【作者】 邹杰；

【导师】 冯江敏；

【作者基本信息】 中国医科大学 ， 内科学， 2008， 博士

【摘要】 目的马兜铃酸肾病（arisolochic acid nephropathy,AAN）是由于服用含有马兜铃酸的中草药所致的肾脏损害。在不同类型的AAN中,以慢性马兜铃酸肾病（chronicaristolochic;acid nephropathy,CAAN）最为多见。进展迅速的小管萎缩、间质纤维化与肾功能损害不平行的早期严重贫血及可伴发肿瘤,是本病的突出特征。至今CAAN的发病机制尚不清楚。近年来研究显示,CAAN在有明显的肾小管损伤、肾间质纤维化之前,肾小管周毛细血管（peritubular capillary,PTC）已有所减少,且PTC减少与肾间质纤维化程度成正相关。因此,如何解决PTC丢失是治疗CAAN的重要环节。骨髓间充质干细胞（Mesenchymal stem cells,MSCs）移植,近年来已成为干细胞领域的研究热点。MSCs不仅支持造血系统,理论上还可向中胚层和外胚层来源的组织分化。最近国内外,体内、体外研究结果均已证明MSCs可向血管内皮细胞（endothelial cells,ECs）分化,为应用MSCs修复PTC,改善肾间质-小管低氧状态,提供了契机。本试验拟从以下三个方面进行研究:①探讨骨髓间充质干细胞是否具有向慢性马兜铃酸肾病肾小管周毛细血管内皮细胞分化的潜能;②骨髓间充质干细胞移植是否具有改善慢性马兜铃酸肾病肾间质缺氧状态的治疗作用;③骨髓间充质干细胞移植是否具有改善慢性马兜铃酸肾病肾间质纤维化的治疗作用。材料与方法第一部分:①MSCs的分离、纯化及培养。于超净台中,取出雄性Wistar大鼠（5只,155±5g,鼠龄6w）双侧股骨,用L-DMEM（低糖DMEM）培养液,冲洗骨髓腔,制备成骨髓单细胞悬液。用1.073的Percoll液来分离获得白色有核细胞层,PBS洗涤后,于1000rpm、4℃,离心30 min。用10%（体积分数）FBS的L-DMEM培养液（加青霉素100U/ml、链霉素100μg/ml）重悬细胞。以1×10⁷ cell/ml接种于25ml塑料培养瓶。培养条件为L-DMEM培养液（组方同上文）、37℃,5%CO₂,100%饱和湿度孵箱静置培养。48-72小时首次换液,弃悬浮细胞。经数次换液,培养10～14d后传代。当贴壁细胞达到90%左右的融合后,予以传代。②MSCs的鉴定。根据细胞表面抗原CD₃₄、CD₁₀₅、CD₂₉、CD₄₄表达情况,以鉴定第5代骨髓源细胞为MSCs后,经消化、洗涤,PBS调整细胞浓度为1×10⁷ cell╱ml后备用。将30只雌性大鼠随机分为3组:①慢性马兜铃酸肾病大鼠模型MSCs非移植组（下文均简称为CAAN组）;②慢性马兜铃酸肾病大鼠模型MSCs移植组（下文均简称为MSCs移植组）;③正常对照组。CAAN组和MSCs移植组各10只雌性大鼠经关木通水煎剂灌胃(关木通原药20g·kg^-1·d^-1,相当于关木通水煎剂10ml·kg^-1·d^-1,2次/日)12周制备CAAN大鼠模型。正常对照组雌性大鼠10只,予饮用水(10ml·kg^-1·d^-1,2次/日)灌胃12周。第12周,MSCs移植组经尾静脉输入1ml MSCs悬液;CAAN组和正常对照组经尾静脉输入1ml生理盐水。第16周,处死全部大鼠留取肾组织标本。本部分用免疫荧光（IF）和Y染色体荧光原位杂交（Y-FISH）法,检测Y染色体和CD34双阳性细胞的分布,观察MSCs移植组大鼠肾组织中MSCs向血管内皮细胞分化情况。第二部分:①MSCs的分离、纯化及培养。于超净台中,取出雄性Wistar大鼠（5只,155±5g,鼠龄6w）双侧股骨,用L-DMEM（低糖DMEM）培养液,冲洗骨髓腔,制备成骨髓单细胞悬液。用1.073的Percoll液来分离获得白色有核细胞层,PBS洗涤后,于1000rpm、4℃,离心30 min。用10%（体积分数）FBS的L-DMEM培养液（加青霉素100U/ml、链霉素100μg/ml）重悬细胞。以1×10⁷ cell╱ml接种于25ml塑料培养瓶。培养条件为L-DMEM培养液（组方同上文）、37℃,5%CO₂,100%饱和湿度孵箱静置培养。48-72小时首次换液,弃悬浮细胞。经数次换液,培养10～14d后传代。当贴壁细胞达到90%左右的融合后,予以传代。②MSCs的鉴定。根据细胞表面抗原CD₃₄、CD₁₀₅、CD₂₉、CD₄₄表达情况,以鉴定第5代骨髓源细胞为MSCs后,经消化、洗涤,PBS调整细胞浓度为1×10⁷ cell/ml后备用。将30只雌性大鼠随机分为3组:①慢性马兜铃酸肾病大鼠模型MSCs非移植组（下文均简称为CAAN组）;②慢性马兜铃酸肾病大鼠模型MSCs移植组（下文均简称为MSCs移植组）;③正常对照组。CAAN组和MSCs移植组各10只雌性大鼠经关木通水煎剂灌胃(关木通原药20g·kg^-1·d^-1,相当于关木通水煎剂10ml·kg^-1·d^-1,2次/日)12周制备CAAN大鼠模型。正常对照组雌性大鼠10只,予饮用水(10ml·kg^-1·d^-1,2次/日)灌胃12周。第12周,MSCs移植组经尾静脉输入1ml MSCs悬液;CAAN组和正常对照组经尾静脉输入1ml生理盐水。第16周,处死全部大鼠,留取血、尿、肾组织标本。处死前留取24小时尿以做24小时尿蛋白定量,眼眦静脉取血作血尿素氮、血清肌酐等生化指标检测。透射电镜、HE染色观察实验各组病理改变情况。应用免疫组织化学技术、Western blotting和RT-PCR等检测方法,观察实验各组大鼠肾组织PTC密度、HIF-1α、VEGF、VEGFR-2表达情况。第三部分:①MSCs的分离、纯化及培养。于超净台中,取出雄性Wistar大鼠（5只,155±5g,鼠龄6w）双侧股骨,用L-DMEM（低糖DMEM）培养液,冲洗骨髓腔,制备成骨髓单细胞悬液。用1.073的Percoll液来分离获得白色有核细胞层,PBS洗涤后,于1000rpm、4℃,离心30 min。用10%（体积分数）FBS的L-DMEM培养液（加青霉素100U/ml、链霉素100μg/ml）重悬细胞。以1×10⁷ cell/ml接种于25ml塑料培养瓶。培养条件为L-DMEM培养液（组方同上文）、37℃,5%CO₂,100%饱和湿度孵箱静置培养。48-72小时首次换液,弃悬浮细胞。经数次换液,培养10～14d后传代。当贴壁细胞达到90%左右的融合后,予以传代。②MSCs的鉴定。根据细胞表面抗原CD₃₄、CD₁₀₅、CD₂₉、CD₄₄表达情况,以鉴定第5代骨髓源细胞为MSCs后,经消化、洗涤,PBS调整细胞浓度为1×10⁷ cell/ml后备用。将30只雌性大鼠随机分为3组:①慢性马兜铃酸肾病大鼠模型MSCs非移植组（下文均简称为CAAN组）;②慢性马兜铃酸肾病大鼠模型MSCs移植组（下文均简称为MSCs移植组）;③正常对照组。CAAN组和MSCs移植组各10只雌性大鼠经关木通水煎剂灌胃(关木通原药20g·kg^-1·d^-1,相当于关木通水煎剂10ml·kg^-1·d^-1,2次/日)12周制备CAAN大鼠模型。正常对照组雌性大鼠10只,予饮用水(10ml·kg^-1·d^-1,2次/日)灌胃12周。第12周,MSCs移植组经尾静脉输入1ml MSCs悬液;CAAN组和正常对照组经尾静脉输入1ml生理盐水。第16周,留取肾组织标本。应用免疫组织化学技术和RT-PCR技术检测结缔组织生长因子（CTGF）、金属蛋白酶组织抑制物-1（TIMP-1）、胶原-Ⅰ（Col-I）的表达情况。观察MSCs移植后,有无改善慢性马兜铃酸肾病肾间质纤维化的治疗作用。结果第一部分:Y-FISH和CD₃₄免疫荧光染色结果。①正常对照组:第16周,雌性大鼠肾组织切片经Y-FISH检测未发现Y染色体阳性（Y⁺,黄绿色荧光亮点）细胞。②CAAN组:第16周,雌性CAAN大鼠肾组织切片经Y-FISH检测未发现Y⁺细胞。③MSCs移植组:第16周,大鼠肾组织切片经Y-FISH检测可见Y⁺细胞;同时,部分Y⁺细胞细胞浆、细胞膜同时可见红色荧光即内皮细胞标准物CD₃₄阳性,即发现Y染色体和CD₃₄双阳性细胞。Y⁺和CD₃₄双阳性细胞占所在视野内皮细胞的比例为（4.98±1.56）%,占所在视野总细胞的比例为（1.12±0.48）%。第二部分:①一般状态观察。CAAN组大鼠一般状态继续恶化,活动明显减少、进食极少、毛色秽暗、严重者出现耸毛、局部脱毛、稀水便加重。逐渐出现毛色暗黄、大便塘等现象;MSCs移植组可见大鼠一般状态明显好转,活动增多、进食增加、毛色恢复光泽、大便成形。②免疫组织化学结果。第16周,CAAN组与MSCs移植组结果分别为:CD₃₄-IOD值（8.26±0.08）×10³、（21.25±0.62）×10³,P＜0.01;PTC密度（5.26±0.78）/0.13mm²、（26.47±1.56）/0.13mm²,P＜0.01;HIF-1α-IOD值（25.27±1.46）×10³、（6.74±0.67）×10³,P＜0.01;VEGF-IOD值（2.75±0.38）×10³、（14.67±1.67）×10³,P＜0.01。③Western blotting结果。MSCs移植组CD₃₄、VEGF、VEGFR-2表达较CAAN组显著增强,P均＜0.01;MSCs移植组HIF-1α表达较CAAN组显著减弱,P＜0.01④RT-PCR结果。MSCs移植组HIF-1αmRNA较CAAN组表达显著减弱;MSCs移植组VEGFmRNA较CAAN组显著增加,P值均＜0.01。⑤MSCs移植组BUN、Scr和24h尿蛋白定量较CAAN组显著降低,P＜0.01。⑥肾组织形态学。HE染色可见CAAN组大部分肾小管萎缩、管壁断裂。肾血管壁增厚、存在广泛玻璃样变。MSCs移植组肾小管上皮细胞肿胀、空泡变性减轻,可见炎性细胞散在分布。电镜可见CAAN组ECs线粒体肿胀、线粒体脊断裂,胞浆空泡和致密颗粒血管基底膜结构不规整,节段性增厚伴有分层,部分ECs与基底膜分离、基底膜断裂、ECs脱落至血管腔内。MSCs移植组ECs线粒体肿胀程度、线粒体脊断裂程度减轻,血管基底膜清晰但是局部仍然厚薄不均。第三部分:①Masson染色。正常对照组肾小球、肾小管形态基本正常。第16周,CAAN组可见大部分肾小管萎缩、管壁断裂,肾间质纤维化面积显著扩大,出现多灶性纤维化。MSCs移植组肾小管上皮细胞肿胀,肾间质纤维化不明显。MSCs移植组较CAAN组肾间质纤维化面积显著减小,P值＜0.01。②免疫组织化学染色结果。第16周,CAAN组与MSCs移植组结果分别为:CTGF-IOD值（31.54±3.14）×10³、（11.28±2.57）×10³,P＜0.01;TIMP-1-IOD值（34.54±2.79）×10³、（16.75±0.74）×10³,P＜0.01;Col-1-IOD值（16.75±1.34）×10³、（8.37±1.12）×10³,P＜0.01;③RT-PCR结果。MSCs移植组较CAAN组HIF-1αmRNA、CTGFmRNA、TIMP-1mRNA的表达显著减弱,两者比较,P值均＜0.01。结论本研究结果表明:1、MSCs移植组大鼠肾脏组织行Y-FISH和CD₃₄荧光染色可见Y染色体和血管内皮细胞标志抗原CD₃₄双阳性细胞,从而证明了MSCs具有在CAAN大鼠肾脏向ECs分化的潜能。2、MSCs移植可增加CAAN大鼠肾脏PTC密度、降低HIF-1α表达,改善肾间质肾小管缺氧状态、并促进VEGF和VEGFR-2表达上调。3、第16周,CAAN大鼠存在明显肾功能恶化、24小时蛋白尿量显著增加。MSCs移植后CAAN大鼠肾功能、24小时蛋白尿量等生化指标得到改善。4、MSCs移植能明显减轻肾间质纤维化面积;降低肾脏纤维化相关因子CTGF、TIMP-1、Col-I的表达。更多还原

【Abstract】 ObjectiveAristolochic Acid Nephropathy（AAN） is a renal disease reported after the introduction of Chinese herbs contained aristolochic acid.The chronic aristolochic acid nephropathy（CAAN） is the commonest AAN.Rapidly progressive tubulointerstitial fibrosis and early,severe anaemia and urothelial malignancy of the upper urinary is the characteristics of AAN.Recently,attention has focused on the mechanism of fibrosis and anaemia in AAN.Pathogenesis of CAAN is not clear yet currently.Peritubular capillary（PTC） loss,with consequent tissue hypoxia and ischemia,play an important role in progressive CAAN.PTC loss contributed to the progressive tubulointerstitial fibrosis.Mesenchymal stem cells（MSCs） have the capability to differentiate cells of endothelial lineage in vitro.MSCs may be a useful source of stem cell therapy.So if MSCs could differentiate into endothelial cells in PTC and repair PTC,MSCs transplantation maybe lessen renal damage and interstitial fibrosis in the rat model of CAAN.Our investigation included three parts:①The differential potentiality study of MSCs into vascular endothelia cells（ECs） in PTC in the rat model of CAAN.②The therapeutic action of MSCs transplantation can repair PTC and improve the condition of hypoxia on the CAAN rats.③The effect of MSCs transplantation to the express of the connective tissue growth factor（CTGF）,tissue inhibitor of matrix metalloproteinase-1（TIMP-1） and collage-Ⅰ（Col-Ⅰ） in the kidney of chronic aristolochic acid nephropathy rats.Materials and MethodsPart 1:Under sterile conditions,the femurs of five male Wistar rats（155±5 g,age 6 weeks） were excised.Bone marrow plugs were extracted from the bones by flushing the bone marrow cavity with a 19G needle and L-DMEM.After mononuclear cells were isolated by centrifugation through 1.073 g/ml Percoll at 1000rpm and 4℃for 30 minutes,the cells were resuspended in DMEM culture medium,plated（1×10⁷ cells per 25 cm² culture flask）,and incubated at 37℃humidified atmosphere with 5%CO₂ for 3 days.The mesenchymal population was isolated on the basis of its ability to adhere to the culture plateau.At 90%confluence,the cells were trypsinized and were passaged to 25 cm² culture flasks at 1:3 ratios.Fifth-passage MSCs were used in all experiments. Fifth-passage MSCs were trypsinized and counted after rinse in phosphate-buffered saline（PBS）.Aliquots of cells were incubated for 30 minutes with PE/FITC labelled monoclonal polyclone against rat CD29,CD34,CD44 and CD105.After being washed with PBS,the stained cells were run on a flow cytometer to identify the purity of MSCs.Thirty female Wistar rats were randomly divided into 3 groups.The Normal Control Group was perfused intragastrically with drinking water for 12 weeks.MSCs Group and CAAN Group were perfused intragastrically with decoction of Caulis aristolochiae manchuriensis for 12 weeks.At weeks 12 after establishing the model of chronic aristolochic acid nephropathy（CAAN） successfully,we injected the MSCs from the male Wistar rat into MSCs Group by caudal vein and injected 0.9%saline solution into CAAN Group and The Normal Control Group respectively.At weeks 16, ten rats respectively in three groups were killed.Their kidneys were takenout. Fluorescence in situ hybridization（FISH） test and immunofluorescence with CY₃-labeled CD34 was used.HE and Masson staining were used to observe the pathology of the kidney. Part 2:Under sterile conditions,the femur of five male Wistar rats（155±5g, age 6 weeks） were excised.Bone marrow plugs were extracted from the bones by flushing the bone marrow cavity with a 19G needle and L-DMEM.After mononuclear cells were isolated by centrifugation through 1.073 g/ml Percoll at 1000rpm and 4℃for 30 minutes,the cells were resuspended in DMEM culture medium,plated（1×10⁷ cells per 25 cm² culture flask）,and incubated at 37℃humidified atmosphere with 5% CO₂ for 3 days.The mesenchymal population was isolated on the basis of its ability to adhere to the culture plateau.At 90%confluence,the cells were trypsinized and were passaged to 25cm² culture flasks at 1:3 ratios.Fifth-passage MSCs were used in all experiments.Fifth-passage MSCs were trypsinized and counted after rinse in phosphate-buffered saline（PBS）.Aliquots of cells were incubated for 30 minutes with PE/FITC labelled monoclonal polyclone against rat CD29,CD34,CD44 and CD105.After being washed with PBS,the stained cells were run on a flow cytometer to identify the purity of MSCs.Thirty female Wistar rats were randomly divided into 3 groups.The Normal Control Group was perfused intragastrically with drinking water for 12 weeks.MSCs Group and CAAN Group were perfused intragastrically with decoction of Caulis aristolochiae manchuriensis for 12 weeks.At weeks 12 after establishing the model of chronic aristolochic acid nephropathy（CAAN） successfully,we injected the MSCs from the male Wistar rat into MSCs Group by caudal vein and injected 0.9%saline solution into CAAN Group and The Normal Control Group respectively.At weeks 16, ten rats respectively in three groups were killed.Before killing the rats,specimens of blood and urine were Collected to detect the blood urea nitrogen（BUN）,serum creatinine（Scr） and urine protein.Their kidneys were takenout.HE staining and electron microscope were used to observe the pathology of the kidney. Immunohistochemistry,Western blotting and RT-PCR used to detect the expression of hypoxia-inducible factor-1α（HIF-1α）,CD34,Vascular endothelial growth factor acceptor 2（VEGFR-2） and Vascular endothelial growth factor（VEGF）. Part 3:Under sterile conditions,the femur of five male Wistar rats（155±5g, age 6 weeks） were excised.Bone marrow plugs were extracted from the bones by flushing the bone marrow cavity with a 19G needle and L-DMEM.After mononuclear cells were isolated by centrifugation through 1.073 g/ml Percoll at 1000rpm and 4℃for 30 minutes,the cells were resuspended in DMEM culture medium,plated（1×10⁷ cells per 25 cm² culture flask）,and incubated at 37℃humidified atmosphere with 5% CO₂ for 3 days.The mesenchymal population was isolated on the basis of its ability to adhere to the culture plateau.At 90%confluence,the cells were trypsinized and were passaged to 25cm² culture flasks at 1:3 ratios.Fifth-passage MSCs were used in all experiments.Fifth-passage MSCs were trypsinized and counted after rinse in phosphate-buffered saline（PBS）.Aliquots of cells were incubated for 30 minutes with PE/FITC labelled monoclonal polyclone against rat CD29,CD34,CD44 and CD105.After being washed with PBS,the stained cells were run on a flow cytometer to identify the purity of MSCs.Thirty female Wistar rats were randomly divided into 3 groups.The Normal Control Group was perfused intragastrically with drinking water for 12 weeks.MSCs Group and CAAN Group were perfused intragastrically with decoction of Caulis aristolochiae manchuriensis for 12 weeks.At weeks 12 after establishing the model of chronic aristolochic acid nephropathy（CAAN） successfully,we injected the MSCs from the male Wistar rat into MSCs Group by caudal vein and injected 0.9%saline solution into CAAN Group and The Normal Control Group respectively.At weeks 16, ten rats respectively in three groups were killed.Immunohistochemistry and RT-PCR were used to detect the expression of connective tissue growth factor（CTGF）,tissue inhibitor of matrix metalloproteinase-1（TIMP-1） and collage-Ⅰ（Col-Ⅰ）.ResultsPart 1:We couldn’t detect the Y chromatin-positive hybrid cells（Y⁺,flavo-green fluorescence brighiened dot） cell in the renal section of the Normal Control Group rat and the CAAN Group rat.Through Y chromosome fluorescence in situ hybridization. Then we detect the MSCs Group,also found the Y⁺ cells,and at the same time part of the Y⁺ cells expressed vascular endothelia cells phenotype CD34,with immunofluorescence by serial seetions.Part 2:At week 16,Scr and urine protein of MSCs Group were significantly lower than those of CAAN Group.The indices of MSCs Group and CAAN Group respectively follows:CD34-IOD[（21.25±0.62）×10³,（8.26±0.08）×10³,P＜0.01].the density of PTC[（26.47±1.56）/0.13mm² and（5.26±0.78）/0.13mm²,P＜0.01],HIF-1α-IOD [（6.74±0.67）×10³,（25.27±1.46）×10³,P＜0.01].The expression of HIF-1αand HIF-1αmRNA of MSCs Group were significantly lower than those of CAAN Group. The density of PTC and expression of VEGF,VEGFR-2 and VEGFmRNA of MSCs Group were significantly higher than those of CAAN Group.There was significantly different between two groups（statistic result respectively showed P＜0.01）.Part3:In the Normal Control Group,no significant abnormality was observed in the renal tissue samples,but severe injury was found in the CAAN Group and renal interstitial fibrosis was obvious.On the contrary,in the MSCs Group rats,there was moderate interstitial edema,no significant interstitial fibrosis.Interstitial fibrosis areas of the CAAN Group was increased,（P value was less than 0.01）.Compared to the CAAN Group rats was obviously improved through electron microscope.The indices of CAAN Group and MSCs group respectively follows:the density of CTGF-IOD [（31.54±3.14）×10³,（11.28±2.57）×10³,P＜0.01],TIMPI-IOD[（34.54±2.79）×10³,（16.75±0.74）×10³,P＜0.01],Col I-IOD[（16.75±1.34）×10³,（8.37±1.12）×10³,P＜0.01].The expression of CTGF,TIMP1and ColⅠmRNA of MSCs Group were significantly lower than those of CAAN Group.There was significantly different between two groups（statistic result respectively showed P＜0.01）.There was significantly different between two groups（statistic result respectively showed P＜0.01）ConclusionThese data suggested that: （1） Y chromosome and CD₃₄ double positive cells could be seen in renal tissue in MSCs Group. MSCs can differentiate into ECs in PTC.（2） MSCs can repair PTC and improve the condition of hypoxia.MSCs transplantation had beneficial effects on CAAN,which was possibly related with the reduction of PTC.MSCs transplantation can significantly improve the renal function of CAAN rats,lessen the level of 24h Urine protein.It expanded a new therapeutic way to CAAN,even to other renal failure.（3） MSCs transplantation can markedly remission renal pathological lesion of CAAN rats,lighten interstitial fibrosis.Decrease the protein and mRNA expression of CTGF,TIMP-1 and Col-Ⅰ.Therefore MSCs transplantation can lessen renal damage and interstitial fibrosis from the two aspects of cell and cytokine.It will provide a beneficial reference to the therapy of CAAN,even to that of other renal tubule interstitial fibrosis.更多还原