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Localization Effect of 131 I-Human Anti-HBs Fab in Nude Mice Models of Human Hepatoma

Guichen WU ; Rongcheng LUO ; Huanxing HAN ; Changxuan YOU ; Xuemei DING ; Aimin LI ; Chuanbin WANG ; Mingjang ZHANG

Chinese Journal of Cancer Biotherapy.2000;7(4):288-290.

Objective: To evaluate the targeting activity in the animal model with human hepatoma, the 131I-human antiHBsAg Fab radioimmunoimaging was explored. Methods: Radioimmunoimagings were taken on different intervals after injection of 131 I-human anti-HBsAg Fab to the nude mice and tissue distribution was measured. The human anti-HBsAg Fab was compared with the murine monoclonal antibodies. Results: The experimental group developed tumor positive images after 3 days of radio-labeled monoclonal antibodies injection, and the peak accumulation of radio-activity on the 5th day.Statistics indicated the tumor/liver ratio of the human anti-HBsAg Fab, murine monoclonal antibodies and the control groups were 5.4,4.0 and 0.9 respectively on the 7th day. Conclusions: Our results suggest that the 131 I-human anti-HB-sAg Fab has a considerable targeting activity, and provide an evidence that it can be used as a novel humanized carrer for targeting therapy of hepatoma.

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Cloning, Expression and Activity of Recombinant Human Angiostatin

Ying JIANG ; Luguo SUN ; Yongli YU

Chinese Journal of Cancer Biotherapy.2000;7(4):285-287.

Objective: To express recombinant human angiostatin for further application in clinic. Methods: The complete encoding eDNA of human angiostatin was isolated from human embryo liver with RT-PCR and expressed in secretory Pichia expression system. Recombinant human angiostatin was purified with heparin sepharose chromatography and its activity was determined in chick embryo chorioallantoic membrane (CAM) and wound healing assays. Results: Expressed in large quantity (yield=5 mg/L) and purified with heparin sepharose, recombinant angiostatin was showed to have a molecular weight of 43 kD in SDS-PAGE and potently inhibit angiogenesis and wound healing. Conclusion: Recombinant human angiostatin was expressed efficiently in a biologically active form.

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Construction of Mutant N-ras/61 Gene Recombinant Vaccinia Virus

Jian ZHANG ; Yueran ZHAO ; Li YOU ; Chunyi GAO ; Zhigang TIAN

Chinese Journal of Cancer Biotherapy.2000;7(4):282-284.

Objective: To construct the recombinant vaccinia virus of mutant N-ras/61 gene and enhance the immunogenecity of mutant N-ras／61 protein produced by the recombinant vaccinia virus. Methods: N-ras／61 gene was inserted into P1108 and transfected into CV-1 cell infected with vaccinia virus by Cell FECTIN. PCR and Western blot were used to identify the recombinants. Results: We get recombinant vaccinia virus rV-N-ras/61 by PCR and tk- selecting. The expression of N-ras gene was detected by Western blot. Conclusion: This study is a test for studing effective vaccine of mutant N-ras/61 gene. The efficacy in vivo of the N-ras／61 vaccine in immunotherapy is under investgation.

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Cloning and Regulating Expression of Human CD34 TRS

Jiangqi LI ; Kunyuan GUO ; Ling ZHOU ; Liannin DUAN ; Jian DU

Chinese Journal of Cancer Biotherapy.2000;7(4):279-281.

Objective: To clone the 5＇-flanking region of the human CD34 gene containing transcriptional regulatory sequence (TRS). Methods: According to the registered 5＇-flanking region of CD34 gene, two pairs of primers were designed and net-PCR was used to amplify 661 bp long TRS of CD34 gene. The CD34 TRS fragment was cloned into reported plasmid pEGFP-1. The role of the regulating the specific expression of recombinant plasmid pCD34 EGFP in hematopoietic and non-hematopoietic cells was observed. Results: Restrictive endonuclease identification and DNA sequencing provedthat the CD34 promoter cloned was consistent with the sequence reported to a large extent. It could induce the EGFP gene to express in hematopoietic cell line K562 specifically, while has no effect on hepatocellular carcinoma cell HepG-2. Conclusion: The cloned CD34 gene TRS has the effect of regulating gene expression specifically. The study established the fundament for the construction of specific gene expression vector used in hematopoietic system cells.

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Cleavage of Oncogene ki-rasG12V mRNA by Ribozyme and It' s Specificity

Guoxiang WU ; Yuqiang FANG ; Guoming XU ; Zhaoshen LI ; Deru LU

Chinese Journal of Cancer Biotherapy.2000;7(4):275-278.

Objective: To design and confirm the cleavage activity of ribozyme Rz217 to oncogene ki-rasG12V messenger RNA and search for a new method for gene therapy targeting oncogene ki-ras. Methods: According to Symon' s principle,design an ribozyme specific for ki-rasc12v mRNA, both the constructs for transcription in vitro of ribozyme Rz217 and ki-ras exonl and the mammalian expression constructs of ribozyme Rz217 were constructed by DNA recombinant technique,ribozyme Rz217 and ki-ras exonl mRNA was obtained by transcription in vitro with T7 and SP6 RNA polymerase. Pancre atic carcinoma cell line PaTu8988 and human hepatocellular carcinomacell line BEL7404 were transfected with Rz217 mammalian expression constructs and the level of endogenous ki-rasG12V mRNA or ki-ras mRNA was determined by semiquantitative RT-PCR. Results: Not only in vitro but also in vivo, ribozyme Rz217 can cleave the mRNA of oncogene ki-ras (G12V) in site-specific manner and can not cleave the mRNA of wild-type ki-ras. Conclusion: Ribozyme Rz217 can cleave oncogene ki-rasc12v mRNA and the cleveage is specific for ki-rasG12V mRNA.

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Cytotoxicity of GS and IL-2 Activated PBMC to Malignant Glioma Cells

Junsheng LUO ; Lixue GU ; Huanjiu XI ; Bingjie WEI ; Xingbo LIU ; Jianwu QIU ; Pengfei ZHANG ; Hongren SHAN

Chinese Journal of Cancer Biotherapy.2000;7(4):273-274.

Objective: To investigate a new method for improving the therapeutic effect on malignant glioma. Methods: A new type of killer cells, named GS-LAK, was induced by means of costimulating the peripheral ginsenoside(GS) and interleukin-2 (IL-2). Comparing with control group-LAK cells, cytotoxicity of GS-LAK cells against malignant glioma cells(BT325) was examined with MTI method. Results: It showed that GS-LAK cells exhibited some advantages over LAKcells in proliferation, cytotoxicity, as well as the utilizing of IL-2. Conclusion: The application of GS-LAK cells mightopen a new prospect to clinical therapeutic approach to malignant glioma.

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The Inhibition of Endostatin on Tumor Growth and Metastasis of Lung Adenocarcinoma LA795 in Mice

Deling LIU ; Jinxu WEN ; Wancheng TONG ; Liheng BEN

Chinese Journal of Cancer Biotherapy.2000;7(4):269-272.

Objective: To evaluate the inhibition of human endostatin on tumor growth and metastasis of lung adenocarcinoma LA795 in mice. Methods: Recombinant human endostatin was purified from pCX expressed endostatin clones. Plasminogen was purified from outdated human plasma by affinity chromatography, and human angiostatin was produced from human plasminogen digested by elastase and purified by affinity chromatography. LA795 cells were inoculated subcutaneously into the dorsa of T739 mice, and the mice were randomized into 3 groups. From the 1Oth day, the first group was given 20 mg/kg of recombinant human endostatin s.c. qd, the second was treated daily s. c. of 7.5 mg/kg of human angiostatin, and the third group received daily s.c. with equal volumes of PBS for 14 days. Volumes of the subcutaneous tumors, lung weights, the number of lung surface metastases and mice life span were observed. The results were analyzed by q-test. Results: The tumor volumes of both the 1 st and the 2nd groups increased slowly. From the 8th day after being treated, the tumor volumes were decreasing. However, in the 3rd group, the tumor volumes increased continuously. The lung weight and the number of lung surface metastases of the 1st and 2nd groups were less than that of the 3rd group. The average survival periods of the 1st and 2nd groups were longer than that of the 3rd group. Conclusion: Human endostatin and angiostatin have strong inhibitory effects both on growth of primary tumor and metastasis of lung adenocarcinoma LA795, and prolongs the survival period of the tumor-bearing mice.

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Effect of Gancyclovir on Proliferation and Differentiation of Chronic MyeloidLeukemia Cells and Research of Its Mechanism

Xiaoping JU ; Fang XIA ; Hong ZHOU ; Jianzen WANG ; Zhengxia HUANG ; Youzhong YUAN ; Jianmin WANG

Chinese Journal of Cancer Biotherapy.2000;7(4):265-268.

Objective: To explore whether gancyclovir (GCV) can inhibit the proliferation and induce the erythro-differ-entiation of the K562 human myeloid leukemia cell line. Methods: 562 cells were cultured with GCV for 4 days to detect cellular changes cloning efficiency, benzidine-positive rate, flow eytometry analysis, and telomerase activity. Results: When 562 cells grew in the medium containing GCV, the cellular growth and division were gradually suppressed,growth fracture decreased and further differentiation towards the cell producing hemoglobins was found. Conclusion: GCVcan inhibit proliferation and induce erythro-differentiation of K562 cells.

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Antitumor Responses Induced by Recombinant Vaccinia Viruses Expressingp53 Antigenic Peptide and B7

Keqiang ZHANG ; Jingqing YANG ; Chunmei HOU ; Peixuan TANG ; Ning MAO

Chinese Journal of Cancer Biotherapy.2000;7(4):261-264.

Objective: To explore antitumor responses induced by recombinant vaccinia viruses expressing a p53 antigenic peptide (rVV-p53M) and enhanced effect of recombinant vaccinia viruses expressing costimulatory molecule B7 (rVVB7). Methods: A 135 Cys to Tyr point mutation p53-transduced P815 mastocytoma (P815-mp53) was used as an experimental tumor and the p53 protein as the model of tumor associated antigen． rVV-p53M and rVV-B7 were used as vaccine to test their induction of CTL and antitumor immunity. Results: Immunization of BABL/c mice with rVV-p53M could elicit specific CTLs, which could specifically lyse P815-mp53 cells. Immunization of mice with rVV-p53M could survive a part of mice challenged with 1×106 P815-mp53. Treatment with rVV-p53M could significantly prolong the survival oftumor-bearing mice. Admixture at 1:1 ratio of rVV-p53 M and rVV-B7 could enhance antitumor responses induced by rVV-p53M. Conclusion: Immunization with recombinant vaceinia virus expressing antigenic peptide is a useful alternative to peptide-based vaccine. Costimulatory molecule B7 can enhance antigenic peptide to induce antitumor responses.

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Density and Affinity of IL-6 Receptors in Human Leukemic Cells

Shuang LIU ; Yongzhi XI ; Siqi GUO ; Nan LIU ; Min TU ; Li JIN ; Xingguo CHEN ; Fanhua KON

Chinese Journal of Cancer Biotherapy.2000;7(4):255-260.

Objective: To make a study of density and affinity of IL-6R in human leukemic cell lines, and discuss the affection of high affinity IL-6R to the targeted treatment of leukemia with IL-6-PE40 fusion protein. Methods: Radial binding assay with scatchard plot and FACS were used to analysis the density and affinity of IL-6R and protein expression of IL-6Rα and β subunits in totally 8 representative human leukemic cell lines. Results: Myelocytie, monocytic and erythrocytic leukemic cell lines U937, HL-60, KG1 and TF1 express high affinity IL-6R, whose average density per cell is 2 502,2 874, 2 319 and 9 329 respectively, however no 125I-IL-6 binding was detected on chronic myelocytic leukemic cell line K562 and lymphoblastic leukemic cell lines such as Raji, CEM and HUT28. These results correlate with those of FACS highly. Conclusion:These observations suggest that acute nonlymphoblastic leukemic cells may be more suitable for targeted treatment with IL-6-PFA0 fusion protein.

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