Introduction: Previous works of our group demonstrated that cellular therapy with adipose tissue-derived stem cells (ASC) of frozen-thawed ovarian autografts preserves the graft morphology and didn’t enhance fibrosis[1]. However, new researchers are necessary to better evaluate the inflammatory response in this graft.
Materials and Methods: Twelve 12-week-old adult female Wistar rats were use. Frozen-thawed ovarian grafts of adult female rats were treated with rat ASC delivery in an acellular matrix (Gelfoam) immediately after an autologous retroperitoneal transplant (n=6). Controls received Gelfoam with vehicle (DMEM low glucose, n=6). The ovarian grafts were retrieved 30 days after transplantation. Quantitative gene expression (qPCR) for inflammatory cytokines (84 genes) was evaluated by Real Time-Polymerase Chain Reaction (RT-PCR). All reactions were performed in triplicate and controlled by negative RT (no enzyme) and no-template controls and all gene expression levels were normalized to the mean of the internal genes. A Fold Regulation reference value of -2 and +2 was considered for expression analyses. Further analyses were done by Ingenuity Pathway Analysis (IPAÒ) software in order to determine the main canoninal pathways and the expression values of the top molecules.
Results and Discussion: There were 36 genes up-regulated and three down-regulated. The ten top molecules up-regulated, in descending order, were CXCL6 (+13.1), IL21 (+10), LTA (+7.6), IL1A (+7.1), CXCL11 (+6.7), CCL19 (+6.4), CCR3 (+6.2), CCR6 (+5.4), LTB (+5.1) and CXCR2 (+5). The top canonical pathways involved were Differential Regulation of Cytokine Production in Macrophages and T Helper Cells (44%), Differential Regulation of Cytokine Production in Intestinal Epithelial Cells (43%), Role of Cytokines in Mediating Communication between Immune Cells (18%), Granulocyte Adhesion and Diapedesis (9%) and Agranulocyte Adhesion and Diapedesis (7%). The top diseases and biofunctions envolved were Inflammatory Response (38 molecules), Inflammatory Disease (37), Immunological Disease (37), Connective Tissue Disorders (31) and Skeletal and Muscular Disorders (31). Tox functions were little involved and represented mainly by hepatic mechanisms such as Increases Liver Damage (11 molecules), Hepatic Cholestasis (10), Hepatic Fibrosis (9), Liver Proliferation (9) and Increases Liver Hepatitis (6).
Conclusion: ASC therapy based on scaffold base-delivery strategy in rat ovarian grafts induces upregulation in 36 genes involved in inflammatory cytokines pathway. New researchers are necessary to evaluate if these findings could compromise the graft quality.
São Paulo Research Foundation (Process numbers 2010/17897-5 and 2012/09469-9); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for post-doctoral scholarship
References:
[1] Damous LL, Nakamuta JS, Saturi de Carvalho AET, Carvalho KC, Soares-Jr JM, Simões MJ, Krieger JE, Baracat EC. Scaffold base-delivery of adipose tissue-derived stem cells in rat frozen-thawed ovarian autografts: preliminary studies in a rat model. J Assist Reprod Genet. 2015 Aug;32(8):1285-94. doi: 10.1007/s10815-015-0527-x. Epub 2015 Jul 24.