| BackgroundThe intestinal ischemia/reperfusion injuries (IRI) have attracted significant attention in clinical practice. The intestine has been recognized as one of the most fragile organs by IRI mechanism in the joint action of several factors. Serious intestinal dysfunction can result in systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). The effective treatments of intestinal ischemia/reperfusion injuries are still not available.Bone marrow-derived mesenchymal stem cells (BM-MSCs) are considered as multipotential progenitor cells of mesodermal origin due to their potential of self-renewal and differentiation. BMSCs have been regarded as a promising cell therapy in regenerative medicine due to its unique properties such as low immunogenicity, multipotential differentiation, great potential in proliferation. Animal studies have confirmed that BM-MSCs can colonize in gastrointestinal tract and participate in the restoration of intestine injury. The application of BM-MSCs in regenerative medicine involves two major mechanisms. It either directly differentiates into specific parenchymal cells or stimulates proliferation of parenchymal cells through paracrine mechanism. However, the precise mechanisms are still under investigation. The current study was designed to study the paracrine mechanism of BM-MSCs in the healing of intestinal IRI injury in rat.Objective1. The methods of isolation, culture, characterization and staining of rat BMSCs were established.2. The IRI rat model was employed, and pathophysiological indices were characterized to confirm the existence of intestinal dysfunction.3. The paracrine patterns of BM-MSCs in intestinal ischemia/reperfusion injuries were investigated.Methods1. BMSCs were isolated, cultured, characterized, and stained.2. The rat intestine IRI model was developed by clamping the superior mesenteric artery. The blood flow of the superior mesenteric artery was blocked for 20, 30, 40, and 50 minutes respectively in order to optimize the clamping schedule by detection of LPS and intestinal damage index in the animals.3. Intestinal epithelial mucosal extracts were obtained from IRI rat or normal rat. The extracts were added into the culture medium, and BM-MSCs were cultured with the conditioned medium respectively.4. The medium was collected at different time points. The levels of TGF-α, VEGF, FGF and IGF-1 in the medium were assayed by ELISA.Results1. The flusk-attached cells obtained from the whole bone marrow culture are consistent with the characteristics of mesenchymal stem cells;2. The rat intestinal IRI model with blood flow blockage of 30 minutes has lower mortality and sufficient injury.3. The contents of all the growth factors detected in conditioned medium with intestinal extracts were significantly higher than in normal medium. The levels of TGF-α, VEGF, FGF in culture medium containing IRI epithelium extracts were significantly higher than that in the medium containing normal intestinal epithelium extracts. The highest levels of VEGF, FGF, and IGF-1 were detected in the medium after 3-, 5-, and 5-days'culture respectively. The IGF-1 level in the medium was stable during the culture.Conclusion1. The whole bone marrow culture is an easy, reliable, and stable method to isolate BM-MSCs .2. Clamping superior mesenteric artery by 30 minutes is a reliable way to establish a rat intestine IRI model.3. BM-MSCs can secret TGF-α, VEGF, FGF, and IGF-1 in the presence of intestinal extracts from IRI rat in the culture. These factors may stimulate the differentiation and proliferation of intestinal epithelial cells through paracrine mechanism. The secretions of some growth factors are time-dependent. |
