| Malignant glioma, the most common intracranial tumor, accounts for 30-50% of primary brain tumor in adults, with high morbidity, mortality and grim prognosis. Because currently available multiple modalities of treatment have not yet substantially changed the natural history of these lethal neoplasms, new therapeutic approaches are actively being investigated. Over the past few years, with the development in understanding the molecular biology and molecular genetics of glioma, gene therapy of tumor has been the hotspot and suicide gene therapy is one of the most favorite strategy used for malignant gliomas.Ideal protocol of brain tumor gene therapy would employ the optimal gene transfer technique with the optical genes transferred. Suicide gene therapy (HSV-tk/GCV) has revealed the encouraging therapeutic effect on glioma in vitro and in vivo animal experiment. However, as far as clinical trials, the results were not satisfactory and the reason might be the poor by-stander effect because of the therapy gene can not distribute in tumor in vivo widely.Antitumor effect of T lymphocytes of glioma patients were decreased, and because of the glioma antigen present ability was decreased owing to poverty cell special surface antigen, antitumor immunity effect was weakened further. According to present view, IL-18 could play its antitumor ability by activating T lymphocytes and natural killer cells, up-regulating expression of MHC and promoting CD4+ helper lymphocytes differentiated to Th1 subset to enhance cell mediated immunity. BMSCs have the ability of immigration and expression of transfected exogenous gene stably which offered the new route of gene therapy against glioma. The experiment planed to employ BMSCs as the therapy gene vector to observe the antitumor effect of BMSCs which infected suicide gene (BMSCs/tk). Furthermore, to enhance antitumor immunity, IL-18 was employed. The study provided a theoretical foundation for the use of BMSCs as vector against glioma. There was no report that BMSCs, as therapy gene vector, were transfected HSV-tk and IL-18 to treat intracranial glioma.1. The initial research of culturing, characterization and labelling bone marrow stromal stem cellsObjective: The purpose of this part is to research how to culture, identity and label bone marrow stromal stem cells(BMSC), which established foundation for further study.Methods: Single-cell suspension obtained from femurs of 2-3-week-old SD rats which were sacrificed and then incubated on plastic dishes in Dulbecco's modified Eagle's medium(DMEM) containing 20% heat-inactivated-fetal bovine serum(FBS) at 37℃with 5% CO2 . 48 hours later, shake the culture bottle slowly and nonadherent cells were removed. Adherent cells were further cultured by exchanging half culture medium every 2-3 days to expend. After 7-8 days of incubation, the bone marrow-derived cells were confluent and expended by a 1:2 split. After in vitro culture-expended 6 passages, adherent bone marrow-derived cells were uniformly fibroblast-like in appearance. Cell growth curve was drown by means of MTT.After subcultured 6 passages and cells were purified, to evaluate the BMSCs characterization, flow cytometry analysis was performed to detect the expression of CD31, CD45, CD90, CD34, CD71, CD44.After subcultured 6 passages, BMSCs, which were at exponential phase of growth, were cultured in flasks which contained several glass coverslips, and after 50% confluent the cells were pulsed for 24 hours with 10μg/ml 5-bromo-2-deoxyuridine (BrdU) (Sigma) in DMEM. Which was followed by fixation with 4% paraformaldehyde for 15 minutes, and then, immunohistochemistry and immunofluorescence were performed and the clips were observed under fluorescence microscope. After being labeled with BrdU, BMSCs were transplanted into rats, brain and the rats were sacrificed 3 weeks later. The brain tissue near the puncture site was made paraffin sections and immunofluorescence was performed.Results: The results showed that at the initial time, there were lots of suspension cells in the culture dishes.48 hours later, there were several fusiform adherent cells. After several semi-exchange culture medium, non-adherent cells were removed and several cell colonies formed. The BMSCs became comparatively homogeneous in appearance and well-distributed as the cells were 6 passages. The cells were relatively elongated or spindle-shaped. Cell growth curve showed that the cell multiplied actively and were at exponential phase of growth 2-3 later. Surface phenotypic characterization of primary BMSCs detected by flow cytometry showed that the percentage of CD31, CD34, CD45, CD44, CD71, CD90 was 5.67%, 4.31%,4.42%, 97.17%, 98.63%, 95.86% respectively. In vitro immunohistochemistry showed that 88.75±4.38% cells could be labeled with BrdU. Immunofluorescence showed that nucleus were labeled obviously and after being transplanted into rat brain 3 weeks later, lots of BMSCs, which were labeled with BrdU, were observed near the puncture site.Conclusions: The results showed that purified BMSCs could be harvested through culturing bone marrow cells and could expended culture in vitro in long-term. BMSCs could be labeled with BrdU high efficiency and stably.2. Tropism for glioma and neural differentiation of bone marrow stromal cellsObjective: The aim was to observe tropism for intracranial glioma of bone marrow stromal cells (BMSCs) and their differentiation in brain of Glioma bearing rats or induced with certain cytokines in vitro.Methods: After being subcultured in vitro 6-7 passages, BMSCs were seeded into flasks which contained glass coverslips coated with polylysine. After cells reached 70% confluence, the coverslips which contained BMSCs were displaced into Neurobasal medium containing 10% heat-inactivated-fetal bovine serum and alltrans retinoic acid (0.5μmol/L) and brain derived nerve growth factor (10ng/mL). BMSCs were cultured without induced cytokines as control group. 4 days later, the coverslips were taken out and fixation in 4% paraformaldehyde for 30 minutes followed by immunofluorescence to observe cells differentiation.In vitro experiment of BMSCs tropism for glioma was performed by means of cylinder experiment and Transwell experiment. C6 cells were cultured and, after C6 were at exponential phase of growth, glioma bearing rats were established. 3 days later, 20 glioma bering rats were selected randomly and were transplanted BMSCs(8×104cells in 2μL PBS)in the contralateral hemisphere. 15 days later, the rats were fixation with 4% paraformaldehyde perfuse through hearts. And then, the brains were removed and pathological sections were made and frozen section were made for differentiation research.Results: The results showed that, after being induced 3 days, observed under inverted microscope form, cells form were changed and some of the cells stretch out long branches and some of which got in touch with the other cells or branches. Several cells were rounding and shedding. The form of BMSCs in control group was not changed. Immunofluorescence showed that, in induced group, GFAP positive ratio was 21.1±6.3%,MAP2 positive ratio was 56.4±13.8%,Nestin positive ratio was 8.3±5.2%;in control group,there were no MAP2 and Nestin positive cells could be found,GFAP positive ratio was 3.9±2.8%。In cylinder experiment, in contrast to 3T3 cells, which remained localized to the area of initial seeding, BMSCs migrated rapidly and interspersed throughout the glioma monolayer, far from the initial site of seeding. Transwell experiment showed that, exposure to cell-free medium (1.1±0.7) or to conditioned medium from 3T3 cells(2.4±0.9) resulting in low levels of migrating BMSCs, there were no statistic difference between the two groups(P>0.05), while exposure to conditioned medium from C6 cells(14.7±4.7) produced significant BMSCs migration, whereas, the largest number of the migration BMSCs was C6 cells(42.7±8.2). Among the four cytokines, PDGF intermediated the maximal migration (32.7±6.3), the 2nd was EGF(26.3±7.4), whereas b-FGF and VEGF had no statistic difference(7.5±2.8, 9.4±3.5). Among all the induced effector, C6 cells induced the obviously immigration (P<0.05).In vivo tropism for glioma experiment showed that there were lots of BMSCs labeled with BrdU located at the boundary of brain tissue and glioma and there were several BMSCs inside glioma. In vivo differentiation immunofluorescence showed that: some of the BMSCs labeled with Hoechst33258 were Nestin positive (8.32±3.41%), some were GFAP positive(32.71±8.66%) and some were MAP2 positive (11.88±5.16%). This suggested that some of the transplanted BMSCs differentiated into neural precursor cells and some differentiated into neuron and gliocyte. In control group2 and control group3, GFAP positive ratio of transplanted BMSCs was 31.19±8.76% and 29.32±7.75% repectively, Nestin positive ratio of transplanted BMSCs was 6.95±3.75% and 7.21±3.31% repectively and MAP2 positive ratio of transplanted BMSCs was 8.76±3.77% and 10.59±5.43% repectively. There was no statistic difference between the three groups (P>0.05).Conclusions: BMSCs displayed extensive tropisum for glioma cells and most of immigration BMSCs located on the boundary of glioma and brain, only few immigration BMSCs were in glioma field. BMSCs, in in vitro induce environment or were transplanted into brain of glioma bearing rats, some of the transplanted BMSCs differentiated into neural precursor cells and some differentiated into neuron and gliocyte.3. In vitro and in vivo study of anti-glioma activity of bone marrow stromal cells transfected with HSV-tkObjective: The purpose of the research was to study the antitumor effect of BMSCs, as vector of therapy gene, after being transfected with tk gene. Methods: After being amplification, AdCMV-tk was used to transfected BMSCs (BMSCs/tk). RT-PCR was performed to detect transcription of tk gene by BMSCs/tk. Flow cytometry and MTT were performed to detect BMSCs/tk cells surface antigen and cell growth curve. Transwell experiment was performed to examine BMSCs/tk tropism for glioma in vitro. Glioma bearing rats were established and BMSCs/tk were transplanted into contralateral hemisphere to observe their tropism for glioma in vivo. Co-culture BMSCs/tk and C6, and then, different effector-target ratio and different GCV dose were introduced followed by cell survival ratio and cell apoptosis ratio examination by means of MTT, TUNEL and Annexin V. Experiment were performed to study by-stander effect of BMSCs/tk in vivo and survival time of glioma bearing rats were recorded after being treated with BMSCs/tk-GCV.Results: The results showed that, the cell surface antigens, cell growth curve and in vitro tropism for glioma of BMSCs were not changed after being infected with AdCMV-tk. RT-PCR results showed that, after being infected with AdCMV-tk 24 hours later, BMSCs/tk could transcribe tk gene obviously and reached its peak at 48 hours. And then, transcription of tk decreased gradually and decreased obviously 1 month later. In vitro by-stander effect experiment showed that, with the increase of effector or GCV dose, kill activity was enhanced obviously. In vivo experiment showed that, by means of TUNEL examination, apoptosis cells number in every view inside glioma was 1.65±1.09 in BMSCs/βgal group, 1.50±1.43 in BMSCs group, 1.45±1.28 in PBS group and 1.30±1.08 in control group and there were no statistic difference between these groups (P>0.05). But the largest apoptosis number was observed in BMSCs/HSV-tk group (8.85±3.33) (P<0.05).Conclusions: After being transfected with AdCMV-tk, BMSCs could transcript tk gene long term. BMSCs/tk displayed extensive tropisum for intracranial glioma and maintained high multiplication ability. BMSCs/tk induced obviously apoptosis of glioma in vitro and in vivo and prolonged survival time of glima bearing rats. 4. In vivo and in vitro study of therapy effect of bone marrow stromal cells co-transfected with HSV-tk and interleukin 18 on gliomaObjective: The purpose of the study was to observe antitumor effect of BMSCs after being transfected with IL-18. Furthermore, antitumor effect of BMSCs, after being co-transfected with HSV-tk and IL-18, were observed through in vitro and in vivo experiments.Methods: After being infected with LXSN/IL-18, BMSCs/IL-18 clone was selected followed by infected with AdCMV/tk, thus, BMSCs/IL-18 and BMSCs/IL-18/tk was harvested. RT-PCR were performed to detect the gene transcription of IL-18 and tk by BMSCs/IL-18 and BMSCs/IL-18/tk. Cells surface antigen and cell growth curve were examined of BMSCs/IL-18 and BMSCs/IL-18/tk through flow cytometry and MTT. Transwell experiment was performed to observe in vitro tropism for glioma. Glioma bearing rats were established and BMSCs/IL-18 and BMSCs/IL-18/tk were transplanted into contralateral hemisphere to observed their tropism for glioma in vivo. To observe by-stander effector of BMSCs/IL-18 and BMSCs/IL-18/tk, co-culture BMSCs/IL-18 and C6 or BMSCs/IL-18/tk and C6, and different effector target ratio and different GCV dose were introduced, and then, cell survival ratio and apoptosis ratio was examined by means of MTT, TUNEL and Annexin V. In vivo experiment was performed to detect BMSCs/IL-18 and BMSCs/IL-18/tk antitumor effect. ELISA test was performed to detect the effect of BMSCs/IL-18 and BMSCs/IL-18/tk on lymphocyte secretion of IFN-γ. Immunohistochemistry was introduced to detect microvessel density, lymphocyte infiltration and cell apoptosis inside glioma after being treated with BMSCs/IL-18 and BMSCs/IL-18/tk. MRI examination was performed to observe the volume change of intracranial glioma after treatment and survival time were recorded.Results: The results showed that the cell surface antigen and tropism for glioma in vitro was not changed after BMSCs were infected with LXSN/IL-18 or LXSN/IL-18 and AdCMV/tk. Cell growth curve showed that BMSCs/IL-18 and BMSCs/IL-18/tk had lower multiply speed than BMSCs. RT-PCR showed that BMSCs/IL-18 and BMSCs/IL-18/tk could transcript IL-18 gene stably and BMSCs/IL-18/tk could transcript tk gene long term. Immunohistochemistry showed that BMSCs/IL-18 and BMSCs/IL-18/tk could express IL-18 stably. In vitro experiment showed that BMSCs/IL-18/tk could display obviously by-stander effect, and with the increase of effectors, by-stander effect was more enhanced. BMSCs/IL-18 did not display by-stander effect. ELISA examination demonstrated that BMSCs/IL-18 and BMSCs/IL-18/tk could increase IFN-γsecretion of lymphocyte and increase IFN-γconcentration of glioma bearing rats serum. In vivo examination showed that BMSCs/IL-18 and BMSCs/IL-18/tk could increase infiltration lymphocytes number and decrease microvessel density and induced glioma cells apoptosis and glioma bearing rats survival time were prolonged obviously after being treated with BMSCs/IL-18/tk.Conclusions: After being transfected with IL-18, BMSCs/IL-18 could transcript and express IL-18 stably. After being transfected with AdCMV-tk, BMSCs/IL-18/tk could transcript tk and IL-18 stably. BMSCs/IL-18 and BMSCs/IL-18/tk displayed extensive tropisum for intracranial glioma and express BMSCs cell surface antigens and maintained high multiplication ability. BMSCs/IL-18/tk could induced obviously apoptosis of glioma in vitro and in vivo, and decrease microvessel density of intracranial glioma, and increase number of infiltration CD4+ and CD8+ lymphocyte inside intracranial glioma. BMSCs/IL-18/tk could prolong survival time of glioma bearing rats obviously. |
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