骨髓干细胞参与小鼠急性肾小管坏死修复的实验研究

【摘要】 目的 观察在生理和病理情况下,骨髓来源的干细胞(BMSC)能否分化成肾小管上皮细胞。 方法 绿色荧光蛋白(GFP)标记的C57BL/6转基因小鼠提供骨髓细胞。同种无荧光标记的C57BL/6小鼠100只分为正常对照组、全身照射组、缺血再灌注组、骨髓移植组、骨髓移植+缺血再灌注组。受体鼠的骨髓重建经血液常规检查和流式细胞仪检测确认,并采用荧光组织化学、免疫组织化学等方法观察绿色荧光标记的BMSC在受体鼠肾脏的分布及数量。 结果 全身致死剂量γ射线照射未造成小鼠肾脏组织结构和生理功能的明显改变。骨髓移植后第56、84天的受体鼠肾小管中有少量GFP阳性细胞的存在[(78.75±5.99)%、(79.58±4.60)%],激光共聚焦显微镜进一步证实这些细胞位于肾小管,并表达肾小管上皮细胞特异性的功能蛋白megalin。 结论 在生理和病理情况下,骨髓干细胞均可以向肾小管上皮细胞转分化,参与肾小管上皮细胞的更新,并且在急性肾小管坏死的病理条件下,骨髓干细胞的肾向转化率与肾脏受损程度有关。     

卡维地洛改善顺铂致肾小管上皮细胞凋亡

目的 观察顺铂致急性肾衰竭(ARF)中肾小管上皮细胞凋亡情况及卡维地洛(carvedilol)对其影响,并探讨其作用机制。方法 雄性Wistar大鼠随机分为盐水对照组、顺铂组、卡维地洛对照组、卡维地洛治疗组。测定BUN、Scr、尿乙酰氨基葡糖苷酶(NAG)、肾组织丙二醛(MDA)与超氧化物歧化酶(SOD)的含量。苏木素-伊红染色(HE)观察肾脏病理改变。原位缺口末端标记法(TUNEL)与DNA电泳观察肾小管上皮细胞的凋亡。Western印迹检测caspase-3的蛋白表达。结果 顺铂组大鼠BUN、Scr、尿NAG升高;肾脏病理改变加重;大量的肾小管上皮细胞凋亡; MDA含量增加,SOD活性降低;caspase-3的蛋白表达增加。上述指标在卡维地洛治疗组均得到改善,BUN从(58.33±19.93)降至(28.74±19.62)mmol/L;Scr从(425.56±97.96)降至(253.90±134.87)μmol/L;NAG从(224.77±75.86)降至(137.52±26.38)U/L;MDA从(18.13±7.01)降至(9.74±1.68)nmol/mg蛋白;SOD从(30.05±12.20)升至(64.67±20.64)U/mg蛋白;caspase-3的蛋白表达从1.94±0.73降至1.25±0.52;细胞凋亡从(42.5±12.6)%降至(23.7±8.4)%;肾组织病理损害也明显改善。结论 肾小管上皮细胞凋亡是顺铂致ARF的重要原因之一。卡维地洛能减轻顺铂的肾毒性,降低肾小管上皮细胞凋亡,其机制可能与减少活性氧(ROS)的产生,部分抑制了caspase依赖的凋亡途径有关。     

新生小鼠足细胞损伤对肾小球发育的影响

目的 探讨新生小鼠中足细胞损伤对肾小球发育的影响及其机制。 方法 于新生ICR小鼠出生后1 d注射嘌呤霉素(puromycin aminonucleoside，PA)，并以注射生理盐水作为对照。观察出生后第2、4、8、12、30、60、90天时肾重/体重、尿蛋白、血压及组织学的改变。应用免疫组化及定量RT-PCR方法测定肾皮质内肾母细胞瘤基因(WT-1)、CD31、血管内皮生长因子(VEGF)及其受体Flk-1、血管生成素(angiopoietin，Ang-1、Ang-2)及其受体Tie-1、Tie-2的表达水平。 结果 注射PA后，新生小鼠肾重、体重均明显低于对照组。出生后第2天(注射PA后1 d)时，肾小球足细胞出现足突广泛融合和微绒毛的脱落；第12天时，肾小球内CD31的表达明显下降，部分肾小球萎缩、发育不良，肾皮质浅层小球成熟指数明显下降；第30天时，原先发育不良的肾小球逐渐被吸收；第60天时，剩余肾小球出现系膜区的扩张和小球节段性硬化。PA鼠在第30天时出现蛋白尿；第60天时血压显著增高。定量RT-PCR显示，第2天时肾皮质Ang-1表达明显上调，Flk-1及Tie-2明显下降。 结论 PA可以在早期损伤的新生ICR小鼠足细胞，改变VEGF、血管生成素系统的表达，导致肾小球毛细血管袢发育不良及在后期产生蛋白尿、高血压和肾小球硬化。     

Alport综合征患者肾组织层粘连蛋白α2链异常分布

目的 观察Alport综合征(AS)患者肾组织层粘连蛋白α2链、α5链和γ1链的分布。方法 采用免疫荧光方法,运用普通荧光和激光共聚焦扫描显微镜观察抗层粘连蛋白α2链、α5链和γ1链单克隆抗体在肾组织中的沉积情况。肾组织标本来自11例AS患者,其中男8例,女3例,年龄11~52岁。10例患者符合X伴性显性遗传(XLAS),1例女性患者符合显性遗传。8例男性XLAS患者肾小球基底膜(GBM)、远端肾小管基底膜均无Ⅳ型胶原α3、5链沉积,表皮基底膜(EBM)无α5(Ⅳ)链沉积;2例女性XLAS患者肾组织α3、5(Ⅳ)链和EBM α5(Ⅳ)链均呈不连续表达,另1例女性患者则同正常肾组织。正常人肾组织标本作为正常对照,9例IgA肾病(IgAN)、6例局灶节段硬化性肾小球病(FSGS)、5例薄基膜病(TBMD)和6例肾小球轻微病变(GML)患者作为疾病对照。结果 正常人肾组织层粘连蛋白α2链主要沉积于肾小球系膜区,层连蛋白α5、γ1链沉积于GBM、所有肾小管基底膜和入球小动脉基底膜。10例XLAS和1例显性遗传AS患者肾组织除了肾小球系膜区外,层粘连蛋白α2链在GBM上亦出现表达。IgAN、TBMD和FSGS患者层粘连蛋白α2链仅在肾小球系膜区沉积。层粘连蛋白α5、γ1链在AS患者和其他肾脏病组织沉积同正常肾组织。结论 层粘连蛋白α2链在AS患者GBM出现异位表达,为继Ⅳ型胶原α链异常之后发现的又一AS基底膜成分的异常,其对于AS可能具有重要的诊断价值。     

他克莫司对糖尿病早期大鼠肾组织巨噬细胞浸润、增殖及活化的影响

目的 研究他克莫司( FK506)对糖尿病早期大鼠肾组织巨噬细胞浸润、增殖及活化的影响及探讨其肾脏保护作用机制.方法 链脲菌素(STZ)腹腔一次性注射建立大鼠糖尿病模型.按数字随机法分为对照组、模型组、他克莫司(0.5、1.0 mg·kg-1·d-1)治疗组,4周后观察大鼠肾质量指数(肾质量/体质量,KWI)、尿白蛋白排泄率(UAER)、肌酐清除率(Ccr)及肾组织病理形态学变化.应用免疫组化单染及双染方法检测肾组织内巨噬细胞表面标志抗原ED-1、增殖细胞核抗原(PCNA)及诱生性一氧化氮合酶(iNOS)的表达.结果 他克莫司1.0组大鼠KWI低于模型组(P＜0.05).他克莫司0.5组与1.0组大鼠UAER水平与肾小球平均体积低于模型组(P＜0.05).他克莫司1.0组肾小管间质损伤指数也明显低于模型组(P＜0.01).免疫组化显示模型组大鼠肾组织ED-1+、PCNA+及iNOS+巨噬细胞数显著高于对照组(P＜0.01);他克莫司0.5与1.0组ED-1+的巨噬细胞数与模型组差异无统计学意义;PCNA+及iNOS+的巨噬细胞数则显著低于模型组(P＜0.01).结论 他克莫司可改善糖尿病早期大鼠肾损害,其机制可能部分与抑制肾组织中巨噬细胞的增殖及活化有关.     

脯氨酸羟化酶2 siRNA对抗霉素诱导肾小管上皮细胞凋亡的影响

目的 探讨是否脯氨酸羟化酶2 (PHD2) siRNA 通过减少缺氧诱导因子(HIF)1的降解而减轻缺氧引起的肾小管上皮细胞凋亡。方法 构建针对人PHD2的表达质粒,将其转染到培养的人肾小管上皮细胞系(HKC)中。用RT-PCR方法观察其对PHD2表达的抑制作用。Western印迹观察HIF-1α的表达。培养的HKC细胞分为对照组、模型组和siRNA组、siRNA组经PHD2 siRNA转染后与模型组同时用抗霉素诱导,用Western印迹方法观察3组细胞HIF-1α的表达,用流式细胞仪观察3组细胞的凋亡情况。 结果 经PHD2 siRNA转染的HKC细胞PHD2 mRNA表达明显下调(P < 0.01),HIF-1α蛋白表达明显上调(P < 0.05)。经抗霉素处理后,模型组和siRNA组HIF-1α蛋白表达均明显高于对照组(P < 0.01),但siRNA组HIF-1α蛋白表达高于模型组(P < 0.05。与模型组相比,siRNA组细胞凋亡明显减轻(P < 0.05)。 结论 通过RNA干扰技术抑制PHD2的表达可以增强HKC细胞HIF-1的蛋白表达,从而增强其在缺氧条件下的抗凋亡能力。     

骨碎补类黄酮对氯化汞所致的急性肾衰竭大鼠模型的保护作用

目的:探讨骨碎补类黄酮提取物(flavonoid fraction,FF)对氯化汞(HgCI2)所致的急性肾衰竭(ARF)大鼠模型的保护作用.方法:利用HgCI2诱导的ARF动物模型,将大鼠随机分成正常对照组、HgCI2(模型组)、HgCI2 FF组、FF组.在实验第24、48、72 h检测血生化指标,光镜观察肾组织改变,免疫组化染色检测增殖细胞核抗原(PCNA)、ED-1在肾组织的表达,同时检测肾组织MDA及GSH含量.结果:HgCI2 FF组肾脏病理改变比HgCI2组减轻,同时血肌酐下降(P<0.05);免疫组化显示PCNA、ED-1在肾间质表达下调,均与HgCI2组有统计学差异(分别P<0.01及P<0.05);FF还可预防肾组织因HgCI2毒性所致的MDA含量升高,GSH下降(P<0.05).结论:FF通过抗氧化作用,减轻HgCI2诱导的急性肾衰竭.     

骨髓间充质干细胞对缺血再灌注损伤肾小管上皮细胞增殖和凋亡的影响

目的：探讨外源性骨髓间充质干细胞（MSCs）移植对缺血再灌注损伤（I/R）后肾小管上皮细胞增殖和凋亡的影响。方法：将雄性SD大鼠MSCs用DAPI标记后注入受体雌性SD大鼠体内。30只受体大鼠随机分为3组：假手术对照组（C组）、MSCs＋I/R组（M组）、DMEM-F12＋I/R组（D组）,每组10只。7d后观察肾功能,肾脏病理改变,采用原位末端标记法检测细胞凋亡指数,免疫组化法检测增殖细胞核抗原（PCNA）的表达,并观察DAPI标记的MSCs在受体大鼠肾脏的分布情况。结果：I/R后第7天,M组在肾功能、肾脏病理改变上,均明显好于D组;肾组织内PCNA＋细胞数和凋亡指数均低于D组。I/R后7d内未发现MSCs定位于肾组织中。结论：外源性MSCs可以促进I/R损伤后肾小管上皮细胞的增殖和减少细胞凋亡,从而有利于肾小管损伤的早期恢复。     

依贝沙坦抑制造影剂诱导的肾小管上皮细胞凋亡

目的 探讨依贝沙坦在造影剂诱导肾小管上皮细胞凋亡中的作用及机制。方法 培养的大鼠肾小管细胞(NRK-52E)分别与不同碘浓度(25、50、100、150 mgI/ml)的安射力(非离子型造影剂)共孵育1 h;安射力(100 mgI/ml)先后刺激NRK-52E细胞0.5、1、2、4 h。与安射力(100 mgI/ml)相同渗透浓度的甘露醇(420 mmol/L)与NRK-52E细胞共孵育1 h作为阳性对照。不同浓度依贝沙坦(0.01、0.1、1 mmol/L)先与NRK-52E细胞共孵育1 h后,安射力(100 mgI/ml)刺激1 h。Hoechst染色和流式AnnexinV-FITC/PI双染法检测细胞凋亡。激光共聚焦显微镜检测细胞内活性氧(ROS)水平。RT-PCR检测Bax、Bcl-2 mRNA的表达。 结果 安射力呈浓度和时间依赖性诱导NRK-52E细胞凋亡。与安射力相同渗透浓度的甘露醇不能明显诱导细胞凋亡。安射力与NRK-52E细胞共孵育后,细胞内ROS产生增多,Bcl-2 mRNA的表达下调,Bax mRNA的表达上调。依贝沙坦呈浓度依赖性抑制安射力诱导的NRK-52E细胞凋亡、细胞内ROS的产生、Bcl-2 mRNA表达下调及Bax mRNA表达上调。细胞内ROS水平与细胞凋亡呈正相关。 结论 安射力呈浓度和时间依赖性诱导NRK-52E细胞凋亡,此作用与细胞内氧化应激及bcl-2 mRNA表达下调、Bax mRNA表达上调相关。依贝沙坦能抑制上述安射力的作用,呈浓度依赖性抑制NRK-52E细胞凋亡。     

大鼠骨髓间充质干细胞移植治疗肾小管间质损伤的实验研究

目的:探讨骨髓间充质干细胞(MSCs)移植对大鼠单侧输尿管梗阻(UUO)后肾小管间质损伤的治疗效果。方法:密度梯度离心法和贴壁细胞培养法结合分离纯化MSCs。取成年雄性Wistar大鼠40只,随机分为移植组(组A,n=15)、对照组(组B,n=15)、假手术组(组C,n=10)。移植组和对照组分别结扎左侧输尿管,制作肾小管间质纤维化模型。移植组于结扎当日肾脏多点注射Brd U标记的含MSCs 1×10~6的DMEM培养基50μl,对照组注射等量DMEM培养基。假手术组开腹但不结扎输尿管。术后第14天处死各组大鼠,行HE染色,观察肾脏病理变化;免疫组化方法测定BrdU阳性细胞在肾脏的分布以及TGF-β_1,Ki-67的表达情况。结果:HE染色显示移植组与对照组肾脏间质损伤严重,两组相比无明显差异,假手术组正常。移植组肾实质内可见Brd U标记的MSCs,向周围组织分散,形态上与肾间质成纤维细胞类似,肾小管上皮细胞中未发现MSCs掺入。移植组较对照组TGF-β_1表达差异无统计学意义。移植组Ki-67表达较对照组有明显增强。结论:UUO大鼠骨髓间充质干细胞移植后,肾小管及间质细胞增生明显增高,提示骨髓间充质干细胞同种异体移植可能通过促进肾脏细胞的增生对肾间质损伤起到一定治疗作用。具体机制尚待进一步研究。     

骨髓间充质干细胞促进大鼠IgA肾病修复

目的 观察骨髓间充质干细胞（MSC）对大鼠IgA肾病有无修复作用，并探讨其可能的机制。 方法 SD大鼠随机分为MSC注射组、生理盐水（NS）组及健康对照组。前两组以牛血清白蛋白(BSA)+葡萄球菌肠毒素B（SEB）＋皮下注射四氯化碳（CCl4）的改良法建立IgA肾病模型。体外连续培养SD大鼠MSC并通过流式细胞仪和成骨成脂细胞诱导分化鉴定MSC，用5-溴脱氧尿嘧啶核苷（BrdU）体外标记培养的MSC。移植后1周及4周分别观察3组的体质量、尿蛋白量（24 h）、肾功能、肾脏病理变化、IgA荧光沉积变化；ELISA法检测尿中的MCP-1、TGF-β1量；RT-PCR法检测肾组织中MCP-1、TGF-β1 mRNA的表达情况；免疫组化观察细胞因子及BrdU标记的MSC在肾组织中的分布情况。 结果 移植后1周，MSC组尿蛋白量（24 h）（36.86±4.78） mg，Scr（53.50± 6.28） μmol/L；NS组尿蛋白量（24 h）(66.98±5.86) mg，Scr (82.50±8.36) μmol/L，两组差异有统计学意义（均P < 0.05）；同时，MSC组MCP-1、TGF-β1在尿中的含量及肾脏中表达均显著低于NS组（均P < 0.05）。移植后4周，MSC组体质量、肾脏病理变化、IgA荧光沉积与NS组差异有统计学意义；MCP-1、TGF-β1在尿中的含量及肾脏中的表达与健康对照组差异无统计学意义。随时间延长，BrdU标记的MSC在肾组织中分布却逐渐减少。 结论 MSC输注可促进大鼠IgA肾病的修复，其作用机制可能并不完全是依赖于MSC的直接分化，而是通过调节肾组织中细胞因子的分泌和（或）其他的功能进行修复。     

体外诱导骨髓间充质干细胞向肾小管上皮样细胞分化

目的 观察不同条件下骨髓间充质干细胞（MSC）体外诱导分化为肾小管上皮样细胞的差异。 方法 抽取SD大鼠的骨髓,经密度梯度离心分离,联合贴壁筛选法获取纯化的MSC。以流式细胞仪鉴定间充质干细胞表面标志。取扩增3代的MSC分组培养：（1）对照组：用含胎牛血清培养基;（2）全反式维甲酸（ATRA）组：胎牛血清+缺血再灌注肾脏匀浆上清+ATRA;（3）联合诱导组：胎牛血清+缺血再灌注肾脏匀浆上清+ATRA+表皮生长因子（EGF）+骨形成蛋白（BMP-7）。诱导7 d后,倒置显微镜下观察细胞形态变化;化学染色检测细胞碱性磷酸酶表达;免疫细胞化学法检测细胞角蛋白18（cytokeratin-18）、E钙黏蛋白(E-cadherin)的表达。 结果 流式细胞仪显示,体外分离培养的第3代MSC,CD44阳性细胞表达率为97.8%±0.9%;CD90阳性细胞表达率为96.8%±1.4%;CD29阳性细胞表达率为97.6%±2.4%;而CD11b/c阳性细胞表达率为13.2%±0.6%; CD34阳性细胞表达率为1.2%±0.5%。诱导7 d后,与对照组长梭形细胞相比,ATRA组部分细胞为圆形、短梭形单层排列;联合诱导组的大部分细胞为圆形、短梭形,细胞密集处呈鹅卵石样排列。碱性磷酸酶染色显示,对照组细胞为阴性;ATRA组部分细胞阳性;联合诱导组阳性细胞数明显增多。免疫细胞化学显示,ATRA组和联合诱导组细胞cytokeratin-18阳性表达率分别为29.47%±1.08%和47.52%±2.13%,显著高于对照组（P ＜ 0.05）;E-cadherin阳性表达率分别为14.88%±2.46%和36.15%±1.13%,也显著高于对照组（P ＜ 0.05）。 结论 在体外模拟的急性肾衰竭微环境中加入ATRA可诱导MSC部分分化为肾小管上皮样细胞。联合EGF、BMP-7共同诱导能进一步促进MSC向肾小管上皮样细胞分化。     

Therapeutic implications of mesenchymal stem cells transfected with hepatocyte growth factor transplanted in rat kidney with unilateral ureteral obstruction

Purpose

The purpose of the study was to establish whether bone marrow mesenchymal stem cells (MSCs) transfected with hepatocyte growth factor (HGF) can migrate and localize in the rat's kidney with unilateral ureteral obstruction (UUO) and contribute to repair of renal fibrosis.

Methods

We separated and cultured bone marrow-derived MSCs of male rats in vitro and transfected them with adenovirus-mediated HGF (Ad-HGF). The expression of HGF was measured with enzyme-linked immunosorbent assay. Sixty female rats were sham operated (n = 24) or subjected to left UUO: Ad-HGF-transfected MSCs, uninfected MSCs, or saline was injected into the rat's tail vein. Kidney tissue was collected at the end of the seventh or 14th day after operation. The distribution of Y chromosome in the kidney after Ad-HGF-transfected MSCs transplantation was determined by an in situ hybridization method. As the hallmark of myofibroblasts, α-smooth muscle actin (expression of which significantly increases in the presence of renal fibrosis) was detected by immunohistochemistry in all UUO rats' left kidney tissue.

Results

Y chromosome-positive cells were found only in the obstructed kidney of the transplantation group. The positive cells were mainly distributed in the tubular cells. The average intensity of immunolabeling for α-smooth muscle actin in the transplanted group significantly decreased compared with sham-transplanted group (P < .05), and the expression in the rats injected with uninfected MSCs was higher than that in the rats with MSCs transfected with HGF (P < .05).

Conclusions

Mesenchymal stem cells transfected with HGF can migrate to the rat kidney with UUO and are mainly distributed in the region of renal tubular epithelial cells. The data indicate that MSCs transfected with HGF contribute to a reduction of renal fibrosis after ureteral obstruction and suggest that this may be exploited therapeutically.     

Distribution of infused umbilical cord mesenchymal stem cells in a rat model of renal interstitial fibrosis

Abstract

Aims: Stem cell transplantation for the treatment of kidney diseases is dependent on choice of transplant pathway. We evaluated the safety of human umbilical cord mesenchymal stem cells through peripheral infusion and their distribution in a rat model of renal interstitial fibrosis (RIF). Method: Cryopreserved umbilical cord mesenchymal stem cells were infused via tail vein injection into rats with unilateral ureteral obstruction and Sham-operated. Blood, kidney, heart, liver, spleen and lung were collected at 14, 21, and 28 days after infusion. Testing included microscopic observation of kidney morphological changes and immunohistochemical testing to identify and count the number of MAB1281 (labeled human cells) positive cells in the heart, liver, spleen, lungs, and kidneys of different treatment groups. Results: There was no significant difference in the Sham-operated group and Sham-operated?+?cell transplantation group at different time points. Human cells were identified mainly in the lungs, spleen, and kidney. The number of human umbilical cord mesenchymal stem cells in the kidney was greater in the unilateral ureteral obstruction?+?cell transplantation group, compared to the Sham-operated?+?cell transplantation group. human umbilical cord mesenchymal stem cells were mainly located in the interstitium of the left kidney. These results suggest that infused mesenchymal stem cells were primed to engraft a damaged kidney, especially damaged renal interstitium. Conclusions: Intravenous infusion of exogenous umbilical cord mesenchymal stem cells is feasible and safe. Infused mesenchymal stem cells can reach damaged kidney tissues with obstructive RIF after a vein graft.     

大鼠供肾转染反义ERK2基因减缓肾移植后慢性移植肾肾病的作用及机制

目的 研究腺病毒介导的反义ERK2(A-dant-ERl(2)基因转染供肾对减缓肾移植后发生慢性移植肾肾病(CAN)的作用及机制.方法 建立大鼠间肾移植模型.按供肾移植前处理方式的不同分为对照组、空载病毒组和基因转染组,每组6只.对照组供肾灌注无菌HTK液0.5 ml,空载病毒组供肾灌注含5x109>pfu LaeZ基因腺病毒(Ad-LacZ)的HTK液0.5 ml,基因转染组供肾灌注含5x 109>pfu Adanti-ERK2的HTK液0.5 ml.肾移植术后24周行移植肾的病理学观察,免疫组织化学法观察肾小管上皮细胞表面标志蛋白E-Cadherin、Vimentin、TβRⅠ的表达以及CD4+、CD8+T淋巴细胞和ED-1+细胞的浸润情况;酶联免疫法检测受者血清中转化生长因子β1>(TGF-β1>)的含量.结果 肾移植术后24周,对照组和空载病毒组移植肾呈CAN表现;肾小球硬化,肾小管萎缩明显,伴严重间质纤维化以及明显的CD4+、CD8+T淋巴细胞和ED-1+细胞浸润;病变区肾小管上皮细胞E-Cadherin表达减少,Vimentin、TβRⅠ表达显著增多.基因转染组移植肾间质内仅有少量CD4+、CD8+T淋巴细胞和ED-1+细胞浸润;肾小管上皮细胞E-Cadherin表达正常.对照组和空载病毒组血清中TGF-β1>,含量明显高于基因转染组.结论 Adanti-ERK2基因转染移植肾可减缓CAN的发生,对移植肾具有保护作用.这种保护机制可能与减少炎症细胞的浸润、下调TGF-β1>,等致纤维化因子的表达以及抑制肾小管上皮细胞向间充质细胞的转化有关.     

Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renal failure

Injury to a target organ can be sensed by bone marrow stem cells that migrate to the site of damage, undergo differentiation, and promote structural and functional repair. This remarkable stem cell capacity prompted an investigation of the potential of mesenchymal and hematopoietic stem cells to cure acute renal failure. The model of renal injury induced in mice by the anticancer agent cisplatin was chosen. Injection of mesenchymal stem cells of male bone marrow origin remarkably protected cisplatin-treated syngeneic female mice from renal function impairment and severe tubular injury. Y chromosome-containing cells localized in the context of the tubular epithelial lining and displayed binding sites for Lens culinaris lectin, indicating that mesenchymal stem cells engraft the damaged kidney and differentiate into tubular epithelial cells, thereby restoring renal structure and function. Mesenchymal stem cells markedly accelerated tubular proliferation in response to cisplatin-induced damage, as revealed by higher numbers of Ki-67-positive cells within the tubuli with respect to cisplatin-treated mice that were given saline. Hematopoietic stem cells failed to exert beneficial effects. These results offer a strong case for exploring the possibility that mesenchymal stem cells by virtue of their renotropic property and tubular regenerative potential may have a role in the treatment of acute renal failure in humans.     

输注供鼠骨髓间充质干细胞延长肾移植大鼠的存活时间

目的 探讨在同种大鼠肾移植中输注供者骨髓间充质干细胞(MSC)对急性排斥反应的影响以及延长受鼠存活时间的作用.方法 取Wistar大鼠骨髓,分离和培养其MSC.以Wistar 大鼠为供者,Lewis大鼠为受者,建立同种大鼠肾移植模型.根据受鼠处理方式的不同,分为低剂量MSC组、高剂量MSC组、CsA组及对照组,低剂量MSC组和高剂量MSC组于移植前后分别多次输注1×106个和t×107个供者MSC,CsA组于术后2d开始腹腔内注入CsA 0.5 mg·kg-1 ·d-1,以腹腔内注射PBS作为对照.移植后,比较各组受鼠的存活时间,观察各组移植肾功能及移植肾组织病理学改变.结果 低剂量MSC组、高剂量MSC组、CsA组及对照组受鼠的存活时间分别为(21.7±7.2)d、(31.2±14.3)d、(34.9±15.7)d及(9.0±2.3)d;低剂量MSC组、高剂量MSC组和CsA组存活时间均明显长于对照组(P＜0.01),而低剂量MSC组存活时间明显短于高剂量MSC组和CsA组(P＜0.05).术后第4天,高剂量MSC组和CsA组移植肾组织形态和结构基本正常;对照组移植肾组织则表现出典型的急性排斥反应,出现广泛间质性浸润,肾小管炎症和片状坏死、出血,肾小球炎症浸润严重;而与对照组相比,低剂量MSC组急性排斥反应的病理表现则要明显减轻.结论 同种肾移植大鼠输注供者MSC后,可以达到有效的免疫调节作用,并且可明显延长大鼠的存活时间,呈MSC剂量依赖性.     

骨髓间充质干细胞自体移植时间对兔急性肾功能衰竭治疗效果的影响

目的 观察骨髓间充质干细胞(MSCs)自体移植对兔急性肾功能衰竭的治疗作用并探讨不同移植时间对其治疗效果的影响.方法 骨髓穿刺抽取新西兰大耳白兔骨髓,分离、培养、扩增MSCs.30只兔通过夹闭双肾动脉90 min后再灌注制作急性肾功能衰竭模型,随机分为移植A组、移植B组和未治疗C组,每组10只.移植A组在恢复血流即刻,移植B组在恢复血流后72 h将BrdU标记的自体MSCs经颈静脉回输移植,未治疗C组仅制作急性肾功能衰竭模型.于造模后21 d处死兔,比较各组的存活率、肾功能及肾组织形态学改变.结果 与未治疗组比较,移植组肾功能较快恢复(P<0.05),肾组织损伤明显改善(P<0.05).移植A组的效果优于移植B组(P<0.05).结论 骨髓间充质干细胞自体移植能有效治疗缺血再灌注损伤引起的急性肾功能衰竭,早期移植的效果好.     

Administered mesenchymal stem cells enhance recovery from ischemia/reperfusion-induced acute renal failure in rats

BACKGROUND: Adult stem cells are promising for the development of novel therapies in regenerative medicine. Acute renal failure (ARF) remains a frequent clinical complication, associated with an unacceptably high mortality rate, in large part due to the ineffectiveness of currently available therapies. The aim of this study was, therefore, to evaluate the therapeutic effectiveness of bone marrow-derived mesenchymal stem cells in a rat model of ischemia/reperfusion (I/R) ARF. METHODS: We used a common I/R model in rats to induce ARF by clamping both renal pedicles for 40 minutes. Mesenchymal stem cells were iron-dextran-labeled for in vivo tracking studies by magnetic resonance imaging (MRI) and kidneys were imaged for mesenchymal stem cells immediately after infusion and at day 3 after ARF. Renal injury was scored on day 3 and cells were additionally tracked by Prussian blue staining. RESULTS: We show in I/R-induced ARF in rats, modeling the most common form of clinical ARF, that infusion of mesenchymal stem cells enhances recovery of renal function. Mesenchymal stem cells were found to be located in the kidney cortex after injection, as demonstrated by MRI. Mesenchymal stem cells-treated animals had both significantly better renal function on days 2 and 3 and better injury scores at day 3 after ARF. Histologically, mesenchymal stem cells were predominantly located in glomerular capillaries, while tubules showed no iron labeling, indicating absent tubular transdifferentiation. CONCLUSION: We conclude that the highly renoprotective capacity of mesenchymal stem cells opens the possibility for a cell-based paradigm shift in the treatment of I/R ARF.     

In vivo effect of bone marrow‐derived mesenchymal stem cells in a rat kidney transplantation model with prolonged cold ischemia

Brain death and prolonged cold ischemia are major contributors to the poorer long‐term outcome of transplants from deceased donor kidney transplants, with an even higher impact if expanded criteria donors (‘marginal organs’) are used. Targeting ischemia‐reperfusion injury‐related intragraft inflammation is an attractive concept to improve the outcome of those grafts. As mesenchymal stem cells (MSCs) express both immunomodulatory and tissue repair properties, we evaluated their therapeutic efficacy in a rat kidney transplant model of prolonged cold ischemia. The in vitro immunomodulatory capacity of bone marrow‐derived rat MSCs was tested in co‐cultures with rat lymph node cells. For in vivo studies, Dark Agouti rat kidneys were cold preserved and transplanted into Lewis rats. Syngeneic Lewis MSCs were administered intravenously. Transplants were harvested on day 3, and inflammation was examined by quantitative RT‐PCR and histology. Similarly to MSCs from other species, rat MSCs in vitro also showed a dose‐dependent immunomodulatory capacity. Most importantly, in vivo administration of MSCs reduced the intragraft gene expression of different pro‐inflammatory cytokines, chemokines, and intercellular adhesion molecule‐1. In addition, fewer antigen‐presenting cells were recruited into the renal allograft. In conclusion, rat MSCs ameliorate inflammation induced by prolonged cold ischemia in kidney transplantation.