Experimental Researches On Human Interferon-β Genetherapy Of Brain Glioma

At present, there is no effective treatment for patients with malignant and recurrent glioma. Patients with malignant glioma have a very poor prognosis, even with treatment including surgical resection, radiation, chemotherapy. Gene therapy has received particular attention as a promising treatment for malignant glioma. The IFNs are a family of natural glycoproteins that consist of IFN-α, β and γ. The antiviral activity of IFNs led to their discovery, but later data revealed that they also control cell growth and differentiation, inhibit expression of oncogenes, and activate T lymphocytes, natural killer cells, and macrophages. Recent research indicates that interferon is implicated in which regulate cell growth, inhibiting the growth of tumor blood vessel and cellular immunity activation. The efficacy of IFN therapy for various malignancies has been investigated for many years. Extensive clinical trials have concluded that the IFNs can be efficacious against many neoplasms. The continuous incubation of different human carcinoma cells withnoncytostatic concentrations of IFN- α or IFN- β down-regulated transcription and protein production of basic fibroblast growth factor, interleukin 8, and collagenase type IV. IFNs are capable of modulating a variety of cellular responses, including cell growth and apoptosis. It was found that IFN-alpha was a rapid and potent inducer of apoptosis in H9 and U-266 cells of hematopoietic cell lines. In recent research IFN- β has multiple biological actions including modulation of gene expression, immunomodulation, slowing of cell proliferation, and alterations in differentiation, human IFN-β has the strongest anti-proliferative activity against human melanoma cell lines. Histological analysis of the injected nodules revealed that the IFN- P gene transfection induced apoptosis of the human melanoma cells. Despite the clinical activity of IFN-β on malignant gliomas, the mechanisms related to inhibiting human malignant glioma growth of interferon-β are not clear.The main research methods and results in our studies as follows: 1. Transfection of glioma SHG44 cell with human IFNβ gene by using liposome. Aliquots of 5X10~4 SHG44 cells were inoculated in each well of a Falcon plate with 1.5 ml of medium and incubated at 37° C for 24 h in a humidified atmosphere of 5% C02. pSV2IFN P (15 nmol of lipid/0. 3 μg of DNA/ml) were added to the medium, and incubation was continued for up to 6 days. The culture medium was collected on days 2 after addition of liposomes, and IFN β concentration in the medium was measured by Western blot. It was confirmed that IFNβ gene can be expressed in SHG44 cells of experimental group byimmunohistochemistry, immunofluorescence and flow cytometry.2. The growth-inhibitory effect of human IFNP gene transfer to glioma SHG44 cells was also evaluated. The biological character of glioma cells in experimental group, empty vector group and control group were examined by Hoechst Stain , electron microscope, flow cytometry and cell growth rate counting. In the Hoechst Stain, The glioma SHG44 cells of control groups have the normal glioma cell morphous. Nuclear fragmentation was seen in glioma SHG44 cells after 48 h of* IFN-0 gene transfection. The characteristic apoptotic cell changes under electron microscope were observed in glioma SHG44 cell.3. The SHG44 cells were injected subcutaneously into the hind limbs of nude mice. After 2 weeks the mice were divided randomly into the following three treatment groups: experimental group, empty vector group and control group. When SHG44 cells grew under the skin of nude mice, the tumor inhibition rate of the injecting pSV2IFN P group reached 55.4%, compared with that of injecting empty vector group or normal saline group. The tumor necrosis rate of injecting pSV2IFN $ group was obviously higher, and the blood vessel numbers were lower. The vessel numbers and tumor necrosis rate in the injecting pSV2IFN P group, injecting group and normal saline group were 6.4, 12. 1 and 11.9 (number/per visual field ), 20.15%, 9.20% and 9.14 % (necrosis area /tumor area) respectively. The detection of apoptotic cells in tissue sections was performed using electron microscope. The glioma growth of nude mice was inhibited. IFN-fl can inhibit the growth of tumor blood vessel and induce glioma cell SHG44 apoptosis.