| ObjectiveGlioma is the most common primary neoplasm of central nervous system ( CNS)in human beings. Because of its high recurrence, pool prognosis and difficulty in total resection. It has become one of the most trouble problems in neu-rosurgery, and it is difficult to get ideal purpose by combining therapy measures of traditional excision with radiotherapy and chemotherapy. In recent years, internal and external investigators have developed the study of anti - tumor activity of TIL to glioma in vivo and in vitro, and have got heartening achievements, and they also have made considerable progress in experimental study on TNF - a therapy to glioma. Experiments in vivo demonstrated that the amount of TIL in tumor was 30 - 40 times of that in normal tissue by infusion of it, and this means TIL can be localized specially at tumor sites, while the content of TIL in tumor constituted a lower percentage of the total amount of infusion, and the therapeutic effect was not satisfied. In this study, we introduced TNF - a gene into TIL cells of human glioma on the basis of separate preparation of TIL cells of human glioma and development of efficient retroviral vectors for gene transfer, and experimental study of its anti-glioma effect in vivo and vitro was performed.Methods1. Separation and amplification of human glioma TIL and determination of its anti - tumor activity. TIL were successfully separated from excised glioma tissues by means of centrifugation, and its amplification in vitro was done, anti -tumor activity was determined.2. Transduction of TNF - a gene into TIL of human glioma by retroviral vectors and study of its biological activity in vitro. The development of retroviral vectors for TNF - a gene transfer and preparation of carrier cells - TIL have allowed transducing marked gene NeoR. and purpose gene TNF - a into TIL cells respectively by use of monoclone tilter virus PLC -2 and PLJC -5. Detect the proliferation of TIL, expression of TNF - a and the anti - tumor activity of glioma TJ8510 cells in vitro.3. In order to expand the anti - tumor effect of TIL in vivo, TNF - a gene was inserted into carrier cells TIL by use of retroviral vectors for TNF - a gene transfer, and its proliferation and artti - tumor activity in vitro were studied. Then in naked mice model of human glioma in vivo, the antitumor effect of TNF- TIL was observed.Results1. TIL were successfully separated from excised glioma tissues of 10 cases by means of centrifugation, and its amplification in vitro was done, anti - tumor activity was determined. Experimental results showed that TIL increased from 1. 1+104-2+106 to 1.1+107 - 1+109 in the presence of IL -2 after culture for 30 days in vitro, increased by an average of 274+45 folds. This indicates that TIL of human glioma can proliferate well in vitro in the presence of IL -2. The killing activity of human glioma TIL to TJ 8510 cells was related to its culture time. The killing activity of TIL was detected after culture 10 days in the presence of IL - 2, but it is not strong, meanwhile the kilh'ng activity of LAK cells was obviously higher than that of TIL; when cultured for 20 days, the killing activity of TIL cells increased rapidly, and that of LAK cells decreased quickly. The difference between them was significant. When cultured for 30 days, the killing activity of cells reached a peak and that of LAK cells decreased continuously. This suggests that TIL of human glioma reacted to IL - 2 slowly with a long latent period, but this was contrary for LAK cells. The anti-tumor activity of TIL in vitro was related to its proliferation potential.2. TIL ceUs containing NeoR ( NeoR - TIL) and TNF - a ( TNF - TIL) were obtained. Safety examination showed that both of them had no foreign virus in their supernatant. It means this method is safe, the cellular proliferation can not be affected considerably by gene transduction, and retroviral vectors for gene transfer can not be alter the functional characteristics of the host cells. TIL is quite suit...
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