OBJECTIVE To develop a rapid, sensitive and specific real-time PCR assay for the detection of WU polyomavirus.METHODS The VP2 gene of WU polyomavirus was selected as target gene.The specific primers and TaqMan probes according to the principle of the TaqMan real-time PCR were designed and synthesized,we optimized the PCR reaction,and detected 394 clinical samples.The sensitivity,specificity,reproducibility and dynamic range of the methods were determined.RESULTS The real-time PCR was established and applied successfully to detect the WU polyomavirus.The real-time PCR assay showed excellent linearity between the log of target input and CT value,demonstrating that the assay had a dynamic range of at least 8 logs between 1.0?100 and 1.0?107 copies;the coefficient of variation of intra-assay and inter-assay was less than 5%.Among these 394 specimens,3(0.76%) were positive for WU polyomavirus.CONCLUSIONS The real-time TaqMan PCR is a rapid,specific and sensitive method to detect WU polyomavirus.It can be used in screening large numbers of samples at the same time and establish the solid technological base for clinical diagnosis and epidemiology study.

Objective To investigate the lentivirus-mediated RNA interference(RNAi) reduced human epidermal growth factor 2 (HER2) gene expression in ovarian cancer chemosensitivity.Methods Specifically targeted short hairpin RNA HER2 gene ( shRNA) lentiviral expression vector and negative control sequences lentiviral vector were constructed.stable expression of HER2-shRNA in SKOV3 cell line ( KD) and negative control cell line (NC) were established.The uninfected SKOV3cell lineserved as blank control(CON)group, sensitivity to changes in three kinds of cells to cisplatin chemotherapy in the HER2 gene expression was detected by RT-PCR and Western blot technique, CCK-8 assay CON and KD cells strains were established xenograft model in nude mice in each group to detect the expression of the HER2 protein, as well as by cisplatin chemotherapy tumor size. Results Expression results showed that a stable cell line HER2-shRNA and negative control cell lines had been successfully establish by fluorescence and flow cytometry.RT-PCR results showed KD cells relative HER2 mRNA expression levels compared with the NC group, the difference was statistically significant (P<0.01), whereas no significant difference when compared with the CON group, HER2 mRNA KD Group inhibition rate was (95.5 ± 0.12)%.After transduction specific HER2-shRNA ovarian cancer tumor cells, HER2 protein expression was significantly reduced, growth had slowed down significantly after cisplatin treatment ( P<0.01 ) .Conclusion Lentivirus-mediated HER2-shRNA can effectively inhibit the expression of the HER2 gene, increase chemosensitivity to cisplatin in ovarian cancer.

Tumor infiltrating lymphocytes(TILs) were isolated by enzymatic digestion and discontinuous gradient centrifugation from 8 human advanced tumors (4 stomach carcinoma, 2 liver cancer, 1 non-small-cell lung carcinoma and 1 colon cancer). These cells were cultured in complete RPMI 1640 medium supplemented with l000U/ml of rhIL2 for 4-6 weeks, till the cell number reach over l09/total, reinfused to the same patients i.v. meanwhile, the patients received 105U of rhIL2 i.m for 5 days. One week before and one month after TIL infusion periphery blood from the patients was collected and the mononuclear cells were isolated. Cytotoxicity against a panel of tumor cell targets by MTT colorometric assay and lymphocyte phenotype by two-color flow cytometry were mornitored. The results showed that there was significant increase in the killing ability to the tested tumor targets to different extent, especially the killing to the target cells which shared the same histological type with the patients tumor. (43 against 1249 lytic units p

Objective:To observe whether the HPV16L1VLP Hela-cell binding-inhibition experiment can assess the immune-protective activities of HPV16L1 antibody in HPV16 prophylactic vaccine or other fields.Methods:C57BL/6 mice were divided into three groups,including pcDNAL1 plasmid,HPV16L1VLP protein,pcDNA3.1 plasmid.Each group of mice was immunized intramuscularly or subcutaneously with certain above antigens,accompanying three times and interval 3 weeks.After 14 days of 3 rd immunization,take blood to make serum for immunochemistry.Results:Hela cell neutralized by the serum IgG of experimental mice can be stained negatively,while the cells of control and non-inoculation groups can be stained positively (brown-yellow color).Conclusion:HPV16LIVLP Hela-cell binding-inhibition assay may be the better way to reflect the neutralizing-protective activities of HPV16L1 antibody in many fields including HPV16 prophylactic vaccine.

Objective To study the effect of levels of 24-hour mean blood pressure(BP)and blood pressure diurnal rhythm on renal function of patients with chronic renal failure(CRF).Methods 24-hour blood pressure monitoring was obtained in patients with CRF(117cases),the GFR was determined periodically.Results The rate of decline of GFR in patients with CRF to a target blood pressure was slower than in patients with CRF not to a target BP(P

Objective To analyze the complications, treatments and prognosis of simultaneous pancreas-kidney transplantation. Methods Forty cases of simultaneous pancreas-kidney transplantation performed between Dec. 1999 and Jan. 2010 in our center were retrospectively analyzed. Results Regarding surgical complications, 4 cases had severe hematuria after operation,which needed clinical intervention, including 1 patient receiving catheterization in duodenum to stop bleeding. Two patients were treated with continuous bladder irrigation, and the remaining one received surgical haemostasis because of donor's duodenum and bladder anastomotic artery hemorrhage.Abdominal hemorrhage occurred in 4 patients, including pancreatic hemorrhage in 3 cases and duodenal muscularis hemorrhage in one case. All of them received surgical treatment for hemostasis.Abdominal infection occurred in 8 cases: one died of multiple organ failure, 2 cases were cured after drainage of abscess, 2 cases underwent surgical removal of abscess, and 3 cases were cured after antibiotic therapy. In one case of postoperative anastomotic leakage, pancreas was resected. Four cases of postoperative ileus were cured by continuous clysis with traditional Chinese medicine. Seven cases had pulmonary bacterial infections, including one cases associated with fungal infection. They were cured by the anti-infective treatment. Other complication included poor healing in 5 cases and urinary infection in 2 cases. After combined simultaneous pancreas-kidney transplantation, 10 patients received reoperation because of surgical complications (14 operations). The re-operation rate was 25 %, including 2 patients (4 operations) for hematuria, 4 patients for abdominal hemorrhage, 2 patients (3 operations) for abdominal infection, 1 patient for pancreatic venous thrombosis, 1 patient for anastomotic leakage, and 1 patient for pancreatic fistula. Conclusion Although simultaneous pancreas-kidney transplantation provides a successful and effective treatment for diabetics with endstage renal disease, surgical complication is still affecting the pancreas and kidney grafts after transplantation.

Objective To explore the technology of excision,and finishing on donative pancreas during combined pancreas-kidney transplantation.Methods We successfully harvested multiple abdominal organs together on 40 cases.Wide surgical exposure was obtained.Cannulas were placed for in situ cooling in portal vein and abdominal aorta,and flushed with HC-A (2000 ml) and UW (1000 ml) with the pressure being 10 cm H2O.The intestine was flushed with 0～4 ( normal saline (1000 ml) and metronidazole (200 ml),the liver,kidney,pancreas,spleen and duodenum were en bloc excised and isolated,and the pancreas and kidney were pruned.Results Excision of donative abdominal organ was successfully performed on all 40 cases.The en bloc warm ischemic time was 3.2 min (2～5 min).The cold ischemic time of pancreas was 10.6 h (8～15 h).The cold ischemic time of kidney was 8.5 h (4～16 h).Post-operation mean withdrawal-insulin time was 9.5 days (4～17 d),FFG 6.7 μmol/L (4.4～10.7 μmol/L),GHbA1c 4.4 ％ (4.1 ％～4.7 ％).Creatine was 87.2 (56～121) μmol/L one month after operation.There were 2 cases of DGF after operation,and the creatine level was returned to the normal within one month after operation.Conclusion Technology of excision,preservation and finishing on donative pancreas for combined pancreas-kidney transplantation was one of the key points for successful transplantation.

Objective To study the incidence of PTEN, p53 and Ki-67 expression in astrocytoma and show the relationship between PTEN, p53 expression and the proliferation activity. Methods The surgical specimens from 68 brain astrocytoma patients were analysed to detect PTEN, p53 and Ki-67 expression with immunohistochemical method. Results The incidence of PTEN, p53 and Ki-67 expression was 54.4 %,45.6 % and 48.5 % respectively in astrocytoma. With the grade of astrocytoma increasing the levels of PTEN protein decreased, on the other hand the levels of p53, Ki-67 increased. There was a negative correlation between PTEN expression and grade of astrocytoma while there was a positive correlation between p53, Ki-67 expression and grade of astrocytoma by using the Spearman Correlation test to analyse the data. The incidence of Ki-67 positive expression was 24.3 % in 37 cases exhibiting PTEN positive staining, whereas the incidence of Ki-67 positive expression was 77.4 % in 31 cases exhibiting PTEN negative staining. In statistics, there was an inverse correlation between PTEN and Ki-67 expression. Conclusion There is an inverse correlation between histopathological grades of astrocytoma and PTEN expression. A positive correlation is found between p53, Ki-67 expression and histopathological grades of astrocytoma. PTEN can inhibit tumor cell proliferation in astrocytoma.

OBJECTIVE:To standardize periooperative prophylactic application of antibiotics. METHODS:According to the characteristics of orthopaedic and parenchyma surgery,classifying evaluation table of typeⅠincision infection risk was designed sci-entifically and rationally. The individual application of antibiotics in surgery patients had been achieved through infection risk evalua-tion. High risk typeⅠincision patients used antibiotics rationally and low risk patients seldom used or didn’t use at all. RESULTS:Through using infection risks classifying table,the rate of antibiotics prophylactic application in typeⅠincision drops from 74.10%to 28.68%,and and the per capita duration of antibiotics prophylactic application shortened from 4.23 d to 2.21 d. The postopera-tive infection rate remained the same. CONCLUSIONS:Through infection risk classifying evaluation,individual application of anti-biotics can be achieved in surgery patients,so as to promote rational use of antibiotics for prophylactic use,reduce antibiotics dos-age and antibiotics abuse under the condition of controllable surgery infection.

Objective To explore the technique of allocation and rebuilt of vessels of donor pancreas during pancreas-kidney transplantation.Methods Multiple abdominal organs were successfully retrieved from 40 donors.Results Excision of donor abdominal organs was successful in all the 40 cases.The average warm ischemic time was no 3.2 min,ranging from 2 min to 5 min.No injury occurred in any of the donor organs or vessels.After transplantation,2 patients lost the transplant,one because of abdominal infection,and the other because of venous thrombosis.Conclusion A good trim or rebuilt of donor pancreas vessels is one of the key points for successful combined pancreas-kidney transplantation.