| Malignant gliomas are the most common primary brain tumors and represent asignificant challenge in the field of neuro-oncology.. Because of their extraordinarymigratory ability and readily penetrate adjacent normal tissue, glioma cells often formtumor microsatellites at distal sites from the main tumor foci. These tumor cellinfiltrations eventually serve as reservoirs for tumor recurrence, which in turncontributes to the lethality of patients with malignat gliomas. Thus, to target thesetumor satellites is critical for the successful therapeutic strategy of gliomas.Because of the chemotaxic characteristics of stem cells, there are some reports onthat NSCs(Neural stem cells) and BMSCs(bone marrow-derived mesenchymal stemcells) have been used as vectors for the immunogene therapy of gliomas in recentyears. However, BMSCs are more readily accessible than NSCs for their unlimitedutilization and from logistic and ethical considerations. BMSCs have been geneticallymodified to express interleukin (IL)-2 or IFN-βwith potent tropism for disseminatingglioma cells and strong antitumor effects in vivo and/or in vitro. All of the aboveattempts, nevertheless, did leave a number of relevant questions, such as theirmigratory characteristic and mechanism, the genomic stability of BMSCs after a longperiod culture and the therapeutic efficacy against glioma as a gene delivery vector.Herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) suicide genetherapy system has been considered as one of the most promising therapeuticstrategies for malignant gliomas theorectically, it can be used for various malignanttumors no matter what kinds of molecular pathological changes they have.HSVtk/GCV viral-directed enzyme prodrug gene therapy causes potent,tumor-selective cytotoxicity. The passage of toxic molecules from HSV-tk~+ cells to neighboring HSV-tk~- cells during GCV therapy is one mechanism that may accountfor this "bystander" cytotoxicity. Even though a failure has been reported from clinicalphaseⅢtrial, but the most important bottleneck of its therapeutic effect is found tobe the limited gene delivery of retrovirus. So in the present study, BMSCs were usedas HSV-TK gene carrier for their chemotactic characteristics. Meanwhile, connexin 43was transfected into glioma cells with lowered expression of connexin for restorationof gap junction intercellular communication and enhancement of bystander effect.Taking the above considerations as a starting point, this study was divided into 3parts. In the first part, Rat BMSCs was obtained by complete marrow cells culture andidentified by their surface antigens CD29(99.67%), CD71 (99.13 %), CD90(98.91%),CD31(1.29%), CD34(3.32%), CD45(3.18%). After a long period culture of BMSCs,soft agar colony assay, serum-dependent assay and cell cycle analysis were used toexamine the stability and biosafety of BMSCs. The results showed that there werestable genotypes in BMSCs and malignant transformation was not found.In the second part, the capability of tracking glioma of rat BMSCs was studiedboth in vitro and in vivo with some different but complementary routes and methods.Three models in vitro, including a central cylinder cell cocultured system, a spheroidcoculture system in matrigel and a transwell migration model, were used to explorethe migratory ability of BMSCs toward glioma. The results from In vitro showed thatalmost every glioma cell line owned its potentiality to induce migration of BMSCs.The efficacy of each glioma cell line, however, was different. Further more, thechemotactic potentiality of BMSCs may be derived from the secreted cytokines ofglioma cells. In vivo, a rat C6 cerebral glioma model was established, followed by aninjection of BMSCs pre-labeled with BrdU into the contralateral hemisphere, carotidartery and femoral vein. The tumor-containing samples were prepared histologicallyto find the distribution pattern of BMSCs in glioma model. The results from in vivostudy displayed the BMSCs gathered mainly in the brain tumor area, and the majorityof BMSCs gathered on the border zone between glioma and normal brain parenchyma including invasive disseminated area and small isolated tumor foci from the mainglioma bed. To illustrate the possible mechanism for the tropism in BMSCs, theexpression of some possible chemotatic factors, CXCR-4, MMP2 and MMP9, wasdetected and a stable expression of these proteins was exihibited from 4 to 40 passageof cultured BMSCs.In the third part, an adenovirus(Ad) vector containing HSVtk gene was transfectedinto rat BMSCs. Meanwhile, a full-length cDNA encoding connexin 43 wastransfected into C6 rat glioma cells. It is supposed that, the GJIC, both in vitro and invivo, can strengthen efficiently the bystander effect of HSVtk/GCV treatment. In vivo.The rats were divided into four groups: C6, CX43-C6, TK-BMSC/C6 andTK-BMSC/CX43-C6. During the observation period of 6 weeks by MRI, the tumorvolumes in CX43-C6 groups were smaller than those in C6 groups, and much smallerin TK-BMSC/C6 and TK-BMSC/CX43-C6 groups than the above groups at the sametime point, and these also contributed to prolonged survival of glioma-bearing rats.The cell proliferation activity indicated by PCNA immunohistochemical staining andapoptosis examined among tumors of all groups further suggested thatTK-BMSC/CX43/GCV system was the best strategy for gene therapy of gliomas dueto their specific killing of tumor satellites at distal sites from main tumor foci.The new findings based on our preliminary study indicate a promising newapproach of using isolated tumor-tropic BMSCs as a mediator for suicide genetherapy of malignat gliomas, especially for disseminated tumor cells around the tumorfoci. With their clinical accessibility, potential for in vitro expansion and modification,and demonstrable migratory activity and tumor tropism, usage of BMSCs may be animportant step forward in the improvement of gene delivery system for certainstrategies of gene therapy of malignant gliomas. However, there are still someproblems exist which should be further investigated.
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