炎症诱导骨髓间充质干细胞条件培养基修复急性辐射引起的小肠上皮干细胞损伤
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摘要
背景:课题组前期发现,炎症预激活的骨髓间充质干细胞条件培养基可促进大鼠小肠急性辐射损伤后的结构和功能修复,该修复作用是否通过调控小肠干细胞得以实现,目前相关研究尚未明确。目的:探究炎症预激活的骨髓间充质干细胞条件培养基对辐射损伤大鼠小肠上皮干细胞的影响,以进一步明确其修复辐射损伤小肠黏膜的机制。方法:(1)分离、培养、鉴定SD幼鼠(中山大学北校区实验中心提供)骨髓间充质干细胞,分别与正常对照和辐射损伤的小肠隐窝细胞株IEC-6在Transwell培养板中共培养24h,预刺激的骨髓间充质干细胞继续单独培养48 h,收集到的上清液分别为正常状态间充质干细胞条件培养基(MSC-CM~(NOR))和辐射炎症预激活间充质干细胞条件培养基(MSC-CM~(IR));(2)将成年SD大鼠(中山大学北校区实验中心提供)随机分为4组:对照组,单纯辐射损伤组,辐射损伤+MSC-CM~(NOR)组,辐射损伤+MSC-CM~(IR)组,每组20只。后3组以14 Gy剂量一次性腹部局部照射制备急性小肠辐射损伤大鼠模型,造模成功后4 h内,单纯辐射损伤组尾静脉注射DMEM-F12培养基,200μL/d,连续注射3 d;辐射损伤+MSC-CM~(NOR)组、辐射损伤+MSC-CM~(IR)组采用胶囊渗透压泵腹腔植入的方式注射MSC-CM~(NOR)和MSC-CM~(IR),胶囊内含2 mL浓缩条件培养基,植入腹腔后以10μL/h的速率匀速释放。于辐射后第1,3,5,7天取小肠组织行免疫组织化学染色、Western blot及qRT-PCR检测。结果与结论:(1)辐射后第3天,位于隐窝基底增殖活跃的Lgr~(5+)小肠上皮干细胞与对照组相比显著减少,几乎难以观察到阳性细胞;而在输注MSC-CM~(IR)后,Lgr~(5+)小肠上皮干细胞与单纯辐射损伤组相比明显增多,MSC-CM~(NOR)组Lgr~(5+)小肠上皮干细胞未见明显增多;(2)辐射损伤后第3天,位于小肠隐窝+4位置相对静止的Bmi1~+小肠上皮干细胞几乎无法观察到;而输注MSC-CM~(IR)后,Bmi1~+小肠上皮干细胞与单纯辐射损伤组相比明显增多,不仅分布在+4细胞位置,还分布在Lgr~(5+)细胞常见的位置,表明Bmi1+细胞在辐射损伤后有可能通过分化为Lgr~(5+)细胞发挥修复效应;(3)Western blot和q RT-PCR进一步验证,MSC-CM~(IR)组的Lgr5表达在同一时间点均明显高于单纯辐射损伤组和MSC-CM~(NOR)组,持续高水平表达后于第7天恢复至正常水平。MSC-CM~(NOR)修复作用较弱,仅在第7天时与单纯辐射损伤组相比差异有显著性意义;(4)结果表明,炎症预激活状态的骨髓间充质干细胞条件培养基具有保护小肠上皮干细胞的作用。
BACKGROUND: Our previous findings indicate that inflammation-activated bone marrow mesenchymal stem cell conditioned medium(MSC-CM) contribute to repairing the structure and function of the small intestine after radiation-induced acute intestinal injury. However, it is unclear whether the repair effect can be achieved by regulating small intestinal stem cells. OBJECTIVE: To investigate the effects of inflammation-activated bone marrow MSC-CM on the small intestinal epithelial stem cells after acute radiation-induced intestinal injury and to further discuss the repairing mechanism. METHODS: Bone marrow mesenchymal stem cells of Sprague-Dawley rats were separated, cultured and identified. Then, the bone marrow mesenchymal stem cells were co-cultured with normal or radiation-induced IEC-6 cell lines in the Transwell system for 24 hours. Inflammation-activated bone marrow mesenchymal stem cells were cultured alone for 48 hours. Non-activated MSC-CM(MSC-CM~(NOR)) and MSC-CM under radiation-induced inflammatory condition(MSC-CM~(IR)) were collected. Adult Sprague-Dawley rats(provided by the Experimental Center of Sun Yat-Sen University North Campus) were randomly divided into four groups with 20 rats in each group: control group, radiation group, radiation+MSC-CM~(NOR) group and radiation+MSC-CM~(IR) group. The rats in the latter three groups were exposed to one-off 14 Gy whole abdominal radiation to make a rat model of acute radiation-induced small intestinal injury. Three-day continuous administration beginning within 4 hours after successful modeling was given via the tail vein and intraperitoneal implantation of Alzet micro-osmotic pumps: EMEM-F12(200 μL/d) for the radiation group, MSC-CM~(NOR) for radiation+MSC-CM~(NOR) group and MSC-CM~(IR) for radiation+MSC-CM~(IR) group. There was 2 mL of concentrated conditioned medium in the pump which was released at a constant rate of 10 μL/h into the abdominal cavity after implantation. Intestinal samples were collected at 1, 3, 5, 7 days after radiation for immunochemistry staining, western blot and qRT-PCR detection. RESULTS AND CONCLUSION:(1) On the 3~(rd) day after radiation, Lgr5 positive cells, which were actively proliferating on the base of crypts, became significantly reduced compared with the normal control group, and there was nearly no existing Lgr5 positive cells. However, after infusion of MSC-CM~(IR), Lgr5 positive intestinal stem cells were significantly increased compared with the radiation group, while in the radiation+MSCNOR group, there was no significant increase in Lgr5 positive intestinal stem cells.(2) On the 3rd day after radiation injury, Bmi1 positive intestinal stem cells were almost invisible. After infusion of MSC-CM~(IR), Bmi1 positive intestinal stem cells increased significantly, and it was observed not only in the +4 cell position but also in the common position used to be Lgr5 stem cells, indicating that Bmi1 stem cells could differentiate into Lgr5 positive cells to act its repairing effect.(3) Western blot and qRT-PCR further confirmed that the radiation+MSC-CM~(IR) group was significantly higher on the Lgr5 expression level than the radiation group and the radiation+MSC-CM~(NOR) group, and it returned to the normal level on the 7~(th) day after the continuous high expression level. The repair effect of radiation+MSC-CM~(NOR) group was weaker, and only on the 7~(th) day, the expression level of Lgr5 was statistically different from the radiation group. To conclude, inflammation-activated bone marrow MSC-CM exert a protective effect on the small intestinal epithelial stem cells after acute radiation-induced intestinal injury
BACKGROUND: Our previous findings indicate that inflammation-activated bone marrow mesenchymal stem cell conditioned medium(MSC-CM) contribute to repairing the structure and function of the small intestine after radiation-induced acute intestinal injury. However, it is unclear whether the repair effect can be achieved by regulating small intestinal stem cells. OBJECTIVE: To investigate the effects of inflammation-activated bone marrow MSC-CM on the small intestinal epithelial stem cells after acute radiation-induced intestinal injury and to further discuss the repairing mechanism. METHODS: Bone marrow mesenchymal stem cells of Sprague-Dawley rats were separated, cultured and identified. Then, the bone marrow mesenchymal stem cells were co-cultured with normal or radiation-induced IEC-6 cell lines in the Transwell system for 24 hours. Inflammation-activated bone marrow mesenchymal stem cells were cultured alone for 48 hours. Non-activated MSC-CM(MSC-CM~(NOR)) and MSC-CM under radiation-induced inflammatory condition(MSC-CM~(IR)) were collected. Adult Sprague-Dawley rats(provided by the Experimental Center of Sun Yat-Sen University North Campus) were randomly divided into four groups with 20 rats in each group: control group, radiation group, radiation+MSC-CM~(NOR) group and radiation+MSC-CM~(IR) group. The rats in the latter three groups were exposed to one-off 14 Gy whole abdominal radiation to make a rat model of acute radiation-induced small intestinal injury. Three-day continuous administration beginning within 4 hours after successful modeling was given via the tail vein and intraperitoneal implantation of Alzet micro-osmotic pumps: EMEM-F12(200 μL/d) for the radiation group, MSC-CM~(NOR) for radiation+MSC-CM~(NOR) group and MSC-CM~(IR) for radiation+MSC-CM~(IR) group. There was 2 mL of concentrated conditioned medium in the pump which was released at a constant rate of 10 μL/h into the abdominal cavity after implantation. Intestinal samples were collected at 1, 3, 5, 7 days after radiation for immunochemistry staining, western blot and qRT-PCR detection. RESULTS AND CONCLUSION:(1) On the 3~(rd) day after radiation, Lgr5 positive cells, which were actively proliferating on the base of crypts, became significantly reduced compared with the normal control group, and there was nearly no existing Lgr5 positive cells. However, after infusion of MSC-CM~(IR), Lgr5 positive intestinal stem cells were significantly increased compared with the radiation group, while in the radiation+MSCNOR group, there was no significant increase in Lgr5 positive intestinal stem cells.(2) On the 3rd day after radiation injury, Bmi1 positive intestinal stem cells were almost invisible. After infusion of MSC-CM~(IR), Bmi1 positive intestinal stem cells increased significantly, and it was observed not only in the +4 cell position but also in the common position used to be Lgr5 stem cells, indicating that Bmi1 stem cells could differentiate into Lgr5 positive cells to act its repairing effect.(3) Western blot and qRT-PCR further confirmed that the radiation+MSC-CM~(IR) group was significantly higher on the Lgr5 expression level than the radiation group and the radiation+MSC-CM~(NOR) group, and it returned to the normal level on the 7~(th) day after the continuous high expression level. The repair effect of radiation+MSC-CM~(NOR) group was weaker, and only on the 7~(th) day, the expression level of Lgr5 was statistically different from the radiation group. To conclude, inflammation-activated bone marrow MSC-CM exert a protective effect on the small intestinal epithelial stem cells after acute radiation-induced intestinal injury
引文
[1]Barker N.Adult intestinal stem cells:critical drivers of epithelial homeostasis and regeneration.Nat Rev Mol Cell Biol.2014;15(1):19-33.
[2]Barker N,van Oudenaarden A,Clevers H.Identifying the stem cell of the intestinal crypt:strategies and pitfalls.Cell Stem Cell.2012;11(4):452-460.
[3]Kim CK,Yang VW,Bialkowska AB.The Role of Intestinal Stem Cells in Epithelial Regeneration Following Radiation-Induced Gut Injury.Curr Stem Cell Rep.2017;3(4):320-332.
[4]Aparicio-Domingo P,Romera-Hernandez M,Karrich JJ,et al.Type 3innate lymphoid cells maintain intestinal epithelial stem cells after tissue damage.J Exp Med.2015;212(11):1783-1791.
[5]Wang LT,Ting CH,Yen ML,et al.Human mesenchymal stem cells(MSCs)for treatment towards immune-and inflammation-mediated diseases:review of current clinical trials.J Biomed Sci.2016;23(1):76.
[6]Sammour I,Somashekar S,Huang J,et al.The Effect of Gender on Mesenchymal Stem Cell(MSC)Efficacy in Neonatal Hyperoxia-Induced Lung Injury.PLoS One.2016;11(10):e0164269.
[7]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.
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[9]Stacey R,Green JT.Radiation-induced small bowel disease:latest developments and clinical guidance.Ther Adv Chronic Dis.2014;5(1):15-29.
[10]Gong W,Guo M,Han Z,et al.Mesenchymal stem cells stimulate intestinal stem cells to repair radiation-induced intestinal injury.Cell Death Dis.2016;7(9):e2387.
[11]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.
[12]Keating A.Mesenchymal stromal cells:new directions.Cell Stem Cell.2012;10(6):709-716.
[13]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.
[14]Marx J.Cancer research.Mutant stem cells may seed cancer.Science.2003;301(5638):1308-1310.
[15]Sell S.On the stem cell origin of cancer.Am J Pathol.2010;176(6):2584-2494.
[16]Stagg J.Mesenchymal stem cells in cancer.Stem Cell Rev.2008;4(2):119-124.
[17]MenaschéP.Stem cells for clinical use in cardiovascular medicine:current limitations and future perspectives.Thromb Haemost.2005;94(4):697-701.
[18]Xin Y,Wang YM,Zhang H,et al.Aging adversely impacts biological properties of human bone marrow-derived mesenchymal stem cells:implications for tissue engineering heart valve construction.Artif Organs.2010;34(3):215-222.
[19]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.
[20]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 U S A.2010;107(42):17933-17938.
[21]van Poll D,Parekkadan B,Cho CH,et al.Mesenchymal stem cell-derived molecules directly modulate hepatocellular death and regeneration in vitro and in vivo.Hepatology.2008;47(5):1634-1643.
[22]Tian H,Biehs B,Warming S,et al.A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable.Nature.2011;478(7368):255-259.
[23]Yan KS,Chia LA,Li X,et al.The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations.Proc Natl Acad Sci U SA.2012;109(2):466-471.
[24]Hua G,Thin TH,Feldman R,et al.Crypt base columnar stem cells in small intestines of mice are radioresistant.Gastroenterology.2012;143(5):1266-1276.
[25]Chang P,Qu Y,Liu Y,et al.Multi-therapeutic effects of human adipose-derived mesenchymal stem cells on radiation-induced intestinal injury.Cell Death Dis.2013;4:e685.
[26]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.
[27]Sadat S,Gehmert S,Song YH,et al.The cardioprotective effect of mesenchymal stem cells is mediated by IGF-I and VEGF.Biochem Biophys Res Commun.2007;363(3):674-679.
[28]Duijvestein M,Wildenberg ME,Welling MM,et al.Pretreatment with interferon-γenhances the therapeutic activity of mesenchymal stromal cells in animal models of colitis.Stem Cells.2011;29(10):1549-1558.
[2]Barker N,van Oudenaarden A,Clevers H.Identifying the stem cell of the intestinal crypt:strategies and pitfalls.Cell Stem Cell.2012;11(4):452-460.
[3]Kim CK,Yang VW,Bialkowska AB.The Role of Intestinal Stem Cells in Epithelial Regeneration Following Radiation-Induced Gut Injury.Curr Stem Cell Rep.2017;3(4):320-332.
[4]Aparicio-Domingo P,Romera-Hernandez M,Karrich JJ,et al.Type 3innate lymphoid cells maintain intestinal epithelial stem cells after tissue damage.J Exp Med.2015;212(11):1783-1791.
[5]Wang LT,Ting CH,Yen ML,et al.Human mesenchymal stem cells(MSCs)for treatment towards immune-and inflammation-mediated diseases:review of current clinical trials.J Biomed Sci.2016;23(1):76.
[6]Sammour I,Somashekar S,Huang J,et al.The Effect of Gender on Mesenchymal Stem Cell(MSC)Efficacy in Neonatal Hyperoxia-Induced Lung Injury.PLoS One.2016;11(10):e0164269.
[7]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.
[8]Citrin D,Cotrim AP,Hyodo F,et al.Radioprotectors and mitigators of radiation-induced normal tissue injury.Oncologist.2010;15(4):360-371.
[9]Stacey R,Green JT.Radiation-induced small bowel disease:latest developments and clinical guidance.Ther Adv Chronic Dis.2014;5(1):15-29.
[10]Gong W,Guo M,Han Z,et al.Mesenchymal stem cells stimulate intestinal stem cells to repair radiation-induced intestinal injury.Cell Death Dis.2016;7(9):e2387.
[11]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.
[12]Keating A.Mesenchymal stromal cells:new directions.Cell Stem Cell.2012;10(6):709-716.
[13]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.
[14]Marx J.Cancer research.Mutant stem cells may seed cancer.Science.2003;301(5638):1308-1310.
[15]Sell S.On the stem cell origin of cancer.Am J Pathol.2010;176(6):2584-2494.
[16]Stagg J.Mesenchymal stem cells in cancer.Stem Cell Rev.2008;4(2):119-124.
[17]MenaschéP.Stem cells for clinical use in cardiovascular medicine:current limitations and future perspectives.Thromb Haemost.2005;94(4):697-701.
[18]Xin Y,Wang YM,Zhang H,et al.Aging adversely impacts biological properties of human bone marrow-derived mesenchymal stem cells:implications for tissue engineering heart valve construction.Artif Organs.2010;34(3):215-222.
[19]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.
[20]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 U S A.2010;107(42):17933-17938.
[21]van Poll D,Parekkadan B,Cho CH,et al.Mesenchymal stem cell-derived molecules directly modulate hepatocellular death and regeneration in vitro and in vivo.Hepatology.2008;47(5):1634-1643.
[22]Tian H,Biehs B,Warming S,et al.A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable.Nature.2011;478(7368):255-259.
[23]Yan KS,Chia LA,Li X,et al.The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations.Proc Natl Acad Sci U SA.2012;109(2):466-471.
[24]Hua G,Thin TH,Feldman R,et al.Crypt base columnar stem cells in small intestines of mice are radioresistant.Gastroenterology.2012;143(5):1266-1276.
[25]Chang P,Qu Y,Liu Y,et al.Multi-therapeutic effects of human adipose-derived mesenchymal stem cells on radiation-induced intestinal injury.Cell Death Dis.2013;4:e685.
[26]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.
[27]Sadat S,Gehmert S,Song YH,et al.The cardioprotective effect of mesenchymal stem cells is mediated by IGF-I and VEGF.Biochem Biophys Res Commun.2007;363(3):674-679.
[28]Duijvestein M,Wildenberg ME,Welling MM,et al.Pretreatment with interferon-γenhances the therapeutic activity of mesenchymal stromal cells in animal models of colitis.Stem Cells.2011;29(10):1549-1558.