Jensen Amanda R, Doster Dominique L, Hunsberger E Bailey, Manning Morenci M, Stokes Samantha M, Barwinska Daria, March Keith L, Yoder Mervin C, Markel Troy A
*Department of Surgery †Section of Pediatric Surgery ‡Department of Cellular and Integrative Physiology §Department of Medicine, Indiana Center for Vascular Biology and Medicine ¶Department of Pediatrics, Section of Neonatology ||Riley Hospital for Children at Indiana University Health #Indiana University School of Medicine, Indianapolis, Indiana.
Shock. 2016 Jul;46(1):75-82. doi: 10.1097/SHK.0000000000000571.
Intestinal ischemia can quickly escalate to bowel necrosis and perforation. Transplantation of stem cells presents a novel treatment modality for this problem. We hypothesized that: human adipose-derived stromal cells (hASCs) would increase survival and mesenteric perfusion to a greater degree compared with differentiated cellular controls following ischemic intestinal injury, and improved outcomes with hASC therapy would be associated with preservation of intestinal histological and tight junction architecture, and lower levels of systemic inflammation following intestinal injury.
hASCs and keratinocytes (differentiated cellular control) were cultured on polystyrene flasks at 37°C in 5% CO2 in air. Adult male C57Bl6J mice were anesthetized and a midline laparotomy performed. The intestines were eviscerated, the small bowel mesenteric root identified, and intestinal ischemia was established by temporarily occluding the superior mesenteric artery for 60 min with a noncrushing vascular clamp. Following ischemia, the clamp was removed, and the intestines were returned to the abdominal cavity. Before abdominal closure, 2 million hASCs or keratinocytes in 250 μL of phosphate-buffered saline (carrier for cells and control solution) were infused into the peritoneum. Animals were allowed to recover for 12 or 24 h (perfusion, histology, cytokine, and immunofluoresence studies), or 7 days (survival studies). Intestinal perfusion was assessed by laser Doppler imaging. Intestinal tissue segments were stained with hematoxylin and eosin, as well as antibodies for the tight junction protein claudin-1. Separate aliquots of intestine, liver, and lung tissue were homogenized and assessed for inflammatory cytokines via multiplex beaded assay.
Animals administered hASCs following intestinal ischemia and reperfusion (I/R) injury had significantly greater 7-day survival and better postischemic recovery of mesenteric perfusion compared with vehicle or keratinocyte therapy. hASCs also abated intestinal mucosal destruction, facilitated preservation of intestinal tight junctions, and decreased the systemic inflammatory response to injury.
Human adipose-derived stromal cells improved survival and mesenteric perfusion and attenuated the mucosal damage associated with intestinal I/R injury. hASCs should be considered as a plausible cell source for novel cellular treatment plans following intestinal ischemia.
肠道缺血可迅速发展为肠坏死和穿孔。干细胞移植为这一问题提供了一种新的治疗方式。我们假设：与缺血性肠损伤后的分化细胞对照相比，人脂肪来源的间充质干细胞（hASCs）能更大程度地提高生存率和肠系膜灌注，hASC治疗改善的结果将与肠道组织学和紧密连接结构的保存以及肠损伤后全身炎症水平降低相关。
将hASCs和角质形成细胞（分化细胞对照）在聚苯乙烯培养瓶中于37°C、5%二氧化碳的空气中培养。成年雄性C57Bl6J小鼠麻醉后行中线剖腹术。取出内脏，识别小肠肠系膜根部，用无损伤血管夹暂时阻断肠系膜上动脉60分钟以建立肠道缺血。缺血后，移除夹子，将肠管放回腹腔。在关闭腹腔前，将200万个hASCs或角质形成细胞溶于250μL磷酸盐缓冲盐水（细胞载体和对照溶液）中注入腹腔。动物恢复12或24小时（灌注、组织学、细胞因子和免疫荧光研究）或7天（生存研究）。通过激光多普勒成像评估肠道灌注。肠道组织切片用苏木精和伊红染色，以及用紧密连接蛋白claudin-1的抗体染色。分别取肠、肝和肺组织的等分试样匀浆，并通过多重珠分析法评估炎症细胞因子。
与载体或角质形成细胞治疗相比，肠道缺血再灌注（I/R）损伤后给予hASCs的动物7天生存率显著更高，缺血后肠系膜灌注恢复更好。hASCs还减轻了肠黏膜破坏，促进了肠道紧密连接的保存，并降低了对损伤的全身炎症反应。
人脂肪来源的间充质干细胞提高了生存率和肠系膜灌注，并减轻了与肠道I/R损伤相关的黏膜损伤。hASCs应被视为肠道缺血后新型细胞治疗方案的一种可行细胞来源。
