Basic Medical Sciences Department, College of Medicine, University of Sharjah, Sharjah 27272, United Arab Emirates.
Department of Applied Biology, College of Science, University of Sharjah, Sharjah 27272, United Arab Emirates.
Int J Mol Sci. 2021 Mar 29;22(7):3514. doi: 10.3390/ijms22073514.
It is evident that depletion of interstitial cells and dysfunction of nitric oxide (NO) pathways are key players in development of several gastrointestinal (GI) motility disorders such as diabetic gastroparesis (DGP). One of the main limitations of DGP research is the lack of isolation methods that are specific to interstitial cells, and therefore conducting functional studies is not feasible. The present study aims (i) to differentiate telomerase transformed mesenchymal stromal cells (iMSCs) into platelet-derived growth factor receptor-α-positive cells (PDGFRα-positive cells) using connective tissue growth factor (CTGF) and L-ascorbic acids; (ii) to investigate the effects of NO donor and inhibitor on the survival rate of differentiated PDGFRα-positive cells; and (iii) to evaluate the impact of increased glucose concentrations, mimicking diabetic hyperglycemia, on the gene expression of neuronal nitric oxide synthase (nNOS). A fibroblastic differentiation-induction medium supplemented with connective tissue growth factor was used to differentiate iMSCs into PDGFRα-positive cells. The medium was changed every day for 21 days to maintain the biological activity of the growth factors. Gene and protein expression, scanning electron and confocal microscopy, and flow cytometry analysis of several markers were conducted to confirm the differentiation process. Methyl tetrazolium cell viability, nitrite measurement assays, and immunostaining were used to investigate the effects of NO on PDGFRα-positive cells. The present study, for the first time, demonstrated the differentiation of iMSCs into PDGFRα-positive cells. The outcomes of the functional studies showed that SNAP (NO donor) increased the survival rate of differentiated PDGFRα-positive cells whereas LNNA (NO inhibitor) attenuated these effects. Further experimentations revealed that hyperglycemia produced a significant increase in expression of nNOS in PDGFRα-positive cells. Differentiation of iMSCs into PDGFRα-positive cells is a novel model to conduct functional studies and to investigate the involvement of NO pathways. This will help in identifying new therapeutic targets for treatment of DGP.
显然，间质细胞耗竭和一氧化氮（NO）途径功能障碍是几种胃肠道（GI）动力障碍发展的关键因素，如糖尿病性胃轻瘫（DGP）。DGP 研究的主要限制之一是缺乏对间质细胞具有特异性的分离方法，因此进行功能研究是不可行的。本研究旨在：（i）使用结缔组织生长因子（CTGF）和 L-抗坏血酸将端粒酶转化的间充质基质细胞（iMSCs）分化为血小板衍生生长因子受体-α阳性细胞（PDGFRα阳性细胞）；（ii）研究一氧化氮供体和抑制剂对分化的 PDGFRα阳性细胞存活率的影响；（iii）评估模拟糖尿病高血糖的葡萄糖浓度增加对神经元型一氧化氮合酶（nNOS）基因表达的影响。使用补充有结缔组织生长因子的成纤维细胞分化诱导培养基将 iMSCs 分化为 PDGFRα阳性细胞。每天更换培养基 21 天，以维持生长因子的生物活性。进行基因和蛋白质表达、扫描电子和共聚焦显微镜以及几种标志物的流式细胞术分析，以确认分化过程。使用甲基噻唑基四唑细胞活力、亚硝酸盐测量测定和免疫染色来研究 NO 对 PDGFRα阳性细胞的影响。本研究首次证明了 iMSCs 向 PDGFRα阳性细胞的分化。功能研究的结果表明，SNAP（NO 供体）增加了分化的 PDGFRα阳性细胞的存活率，而 LNNA（NO 抑制剂）减弱了这些作用。进一步的实验表明，高血糖导致 PDGFRα阳性细胞中 nNOS 的表达显著增加。将 iMSCs 分化为 PDGFRα阳性细胞是一种进行功能研究和研究 NO 途径参与的新模型。这将有助于确定治疗 DGP 的新治疗靶点。
