| Objective: Malignant gliomas are the most common brain tumors in humans, and current treatments fail to provide long-term management of these tumors. The prognosis for patients with high-grade glioma remains poor: survival for less than 1 year, even following surgery and adjuvant therapies such as chemotherapy and radiation therapy. This disease clearly requires development of more effective therapeutic modalities that will improve long-term control and survival. The development of gene therapy techniques has provided a new and promising avenue of research into more efficacious treatments for gliomas and other cancers. Immune gene therapy, which may up-regulate the immune system and create an immunostimulatory tumor microenvironment, is thought an effective treatment for tumor. Cytokine-mediated immune gene therapy is designed to trigger an effective antitumor immune response and to achieve complete regression of established neoplasms. Direct transfer of cytokine genes into tumor cells can result in sustained local release of the cytokines and consequently induce potent immune responses against peripheral tumors. The classical conception that the CNS is an immunologically privileged site was contradictory with such approaches for the treatment of brain tumors. On the other hand, recent studies showed that the CNS could be accessible by the systemic immune system through cervical lymph nodes and the blood-brain barrier. These reports demonstrated that CNS-derived antigens were presented to peripheral immunocompetent cells, and the activated lymphocytes migrated into the CNS. Although an immune response within the CNS may be different from that in other organs, privileged immunity in the CNS is not absolute, and brain tumors are possibly subjected to cytokine-mediated immune gene therapy. However, difficulties in complete elimination brain tumors indicate that improvements in the efficacy of the induction of immune responses will be necessary before maximal effects can be achieved. Enforcement of systemic immunity by facilitated antigen presentation in the periphery and by local secretion of cytokines with chemotactic activity can achieve better antitumor effects against brain tumors. Several reports have shown that cytokines such as IL-2, IL-4, IL-12, or granulocyte macrophage-colony stimulating factor secreted from brain tumors could generate antitumor immunity and suppress the growth of cytokine-producing tumors even in the CNS. IL-18 is a newly identified cytokine that mediates many important biological functions including induction of IFN-γ, enhancement of NK cell activity, stimulation of proliferation of activated T cells, regulation of FasL-dependent apoptosis and promotion of Th1 response. IL-18 was shown to induce the immune defense against tumors in various animal models. Transfection of IL-18 gene into the tumor cell may augment the production of tumor-suppressive cytokines, rescue the CTL against the tumor cells, and enhance NK cell activity. IL-18 exhibits anti-tumor activity in various tumor models. Protective immunity was induced by the immunization with colon carcinoma cells genetically modified to express IL-18. IL-18 also acts as an angiogenesis suppressor in vitro and in vivo. The biologic functions of IL-18 mentioned above make it attractive to be used in potentiating the antirumor. IL-24/MDA-7 (The melanoma differentiation associated gene-7, mda-7) is a dual-function molecule with tumor suppression and cytokine functions. Initial gene transfer studies convincingly demonstrated potent anti-tumor effects of mda-7. Further studies showed that the mechanism of anti-tumor activity was due to induction of apoptosis. Most striking was the tumor-selective killing by mda-7 gene transfer—normal cells were unaffected by Adenoviral delivery of mda-7 (Ad-mda7). A variety of molecules implicated in apoptosis and intracellular signaling are regulated by Ad-mda7 transduction. Different apoptosis effector proteins are regulated in differenttumor types, suggesting that Ad-mda7 may regulate various signaling pathways. Indeed, loss of MDA-7/IL-24 protein expression correlates strongly with melanoma tumor invasion and disease progression. The ''bystander''effects proposed for MDA-7/IL-24 protein include immune stimulation, antiangiogenesis and receptor-mediated cytotoxicity. Thus, mda-7 is a unique multifunctional cytokine in the IL-10 family and may have potent anti-tumor utility in a clinical setting. In the present study, to explore the anti-tumor effects against brain tumors of IL-18, IL-24 genes, the two genes were transfected into rat C6 glioma cell respectively by retrovirus. Then we accessed whether IL-18, IL-24 genes can suppress the proliferation in vitro and tumerigenicity in vivo of rat C6 glioma cell and investigated the mechanism. The study is a basic investigation for the immune gene therapy using IL-18, IL-24. Material and methods 1 Retrovirus-mediated IL-18 gene expression in rat C6 glioma cell 1.1 Establishment of C6/IL-18 cell line Plasmid LXSN/IL-18 and two packaging cellsψ2(ecotropic), PA317(amphotropic) were obtained kindly from Professor Yasuo Iwadate (Chiba Cancer Center Hospital and Research Institute, Chiba, Japan). C6 cell line was reserved in Cell Biology Division, Institute of Basic Medicine, Hebei Medical University. SD rat(Sprague Dawley rat)was provided by Animal Center of Hebei Medical University. 1.1.1 Obtaining recombinant retrovirus Recombinant retrovirus was obtained by transfecting retroviral vector (LXSN/IL-18) intoψ2 and PA317 packaging cells, then used directly to infect C6 cell. 1.1.2 Retrovirus infected C6 cells The C6 cells was incubated with supernatant containing LXSN/IL-18 for 24h, then screened by G418(400mg/l), and 14 days later, G418-resistant cell clones were picked up and cultured for 14-20 days, then analyzed for transgene expression. 1.1.3 FCM for the quantification of IL-18 expression The cells of different G418-resistant cell clones were incubated with anti-IL-18 antibody and FITC –Ig G. The quantification of IL-18 protein expression was measuredby FCM. The cell clone with the highest quantification was named C6/IL-18 cell line and was used in following study. 1.2 Immunocytochemistry for IL-18 expression After fixed with 4% glutaral, the adhere C6/IL-18 cells was processed for immunocytochemistry using a rabbit polyclonal anti-IL-18 antibody. 1.3 RT-PCR for IL-18 mRNA expression Total RNA of C6/IL-18 cells was extracted by Trizol Reagent, according to the instructions of the manufacturer, and performed RT-PCR. Primers for IL-18 amplification were 5'-CACAGATGAGTTGGGGACT-3'(sense) and 5'-AGTCAGAGATGGTAGAATTTCTG -3'(antisense) with an expected PCR product of 632bp. Cycling conditions were 50 min of reverse transcription at 370C, 5min, then at 960C for 5min, 30s of denaturation at 960C, 30s of anneal at 59.50C, 45s of extension at 720C, there are 35 cycles totally. PCR products were visualized by electrophoresis in a agarose gel. 1.4 ELISA detected the IL-18 activity After grinded ,filtered and centrifuged, the single cell suspensions of splenocytes was harvested and incubated in 96-well plate for 24h with 200μl supernatant of C6/IL-18. The amount of IFN-γconcentration in the spleen cells supernatant was tested using a rat IFN-γ-specific ELISA kit. 1.5 MTT tested C6/IL-18 growth in vitro C6/IL-18 cells were incubated in 96-cell plate for 6 days. Microplate reader detected the cell growth every day. Before detected, the cells in each well was labeled by 20μl MTT for 4 h at 370C. Till the complete dissolving of formazan precipitates in 150μl dimethylsulfoxide, absorbance was measured at 570 nm in a microplate reader. 1.6 FCM for proliferation index (PI) of IL-18 The DNA of C6/IL-18 was stained by EB. The amount of DNA in C6/IL-18 was detected by FCM, after it was stained by EB. The proportion data of G0/1,S,G2M was analyzed by DNA cell cycle sofeware. The PI of C6/IL-18 was calculated according to the following formula: PI(%)=(S+G2M)/(G0/1+S+G2M)×100%。1.7 Establishment intracranial tumor model SD rats (male, 250-350g)were placed in a stereotactic frame, 1×109/L C6 cells in 10μL serum-free culture medium were injected into the right striatum with a microsyringe for 10 min (with respect to Bregma:1mm anterior, 3-3.5mm right lateral, 6mm depth.). Bone wax was used to seal the burr-hole and avoid the spreading of cells outside the needle track.. MRI study was performed 20 days later and the volume of tumor was calculated according to the following formula: Tumor volume (mm3)=(长径×宽径×2 å€å±‚æ•°)×π/6。2 Retrovirus-mediated IL-24 gene expression in rat C6 glioma cell 2.1 Establishment of C6/IL-24 cell line Plasmid LXSN/IL-24 and two packaging cellsψ2(ecotropic) , PA317(amphotropic) were obtained kindly from Professor Yasuo Iwadate (Chiba Cancer Center Hospital and Research Institute, Chiba, Japan). C6 cell line was reserved in Cell Biology Division, Institute of Basic Medicine, Hebei Medical University. SD rat(Sprague Dawley rat)was provided kindly by Animal Center of Hebei Medical University. 2.1.1 Obtaining recombinant retrovirus(The same as 1.1.1 ) 2.1.2 Retrovirus infected C6 cells(The same as 1.1.2 ) 2.1.3 Semi-quantity RT-PCR for IL-24 expression Total RNA of different G424-resistant cell clones was extracted by Trizol Reagent, according to Primers for amplification of IL-24, 5'-CACAGATGAGTTGGGGACT -3'(sense) and 5'-AGTCAGAGATGGTAGAATTTCTG -3'(anti –sense ), with an expected PCR product of 553bp. Cycling conditions were 50 min of reverse transcription at 370C, 5min, then at 960C for 5min, 30s of denaturation at 960C, 30s of anneal at 560C, 30s of extension at 720C, there are 35 cycles totally. PCR products were visualized by electrophoresis in a agarose gel. Densities of the amplifiedβ-actin and IL-24 were analyzed using Get Pro 3.1 software. Results were expressed as a tatio of quantification IL-24 product over β-actin product. The cell clone with highest quantification was named C6/IL-24 cell line and used in following study. 2.2 MTT test for C6/IL-24 growth in vitro (the same as 1.5) 2.3 FCM for proliferation index (PI) of IL-24(the same as 1.6)2.4 Establishment intracranial tumor model SD rats (male, 250-350g) were placed in a stereotactic frame, 1×109/L C6 or C6/IL-24 cells in 10μl serum-free culture medium were injected into the right striatum with a microsyringe for 10 min (with respect to Bregma:1mm anterior, 3-3.5mm right lateral, 6mm depth.). Bone wax was used to seal the burr-hole and avoid the spreading of cells outside the needle track.. MRI study was performed 20 days later and the volume of tumor was calculated according to the following formula: Tumor volume (mm3)=(长径×宽径×2 å€å±‚æ•°)×π/6。3 The tumorigenicity of C6/IL-18, C6/IL-24 in vivo 3.1 Establishment intracranial tumor model 23 SD rats (male, 250-350g) was divided randomly into 3 groups. Group 1 includes 9 rats rejected with C6/IL-18 cells. Group 2 includes 9 rats rejected with C6/IL-24 cells. Control group includes 5 rats rejected with C6 cells. The method of establishing intracranial tumor model was same as 1.7. 3.2 Histopathological and Immunohistochemical examination 3.2.1 All rats were sacrificed 21 days after injected tumor cells; the tumors were resected and fixed with 10% formalin and embedded in pataffin. The 4-5μm thick sections were cut from paraffin mocks and stained with hematoxylin-eosin(HE), according to standard products and incubated with antibody(including anti-PCNA, anti–CD34 ,anti-CD45RO, anti-CD20) for immunocytochemistry. (SP method) 3.2.2 PCNA expression The positive cell expressing PCNA showed brown material in nucleus by immunocytochemistry examination. According to Pedersen; in every visual field, +, 1-5% positive cells; ++, 5-50% positive cells; +++: >50% positive cells. 3.2.3 Endothelial marker CD34 expression To analyze the microvessel formation in tumors, sections were stained with anti-CD34 antibody and subsequently with the SP method. Positively vascular endothelial cells stained brown. The microvessel density (MVD) was determined according to the following methods. Briefly, regions of highest vessel density were scanned at low magnification (x100) and counted at high magnification (x200). Threesuch fields were examined in each tumor section, and the mean MVD value was recorded. Any endothelial cell or endothelial cell cluster that was clearly separated from adjacent microvessels was considered a single, countable microvessel. 3.2.4 CD45RO,CD20 expression The tumor-infiltrating lymphocyte cell expressing CD45RO, CD20 positively showed brown material in cytoplasm by immunocytochemistry examination 4 The investigation of IL-18 and IL-24 genes expression in rat astoglial cell 4.1 Isolation of rat astroglial cell The cerebral cortex of temple and parietal lobe from the newly born SD rats was dissected on the sterility condition. These tissues were digested by 0.25% trypsin filtered by meshwork. 5×106 cells were incubated in 75ml flask. After 9 days, the cells became 60%-70% confluence. Shaked the flask and rinsed the cells by NS and EDTA, the astroglial cells were digested by 0.25% trypsin and divided into 2 flasks. After continuous cultured for 24h, the cells were fixed with 4% glutaraldehyde, labeled with GFAP antibody, and identified using Immunocytochemical staining. 4.2 RT-PCR for IL-18, IL-24 expression in rat astroglial cell Total RNA of rat astoglial cell, C6, IL-18, IL-24 cells were extracted with Trizol Reagent. (C6 cell lies was negative control, C6/IL-18 and C6/IL-24 cells was positive control), and then performed RT-PCR. The primers and cycling conditions were same as 1.3 and 2.2.3. 4.3 Immunocytochemistry for IL-18 protein expression in rat astroglial cell The adhere astroglial cells were fixed with 4% glutaral, and then was processed for immucytochemistry by anti–IL-18 antibody (SABC method). (C6 cell was used as negative control; C6/IL-18 cell was used as positive control) Results 1 Retrovirus-mediated IL-18 gene expression in rat C6 glioma cell 1.1 The IL-18 protein expression in C6/IL-18 IL-18 gene was tranfectedinto C6 Cells by retrovirus vector, eight G418-resistant cell clones was obtained. The No.2 cell clone with the highest quantity of IL-18 protein expression was named C6/IL-18 cell and used in following study. The immunocytochemistry showed IL-18 protein expressed in cytoplasm of C6/IL-18. 1.2 IL-18 mRNA expression in C6/IL-18 RT-PCR result showed: the IL-18 mRNA expression was positive. 1.3 Activity examination of secreted IL-18 from C6/IL-18 Stimulated by supernatant of C6/IL-18, rat splenocytes could secrete IFN-γ.The amount measured by ELISA was 15.0 pg/ml, was higher than control group (5.0pg/ml,p<0.01), revealed that C6/IL-18 cell could secret IL-18 protein with cytokine function. 1.4 The growth of C6/IL-18 in vitro Analysis by MTT, C6/IL-18 cell could grow in vitro, however, the growth capacity was lower than parent C6 cell. 1.5 PI of C6/IL-18 tested by FCM PI of the C6/IL-18 was (28.68±1.50)%, was lower than C6 cells(39.34±1.09)%. 1.6 Tumorigenicity of C6/IL-18 in vivo MRI showed: among 9 rats injected with C6/IL-18 cells, 4 rats had no tumor formation, while the 5 rats injectal with C6 cells, all had tumor formation. The average tumor volume of 4 C6/IL-18 animal models was (48.3±13.6 mm3), smaller than that of 5 C6 animal models(213.6±26.4 mm3),P<0.05, implied that IL-18 could inhibit the cell growth in vivo. 2 Retrovirus-mediated IL-24 gene expression in rat C6 glioma cell 2.1 The IL-24 protein expression in C6/IL-24 IL-24 gene was tranfected into C6 Cells by retrovirus vector, 4 G418-resistant cell clones was obtained. RT-PCR result showed: the IL-24 mRNA expression in four different cell clones were positive. The No.4 cell clone with highest quantification was named C6/IL-24 cell line and used in following study. 2.2 .The growth of C6/IL-24 in vitro Analysis by MTT, C6/IL-24 cell could grow in vitro, however, the growth capacity was lower than parent C6 cell. 2.3 PI of C6/IL-24 tested by FCM PI of the C6/IL-24 was (29.71±0.89)%, was lower than C6 cells(39.34±1.09)%. 2.4 Tumorigenicity of C6/IL-24 in vivo MRI showed: among 9 rats injected with C6/IL-24 cells, 3 rats had no tumor formation, while the 6 rats injected with C6 cells, all had tumor formation. The average tumor volume of 6 C6/IL-24 animal models was (48.3±13.6 mm3), smaller than that of 5 C6 animal models(213.6±26.4 mm3),P<0.05, implied that IL-24 could inhibit the cell growth in vivo. 3 The tumorigenicity of C6/IL-18, C6/IL-24 in vivo 3.1 Histological analysis. The tumor cells in tumor tissue formed by C6 cell spread closely and grew actively. The nuclei with much abnormal nrclear fission were heteromophism. There were lots of neovascolarity in the tumor tissue and few inflammatory infiltration cells around the tumor tissue. The tumor cells in tumor tissue formed by C6/IL-18, C6/IL-24 cells were decayed. The nuclei were karyopycnosis. There were less neovascolarity in the tumor tissue and lots of inflammatory infiltration cell around the tumor tissue compared with the C6 cell group. 3.2 PCNA expression PCNA expression of tumor tissue formed by C6 was +++,formed by C6/IL-18 and C6/IL-24 was ++. 3.3 MVD calculation MVD of tumor tissue formed by C6/IL-18, C6/IL-24 were 24.70±7.10 and 21.10±3.48, and were much less than mirovessude in the tumor tissue formed by C6 cell. (55.42±9.54, P<0.01, P<0.01). The result revealed a significant reduction of number of endothelial cells in C6/IL-18, C6/IL-24 tumor tissue. 3.4 The CD45RO, CD20 expression in tumor–infiltrating lymphocyte(TIL) The CD45RO expression of TIL in tumor tissure formed by C6/IL-18, C6/IL-24 was positive and the CD20 expression of TIL was regative, indicated that most of TILs was T lymphocyte. 4 The investigation of IL-18 and IL-24 genes expression in rat astoglial cell 4.1 Isolation of rat astroglial cell. Incubated for 24h-48h, the cells isolated from the cerebral cortex of SD rats began to adhere. Incubated for 5-6 days,...
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