炎症预激活骨髓间充质干细胞条件培养基调控急性辐射损伤小肠炎症反应的作用
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摘要
背景:骨髓间充质干细胞具有强大的免疫调节和抗炎能力。前期实验发现,炎症预激活骨髓间充质干细胞条件培养基可促进急性辐射小肠损伤结构和功能的修复,但其对急性辐射小肠损伤炎症反应的作用机制尚未完全明确。目的:探讨炎症预激活骨髓间充质干细胞条件培养基对急性辐射小肠损伤炎症反应的影响。方法:随机将80只SD大鼠(中山大学北校区实验中心提供)分为对照组、单纯辐射损伤组、辐射损伤+正常间充质干细胞条件培养基治疗组(IR+MSC-CM~(IEC-6(NOR))组)、辐射损伤+辐射炎症预激活间充质干细胞条件培养基治疗组(IR+MSC-CMIEC-6(IR)组),每组20只。使用尾静脉联合腹腔给药,辐射后第1, 3, 5, 7天,取小肠组织行苏木精-伊红染色观察小肠组织学变化,取小肠组织及血清采用ELISA检测炎症因子水平,辐射后第3天取小肠肠系膜淋巴结组织用流式细胞仪检测CD4~+Foxp3~+Treg细胞数量百分比。结果与结论:①与甲单纯辐射损伤组和正常间充质干细胞条件培养基治疗组相比,辐射炎症预激活间充质干细胞条件培养基组小肠上皮绒毛及隐窝结构明显改善;小肠组织中前炎症因子如白细胞介素1β、白细胞介素6和肿瘤坏死因子。水平显著减少,抗炎因子白细胞介素10水平显著升高;血清中前炎因子ActivinA、白细胞介素1β、白细胞介素6和肿瘤坏死因子α水平显著减少,抗炎因子白细胞介素10水平显著升高,以辐射后第3天最为明显;小肠肠系膜淋巴结中CD4~+Foxp3~+Treg细胞数量百分比显著升高,差异均有显著性意义;②上述结果表明,辐射炎症预激活间充质干细胞条件培养基能调控前炎因子和抗炎因子的平衡,增加小肠肠系膜淋巴结中CD4~+Foxp3~+Treg细胞数量,抑制急性辐射小肠损伤全身水平及小肠黏膜的炎症反应,促进小肠黏膜损伤的修复。
BACKGROUND: Bone marrow mesenchymal stem cells(MSCs) have powerful anti-inflammatory and immunomodulatory properties. Our previous experiments have demonstrated that conditioned medium from bone marrow MSCs with inflammatory activation(MSC-CM~(IEC-6(IR)))could improve the structural and functional repair of the small intestine after acute radiation-induced intestinal injury, but its effects on the inflammatory response to acute radiation-induced intestinal injury have not been elucidated.OBJECTIVE: To investigate the effects of MSC-CM on the inflammatory response to acute radiation-induced intestinal injury.METHODS: Eighty Sprague-Dawley rats(provided by the experimental center of the North Campus of Sun Yat-Sen University in China) were randomly divided into four groups(n=20/group): control group, irradiation(IR) group, irradiation~+MSC-CM~(IEC-6(NOR)) group(IR~+MSC-CMIEC-6(NOR group) and irradiation~+MSC-CM~(IEC-6(IR)) group(IR~+MSC-CM~(IEC-6(IR)) group), followed by continuous administration via the tail vein and abdominal cavity. Intestinal tissue and serum samples were collected 1, 3, 5, 7 days after radiation for hematoxylin-eosin staining and analysis of the concentrations of inflammatory factors using ELISA. Mesenteric lymph nodes were collected 3 days after radiation for analysis of the percentage of CD4~+Foxp3~+Treg cells using flow cytometry.RESULTS AND CONCLUSION: Compared with the IR group and IR~+MSC-CM~(IEC-6(NOR)) group, the intestinal structure(small intestine epithelial villi and crypt) was significantly improved and the pro-inflammatory factors such as interleukin-1 β, interleukin-6 and tumor necrosis factor-a in the small intestine were significantly decreased in the IR~+MSC-CM~(IEC-6(IR)) group, whereas the level of anti-inflammatory factor interleukin-10 was significantly increased. Similarly, the levels of pro-inflammatory factors such as Activin A, interleukin-1β, interleukin-6 and tumor necrosis factor-a in the serum samples were significantly decreased and the level of anti-inflammatory factor IL-10 was significantly increased. The levels of inflammatory factors changed most significantly at 3 days after irradiation. The number of CD4~+Foxp3~+Treg cells in mesenteric lymph nodes in the IR~+MSC-CMIEO-6(IR) group was significantly higher than that in the control group and IR~+MSC-CMIEC-6(IOR)group. These results suggest that MSC-CM~(IEC-6(IR)) can regulate the balance of pro-inflammatory factors and anti-inflammatory factors, and increase the number of CD4~+Foxp3~+ Treg cells in mesenteric lymph nodes, thus inhibiting the inflammatory response at systemic and mucosal levels in acute radiation-induced intestinal injury and promoting the repair of small intestinal mucosal injury.
BACKGROUND: Bone marrow mesenchymal stem cells(MSCs) have powerful anti-inflammatory and immunomodulatory properties. Our previous experiments have demonstrated that conditioned medium from bone marrow MSCs with inflammatory activation(MSC-CM~(IEC-6(IR)))could improve the structural and functional repair of the small intestine after acute radiation-induced intestinal injury, but its effects on the inflammatory response to acute radiation-induced intestinal injury have not been elucidated.OBJECTIVE: To investigate the effects of MSC-CM on the inflammatory response to acute radiation-induced intestinal injury.METHODS: Eighty Sprague-Dawley rats(provided by the experimental center of the North Campus of Sun Yat-Sen University in China) were randomly divided into four groups(n=20/group): control group, irradiation(IR) group, irradiation~+MSC-CM~(IEC-6(NOR)) group(IR~+MSC-CMIEC-6(NOR group) and irradiation~+MSC-CM~(IEC-6(IR)) group(IR~+MSC-CM~(IEC-6(IR)) group), followed by continuous administration via the tail vein and abdominal cavity. Intestinal tissue and serum samples were collected 1, 3, 5, 7 days after radiation for hematoxylin-eosin staining and analysis of the concentrations of inflammatory factors using ELISA. Mesenteric lymph nodes were collected 3 days after radiation for analysis of the percentage of CD4~+Foxp3~+Treg cells using flow cytometry.RESULTS AND CONCLUSION: Compared with the IR group and IR~+MSC-CM~(IEC-6(NOR)) group, the intestinal structure(small intestine epithelial villi and crypt) was significantly improved and the pro-inflammatory factors such as interleukin-1 β, interleukin-6 and tumor necrosis factor-a in the small intestine were significantly decreased in the IR~+MSC-CM~(IEC-6(IR)) group, whereas the level of anti-inflammatory factor interleukin-10 was significantly increased. Similarly, the levels of pro-inflammatory factors such as Activin A, interleukin-1β, interleukin-6 and tumor necrosis factor-a in the serum samples were significantly decreased and the level of anti-inflammatory factor IL-10 was significantly increased. The levels of inflammatory factors changed most significantly at 3 days after irradiation. The number of CD4~+Foxp3~+Treg cells in mesenteric lymph nodes in the IR~+MSC-CMIEO-6(IR) group was significantly higher than that in the control group and IR~+MSC-CMIEC-6(IOR)group. These results suggest that MSC-CM~(IEC-6(IR)) can regulate the balance of pro-inflammatory factors and anti-inflammatory factors, and increase the number of CD4~+Foxp3~+ Treg cells in mesenteric lymph nodes, thus inhibiting the inflammatory response at systemic and mucosal levels in acute radiation-induced intestinal injury and promoting the repair of small intestinal mucosal injury.
引文
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[2]Martin E,Pointreau Y,Roche-Forestier S,et al.Normal tissue tolerance to external beam radiation therapy:small bowel.Cancer Radiother.2010;14(4-5):350-353.
[3]Keating A.Mesenchymal stromal cells:new directions.Cell Stem Cell.2012;10(6):709-716.
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[5]Canavan JB,ScottàC,Vossenk?mper A,et al.Developing in vitro expanded CD45RA+regulatory T cells as an adoptive cell therapy for Crohn's disease.Gut.2016;65(4):584-594.
[6]Mao F,Wu Y,Tang X,et al.Human umbilical cord mesenchymal stem cells alleviate inflammatory bowel disease through the regulation of15-LOX-1 in macrophages.Biotechnol Lett.2017;39(6):929-938.
[7]Di Trapani M,Bassi G,Midolo M,et al.Differential and transferable modulatory effects of mesenchymal stromal cell-derived extracellular vesicles on T,B and NK cell functions.Sci Rep.2016;6:24120.
[8]De Castro LL,Xisto DG,Kitoko JZ,et al.Human adipose tissue mesenchymal stromal cells and their extracellular vesicles act differentially on lung mechanics and inflammation in experimental allergic asthma.Stem Cell Res Ther.2017;8(1):151.
[9]Thorpe DW,Stringer AM,Gibson RJ.Chemotherapy-induced mucositis:the role of the gastrointestinal microbiome and toll-like receptors.Exp Biol Med(Maywood).2013;238(1):1-6.
[10]刘婉薇,陈韵,郑跃,等.炎症预激活骨髓间充质干细胞条件培养基修复小肠黏膜急性辐射损伤[J].中国组织工程研究,2015,19(10):1544-1550.
[11]Dalal J,Gandy K,Domen J.Role of mesenchymal stem cell therapy in Crohn's disease.Pediatr Res.2012;71(4 Pt 2):445-451.
[12]Zhao K,Lou R,Huang F,et al.Immunomodulation effects of mesenchymal stromal cells on acute graft-versus-host disease after hematopoietic stem cell transplantation.Biol Blood Marrow Transplant.2015;21(1):97-104.
[13]Shrestha B,Coykendall K,Li Y,et al.Repair of injured spinal cord using biomaterial scaffolds and stem cells.Stem Cell Res Ther.2014;5(4):91.
[14]Gonzalez-Rey E,Anderson P,González MA,et al.Human adult stem cells derived from adipose tissue protect against experimental colitis and sepsis.Gut.2009;58(7):929-939.
[15]Fuenzalida P,Kurte M,Fernández-O'ryan C,et al.Toll-like receptor 3pre-conditioning increases the therapeutic efficacy of umbilical cord mesenchymal stromal cells in a dextran sulfate sodium-induced colitis model.Cytotherapy.2016;18(5):630-641.
[16]Chen Y,Song Y,Miao H,et al.Gene delivery with IFN-γ-expression plasmids enhances the therapeutic effects of MSCs on DSS-induced mouse colitis.Inflamm Res.2015;64(9):671-681.
[17]Raicevic G,Rouas R,Najar M,et al.Inflammation modifies the pattern and the function of Toll-like receptors expressed by human mesenchymal stromal cells.Hum Immunol.2010;71(3):235-244.
[18]Raicevic G,Rouas R,Najar M,et al.Inflammation modifies the pattern and the function of Toll-like receptors expressed by human mesenchymal stromal cells.Dev Growth Differ.2014;56(3):233-244.
[19]Le Blanc K,Mougiakakos D.Multipotent mesenchymal stromal cells and the innate immune system.Nat Rev Immunol.2012;12(5):383-396.
[20]Liu L,Song H,Duan H,et al.TSG-6 secreted by human umbilical cord-MSCs attenuates severe burn-induced excessive inflammation via inhibiting activations of P38 and JNK signaling.Sci Rep.2016;6:30121.
[21]郑跃,陈浩,沙卫红,等.缺氧诱导骨髓间充质干细胞条件培养基体外修复辐射损伤小肠上皮细胞变化[J].中国组织工程研究,2015,19(45):7230-7236.
[22]Gao F,Chiu SM,Motan DA,et al.Mesenchymal stem cells and immunomodulation:current status and future prospects.Cell Death Dis.2016;7:e2062.
[23]Burr SP,Dazzi F,Garden OA.Mesenchymal stromal cells and regulatory T cells:the Yin and Yang of peripheral tolerance.Immunol Cell Biol.2013;91(1):12-18.
[24]Wang Y,Liu XP,Zhao ZB,et al.Expression of CD4+forkhead box P3(FOXP3)+regulatory T cells in inflammatory bowel disease.J Dig Dis.2011;12(4):286-294.
[2]Martin E,Pointreau Y,Roche-Forestier S,et al.Normal tissue tolerance to external beam radiation therapy:small bowel.Cancer Radiother.2010;14(4-5):350-353.
[3]Keating A.Mesenchymal stromal cells:new directions.Cell Stem Cell.2012;10(6):709-716.
[4]Sémont A,Mouiseddine M,Fran?ois A,et al.Mesenchymal stem cells improve small intestinal integrity through regulation of endogenous epithelial cell homeostasis.Cell Death Differ.2010;17(6):952-961.
[5]Canavan JB,ScottàC,Vossenk?mper A,et al.Developing in vitro expanded CD45RA+regulatory T cells as an adoptive cell therapy for Crohn's disease.Gut.2016;65(4):584-594.
[6]Mao F,Wu Y,Tang X,et al.Human umbilical cord mesenchymal stem cells alleviate inflammatory bowel disease through the regulation of15-LOX-1 in macrophages.Biotechnol Lett.2017;39(6):929-938.
[7]Di Trapani M,Bassi G,Midolo M,et al.Differential and transferable modulatory effects of mesenchymal stromal cell-derived extracellular vesicles on T,B and NK cell functions.Sci Rep.2016;6:24120.
[8]De Castro LL,Xisto DG,Kitoko JZ,et al.Human adipose tissue mesenchymal stromal cells and their extracellular vesicles act differentially on lung mechanics and inflammation in experimental allergic asthma.Stem Cell Res Ther.2017;8(1):151.
[9]Thorpe DW,Stringer AM,Gibson RJ.Chemotherapy-induced mucositis:the role of the gastrointestinal microbiome and toll-like receptors.Exp Biol Med(Maywood).2013;238(1):1-6.
[10]刘婉薇,陈韵,郑跃,等.炎症预激活骨髓间充质干细胞条件培养基修复小肠黏膜急性辐射损伤[J].中国组织工程研究,2015,19(10):1544-1550.
[11]Dalal J,Gandy K,Domen J.Role of mesenchymal stem cell therapy in Crohn's disease.Pediatr Res.2012;71(4 Pt 2):445-451.
[12]Zhao K,Lou R,Huang F,et al.Immunomodulation effects of mesenchymal stromal cells on acute graft-versus-host disease after hematopoietic stem cell transplantation.Biol Blood Marrow Transplant.2015;21(1):97-104.
[13]Shrestha B,Coykendall K,Li Y,et al.Repair of injured spinal cord using biomaterial scaffolds and stem cells.Stem Cell Res Ther.2014;5(4):91.
[14]Gonzalez-Rey E,Anderson P,González MA,et al.Human adult stem cells derived from adipose tissue protect against experimental colitis and sepsis.Gut.2009;58(7):929-939.
[15]Fuenzalida P,Kurte M,Fernández-O'ryan C,et al.Toll-like receptor 3pre-conditioning increases the therapeutic efficacy of umbilical cord mesenchymal stromal cells in a dextran sulfate sodium-induced colitis model.Cytotherapy.2016;18(5):630-641.
[16]Chen Y,Song Y,Miao H,et al.Gene delivery with IFN-γ-expression plasmids enhances the therapeutic effects of MSCs on DSS-induced mouse colitis.Inflamm Res.2015;64(9):671-681.
[17]Raicevic G,Rouas R,Najar M,et al.Inflammation modifies the pattern and the function of Toll-like receptors expressed by human mesenchymal stromal cells.Hum Immunol.2010;71(3):235-244.
[18]Raicevic G,Rouas R,Najar M,et al.Inflammation modifies the pattern and the function of Toll-like receptors expressed by human mesenchymal stromal cells.Dev Growth Differ.2014;56(3):233-244.
[19]Le Blanc K,Mougiakakos D.Multipotent mesenchymal stromal cells and the innate immune system.Nat Rev Immunol.2012;12(5):383-396.
[20]Liu L,Song H,Duan H,et al.TSG-6 secreted by human umbilical cord-MSCs attenuates severe burn-induced excessive inflammation via inhibiting activations of P38 and JNK signaling.Sci Rep.2016;6:30121.
[21]郑跃,陈浩,沙卫红,等.缺氧诱导骨髓间充质干细胞条件培养基体外修复辐射损伤小肠上皮细胞变化[J].中国组织工程研究,2015,19(45):7230-7236.
[22]Gao F,Chiu SM,Motan DA,et al.Mesenchymal stem cells and immunomodulation:current status and future prospects.Cell Death Dis.2016;7:e2062.
[23]Burr SP,Dazzi F,Garden OA.Mesenchymal stromal cells and regulatory T cells:the Yin and Yang of peripheral tolerance.Immunol Cell Biol.2013;91(1):12-18.
[24]Wang Y,Liu XP,Zhao ZB,et al.Expression of CD4+forkhead box P3(FOXP3)+regulatory T cells in inflammatory bowel disease.J Dig Dis.2011;12(4):286-294.
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