Objective To establish a rapid monitoring method of the three common bacteria in mice frozen resources, such as embryo, sperm, etc. Methods To extract DNA of the three positive bacteria( Staphylococcus auerus, Klebsiella?pneumoniae and β?hemolyticstreptococcus) , and establish PCR monitoring method of the three positive strains through designing primer and refining PCR condition. Then extract total DNA of the frozen resources, detect the DNA according to the PCR condition of the three positive bacteria, some samples were detect by fluorescence quantitative PCR at the same time. Results ①successfully establish a PCR detection method of the three positive bacteria, the minimum detectable concentration of Staphylococcus auerus, Klebsiella pneumoniae and β?hemolytic streptococcus is 4?19 × 10 -5 ng/μL, 1?98 × 10 -5 ng/μL and 1?07 × 10 -3 ng/μL. ②Proved that the three bacteria doesn ’ t exist in the sample by normal PCR and fluorescence quantitative PCR methods. Conclusions Establising a rapid monitoring method of common pathogens of frozen embryo and sperm in mice.

Objective To investigate the changes of average body weight gain and serum biochemical indexes of C57BL/6J mice (B6 mouse) and their offspings after frozen-thawed embryo transfer of B6 mice.Methods The mice were divided into three groups in this study.In the experimental group I (E-I,30 males and 20 females),2-cell embryos after in-vitro fertilization were collected,and cryopreserved by EFS method,then obtained the offsprings after transplantation of the recovered embryos to oviduct of recipient mice (ICR mouse).In the experimental group II (E-II,26 males and 17 females),when the mice from E-I grew to maturity,the offsprings were obtained from natural mating of mice from E-I.In the control group (20 males and 20 females),the offsprings came from conventional feeding and natural mating.The three groups of mice were raised to 16 weeks old,weighing the body weight at a regular time intervals,and the serum biochemical indexes were obtained from 16-week-old mice.Then the changes of average body weight and serum biochemical indexes of the mice were analyzed.Results The average body weight of E-I mice was significantly higher than that of control group at each week-age (P<0.01).The average body weight of E-II female mice was significantly higher than that of the control group in 12-16-week old mice (P<0.01),but the average body weight of E-II male mice showed no significant differences compared with the control group except for few weeks.The serum biochemical indexes of E-I and E-II mice were changed in all items except for AST,TP and Ca.Conclusions There are some effects on the average body weight gain and serum biochemical indexes of C57BL/6J mice and their offspings after frozen-thawed embryo transfer.

BACKGROUND: Hemorrhagic cystitis remains a common complication of hematopoietlc stem cell transplantation.Granulocyte-macrophage colony stimulating factor (GM-CSF) affects proliferation and differentiation of hematopoietic stem/progenitor cells, adjusts functions of monocytes, granulocytes, lymphocytes and endothelial cells.OBJECTIVE: To investigate the protective effects of GM-CSF bladder irrigation in hemorrhagic cystitis after allogeneic hematopoietic stem call transplantation.DESIGN: Case analysis.PARTICIPANTS: A total of 15 hematopathy patients undergoing allogenic hematopoietic stem cell transplantation at the Zhongshan Hospital of Sun Yat-sen University from January 2004 to August 2006 (routine treatment group). A total of 16 hematopathy patients undergoing allogenic hematopoietic stem cell transplantation from September 2006 to December 2008 (GM-CSF group).METHODS: In the routine treatment group, patients received mesna, hydration, alkalization and forced diuresis in the prevention of hemorrhagic cystitis. In the GM-CSF group, GM-CSF was infused into the bladder in addition to mesna,hydration, alkalization and forced diuresis in the prevention of hemorrhagic cystitis 24 hours before cyclophosphamide treatment. Catheter was extracted 3 days following cyclophosphamide withdraw. Following washing with saline, the bladder was emptied. 10 mL of saline and 5 mL of lidocaine were added into 300 μg of GM-CSF. The mixture was infused into the bladder for 60-120 minutes.MAIN OUTCOME MEASURES: The following parameters were measured: occurrence of hemorrhagic cystitis and its correlation to graft versus host disease, as well as the occurrence of cytomegalovirus infection and urinary system infection.RESULTS: Compared with routine treatment group, the occurrence rate of hemorrhagic cystitis was significantly decreased in the GM-CSF group (x2=4.39, P < 0.05), mean duration of hemorrhagic cystitis and duration of hospitalization were significantly shortened (t=3.97, P < 0.05; t=3.13, P < 0.05), and the occurrence rate of over grade HI hemorrhagic cystitis was significantly reduced (x2=5.04, P < 0.05). Cystitis degree was associated with degree and duration of graft-versus-host disease (r = 0.76).Compared with the routine treatment group, cytomegalovirus infection rate was slightly decreased in the GM-CSF group (x2=0.28, P> 0.05), and occurrence rate of over grade Ⅲ hemorrhagic cystitis was higher in patients with cytomegalovirus infection.Compared with the routine treatment group, the occurrence rate of urinary system infection was slightly reduced in the GM-CSF group (x2=0.28, P > 0.05).CONCLUSION: GM-CSF bladder irrigation is well tolerated and often effective, and should be considered as a preparative regimen of hemorrhagic cystitis after allogeneic hematopoietic stem call transplantation.

Heparinase II (Hep II) from Flavobacterium heparinum is an enzyme that could specifically cleave certain sequence of heparin and heparan sulfate. In this work, fermentation conditions of recombinant heparinase II (His-Hep II) producing bacteria were optimized, including initial induction time, inducer (IPTG) concentration, induction temperature and induction time. The optimum conditions were as follows: cultivating recombinant bacteria to exponential prophase under 37 degrees C, then adding IPTG to a final concentration of 0.3 g/L, finally cultivating recombinant bacteria under 20 degrees C for 10 h. The total crude enzyme activity reached 570 U/L. Based on these results, high cell density fermentation of recombinant bacteria was studied. The final OD600 could reach 98 and the total crude enzyme activity of His-Hep II increased to 9 436 U/L.
Fermentation ; Flavobacterium ; metabolism ; Microbiological Techniques ; Polysaccharide-Lyases ; biosynthesis ; Recombinant Proteins ; biosynthesis

Objective To investigate the efficacy and tolerability of dose-dense chemotherapy with cisplatin plus 5-fluorouracil( PF regimen)in distant metastatic nasopharyngeal carcinoma( NPC)patients. Methods From April 1,2008 to April 30,2014,168 patients were assigned to traditional group(n = 83) and dose-dense group(n = 85)using digital random table in 1 : 1 ratio. All patients received PF regimen,and once every 28 days in traditional group and once every 14 days in dose-dense group. The primary endpoint was progression-free survival(PFS)and the secondary endpoint was overall survival(OS),toxicity and response rate. Results The median PFS,median OS,1,2,3-year survival rate,complete response rate and objective response rate were significantly improved in dose-dense group which were 13. 3 months,20. 2 months,80. 2% , 36. 0% ,16. 1% ,16. 5% ,84. 7% ,and those in control group were 10. 0 months,16. 1 months,59. 6% , 10. 1% ,0,3. 6% ,54. 2% respectively(χ2 = 24. 47,P = 0. 000;χ2 = 16. 65,P = 0. 000;χ2 = 8. 41,P =0. 004;χ2 = 16. 96,P = 0. 000;χ2 = 14. 91,P = 0. 000;χ2 = 7. 63,P = 0. 006;χ2 = 18. 47,P = 0. 000). The rates of grade 3-4 adverse events in dose-dense and traditional group were 38. 8% and 6. 0%(χ2 = 25. 81, P = 0. 000). Conclusion Dose-dense chemotherapy of PF is more efficient and acceptable toxic than the tra-ditional one. It can be a new treatment option for patients with distant metastases of NPC.

Objective To investigate the efficacy and safety of low dose amphotericin B for treating invasive fungal infection (IFI) in hematologic malignancies.Methods Ninety-eight patients with hematologic diseases who visited our hospital from January 2008 to June 2012 were randomly divided into the control group (n =47) and the treatment group(n =51).Patients in the control group were treated with amphotericin B,50-60 mg per day and patients in treatment group were given amphotericin B,25-30 mg per day.Clinical efficacy and side effects were compared between the two groups.Results There was no significant difference on the median cumulative dose between the control group and the treatment group(725 (175,1595) mg vs 735 (225,1485) mg,P =0.834).But there was significant difference on median treatment days between the control group and the treatment group(19(8,34) d vs 29(11,58) d,P =0.000).After treatment for 14 days,the patients who can be evaluated for efficacy were 37 and 48 cases respectively in the control group and in the treatment group,and there was no significant difference on the total efficacy rate,the rate of progress and the rate of fever between the control group and the treatment group (all P ＞ 0.05).There were 14 patients and 6 patients terminated treatment because of adverse reaction,and the difference was significant (29.8％ vs 11.8％,P =0.027).The side effect rates of hepatic and renal impairment in the control group was significantly higher than that in the treatment group(27.7％ (13/47) vs 11.8％ (6/51),P =0.047).Conclusion The efficacy of 25-30 mg per day's amphotericin B treatment is not lower than amphotericin B at 50-60 mg per day on IFI in hematologic malignancies.It is not only relatively safe and less expensive,but also operability and practicality in the treatment of IFI.

Objective:To study and compare the prognosis of different pathological subtypes of renal cell carcinoma (RCC).Methods:Clinicopathological and prognostic data of 1 346 cases of postoperative renal cell carcinoma during July 2002 to June 2014 in West China Hospital were collected retrospectively.There were 839 males and 507 females, aged (55.1±13.4)years, including 1 120 cases of clear cell RCC, 62 cases of papillary RCC, 79 cases of chromophobe RCC and 85 cases of the other pathological types respectively. ECOG 0 and ≥1 were 911 and 435 cases, with; T 1, T 2, T 3 and T 4 of 1 019, 177, 102 and 48 cases respectively; WHO nuclear grade for well, intermediate, poor differentiation and unknown were 587, 530, 85 and 144 cases separately.Tumor size <5cm, 5-10cm, ≥10cm and unknown were 685, 541, 104 and 16 cases.Combined with necrosis or sacromatoid differentiation were 200/1 146 and 27/1 319 cases separately. Meanwhile, data of 80 439 cases from Surveillance, Epidemiology, and End Results Program (SEER) were also collected.There were 51 371 males and 29 068 females, aged (60.9±12.4) years; , with 66 261, 8 680, 5 022 and 476 cases of White, Black, Asian, American native, or unknown race separately. There were 62 600 of clear cell RCC, 12 170 of papillary RCC, 4 354 of chromophobe RCC and 1 315 of other pathological types, with T 1, T 2, T 3 and T 4 of 55 332, 8 687, 15 516 and 904 cases respectively; WHO nuclear grade for well, intermediate and poor differentiation were 52 323, 22 700 and 5 416 cases separately.Tumor size <5cm, 5-10cm, ≥10cm were 46 741, 25 760 and 7 938 cases respectively. Kaplan-Meier survival analysis were performed on these two group of cases, with different factors between subgroups (gender, age, pathological types, tumor stage, size and nuclear grade) evaluated by log-rank test. To evaluate accuracy of outcome prediction models of SSIGN, Leibovich and UISS score, concordance index of these models were evaluated. Results:In 1 346 cases of our cohort, those with chromophobe RCC were well prognostic, survival were relatively better in clear cell RCC than that of papillary RCC, and worst prognosis were demonstrated in those with other types of RCC (5 year overall survival rate: 97.5%, 87.9%, 79.7% and 68.4% separately). Poor prognosis were seen in those older than 50 years, with poor T stage or nuclear grade, large tumor size and tumors with necrosis or sacromatoid differentiation ( P<0.05). In 80 439 seer cases, the best prognosis was also seen in chromophobe RCC and the worst in other type of RCC separately (5 year overall survival rate: 96.3% and 85.3%). In addition, longer survival was seen in papillary RCC than clear cell RCC (5 year overall survival rate: 92.5% and 88.9%). However, similar results with our cohort were seen in Asian and American native subgroup of SEER cases (95.1%, 88.6%, 86.7%, 80.2% for chromophobe, clear cell, papillary and other types of RCC respectively). Poor prognosis were seen in those older than 50 years, males, Asian/ American Indian, poor T stage or nuclear grade and large tumor size ( P<0.05). Concordance index for SSIGN, Leibovich and UISS models in our cohort were 0.763-0.781, 0.725-0.752 and 0.641-0.660, respectively. The chromophobe RCC subgroup was relative better based on predictive value of prognosis models(c-index of UISS of 0.670-0.781, SSIGN and Leibovich of 0.733-0.903). Conclusions:In Asian RCC population, prognosis of chromophobe RCC is best, clear cell RCC is slightly better than papillary RCC, and the prognosis of other types of RCC is the worst. Concordance index of SSIGN and Leibovich in our cohort were higher than that of UISS, and the use value for predictive model was better in the chromophobe RCC subgroup.

Objective To evaluate the benefit of radical prostatectomy (RP) in patients with lymph node-positive prostate cancer.Methods A systematic review of the studies about radical prostatectomy for the prognosis of node-positive prostate cancer was performed.An electronic search was completed on the basis of PubMed,Embase,Cochrane library,China Biology Medicine disc (CBM),China National Knowledge Infrastructure (CNKI),VIP and Wanfang database from inception up to November 2018.The outcomes are overall survival (OS) and cancer-specific survival (CSS).Results Six studies incorporating 7 890 patients were eligible for the present meta-analysis.6 247 patients underwent RP,the remaining 1 643 patients did not undergo RP.Lymph node-positive patients treated with RP had improved OS (HR =0.55,95％ CI 0.49-0.62,P ＜0.001) and CSS (HR =0.49,95％ CI 0.42-0.57,P ＜ 0.001).Conclusions Radical prostatectomy may be a beneficial option for patients with lymph node metastases at initial diagnosis,which also improve the OS and CSS.More randomized controlled trials are needed to give more evidence further.

Objective To report clinical application of free chimeric vascularized fibular graft combined with sural flap for reconstruction of composite extremity defects after open fracture.Methods From June 2010 to July 2014,free chimeric vascularized fibular grafts and sural flaps were used to treat 4 patients with composite extremity defects at Department of Orthopaedics,The Sixth People's Hospital.They were 3 men and one woman,aged from 39 to 61 years(average,48.5 years).There were 2 cases of soft tissue defects on the forearm complicated with radial defect,one case of soft tissue defects on the forearm complicated with ulnar shaft defect,and one case of soft tissue defects on the leg complicated with tibial defect.The length of bone defect ranged from 8 cm to 18 cm (average,13.0 cm);the size of soft tissue defects ranged from 22 cm × 6cmto23cm × 15 cm (average,22.3cm × 9.7cm).Results The area of flap ranged from 25 cm × 9 cm to 26 cm × 18 cm (average,25 cm × 13 cm);the length of fibular graft ranged from 8 cm to 18 cm (average,13 cm).The 4 patients were followed up for 8 to 42 months (average,20.5 months).All the chimeric flaps survived.All the fractures united after an average of 8.5 months.The last follow-ups revealed no refracture.All the patients were satisfied with the outcomes.Conclusion Free chimeric vascularized fibular graft combined with sural flap is a reliable choice for reconstruction of composite extremity defects after open fracture.