Diabetes Mellitus Type ۲ Sera Increased Exosome Secretion Pathway Activity in Human Marrow MesenchemalStem Cell In Vitro

Background: Exosomes, nano-sized cell-derived membranevesicles (۳۰-۱۲۰ nm), secreted by many cell types. It was recentlydemonstrated that exosomes contribute in cell-to-cellcommunication by transfer of bio-materials. Exosomes participatein physiological and pathological processes. Previous studiesdescribed adverse effects of diabetes mellitus type ۲ (DM۲)on stem cell function and biology. It was revealed that humanmarrow mesenchemal stem cells (hMSCs) were more sensitiveto a persistent diabetic condition which associated with changesin their secretome. In the present study we aimed to study the effect of serum from type ۲ diabetic mellitus patients on theexosome secretion pathway in human mesenchymal stem cellsin vitro.Materials and Methods: For in vitro assays, hMSCs wereclassified into two groups as follows; Control: cells receivedDMEM/LG and ۱۰% sera from healthy subjects; Diabeticgroups treated with DMEM/LG and ۱۰% diabetic sera over aperiod of ۷ days. To investigate expression of Alix, CD۶۳, Rab۲۷a,Rab۲۷b, and Rab۸b genes, we performed real -time PCR(Rotor-Gene ۳۰۰۰, Corbett Robotics) using SYBR Green PCRMaster Mix (Cat no: YT۲۵۵۱, Iran). To further confirmation ofCD۶۳ level, protein level of CD۶۳ was examined using westernblotting method and densitometry analysis was performedwith ImageJ software ver.۱.۴۴p (NIH). Furthermore, exosomesecretion quantified by acetylcholinesterase (ACE) assay usingkit (Cat No: BXC۰۸۰۱; Biorexfars). Choline esterase activitywas calculated by following formula; Activity (U/l) = ΔAbs/min × ۶۵۸۰۰. Data are expressed as mean ± SD. Student’s t-testwas used to calculate the significance of differences betweengroups. Values of P<۰.۰۵ were considered statistically significant.Results: Compared to control group, the mRNA level of Alix,CD۶۳, Rab۲۷a, and Rab۸b genes in diabetic group was significantlyincreased (pcontrol versus diabetic <۰.۰۱). Furthermore,mRNA level of Rab۲۷b was enhanced as compared to controlgroup (P<۰.۰۵). Western blotting analysis showed that the proteinlevel of CD۶۳ was significantly enhanced in diabetic group(pcontrol versus diabetic <۰.۰۱). Acetylcholinesterase activitywas significantly increased in diabetic group (pcontrol versusdiabetic <۰.۰۰۱).Conclusion: The present study provides the evidence of a specificmechanism that stem cell biology was altered throughexosome signaling pathway in diabetic mellitus. As a result,we consider diabetic hMSC release more exosomes throughupregulation of genes involved in exosome secretion pathway.

کلیدواژه ها:

Exosome ، Human Marrow Mesenchemal Stem Cell ، Dibetes Mellitus ، Rab