Culture and Identification of Monoclonal Neural Stem Cells Derived from Cerebral Cortex

Summary： To isolate and culture the purified monoclonal neural stem cells from the cerebral cortex of new born mice, new-born mice cerebral cortex was isolated and dissociated to single-cell suspension by mechanical trituration. The dissociated single cells were cultured in serum-free medium. After the formation of neurospheres, single-cell clone culture was performed by limiting dilution and the proliferated single-cell clones were harvested for subculture. Immunocytochemistry was used to detect the specific marker of neuroepithelial stem cells （Nestin） of the primary and monoclonal neurospheres. In the differentiated cells we detected the specific antigen of NF-200 and GFAP. Our results showed that the primary neurospheres expressed Nestin antigen positively. By limiting dilution, we cultured the cell lines from single-cell clone and the monoclonal neurospheres expressed Nestin and had capabilities of self-renewal, proliferation and the potentiality of differentiation into neurons and glial cells. It is concluded that monoclonal neural stem cells which have the ability of proliferation and multi-directional differentiation can be isolated and cultured from the cerebral cortex of new-born mice by limiting dilution.

CYTOTOXICITY OF INDIRECT IMMUNOTOXIN MEDIATED BY ANTI-GASTRIC CANCER MONOCLONAL ANTIBODIES ON TUMOR CELLS

In the present study, an indirect assay was employed to investigate 5 anti-gastric cancer monoclonal antibodies for their cytotoxic potential as ricin A chain-containing immunotoxins. The tumor cell, were treated with dilutions of tested antibody followed by ricin A chain coupled to goat anti-mouse immunoglobulin. The cytotoxic effect was determined with tetrazolium colorimetric assay. The results showed that among the 5 antibodies chosen, MGb2 and MG7 could be well used for preparation of effective A chain immunotoxins.

IMMUNOELECTRON MICROSCOPIC STUDY ON THE LOCALIZATION OF MONOCLONAL ANTIBODIES ON GASTRIC CANCER CELLS

The localization of three monoclonal antibodies (MAb) against gastric cancer was studied on two human gastric cancer cell lines by immunoelectron microscopic technique. It has shown that the corresponding antigens of MAb 3G9 and 3H11 were distributed on the microvilli (M) and non-microvillus (NM) plasma membrane of target cells, with various M to NM ratios depending on the MAbs and target cells used. However, the corresponding antigens of MAb PD4 was only localized on the surface of round or finger-like bulges of target cells and never on the microvilli and non-microvillous plasma membrane. Since the nature and function of these tumor antigens have not been identified yet, the implication of the different distributions of these antigens remians to be clarifated.

THE STUDIES ON MONOCLONAL ANTIBODY SZ-39 AGAINST HUMAN GLIOMA CELLS

A hybridoma cell line SZ-39 secreting monoclonal antibody against the human glioma cell has been established by a fusion between NS-1 myeloma cells and spleen cells from mice immunized with human glioma cell lines. Monoclonal antibody (McAb) SZ-39 was analyzed by ELISA, quantitative absorption, indirect immunofluorescence and ABC immunohistology. McAb SZ-39 strongly bound to 9/10 glioma cell lines, 17/20 glioma tissues, weakly bound to one liver cancer cell line and 1/2 lung cancer line, but they did not band with other tested human cancer linse. NcAb SZ-39 have no cross-reaction with lymphocyte, ABC red blood cells, white blood cells, blood platelet, normal bone marrow cells, fibroblast cells and 12 normal human tissues.The result indicated the antigen recognized by McAb SZ-39 may be a glioma-associated antigen <GAA). This GAA was analyzed by means of Western blotting. It was a MW 180 Kd glycopro-tein. The 131I-McAb SZ-39 specifically localized in human glioma xenografted in nude mice that indicate it may be useful in radioimmunoimaging and as a target for immunotherapy on human glioma.

SELECTIVE KILLING OF GASTRIC CANCER CELLS BY MONOCLONAL ANTIBODY CONJUGATED WITH A CHAIN OF RICIN

A specific cytotoxic agent against gastric cancer was constructed by covalently coupling the ricin A chain to monoclonal artibody, MGb2. MGb2 was modified by SPDP to introduce the 3-(2-pyridylthio) propionyl radical and then treated with a reduced A chain to give a disulfide linked conjugate that retained the original binding specificity of the antibody moiety. The conjugate obtained retained the activity of the antibody and the biological activity of the A chain well.

USE OF THE POLYMERASE CHAIN REACTION (PCR) TO DETECT MONOCLONALITY OF B CELL LYMPHO-PROLIFERATIVE DISORDERS

A technique has been developed using PCR to detect monoclonality of B-lymphoproliferative disorders. DNA was extracted from the blood, tissue and paraffin embedded sections by biochemical means or boiling. Forty cases of B-non Hodgkin's lymphoma (NHL), 15 cases of T-NHL, 8 cases of chronic lymphocytic leukemia, 17 cases of reactive lymphadenopathy and 12 cases of various non-lym-phocytic tumor were examined. Monoclonality of B-lymphocytes was detected in 86-92% of cases with B-lymphoproliferative diseases, but none in T-NHL, reactive disorders and non-lymphatic tumors. This technique provides a new molecular biologic method to diagnose malignant B-lymphoproliferative dicor-ders. It may be useful in Ig gene rearrangement study, differential diagnosis and retrospective investigation of lymphoproliferative disorders.

THE ALTERNATIVE PATHWAY OF HUMAN T CELL ACTIVATION BY MONOCLONAL ANTIBODIES(A COMPARATIVE STUDY BETWEEN NORMAL INDIVIDUALS AND CANCER PATIENTS)

This paper described T cell proliferative response by an alternative pathway in normal subjects and In patients with malignant diseases. Two McAbs, Anti-CCTl and Lo-CD2-act recognizing two distinct epitopes on E-receptor (CD2) were used to costimulate PBMC. Proliferative responsiveness was measured by 3H-thymidine incorporation. It was found that 82% of 72 nonnal subjects gave proliferative response whereas only 23% of the 93 patients did. The average cpm±SD in patients with bladder cancer (118±2314), kidney cancer (1619±2719) or lymphoma (2518±4057) was significantly lower than that in normal subjects (4935±2314), (P<0.001). These results indicate that T cell proliferation through the alternative pathway was significantly depressed in patients with cancer, and this can be used as a new parameter to monitor the immune status of cancer patients.