Objective:To investigate the immunogenicity of pORF5 plasmid protein,and further to screen and identify its im-munodominant domian.Methods: 10 different fragments of pORF5 gene including full length were amplified from the DNA of Chlamydia trachomatis serovar D by PCR and cloned into appropriate site of pGEX-6p vector to construct recombinant vectors after digested with BamHⅠand NotⅠrestriction endonucleases.After identification by PCR and sequencing,the recombinant plasmids were transformed into XL1 Blue E.coli to express the GST fusion proteins.ELISA and Western blot were carried out to identify the immunogenicity and immunoreaction of pORF5 plasmid protein.10 different fragments were reacted with sera from patients urogenitally infected with Chlamydia trachomatis, mouse polyclonal antibodies and mouse monoclonal antibodies of pORF5 plasmid protein with ELISA method.Results: pORF5 plasmid protein displayed strong immunogenicity and could induce a strong antibody response in human.The reactivity of human antibodies almost completely disappeared,when the native structure of pORF5 plasmid protein was de-stroyed.F6 that only lacked the N-terminal 66 amino acids was recognized by antibodies in ELISA as strongly as the whole pORF5 plasmid protein was.However,no other fragments were significantly recognized although there was a minimal reactivity of F2 and F3 with antibodies.Conclusion:pORF5 plasmid protein was an immunodominant antigen containing conformation-dependent epitope,and the C-terminal three quarters of pORF5 amino acid sequence was required for maintaining its immune dominance and conformation.The significance of the above findings lay a foundation for the further study on pORF5 protein function and vaccine development.

Objective To analyze the protein expression profile of HeLa cells transfected with pORF5 gene of Chlamydia trachomatis. Methods A lentiviral expression vector containing pORF5 gene was constructed. The lentiviral expression vector and helper plasmids were co-transfected into 293T cells to construct the recombinant lentivirus, which was used to infect HeLa cells. HeLa cells transfected with pORF5 gene and control HeLa cells were sorted out by flow cytometry. The isobaric tags for relative and absolute quantitation ( iTRAQ) approach combined with nano-liquid chromatography-tandem mass spec-trometry ( NanoLC-MS/MS) analysis was performed to understand protein expression profiles and to iden-tify and quantify the differentially expressed proteins in the pORF5-transfected HeLa cells ( pORF5-Hela) and the control HeLa cells. Quantitative real-time PCR ( qRT-PCR ) and Western blot analysis were performed to detect the expression of some proteins at mRNA and protein levels, respectively. Results HeLa cell line stably transfected with pORF5 gene and control HeLa cell line were constructed successful-ly. Totally 314 proteins were differentially expressed between the pORF5-HeLa and control HeLa cells, 159 of which showed increased expression and the other 155 showed decreased expression in pORF5-HeLa cells. The differentially expressed proteins were involved in many processes, such as metabolic process, immune response, biological adhesion and so on. Results of qRT-PCR showed that the expression of HIST1H1C(histone H1. 2C), HBA1(hemoglobin subunit alpha), PARK7(parkinson disease protein 7), HMGB1(high mobility group protein B1) and HMGB2 at mRNA level in pORF5-HeLa cells were up-regulated, while the expression of CLIC1 ( chloride intracellular channel protein 1 ) , KRT7 ( typeⅡ cy-toskeletal 7), SFN(14-3-3 protein sigma) and CDKN2A(cyclin-dependent kinase inhibitor 2A) were down-regulated. Western blot analysis confirmed the enhanced expression of HMGB1 and PRAK7 at pro-tein level. The results of qRT-PCR and Western blot analysis were consistent with proteomic data. Con-clusion Expression profiles for differentially expressed proteins between pORF5-HeLa and control HeLa cells were established successfully. The differentially expressed proteins regulated by pORF5 gene were found to be related to cell metabolism, proliferation, adhesion and so on, suggesting that pORF5 might promote the growth and proliferation of Ct by regulating protein expression and biological behavior of host cells.

Objective To investigate the application value of the modified Overlap method in digestive tract reconstruction of totally laparoscopic total gastrectomy (TLTG).Methods The retrospective cohort study was conducted.The clinicopathological data of 50 patients with gastric cancer who underwent TLTG with Overlap anastomosis between January 2016 and December 2016 in the Tangdu Hospital of the Fourth Military Medical University were collected.Twenty-six patients using classic Overlap method and 24 patients using modified Overlap method were respectively allocated into the classic Overlap group and modified Overlap group.All the patients underwent D2 lymph node dissection.Patients in the classic Overlap group underwent totally laparoscopic catastalsis side-to-side esophagojejunostomy.During digestive tract reconstruction in the modified Overlap group,there was no esophageal transection before anastomosis,and gastric fundus traction fully exposed to the lower esophagus.Esophagus was spun anticlockwise,and a hole was opened at the left posterior esophageal wall.Transection of jejunum was 25 cm away from Treitz ligment,and opening a hole at mesenteric margin was 6 cm away from distal jejunum to transected end of jejunum.Esophagus-distal jejunum side-to-side anastomosis was done using 60 mm linear stapler,and then laterally closing openings and transecting esophagus.Observation indicators:(1) intra-and post-operative recovery:total operation time,time of esophagus-jejunum anastomosis,volume of intraoperative blood loss,number of lymph node dissected,time to anal exsufflation,cases with complications and duration of postoperative hospital stay;(2) follow-up and survival.Follow-up using outpatient examination and telephone interview was performed to detect the postoperative tumor-free survival and tumor recurrence or metastasis up to March 2017.Measurement data with normal distribution were represented as (x)±s and comparison between groups was analyzed using the independent-sample t test.Comparison of count data was analyzed using the chi-square test or Fisher exact probability.Results (1) Intra-and post-operative recovery:all the 50 patients underwent successful TLTG using Overlap method,without conversion to open surgery.Total operation time and time of esophagus-jejunum anastomosis were respectively (278.6± 14.9) minutes,(46.5 ± 4.4) minutes in the classic Overlap group and (253.3 ± 12.8) minutes,(20.4 ± 2.3) minutes in the modified Overlap group,with statistically significant differences between the 2 groups (t =5.459,22.482,P＜0.05).Volume of intraoperative blood loss,number of lymph node dissected,time to anal exsufflation,cases with complications and duration of postoperative hospital stay were respectively (73±25) mL,34±6,(2.7± 1.0) days,2,(9.7± 1.6) days in the classic Overlap group and (71 ± 22) mL,35± 5,(2.6± 1.3) days,2,(9.8± 1.5) days in the modified Overlap group,with no statistically significant difference between the 2 groups (t =0.175,-0.616,0.293,-0.217,P＞ 0.05).Two patients in the classic Overlap group were respectively complicated with esophagus-jejunum anastomotic fistula and pancreatic leakage,2 patients in the modified Overlap group were respectively complicated with pulmonary infection and subcutaneous emphysema,and they were improved by symptomatic treatment.(2) Follow-up and survival:41 of 50 patients were followed up for 3-15 months,with a median time of 7 months,including 20 in the classic Overlap group and 21 in the modified Overlap group.During follow-up,patients had tumor-free survival,without tumor recurrence and metastasis.Conclusion Compared with classic Overlap method,the modified Overlap method can simplify the anastomotic procedures,shorten operation time and achieve similar efficacy,and it is also a simple and effective method for digestive tract reconstruction after TLTG.

OBJECTIVETo evaluate the effect of antiviral therapy on the quality of life (QOL) of patients with chronic hepatitis C (CHC) and cirrhosis during the 5-year period following splenectomy to treat hypersplenism.

METHODSData of patients with CHC and cirrhosis who had undergone treatment for hypersplenism were retrospectively selected from the hospital database of medical records. The patients were first grouped according to the hypersplenism treatment: splenectomy (group A, 28 cases) and conservative/non-operative (group B, 30 cases). Sub-grouping was carried out according to the CHC treatment: interferon-alpha-2a and ribavirin (15 cases in the A1 group, and 19 cases in the B1 group) and non-antiviral (13 cases in the A2 group, and 11 cases in the B2 group). To determine the intergroup differences in QOL during the 5-year period following the hypersplenism treatment, the QOL was assessed by chronic liver disease questionnaire (CLDQ), listing of specific symptoms (SS), and the World Health Organization QOL scale (WHOQOL-BREF).

RESULTSBetween-group statistical comparison of the subjective feeling, physiological status, mental state, and social life relationship of the patients showed no significant differences among the patients who received splenectomy compared to those who received the conservative treatment. However, the QOL of splenectomy-treated patients who received non-antiviral CHC treatment was worse than that of the patients who were given conservative treatment for the hypersplenism and antiviral therapy for the CHC. The patients who received splenectomy and antiviral therapy had better QOL than the other patient group(3.69 +/- 0.75 vs 2.15 +/- 0.98, P = 0.0003).

CONCLUSIONSplenectomy followed by antiviral therapy may improve the QOL of patients with CHC-related cirrhosis and hypersplenism.

Adult ; Antiviral Agents ; therapeutic use ; Female ; Hepatitis C, Chronic ; complications ; drug therapy ; Humans ; Liver Cirrhosis ; drug therapy ; virology ; Male ; Middle Aged ; Quality of Life ; Retrospective Studies ; Splenectomy ; Treatment Outcome