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.

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.

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.

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 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.

The graft-versus-tumor (GVT) effect of T cells induced by tumor antigen-pulsed CD8α(+) dendritic cells (DCs) in vitro was investigated in this study. Immature CD8α(+) DCs were prepared from C57BL/6 (H-2(b)) bone marrow cells by using a cytokine cocktail. On the 3rd day of culture, CD8α(+) DCs were pulsed by allogeneic (Balb/c, H-2(d)) EL9611 leukemia antigen, or RM-1 syngeneic prostate cancer antigen, with the concentration series of 0, 2.5, 5.0, 10.0, 20.0 μg/mL, respectively, then antigen-loaded immature CD8α(+) DCs were co-cultured with syngeneic T cells according to the DC/T ratio of 1:1, 2:1 and 4:1. T cell proliferation was measured by MTT assay. Cytokines including interferon gamma (IFN-γ) and interleukin-10 (IL-10) in CD8α(+) DCs and T co-culture supernatant were detected by using ELISA. Cytotoxic effect of antigen-specific T cells was tested by LDH release assay. Conventional mature DCs (mDCs) induced from C57BL/6 (H-2(b)) bone marrow cells by using granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) served as a control. The results showed that the proliferative activity of T cells stimulated by CD8α(+) DCs loaded with allogeneic or syngeneic tumor antigen was augmented with the CD8α(+) DC/T ratio increased (P<0.05). When antigen concentration ≤ 5 μg/mL and CD8α(+) DC/T ratio ≤ 2:1, the ability of CD8α(+) DCs to stimulate T cell proliferation was higher than mDC control in allogeneic tumor antigen-pulsed groups (P<0.05), but not in syngeneic tumor antigen-pulsed groups (P>0.05). The level of IFN-γ and IL-10 in CD8α(+) DCs and T cell co-culture supernatant were increased in both allogeneic and syngeneic antigen-pulsed groups (P<0.05), and the cytokine level was higher in allogeneic antigen-pulsed groups than in syngeneic antigen groups when the CD8α(+) DC/T was 1:1 or 2:1 (P<0.05). There existed a negative correlation between the level of IL-10 and T cell proliferation. T cell cytotoxicity assay showed that when CD8α(+) DCs were pulsed with allogeneic tumor antigen, the maximal T cell killing efficiency could reach (100±7.7)%, whereas syngeneic tumor antigen-pulsed group had only (65.0±3.4)%. It was concluded that syngeneic and allogeneic tumor antigen-pulsed immature CD8α(+) DCs could stimulate T cells to exert the GVT effect in vitro, and the GVT effect was more obvious with allogeneic tumor antigen than with syngeneic tumor antigen. The optimal condition was low allogeneic tumor antigen pulsation (≤ 5 μg/mL) and low CD8α(+) DC/T ratio (1:1 and 2:1).

AIM: To clone and express a human monoclonal anti-D Fab fragment in E. coli and make benefits for the expression of the whole immunoglobulin molecules of anti-D. METHODS: The gene of anti-D Fab fragment was cloned into the phagemid vector pComb3. After analyzing by PCR and restriction site analysis, the recombinant was expressed in E. coli and the expressed protein was analyzed by SDS-PAGE and ELISA. RESULTS: The result of SDS-PAGE confirmed that E.coli expressed a 48 kD protein. The ELISA result demonstrated that the cell culture supernatant reacted with Rh+ group O human erythrocytes, but was not recognized by Rh-group O human erythrocytes. CONCLUSION: Expressed Fab fragment has the antigenic specificity for human erythrocytes.

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 find out the gene structure and diversity of protate specific membrane antigen(PSMA) alternative spliced variants, and probe into the pathogenesis of prostate cancer.METHODS: 5'-RACE and 3'-RACE methods were used to amplify the 5' and 3'end of alternative spliced variant and then those viariants were sequenced for analyzing the gene stucture and diversity of PSMA alternative spliced variants of prostate cancer tissues.RESULTS: Four new alternative spliced variants of PSMA were discovered from prostate cancer tissues.Compared with reported PSMA alternative spliced variants,different insertions and deletions existed in different sites of those new variants.CONCLUSION: The discovery of the new variants confirms the diversity of PSMA spliced variants and provides the clues for seeking the target of diagnosis and therapy of prostate cancer.

AIM:To study the blocking effect of shRNA on the expression of PSMA gene in LNCaP cell line by using shRNA eukaryotic expression vector.METHODS:Three pairs of DNA templates coding shRNA,synthesized against PSMA and cloned into the vector pSilencer 2.1-U6-neo,which was named pSilencer 2.1-U6-neo-shRNA,were identified by restriction endonuclease digestion analysis and DNA sequencing.LNCaP cells were then transfected with these three pSilencer 2.1-U6-neo-shRNAs and the negative control pSilencer 2.1-U6-neo-NC.After G418 selection,the cells were selected and the interfering effect was detected by RT-PCR and Western blotting.The biological behaviours of the transfected LNCaP cells were also tested.RESULTS:Restriction endonuclease digestion analysis and DNA sequencing results all showed that the 3 target segments were cloned into pSilencer 2.1-U6-neo vector respectively.After transfected into LNCaP cells,the inhibitory ratio of PSMA mRNA was 33.15%,9.26% and 41.97% respectively,and that of PSMA protein was 26.26%,6.47%,40.69% respectively.The p-shRNA3 was chosen to test the cell growth and its invasive power in vitro.The results showed that after interfering,the invasiveness of LNCaP cells were enhanced.CONCLUSION:The vector-based shRNA on PSMA gene effectively knocks down the PSMA gene expression.The successful construction of PSMA shRNA makes it possible for further study of the interaction between PSMA and prostate cancer.