parathyroid cells only at 24 h time point in vitro (P<0.05). (3) Expression of GATA-3, FGFR3, Klotho and PCNA was significantly increased and FGFRl was signiticantly decreased in parathyroid tissue of SHPT-patients as compared to healthy people. (4) Positive cell rate of GATA-3 was positively correlated with iPTH (r~2=0.1901, P=0.0425) and PCNA (r~2=0.2584, P=0.0025). Klotho was positively correlated with FGFRI and FGFR3 (r~2=0.2046, P=0.0082;r~2=0.2833, P=0.0014). PCNA was negatively correlated with FGFR1 (r~2=0.1292, P=0.0399) and positively correlated with FGFR3 (r~2=0.1226, P=0.0457). FGFR1 was negatively correlated with serum phosphate (r~2=0.2329, P=0.0044) and positively correlated with serum calcium (r~2=0.1422, P=0.0305). Conclusions FGF23 level is positively correlated with iPTH level in MHD patients. FGF23 can inhibit iPTH secretion of parathyroid cells in a weak and short way, which may be associated with the proliferation of GATA-3 positive cells and parathyroid cells, the up-regulation of FGFR3 and the down-regulation of FGFR1 expression.