Bone Mesenchymal Stem Cells with Cytosine Deamimase Gene Transducted in Migrat to Glioma in Vivio
|School||Dalian Medical University|
|Keywords||Cytosine deamimase gene Bone mesenchymal stem cells Glioma|
Objective: More and more studies show that bone mesenchymal stem cells(MSCs) in the body has powerful migration force and tumor tendency. But few reported the migration force and tumor tendency of bone mesenchymal stem cells(MSCs) carrying exogenous genes. Cytosine deamimase gene(CD gene) is a kind of tumor therapy genes widely concerned recent years. This experiment structured SD rats intracranial C6 glioma models, injected bone mesenchymal stem cells with CD gene transducted in(MSCs-CD/eGFP) and stamped by green fluorescent protein through tumor-burdened side internal carotid artery , the opposite hemisphere and ipsilateral gliomas inside into SD rats intracranial C6 glioma models, taked the brain after fixed by perfusion, sliced brain tissues into sections, observe the distribution of MSCs-CD/eGFP marked by eGFP in the tissue slice under fluorescence microscope, exploring the different glioma tendency of the three transplantation ways, and exploring a kind of idea transplant approach for clinical application.Methods: Structured SD rats intracranial C6 glioma models by intracranial stereotactic inoculation method. Preoperative 12 hours keeped the mouses off food and water, anesthetized rats with 10% hydration chlorine aldehyde (3ml/kg) by intraperitoneal injection, fixed the heads by otaxic instrument, symmetrically fixed by inserting two auricular needles into external auditory canals deeply, cutted the head hair, paved hole cloth after sterilized by iodine and alcohol, cutted the scalp about 1.5cm longitudinally along the sagittal line, separated to exposure parietal(cutted the scalp breadthwise after about 1.5cm to the ligature of two palpebral fissure, exposured bregma), fixed position according to coordinate, drilled by cranial drill, aperture 2.0 mm, go right to the endocranium. The depth of needling microsyringe was 60.mm, then return needled 1.0 mm. Injected 1×10~6cells/15μl into target section within 10min, retaining needle 5min, made the cells fully deposited. Slowly pulled needle, closed bone hole with bones wax, washed operative field with physiological saline, sutured scalp incision with Suture-line One.6days after models structured, selected 8 randomly from the 24 rats, anesthetized rats with 10 % hydration chlorine aldehyde (3ml/kg) by intraperitoneal injection, separately MRI scanned at sagittal view, coronal view and horizontal position, enhanced scan by injecting 0.2mmol/kgGadodiamide through caudal vein after conventional scanning, observed the tumor growth. Tumor age 7 days, respectively inoculated 1×10~6MSCs - CD/eGFP from tumor-burdened side internal carotid artery (group A), the opposite hemisphere(group B) and ipsilateral gliomas inside (group C). Eight days after cells inoculation, taked the brain after fixed by perfusion, sliced brain tissues into sections, observe the distribution of MSCs-CD/eGFP marked by eGFP in the tissue slice under fluorescence microscope.Results: 6 days after SD rats intracranial C6 glioma models structured successfully, we discovered round or oval tumor masses formatted in the intracranial C6 glioma cells inoculation area by MRI scan, showing invasive growth, edema existed around partial tumor masses, had obvious mass effect, some tumor had necroticareas, tumor masses exhibited heterogeneous enhancement. Inoculated MSCs - CD/eGFP at the 7th day after SD rats intracranial C6 glioma models structured successfully, at the 8th day after inoculated MSCs-CD/eGFP, obtained the brain after fixed, sliced the brains into sections, observed under the fluorescence microscope, MSCs - CD/eGFP distributed in vivo and the edge spot of the gliomas whether from the tumor-burdened lateral internal carotid artery, the contralateral brain caudate nucleus or the ipsilateral gliomas, while no MSCs - CD/eGFP in fine lateral brains and normal brains. Injected MSCs - CD/eGFP from the tumor-burdened lateral internal carotid artery, MSCs - CD/eGFP distributed in vivo of the gliomas uniformly, rarely migrated to the junction of the tumor and normal tissues. Injected MSCs-CD/eGFP into brains, whether from the contralateral brain caudate nucleus(Fig4) or the ipsilateral gliomas, MSCs-CD/eGFP distributed in vivo and the edge spot of the gliomas, mainly distributed in glioma tissues’irregular edges, as bag round shape. Injected from the contralateral brain, MSCs - CD/eGFP mainly distributed in the proximal ends of the vaccination site of tumors, distal ends rare.Conclusions: the rate of successfully structuring SD rats intracranial C6 glioma models was 100%. Bone mesenchymal stem cells with CD gene transducted in(MSCs-CD/eGFP) still have powerful migration force and tumor tendency in vivo, the MSCs-CD/eGFP mainly distributed at the inside or the edge of the tumor tissues, was a kind of ideal cell carriers for researching glioma clinical gene therapy, laid a good foundation for further discussing the effect of bone mesenchymal stem cells with CD gene transducted in for restraining C6 gliomas cells in vivio.