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BACKGROUND: Rejection is the main cause of the failure in small bowel transplant. Chemotatic factor RANTES and receptor mediated cellular immunity are very important in acute rejection.OBJECTIVE: To explore the immunosuppressive effect of early adopting chemokine receptor antagonist, Met-RANTES after small bowel transplant on acute allograft rejection and its coordinative effect with Tacrolimus (FK506).DESIGN: Randomized complete-block design, controlled animal experiment.SETTING: Department of General Surgery, the 451 Hospital of Chinese PLA; Laboratory of Department of Gastrointestinal Surgery, Xijing Hospital, Fourth Military Medical University of Chinese PLA; Electronic Microscope Center, School of Basic Medicine, Fourth Military Medical University of Chinese PLA.MATERIALS: This study was carried out in the Laboratory of Department of Gastrointestinal Surgery, Xijing Hospital,Fourth Military Medical University of Chinese PLA from September 2003 to March 2005. Totally 192 animals including 96 SD rats (donors) and 96 Wistar rats (recipients) were involved in this study. Heterotopic segmental small bowel transplantation was performed.METHODS: The transplant rats were divided into 4 groups averagely by the randomized complete block design: control group (allogeneic small bowel transplant untreated group), Met-RANTES group(200 μg/d, 0-7 days, i.p.), FK506 group [0.5 mg/(kg·d) ,0-7 days,i.p.], Met-RANTES + FK506 group [Met-RANTES, 200 μg/d,0-7 days,i.p.+ FK506 0.5 mg/(kg ·d),0-7 days, i.p.]. Rats in the latter 3 groups were intraperitoneally administrated after transplant within 7 days successively.Rats in the control group were not given any treatments before and after transplant. Postoperatively, gross status,survival time and immunocyte infiltration were observed. Pathological examination was conducted in 6 rats of each group on postoperative days 3, 5 and 7. Fluorescent staining and successive quantitative measurement were conducted to detect the expressions of intragraft RANTES, CD4+, CD8+ and CD25+ T lymphocyte. Survival duration of the rest 6 rats of each group was observed for 5 weeks.MAIN OUTCOME MEASURES: ① Survival time of rats in each group following transplant. ② Pathological changes of small bowel intragraft of rats in each group. ③ RANTES and T lymphocyte expressions of rats in each group.RESULTS: Following transplantation, 96 Wistar rats (recipient) were all involved in the final analysis. ①Compared with control group, the survival time of rats in Met-RANTES group, FK506 group, Met-RANTES + FK506 group was significantly longer (P ＜ 0.01). In addition, rats in Met-RANTES + FK506 group survived the longest. There were significant differences in survival rate as compared with Met-RANTES group and FK506 group (P ＜ 0.01). ②All rats in the control group died of acute rejection and infection. Histopathologic examination showed mild, moderate and severe rejection on the postoperative days 3,5 and 7, respectively. No obvious rejection was found in the rats in the Met-RANTES group, FK56 group and Met-RANTES+FK506 group on the postoperative days 3,5 and 7. ③Postoperatively, intragraft RANTES expression of rats was significantly higher in each time period in control group than in the other 3 groups (P ＜ 0.01), and its dynamic change was positively correlated with the process of acute rejection; The expression of intragraft RANTES, CD4+, CD8+ and CD25+ T lymphocytes of rats was significantly lower, respectively, in the Met-RANTES group and Met-RANTES+FK56 group than in the control group (P ＜ 0.01).CONCLUSION: Met-RANTES may obviously suppress acute allograft rejection in small bowel transplant, effectively protect the function of grafts, and significantly prolong the survival time of the recipients. In addition, Met-RANTES may enhance the immunosuppressive function of small dose of FK506[0.5 mg/(kg · d)].
ABSTRACT
Objective To construct a mammalian vector encoding angiostatin kringle 5 (K5) under the control of ?-fetoprotein (AFP) enhancer and albumin promoter, and to observe the expression of angiostatin by introducting angiostatin gene into hepatocellular carcinoma cells through gene transfection. Methods Angiostatin cDNA was amplified from normal human eukaryotic cells by using RT-PCR. Meanwhile, AFP enhancer and albumin promoter sequences were directed cloned and were inserted into vector pcDNA3.1. The recombinant vector of pcDNA3.1-AFAB-angiostatin K5-His was constructed, which contained the angiostatin K5 cDNA sequence that was under the control of the AFP enhancer and promoter. Angiostatin K5 cDNA was introduced into human AFP positive hepatocellular carcinoma cell lines with the transfected cultured cells that were mediated with Lipofectamine 2000. The expression of angiostatin K5 was analyzed by Western blot and the protein was dectected with anti-His antibody. Results The 500-base pair of angiostatin K5 was in accordance with the expected sequence and the recombinant vector of pcDNA3.1-AFAB-angiostatin K5-His was also confirmed as the anticipated sequence. The expression of angiostatin K5 in AFP positive hepatocellular carcinoma cells was detected both by SDS-PAGE and Western blot. Conclusion Efficient construction and expression of angiostatin K5 to AFP positive cells make it possible for antiangiogenesis therapy of human hepatocellular carcinomas, which may provide a promising approach.
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Objective To investigate the expression of tumor associated glycoprotein-72 (TAG-72) in human breast cancer cells. Methods Two breast cancer cell lines MCF-7 and Bcap-37 were cultured and prepared. TAG-72 expressions in MCF-7 and Bcap-37 were detected immunochemically with anti-TAG-72 single chain variable fragment (scFv). Results TAG-72 was positively expressed in MCF-7 cells but negatively expressed in Bcap-37 cells. Conclusion Tumor associated antigen TAG-72 is expressed in certain human breast cancer cells, which indicate TAG-72 may be used as a tumor maker and anti-TAG-72 scFv may play a role in the diagnosis and treatment of breast cancer.
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Aim To study relationship between expressions of apoptosis-related gene bax and bcl-x in heopatocellular carcinoma (HCC) tissues and its clinical feature. Mothods The expression of Bax and Bcl-x were determined by immunohistochemistry with EnvisionTM system. Results Bcl-x and Bax proteins were detectable in 20 and 19 out of 40 HCCs, respectively. Their expressions in HCC cells were lower than those in cirrhosis(P＜ 0.05), The expression of Bax and Bcl-x in highly or intermediatly differentiated HCC cells were highed than that in low-diffe-rehtiated HCC cells suggesting that there was significant relationship among bcl-x and bax expressions and differential degree， clinical stages of HCC cells， and AFP level. However, the expression of this proteins showed no relationship with patient's sex and age(＞ 60 or ＜ 60 years old) (＞ 0.05). Conclusion Apoptosis-related genes bcl-x and bax participate partially in apoptotic regulation of HCC cells, and show that their expressions have correlation with differential degree, clinical stage of HCC cells and AFP levels.
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Objective To generate specific targeting T cells against gastrointestinal cancer, a eukaryotic expression vector encoding carcinoembryonic antigen(CEA) specific single chain variable fragment(scFv) fused to the transmembrane and intracellular domains of the signal transducing chain of CD3? was constructed. Methods Anti CEA scFv cDNA was obtained by RT PCR. The transmembrane and intracellular domains of CD3?cDNA was amplified from human T lymphocyte using RT PCR to clone into a eukaryotic expression vector, and anti CEA scFv cDNA was inserted upstream of CD3?. Finally, the eukaryotic expression vector containing scFv CD3? pcDNA3.0 was constructed and sequenced. Results A 810 base pair of anti CEA scFv was in accordance with sequence concerned, a 438 base pair of cDNA of the transmembrane and intracellular domains of CD3? was confirmed as sequence concerning of GenBank. Further identification was carried out through agarose electrophoresis after enzyme digestion. Conclusion We constructed a fusion molecular with both CEA targeting and CD3? signal transduction perporties, which lay a good foundation for generation of modified cytotoxic T lymphocytes against gastrointestinal tumors.