Zhang Guimei

Effect of Diallyl Trisulfide on the Activation of T Cell and Macrophage-mediated Cytotoxicity

SummaryAt high concentration (50 μg/ml), diallyl trisulfide (DATS) had an inhibitory effect on T cell activation (compared with control group,P<0.05). But at appropriate concentrations (3.125–12. 5 μg/ml), DATS augmented the activation of T lymphocytes by Con A (compared with control group,P<0. 01). The augmentation of T cell activation by DATS was related to its inhibitory effect on the production of nitric oxide (NO) by macrophages. In a wide range of concentrations (1–100 μg/ml), DATS can inhibit the production of NO by macrophages (P<0.05,P<0.01). In addition, DATS can antagonize the inhibition of tumor-derived immunosuppressive factors produced by S180 cells and Ehrlich ascitic cancer cells on the activation of T cells, and reduce the inhibitory rate significantly (P < 0.01). DATS, despite its inhibition of the production of NO by macrophages, can significantly enhance the production of hydrogen peroxide (H2O2) by macrophages. When macrophages were pretreated with DATS for 24 h, the cytotoxicity % of macrophages to three tumor cell lines was significantly higher than that in corresponding control group (P<0.05,P<0.01). In the presence of both DATS and LPS, the cytotoxicity of macrophages was further enhanced so that the cytotoxicity % of macrophages to tumor cells was significantly higher than either that in the presence of DATS alone or that in the presence of LPS alone (P<0. 05,P<0. 01). These results indicate that DATS can augment the activation of T cells and enhance the anti-tumor function of macrophage, suggesting that DATS may be potentially useful in tumor therapy.

Construction of expressing plasmids of recombinant FN polypeptides with bifunctional-domain and the characterization of the products expressed in E. coli.

SummaryTwo expressing plasmids have been constructed and used to express two bifurictional-domain recombinant polypeptides of human fibronectin (FN) inE. coli. One was CH50 (Pro1239-Ser1515 of FN linked with Ala1690-Thr1960 of FN through Met) and the other was CH56 (Pro1239-Thr1960 of FN). Both of two polypeptides were capable of binding heparin and were purified by heparin-agarose affinity chromatography. The purified products were capable of binding cells. The production of CH50 and CH56 polypeptides provided a fundamental basis for further study of the anti-metastatic function of recombinant fibronectin polypeptides.

Comparative study on the inhibitory effect of recombinant FN polypeptide CH50 and CH56 on the metastasis of melanoma cells

SummaryOn the basis of preparation of anti-metastatic recombinant FN polypeptides, CH50 and CH56, we further studied the function of these polypeptides. The capacity of CH50 binding with melanoma cells (ED50 30 mM) was higher than that of CH56 (ED50 45 mM). Both of the polypeptides could significantly suppress the binding of melanoma B16 cells to laminin. There was no significant difference in the inhibitory effect between two polypeptides. In the experimental metastasis of melanoma cells, both of CH50 and CH56 could significantly inhibit the metastasis of the tumor cells, and reduce the number of lung metastasis by about 80 %. Our results suggest that III-11 and ED-A repeats influenced, to some extent, the binding capacity of bifunctional-domain polypeptide to cells, but did not affect the inhibition of the polypeptide on the metastasis of melanoma cells. The presence and connection of cell I and Hep II domains are the elements which determine the ability of recombinant FN polypeptides to inhibit the metastasis of tumor cells.

Augmentation of recombinant fibronectin polypeptide CH50 on the antitumor function of macrophages

SummaryWe prepared an anti-metastatic polypeptide, recombinant fibronectin polypeptide CH50, and finished the preliminary identification of its functions. In this paper, we studied the effect of this polypeptide on the function of macrophages. CH50 can significantly augment the production of nitric oxide (NO) by macrophages in a dose-dependent manner. The continuous presence of CH50 had a much stronger effect. In the presence of CH50, the cytotoxicity of macrophages to melanoma B16/F1 cells was significantly enhanced, and a stronger effect was obtained if CH50 was present continuously. CH50 polypeptide and IFN-γ have a synergistic effect on the production of NO by macrophages and the cytotoxicity of macrophages on tumor cells. In thein vivo experiments, CH50 can inhibit the growth of tumor cells, and have a better effect in the presence of IFN-7. Our results suggest that recombinant fibronectin polypeptide CH50 has two functions: one is to inhibit the metastasis of tumor cells, and the other one is to augment the function of macrophages. And this polypeptide will be potentially useful in tumor therapy.

Effect of polypeptide CH50 on macrophage activationin vivo and antitumor function

SummaryThe main features of CH50, a recombinant polypeptide of human fibronectin, activating macrophagesin vivo and its anti-tumor function were investigated. After injection of CH50 and (or) transfection of IFN-γ genein vivo, several kinds of factors produced by macrophages were determined and the growth of tumorin vivo was measured. CH50 could enhance the production of such factors as NO, TNF and IL-1 by macrophages, but the activation of macrophages was relatively slow when CH50 was used in vivo alone. CH50 and IFN-γ could synergistically activate macrophages rapidly in vivo no matter whether the injection of CH50 or the transfection of IFN-γ gene was performed first. Injection of CH50 alone inhibited the formation of tumor nodes in a dose-dependent manner. Low dose of CH50 could strongly inhibit the formation of tumor nodes less than 1 mm, while high dose of CH50 could inhibit those more than 1 mm. A stronger inhibition on the growth of tumor in vivo was obtained by the synergistic effect of CH50 and IFN-γ. CH50 and IFN-γ, as double-signal factors for activation of macrophages, will be potentially useful in tumortherapy.

Construction of eukaryotic expressing vector pCH503 of CH50 and its chemotaxis and anti-tumor function by expressionin vivo in mice

SummaryAn eukaryotic expressing vector that expresses CH50, a recombinant polypeptide of human fibronectin, in mice was constructed, and its chemotactic and anti-tumor function byin vivo gene transfection was investigated. The plasmid was constructed by recombination techniques. The cDNA fragment coding CH50 polypeptide from a prokaryotic expressing vector of CH50 was ligated with 5′-terminal noncoding region and coding region of signal peptide of mouse IFN-7 cDNA at 5′ side and 3′-terminal noncoden region of human FN cDNA at 3′ side. The recombinant cDNA was inserted into plasmid pREP8. The resulted expressing plasmid was designated as pCH503. The macrophages transfected with pCH503in vivo and culturedin vitro could produce CH50. The expressed product was identified by heparin-affinity chromatography and SDS-PAGE. By counting and Giemsa-staining of coeliac cells and histotomy and staining of muscle tissue, the chemotaxis on immune cells was observed after transfection of pCH503 either in peritoneal cavity or in muscle. The inhibition of gene transfection of pCH503 on melanoma was observed in mice. The number of melanoma nodes in mice was reduced by 50%–60% after coeliac transfection with pCH503. The pCH503, an eukaryotic expressing vector of CH50, can expressin vivo in mice. The transfection of pCH503in vivo has the chemotaxis on immune cells and can inhibit the formation of tumor nodes, suggesting that plasmid pCH503 is potentially useful in combined treatment of tumor.

Inhibitory effect of recombinant FN polypeptide CH50 on the metastasis and tumor growth.

SummaryThe inhibition of CH50, a recombinant polypeptide of human fibronectin, on the formation of tumor metastasesin vivo was studied by inoculation of melanoma B16/F1 cells by hypodermic or intraperitoneal injection, and the size and amount of tumor nodes after therapy were measured. In the treatment of hypodermic tumor, local injection of CH50 produced much better efficacy than distance injection of CH50 did. The inhibitory effect of CH50 on the growth of tumor reached 50 %. In the treatment of peritoneal metastasis, the inhibition of CH50 on metastases smaller than 1 mm was above 80 %, and above 50 % on metastases larger than 1 mm. A better efficacy was achieved if CH50 was used in combination with hydroxycamptothecine (HCPT), a chemotherapeutic agent. CH50 displayed a strong inhibitory effect on the formation of small metastasis and growth of larger nodes of tumor, suggesting that CH50 plays a very important role in combined treatment of tumor therapy.

Effect of fragment Asp1961—Glu1978 in fibronectin on the expression of triple-domain polypeptide inE. coli

SummaryTwo plasmids were constructed and used to express two triple-domain recombinant polypeptide of human fibronectin (FN). The cDNAs in plasmids code for two polypeptides, CH62 (Pro1239-Ser1515 of FN linked with Ala1690-Val2049 through Met) and CH63 (CH62 without Ile1850-Glu1978). The expression level of CH62 inE. coli was very low, but that of CH63 was very high. The results suggests that Asp1961 -Glu1978 in FN is a key sequence influencing the expression of triple-domain polypeptide inE. Coli. After being dissolved and renatured, CH63 can be purified by heparin-agarose affinity chromatography. Both of the cell-binding domains in the recombinant polypeptide were functional. The production of CH63 provides a fundamental basis for further study of recombinant products with better anti-metastasis function.

Investigation on the purification and expression of cell I — Hep I recombinant FN polypeptide inE. Coli

SummaryAn anti-metastatic polypeptide, bifunctional-domain (Cell I -Hep I) recombinant polypeptide of human fibronectin, was expressedin E. coli and purified. The expression level was found to be about 20%– 30 % of the total cell proteins. In BL21 (DE3)/T7, anE. coli expressing system, lactose can be used as an inducer to substitute IPTG thereby reducing the cost by several hundredfold and it is suitable for the large-scale preparation of recombinant FN polypeptide. Cell I -Hep 1 fragment is an alkaline polypeptide. In BL21(DE3)/T7 expressing system, better isolation was achieved if DEAE-52, instead of CM-52, was used for ion-exchange chromatography. The purified product was obtained after heparin-agarose affinity chromatography following ion-exchange chromatography.

Effect of “Re Du Qing” on the activation, proliferation and membrane fluidity of lymphocytes

SummaryEffects of Chinese Medicinal Preparation “Re Du Qing” (RDQ) on the activation, proliferation and membrane fluidity of T lymphocytes from human peripheral blood Were studied by means of3H-TdR incorporation and DPH fluorescence polarization. The results showed that “RDQ” can:1) significantly inhibit the activation of T lymphocytes; 2) restrain the proliferation of activated T lymphoblasts in the presence of exogenous interleukin-2 (IL-2); and 3) increase the membrane fluidity of T lymphocytes and antagonize the decreased fluidity of lymphocyte membrane mediated by Con A or PHA. The functional abnormalities of T lymphocytes in some autoimmune diseases such as arthritis and the usefulness of RDQ in the treatment of these diseases were also discussed.