Objective To construct the eukaryotic expression vectors of fibroblast growth factor receptor 3(FGFR3) MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN, and to detect their expressions in human chronic myeloid leukemia(CML)K562 cell line.Methods The full-length FGFR3 (fgfr3-WT)and dominant negative FGFR3 (fgfr3-DN)were amplified by polymerase chain reaction (PCR). The two genes were respectively digested with EcoRⅠand BamHⅠ,and then ligated into MSCV/puro to construct the recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN which were tranduced into K562 cells by LipofectaminTM 2000 after PCR,double digestion and DNA sequencing.The expressions of FGFR3 protein in K562 cells were detected by Western blotting and flow cytometry. Results The recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN were amplified by PCR method, and the results showed fgfr3-WT of 2 400 bp and fgfr3-DN of 1 200 bp had been successfully cloned into MSCV-puro vector. The 2 400 bp fragment was oblained after double digestion of recombinant plasmid.The sequencing results showed that the size of fgfr3-WT was 2 400 bp which was the same as the sequence from GeneBank.Fgfr3-DN of 1 200 bp was also in conformity with the expected sequence.Compared with control (K562 MSCV)group,the expression level of FGFR3-WT in MSCV/puro-fgfr3-WT transfection (K562-WT)group was increased to above 10 times.There was high expression of FGFR3-DN in MSCV/puro-FGFR3-DN transfection (K562-DN)group,but there was no expressions in control(K562 MSCV)group and K562-WT group.The flow cytometry results showed that the high expressions of FGFR3-WT were in 57.5% cells in K562-DN group and the high expressions of FGFR3-DN were in 41.5% cells in K562-DN group. Conclusion The K562 cell lines highly expressing FGFR3-WT and FGFR3-DN are constructed successfully.

Objective To evaluate the clinical feature and potential reasons of delayed papillary bleeding after endoscopic retrograde cholangiopancreatography (ERCP),and search for effective hemostasis and strategies.Methods A total of 76 patients with post-ERCP bleeding underwent endoscopic treatment in the Eastern Hepatobiliary Hospital from August 2000 to August 2016.Clinical data,haemostatic methods,and treatment outcomes of patients were retrospectively analyzed.Results Delayed papillary hemorrhage mostly occmred within 48 hours after ERCP (67.2％,45/67),with main manifestations of hematemesis,bloody stool,and bile.The lowest incidence of delayed bleeding was detected after endoscopic papillary balloon dilation (EPBD,0.1％),which was followed by papillary precut (0.6％) and endoscopic sphincterotomy (EST,0.9％).And EST+EPBD had the highest incidence of delayed post-ERCP papillary hemorrhage (2.4％).The most bleeding site was the left side of the incision (67.1％,51/76).Emergent endoscopic interventions were applied in all patients with success of hemostasis in 71 out of 76 (93.4％),and injection with diluted epinephrine,electric coagulation,hemoclipping,and metal stenting were used sequentially for hemostasis.Among the 71 successful cases of hemostasis,66 patients were performed endoscopic hemostasis for once,4 patients took twice,and 1 case took thrice.Endoscopic hemoclipping was the most commonly used method with successful rate of 76.9％ (50/65) for hemostasis.Conclusion Precut papillotomy is safe and effective,and its complication occurrence rate is similar to that of EST.Hemorrhage should be prevented and timely dealt with in small/median EST and/or EPBD.Once hemorrhage is suspected clinically,endoscopic inventions should be applied timely,and hemoclipping is a safe and effective method.

Objective To evaluate the feasibility and the safety of endoscopic closure for acute colon perforations with novel successive endoclips in animal model. Methods Approximately 1. 5 to 2 cm colonic full-thickness resections were created with an electrotome in 3 Ba-Ma pigs that were under general anesthesia and were closed with the conventional endoclips and novel successive endoclips respectively. The procedure time and the efficacy of each endoclip were recorded. After the procedure,the general condition and physiological indicators were carefully monitored. After a follow-up of 1 week,the pigs were euthanized for an endoscopic observation of the healing condition and the residual endoclips. A postmortem examination was performed to observe the abdominal infection and incision condition. Transverse sections of the colon across the site of perforation were taken for histopathologic examination to assess the healing process. Results All the colonic perforation models in three pigs were established successfully. Endoscopic closure for acute colon perforation with two kinds of endoclips was technically successful in all 3 pigs. The mean time of the procedure with successive endoclip was less than that with the conventional endoclip(54. 0 seconds VS 91. 9 seconds,P<0. 001),but the successful release rate of the successive endoclip was lower than that of the conventional endoclip without significant difference[62. 5%(15/ 24)VS 14/ 16,P= 0. 17].All the ani-mals survived without infection and hemorrhage. Five perforations demonstrated signs of healing,whereas one closed with the novel successive endoclips failed to heal completely. Conclusion Endoscopic closure for a-cute colon perforations with novel successive endoclips is effective and safe. The novel successive endoclip has the advantage of saving time,but it needs to be further improved and perfected to satisfy the clinical need.

Objective To evaluate the long-term outcome of fully covered self-expandable metal stents(FCSEMS)for the treatment of benign biliary strictures(BBS). Methods Between June 2008 and September 2013, 68 patients with BBS receiving endoscopic retrograde cholangiopancreatography and FCSEMS placement were retrospectively enrolled. Data of endoscopic treatment, stricture resolution and recurrence were collected, and related risk factors were analyzed. Results FCSEMSs were successfully placed in all patients and removed in 93.4%(57/61). The median stent duration was 9.0(range 0.2-37.1)months. Stricture resolution occurred in 74.2%(46/62)patients. During median follow-up of 54.0 (range 2.5-96.0)months,stricture recurrences were seen in 16.7%(6/36)patients. Multivariate analysis revealed that distance between stricture and hepatic bifurcation of less than 1.5 cm(P=0.034,OR=6.395, 95%CI:1.153-35.464), and stent migration(P= 0.024, OR= 0.153, 95%CI:0.030-0.782)were significant risk factors for stricture resolution. Meanwhile, the stricture length longer than 1.0 cm(P=0.028, HR = 6.766, 95% CI:1.233-37.122)was a significant risk factor for stricture recurrence. Conclusion Endoscopic treatment combined with FCSEMS can achieve excellent efficacy in resolving BBS with low recurrence rate. However, location and length of BBS, as well as stent migration may impair its effectiveness.

Objective To detect cancer-related biomarkers for early diagnosis of biliary tract carcinoma. Methods Bile and serums were obtained from patients with histopathologically proven malignant or benign biliary diseases, and clinical data of patients were recorded. Two-dimensional liquid chromatography tandem mass spectrometry(2D-LC-MS/MS)was applied to detect the differential protein profiles in the bile. Western blot was used to validate the results in the biles and serums. Results Ten differential protein profiles were detected in 4 protein groups. Among them, the expression of haptoglobin significantly increased in the bile of patients with malignancy. This result was further verified using Western blot. Conclusion Haptoglobin may be a potential biomarker for biliary tract carcinoma.