Effects of Gene Tranfection with CH50 Polypeptide on the Invasion Ability of Bladder Cancer Cell Line BIU-87 / 华中科技大学学报（医学）（英德文版）

Summary: The expression of CH50 polypoptide in bladder cancer cell line BIU-87 and the effects on the invasion ability of BIU-87 were investigated. The eukaryotic expressing vector pCH510 of polypeptide CH50 was introduced into BIU-87 cells by gene transfection in vitro. The expression of CH50 polypeptide was detected by using immunohistochemical S-P method. The expression of the transfected gene was identified by RT-PCR. Cell invasion assay kit was applied to detect the effect of CH50 polypeptide on the invasion ability of BIU-87. The results showed that the BIU-87 cells transfected with pCH510 could express the CH50 polypeptide, while in the control group, no CH50 polypeptide was detectable. In the transfection group, the invasion ability of BIU-87 in vitro was lower than in control group (P<0.05). It was concluded that CH50 polypeptide was successfully expressed in BIU-87 cells by gene transfection, by which the in vitro invasion ability of BIU-87 was inhibited.

Investigation on the effects of soluble programmed death-1 (sPD-1) enhancing anti-tumor immune response / 华中科技大学学报（医学）（英德文版）

By using semi-quantitative RT-PCR method, it was found that PD-L1 mRNA but not PD-L2 mRNA was expressed in H22 hepatoma cells and both PD-L1 and PD-L2 mRNAs were expressed in tumor tissues of tumor-bearing mice and upregulated as compared with muscle tissues in normal mice and H22 hepatoma cells. PD-L1 and PD-L2 were also expressed on the surface of the activated T cells. The soluble recombinant sPD-1 expressed from the constructed eukaryotic expression vector could enhance the lysis of tumor cells by lymphocytes stimulated specifically with antigen. The expresssion of sPD-1 by local gene therapy on the inoculation site of H22 hepatoma cells could inhibit the growth of tumor. The results of this study indicate that expression of soluble receptor of negative costimulatory molecules could reduce the inhibitory effect on T cells in tumor microenvironment and enhance the cytotoxicity of T cells on tumor cells. This possibly provides a new method of improving efficacy of tumor gene therapy.

Inhibitory effect of recombinant endostatin on angiogenesis and tumor growth of hepatoma / 华中科技大学学报（医学）（英德文版）

To study the influence of recombinant endostatin on angiogenesis and tumor growth of mice H22 hepatoma, tumor models were constructed by injecting H22 hepatoma cells into the leg muscle of mice. Recombinant endostatin was produced by gene engineering in E. coli. The recombinant protein was injected subcutaneously to treat transplanted hepatoma faraway. The weight of tumors was measured, and the changes of necrosis of tumor cells and vessel density were observed by immunohistochemistry. The results suggested that the growth of hepatoma models transplanted in the muscle of legs was suppressed by recombinant endostatin. The density of vascularity was decreased, but the necrosis of tumor cells increased. The inhibitory effect of recombinant endostatin on angiogenesis and tumor growth of hepatoma was not affected after chemotherapy.

Hsp70-H22 tumor antigen peptide complex activated dendritic cell in the induction of antitumor immunity / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of reduction in tumor antigen peptide dose by dendritic cell (DC)-presenting so as to elucidate the characteristics of modifying DC by heat shock protein (Hsp70) and antigen peptide.</p><p><b>METHODS</b>Antigen peptide bound to Hsp70 was used to modify DC in vitro. The metabolism of the modified DC and the cytokine secreted thereby was determined. Then the activation of lymphocytes by the modified DC and Hsp70-H22 peptide was tested. The cytotoxicity of the activated lymphocytes to H22 tumor cells and the inhibition of tumor in mice by DC injection and Hsp70-H22 peptide was tested.</p><p><b>RESULTS</b>0.15 micro g of H22 peptide bound to Hsp70 could mature 2 x 10(5) DC. 4 x 10(3) matured DC could activate 2 x 10(6) lymphocytes. The same amount of lymphocyte could be activated to produce similar cytotoxicity to tumor cells by either DC modified by 0.003 micro g of peptides bound with Hsp70 or by direct stimulation with 0.15 micro g of peptides bound to Hsp70. The dose of peptide could be reduced to 1/50 if the modified DC injection was used instead of direct Hsp70-peptide injection. Peptide from the normal hepatocytes, if bound to Hsp70, could not mature DC, nor could it activate lymphocytes through DC.</p><p><b>CONCLUSION</b>The dose of Hsp70-H22 peptides can be reduced significantly by DC-presenting to activate lymphocytes. Peptides from normal cells, being unable to activate the lymphocytes by either Hsp70-presenting or DC-presenting, have little to offer in the induction of autoimmunity.</p>

Inducement of specific CTLs by antigen-peptides from human leukemia cells and their cytotoxicity to leukemia cells / 华中科技大学学报（医学）（英德文版）

To investigate the inducement of cytotoxic T lymphocytes (CTLs) by antigen peptides mixture from different leukemia cells and the cross-reaction of the mixtures from different cell lines, antigen peptides mixtures were prepared from different leukemia cell lines respectively and then bound with Hsp70 in vitro. Activation and proliferation of PBMC were observed after stimulation with different Hsp70-peptide complexes. The ratio of CD8+ in proliferative cells was analyzed by flow cytometry. The cytotoxicity of the activated PBMC to different target cells was assayed. The results showed that the antigen peptides from different leukemia cell lines, bound with Hsp70, could activate PBMC effectively, and stimulate the activated PBMC to proliferate. The proliferative PBMC had specific cytotoxicity to corresponding leukemia cells. CD8+ cells, accounting for a high proportion in proliferative cells, had a specific cytotoxicity to leukemia cells from which antigen peptides were prepared, suggesting that these CD8+ cells were CTLs specific to leukemia cells. CTLs activated by Hut78-peptides or Molt4-peptides had a significantly stronger cytotoxicity to Hut78 cells, Molt-1 cells and Jurkat cells than that of CTLs activated by HL-60-peptides (P < 0.05). And the cytotoxicity of CTLs activated by Hut78/Molt4-peptides to Jurkat cells was significantly stronger than that of CTLs activated by either Hut78-peptides or Molt4-peptides alone (P < 0.05). It is concluded that antigen peptides mixtures from leukemia cells can induce specific antitumor CTLs. There exists cross-reactivity among antigen peptides mixtures from different cell lines of the same type leukemia and more cross-reactive antigen peptides could be obtained from more cell lines, suggesting that antigen peptides mixture with broad antigenic spectrum could be prepared by using multiple leukemia cell lines.

Tumor antigen peptides from HL-60 cells induce specific immune response / 中华血液学杂志

<p><b>OBJECTIVE</b>To prepare tumor antigen peptides from HL-60 cells and to induce specific immune response.</p><p><b>METHODS</b>HL-60 antigen peptides were obtained using techniques including freezing and thawing, heat precipitation and acid precipitation. The stimulating effect of the in vitro Hsp70 binding HL-60 peptides on PBMC and the proliferation of stimulated PBMC were observed by T cell activation test. The cytotoxicity of proliferated PBMC is detected by incubating HL-60 cells or K562 cells with PBMC respectively.</p><p><b>RESULTS</b>The obtained tumor antigen peptides were a peptides mixture. The mixed peptides could activate PBMC and cause PBMC proliferation in vitro after presented by Hsp70. The proliferated PBMC showed specific cytotoxicity to HL-60 cells but not to K562 cells.</p><p><b>CONCLUSION</b>The method for preparing of human leukemia tumor antigen peptides used in this paper is simple and easy; the obtained antigen peptides can induce specific immune response in vitro.</p>

Construction and Expression of Eukaryotic Expressing Vector pCH510 of Polypeptide CH50 and Its Chemotaxis and Antitumor Function by in vivo Transfection / 华中科技大学学报（医学）（英德文版）

To construct an eukaryotic expressing vector that expresses CH50, a recombinant CellⅠ-HepⅡ bifunctional-domain polypeptide of human fibronectin, and to investigate the chemotaxis to immune cells and the inhibitory effect on the growth of tumor by the expression of the plasmid in vivo, the plasmid was constructed by DNA recombination. Gene transfection was performed in vitro and in vivo. The expressed product was identified by Western blot. The chemotaxis after gene transfection in vivo was observed by histotomy and staining of muscle tissues. The inhibition of gene transfection on solid tumor was observed in mice. The results showed that plasmid pCH510 was constructed by the recombination of the 5′-terminal noncoding region and signal peptide coding region of human fibronectin cDNA and cDNA fragment coding CH50 polypeptide with a 3′-terminal noncoding region of human FN cDNA, and the insertion of the recombinated fragment into plasmid pcDNA3.1. After transfection with plasmid pCH510, NIH3T3 cells could produce CH50 polypeptide. The transfection of plasmid pCH510 by the injection in muscle of mouse could produce the effects of chemotaxis on immune cells and the inhibition on the growth of solid tumor. It is concluded that plasmid pCH510 can express in cells and in vivo in mouse. The expression of the plasmid in vivo has a chemotactic effect on immune cells and can inhibit the growth of solid tumor.

Construction and Expression of Eukaryotic Expressing Vector pCH510 of Polypeptide CH50 and Its Chemotaxis and Antitumor Function by in vivo Transfection / 华中科技大学学报（医学）（英德文版）

To construct an eukaryotic expressing vector that expresses CH50, a recombinant CellⅠ-HepⅡ bifunctional-domain polypeptide of human fibronectin, and to investigate the chemotaxis to immune cells and the inhibitory effect on the growth of tumor by the expression of the plasmid in vivo, the plasmid was constructed by DNA recombination. Gene transfection was performed in vitro and in vivo. The expressed product was identified by Western blot. The chemotaxis after gene transfection in vivo was observed by histotomy and staining of muscle tissues. The inhibition of gene transfection on solid tumor was observed in mice. The results showed that plasmid pCH510 was constructed by the recombination of the 5′-terminal noncoding region and signal peptide coding region of human fibronectin cDNA and cDNA fragment coding CH50 polypeptide with a 3′-terminal noncoding region of human FN cDNA, and the insertion of the recombinated fragment into plasmid pcDNA3.1. After transfection with plasmid pCH510, NIH3T3 cells could produce CH50 polypeptide. The transfection of plasmid pCH510 by the injection in muscle of mouse could produce the effects of chemotaxis on immune cells and the inhibition on the growth of solid tumor. It is concluded that plasmid pCH510 can express in cells and in vivo in mouse. The expression of the plasmid in vivo has a chemotactic effect on immune cells and can inhibit the growth of solid tumor.

Antitumor effect of DC vaccine modified by Hsp70-H22 tumor peptide complex in vitro and in vivo / 中国免疫学杂志

Objective:To study the specific antitumor effect of DC modified by Hsp70 tumor peptide complexes in vitro and in vivo.Methods:The tumor antigen peptides were acquired from H22 liver cancer cells and bound Hsp70 in vitro by using biochemical technique;the mouse marrow cells were cultured with induction of rmGM CSF and rmIL 4 by using cell culture technique;mouse spleen lymphocytes was stimulated.The cultured DC cells were harvested and activation of lymphocytes was detected by MTT test and cytotoxicity of stimulated and proliferated lymphocytes to H22 tumor cells and Ehrilich ascites carcinoma cells was tested;The inhibitation to tumor was observed in vivo,after stimulated DCs were injected in mice inoculated by tumor cells.Results:DCs could become mature with the effect of Hsp70 H22 peptide complexes and secret IL 12?TNF ??IL 1? and effectively activate lymphocyte;The activated and proliferated lymphocytes could specifically kill H22 cells but not Ehrilich ascites carcinoma cells in vitro;DCs modified by Hsp70 H22 peptide complexes could become one useful kind of vaccines to inhibit H22 tumor growth in vivo.Conclusion:DCs orignied from marrow cells can be effectively modified by Hsp70 H22 peptide complexes,these modified DCs can specifically activate lymphocytes in vitro and effectively induce antitumor immune response.

Improvement of MTT assay by 2-chloroadenosine in activation test and cytotoxicity test of lymphocyte / 中国免疫学杂志

Objective:To sutdy the effect of 2 chloroadenosine(2 ClA),which is specifically cytotoxic to macrophages,on MTT assay in activation test and cytotoxicity test of lymphocyte.Methods:Using cell culture technique,mouse splenic lymphocytes and peritoneal macrophages were cultured.Lymphocyte activation and specific cytotoxicity to tumor cells and toxicity of 2 ClA to macrophages were measured by MTT assay in the presence or absence of 2 ClA.Results:2 ClA had a strong cytotoxic effect on macrophages.When the activation test and cytotoxicity test of lymphocyte were measured by MTT assay,the optical density values of 2 ClA group was lower than that of control group,and statistic analysis showed P

The Prevention and Therapeutic Effects of Hsp70-Antigen Peptide Complexes on B16 Tumor Pulmonary Metastasis in Mice / 中国肿瘤生物治疗杂志

Objective: To explore the preventive and therapeutic effects of Hsp70-antigen peptide complexes (HACs) on pulmonary metastasis of malanoma B16 cells in mice. Methods: Antigen peptide mixtures were prepared from massive tumors and metastatic foci respctively, and bound with Hsp70 in vitro. The HACs were used to mice to prevent the formation of micrometastatic foci after the injection of B16 cells through tail vein, or treat the existent micrometastatic foci in lung The number of metastatic foci was counted and the cytotoxicity of splenocytes to B16 cells was determined. Results: After immunization with HACs, the number of metastatic foci in mice decreased significantly (P

Influence of Chemotherapeutic Drugs on Immunocytes in vitro and in vivo / 中国肿瘤生物治疗杂志

Objective: To invistigate the relationship between time and efficacy of the linkage of tumor biotherapy after chemotherapy by studying the influence of chemotherapeutic drugs on immunocytes.Methods: The cytotoxicity of chemotherapeutic drugs to tumor cells, mouse peritoneal macrophages and spleen lymphocytes was observed by cell culture technique. The influence of chemotherapeutic drugs to the metabolism and activation of macrophages and lymphocytes at different time after peritoneal injection of drugs was observed. The mice were inoculated with tumor cells two days after the injection of drugs, and the growth of tumor was measured 14 days after inoculation by anatomizing mice. Results: Chemotherapeutic drugs had cytotoxicity in vitro to different cells, suppressed the function of immunocytes, and decreased the number of immunocytes in vivo. After injection of drugs, the number of immunocytes was the lowest on the third day and recovered to the nomal level on the 10th day. If drugs was injected two days before the inoculation of tumor cells, the growth of tumor became faster than control group. Conclusions: Chemotherapy not only decreases the number of immunocytes but also suppresses the function of immunocytes, and it can promote the growth of tumor after its cytotoxicity disappeared. So it is not good that biotherapy, which depends on immunocyte to kill tumor cells, is used immediately after chemotherapy and it is also not good for using biotherapy with a long interval after chemotherapy . It is good time to use biotherapy when the number of immunocytes is lowest or the recovery just starts.

The Influence of Chemotherapeutic Dosage to Tumor Chemotherapy Linked with Biotherapy / 中国肿瘤生物治疗杂志

Objective: To investigate the effect of the dosageof chemotherapeutic agent on tumor chemotherapy linked with biotherapy and provide experimentalevidence for the dose choice of chemical drugs in the combination of chemotherapy and biotherapy.Methods: The high- and low-dosage of MMC was determined by injection of mice with different dosages of MMC. Mice inoculated with H22 hepatic carcinoma cells were treated with different dosages of MMC followed by three different kinds of biotherapy: transfection with plasmid pCH510 in vivo, immunization with Hsp70-tumor antigen peptide complexes and the combination of these two elements. Results: By toxicity test of MMC to mice, it was determined that 100 ?g of MMC was high dosage and 50 ?g was low dosage. The curative effect was significantly improved if chemotherapy was followed by different elements of biotherapy. Better efficacy was obtained when biotherapy elements were used to follow the chemotherapy with high dosage of MMC. In the case of low dosage of MMC, no difference could be observed in curative effect of three different kinds of biotherapy. When high dosage of MMC was used, the curative effect of three different kinds of biotherapy was signiferently different. The best efficacy was obtained if chemotherapy was followed by the combination of two biotherapy elements, transfection with plasmid pCH510 in vivo and immunization with Hsp70-tumor antigen peptide complexes. Conclusions: Using different chemical dosages, the curative effect of chemotherapy linked with biotherapy is different. In the case of high dose, the chemotherapy linking with biotherapy can reach more better efficacy.

Effect of Polypeptide CH50 on Macrophage Activation in vivo and Its Antitu- mor Function / 中国肿瘤生物治疗杂志

Objective: To study the main features of CH50, a recombinant polypeptide of human fibronectin, to activate macrophages in vivo and its anti-tumor function. Methods: CH50 was injected and IFN-? gene was transfected in mice several kinds of factors produced by marcrophages were determined. The growth of tumor in force was also measured. Results: CH50 could enhance the production of such factors as NO, TNF and IL-1 by macrophages, but the activation of macrophages was rela- tively slow when CH50 was used in vivo alone. CH50 and IFN-? could synergistically activate macrophages rapidly in vivo no matter whether the injection of CH50 or the transfection of IFN-? gene was Performed first.Injection of CH50 alone inhibited the formation of tumor nodes in a dose-dependent manner. There was strong inhibition of low dosage of CH50 on the formation of tu- mor nodes smaller than 1 mm, and high dosage of CH50 on those bigger than 1 mm. A stronger inhibition on the growth of tumor in vivo was obtained by the synergistic effect of CH50 and IFN-?. Conclusion: CH50 and IFN-?, as double-signal factors for activation of macrophages, will be potentially useful in tumor therapy.

Role of plasmid DNA and in combination with IFN-? or with CH50 in activation of macrophages immunity / 中国免疫学杂志

Objective:To investigate the effect of eucaryotic expression plasmid DNA on activation of macrophage and the mechanism Methods:NO production by macrophages,the expression of surface molecules on macrophage by FACS and the cytotoxicity of macrophage were measured by Griess method,FACS and MTT respectively Results:The results showed that macrophages were activated to produce NO by plasmid pCH510 and pcDNA3 1 respectively in vitro or in vivo The expression of MHC Ⅱand B7 1 on macrophage and the cytotoxicity of macrophage was increased Plasmids DNA in combination with IFN ? but not with CH50 augmented the activation of macrophage in vitro In contrast, the activation of the macrophage were enhanced when the stimulated macrophages by plasmid DNA in vivo were treated by addition of CH50, but not IFN ? Conclusion:The results suggested the plasmid DNA could activate macrophage in vitro or in vivo but the mechanism of activation was different In vitro, the plasmid DNA directly activated macrophage and had synergistic effect with IFN ?,while in vivo macrophages were mainly indirectly activated and the activation of macrophages was enhanced in the presence of CH50 When eucaryotic expression plasmid pCH510 was used in gene therapy of tumor, besides the function of its products expressed, the plasmids showed the significant role in tumortherapy by its direct immune activation of macrophages