脂肪干细胞移植修复急性挤压伤性肾损伤
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摘要
背景:脂肪干细胞具有易获取、易分离、创伤小、增殖速度快等优点。目前肾损伤的治疗手段较局限,脂肪干细胞可能会为其提供一个新的治疗途径。目的:探讨脂肪干细胞移植对挤压伤性急性肾损伤大鼠肾功能的影响及其机制。方法:体外复苏冻存的鼠脂肪干细胞并制备脂肪干细胞悬液,使用PKH-26对脂肪干细胞进行荧光标记。从66只SD大鼠(北京维通利华动物实验技术有限公司提供)中随机选取20只大鼠作为正常对照组;余46只大鼠钳夹双后肢近端建立挤压伤所致急性肾损伤病理模型,最终40只大鼠建模成功,随机分为模型组和脂肪干细胞组,每组20只。造模成功后6 h,模型组大鼠尾静脉注射20μL生理盐水,脂肪干细胞组大鼠尾静脉注射20μL PKH-26标记的脂肪干细胞(细胞浓度为3×106 L-1),1次/d,连续3 d。移植后第1,3,14,21天检测各组大鼠血清肌酐和尿素氮水平。移植后第3,21天,取各组大鼠左肾组织进行苏木精-伊红染色、TUNEL染色、RT-PCR及Western blot检测。结果与结论:(1)细胞移植后第1,3,14,21天,模型组大鼠血清肌酐和尿素氮水平均显著高于正常对照组(P <0.05),脂肪干细胞移植组大鼠血清肌酐和尿素氮水平显著低于模型组(P <0.05);(2)细胞移植后第3,21天,脂肪干细胞组和模型组大鼠肾脏损伤评分、肾脏细胞凋亡率显著高于正常对照组(P <0.05),脂肪干细胞组肾脏损伤评分、肾脏细胞凋亡率显著低于模型组(P <0.05);(3)细胞移植后第3,21天,模型组及脂肪干细胞组肾组织中bax及Caspase-3基因和蛋白相对表达水平均高于正常对照组(P<0.01);脂肪干细胞组肾组织中bax及Caspase-3基因和蛋白相对表达水平显著低于模型组(P<0.05);(4)结果表明,脂肪干细胞移植对挤压伤所致急性肾损伤具有明显的修复作用,其机制可能与脂肪干细胞参与调节bax和Caspase-3表达有关。
BACKGROUND: Adipose-derived stem cells have the advantages of easy access, easy separation, small trauma, and rapid proliferation. Current treatments for kidney injury are more limited, and adipose-derived stem cells may provide a new treatment route. OBJECTIVE: To investigate the effects of adipose-derived stem cell transplantation on the kidney function of rats with acute kidney injury induced by crush injury in rats. METHODS: Cryopreserved adipose-derived stem cells were recovered in vitro and cultured to prepare cell suspension following labeling with PKH-26. Twenty rats were randomly selected from 66 experimental Sprague-Dawley rats(provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.) as normal control group. In the 40 of the remaining 46 rats, a pathological model of acute kidney injury caused by compression injury was successfully established by the use of forceps to double the proximal hind limbs. The 40 rat models were divided into model group and cell transplantation group, with 20 rats in each group. After 6 hours of modeling, the rats in the model group were given intravenous injection of normal saline(20 μL), and the rats in the cell group were given intravenous injection of PKH-26-labeled adipose-derived stem cells(20 μL, 3×106/L), once a day for 3 continuous days. The levels of serum creatinine and urea nitrogen were measured in each group at 1, 3, 14 and 21 days after transplantation. The left kidney of the rats in each group was observed using hematoxylin-eosin staining, TUNEL staining, RT-PCR and western blot assay at 3 and 21 days after cell transplantation. RESULTS AND CONCLUSION:(1) The levels of creatinine and urea nitrogen in the serum of rats at 1, 3, 14 and 21 days after cell transplantation were significantly higher in the model group than the normal control group(P < 0.05) and cell transplantation group(P < 0.05).(2) At 3 and 21 days after cell transplantation, the scores on the kidney injury and apoptotic rate of kidney cells were ranked as follows: model group > cell transplantation group > normal control group, and there were significant differences between groups(P < 0.05).(3) At 3 and 21 days after cell transplantation, the expressions of bax and Caspase-3 in the kidney tissue at mRNA and protein levels were significantly higher in the model group and cell transplantation than the normal control group(P < 0.01) as well as significantly higher in the cell transplantation group than the model group(P < 0.05). To conclude, adipose-derived stem cell transplantation has obvious repairing effect on acute kidney injury caused by crush injury, and its mechanism may be related to the involvement of adipose-derived stem cells in regulating the expression of bax and Caspase-3.
BACKGROUND: Adipose-derived stem cells have the advantages of easy access, easy separation, small trauma, and rapid proliferation. Current treatments for kidney injury are more limited, and adipose-derived stem cells may provide a new treatment route. OBJECTIVE: To investigate the effects of adipose-derived stem cell transplantation on the kidney function of rats with acute kidney injury induced by crush injury in rats. METHODS: Cryopreserved adipose-derived stem cells were recovered in vitro and cultured to prepare cell suspension following labeling with PKH-26. Twenty rats were randomly selected from 66 experimental Sprague-Dawley rats(provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.) as normal control group. In the 40 of the remaining 46 rats, a pathological model of acute kidney injury caused by compression injury was successfully established by the use of forceps to double the proximal hind limbs. The 40 rat models were divided into model group and cell transplantation group, with 20 rats in each group. After 6 hours of modeling, the rats in the model group were given intravenous injection of normal saline(20 μL), and the rats in the cell group were given intravenous injection of PKH-26-labeled adipose-derived stem cells(20 μL, 3×106/L), once a day for 3 continuous days. The levels of serum creatinine and urea nitrogen were measured in each group at 1, 3, 14 and 21 days after transplantation. The left kidney of the rats in each group was observed using hematoxylin-eosin staining, TUNEL staining, RT-PCR and western blot assay at 3 and 21 days after cell transplantation. RESULTS AND CONCLUSION:(1) The levels of creatinine and urea nitrogen in the serum of rats at 1, 3, 14 and 21 days after cell transplantation were significantly higher in the model group than the normal control group(P < 0.05) and cell transplantation group(P < 0.05).(2) At 3 and 21 days after cell transplantation, the scores on the kidney injury and apoptotic rate of kidney cells were ranked as follows: model group > cell transplantation group > normal control group, and there were significant differences between groups(P < 0.05).(3) At 3 and 21 days after cell transplantation, the expressions of bax and Caspase-3 in the kidney tissue at mRNA and protein levels were significantly higher in the model group and cell transplantation than the normal control group(P < 0.01) as well as significantly higher in the cell transplantation group than the model group(P < 0.05). To conclude, adipose-derived stem cell transplantation has obvious repairing effect on acute kidney injury caused by crush injury, and its mechanism may be related to the involvement of adipose-derived stem cells in regulating the expression of bax and Caspase-3.
引文
[1]曾筱茜,王婷立,付平.地震挤压伤中急性肾损伤的临床特征分析[J].西部医学,2014,26(6):715-718.
[2]潘鑫,邹圣强.挤压综合征研究进展[J].中国急救复苏与灾害医学杂志,2014,9(8):764-766.
[3]高阳,樊毫军,刘子泉,等.肢体挤压时长与急性肾损伤关系的动物实验研究[J].中国急救复苏与灾害医学杂志,2015,10(8):712-715.
[4]Tang Y,Gan X,Cheheltani R,et al.Targeted delivery of vascular endothelial growth factor improves stem cell therapy in a rat myocardial infarction model.Nanomedicine.2014;10(8):1711-1718.
[5]Atashi F,Modarressi A,Pepper MS.The role of reactive oxygen species in mesenchymal stem cell adipogenic and osteogenic differentiation:a review.Stem Cells Dev.2015;24(10):1150-1163.
[6]Jeong H,Yim HW,Cho YS,et al.Efficacy and safety of stem cell therapies for patients with stroke:a systematic review and single arm meta-analysis.Int J Stem Cells.2014;7(2):63-69.
[7]Bruin JE,Saber N,Braun N,et al.Treating diet-induced diabetes and obesity with human embryonic stem cell-derived pancreatic progenitor cells and antidiabetic drugs.Stem Cell Reports.2015;4(4):605-620.
[8]Morigi M,De Coppi P.Cell therapy for kidney injury:different options and mechanisms--mesenchymal and amniotic fluid stem cells.Nephron Exp Nephrol.2014;126(2):59.
[9]Ma T,Sun J,Zhao Z,et al.A brief review:adipose-derived stem cells and their therapeutic potential in cardiovascular diseases.Stem Cell Res Ther.2017;8(1):124.
[10]Tabatabaei Qomi R,Sheykhhasan M.Adipose-derived stromal cell in regenerative medicine:A review.World J Stem Cells.2017;9(8):107-117.
[11]Saidi RF,Rajeshkumar B,Shariftabrizi A,et al.Human adiposederived mesenchymal stem cells attenuate liver ischemiareperfusion injury and promote liver regeneration.Surgery.2014;156(5):1225-1231.
[12]Chen L,Qin F,Ge M,et al.Application of adipose-derived stem cells in heart disease.J Cardiovasc Transl Res.2014;7(7):651-663.
[13]刘少鹏.脂肪干细胞(ASCs)在急性肾损伤(AKI)动物模型治疗中的作用研究进展[J].复旦学报(医学版),2014,41(3):400-404.
[14]郑凯,吴卫真,谭建明,等.骨髓间充质干细胞治疗兔挤压伤后急性肾损伤[J].中华创伤杂志,2015,31(2):173-175.
[15]张春梅,唐华,陈蓉,等.挤压综合征性肾损伤时HIF-1α变化及与肾血流量和血液流变学的关系的研究[J].现代预防医学,2015,42(3):504-507.
[16]王捷荣,李咏梅.窒息性肾损伤新生儿尿神经生长因子-1、视黄醇结合蛋白、血肌酐水平变化的临床意义[J].中国临床医生,2016,44(8):90-92.
[17]Sheashaa H,Lotfy A,Elhusseini F,et al.Protective effect of adipose-derived mesenchymal stem cells against acute kidney injury induced by ischemia-reperfusion in Sprague-Dawley rats.Exp Ther Med.2016;11(5):1573-1580.
[18]Yao W,Hu Q,Ma Y,et al.Human adipose-derived mesenchymal stem cells repair cisplatin-induced acute kidney injury through antiapoptotic pathways.Exp Ther Med.2015;10(2):468-476.
[19]Overath JM,Gauer S,Obermüller N,et al.Short-term preconditioning enhances the therapeutic potential of adiposederived stromal/stem cell-conditioned medium in cisplatin-induced acute kidney injury.Exp Cell Res.2016;342(2):175-183.
[20]Tsuji W,Rubin JP,Marra KG.Adipose-derived stem cells:Implications in tissue regeneration.World J Stem Cells.2014;6(3):312-321.
[21]刘少鹏,俞小芳,钟一红,等.经肾动脉移植脂肪干细胞对大鼠急性缺血性肾损伤的治疗作用研究[J].中华肾脏病杂志,2013,29(10):768-774.
[22]Wu R,Tang S,Wang M,et al.MicroRNA-497 Induces Apoptosis and Suppresses Proliferation via the Bcl-2/Bax-Caspase9-Caspase3 Pathway and Cyclin D2 Protein in HUVECs.PLoS One.2016;11(12):e0167052.
[23]Jing D,Bai H,Yin S.Renoprotective effects of emodin against diabetic nephropathy in rat models are mediated via PI3K/Akt/GSK-3βand Bax/caspase-3 signaling pathways.Exp Ther Med.2017;14(5):5163-5169.
[24]柏合,刘勇,李洪志,等.姜黄素对2型糖尿病肾病大鼠肾脏的保护作用探究[J].光明中医,2017,32(11):1575-1577.
[25]吴暄,郭义峰.肾脏损伤后成体干细胞修复的研究进展[J].中国医师杂志,2017,19(10):1594-1598.
[26]马华林,徐莹,张蓉蓉,等.间充质干细胞对内毒素诱导急性肾损伤的作用[J].暨南大学学报(自然科学与医学版),2017,38(3):253-258.
[27]黄克静,黄婷,胡文兵,等.脂肪间充质干细胞对肾缺血再灌注损伤的保护作用[J].临床肾脏病杂志,2017,17(2):115-119.
[28]汪娟,潘勇军,张杰.人胎盘间充质干细胞条件培养基对急性缺血再灌注肾损伤的修复作用[J].创伤外科杂志,2017,19(12):924-928.
[29]习小庆,胡红林,邹丛,等.Tet-On调控NICD稳定表达的骨髓间充质干细胞治疗肾缺血再灌注损伤[J].中国组织工程研究,2018,22(25):4035-4040.
[30]Lo CY,Weil BR,Palka BA,et al.Cell surface glycoengineering improves selectin-mediated adhesion of mesenchymal stem cells(MSCs)and cardiosphere-derived cells(CDCs):Pilot validation in porcine ischemia-reperfusion model.Biomaterials.2016;74:19-30.
[31]刘倩,张玉静,刘静,等.脂联素与鼠脂肪干细胞移植治疗大鼠肾病综合征[J].中国组织工程研究,2018,22(13):2109-2113.
[32]汪九龄,何波,储瑞亮,等.大鼠急性脊髓损伤后肾脏的病理变化及其意义[J].中华创伤杂志,2017,33(5):459-464.
[33]薛建新,李潇,贾瑞鹏,等.Ang-1基因转染的内皮祖细胞移植对大鼠肾脏缺血再灌注后的保护作用[J].国际泌尿系统杂志,2017,37(5):755-760.
[34]唐海林,王志刚,李巧,等.超声辐照微泡促骨髓间充质干细胞修复大鼠急性肾损伤的实验研究[J].中华医学超声杂志(电子版),2015,12(8):652-656.
[35]王志红,林芸,陈为民,等.骨髓间充质干细胞修复衰老大鼠肾脏损伤的研究[J].创伤与急诊电子杂志,2016,4(4):191-195,199.
[2]潘鑫,邹圣强.挤压综合征研究进展[J].中国急救复苏与灾害医学杂志,2014,9(8):764-766.
[3]高阳,樊毫军,刘子泉,等.肢体挤压时长与急性肾损伤关系的动物实验研究[J].中国急救复苏与灾害医学杂志,2015,10(8):712-715.
[4]Tang Y,Gan X,Cheheltani R,et al.Targeted delivery of vascular endothelial growth factor improves stem cell therapy in a rat myocardial infarction model.Nanomedicine.2014;10(8):1711-1718.
[5]Atashi F,Modarressi A,Pepper MS.The role of reactive oxygen species in mesenchymal stem cell adipogenic and osteogenic differentiation:a review.Stem Cells Dev.2015;24(10):1150-1163.
[6]Jeong H,Yim HW,Cho YS,et al.Efficacy and safety of stem cell therapies for patients with stroke:a systematic review and single arm meta-analysis.Int J Stem Cells.2014;7(2):63-69.
[7]Bruin JE,Saber N,Braun N,et al.Treating diet-induced diabetes and obesity with human embryonic stem cell-derived pancreatic progenitor cells and antidiabetic drugs.Stem Cell Reports.2015;4(4):605-620.
[8]Morigi M,De Coppi P.Cell therapy for kidney injury:different options and mechanisms--mesenchymal and amniotic fluid stem cells.Nephron Exp Nephrol.2014;126(2):59.
[9]Ma T,Sun J,Zhao Z,et al.A brief review:adipose-derived stem cells and their therapeutic potential in cardiovascular diseases.Stem Cell Res Ther.2017;8(1):124.
[10]Tabatabaei Qomi R,Sheykhhasan M.Adipose-derived stromal cell in regenerative medicine:A review.World J Stem Cells.2017;9(8):107-117.
[11]Saidi RF,Rajeshkumar B,Shariftabrizi A,et al.Human adiposederived mesenchymal stem cells attenuate liver ischemiareperfusion injury and promote liver regeneration.Surgery.2014;156(5):1225-1231.
[12]Chen L,Qin F,Ge M,et al.Application of adipose-derived stem cells in heart disease.J Cardiovasc Transl Res.2014;7(7):651-663.
[13]刘少鹏.脂肪干细胞(ASCs)在急性肾损伤(AKI)动物模型治疗中的作用研究进展[J].复旦学报(医学版),2014,41(3):400-404.
[14]郑凯,吴卫真,谭建明,等.骨髓间充质干细胞治疗兔挤压伤后急性肾损伤[J].中华创伤杂志,2015,31(2):173-175.
[15]张春梅,唐华,陈蓉,等.挤压综合征性肾损伤时HIF-1α变化及与肾血流量和血液流变学的关系的研究[J].现代预防医学,2015,42(3):504-507.
[16]王捷荣,李咏梅.窒息性肾损伤新生儿尿神经生长因子-1、视黄醇结合蛋白、血肌酐水平变化的临床意义[J].中国临床医生,2016,44(8):90-92.
[17]Sheashaa H,Lotfy A,Elhusseini F,et al.Protective effect of adipose-derived mesenchymal stem cells against acute kidney injury induced by ischemia-reperfusion in Sprague-Dawley rats.Exp Ther Med.2016;11(5):1573-1580.
[18]Yao W,Hu Q,Ma Y,et al.Human adipose-derived mesenchymal stem cells repair cisplatin-induced acute kidney injury through antiapoptotic pathways.Exp Ther Med.2015;10(2):468-476.
[19]Overath JM,Gauer S,Obermüller N,et al.Short-term preconditioning enhances the therapeutic potential of adiposederived stromal/stem cell-conditioned medium in cisplatin-induced acute kidney injury.Exp Cell Res.2016;342(2):175-183.
[20]Tsuji W,Rubin JP,Marra KG.Adipose-derived stem cells:Implications in tissue regeneration.World J Stem Cells.2014;6(3):312-321.
[21]刘少鹏,俞小芳,钟一红,等.经肾动脉移植脂肪干细胞对大鼠急性缺血性肾损伤的治疗作用研究[J].中华肾脏病杂志,2013,29(10):768-774.
[22]Wu R,Tang S,Wang M,et al.MicroRNA-497 Induces Apoptosis and Suppresses Proliferation via the Bcl-2/Bax-Caspase9-Caspase3 Pathway and Cyclin D2 Protein in HUVECs.PLoS One.2016;11(12):e0167052.
[23]Jing D,Bai H,Yin S.Renoprotective effects of emodin against diabetic nephropathy in rat models are mediated via PI3K/Akt/GSK-3βand Bax/caspase-3 signaling pathways.Exp Ther Med.2017;14(5):5163-5169.
[24]柏合,刘勇,李洪志,等.姜黄素对2型糖尿病肾病大鼠肾脏的保护作用探究[J].光明中医,2017,32(11):1575-1577.
[25]吴暄,郭义峰.肾脏损伤后成体干细胞修复的研究进展[J].中国医师杂志,2017,19(10):1594-1598.
[26]马华林,徐莹,张蓉蓉,等.间充质干细胞对内毒素诱导急性肾损伤的作用[J].暨南大学学报(自然科学与医学版),2017,38(3):253-258.
[27]黄克静,黄婷,胡文兵,等.脂肪间充质干细胞对肾缺血再灌注损伤的保护作用[J].临床肾脏病杂志,2017,17(2):115-119.
[28]汪娟,潘勇军,张杰.人胎盘间充质干细胞条件培养基对急性缺血再灌注肾损伤的修复作用[J].创伤外科杂志,2017,19(12):924-928.
[29]习小庆,胡红林,邹丛,等.Tet-On调控NICD稳定表达的骨髓间充质干细胞治疗肾缺血再灌注损伤[J].中国组织工程研究,2018,22(25):4035-4040.
[30]Lo CY,Weil BR,Palka BA,et al.Cell surface glycoengineering improves selectin-mediated adhesion of mesenchymal stem cells(MSCs)and cardiosphere-derived cells(CDCs):Pilot validation in porcine ischemia-reperfusion model.Biomaterials.2016;74:19-30.
[31]刘倩,张玉静,刘静,等.脂联素与鼠脂肪干细胞移植治疗大鼠肾病综合征[J].中国组织工程研究,2018,22(13):2109-2113.
[32]汪九龄,何波,储瑞亮,等.大鼠急性脊髓损伤后肾脏的病理变化及其意义[J].中华创伤杂志,2017,33(5):459-464.
[33]薛建新,李潇,贾瑞鹏,等.Ang-1基因转染的内皮祖细胞移植对大鼠肾脏缺血再灌注后的保护作用[J].国际泌尿系统杂志,2017,37(5):755-760.
[34]唐海林,王志刚,李巧,等.超声辐照微泡促骨髓间充质干细胞修复大鼠急性肾损伤的实验研究[J].中华医学超声杂志(电子版),2015,12(8):652-656.
[35]王志红,林芸,陈为民,等.骨髓间充质干细胞修复衰老大鼠肾脏损伤的研究[J].创伤与急诊电子杂志,2016,4(4):191-195,199.
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