炎症激活骨髓间充质干细胞条件培养基对辐射损伤IEC-6细胞的修复
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摘要
背景:间充质干细胞条件培养基是干细胞移植理想的替代方案,课题组前期研究表明炎症预激活的间充质干细胞条件培养基能促进辐射损伤小肠组织学结构和功能的修复,改善急性辐射损伤大鼠生存状态,但其潜在的细胞机制未进一步探讨。目的:观察炎症预激活的间充质干细胞条件培养基对辐射损伤小肠上皮细胞增殖、凋亡的影响,探讨预激活间充质干细胞条件培养基修复小肠黏膜的细胞机制。方法:小肠隐窝IEC-6细胞分为对照组、辐射损伤组、辐射损伤+MSC-CM~(IEC-6(NOR))组和辐射损伤+MSC-CM~(IEC-6(IR))组,后3组以10 Gy X射线一次性照射IEC-6细胞建立体外急性小肠辐射损伤模型,对照组及辐射损伤组加入DMEM-F12培养基,辐射损伤+MSC-CM~(IEC-6(NOR))组加入正常状态间充质干细胞条件培养基,辐射损伤+MSC-CM~(IEC-6(IR))组加入炎症激活状态间充质干细胞条件培养基。照射后3 d收集细胞行免疫荧光PCNA染色观察增殖改变,行TUNEL凋亡染色及Western blot蛋白免疫印迹法观察凋亡改变。结果与结论:与辐射损伤组相比,辐射损伤+MSC-CM~(IEC-6(IR))组IEC-6细胞PCNA阳性细胞数明显增加(P<0.05),TUNEL阳性细胞数下降(P<0.05),凋亡相关因子Caspases-3蛋白表达明显下降(P<0.05),而辐射损伤+MSC-CM~(IEC-6(NOR))组的各项指标变化无统计学意义(P>0.05)。上述结果表明,与正常状态的间充质干细胞条件培养基相比,炎症激活的间充质干细胞条件培养基显著促进辐射损伤肠上皮细胞的增殖,并抑制其凋亡,促进辐射损伤小肠上皮修复。
BACKGROUND:Conditioned medium from mesenchymal stem cells(MSC-CM)may represent a promising alternative to MSCs transplantation.Our previous study has demonstrated that MSC-CM with inflammatory activation improves the structural and functional restoration of the small intestine after radiation-induced intestinal injury,improve the survival status of rats with acute radiation injury,but its potential cellular mechanism has not been further explored.OBJECTIVE:To observe the effect of MSC-CM with inflammatory activation on the proliferation and apoptosis of intestinal epithelial cells(IEC-6)after radiation injury and to investigate the cellular mechanism of pre-activated MSC-CM in repairing the small intestinal mucosa.METHODS:IEC-6 cells were divided into four groups:control group,radiation injury group,normal MSC-CM(MSC-CM~(IEC-6(NOR)))group and inflammatory pre-activated MSC-CM(MSC-CM~(IEC-6(IR)))group.IEC-6 cells in the latter three groups were exposed to 10 Gy X-ray irradiation and cultured in DMEM-F12 medium,MSC-CM~(IEC-6(IR))and MSC-CM~(IEC-6(NOR))respectively.Cells in the control group were only cultured in DMEM-12 medium.Cultured cells were collected at 3 days after radiation to observe the proliferation of IEC-6 cells by using proliferating cell nuclear antigen immunofluorescence staining,and to observe the apoptosis by using TUNEL apoptosis staining and western blot assay.RESULTS AND CONCLUSION:Compared with the radiation injury group,in the MSC-CM~(IEC-6(IR))group,the number of cells positive for proliferating cell nuclear antigen increased significantly(P<0.05),the number of TUNEL positive cells decreased significantly(P<0.05),and the expression of Caspases-3 decreased significantly(P<0.05),However,there was no significant difference between the MSC-CM~(IEC-6(NOR))group and radiation injury group(P>0.05).Taken together,MSC-CM~(IEC-6(IR)),but not non-activated MSC-CM,significantly promotes the proliferation and reduces apoptosis of intestinal epithelial cells after radiation injury and therefore repair the injured intestinal tissue.
BACKGROUND:Conditioned medium from mesenchymal stem cells(MSC-CM)may represent a promising alternative to MSCs transplantation.Our previous study has demonstrated that MSC-CM with inflammatory activation improves the structural and functional restoration of the small intestine after radiation-induced intestinal injury,improve the survival status of rats with acute radiation injury,but its potential cellular mechanism has not been further explored.OBJECTIVE:To observe the effect of MSC-CM with inflammatory activation on the proliferation and apoptosis of intestinal epithelial cells(IEC-6)after radiation injury and to investigate the cellular mechanism of pre-activated MSC-CM in repairing the small intestinal mucosa.METHODS:IEC-6 cells were divided into four groups:control group,radiation injury group,normal MSC-CM(MSC-CM~(IEC-6(NOR)))group and inflammatory pre-activated MSC-CM(MSC-CM~(IEC-6(IR)))group.IEC-6 cells in the latter three groups were exposed to 10 Gy X-ray irradiation and cultured in DMEM-F12 medium,MSC-CM~(IEC-6(IR))and MSC-CM~(IEC-6(NOR))respectively.Cells in the control group were only cultured in DMEM-12 medium.Cultured cells were collected at 3 days after radiation to observe the proliferation of IEC-6 cells by using proliferating cell nuclear antigen immunofluorescence staining,and to observe the apoptosis by using TUNEL apoptosis staining and western blot assay.RESULTS AND CONCLUSION:Compared with the radiation injury group,in the MSC-CM~(IEC-6(IR))group,the number of cells positive for proliferating cell nuclear antigen increased significantly(P<0.05),the number of TUNEL positive cells decreased significantly(P<0.05),and the expression of Caspases-3 decreased significantly(P<0.05),However,there was no significant difference between the MSC-CM~(IEC-6(NOR))group and radiation injury group(P>0.05).Taken together,MSC-CM~(IEC-6(IR)),but not non-activated MSC-CM,significantly promotes the proliferation and reduces apoptosis of intestinal epithelial cells after radiation injury and therefore repair the injured intestinal tissue.
引文
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[22]Jiao X,Cai J,Yu X,et al.Paracrine Activation of the Wnt/β-Catenin Pathway by Bone Marrow Stem Cell Attenuates Cisplatin-Induced Kidney Injury.Cell Physiol Biochem.2017;44(5):1980-1994.
[23]Zhou X,Gu J,Gu Y,et al.Human umbilical cord-derived mesenchymal stem cells improve learning and memory function in hypoxic-ischemic brain-damaged rats via an IL-8-mediated secretion mechanism rather than differentiation pattern induction.Cell Physiol Biochem.2015;35(6):2383-2401
[24]Nazemian V,Manaheji H,Sharifi AM,et al.Long term treatment by mesenchymal stem cells conditioned medium modulates cellular,molecular and behavioral aspects of adjuvant-induced arthritis.Cell Mol Biol(Noisy-le-grand).2018;64(1):19-26.
[25]Angoulvant D,Ivanes F,Ferrera R,et al.Mesenchymal stem cell conditioned media attenuates in vitro and ex vivo myocardial reperfusion injury.J Heart Lung Transplant.2011;30(1):95-102.
[26]Osugi M,Katagiri W,Yoshimi R,Inukai T,Hibi H,Ueda M.Conditioned Media from Mesenchymal Stem Cells Enhanced Bone Regeneration in Rat Calvarial Bone Defects.Tissue Eng Part A 2012;18(13-14):1479-1489.
[27]Yuen WW,Du NR,Chan CH,et al.Mimicking nature by codelivery of stimulant and inhibitor to create temporally stable and spatially restricted angiogenic zones.Proc Natl Acad Sci US A.2010;107(42):17933-17938.
[28]Luo Y,Wang Y,Poynter JA,et al.Pretreating mesenchymal stem cells with interleukin-1βand transforming growth factor-βsynergistically increases vascular endothelial growth factor production and improves mesenchymal stem cell-mediated myocardial protection after acute ischemia.Surgery.2012;151(3):353-363.
[29]Li C,Li G,Liu M,et al.Paracrine effect of inflammatory cytokine-activated bone marrow mesenchymal stem cells and its role in osteoblast function.J Biosci Bioeng.2016;121(2):213-219.
[2]Kudo K,Liu Y,Takhashi K,et al.Transplantation of mesenchymal stem cells to prevent radiation-induced intestinal injury in mice.J Radiat Res.2010;51:73-79.
[3]Han YM,Park JM,Choi YS,et al.The efficacy of human placenta-derived mesenchymal stem cells on radiation enteropathy along with proteomic biomarkers predicting a favorable response.Stem Cell Res Ther.2017;8(1):105.
[4]Chang YH,Lin LM,Lou CW,Chou CK and Ch'ang HJ.Bone marrow transplantation rescues intestinal mucosa after whole body radiation via paracrine mechanisms.Radiother Oncol.2012;105:371-377.
[5]Narita T,Suzuki K.Bone marrow-derived mesenchymal stem cells for the treatment of heart failure.Heart Fail Rev.2015;20:53-68.
[6]Samper E,Diez-Juan A,Montero JA,et al.Cardiac cell therapy:boosting mesenchymal stem cells effects.Stem Cell Rev.2013;9(3):266-280.
[7]Wu R,Hu X,Wang J.Concise Review:Optimized strategies for stem cell-based therapy in myocardial repair:Clinical translatability and potential limitation.Stem Cells 2018;36:482-500.
[8]Lytle NK,Barber AG,Reya T.Stem cell fate in cancer growth,progression and therapy resistance.Nat Rev Cancer.2018;18(11):669-680.
[9]Khubutiya MS,Vagabov AV,Temnov AA,et al.Paracrine mechanisms of proliferative,anti-apoptotic and anti-inflammatory effects of mesenchymal stromal cells in models of acute organ injury.Cytotherapy.2014;16(5):579-585.
[10]Gao Z,Zhang Q,Han Y,et al.Mesenchymal stromal cell-conditioned medium prevents radiation-induced small intestine injury in mice.Cytotherapy.2012;14(3):267-273.
[11]Ren H,Zhang Q,Wang J,et al.Comparative Effects of Umbilical Cord-and Menstrual Blood-Derived MSCs in Repairing Acute Lung Injury.Stem Cells Int.2018;2018:7873625.
[12]Lu Z,Chen Y,Dunstan C,et al.Priming Adipose Stem Cells with Tumor Necrosis Factor-Alpha Preconditioning Potentiates Their Exosome Efficacy for Bone Regeneration.Tissue Eng Part A.2017;23(21-22):1212-1220.
[13]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.
[14]Ishihara H,Tanaka I,Yakumaru H,et al.Pharmaceutical drugs supporting regeneration of small-intestinal mucosa severely damaged by ionizing radiation in mice.J Radiat Res.2013;54(6):1057-1064.
[15]Kiang JG,Smith JT,Anderson MN,et al.Hemorrhage enhances cytokine,complement component 3,and caspase-3,and regulates micro RNAs associated with intestinal damage after whole-body gamma-irradiation in combined injury.PloS one.2017;12(9):e0184393.
[16]邓庆先,贺湘英,黄永坤,等.谷氨酰胺对新生鼠坏死性小肠结肠炎增殖细胞核抗原的影响[J].临床儿科杂志,2015,33(3):276-279.
[17]Wang H,Sun RT,Li Y,et al.HGF Gene Modification in Mesenchymal Stem Cells Reduces Radiation-Induced Intestinal Injury by Modulating Immunity.PloS one.2015;10(5):e0124420.
[18]Matsuzaki-Horibuchi S,Yasuda T,Sakaguchi N,et al.Cell-permeable intrinsic cellular inhibitors of apoptosis protect and rescue intestinal epithelial cells from radiation-induced cell death.J Radiat Res.2015;56(1):100-113.
[19]Gurley KE,Ashley AK,Moser RD,et al.Synergy between Prkdc and Trp53 regulates stem cell proliferation and GI-ARS after irradiation.Cell Death Differ.2017;24(11):1853-1860.
[20]Hauer-Jensen M,Wang J,Boerma M,et al.Radiation damage to the gastrointestinal tract:mechanisms,diagnosis,and management.Curr Opin Support Palliat Care.2007;1(1):23-29.
[21]Gu J,Liu S,Mu N,et al.A DPP-IV-resistant glucagon-like peptide-2 dimer with enhanced activity against radiation-induced intestinal injury.J Control Release.2017;260:32-45.
[22]Jiao X,Cai J,Yu X,et al.Paracrine Activation of the Wnt/β-Catenin Pathway by Bone Marrow Stem Cell Attenuates Cisplatin-Induced Kidney Injury.Cell Physiol Biochem.2017;44(5):1980-1994.
[23]Zhou X,Gu J,Gu Y,et al.Human umbilical cord-derived mesenchymal stem cells improve learning and memory function in hypoxic-ischemic brain-damaged rats via an IL-8-mediated secretion mechanism rather than differentiation pattern induction.Cell Physiol Biochem.2015;35(6):2383-2401
[24]Nazemian V,Manaheji H,Sharifi AM,et al.Long term treatment by mesenchymal stem cells conditioned medium modulates cellular,molecular and behavioral aspects of adjuvant-induced arthritis.Cell Mol Biol(Noisy-le-grand).2018;64(1):19-26.
[25]Angoulvant D,Ivanes F,Ferrera R,et al.Mesenchymal stem cell conditioned media attenuates in vitro and ex vivo myocardial reperfusion injury.J Heart Lung Transplant.2011;30(1):95-102.
[26]Osugi M,Katagiri W,Yoshimi R,Inukai T,Hibi H,Ueda M.Conditioned Media from Mesenchymal Stem Cells Enhanced Bone Regeneration in Rat Calvarial Bone Defects.Tissue Eng Part A 2012;18(13-14):1479-1489.
[27]Yuen WW,Du NR,Chan CH,et al.Mimicking nature by codelivery of stimulant and inhibitor to create temporally stable and spatially restricted angiogenic zones.Proc Natl Acad Sci US A.2010;107(42):17933-17938.
[28]Luo Y,Wang Y,Poynter JA,et al.Pretreating mesenchymal stem cells with interleukin-1βand transforming growth factor-βsynergistically increases vascular endothelial growth factor production and improves mesenchymal stem cell-mediated myocardial protection after acute ischemia.Surgery.2012;151(3):353-363.
[29]Li C,Li G,Liu M,et al.Paracrine effect of inflammatory cytokine-activated bone marrow mesenchymal stem cells and its role in osteoblast function.J Biosci Bioeng.2016;121(2):213-219.