脂联素基因修饰脂肪干细胞移植治疗大鼠肾病综合征
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摘要
背景:脂联素基因表达在肾病综合征的发病过程中有着重要的作用,移植脂肪干细胞可以对肾病综合征大鼠起到一定的治疗效果。目的:探讨脂联素基因转染脂肪干细胞移植对肾病综合征大鼠肾功能及凝血功能的改善作用。方法:(1)在体外复苏培养脂肪干细胞并制备其细胞悬液,采用CM-Dil进行标记,取第3代脂肪干细胞进行脂联素基因电转染,Western blot检测转染第3天和第28天细胞中脂联素蛋白的相对表达水平;(2)从80只SD大鼠(北京维通利华动物实验技术有限公司提供)中随机取20只不予任何处理作为正常对照组,其余60只SD大鼠静脉内注射阿霉素建立肾病综合征大鼠模型,随机分为3组:模型组、脂肪干细胞组以及脂联素修饰脂肪干细胞组,正常对照组和模型组大鼠经尾静脉注射20μL PBS,脂肪干细胞组、脂联素修饰脂肪干细胞组大鼠经尾静脉注射20μL转染空载质粒的脂肪干细胞悬液或脂联素修饰的脂肪干细胞悬液,1次/d,连续7 d;(3)移植4周后,全自动生化分析仪检测血清生化指标和凝血指标,苏木精-伊红染色观察肾组织病理变化,RT-PCR、Western blot检测肾组织中乙酰肝素酶及脂联素的表达。结果与结论:(1)Western blot检测显示脂肪干细胞中脂联素高表达可持续28 d;(2)脂联素修饰脂肪干细胞组的病理改善明显优于脂肪干细胞组,脂肪干细胞组较模型组稍有改善;(3)脂肪干细胞组及脂联素修饰脂肪干细胞组的24 h尿蛋白定量及血清总胆固醇、三酰甘油、低密度脂蛋白、肌酐、尿素氮水平均明显低于模型组(P <0.05),而血清总蛋白、白蛋白、高密度脂蛋白水平则明显高于模型组(P <0.05)。脂联素修饰脂肪干细胞组各项生理生化指标均优于脂肪干细胞组,差异有显著性意义(P <0.05)。脂肪干细胞组、脂联素修饰脂肪干细胞组大鼠的血液高凝状态较模型组明显缓解,脂联素修饰脂肪干细胞组缓解更明显,各组间差异有显著性意义(P <0.05);(4)与模型组相比,脂肪干细胞组、脂联素修饰脂肪干细胞组大鼠肾组织中乙酰肝素酶的mRNA和蛋白表达水平明显降低(P <0.05),而脂联素表达水平明显升高(P <0.05)。脂联素修饰脂肪干细胞组乙酰肝素酶和脂联素m RNA和蛋白表达水平均优于脂肪干细胞组(P <0.05);(5)结果表明,脂联素基因修饰的脂肪干细胞移植可以显著改善肾病综合征大鼠的肾功能、血液高凝状态及高脂血症,该作用可能与乙酰肝素酶和脂联素的表达水平有关。
BACKGROUND:The expression of adiponectin gene plays an important role in the pathogenesis of nephrotic syndrome. Transplantation of adipose stem cells can has a certain therapeutic effect in rats with nephrotic syndrome.OBJECTIVE:To investigate the effect of adiponectin gene-transfected adipose stem cell transplantation on renal function and coagulation function in rats with nephrotic syndrome.METHODS:(1) Adipose stem cells were cultured in vitro and the cell suspension was prepared. CM-Dil was used as a marker. The third generation of adipose stem cells was used for electrotransfection using adiponectin gene. Western blot was used to detect the relative expression level of adiponectin in the cells at 3 and 28 days of transfection.(2) The random 20 of 80 Sprague-Dawley rats(provided by Beijing Vitone Lihua Animal Experimental Technology Co., Ltd., China) were taken as normal control group without any treatment, and the remaining60 rats were intravenously injected with doxorubicin to establish nephrotic syndrome models. These rat models were randomized into three groups:model group, adipose stem cell group and adiponectin-transfected adipose stem cell group. Normal control group and model group rats were injected 20 μL of PBS via the tail vein, and rats in the adipose stem cell group and adiponectin-transfected adipose stem cell group were intravenously injected with 20 μL of adipose stem cell suspension and adiponectin-transfected adipose stem cell suspension,respectively, once a day, for 7 continuous days.(3) At 4 weeks after transplantation, a full-automatic biochemical analyzer was used to detect serum biochemical indicators and coagulation indicators, hematoxylin-eosin staining used to observe renal histopathological changes,RT-PCR and Western blue used to detect the expression of heparanase and adiponectin in the kidney tissue.RESULTS AND CONCLUSION:(1) The high expression of adiponectin in adipose stem cells continued for 28 days.(2) The pathological improvement of adiponectin-transfected adipose stem cell group was superior to that of the adipose stem cell group, and the pathological manifestation in the adipose stem cell group was also slightly improved compared to the model group.(3) Serum levels of total cholesterol,triglyceride, low-density lipoprotein, creatinine, and 24-hour urea nitrogen level were significantly lower in the adipose stem cell group and adiponectin-transfected adipose stem cell group than in the model group(P < 0.05), while the serum levels of total protein, albumin, and high-density lipoprotein were significantly higher than the model group(P < 0.05). The physiological and biochemical indicators of adiponectin-transfected adipose stem cell group were superior to those of adipose stem cell group, and the difference was significant(P <0.05). The hypercoagulant state of blood in the adipose stem cell group and adiponectin-transfected adipose stem cell group was significantly reduced compared to the model group, and this hypercoagulant state in adiponectin-transfected adipose stem cell group was significantly relieved(P < 0.05).(4) Compared with the model group, the expression levels of heparanase mR NA and protein in the kidney tissue of adipose stem cell group and adiponectin-transfected adipose stem cell group were significantly reduced(P < 0.05), and the expression level of adiponectin increased significantly(P < 0.05). The mRNA and protein expressions of heparanase and adiponectin in the adiponectin-transfected adipose stem cell group were on average superior to those in the adipose stem cell group(P < 0.05). To conclude,adiponectin-transfected adipose stem cell transplantation could significantly improve renal function, blood hypercoagulability and hyperlipidemia in rats with nephrotic syndrome. This effect may be related to the expression levels of heparanase and adiponectin
BACKGROUND:The expression of adiponectin gene plays an important role in the pathogenesis of nephrotic syndrome. Transplantation of adipose stem cells can has a certain therapeutic effect in rats with nephrotic syndrome.OBJECTIVE:To investigate the effect of adiponectin gene-transfected adipose stem cell transplantation on renal function and coagulation function in rats with nephrotic syndrome.METHODS:(1) Adipose stem cells were cultured in vitro and the cell suspension was prepared. CM-Dil was used as a marker. The third generation of adipose stem cells was used for electrotransfection using adiponectin gene. Western blot was used to detect the relative expression level of adiponectin in the cells at 3 and 28 days of transfection.(2) The random 20 of 80 Sprague-Dawley rats(provided by Beijing Vitone Lihua Animal Experimental Technology Co., Ltd., China) were taken as normal control group without any treatment, and the remaining60 rats were intravenously injected with doxorubicin to establish nephrotic syndrome models. These rat models were randomized into three groups:model group, adipose stem cell group and adiponectin-transfected adipose stem cell group. Normal control group and model group rats were injected 20 μL of PBS via the tail vein, and rats in the adipose stem cell group and adiponectin-transfected adipose stem cell group were intravenously injected with 20 μL of adipose stem cell suspension and adiponectin-transfected adipose stem cell suspension,respectively, once a day, for 7 continuous days.(3) At 4 weeks after transplantation, a full-automatic biochemical analyzer was used to detect serum biochemical indicators and coagulation indicators, hematoxylin-eosin staining used to observe renal histopathological changes,RT-PCR and Western blue used to detect the expression of heparanase and adiponectin in the kidney tissue.RESULTS AND CONCLUSION:(1) The high expression of adiponectin in adipose stem cells continued for 28 days.(2) The pathological improvement of adiponectin-transfected adipose stem cell group was superior to that of the adipose stem cell group, and the pathological manifestation in the adipose stem cell group was also slightly improved compared to the model group.(3) Serum levels of total cholesterol,triglyceride, low-density lipoprotein, creatinine, and 24-hour urea nitrogen level were significantly lower in the adipose stem cell group and adiponectin-transfected adipose stem cell group than in the model group(P < 0.05), while the serum levels of total protein, albumin, and high-density lipoprotein were significantly higher than the model group(P < 0.05). The physiological and biochemical indicators of adiponectin-transfected adipose stem cell group were superior to those of adipose stem cell group, and the difference was significant(P <0.05). The hypercoagulant state of blood in the adipose stem cell group and adiponectin-transfected adipose stem cell group was significantly reduced compared to the model group, and this hypercoagulant state in adiponectin-transfected adipose stem cell group was significantly relieved(P < 0.05).(4) Compared with the model group, the expression levels of heparanase mR NA and protein in the kidney tissue of adipose stem cell group and adiponectin-transfected adipose stem cell group were significantly reduced(P < 0.05), and the expression level of adiponectin increased significantly(P < 0.05). The mRNA and protein expressions of heparanase and adiponectin in the adiponectin-transfected adipose stem cell group were on average superior to those in the adipose stem cell group(P < 0.05). To conclude,adiponectin-transfected adipose stem cell transplantation could significantly improve renal function, blood hypercoagulability and hyperlipidemia in rats with nephrotic syndrome. This effect may be related to the expression levels of heparanase and adiponectin
引文
[1]管娜.儿童原发性肾病综合征治疗进展[J].中华儿科杂志,2017,55(8):638-640.
[2]郑秉暄,李金存,王慧,等.原发性肾病综合征高脂血症发生机制的研究进展[J].现代生物医学进展,2014,14(8):1572-1575.
[3]陈琪,朱彩风,朱斌.原发性肾病综合征的治疗机制研究进展[J].温州医科大学学报,2014,44(1):71-74.
[4]Kishida K,Funahashi T,Shimomura I.Adiponectin as a routine clinical biomarker.Best Pract Res Clin Endocrinol Metab.2014;28(1):119-130.
[5]Lim S,Quon MJ,Koh KK.Modulation of adiponectin as a potential therapeutic strategy.Atherosclerosis.2014;233(2):721-728.
[6]Lippi G,Meschi T,Mattiuzzi C,et al.Adiponectin and migraine:systematic review of clinical evidence.Neurol Sci.2014;35(8):1167-1171.
[7]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.
[8]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.
[9]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.
[10]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.
[11]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.
[12]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.
[13]Tabatabaei Qomi R,Sheykhhasan M.Adipose-derived stromal cell in regenerative medicine:A review.World J Stem Cells.2017;9(8):107-117.
[14]Saidi RF,Rajeshkumar B,Shariftabrizi A,et al.Human adipose-derived mesenchymal stem cells attenuate liver ischemia-reperfusion injury and promote liver regeneration.Surgery.2014;156(5):1225-1231.
[15]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.
[16]Kho MC,Park JH,Han BH,et al.Plantago asiatica L.Ameliorates Puromycin Aminonucleoside-Induced Nephrotic Syndrome by Suppressing Inflammation and Apoptosis.Nutrients.2017;9(4):E386.
[17]高大维,杨鹏.血管内皮生长因子基因修饰羊膜间充质干细胞移植治疗肾病综合征[J].中国组织工程研究,2018,22(1):83-88.
[18]Chru?ciel P,Sahebkar A,Rembek-Wieliczko M,et al.Impact of statin therapy on plasma adiponectin concentrations:A systematic review and meta-analysis of 43 randomized controlled trial arms.Atherosclerosis.2016;253:194-208.
[19]Kawwass JF,Summer R,Kallen CB.Direct effects of leptin and adiponectin on peripheral reproductive tissues:a critical review.Mol Hum Reprod.2015;21(8):617-632.
[20]程捷瑶,王美娟,马红,等.脂联素通过腺苷酸激活蛋白激酶途径抑制大鼠肝星状细胞氧化应激并调控转化生长因子β1和Ⅰ型胶原表达[J].中华肝脏病杂志,2015,23(1):69-72.
[21]王林,解汝娟,刘晓刚.脂代谢异常对肾病的影响[J].现代生物医学进展,2015,15(13):2577-2580.
[22]朱磊,解德琼,张臣丽,等.肾病综合征患者外周血T细胞亚群及血清脂联素水平与患者激素治疗效果的关系[J].河北医学,2017,23(10):1646-1649.
[23]陈海天,叶景光,吴淑端,等.妊娠期糖尿病孕妇胎盘组织中脂联素的表达及滋养细胞增殖的变化[J].中华围产医学杂志,2014,17(11):760-763.
[24]陈海天,王冬昱,耿慧珍,等.脂联素对体外高糖诱导的滋养细胞增殖的抑制作用[J].中华妇产科杂志,2016,51(3):204-208.
[25]屠叶平,施辉.脂联素和胱抑素C在糖尿病肾病早期诊断中的临床意义[J].现代中西医结合杂志,2014,23(20):2191-2194.
[26]杨笑云,常宝成,单春艳,等.“亚临床”2型糖尿病肾病患者尿血管紧张素原水平及相关因素分析[J].中华内分泌代谢杂志,2015,31(5):395-399.
[27]王丽,郭宏,王曦,等.不同分期老年2型糖尿病肾病患者外周血调节性和辅助性T细胞的表达[J].中国老年学杂志,2014,34(18):5142-5143.
[28]李美花.盐酸川芎嗪联合螺内酯治疗糖尿病肾病的疗效观察[J].中国实用医刊,2016,43(3):127-128.
[29]潘锡正,林芳.肾小球电荷屏障研究的新进展[J].医学综述,2014,20(12):2138-2140.
[30]郭倩,张肖,刘利飞,等.化瘀通络中药对糖尿病肾病大鼠肾小球电荷屏障的影响[J].中医杂志,2016,57(8):690-695.
[31]刘增波,梅长林,胡惠民,等.乙酰肝素酶对脓毒症肾小球内皮细胞糖萼的影响[J].中华肾脏病杂志,2014,30(7):524-529.
[2]郑秉暄,李金存,王慧,等.原发性肾病综合征高脂血症发生机制的研究进展[J].现代生物医学进展,2014,14(8):1572-1575.
[3]陈琪,朱彩风,朱斌.原发性肾病综合征的治疗机制研究进展[J].温州医科大学学报,2014,44(1):71-74.
[4]Kishida K,Funahashi T,Shimomura I.Adiponectin as a routine clinical biomarker.Best Pract Res Clin Endocrinol Metab.2014;28(1):119-130.
[5]Lim S,Quon MJ,Koh KK.Modulation of adiponectin as a potential therapeutic strategy.Atherosclerosis.2014;233(2):721-728.
[6]Lippi G,Meschi T,Mattiuzzi C,et al.Adiponectin and migraine:systematic review of clinical evidence.Neurol Sci.2014;35(8):1167-1171.
[7]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.
[8]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.
[9]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.
[10]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.
[11]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.
[12]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.
[13]Tabatabaei Qomi R,Sheykhhasan M.Adipose-derived stromal cell in regenerative medicine:A review.World J Stem Cells.2017;9(8):107-117.
[14]Saidi RF,Rajeshkumar B,Shariftabrizi A,et al.Human adipose-derived mesenchymal stem cells attenuate liver ischemia-reperfusion injury and promote liver regeneration.Surgery.2014;156(5):1225-1231.
[15]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.
[16]Kho MC,Park JH,Han BH,et al.Plantago asiatica L.Ameliorates Puromycin Aminonucleoside-Induced Nephrotic Syndrome by Suppressing Inflammation and Apoptosis.Nutrients.2017;9(4):E386.
[17]高大维,杨鹏.血管内皮生长因子基因修饰羊膜间充质干细胞移植治疗肾病综合征[J].中国组织工程研究,2018,22(1):83-88.
[18]Chru?ciel P,Sahebkar A,Rembek-Wieliczko M,et al.Impact of statin therapy on plasma adiponectin concentrations:A systematic review and meta-analysis of 43 randomized controlled trial arms.Atherosclerosis.2016;253:194-208.
[19]Kawwass JF,Summer R,Kallen CB.Direct effects of leptin and adiponectin on peripheral reproductive tissues:a critical review.Mol Hum Reprod.2015;21(8):617-632.
[20]程捷瑶,王美娟,马红,等.脂联素通过腺苷酸激活蛋白激酶途径抑制大鼠肝星状细胞氧化应激并调控转化生长因子β1和Ⅰ型胶原表达[J].中华肝脏病杂志,2015,23(1):69-72.
[21]王林,解汝娟,刘晓刚.脂代谢异常对肾病的影响[J].现代生物医学进展,2015,15(13):2577-2580.
[22]朱磊,解德琼,张臣丽,等.肾病综合征患者外周血T细胞亚群及血清脂联素水平与患者激素治疗效果的关系[J].河北医学,2017,23(10):1646-1649.
[23]陈海天,叶景光,吴淑端,等.妊娠期糖尿病孕妇胎盘组织中脂联素的表达及滋养细胞增殖的变化[J].中华围产医学杂志,2014,17(11):760-763.
[24]陈海天,王冬昱,耿慧珍,等.脂联素对体外高糖诱导的滋养细胞增殖的抑制作用[J].中华妇产科杂志,2016,51(3):204-208.
[25]屠叶平,施辉.脂联素和胱抑素C在糖尿病肾病早期诊断中的临床意义[J].现代中西医结合杂志,2014,23(20):2191-2194.
[26]杨笑云,常宝成,单春艳,等.“亚临床”2型糖尿病肾病患者尿血管紧张素原水平及相关因素分析[J].中华内分泌代谢杂志,2015,31(5):395-399.
[27]王丽,郭宏,王曦,等.不同分期老年2型糖尿病肾病患者外周血调节性和辅助性T细胞的表达[J].中国老年学杂志,2014,34(18):5142-5143.
[28]李美花.盐酸川芎嗪联合螺内酯治疗糖尿病肾病的疗效观察[J].中国实用医刊,2016,43(3):127-128.
[29]潘锡正,林芳.肾小球电荷屏障研究的新进展[J].医学综述,2014,20(12):2138-2140.
[30]郭倩,张肖,刘利飞,等.化瘀通络中药对糖尿病肾病大鼠肾小球电荷屏障的影响[J].中医杂志,2016,57(8):690-695.
[31]刘增波,梅长林,胡惠民,等.乙酰肝素酶对脓毒症肾小球内皮细胞糖萼的影响[J].中华肾脏病杂志,2014,30(7):524-529.