Bone Marrow Stroma Stem Cells Express Murine Interleukin-18 To Suppress The Growth Of Glioma In Rats

Glioma is the most common primary intracranial tumor in humans.Despite advances in surgical techniques and adjuvant radio- and chemotherapies,the prognosis for patients with gliomas remains poor.Its extensive ability to proliferate, invade,and "microsatellites"migrate to distal sites from the primary tumor mass is responsible for tumor recurrence.Therefore,new strategys should be taken to eliminate the viable intracranial neoplastic cells and viable glioma microsatellites that have migrated from the primary tumor mass after surgical resection.Following with the development and application of biotechnology,transgenic technology is becoming a new direction.Nowdays,most strategys of gene therapy are consist of suicide gene,immunogene and destroy tumor vessel or combine with them.Each one of them have their own merits ang defects,but the key of successful carry out gene therapy are objective gene can migrate to the tumor mass and eliminate the relic tumor cells by persist and effectively expressing the objective gene.Viral vectors can not migrate to the tumor bed and carry little dosage of gene are responsible for the unsatisfactory results in the present days.So researchers are exploring a new promising strategy to overcome glioma using gene modified stem cells. Recently,some researches have shown that bone marrow stroma stem cells(BMSCs) can be promoted proliferation by secrecting cytokine in the microenvironment of tumors,BMSCs transfected with exogenous gene were mainly localized in the borderline of tumors and stable expressed the objective gene.The migrate ability of BMSCs were associated with the degree of tumors and can tracing the microsatellites;it more easier for BMSCs to migrate to the tumor tissue when transplanted into the brains,BMSCs can differentiate into functional neurocyte by synthesis and secreting various kinds of somatomedin and interact with the damaged brains to fulfil self reparation and reconstruction after operation.One of the secreting nerve growth factors can not only inhibit the apoptosis of neurons,but also induce glioma cell differentiation and direct repress the growth of glioma.Apart from these, BMSCs can secreting considerable amount of angiopoietin-1 which can suppress the growth of glioma and prevent the leakage of tumor vessel to release the oedema of brains.Besides,it is easy for BMSCs to combine with host cells and have little immunological rejection or oncogenicity.In a word,it is becoming a hot spot and a promossing way by using BMSCs as a vector for gene therapy.Interleukin-18 is one of a new cytokines were found in recent years,possessed many kind of biologic activity which can induce generate interferon-γ,play an important role in primarily and secondly immunization of tumors,compared with other cytokines,Interleukin-18 can more effectively anti-tumor and have less side effects.Interleukin-18 can promote T helper cell-1(Th1) multiplication by stimμlating Th1 cells to secret Interleukin-12(IL-2),Granulocyte-macropHage colony stimutaing factor(GM-SCF),interferon-γ(INF-γ);and can make new born CD4+T change into Th1 and enhance the cytotoxic effect of Th1 via Fas ligand,and promote the cytotoxic effect of nature killer cells via Fas-Fas-1 ligand or enhance the traditional kill activity of nature killer cells via porforin,furthermore,IL-18 also can repress the formation of tumor vessel.A lot of animal models of tumor have confirmed that IL-18 can effectively suppress the growth of primary tumor and metastasis by systematic using.In the present study,we utilize the tumors tropism of BMSCs and the anti-tumor effects of IL-18,construct the replication defective adenovirus vector expressed mIL-18 transfected into BMSCs and then transplanted into the Intracalvarium of glioma model rats which were established by 9Lcells/Fisher344 rats.Observe the effects of BMSCs express mIL-18 suppress the growth of glioma in rats and aim to explore a new strategy for the treatment of glioma.Chapterâ… :Construction and package of recombinate adenovirus vectors expressing mIL-18Objectives:Construct mIL-18 expressing recombinate adenovirus vector and undertake package,assessment,purification,determine the titre,for establish the foundation of subsequent research.Methods:Restriction enzyme Hindâ…¢and EcoRâ…¤digested the eukaryotic expression plasmid Pcr3.1uni-mIL-18 and shuttle plasmid pAdTrack-CMV respectively.The resulting fragments were separated by agarose electrophoresis.Subsequently,two DNA fragments were religated using T4 DNA ligase.The new plasmids were identified by gene sequencing and Restriction enzyme Hindâ…¢and EcoRâ…¤. pADTrack-IL18 was then cut by Pmeâ… and the fragment of interest cloned by homologous recombination in the competent BJ5183 cells,which were prepared by transforming pAdEasy-1 bacteria using the way of Calcium chloride.And then packaged using human embryonic kidney 293(HEK293) cells,after linearization by Pacâ… .The recon were identified by polymerase chain reaction(PCR),purified by cesium chloride(CsCL),determine the viral titre by double dilution. Results:Successfully constructed the replication defective adenovirus vector pAd-mIL-18 and fulfilled the package by HEK293 cells.The virus contained the full sequence of mIL-18 and the virus titre were 2.45×10¹⁰PFU/ml.Conclusions:(1) Construct replication defective adenovirus vector by homology recombination in bacterium using "two footwork" have the merits of high achievement ratio, convenient,shortcut,and save time.(2) The technology of packaging recombine adenovirus is mature,stable and reliable using HEK293 cells.(3) Double dilution are both easy and reliable for determine the titre of virus;and the virus purified by CsCL can meet the experiment requirement both in vitro and in vivo.Chapterâ…¡,Proliferate and identify bone marrow stroma stem cells of Fisher 344 rats in vitroObjectives:To establish a simple and feasible method to culture and proliferate bone marrow stroma stem cells of Fisher 344 rats in vitro.To analyze their biological characterizations such as cell phenotype,generation cycle,growth curve, ultramicrostructure.Methods:Bone marrow stroma stem cells of Fisher 344 rats were isolated by gradient density centrifugation and cultured in conditioned medium.Morphological observations were performed with inverted phase contrast microscope,growth curves of BMSCs were drawn by MTT assay,cell phenotype and generation cycle were detected by flow cytometry,cell ultramicrostructures were determined by electron microscope.Results:Higher purity of BMSCs could be isolated by gradient density centrifugation. The typical morphology of BMSCs was with long spindle shape,little volume and large caryoplasmic ratio.In addition,most of them grew adherently,with obvious syntropy,and arrayed in whirlpool.The cell survival ratio was above 95%.The positive expression ratio of cell phenotype was various as following respectively: CD29,95.3%;CD34,1.8%;CD44,94.7%;CD45,0.8%;CD71,96.2%;The grew curve of BMSCs was "S" shaped.The ratio of cells in S+G2+M stage was 17.1%by analysis of generation cycle,which demonstrated that only a part of BMSCs remained proliferation ability on a certain stage.Many evections and pores were observed on BMSCs' surface,while abundant cytoplasm,prosperous rough endoplasmic reticulum and plenty of protein excretion were found inside BMSCs which also demonstrated the proliferation ability of BMSCs was strong.Conclusions:(1) Higher purity of BMSCs can be isolated by gradient density centrifugation.(2) They can express the phenotype of BMSCs instead of haemopoietic stem cell.(3) Most of them in the resting state,but BMSCs have the strong reproductive activity so they can be generous cultured and Proliferated in vitro.Chapterâ…¢,The effects of suppressed the growth of glioma by BMSCs expressing Ad-mIL-18 in rat modelsObjectives:To observe the efficiency of infection,influence of the cells' biology character and objective gene expression of BMSCs by replication defective adenovirus Ad-mIL-18,then explore the effects and mechanism of suppress the growth of glioma using BMSCs-mIL-18.Methods:BMSCs were infected with Ad-mIL-18 at various multiplicity of infection (MOI):control(a),50(b),100(c),200(d),the infection efficiency were performed by microscope fluorescence;the proliferation were determined by MTT,BMSCs differentiated into neuron like cells were induced byβ-mercaptoethanol(β-ME), mIL-18 mRNA and cytokine expression were meseaured by RT-PCR and ELISA respectively 3days after the infection.Tumor-bearing rats were random divided into normal saline group(n=9),BMSCs transplantation group(n=10),BMSCs-GFP transplantation group(n=10) and BMSCs-mIL-18 transplantation group(n=10),after 3 days of the 9L/Fisher 344 rats glioma models were established by stereotaxis equipment.And then treated with intratumoral inoculations of saline(10μl;n=9); 1×10⁶ BMSCs(n=10);1×10⁶ BMSCs-GFP(n=10),1×10⁶ BMSCs-mIL-18(n=10) in 10μl of virus-free DMEM.Subsequently,explored the neuroethology change, survived time,tumor volume were detected using magnetic resonance imaging (MRI),mIL-18 mRNA and cytokine expression in tumors mass were also meseaured by RT-PCR and ELISA respectively,CD4⁺,CD8⁺ cells of the brains were examined by immunohistochemical method two weeks latter of the treatment.To explore whether the immunological memory effects were formed or not,rats survived more 2 month in the group treated with BMSCs-mIL-18 were inoculated with the same number of 9L cells into the same location.Results:Green fluorescence can be detected after 24h of BMSCs infected with Ad-mIL-18, the infection rate were various according with the MOI:a(0),b(55%),c (85%),d(88%),but occurred cell pathology phenomenon in d group in 7days,the strongest expression of fluorescence also in 7days,and the fluorescent can still be seen in 28days.When the dose of MOI reached 200,the growth suppression of BMSCs were appeared.β-mercaptoethanol can induce BMSCs differentiated into neuron like cells.BMSCs can express mIL-18 mRNA after infected with Ad-mIL-18 in vitro,two weeks latter the content of cytokine were various as follows:group a(MOI=50;45.6±5.3ng/10⁶cells/72h);group b(MOI=100;89.2±4.9ng/10⁶cells/72);group c(MOI=200;90.8±4.1ng/10⁶cells/72h).There were significant difference when group a versus group b and c(P＜0.01,respectively);however there were not significance difference when group b versus group c(P=0.488).The contents of mIL-18 in vivo were as follows:saline group 1.6±0.3pg/mg;BMSCs group:1.8±0.1pg /mg;BMSCs-GFP group:1.8±0.1pg/mg;BMSCs-mIL-18 group 53.84±3.3pg/mg. BMSCs-mIL-18 group versus saline group,BMSCs group,BMSCs-GFP group(P＜0.01 respectively);saline group versus BMSCs group(P=0.755),saline group versus BMSCs-GFP group,P=0.740).The results of survival analysis shown that survived time was significantly longer in the BMSCs-mIL-18 treated group than other groups (P＜0.01,BMSCs-mIL-18 versus BMSCs-GFP;P＜0.01,BMSCs-mIL-18 versus BMSCs-mock;P＜0.01,BMSCs-mIL-18 versus saline;P=0.919,BMSCs-GFP versus BMSCs-mock;Log-rank),with 40%of treated rats survived beyond 60 days. The results of tumor volume suggested that BMSCs-mIL-18 group versus normal saline group,BMSCs group,BMSCs-GFP group respectively(P＜0.01) at 7days and 14days time points;however there were not significant difference when BMSCs group versus BMSCs-GFP group(in 7days P=0.725;14days P=0.620).at 21days, all rats were died only in normal saline group,and there were significant difference when BMSCs-mIL-18 group versus BMSCs and BMSCs-GFP group respectively(P＜0.01);however there were not significant difference when BMSCs group versus BMSCs-GFP group(P=0.839);in 28days,rats only in BMSCs-mIL-18 group were survival,and the volume of tumors were still relative small,were 51.30±2.56 mm³ in 28days and 59.57±2.50 mm³ in 56days.The results of immunohistochemical shown that a large number of CD4⁺ and CD8⁺ cells were detected in and around the tumor mass,however there were negligible CD4⁺ and CD8⁺ cells in BMSCs-GFP groups.The BMSCs-mIL-18 treated survivors remained alive beyond 90 days after rechallenge.Conclusions:(1) replication defective adenovirus vectors pAd-mIL18 can effectively infected BMSCs and there not obvious influence of the cells' biology character.(2) BMSCs infected with pAd-mIL-18 can not only express the mRNA of mIL-18, but also can secret cytokine.(3) The 9L/Fisher344 rats glioma models established with Horseley-Clarke technique are reliable and suit for the research of gene and immune therapy.(4) transplantation of BMSCs-mIL-18 can effectively suppressed the growth of Fisher344 rats glioma.(5) CD8⁺T,CD4⁺T lymph cells infiltration maybe is one of the mechanism of BMSCs-mIL-18 suppressed the growth of Fisher344 rats glioma.