Objective To express the recombinant mouse histidyl-tRNA synthetase (HARS) and maltose binding protein (MBP) gene in Escherichia coli and obtain the purified protein which possesses antigen specificity.Methods Total RNA was extracted from the myocytes of C57BL/6 mouse and reversely transcripted to cDNA.The gene of N-terminal origin of 591 base pairs was amplified,then cloned into pMALc-5e vector.The recombinant plasmid was transformed into Rosetta-gami B,then IPTG was used to induce the expression of HARS-MBP.Fusion protein was purified by affinity chromatograph.The molecular weight (MW) of HARS-MBP was roughly determined by SDS-PAGE.The antigen specificity was identified by Western blotting using anti-Jo-1 serum from patients,commercial anti-HARS and anti-MBP antibodies.Results The recombinant HARS-MBP protein gene was efficiently expressed in Escherichia coli,and the MW was consistent with predicted MW of 66 000.The fusion protein was specifically combined with its antibody.Conclusion The HARS-MBP fusion protein could be efficiently and steadily synthesized in Escherichia coli,which shows satisfactory antigen specificity and provides the key requirement for making a deep study of HARS in the pathogenesis of idiopathic inflammatory myopathy(IIM) and animal modeling of IIM.

Background Studies showed that activation of microglia-derived nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a key role in the neurodegenerative diseases and neural cell death in central nervous system.The effect of NADPH on cone degeneration have been determined in rd rats,its role in rod degeneration is relatively less studied.Objective This study was to study the expression of NADPH oxidase in the retinal degenerative process in rd mice and further explore its role in the photoreceptor degeneration.Methods rd Mice at postnatal day 8 (P8),P10,P12,P14,P16 and P18 were collected.The mice were sacrificed,and retinal sections,RNAs and proteins were prepared in above-mentioned time points.The expressions of the gp91 phox,a major subunit of NADPH oxidase,in transcript level and protein level in the retinas were semi-quantitatively detected by real-time PCR and Western blot respectively.Expression of gp91phox was localized in the rd retinas as ageing by immunohistochemstry,and the co-expression of gp91phox with CD11b,a specific marker of microglial cells,was assayed by immunofluorescent double labeling.The C57BL/6N mice were served as controls.The use and care of the animals complied with the Guideline of ARVO.Results Real-time PCR showed that gp91phox mRNA was not expressed in the retinas of C57BL/6N mice.Gp91phox mRNA was found to have less expressed in retinas of P8 rd mice.With aging,the expression level of gp91phox mRNA (gp91phox mRNA/β-actin) in rd mouse retinas was gradually increased with the highest level in P14 mice(1.136±0.370).A significant difference was seen in the gp91 phox mRNA expression among various groups of mice (F=17.81,P =0.00),and gp91phox mRNA expression was significantly elevated in P10,P12,P14,P16 and P18 rd mice compared with P8 rd mice(all at P＜0.05).The expression level of gp91phox protein (A value) in the retinas presented with a similar trend in the rd mice,with a significant difference among the various ages of rd mice and C57BL/6N mice (F =354.00,P＜0.01).The expression level of gp91 phox protein was increased in the rd mice in comparison with the C57BL/6N mice (all at P＜0.05).Immunochemistry revealed that the positive response cells for gp91phox increased in the inner layers of retinas in P10 rd mice and peaked in P14 mice.Immunofluorescent double labeling exhibited that gp91phox were seen to present a co-expression with CD11b,showing an orange fluorescence.Conclusions Expression of NADPH oxidase in the rctinas in the rd mice up-regulates and is parallels to the microglial activation and photoreceptor degeneration,suggesting that NADPH oxidase plays a role in the retinal dystrophy associated with microglial activation.

Objective To present the experience of laparoscopic extraperitoneal radical prostatectomy and evaluate its safety and efficacy. Methods A total of 91 patients diagnosed with localized prostate carcinoma were admitted from February 2003 to June 2008. The level of serum PSA ranged from 7. 5 - 47. 0 ng/ml(mean 14. 0 ng/ml). The volume of the prostate ranged from 35 - 75 ml(mean 52 ml). Biopsy was performed before the operation and the pathological results revealed prostate carcinoma with Gleason score no more than 8. CT, MR and ECT revealed there was no lymph node or seminal vesicle involvement and there was no bone metastasis. The procedures were performed with an-tegrade techniques and pelvic lymphadenectomies were performed in 32 cases and nerve-sparings were performed in 11 cases. Results The operation duration ranged from 105 - 270 min (mean 173 min). Intraoperative blood loss was 110 - 1200 ml(mean 315 ml). Incontinence occurred in 19 cases in early stage and 18 cases recovered within 3 months. Positive surgical margin occurred in 11 cases. There was no complication of urethra stricture during 3 - 30 months' follow-up. No lymph node was involved in 32 cases with pelvic lymphadectomy. Five of the 11 cases received nerve-sparing prostatectomy had normal erectile function during the follow-up. Conclusions Laparoscopic extraperitoneal radical prostatectomy is a safe, effective and efficient surgical procedure with the minimal invasion, less morbidity and rapid recovery. Laparoscopic radical prostatectomy is emerging as an alternative to open radical prostatectomy.

Objective: To study the biological effects of the HPV16Z-Hsp65-E6/E7 fusion protein vaccine on the tumor associated with HPV16 infection. Methods: We tested the cellular immune responsive intensity to the vaccine by the lymphocyte proliferation and CTL response, and studied the therapeutic effect of the vaccine on mouse TC-1 cell transplanted cancer in vivo and the influence on mouse lifetime. Results: The spleen lymphocytes from the C57BL/6 mouse immunized by the Hsp65-E6/E7 vaccine could proliferate obviously in the presence of the protein and TC-1 tumor cell could be lysed specifically by immune activated lymphocytes in vitro. This animal therapeutic experiment in vivo showed that the vaccine suppressed the growth of TC-1 cell transplanted tumor remarkably. Conclusion: The recombined vaccine can induce specific cellular immune response in vitro and suppress HPV16 positive TC-1 tumor cell growth obviously in vivo.

Objective　To evaluate the technique and clinical outcomes of modified transperitoneal laparoscopic radical prostatectomy.　Methods　A total of 285 patients received the operation with mean age of 67 years (50-76 years) from January 2008 to April 2012.Mean level of PSA was 15.7 μg/L (1.8 -50.0 μg/L),and mean prostatic volume was 44 ml (26 -74 ml). No lymph node or seminal vesicle involvement was found by CT or MR and radionuclide bone scan revealed no metastasis.271 cases were confirmed diagnosis by prostatic biopsy and 14 were detected through pathological studies of TURP specimens.Gleason score ranged from 6 to 8.14 cases were in clinical stage T1b,29 cases in T1c,214 cases in T2 and 28 cases in T3a.Transperitoneal approach and modified technique involving bladder neck dissection,nervesparing technique and vesicoureteral anastomosis were applied on patients.　Results　Mean operative time was 105 min (55 -150 min).Mean intraoperative estimated blood loss was 240 ml (50-800 ml).Rectal injures occurred in 2 cases and were repaired under laparoscopy.Drainage tube and urinary catheter were removed 48 -72 h and 5 -8 d postoperatively.Postoperative hospital stay was 7 d (5 - 11 d).Positive surgical margin was present in 58 patients.Mean follow-up time was 29 months (3 -50 months).Complete continence were found in 208 patients immediately after catheter removal.68 patient recovered continence within 3 months and 9 patients remained incontinence 3 months after surgery. Normal erection presented in 42 of the 57 cases with nerve-sparing.　Conclusions　Transperitoneal laparoscopic radical prostatectomy is safe and efficient.Higher efficiency and lower complication rate have been achieved through modified laparoscopic technique involving bladder neck dissection,nerve-sparing technique and vesicoureteral anastomosis.

Objective: To develop a therapeutic adjuvantfree protein vaccine against HPV16 which is closely related to cervical cancer of China. Method: First the E6/E7 gene by PCR technology from HPV16z virus strain was isolated in the highrisk cervical cancer area of Shanxi province of China in1990s, and again got the gene segment of Hsp65 from BCG by the same method, mutated the transforming codes in sequences of HPV 16 E6/E7 genes and thus constructed the expression vector pET28aHsp65E6/E7, expressed the Hsp65E6/E7 fusion protein in E.coli BL21(DE3) strain and researched optimal protein purification procedures. Results: The expression vector pET28aHsp65E6/E7 was constructed successfully and E6/E7 gene was mutated correctly. Hsp65E6/E7 fusion protein was renatured and purified on the affinity chromatography column simultaneously. The protein purity achieved 95% after the anionic exchange chromatograph purification. conclusions: This research laid a foundation for further functional study of the therapeutic adjuvantfree protein vaccine——Hsp65E6/E7.

Objective To evaluate the technique and efficacy of retroperitoneal laparoscopic partial nephrectomy. Methods From June 2002 to December 2009, 113 cases of renal tumor received retroperitoneal laparoscopic partial nephrectomy. The age ranged from 26 to 73 years. The tumor located in left side in 51 cases and right side in 62 cases with the mean diameter of 3.7 cm(1.2-6.3cm). During the procedure, the renal artery was separated and then clamped with bulldog. The renal parenchymal was incised with cold endoscissor and the tumor was totally removed. Pelvicalyceal repairing and parenchymal hemostasis were then performed. Renal defect closure was achieved with running suture or horizontal mattress suture. Results All the procedures were completed successfully.There was no open conversion. The mean operation time was 85 min(60- 125 min), the mean warm ischemic time was 24 min(19-43 min). The pathology studies revealed 87 cases of clear cell carcinoma, 9 cases of papillary renal cell carcinoma, 7 cases of chromophobe cell carcinoma, 6 cases of perivascular epithelioid renal cell tumor and 4 cases of renal oncocytoma. The surgical margin was negative in all cases. There was no complication of urine leakage. Gross hematuria occurred in 2 cases.During 3-41 months of following up, there was no recurrence. Conclusion Retroperitoneal laparo-scopic partial nephrectomy is safe and effective for the treatment of renal tumor, which becomes an alternative treatment to open procedure.

OBJECTIVETo study the technique and clinical outcomes of laparoscopic radical prostatectomy for high risk prostate cancer.

METHODSA total of 65 patients with high risk prostate cancer were treated with surgery in the First Affiliated Hospital of Nanjing Medical University from January 2011 to June 2013. The mean age was 67 years (range 45-75 years). The mean preoperative prostate specific antigen (PSA) level was 26.7 µg/L (range 11.2-65.5 µg/L). The transrectal biopsy revealed Gleason score of 3+3 in 4 patients, Gleason 3+4 in 27 patients, Gleason 4+3 in 11 patients, Gleason 4+4 in 21 patients and Gleason 4+5 in 2 patients. The bone metastasis was excluded by scintigraphy examination. The surgical procedures were performed through transperitoneal approach. Extended pelvic lymph nodes dissection was performed after the removal of the prostate. Adjuvant radiotherapy or hormonal therapy was administrated according to the pathological results. Serum PSA was detected every 1 to 2 month and urinary continence was evaluated every 3 month in the first year, and then serum PSA was detected every 2 to 3 month.

RESULTSThe mean operative time was (134±21) minutes and the median blood loss was (300±146) ml. Bladder neck reconstruction was performed in 15 cases. The drainage was removed on postoperative day 4 and the catheter was removed on day 7. Pathologic results demonstrated pT2 in 25 patients, pT3a in 28 patients, pT3b in 9 patients and pT4 in 3 patients. Positive surgical margin was presented in 15 patients. A median of 19 lymph nodes (range 11-24 nodes) were retrieved during lymphadenectomy and 11 patients had lymph nodes metastasis with a total of 19 positive nodes. Forty-three patients recovered continence after the removal of catheter. Eleven patients received adjuvant hormonal therapy and 19 patients received adjuvant radiation therapy. With the median of 20 months follow-up (range 12-30 months), 5 patients got biochemical recurrence.

CONCLUSIONSLaparoscopic radical prostatectomy with extended lymph nodes dissection for high risk prostate cancer is safe and technical feasible. It provides accurate information on tumor stage and grade. It is an important component of multimodality for the treatment of high risk prostate cancer.

Aged ; Biopsy ; Humans ; Laparoscopy ; Lymph Node Excision ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Grading ; Postoperative Period ; Prostate-Specific Antigen ; blood ; Prostatectomy ; Prostatic Neoplasms ; diagnosis ; surgery

BACKGROUNDOver the past two decades, the clinical presentation of renal masses has evolved, where the rising incidence of small renal masses (SRMs) and concomitant minimal invasive treatments have led to noteworthy changes in paradigm of kidney cancer. This study was to perform a proportional meta-analysis of observational studies on perioperative complications and oncological outcomes of partial nephrectomy (PN) and radiofrequency ablation (RFA).

METHODSThe US National Library of Medicine's life science database (Medline) and the Web of Science were exhaustly searched before August 1, 2013. Clinical stage 1 SRMs that were treated with PN or RFA were included, and perioperative complications and oncological outcomes of a total of 9 565 patients were analyzed.

RESULTSPatients who underwent RFA were significantly older (P < 0.001). In the subanalysis of stage T1 tumors, the major complication rate of PN was greater than that of RFA (laparoscopic partial nephrectomy (LPN)/robotic partial nephrectomy (RPN): 7.2%, open partial nephrectomy (OPN): 7.9%, RFA: 3.1%, both P < 0.001). Minor complications occurred more frequently after RFA (RFA: 13.8%, LPN/RPN: 7.5%, OPN: 9.5%, both P < 0.001). By multivariate analysis, the relative risks for minor complications of RFA, compared with LPN and OPN, were 1.7-fold and 1.5-fold greater (both P < 0.01), respectively. Patients treated with RFA had a greater local progression rate than those treated by PN (RFA: 4.6%, LPN/RPN: 1.2%, OPN: 1.9%, both P < 0.001). By multivariate analysis, the local tumor progression for RFA versus LPN/RPN and OPN were 4.5-fold and 3.1-fold greater, respectively (both P < 0.001).

CONCLUSIONSThe current data illustrate that both PN and RFA are viable strategies for the treatment of SRMs. Compared with PN, RFA showed a greater risk of local tumor progression but a lower major complication rate, which is considered better for poor candidates. PN is with no doubt the golden treatment for SRMs, and LPN has been widely accepted as the first option for nephron-sparing surgery by experienced urologists. RFA may be the best option for select patients with significant comorbidity.

Catheter Ablation ; adverse effects ; methods ; Humans ; Kidney Neoplasms ; surgery ; therapy ; Nephrectomy ; adverse effects ; methods

Objective:To investigate the epidemiological characteristics, diagnosis, treat-ment and prognosis of gallbladder cancer in China from 2010 to 2017.Methods:The single disease retrospective registration cohort study was conducted. Based on the concept of the real world study, the clinicopathological data, from multicenter retrospective clinical data database of gallbladder cancer of Chinese Research Group of Gallbladder Cancer (CRGGC), of 6 159 patients with gallbladder cancer who were admitted to 42 hospitals from January 2010 to December 2017 were collected. Observation indicators: (1) case resources; (2) age and sex distribution; (3) diagnosis; (4) surgical treatment and prognosis; (5) multimodality therapy and prognosis. The follow-up data of the 42 hospitals were collected and analyzed by the CRGGC. The main outcome indicator was the overall survival time from date of operation for surgical patients or date of diagnosis for non-surgical patients to the end of outcome event or the last follow-up. Measurement data with normal distribu-tion were represented as Mean±SD, and comparison between groups was conducted using the t test. Measurement data with skewed distribution were represented as M( Q1, Q3) or M(range), and com-parison between groups was conducted using the U test. Count data were described as absolute numbers or percentages, and comparison between groups was conducted using the chi-square test. Univariate analysis was performed using the Logistic forced regression model, and variables with P<0.1 in the univariate analysis were included for multivariate analysis. Multivariate analysis was performed using the Logistic stepwise regression model. The life table method was used to calculate survival rates and the Kaplan-Meier method was used to draw survival curves. Log-rank test was used for survival analysis. Results:(1) Case resources: of the 42 hospitals, there were 35 class A of tertiary hospitals and 7 class B of tertiary hospitals, 16 hospitals with high admission of gallbladder cancer and 26 hospitals with low admission of gallbladder cancer, respectively. Geographical distribution of the 42 hospitals: there were 9 hospitals in central China, 5 hospitals in northeast China, 22 hospitals in eastern China and 6 hospitals in western China. Geographical distribution of the 6 159 patients: there were 2 154 cases(34.973%) from central China, 705 cases(11.447%) from northeast China, 1 969 cases(31.969%) from eastern China and 1 331 cases(21.611%) from western China. The total average number of cases undergoing diagnosis and treatment in hospitals of the 6 159 patients was 18.3±4.5 per year, in which the average number of cases undergoing diagnosis and treatment in hospitals of 4 974 patients(80.760%) from hospitals with high admission of gallbladder cancer was 38.8±8.9 per year and the average number of cases undergoing diagnosis and treatment in hospitals of 1 185 patients(19.240%) from hospitals with low admission of gallbladder cancer was 5.7±1.9 per year. (2) Age and sex distribution: the age of 6 159 patients diagnosed as gallbladder cancer was 64(56,71) years, in which the age of 2 247 male patients(36.483%) diagnosed as gallbladder cancer was 64(58,71)years and the age of 3 912 female patients(63.517%) diagnosed as gallbladder cancer was 63(55,71)years. The sex ratio of female to male was 1.74:1. Of 6 159 patients, 3 886 cases(63.095%) were diagnosed as gallbladder cancer at 56 to 75 years old. There was a significant difference on age at diagnosis between male and female patients ( Z=-3.99, P<0.001). (3) Diagnosis: of 6 159 patients, 2 503 cases(40.640%) were initially diagnosed as gallbladder cancer and 3 656 cases(59.360%) were initially diagnosed as non-gallbladder cancer. There were 2 110 patients(34.259%) not undergoing surgical treatment, of which 200 cases(9.479%) were initially diagnosed as gallbladder cancer and 1 910 cases(90.521%) were initially diagnosed as non-gallbladder cancer. There were 4 049 patients(65.741%) undergoing surgical treatment, of which 2 303 cases(56.878%) were initially diagnosed as gallbladder cancer and 1 746 cases(43.122%) were initial diagnosed as non-gallbladder cancer. Of the 1 746 patients who were initially diagnosed as non-gallbladder cancer, there were 774 cases(19.116%) diagnosed as gallbladder cancer during operation and 972 cases(24.006%) diagnosed as gallbladder cancer after operation. Of 6 159 patients, there were 2 521 cases(40.932%), 2 335 cases(37.912%) and 1 114 cases(18.087%) undergoing ultrasound, computed tomography (CT) or magnetic resonance imaging (MRI) examination before initial diagnosis, respec-tively, and there were 3 259 cases(52.914%), 3 172 cases(51.502%) and 4 016 cases(65.205%) undergoing serum carcinoembryonic antigen, CA19-9 or CA125 examination before initially diagnosis, respectively. One patient may underwent multiple examinations. Results of univariate analysis showed that geographical distribution of hospitals (eastern China or western China), age ≥72 years, gallbladder cancer annual admission of hospitals, whether undergoing ultrasound, CT, MRI, serum carcinoembryonic antigen, CA19-9 or CA125 examination before initially diagnosis were related factors influencing initial diagnosis of gallbladder cancer patients ( odds ratio=1.45, 1.98, 0.69, 0.68, 2.43, 0.41, 1.63, 0.41, 0.39, 0.42, 95% confidence interval as 1.21-1.74, 1.64-2.40, 0.59-0.80, 0.60-0.78, 2.19-2.70, 0.37-0.45, 1.43-1.86, 0.37-0.45, 0.35-0.43, 0.38-0.47, P<0.05). Results of multivariate analysis showed that geographical distribution of hospitals (eastern China or western China), sex, age ≥72 years, gallbladder cancer annual admission of hospitals and cases undergoing ultrasound, CT, serum CA19-9 examination before initially diagnosis were indepen-dent influencing factors influencing initial diagnosis of gallbladder cancer patients ( odds ratio=1.36, 1.42, 0.89, 0.67, 1.85, 1.56, 1.57, 0.39, 95% confidence interval as 1.13-1.64, 1.16-1.73, 0.79-0.99, 0.57-0.78, 1.60-2.14, 1.38-1.77, 1.38-1.79, 0.35-0.43, P<0.05). (4) Surgical treatment and prognosis. Of the 4 049 patients undergoing surgical treatment, there were 2 447 cases(60.435%) with complete pathological staging data and follow-up data. Cases with pathological staging as stage 0, stage Ⅰ, stage Ⅱ, stage Ⅲa, stage Ⅲb, stage Ⅳa and stage Ⅳb were 85(3.474%), 201(8.214%), 71(2.902%), 890(36.371%), 382(15.611%), 33(1.348%) and 785(32.080%), respectively. The median follow-up time and median postoperative overall survival time of the 2 447 cases were 55.75 months (95% confidence interval as 52.78-58.35) and 23.46 months (95% confidence interval as 21.23-25.71), respectively. There was a significant difference in the overall survival between cases with pathological staging as stage 0, stage Ⅰ, stage Ⅱ, stage Ⅲa, stage Ⅲb, stage Ⅳa and stage Ⅳb ( χ2=512.47, P<0.001). Of the 4 049 patients undergoing surgical treatment, there were 2 988 cases(73.796%) with resectable tumor, 177 cases(4.371%) with unresectable tumor and 884 cases(21.833%) with tumor unassessable for resectabi-lity. Of the 2 988 cases with resectable tumor, there were 2 036 cases(68.139%) undergoing radical resection, 504 cases(16.867%) undergoing non-radical resection and 448 cases(14.994%) with operation unassessable for curative effect. Of the 2 447 cases with complete pathological staging data and follow-up data who underwent surgical treatment, there were 53 cases(2.166%) with unresectable tumor, 300 cases(12.260%) with resectable tumor and receiving non-radical resection, 1 441 cases(58.888%) with resectable tumor and receiving radical resection, 653 cases(26.686%) with resectable tumor and receiving operation unassessable for curative effect. There were 733 cases not undergoing surgical treatment with complete pathological staging data and follow-up data. There was a significant difference in the overall survival between cases not undergoing surgical treatment, cases undergoing surgical treatment for unresectable tumor, cases undergoing non-radical resection for resectable tumor and cases undergoing radical resection for resectable tumor ( χ2=121.04, P<0.001). (5) Multimodality therapy and prognosis: of 6 159 patients, there were 541 cases(8.784%) under-going postoperative adjuvant chemotherapy and advanced chemotherapy, 76 cases(1.234%) under-going radiotherapy. There were 1 170 advanced gallbladder cancer (pathological staging ≥stage Ⅲa) patients undergoing radical resection, including 126 cases(10.769%) with post-operative adjuvant chemotherapy and 1 044 cases(89.231%) without postoperative adjuvant chemo-therapy. There was no significant difference in the overall survival between cases with post-operative adjuvant chemotherapy and cases without postoperative adjuvant chemotherapy ( χ2=0.23, P=0.629). There were 658 patients with pathological staging as stage Ⅲa who underwent radical resection, including 66 cases(10.030%) with postoperative adjuvant chemotherapy and 592 cases(89.970%) without postoperative adjuvant chemotherapy. There was no significant difference in the overall survival between cases with postoperative adjuvant chemotherapy and cases without postoperative adjuvant chemotherapy ( χ2=0.05, P=0.817). There were 512 patients with pathological staging ≥stage Ⅲb who underwent radical resection, including 60 cases(11.719%) with postoperative adjuvant chemotherapy and 452 cases(88.281%) without postoperative adjuvant chemotherapy. There was no significant difference in the overall survival between cases with postoperative adjuvant chemo-therapy and cases without post-operative adjuvant chemo-therapy ( χ2=1.50, P=0.220). Conclusions:There are more women than men with gallbladder cancer in China and more than half of patients are diagnosed at the age of 56 to 75 years. Cases undergoing ultrasound, CT, serum CA19-9 examination before initial diagnosis are independent influencing factors influencing initial diagnosis of gallbladder cancer patients. Preoperative resectability evaluation can improve the therapy strategy and patient prognosis. Adjuvant chemotherapy for gallbladder cancer is not standardized and in low proportion in China.