ABSTRACT
Objetivo. Desarrollar un modelo animal de neuroblastoma (NB) que posibilite estudios relacionados con la inmunidad tumoral. Material y métodos. Se utilizaron dos tipos de células NB. La línea 36769 procedía del ratón TH-MYCN+ en el que la sobreexpresión del gen MYCN está gobernada por el promotor de la tirosín hidroxilasa de rata. La línea 4040 procedía de ratones TH-MYCN+/ALK+, que además expresan una mutación activadora del gen ALK. De cada tipo celular se implantaron 1x106 neurosferas en ratones 129/SVJ (mismo fondo genetico que los donantes, n=8), mediante inyección ortotópica en glándula suprarrenal izquierda por abordaje intraperitoneal, a través de laparotomía transversa supraumbilical. Se realizó seguimiento clínico diario postquirúrgico de los animales hasta su sacrificio a las 4 semanas. La presencia de tumor se confirmó macroscópicamente. La pieza tumoral se extirpó y se procesó para estudios de inmunidad celular y mediadores moleculares de tolerancia. Se investigó la existencia de metástasis por citometría de flujo en bazo, médula ósea y sangre periférica. Resultados. 1) En todos los ratones trasplantados se generó NB ortotópico. 2) La pieza tumoral se encontró infiltrada por diversas subpoblaciones inmunes, con inmunofenotipo efector, regulador y supresor, similar a la situación descrita en los NB humanos. Además, los mediadores moleculares del microambiente apuntan a un estado de tolerancia protumoral. Conclusiones. La implantación ortotópica de neurosferas NB en ratones singénicos nos ha permitido generar un modelo de NB en el que ha sido posible estudiar la inmunidad tumoral
Aim. To develop a NB animal model which makes possible studies related to tumor immunity. Materials and methods. Two types of NB cells were used. Cell line 36769 was derived from TH-MYCN+ mouse in which overexpression of the MYCN gene is governed by rat tyrosine hydroxylase promotor. Cell line 4040 was derived from TH-MYCN/ALK mice, which in addition express an activating mutation of ALK gene. For each cell type, 1x106 neurospheres were implanted in 129/SVJ mice (with the same genetic background as donors, n=8), via orthotopic injection in the left suprarenal gland by intraperitoneal approach, through a transverse supraumbilical laparotomy. Daily postsurgical clinical follow-up of the animals was done until they were sacrificed at four weeks. The tumor presence was macroscopically confirmed. The tumoral sample was excised and was processed for cellular immunity and molecular tolerance mediators studies. The existence of metastasis was investigated by flow cytometry in the spleen, bone marrow and peripheral blood. Results. 1) Orthotopic Neuroblastoma was generated in all the transplanted mice. 2) The tumors were infiltrated by several immune subpopulations, with effector, regulatory and suppressor inmunophenotype. This was similar to the inmunophenotype described in human NB. Furthermore, the molecular mediators of the environment point to a state of protumoral tolerance. Conclusion. The orthotopic implantation of NB neurospheres in syngeneic mice has allowed us to generate a NB model in which it has been possible to study the tumor immunity
Subject(s)
Humans , Mice , Animals , Neuroblastoma/immunology , Sympathetic Nervous System/immunology , Adrenal Gland Neoplasms/immunology , Disease Models, AnimalABSTRACT
Peripheral nerves have the intrinsic capacity of self-regeneration after traumatic injury but the extent of the regeneration is often very poor. Increasing evidence demonstrates that mesenchymal stem/stromal cells (MSCs) may play an important role in tissue regeneration through the secretion of soluble trophic factors that enhance and assist in repair by paracrine activation of surrounding cells. In the present study, the therapeutic value of a population of umbilical cord tissue-derived MSCs, obtained by a proprietary method (UCX(®)), was evaluated on end-to-end rat sciatic nerve repair. Furthermore, in order to promote both, end-to-end nerve fiber contacts and MSC cell-cell interaction, as well as reduce the flush away effect of the cells after administration, a commercially available haemostatic sealant, Floseal(®), was used as vehicle. Both, functional and morphologic recoveries were evaluated along the healing period using extensor postural thrust (EPT), withdrawal reflex latency (WRL), ankle kinematics analysis, and either histological analysis or stereology, in the hyper-acute, acute and chronic phases of healing. The histological analysis of the hyper-acute and acute phase studies revealed that in the group treated with UCX(®) alone the Wallerian degeneration was improved for the subsequent process of regeneration, the fiber organization was higher, and the extent of fibrosis was lower. The chronic phase experimental groups revealed that treatment with UCX(®) induced an increased number of regenerated fibers and thickening of the myelin sheet. Kinematics analysis showed that the ankle joint angle determined for untreated animals was significantly different from any of the treated groups at the instant of initial contact (IC). At opposite toe off (OT) and heel rise (HR), differences were found between untreated animals and the groups treated with either uCx(®) alone or UCX(®) administered with Floseal(®). Overall, the UCX(®) application presented positive effects in functional and morphologic recovery, in both the acute and chronic phases of the regeneration process. Kinematics analysis has revealed positive synergistic effects brought by Floseal(®) as vehicle for MSCs.
