AIM: To investigate the effect of norcantharidin(NCTD)on proliferation and invasion of human breast cancer cell line SKBR3 in vitro and its anticancer mechanisms.METHODS: MTT assay was used to determine SKBR3 cell proliferation. Light and FACScan were used to detect apoptosis and cell cycle. The invasiveness of SKBR3 was evaluated by the adhesion test,Matrigel experiment and the crossing-river test.RESULTS: NCTD had inhibitive effects on growth of SKBR3 cells in a dose and time-dependent manner, with the IC50 value of 12.5 mg/L at 24 h.The cells treated with 10 mg/L NCTD for 24 h and 48 h showed typical apoptotic morphology and hypodiploid peak before G1 phase. The cell cycle was arrested at G2/M phase. The apoptosis percentage was up to 3.44% and 6.17%, and the G2/M percentage was up to 35.82% and 38.70%. NCTD also could inhibit obviously the adhesion, movement and invasive capability simulating human basement membrane of SKBR3. Its effect was also in a dose-dependent manner. In the NCTD-treated group, crossing-river time was prolonged significantly and passing-membrane cells markedly decreased. CONCLUSION: NCTD in vitro inhibits not only the proliferation and growth of human breast cancer cells but also invasion and metastasis of the cells at relatively low concentration. NCTD shows prominent anti-tumor effects.

Objective To evaluate the influence of transcatheter arterial chemoembolization (TACE) with Alginate microsphere-Adriamycin on angiogenesis in VX2 liver tumor. Methods Thirty New Zealand rabbits were randomly divided into 5 groups (each n=6), and VX2 carcinoma was implanted in the left lobes of the livers. TACE was performed with Alginate microsphere (Group A), Alginate microsphere-Adriamycin (Group B), Lipiodol (Group C), Lipiodol-Adriamycin (Group D), and control group (Group E), respectively. Three weeks later, the animals were killed and the samples were evaluated with immunohistochemical reaction to examine the VEGF expression and MVD count. Results The positive rate of VEGF expression was 66.67%, 50.00%, 100%, 83.33% and 66.67% respectively in five groups (P>0.05). MVD count was 55.36±7.02, 41.27±8.45, 82.42±6.23, 67.81±11.42 and 62.46±7.54 respectively in five groups. MVD value of group C was higher than that of group A and group B (P<0.05); of group B was lower than that of group D (P<0.05). Spearman rank correlation analysis showed a correlation coefficient of 0.726 between VEGF and MVD (P<0.01). Conclusion TACE with Alginate microsphere-Adriamycin can reduce VEGF expression and MVD of VX2 liver tumor in rabbits, but the possibility of tumor blood vessels rapid occlusion and therefore resulting in tumor necrosis can not be ruled out.

ObjectiveTo assess the values of 320-detector row dynamic volume CT angiography (CTA) and transthoracic echocardiography (TTE) in follow up of coronary artery aneurysm (CAA) caused by Kawasaki disease (KD).Methods320-de-tector row CTA and TTE were applied in long-term follow-up of 8 patients with CAA caused by KD.ResultsIn 8 patients, the mean age at onset was 41.63±22.70 months and the mean follow up time was 43.50±10.99 months. In acute phase, 3 cases of giant coronary artery aneurysms (GCAA) and 5 cases of mid-small CAA were diagnosed by TTE. A total of 16/32 arteries (50%) were involved. At the end of follow-up, 3 cases of GCAA and 2 cases of mid-small CAA were still diagnosed by TTE, and small CAAs were regressed in another 3 cases. A total of 6/32 arteries (18.75%) were involved. Simultaneously at the end of follow-up, a total of 7/32 arteries (21.9%) were involved by 320-detector row CTA. The distribution was consistent with that of TTE. Mean-while, there were one case of left circumlfex artery, one case of GCAA at distal of the right coronary artery, 2 cases of thrombus, 1 case of coronary stenosis and 2 cases of calciifcation.ConclusionsCAA caused by KD may be persistent for a long time. The thrombus, stenosis, and calciifcation of coronary can occurr at late phase in GCAA. TTE is sensitive and reliable to detect proxi-mal and middle segment of coronary lesions, but has limitations in detection of distal segment of coronary arteries. 320-detector row CTA has more comprehensively view of each coronary artery lesions and is especially sensitive and reliable to detect coro-nary thrombosis, calciifcation and narrowing in proximal and distal coronary arteries after acute phase.

The lumber ligaments play an important role in spinal bsomechanics. The results of three-dimensional finite element analysis showed that one of functions of lumbar ligaments is transmission of the tensile load between the lumbar vertebrae. The anterior longitudinal ligament is loaded in extension of lumbar spine and the resistance to the tensile load in flexion is provided by other ligaments. These ligaments are subject to much more tension with degsneration of the intervertebral disc so that a series of pathological changes occur. Relevant significance in clinical aspect is also discussed.

Objective To investigate the effects of contrast dose and region of interest (ROI) depth on quantitative analysis of liver by contrast-enhanced ultrasound (CEUS) during clinical application.Methods After bolus injection of contrast agent,the change of quantitative parameters [including echo mean(EM),rise time(RT),peak intensity(PI),mean transit time(MTT),area under the curve(AUC),time from peak to one half(TPH),wash in slope(WIS),time to peak(TTP)] of time-intensity curves were analyzed based on groups from different doses (1.0 ml and 1.6 ml) and different depth (＜30 mm,30-60 mm,and ≥60 mm).Results MTT and TPH were increased with dose increasing from 1.0 ml to 1.6 ml (P＜0.05).With the dose 1.0 ml,TPH,WIS,PI,AUC and MTT showed significant difference when the depth of the ROI changes (P ＜0.05),with the depth increased,TPH,WIS,PI,and AUC all decreases and MTT increases.For all the other parameters,no significant changes were found (P ＞0.05).Conclusions CEUS and its imaging process can directly influence the accuracy of the parameters from the quantitative analysis.Standardization of contrast agent with predefined dose and depth can potentially facilitate future clinical studies in liver CEUS.

AIM: To discuss the relationship between prostate specific membrane antigen(PSMA) and prostate cancer and to seek a target for diagnosis and therapy of prostate cancer.METHODS: A pair of primers was designed according to the published PSMA mRNA sequence.Total RNA was extracted from prostate cancer tissues and was reversely transcribed into cDNA,which was used as a template for PCR to amplify the PSMA gene.The recombinant was sequenced and the result was analyzed by BLAST.The PSMA5 gene specific primers were designed to identify its expression in different cells and prostate tissues.RESULTS: A new alternatively spliced variant of PSMA named PSMA5 was discovered when sequencing the recombinant.PSMA5 showed well pathological tissue-specificity,and its expression rate in prostate cancer,benign prostatic hyperplasia of prostate,and normal prostate tissue were 92.6%,78.8% and 10.0%,respectively.It expressed specifically in Pca cell line LNCaP,not in cell lines of PC3,bladder carcinoma,renal carcinoma,or hepatoma.CONCLUSION: A new alternative spliced variant of PSMA named PSMA5 was discovered,which was well correlated with prostate cancer and benign prostatic hyperplasia.This finding may give a new clue to the evolution of prostate cancer and may provide a target for the diagnosis and therapy of prostate cancer.

AIM: To obtain eukaryotic expression vector of Chinese prostate-specific membrane antigen. METHODS: Chinese prostate-specific membrane antigen (PSMA) cDNA was amplified by RT-PCR from prostate cancer tissues, then cloned into eukaryotic expression vector pcDNA3 0 and sequenced. RESULTS: Seven bases in Chinese PSMA cDNA sequence were found different from those reported by Israeli, which lead to two different amino acids. CONCLUSION: We have obtained the PSMA cDNA, and the recombinant eukaryotic expression vector was successfully constructed. The study lays foundation for DCs vaccine modified by PSMA gene for the treatment of prostate neoplasms.

Objective To construct the murine allogeneic acute GVHD model.Methods C57BL/6 (H-2b) mice were used as the donors and Balb/c (H-2d) mice as the recipients in allogeneic bone marrow transplantation (BMT).Groups were set as total body radiation (TBI) control group (n =4),GVHD group (n =10),simple BM transplantation group (n =10) and normal control group (n =4).For TBI control group,mice were subjected to TBI but did not receive BMT after radiation.For GVHD group,5 days before TBI,gentamycin (320 mg/L) and erythromycin (250 mg/L) were added into the drinking water,and on the day of transplantation,mice received one total dose of 8.0 Gy 60Coγ TBI,and within 5 h,2 × 106 C57BL/6 BM cells and 1 × 107 C57BL/6 spleen cells were transfused per mouse via the tail vein.For simple BMT group,the pretreatment was the same as GVHD group,and mice received only 2 × 106 C57BL/6 BM cells per mouse via the tail vein.The mental status,activity,posture,fur,weight,and stool were observed after transplantation.Survival time of each mouse was recorded,survival rate was calculated,and survival curve was drawn.Pathological examination was done for the liver,skin,small intestine and BM on the brink of death.Results The median survival time (MST) in TBI control group,GVHD group and BMT group was (9.0 ± 0.7),(32.0 ± 3.2) and ( 17.5 ± 1.6) days respectively,and there was significant difference between every two groups (P ＜ 0.01 ).Pathological examination in TBI control group showedhematopoiesis exhaustion.GVHD group showed acute GVHD symptoms 10-13 days after allo-BMT,and the pathological changes of the skin,liver and small intestine corresponded to those of Ⅰ to Ⅱ degree of GVHD.Simple BMT group also showed acute GVHD symptoms 10-13 days after alloBMT,but their GVHD manifestation and histological changes were less serious and only 0 to Ⅰ degree of GVHD could be seen.ConclusionStable acute GVHD model can be constructed by transfusion of allogeneic BM cells and spleen cells into Balb/c mice after lethal TBI.

Objective To observe the function of immature CD8α+ dentritic cells (DCs) in vitro. Methods The bone marrow and spleen of C57BL/6(H-2b) and Balb/c (H-2d) mice were got to prepare immature CD8α+ DCs and spleen lymphocytes,and treated by mytomycin. MTT test was used.MLR group, MLR plus variable density syngeneic CD8α+ DC group, MLR plus variable density allogeneic CD8α+ DC group,MLR plus variable density CD8α+ DC supernatant group,CD8α+ DC plus syngeneic T cell group and negative control group were established. MLR group was set up by responder cell ratio of 0.2,0.5,0.8,1.0,to build the MLR plus syngeneic and allogeneic CD8α+ DC experimental groups. Culture supernatant from different density (1 × 105/ml - 5 × 106/ml) of CD8α+DCs was added into MLR to build CD8α+ DC supernatant group. CD8α+ DCs were co-cultured with syngeneic T cells to build CD8α+ DCs plus syngeneic T cells group. 2 × 105/well responder cells served as the negative control group. ELISA was used to detect the concentrations of IFN-γ and IL-10 in the DCs could both suppress MLR (P＜0. 05), and the difference was not statistically significant (P＞0. 05). When CD8α+ DCs were increased, the suppressive effect was enhanced. When CD8α+ DC/responder cell ratio ＞0. 2, the inhibitory effect could be observed, and this effect reached the peak when the ratio was 1.0. The CD8α+ DCs had weak ability to stimulate syngeneic lymphocyte proliferation in vitro, and certain stimulating effect could be seen only when CD8α+ DC/responder cell ratio ＞2 (P＜0. 05). Its culture supernatant also showed suppressive effect (P＜0. 05), and the supernatant with a cell density of 5 × 105/ml showed the maximum effect. IL-10 concentration in the concentration was 1.0 ± 1.2 pg/ml. Conclusion The in vitro function of immature CD8α+ DCs was immunosuppression/tolerance,and they could secret high level of IL-10. The CD8α+ DCs and their culture supernatant could suppress MLR in vitro.

Objective To compare the effect of perineal cleansing with the potassium permanganate or sterile water on mid- stream urine culture. Methods Mid- stream specimens of urine were obtained from inpatients in our hospital between January 2002 and December 2006. All these patients may be diag-nosed as urinary tract infection. The urine specimens were divided into the potassium permanganate group (n=1572, the sterilization group) and the sterile water group (n=544). The change of positive and contami-nation rate of mid-stream urine culture from the specimens was observed. More than two kinds of germs in one urine specimen were defined as contamination. Results 830 patients with urinary tract infection had been enrolled. 2116 specimens were collected and 531 strains of causative organism were detected. The positive rate of the sterilization group and the sterile water group was 20.04% and 39.71%, respectively,and such difference was significant. The rate of identical causative organism from the same patient whose spec-imen was cultivated twice in the sterilization group was 0.012% and the rate was 0.105% in the sterile water group. The difference was significant. The rate of different or one kind of causative organism from the same patient whose specimen was cultivated twice in these two groups hadn't significant deviation. The contami-nation rate of the sterilization group (0.028%) was significantly higher than that of the sterile water group (0.007%). Conclusions Perineal cleansing with sterile water can reduce the false negative rate of mid-stream urine culture without increasing the contamination rate. Potassium permanganate sterilization is re-sponsible for the high false-negative in mid-stream urine culture.