Objectives To prepare the HL-60 cell vaccine expressing heat shock protein 70 (HSP70) of Bacille calmette-Guérin (BCG), so as to study its anti-tumor effect and mechanism. Methods The whole BCG HSP70 gene was amplified from BCG genome by polymerase chain reaction (PCR) and sub-cloned into the polyclone endonuclease sites in pDisplay. The recom-binant vector of pDisplay-HSP70 was verified by sequencing. Then the HL-60 cell vaccine expressing the protein onto the cell surface was prepared by lipofectamine transfection. To detect the immunogenicity of HL-60 cells expressing HSP70, the test groups were divided into three subgroups, HL60-wt, HL60-pDisplay, and HL60-HSP70 respectively. Each group was cultured with peripheral blood T cells for 72 h, then the proliferation indices of T cells were assayed by CFSE-staining method, and IFN-γwere tested by enzyme-linked immunosorbent assay (ELISA). The HL-60 cells of different groups were cultured with peripher-al blood T cells for 6d. The wild-type HL-60 cells were added and co-cultured for another 12h. Cytotoxicity assay was measured by LDH release. Results (1) The fragment of BCG HSP70 was consistent with the theoretical value. DNA sequencing showed that the recombinant vector of pDisplay-HSP70 was correctly constructed. (2) BCG HSP70 expressed onto the HL-60 cells sur-face. (3) Detection of the immunogenicity: ①The most significant T cell proliferation was observed in the group of HSP70-transfected HL-60 cells (P<0.05). There was no difference between the HL60-wt group and HL60-pDisplay group (P>0.05).②The contents of IFN-γof the HSP70-HL60 group was the highest.③The inhibiting activity of CTLs on HL-60 cells in the group of HSP70-transfected HL-60 cells was more significant than that of wide-type and pDisplay--transfected HL-60 cells. And with the increase of the E:T ratio, the inhibiting activity of CTLs in the HSP70-HL60 group was rising. Conclusions The recombinant eukaryotic expression vector (pDisplay-HSP70) of BCG HSP70 was successfully constructed. And the HL-60 cell vaccine expressing BCG HSP70 onto its surface was successfully prepared. The results showed that gene transfection of BCG HSP70 could significantly enhance the immunogenicity of HL-60 cells.

OBJECTIVE:To provide reference for imipenem-resistant Pseudomonas aeruginosa infection control. METHODS:114 patients infected with imipenem-resistant P. aeruginosa were selected from 3 tertiary hospitals in Zhoushan during Feb. 2013 to Feb. 2014. 114 strains of P. aeruginosa were isolated from clinical specimens,and drug resistance characteristics and carbapene-mase-producing gene diversity were analyzed. 101 inpatients with imipenem-resistant P. aeruginosa infection were included in con-trol group;univariate and multivariate Logistic regression analysis were adopted to explore the risk factors of imipenem-resistant P. aeruginosa infection. RESULTS:114 strains were sensitive to polymyxin B,and had different levels of resistance to other 9 kinds of antibiotics. Carbapenemase-producing gene were mainly IMP and VIM type gene. Long-term hospitalization,mechanical ventila-tion,used imipenem and early combined use of antibiotics were risk factors of imipenem-resistant P. aeruginosa infection. CON-CLUSIONS:In Zhoushan area,imipenem-resistant P. aeruginosa shows serious drug resistance. To avoid long-term hospitalization and early combined use of antibiotics can reduce imipenem-resistant P. aeruginosa infection.

Objective To evaluate the clinical value of cerebrospinal fluid ADA,IL-23 joint detection in the diagnosis of tuberculous meningitis related diseases.Methods 253 cases with tuberculous meningitis related diseases were selected as the research subjects.According to the diagnosis,they were divided into tuberculous meningitis group (meningitis group,138 cases ),tuberculous meningitis complicated with hydrocephalus group (hydrocephalus group,35 cases)and control group(80 cases).All patients after admission received lumbar puncture, the part of cerebrospinal fluid specimens were inspected,the cerebrospinal fluid ADA,IL -23 joint test was conducted.Meningitis group and hydrocephalus group were given anti -TB drugs (INH,RFP and PZA +sm ) combined with chemotherapy.3 months after treatment,the meningitis group and hydrocephalus group received lumbar puncture cerebrospinal fluid ADA,IL -23 joint test again.The levels of ADA,IL -23 in cerebrospinal fluid were compared.Results Before treatment,cerebrospinal fluid ADA,IL-23 levels in the meningitis group were (12.64 ± 5.54)u/L and (48.38 ±10.78)pg/mL,those in the hydrocephalus group were (15.81 ±6.92)u/L and (77.21 ± 13.42 mm)pg/mL,which in the control group were (3.21 ±2.20)u/L and (9.05 ±3.89)pg/mL,ADA,IL-23 levels in meningitis group and hydrocephalus group before treatment were significantly higher than the control group (F=117.24,724.97,P<0.001).Spearma analysis showed that each group of cerebrospinal fluid ADA and IL-23 had no correlation.After treatment,the cerebrospinal fluid ADA,IL-23 levels in the meningitis group were (3.79 ± 3.13)u/L and (13.46 ±6.62)pg/mL,which in the hydrocephalus group were (6.42 ±4.35)u/L and (25.42 ± 8.54)pg/mL,the meningitis group before and after treatment had statistically significant differences in cerebrospinal fluid ADA,IL-23 (t=16.34,32.43,all P<0.001);hydrocephalus group before and after treatment had statistically significant differences in cerebrospinal fluid ADA,IL-23 (t=6.80,19.26,all P<0.001 ).Conclusion Cerebro-spinal fluid ADA,IL-23 joint detection in the early diagnosis of tuberculous meningitis related diseases and clinical observation has high clinical value.

Tethered cord syndrome is a severe and congenital disease. Resecting the diseased ilium terminal(FT), eystis, lipoma is the main treatment for the disease, relieving the injury to the eonus medullaris. Why the FT is diseased and thickened? Is it necessary to eut internal FT or external FT? What is the injury mechanism of the low-positional or normal positional oonus medullaris? To study the component and structure of the conus medullaris and FT may give definite answers.

Objective To access the clinical value of interstitial brachytherpay in patients with advanced carcinoma. Methods 6 patients(1 case of hepatic cancer,3 cases of prostate cancer,2 cases of local recurrence of rectal cancer)received interstitial high dose rate(Ir-192)brachytherpay with afterloading technique(6.0 Gy/day in 4 days) Tubes were inserted with laparoscopic guidance to minimize the risk of tube misplacement. Results The size of the tumors was reduced and the local control rate was encouraging(6/6). one patient with local recurrence of rectal cancer aquired CR,the ofher five cases acquired PR. Conclusions Interstitial tube insertion with laparoscopic guidance to treat advanced carcinoma in afterloading technique is a simple,minimally invasive,safe and economical method,especially fit for elder patients who intolerate or disagree to operation.

Objective To explore the method and result of endoscopic thyroidectomy. Methods Thyroidectomy was performed endoscopically in 4 cases of thyroidoma. Results 4 patients underwent endoscopic thyroidectomy, whose operation time was 52,63,70,75 minutes respectively. An average blood loss during operation was 35ml and no complication occurred. They were discharged 4 days～5 days after operation. Conclusions The endoscopic thyroidectomy has the advantages of minimal invasion, less bleeding, less complication and quicker recovery.

Objective To study the feasibility of endoscopic thyroidectomy with bipolar electrocoagulation. Methods Endoscopic partial thyroidectomy via precordial approach, mainly by using bipolar electrocoagulation and scissors, was accomplished in 21 patients with thyroid tumors with a diameter of 0.7～5.5 cm. Results The operation was accomplished endoscopically in 20 patients, whereas a conversion to open thyroidectomy was conducted in 1 patient. The mean incision length was 22 mm, the operation time was 69?47 min, and the blood loss was 55?41 ml. Postoperative analgesic treatment was required in 2 patients and no serious complications happened. Conclusions Endoscopic thyroidectomy with bipolar electrocoagulation is feasible in the absence of ultrasonic scalpel.

Objective To evaluate the feasibility of hand-assisted laparoscopic colorectal resection. Methods Clinical data of 21 cases of hand-assisted laparoscopic colorectal resection in this hospital from October 2003 to August 2004 were retrospectively reviewed. Results The hand-assisted laparoscopic resection was accomplished in 20 cases. The mean operation time was 144 min, the mean blood loss was 120 ml, the mean number of dissected lymph nodes was 8.5, and the mean time to intestinal function recovery, 69 h. Postoperative abdominal bleeding was found in 1 case, which was cured with conservative therapy. A conversion to open surgery was needed in 1 case. Conclusions Hand-assisted laparoscopic colorectal resection safe, feasible, and radical, with advantages of fewer complications and simplicity of performance.

Objective To culture the acute leukemia cells in vitro,and to prepare cancer vaccine expressing heat shock protein 70 (HSP70) of Bacille Calemette-Geérin(BCG) onto the cell surface,so as to study its anti-tumor effect and mechanism.Methods Acute myeloid leukemia (AML) cells were cultured in a serum-free Stemspan(H) culture supplemented with cytokines [stem cell factor(SCF),flt-3 ligand (FL),interleukin (IL)-3 and IL-6] in vitro.And B-lineage acute lymphoblastic leukemia (B-ALL) cells were cultured in a Iscove modified medium(IMDM) culture supplemented with cytokines (SCF,FL,IL-3 and IL-7) in vitro.Cellular morphology was observed by the microscopy and immunophenotype determination was used to verify the biological characteristics of acute leukemia cells after culture.Lipofectamine 2000 was used to transfect the pDisplay-HSP70 plasmid into acute leukemia cells.The expression of HSP70 on the cell surface was detected by fluorescene microscope.Then the immunogenicity of the leukemia cells expressing HSP70 were detected.The experimental groups were divided into 3 subgroups:the wide-type acute leukemia cells (wt-LC group),the pDisplay-leukemia cells (pDisplay-LC group),and the pDisplay-HSP70-leukemia cells (HSP70-LC group),respectively.The leukemia cells in different groups were cultured with autologous peripheral blood T cells for 72 hours.The proliferation indices of T cells were assayed by carboxyfluorescein diacetate succinimidyl ester (CFSE)-staining method,and the contents of interferon-γ(IFN-γ) were tested by enzyme-linked immunosorbent assay (ELISA).The leukemia cells in different groups were cultured with autologous peripheral blood T cells,and after 6 days,the fresh acute leukemia cells were added [in the different ratios of cytotoxicity T lymphocyte (CTL):leukemia cells were 10 ∶ 1,20 ∶ 1,40 ∶ 1 and 80 ∶ 1] and continued to be cultured for another 12 hours.Cytotoxicity assay was measured by lactate dehydrogenase (LDH) release.Results After short term culture in vitro,the leukemia cells were in colony-like suspension and maintain the proliferation characteristics were maintained.The cell proliferation was rapidly cultured for about 10 days and then was gradually slowed down.But there was no difference between the day 10 and day 0 in the expressions of CD13 and CD33 in fifteen cases of AML cells (P ＞ 0.05).Equally,there was no difference between the day 10 and day 0 in the expressions of CD19,CD10 and CD22 in fifteen cases of B-ALL cells (P ＞ 0.05).After BCG HSP70 gene transfection,the yellow-green fluorescence on the leukemia cells surface was observed under the confocal microscope.Detection of the immunogenicity:(1) Autologous T cell proliferation:the most significant T cell proliferation was observed in the group of HSP70-transfected leukemia cells (t =17.89,19.58,all P ＜0.05).There was no difference between the wt-LC group and pDisplay-LC group (P ＞ 0.05).(2) The contents of cytokines:the IFN-γ level in the group of HSP70-transfected leukemia cells was higher than those of wide-type acute leukemia cells and the pDisplay-transfected ones (t =24.72,24.81,all P ＜ 0.05).(3) Cytotoicity of CTL:the killing rate in HSP70-transfected leukemia cells was significantly higher than those of wide-type acute leukemia cells and pDisplay——transfected ones(F =13.66,P ＜ 0.05).And with the increase of the ratio from 10 ∶ 1 to 80 ∶ 1,the inhibiting activity of CTL in the HSP70-LC group was raising(F =19.69,P ＜ 0.05).Conclusions Fresh acute leukemia cells can be successfully cultured in vitro.Short-term culture can significantly increase the number of leukemia cells,but has little effect on surface antigen expression.So,the biological characteristics of the leukemia cells can be maintained.The leukemia cells vaccine expressing BCG HSP70 onto its surface was successfully prepared,and gene transfection of BCG HSP70 can significantly enhance the immunogenicity of leukemia cells.

Objective:To study the relationship betwen the mechanism of autoimmune disease and CD4+CD25+ T cell population in NIK mutated mice-aly mice.Methods:NIK mutated mice-aly/aly mice were used as model,aly/+mice as NIK normal control;cell populations were determined by FACS and the thymus structure were analyzed by immunohistochemistry.Results:The CD4+CD25+CD8- population were remarkably decreased in aly mice;and the UEA-1 positive cells were absent in aly mice.Conclusion:The autoimmune disease in aly mice might be the result of deceased the CD4+CD25+ population;the UEA-1 positive cells might play an important role in the development of CD4+CD25+ population. [