BACKGROUND: The transplantation of allogeneic cartilage has local immunological rejection, and it is necessary to further reduce the rejection to promote its application in clinic, thus it is significant to perform a series of experiments to induce local immune privilege.OBJECTIVE: To observe the in vivo growth of tissue engineered allogeneic cartilage reconstructed by chondrocytes transfected with recombinant retroviral vector pLNCX2-FasL.DESIGN: A randomized controlled observation.SETTING: Shanghai Jiao Tong University.MATERIALS: Thirty-six allogeneic New Zealand rabbits as recipients and 45 1-week-old chinchillas as donors, either sex,were purchased by the experimental animal center of Chinese Academy of Sciences. Amphotropic recombinant retrovirus coated cell line PT67 was purchased from Clontech Company; Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), G418 and Polybrene were bought from GIBCO BRL.METHODS: The experiment was carried out in original Shanghai Second Medical University from January 2000 to July 2005. The New Zealand rabbits were randomly divided into three groups: FasL-transfected group (n =12), untransfected group (n =12) and blank control group (n =12). The rabbit allogeneic cartilages were constructed by the compound of pLNCX2-FasL transfected chondrocytes and tissue engineered material of pluronic F-127. ① Gross observation and mass changes of the grafts: Corresponding materials were infused subcutaneously, the grafts were removed at 1, 2 and 3 months after transplantation for gross observation and the mass changes. ② Staining observation: The grafts were removed at 1, 2 and 3 months after transplantation, then prepared into sections, and observed by hematoxylin and eosin (HE), Safranin'O and Masson's trichrome stainings. ③ Antibody detection: Blood samples (1 mL) were collected at 1 and 2 months after transplantation, the chondrocytes of the chinchillas were lysed freezingly with lysis antigen as the mixed antigen, and separated by electrophoresis in agarose medium, then acted with serum of recipient to observe whether corresponding antibody generated. ④ Complement dependent cytotoxicity (CDC) test: The chondrocytes of chinchillas were prepared into cell suspension (2×109/L), and then seeded into 96-well plate, attached grew for 24 hours, then recipient serum was added for the CDC test, and the percentage of apoptotic cells was counted under microscope.MAIN OUTCOME MEASURES: ① Gross observation and mass changes of the grafts;② Histological changes; ③ Results of the antibody detection; ④ Percentage of apoptotic cells.RESULTS: All the 81 rabbits were involved in the analysis of results. ① Gross observation and mass changes of the grafts: Two weeks after inoculation, there were obvious nod formations at the inoculated sites, but no nod formed in the blank control group. The new cartilage tissues became smaller gradually and completely disappeared at 4 months in the untransfected group, whereas those in the FasL-transfected group became smaller, but still existed after 7 months. The masses of grafts in the FasL-transfected group were higher than those in the untransfected group (P ＜ 0.05). ②Histological observation: Plenty of lymphocytic infiltrations around cartilage tissue could be observed in the untransfected group, and obviously decreased in the FasL-transfected group. No lymphocyte was observed inside the chondrocytes.Masson's trichrome staining was performed, and it was observed under light microscope that the small white parts in the middle were immature chondrocytes, and there were green collagen around most of the mature chondrocytes. Safranin O staining showed strong positive reaction, suggested that there were rich glycosaminoglycan in matrix. ③ Antibody detection: The chondrocytes of the chinchillas were lysed freezingly with lysis antigen as the mixed antigen, then acted with serum of recipient, and the results showed that no corresponding antibody generated. ④ Percentage of apoptotic cells: The percentages of serum CDC apoptotic cells in the FasL can ransfected group, untransfected group and blank control group were 5%, 6% and 1%, which were all negative.CONCLUSION: Rabbit allogeneic chondrocytes transfected with recombinant retroviral vector pLNCX2-FasL can reconstruct tissue engineered cartilage, and can postpone the degeneration by 3 months.

BACKGROUND: At present,enhanced green fluorescent protein(EGFP)is proved to be the best labeled molecule,with unique advantages,such as high fluorescence specificity,easy to be detected,and so on.Recombined retroviral vector EGFP-pLNCX2,which can stably express EGFP,can be construct using gene-engineering technique.Transfecting allogenic chondrocytes is indeed very useful to investigate the target gene expression and process of constructing tissue engineered cartilage in vivo.OBJECTIVE:To construct recombined retroviral vector EGFP-pLNCX2 which can stably express EGFP,and investigate optimal conditions for retrovirus transfection of chondrocytes.DESIGN:Randomized controlled observation.SETTING:Shanghai Jiao Tong University.MATERIALS:Thirty New Zealand rabbits of either gender,1 week of age,were purchased from Experimental Animal Center of Chinese Academy of Sciences.Amphotropic retrovirus package cell line PT67 pLNCX2 and pEGEP-C1 were purchased from Clontech Company;NIH 3T3 cell line was purchased from ATCC Company;DH5a Bacterium coli was preserved by the laboratory of Shanghai Jiao Tong University;Retroviral vector pLNCX2 was purchased from Clontech Company;pEGFP-C1 plasmid with EGFP were donated by professor Cong Xiao-qian from Chinese Academy of Sciences.METHODS:This experiment was carried out in the Shanghai Jiao Tong University in August 2005.Chondrocytes of New Zealand rabbits were isolated and cultured.The recombinant retroviral vector EGFP-pLNCX2,which can stably express EGFP,was constructed to transfect cultured chondrocytes from New Zealand rabbits by using gene engineering technique.Transfection results were observed under fluorescence microscope.Altogether 6×105 chondrocytes incubated in 10 cm-diameter flat plate were used to transfect retrovirus-EGFP immediately and at 12,24 and 48 hours after inoculation.One week later,EGFP expression efficiency was measured with flow cytometer,and best occasion for retrovirus transfecting primary chondrocytes.Enzyme-digested chondrocytes were inoculated for 24 hours to transfect retrovirus.After 250 mg/L G418 was added,chondrocytes were screened on the 2nd,3rd,4th,5th and 6th days separately.After phosphate buffer solution(PBS)washings,the transfection efficiency of chondrocytes was detected by flow cytometer,and the best occasion for G418 screening was observed.MAIN OUTCOME MEASURES:①EGFP-pLNCX2 transfection efficiency.②The best occasions for retrovirus transfecting primary chondrocytes and G418 screening.RESULTS: ①Retroviral vector EGFP-pLNCX2 transfected primary chondrocytes of rabbits,and high expression of transfected chondrocytes could be obtained through preliminary screening of G418.After being screened and expressing EGFP,chondrocytes kept normal morphology,with pseudopod adhering to the wall and matrix secreting vigorously.②The best occasion for retrovirus transfecting primary chondrocytes was at 24 hours after cell inoculation.The transfection efficiency determined with flow cytometer was 19.14% on the 5th day.The best occasion for G418 screening was on the 5th day after culture.Transfection efficiency of G418 screening was 55.75% on the 7th day.CONCLUSION:Recombinant retroviral vector EGFP-pLNCX2 can effectively transfect chondrocytes.The best occasion for retrovirus transfecting primary chondrocytes is at 24 hours after inoculation, and the best occasion for G418 screening is on the 5th day after culture.

Objective To determine the consistency between quantitative CT perfusion measurements of colorectal cancer obtained from single section with maximal tumor dimension and from average of whole tumor,and compare intra-and inter-observer consistency of the two analysis methods.Methods Twenty-two patients with histologically proven colorectal cancer were examined prospectively with 256-slice CT and the whole tumor perfusion images were obtained.Perfusion parameters were obtained from region of interest (ROI) inserted in single section showing maximal tumor dimension,then from ROI inserted in all tumor-containing sections by two radiologists.Consistency between values of blood flow (BF),blood volume (BV) and time to peak (TTP) calculated by two methods was assessed.Intra-observer consistency was evaluated by comparing repeated measurements done by the same radiologist using both methods after 3 months.Perfusion measurements were done by another radiologist independently to assess inter-observer consistency of both methods.The results from different methods were compared using paired t test and Bland-Altmnan plot.Results Twenty-two patients were examined successfully.The perfusion parameters BF,BV and TTP obtained by whole tumor perfusion and single-section analysis were (35.59 ± 14.59) ml · min-1 · 100 g-1,(17.55 ±4.21) ml · 100 g-1,(21.30 ±7.57) s and (34.64 ± 13.29)ml· min-1 · 100 g-1,(17.61 ±6.39)ml± · 100 g-1,(19.82 ±9.01)s,respectively.No significant differences were observed between the means of the perfusion parameters (BF,BV,TTP)calculated by the two methods (t =0.218,-0.033,-0.668,P ＞ 0.05,respectively).The intra-observer 95％ limits of consistency of perfusion parameters were BF-5.3％ to 10.0％,BV-13.8％ to 10.8％,TTP-15.0％ to 12.6％ with whole tumor analysis,respectively; BF-14.3％ to 16.5％,BV-24.2％ to 22.2％,TTP-19.0％ to 16.1％ with single section analysis,respectively.The inter-observer 95％ limits of consistency of perfusion parameters were BF-8.0％ to 8.3％,BV-10.9％ to 11.5％,TTP -14.5％ to 11.1％ with whole volume analysis,respectively; BF-10.2％ to 14.1％,BV-19.0％ to 17.6％,TTP-22.0％ to 24.0％ with single section analysis,respectively.Conclusion There was no statistically different between the single section and whole volume analysis of tumor perfusion CT.The whole volume perfusion analysis apparently improves intra-and inter-observer consistency and can reflect the whole tumor angiogenesis more accurately and repeatedly.

Objective To explore the characteristics of CT image and pathological changes of intrahepatic lymphatic stasis(ILS) caused by hepatic diseases.Methods Clinical data and CT images in contrast-enhanced arterial phase and portal venous phase in 36 cases with ILS saused by hepatic cancer,cirrhosis,hepatic metastatic tumors,hepatitis and hepatic contusion were analyzed retrospectively.The etiological mechanism of ILS was also discussed.Results On contrast-enhanced SCT scan,low-density line or annular line around the branches of hepatic vein,portal vein and/or intrahepatic inferior vena cava were found in all case,they presented as a kind of "halo sign" around the blood vessel.Conclusion The CT finding of ILS in hepatic diseases is quite characteristic and it is of great value in diagnosis of ILS.

Objective To observe the role of dual-source CT (DSCT) in the diagnosis of coronary artery fistula. Methods Nine patients with coronary artery fistula were examined with dual-source CT coronary artery angiography. Then the source images were post processed using volume rendering (VR), multiple planar reformation (MPR), maximum intensity projection (MIP) and curved planar reformation (CPR), and sequential segmental analysis of the intracardiac and extracardiac anomalies was performed. Results Coronary artery fistula in all the 9 patients were accurately displayed with DSCT, including 2 with left main trunk to right ventricle fistula, 7 with left main trunk and (or) left anterior descending artery to main pulmonary trunk fistula, 5 with complex coronary-pulmonary artery fistula. Conclusion Dual-source CT coronary artery angiography is convenient, fast, non-invasive, and may be the preferable method for diagnosis of coronary artery fistula.

Objective To investigate the effect of CD40/CD40 ligand on the genesis and development of coronary artery disease (CAD),and the inhibitory effect of cyclosporine A (CsA) on CD40/CD40 ligand. Methods A total of 71 patients were divided into four groups:acute myocardial infarction group (AMI, n=19), unstable angina pectoris group (UAP, n=18), stable angina pectoris group (SAP, n=17) and normal control group (N, n=17). Flow cytometry was used to detect the expres?sion of CD40 and CD40L in peripheral blood mononuclear cells (PBMCs) of four groups. The group in which CD40 and CD40L were produced at the highest level was chosen, and a series concentrations of CsA(H1:0 mg/L, H2:0.01 mg/L, H3:0.1 mg/L, H4:1 mg/L)were used to treat the cells. Then the expressions of CD40 and CD40L were measured by flow cytome?try. Results Compared with N group,the expression of CD40 was significant higher in other groups (P<0.05). The expres?sion of CD40 was significant higher in UAP group and AMI group than that of SAP group. There was no significant difference in the expression of CD40 between UAP group and AMI group (P>0.05). The expression of CD40L was elevated and fol?lowed by different severity of CAD. There was significant difference in the expression of CD40L between groups (P<0.05) . AMI group showed the highest expression of CD40 and CD40L. After being treated with CsA, the expression of CD40 was higher in H1 group than that of H3 group and H4 group (P<0.05). The expression of CD40L was significantly higher in H1 group than that of other three groups (P < 0.05). Conclusion CD40 and CD40L may be involved in the development of CAD. Moreover, it might be restrained by CsA via regulation of CD40/CD40L.

Objective To investigate the relationship between vascular lesion and serological changes in patients with coronary heart disease (CHD) complicated with obstructive sleep apnea hypopnea syndrome (OSAHS). Methods According to the standard, a total of 168 patients of OSAHS complicated with CHD were selected in this study. Those patients were divided into 3 groups according to the apnea hypopnea index (AHI) level:light group (AHI, 5-14/h), moderate group (AHI, 15-30/h) and severe group (AHI,>30/h). Syntax scores were performed on three groups according to coronary angiography results. The data of hemoglobin (Hb), platelet count (PLT), fibrinogen (FIB), D-Dimer (DD), total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL), triacylglycerol (TG), alanine aminotransferase (ALT), aspartate transaminase (AST), uric acid (UA), creatinine (Cr) and echocardiographic examination index were collected and analyzed in three groups. Results The Syntax score was significantly higher in severe group than that in mild and moderate groups (P<0.05). There was no significant difference in the Syntax score between mild and moderate groups. The levels of Hb, FIB, LDL, pulmonary arterial pressure (PAP), and right ventricle transverse diameter (RVD) were significantly lower in the mild and moderate groups than those in the severe group (P<0.01). The left ventricular ejection fraction (LVEF) was significantly lower in the severe group than that in the mild and moderate groups (P < 0.01). There were no significant differences in levels of Hb, FIB, LDL, PAP, RVD and LVEF between mild group and moderate group (P > 0.05). Conclusion The serology and cardiac structure can change gradually in severe OSAHS patients, and the coronary artery lesion will be more complex. Therefore, the clinical treatment should pay attention to screening for OSAHS in patients with coronary heart disease.

Objective To determine the diagnostic accuracy of dual- source computed tomography coronary angiography (DSCT-CA) for detecting various degrees of coronary artery stenosis. Methods A total of 278 patients with coronary atherosclerotic heart disease (CHD) were selected for DSCT-CA examination. The coronary angiography (CAG) examination was carried out within 30 days in these patients. The result of CAG was used as the gold standard. The coronary artery stenosis of 50% and 75% was used as the sector. Based on the data of patients, DSCT-CA was calculated for quantitative assessment of the sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of coronary stenosis for the coronary blood vessels and coronary artery segments. Kappa consistence test was used to analyze the results of two methods. Results The 50%and 75%of coronary artery stenosis was used for the sector, based on the patients, the sensitivities of DSCT-CA were 99.6% and 99.1%, specificities were 84.2%, 89.6%, diagnostic accuracies were 98.6%and 97.5%, and the Kappa values were 0.88 and 0.91. Based on coronary vessels, the sensitivities were 92.0%and 96.9%, specificities were 89.9%and 94.8%, diagnostic accuracies were 91.0%and 95.8%, and the Kappa values were 0.88 and 0.92. Based on coronary artery segments, the sensitivities were 97.3%, 96.5%, specificities were 93.3%, 98.1%, diagnostic accuracies were 94.2%and 97.9%, and the Kappa values were 0.84 and 0.91. Conclusion Based on patient’s coronary artery analysis, DSCT-CA can accurately assess the different degrees of coronary stenosis, but based on the analysis of the coronary artery segment, the false-positive results may occur in the diagnosis of DACT-CA, which
suggests that DSCT-CA can partly replace the CAG, still cannot completely replace the CAG.

Objective: To observe the effect of mild-warm moxa stick moxibustion on fatigue, immune state and quality of life (QOL) in sub-health population. Methods: A total of 120 cases were randomly allocated into a moxibustion group (n=60) and a Chinese patent medicine (CPM) group (n=60). People in the moxibustion group were intervened with mild-warm moxa stick moxibustion, once a day; while people in the control group took oral Bu Zhong Yi Qi Wan (Spleen/stomach-supplementing and Qi-boosting Pill), 3 doses a day. Two months made up a course of treatment. The fatigue, immune state and QOL were then observed after one treatment course, followed by a 12-month follow-up. Results: A total of 115 cases completed the clinical trial. After intervention, the fatigue scores, immune parameters and QOL scores were significantly increased in both groups, showing statistical significances (P<0.05). There were between-group significant differences in fatigue score, levels of immunoglobulin M (IgM) and IgG, natural killer (NK) cell activity and QOL score (P<0.05). After the 12-month follow-up, there were still significant between-group differences in fatigue score, physical health, psychological, social relationships and overall perception of QOL and overall perception of health (P<0.05). Conclusion: Mild-warm moxibustion can markedly improve the fatigue, immune state and QOL in sub-health population.

Two highly conserved and with abundant quantity of lipoproteins in outer membrane of pathogenic species,but not in saprophytic species of leptospira ,LipL32 and LipL21,were selected to construct the fusion gene DNA vaccine pVAX1/LipL21-LipL32,and its ability to induce immune responses in BALB/c mice inoculated with this recombinant DNA vaccine was investigated in the present study.Expression of the fusion-protein LiPL21-LiPL32 was demonstrated in HEK293 cells following transfection with the fusion gene DNA vaccine and the immune responses induced after intramuscular inoculation with this DNA vaccine in BALB/c mice was then evaluated by microscopic agglutination test (MAT),meanwhile the ELISA assay was used to detect the cytokines induced.It was demonstrated that significant level of specific antibodies agglutinating antigens of Leptospira interrogans could be detected by MAT after DNA vaccine inoculation.The production of cytokines IL-10 and TNF-β in mice inoculated with DNA vaccine pVAX1/LipL21-LipL32 was significantly increased in comparison with that of the group inoculated with pVAX1 alone.These results indicate that the recombinant DNA vaccine pVAX1/LipL21-LipL32 may be of potential value to design and develop new generation of vaccines against leptospirosis.