超声介导的微泡破坏促进MSCs归巢并修复糖尿病肾病的实验研究
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摘要
研究背景：
糖尿病肾病(diabetic nephropathy, DN)是终末期肾脏疾病(end-stage renal disease,ESRD)的主要原因，与ESRD较高的死亡率密切相关。目前广泛认为小管间质损害是ESRD可靠的预测因子并与肾功能障碍密切相关。微量白蛋白尿是糖尿病肾病的早期征象。现有许多证据表明对糖尿病肾病进行早期干预和精确调控可以减缓，甚至逆转糖尿病肾病的进程。因此，对早期糖尿病肾病进行及早干预、提高治疗物质在肾间质的聚集是目前医学领域亟待解决的问题。目前治疗糖尿病肾病的常用方法诸如调控血糖、胰腺和肾脏移植、透析治疗等虽然能改善肾功能，却因当前药物治疗和治疗技术的有限性，导致并发症多、生存质量不高，效果并不显著。
骨髓来源的间充质干细胞(bone marrow-derived mesenchymal stem cells, BM-MSCs)移植治疗是近年来国内外研究的重点，其易于分离提取，具有高度的扩增潜能，有良好的基因稳定性、组织相容性好、不涉及伦理道德问题，为受损组织和器官的修复开辟了一条新途径。MSCs能够分化为功能性的胰岛素生成细胞，逆转糖尿病大鼠高血糖状态，也能够分化为肾脏细胞修复受损肾脏。因而，MSCs移植治疗被认为是改善高血糖和肾功能的、具有极大吸引力的治疗手段。但骨髓中MSCs含量极少，远远小于肾脏修复所需要细胞的量，加上目前干细胞移植治疗移植后细胞存活率较低、干细胞靶向归巢能力欠佳等问题，均极大地限制了其应用。因此，如何调控骨髓干细胞的肾向性、提高干细胞靶向归巢能力和移植的有效性是干细胞移植治疗肾脏疾病的关键。
超声介导的微泡破坏(Ultrasound-mediated microbubble destruction)作为一种新的、极具前景的治疗方法应用于多个领域，例如将药物和基因传递到心血管系统，开放血脑屏障和血瘤屏障，增强毛细血管和血管通透性以及促进干细胞归巢等。肾脏是高滤过器官，对生物学效应较为敏感，对于肾脏疾病的干细胞移植治疗，超声介导的微泡破坏作用是否同样有效呢？研究显示，超声基因转染治疗仪(1.0W/cm~2)联合自制微泡能有效促进MSCs归巢至肾脏，使急性肾损伤大鼠肾功能得到明显改善。该研究还发现，超声+微泡组大鼠肾脏ICAM-1mRNA表达高于对照组、超声+微泡+MSCs组大鼠肾脏ICAM-1mRNA表达高于单纯MSCs移植组，说明超声介导的微泡破坏可以增加MSCs的靶向黏附、提高MSCs归巢至肾脏的能力，进而更有效地实现MSCs靶向移植治疗肾脏疾病的效果。
然而，关于慢性肾脏疾病的干细胞治疗，超声介导的微泡破坏作用对其影响的研究较少，本实验尝试利用超声介导的微泡靶向破坏作用，经静脉移植MSCs治疗链脲佐菌素(streptozotocin, STZ)诱导的1型糖尿病肾病大鼠，并试图探索其安全性及可能机制，期望能提供一种新的、非侵入性的方法，对糖尿病肾病患者给予系统性药物治疗的前提下选择性增强干细胞在肾脏的聚集，最大化提高糖尿病肾病的治疗效果，为提高干细胞无创、有效地靶向归巢及减缓疾病进程、或是逆转糖尿病肾病病程进展提供一种新思路和新方法。
研究目的：
1.探讨诊断超声介导的微泡破坏促进经静脉移植的同源异体MSCs归巢糖尿病肾病大鼠肾脏的可行性和安全性；
2.探讨在诊断超声介导的微泡破坏作用下，经静脉移植的MSCs对修复大鼠糖尿病肾病的有效性；
3.初步探讨诊断超声介导的微泡破坏技术对促进静脉移植的MSCs靶向归巢并修复大鼠糖尿病肾病的可能机制。
研究方法：
1.采用全骨髓培养法对SD (Sprague Dawley)大鼠骨髓MSCs进行分离、培养、及纯化，检测其生物学特性。流式细胞仪检测细胞表面标记分子，成脂、成骨诱导分化进行细胞鉴定。为便于对体内干细胞的追踪，采用携带增强型绿色荧光蛋白的慢病毒对培养的MSCs进行转染，并观察转染后MSCs表达绿色荧光情况及细胞毒性反应。
2.腹腔单次注射60mg/Kg SZT建立稳定的大鼠1型早期糖尿病肾病模型，随机血糖浓度和尿白蛋白排泄率(urinary albumin excretion rate, UAER)评价模型是否成功建立。
3.①观察eGFP标记的MSCs归巢肾脏的情况。模型大鼠被随机分为MSCs组和超声+微泡+MSCs组，两组均经尾静脉移植MSCs，72h后采用激光共聚焦显微镜观察eGFP标记的MSCs在肾脏的定植情况，观察MSCs在MSCs组与超声+微泡+MSCs组肾脏内的荧光分布并进行阳性细胞计数和统计学比较。②超声介导的微泡破坏对糖尿病肾病模型大鼠肾脏通透性的影响。模型大鼠被随机分为三个批次，每一批次均分为对照组(未处理组)、超声组、微泡组与超声+微泡组。第一批次大鼠在注射伊文思蓝(evans blue, EB)同时接受相应处理，1小时后检测EB含量、激光共聚焦显微镜及透射电镜观察各组毛细血管通透性改变，实时荧光定量PCR (Real-Time PCR)和蛋白质印迹法(Western blot)检测E选择素(E-selectin)的mRNA和蛋白表达，肾脏组织学观察超声介导的微泡破坏对组织结构的可能损害；第二批次大鼠(处理同第一批次)24小时后检测UAER评价超声介导的微泡破坏对肾脏滤过功能可能的影响；第三批次大鼠(恢复组)接受相应处理后24小时再接受EB注射，观察毛细血管通透性的恢复。
4.模型大鼠被随机分为正常非糖尿病肾病组、未治疗组、超声+微泡组、MSCs组与超声+微泡+MSCs组。给予相应处理后1、2、4、8周每周固定时间点检测各组随机血糖浓度。8周后，检测大鼠UAER和血浆胰岛素水平、透射电镜观察肾小球和肾小管超微结构、肾脏PAS染色和胰腺HE染色观察肾脏和胰腺病理结构，胰腺组织免疫荧光双标技术观察胰岛β细胞和α细胞分布及数量恢复，酶联免疫吸附测定(Enzyme-linkedimmunosorbent assay, ELISA)检测抗炎因子白细胞介素10(IL-10)的蛋白表达，免疫组织化学法(Immunohistochemistry, IHC)检测促纤维化因子TGF-β1在肾内的分布和表达，并进行半定量分析。
结果：
1.培养的MSCs贴壁生长，形态以梭形为主；细胞表面高表达CD44(99.44%)和CD90(99.37%)，几乎不表达CD34(1.17%)和CD45(7.12%)。成脂诱导培养1周后油红O染色显示胞浆红色脂滴形成，成骨诱导2周后茜素红S染色显示红色钙结节形成。选择感染复数(Multiplicity of infection, MOI)为10转染MSCs，转染后72h荧光显微镜下观察，绝大部分MSCs呈现明亮的绿色荧光，eGFP转染阳性率约90%。转染后MSCs形态仍为梭形，生长良好，未见明显毒性反应，传代后荧光表达稳定。
2. STZ注射3天后，大鼠逐渐出现多饮、多食、多尿、精神萎靡、活动减少、反应迟钝、鼠毛稀疏、晦暗无光泽等体征，连续3天监测随机血糖浓度均大于16.7mmol/L，提示1型糖尿病模型建立成功。糖尿病模型成功建立后4周，血糖值稳定在20.8-33.8mmol/L之间，大鼠出现微量白蛋白尿，UAER值从(18.64±6.43μg/mg，对照组)升高至(46.79±15.42μg/mg，糖尿病肾病组)。胰腺及肾脏病理学均提示早期糖尿病肾病模型成功复制。
3.①荧光显微镜计数并比较MSCs组与超声+微泡+MSCs组肾内eGFP阳性细胞数，分别为(5.7±0.8)个，(18.3±2.9)个，差异有统计学意义(P＜0.01)。eGFP标记的MSCs主要位于肾间质毛细血管外、管周区域，少数位于肾小球。②超声联合微泡组肾内EB蓝染面积最明显，表面与冠状面蓝染面积分别为(49.12±7.31)%和(37.99±4.36)%，超声组肾内有轻微蓝染，表面与冠状面蓝染面积分别为(9.37±3.45)%和(10.10±3.24)%，对照组和微泡组未见蓝染。③超声联合微泡组的EB含量(37.267±4.948μg/g)明显高于对照组(13.942±2.848μg/g)、超声组(14.126±2.570μg/g)、微泡组(13.969±2.076μg/g)和恢复组(14.658±3.916μg/g)。④激光共聚焦结果显示超声+微泡组肾间质毛细血管周围EB渗出较其他组为明显，似呈“渔网状”，余各组未见EB渗出。⑤透射电镜观察到超声辐照后间质毛细血管壁变薄，变得不连续、不光滑，对照组、超声组和微泡组的间质毛细血管保持完整。⑥超声+微泡组E-selectin的mRNA和蛋白表达明显高于对照组、超声组和微泡组。⑦肾脏HE染色后观察，各组肾脏组织没有出血、坏死和肾脏结构的改变。⑧各组之间UAER值没有统计学差异。⑨各组未见血尿。
4.①细胞治疗后1周，MSCs组和超声+微泡+MSCs组血糖浓度明显降低。细胞移植后第2周，血糖浓度降到最低水平并持续至整个实验阶段。细胞治疗后8周，MSCs组和超声+微泡+MSCs组，血糖浓度无明显差异(23.8±5.5mmol/L&24.0±4.6mmol/L,P=0.395)。未治疗组和超声+微泡组直至实验结束仍保持较高血糖水平，浓度分别为(28.4±6.2) mmol/L及(27.9±6.5) mmol/L。②细胞治疗后8周，与正常组(25.27±2.34mIU/L)相比，未治疗组和超声+微泡组的血浆胰岛素水平明显降低，分别为(16.42±2.17) mIU/L和(16.67±2.33) mIU/L，二者之间无明显统计学差异，而MSCs组和超声+微泡+MSCs组的血浆胰岛素水平有所上升，分别为(19.31±1.68) mIU/L和(19.53±1.59) mIU/L，二者之间也无明显统计学差异。③细胞治疗后8周，未治疗组和超声+微泡组的UAER值均保持较高水平，分别为(643.25±204.58) μg/mg和(637.29±212.24) μg/mg。该值约4倍高于同时期正常组大鼠UAER值(128.57±36.93μg/mg)。MSCs组和超声+微泡+MSCs组的UAER值明显降低，分别为(302.41±49.21) μg/mg和(252.83±39.58) μg/mg。尽管MSCs组和超声+微泡+MSCs组的UAER值均未降至正常水平，但超声+微泡+MSCs组的UAER水平明显低于MSCs组。④MSCs治疗后8周，未治疗组和超声+微泡组大鼠肾小球基底膜增厚，细胞外基质增生，系膜区增宽，足突广泛融合、增宽；肾小管上皮细胞胞浆内可见髓鞘样结构，小管上皮细胞微绒毛肿胀，线粒体肿胀并伴大量空泡变。MSCs组大鼠肾小球基底膜增厚、细胞外基质增生以及系膜区增宽情况减轻，小部分足突融合，大部分修复；肾小管上皮细胞部分线粒体肿胀伴少量空泡变性。超声+微泡+MSCs组大鼠肾小球基底膜大部分未见明显增厚，细胞外基质增生明显减轻，系膜区未见增宽，足突融合明显好转；肾小管上皮细胞线粒体轻度肿胀，未见明显空泡变性。⑤肾脏PAS染色显示，至整个实验结束，未治疗组与超声+微泡组的肾小球和肾小管在光镜下有类似改变，均表现为肾小球硬化、体积增大、基底膜增厚、系膜区增宽、系膜基质增生和肾小管扩张，肾小管上皮细胞空泡变性。MSCs组和超声+微泡+MSCs组上述情况均明显好转，MSCs组仅有一小部分肾小球硬化和肾小管扩张，超声+微泡+MSCs组光镜下可观察到几乎正常的肾小球和肾小管结构。⑥胰腺HE染色和胰腺组织免疫荧光双标检查均显示，未治疗组和超声+微泡组胰岛和胰岛β细胞大面积破坏，与正常组胰岛或胰岛β细胞相比，胰岛形态不规则、体积减小、胰岛素生成细胞数量减少并向周边分布，而胰高血糖素生成细胞数量增多并向中央分布。MSCs移植治疗后8周，MSCs组和超声+微泡+MSCs组胰岛或胰岛β细胞无论在数量、结构或是体积上均明显改善，经治疗后的胰岛素生成细胞重新向胰岛中央分布，胰高血糖素生成细胞重回周边分布。⑦免疫组织化学法检测TGF-β1在正常组中有少量表达，在未治疗组和超声+微泡组中强表达，MSCs组表达次之，超声+微泡+MSCs组弱表达，半定量分析后显示，超声+微泡+MSCs组与MSCs组相比，差异有统计学意义(P＜0.05)。⑧ELISA法检测IL-10在正常肾组织中高表达，未治疗组和超声+微泡组中表达明显降低，而MSCs组表达增加，超声+微泡+MSCs组表达进一步增加。
结论：
1.成功实现大鼠MSCs的分离、培养及纯化，骨髓贴壁法是获取、纯化、扩增MSCs简便、易行的方法。通过生长曲线、细胞周期、透射电镜等生物学特性检测，提示培养的MSCs呈低分化状态，有较强的自我更新能力。通过流式细胞仪检测细胞表面标记分子，以及体外成脂、成骨诱导分化，初步判定所获细胞为MSCs。
2.腹腔单次注射链脲霉素(60mg/Kg)可建立稳定的大鼠1型糖尿病肾病模型，随机血糖浓度和UAER可有效评价模型是否成功建立。
3. eGFP转染MSCs可有效示踪MSCs在靶器官的分布，按照MOI=10转染细胞，细胞转染后无明显毒性且荧光表达稳定，其方法简便，操作便捷，可应用于MSCs的标记。
4.诊断超声介导的微泡破坏技术可增加糖尿病肾病大鼠肾间质毛细血管通透性，为经静脉移植的MSCs归巢并修复受损肾脏提供了研究基础。其可能机制为超声介导的微泡破坏所产生的空化效应可刺激超声辐照区域肾间质毛细血管的破裂及局部炎症发生，上调E-selectin炎症因子的表达，改变微环境，进而增加移植的MSCs向受损肾脏聚集与归巢。
5.超声介导的微泡破坏技术在合适的参数下对肾脏可能是安全的，对大鼠肾脏滤过功能没有影响。肾间质毛细血管通透性的增加是可逆的，24小时后可恢复到正常水平。
6.诊断超声介导的微泡破坏产生的空化效应通过增加间质毛细血管通透性、增加炎症因子E-selectin的表达，局部改变微环境，促进更多的外源性MSCs黏附、聚集、归巢至肾间质，抑制促纤维化因子TGF-β1表达，减轻肾小管和间质损害，阻止糖尿病肾病的进程。
7. MSCs移植通过旁分泌效应产生抗炎因子IL-10，调节炎性细胞因子/抗炎细胞因子的产生，抑制机体炎症反应，从而减轻肾小球增大和肾小管扩张程度，延缓肾损害，保护肾脏。
8. MSCs移植降低血糖浓度，促进胰岛恢复，有效遏制糖尿病肾病的进程，对促进肾脏修复产生积极作用。
Background：
As one of the most detrimental long-term complications of diabetes mellitus (DM),diabetic nephropathy (DN) has evolved as a leading cause of end-stage renal disease (ESRD)around the world, which is highly associated with premature morbidity and mortality ofdiabetic patients. Traditionally DN is considered to be part of the microvascular complicationsof DM, while advanced DN is also characterized by tubulointerstitial fibrosis. It is widelyaccepted that tubulointerstitial damage correlates with the degree of renal dysfunction and thatit is a reliable predictor for ESRD. Microalbuminuria is an early sign of diabetic nephropathy.There is now strong evidence that early intervention and tight control have the potential toslow, or perhaps even reverse the progression of early diabetic nephropathy. Consequently,early prevention of DN is an urgent medical issue at present. At the moment, transplantationof pancreatic islet and kidney is the most preferred cell replacement therapy to DN. However,the scarcity of transplantable donors and the need for lifelong immunosuppression limit thewidespread use of the curative therapy. New antifibrotic therapies aimed at inhibition ofprofibrotic mediators are rapidly progressing but require a large amount of antibodies, as wellas will need to be tested on top of the existing therapy of renin-angiotensin system inhibition.The sustained hyperglycemic state accelerates the progress of early diabetic nephropathy inboth type1and type2diabetic patients. Meticulous control of blood glucose decreases therisk of developing nephropathy but is not always feasible due to limitations in the currentlyavailable drugs and therapeutic techniques.
Mesenchymal stem cells (MSCs) are the stromal component of bone marrow (BM) andeasily obtained from BM, have the potential to differentiate into several cell types, and showimmunomodulatory properties. MSCs are capable of differentiate into functional insulin- producing cells in vitro, which can reverse hyperglycemia in diabetes rats. MSCs also havethe potential to differentiate into renal cells in vivo, which can repair the destroyed kidney.Therefore, MSCs can be regarded as an attractive strategy to ameliorate hyperglycemia andimprove renal function though intracardiac injection, intra arterial or intravenous injection,and intrarenal injection. On the other hand, their utility for targeting tissue in living animalshas proved to be limited. For instance, MSCs transplantation usually resulted in aninsufficient number of engrafted MSCs in injury site. In view of the drawback, we havedeveloped a technique that applies ultrasound-mediated microbubble destruction to promotehoming of MSCs to impaired kidney.
Ultrasound microbubble contrast agent has been developed as a method to enhanceconventional ultrasound imaging in clinical ultrasonography, by destroying intravascularmicrobubbles to characterize refill kinetics. Furthermore, ultrasound-mediated microbubbledestruction is a new and promising technique applied in many fields for drug and genedelivery to cardiovascular system, blood-brain barrier and blood-tumor barrier opening,capillary and vascular permeability enhancement, and stem cells homing. Kidney, as a highefficiency filter, appears to be especially sensitive to the bioeffects due to the relatively highblood pressure in the capillaries of the glomeruli. Microbubble destruction by ultrasound genetransfection treatment (1.0W/cm2) promoted renal recovery in acute kidney injury in rats. Theresults from RT-PCR revealed that the US+MB and US+MB+MSCs groups markedlyincreased the level of inter-cellular adhesion molecule1(ICAM-1) messenger ribonucleicacid (mRNA) compared with the control group and the MSCs group (p＜0.05).
So far, no studies have been reported whether this technique provides an equalcontribution to diabetic kidney disease which acts as a complication of primary disease. Basedon these previous findings, we attempt to explore whether this technique is implemented toincrease interstitial capillary permeability of DN. It is possible to take advantage of thistechnique to selectively increase drug and gene deliver to tubulointerstitial area, to enhancetherapeutic effect, to slow down disease progression, or perhaps even reverse the progressionof DN in later work.
Objevtives：
1. To study the feasibility and safety of the improvement of targeted homing of bonemarrow derived mesenchymal stem cells by diagnostic ultrasound-mediated microbubbles destruction to the early diabetic nephropathy rats.
2. To study the efficacy of intravenous MSCs implantation by diagnostic ultrasound-mediated mirobubbles destruction on the improvement of diabetic nephropathy of rats.
3. To study the preliminary mechanisms of the improvement of diabetic nephropathy ofrats by MSCs transplantation by diagnostic ultrasound-mediated microbubbles destruction.
Methods：
1. Adherent culture method was used in the isolation and cultivation of MSCs. Themorphological and other characterics were detected. Flow cytometry (FCM) was applied todetect the expression of surface marker molecules on the stem cells. Osteogenic andadipogenesis induction of MSCs were performed.
2. All rats were received a single intraperitoneal injection of60mg/kg streptozotocin(STZ, Sigma) immediately after dissolving in0.1M citrate buffer at pH4.5. Three days afterSTZ injection, diabetes was confirmed by random blood glucose concentrations of greaterthan16.7mmol/L for3consecutive days.4weeks after onset diabetes, diabetic rats presentedmild microalbuminuria (an early sign of DN) and were considered to be diabetic nephropathy.
3. MSCs (1×106cells) were transfected with lentiviral vectors carrying eGFP to track theengrafted cells in kidneys. DN rats (n=10) were equally divided into eGFP-labeled MSCsgroup and eGFP-labeled MSCs+US+MB group. Three days after cells transplantation, ratswere killed by anesthetic overdose. Kidneys were separated and immediately snap-frozen inliquid nitrogen for frozen sections (5μm in thickness). The survival of implanted cells wasobserved and counted in frozen sections under a laser scanning confocal microscope (LSCM).Rats were randomly assigned into three batches. The first batch (88rats) was equally dividedinto four groups: control group received Evans blue (EB,2%in saline,50mg/kg) injectiononly; US group received EB injection, followed by0.5mL of PBS injection (an equivalentvolume of microbubble injection) and ultrasonic irradiation; MB group received EB injection,followed by microbubble injection (0.05mL/kg) without ultrasonic irradiation; US+MB groupreceived EB injection, followed by microbubble injection and ultrasonic irradiation. Beforeinjection, the bottle filled with microbubbles was shaken45s to form microbubblessuspension using a custom-built oscillation apparatus. Microbubbles were injected into lateraltail veins through26-gauge needles connected with a1mL syringe via a15cm length catheter,controlled by a syringe pump within30s and followed by0.5mL saline to wash the tube. A blanket covered the treated rats for1h.1h after treatment, the change of state of endothelialcells after ultrasonic irradiation was assessed by E-selectin mRNA and protein expression.Renal interstital capillary permeability was assessed by EB content assay, confocal laserscanning microscope (CLSM) and transmission electron microscope (TEM) examination. Thepossible injury in renal structure was evaluated by renal histology. To assess the impact ofultrasound-mediated microbubble destruction on kidney, urine samples of the third batch (32rats) which had the same grouping (four groups, n=8/group) and treatments as the first batchwere collected after24h post-treatment and measured for red blood cells, urinary albumin,and urinary creatinine. Urinary albumin excretion rate (UAER) was determined using urinaryalbumin-to-urinary creatinine ratio. Additionally, to assess the permeability recovery ofkidneys after ultrasound-mediated microbubble destruction, the second batch (32rats) withthe same grouping as above (four groups, n=8/group) received microbubbles injection, orultrasonic irradiation, or microbubble-ultrasound interaction, and then received EB injectionafter24h (Recovery group).
4. Random blood glucose concentrations were measured at1,2,4,8weeks after MSCstransplantation. UAER values and plasma insulin levels were detected at8weeks after MSCstransplantation. In addition, the ultrastructure of glomeruli and tubules was observed bytransmission electron microscope. The histological structure of pancreas and kidney wasobserved by HE and PAS staining, respectively. The distribution and quantity of islets β/α cellwere observed by pancreas double-label immunohistofluorescence. IL-10protein expressionin kidney was detected by ELISA. The distribution, expression, and quantitative analysis ofTGF-β1in kidney were assessed by immunohistochemistry.
Results：
1. Most of cultured MSCs were spindle shaped, attached to the culture dish tightly,proliferated in culture medium, and became more uniform after several passages. Theintracellular lipid droplets stained with oil red O staining and the calcium depositions stainedwith alizarin red S staining were observed after1week and2week of treatment, respectively.Flow cytometry analysis showed that these expanded MSCs were positive for CD44(99.44%),CD90(99.37%), and negative for the leukocyte common antigen CD34(1.17%), CD45(7.12%).
2. Three days after STZ injection, diabetes was confirmed by random blood glucose concentrations of greater than16.7mmol/L for3consecutive days.4weeks after onsetdiabetes, diabetic rats presented mild microalbuminuria (an early sign of DN) and wereconsidered to be diabetic nephropathy.
3.①The number ofeGFP-labeled MSCs in tubulointerstitial area of US+MB+MSCsgroup (18.3±2.9) was much more than that of MSCs group (5.7±0.8) under CLSM(P=0.0006). eGFP-labeled MSCs were mostly localized in outer region of interstitial capillary,the peritubular area and a small number of eGFP-MSCs located in glomeruli.②There was abit of blue staining in US group which the percentage of blue staining of renal surface andcoronal plane was (9.37±3.45%) and (10.10±3.24%), respectively, but no visible staining incontrol group and MB group. The percentage of blue staining of renal surface and coronalplane in US+MB group was (49.12±7.31%) and (37.99±4.36%), respectively.③EB contentwas significantly higher in US+MB group (37.267±4.948μg/g) than that in control group(13.942±2.848μg/g, P＜0.01), US group (14.126±2.570μg/g, P＜0.01), MB group(13.969±2.076μg/g, P＜0.01), and recovery group (14.658±3.916μg/g, P＜0.01).④Renalinterstitial capillary structure maintained intact and clear, and there was no EB extravasationin renal interstitium in control group, US group, and MB group. While the extravasation ofred EB fluorescence around interstitial capillary was evident in US+MB group, causing“fishing net” graph.⑤A part of interstitial capillary walls became thinned, discontinuous,and unsmoothed in US+MB group and they kept intact in control group, US group, and MBgroup.⑥The increase of E-selectin mRNA and protein expression were apparently morepronounced in US+MB group than in control group, US group, and MB group (P＜0.01,respectively).⑦Hematoxylin-eosin staining confirmed that there was no hemorrhage andnecrosis in renal structure in all groups.⑧There was no significant difference in UAER valuebetween US+MB group and Control group (60.12±25.67μg/mg creatinine&55.99±20.55μg/mg creatinine, P＞0.05), which was similar to that of US+MB group and US group(60.12±25.67μg/mg creatinine&57.43±18.40μg/mg creatinine, P＞0.05), US+MB groupand MB group (60.12±25.67μg/mg creatinine&53.86±22.42μg/mg creatinine, P＞0.05).⑨Hematuria was not detected in any group.
4.①Within1week after cell therapy, blood glucose concentrations significantlydecreased in MSCs group (22.5±5.3mmol/L) and US+MB+MSCs group (22.1±5.0mmol/L),reached the lowest levels at2weeks and lasted at least during the observation period. In contrast, untreated PBS group (28.4±6.2mmol/L) and US+MB group (27.9±6.5mmol/L)remained a high level of blood glucose until the end of the experiment. No obvious differencewas found in blood glucose concentration at the end of the experiment between MSCs group(23.8±5.5mmol/L) and US+MB+MSCs group (24.0±4.6mmol/L), P=0.395, which was thesame as that between PBS group (28.4±6.2mmol/L) and US+MB group (27.9±6.5mmol/L),P=0.816.②At the end of the experiment, compared with Normal group (25.27±2.34mIU/L),plasma insulin levels decreased in PBS group (16.42±2.17mIU/L) and US+MB group(16.67±2.33mIU/L), and improved in MSCs group (19.31±1.68mIU/L) and US+MB+MSCsgroup (19.53±1.59mIU/L). There was no significant difference in plasma insulin levelsbetween MSCs group and US+MB+MSCs group (P=0.574), which was the same as thatbetween PBS group and US+MB group (P=0.564).③At the end of the experiment, UAERvalues maintained a high level in PBS group (643.25±204.58μg/mg) and US+MB group(637.29±212.24μg/mg) and both of them were more than fourfold higher than that ofage-matched normal control rats (128.57±36.93μg/mg). As a comparison, UAER valuesreduced in MSCs group (302.41±49.21μg/mg) and US+MB+MSCs group (252.83±39.58μg/mg). Furthermore, UAER values were significantly milder in US+MB+MSCs group(252.83±39.58μg/mg) than that in MSCs groups (302.41±49.21μg/mg).④PBS group andUS+MB group rats showed glomerular basement membrane thickening, extracellular matrixdeposition, glomerular mesangial expansion and podocyte fusion in glomerular ultrastructureunder transmission electron microscope. There were myelin-like structures in renal tubularepithelial cells, microvilli swelling in tubule epithelial cells, and mitochondrial swelling witha large number of vacuolation in tubular ultrastructure under TEM. There was a significantimprovement in MSCs group and US+MB+MSCs gorup, especially in US+MB+MSCs group.⑤Renal histological analysis showed that there have similar changes both in PBS group andUS+MB group in glomeruli and tubule which displayed glomerular sclerosis, mesangialexpansion, and tubular dilatation observed by light microscope at the end of experiment.However, the extent of such changes of glomeruli and tubule was extenuated in MSCs groupand US+MB+MSCs group. Only a small part of glomerular hyalinosis and tubular dilatationwere observed in MSCs group. Further improvements in structure or even nearly normalstructure of glomeruli and tubule were observed in US+MB+MSCs group.⑥At the end ofthe experiment, PBS group and US+MB group showed a massive destruction of pancreatic islets/β cells observed by pancreatic pathology and double-label immunohistofluorescence.Compared to normal pancreatic islets/β cells, morphological irregularity, volume reduction,less insulin-and more glucagon-positive cells were observed in PBS group and US+MBgroup. As expected, MSCs group and US+MB+MSCs group exhibited a good recovery onamount, architecture and volume of pancreatic islets/β cells.⑦TGF-β1protein expressiondetected by immunohistochemistry was markedly increased in glomeruli and interstitialspaces in PBS group and US+MB group, compared with Normal group. MSCs therapydecreased TGF-β1protein expression in glomeruli and interstitial spaces. Furthermore,TGF-β1protein expression in glomeruli and interstitial spaces was significantly lower inUS+MB+MSCs group than that in MSCs group.⑧IL-10protein expression in renal tissuedetected by ELISA was markedly decreased in PBS group and US+MB group, compared withNormal group. MSCs therapy increased IL-10protein expression. Furthermore, IL-10proteinexpression was significantly higher in US+MB+MSCs group than that in MSCs group.
Conclusions：
1. Purified MSCs can be obtained via the adherent culture methods using morphologicalobservation, surface marker identification and inducing differentiation. It is a simple availablemethod for the isolation and culture of MSCs.
2. Three days after a single intraperitoneal injection of60mg/kg STZ injection, diabeteswas confirmed by random blood glucose concentrations of greater than16.7mmol/L for3consecutive days.4weeks after onset diabetes, diabetic rats presented mild microalbuminuria(an early sign of DN) and were considered to be diabetic nephropathy.
3. eGFP labeled MSCs could efficiently track the distribution of MSCs in organs.
4. Ultrasound-mediated microbubble destruction locally increased renal interstitialcapillary permeability in early diabetic nephropathy rats, which should be considered anenhancement therapy for drug, gene, antifibrotic agent, or stem cells delivery to kidney in thefuture. The underlying mechanism may be that ultrasound-mediated microbubble cavitationruptured interstitial capillary walls, which promoting therapeutic substances to cross throughdiscontinuous walls, enter renal interstitium, and maximize therapeutic effect. The mild injuryof interstitial capillary after cavitation effect caused the up-regulation of E-selectin, whichperhaps promoted the homing of transplanted stem cells to DN. This technology perhapsprovides a new idea for personalized cell therapy to DN by using stem cells.
5. Microbubble insonation and inertial cavitation may be safe to kidney underappropriate parameters. The increased permeability in renal interstitial capillary is reversibleand EB content returned to the control group level after24h.
6. The interstitial capillary permeability and E-selectin mRNA and protein expressionwere increased, and the microenvironment was changed by ultrasound-mediated microbubbledestruction, which facilitated the homing and gathering of MSCs to kidney, inhibited TGF-β1expression, reduce the damage to the tubules and interstitium, and prevented the process ofdiabetic nephropathy.
7. The increased expression of IL-10and inhibition of TGF-β1in US+MB+MSCs groupmay be induced by paracrine effect of the implanted MSCs, which augmented the adhension,homging and gathering ability to kidney, inhibited the inflammatory response, reduced thedegree of renal hypertrophy, and improved renal function.
8. MSCs transplantation reduced blood glucose concentrations, promoted islets recovery,and prevented the process of diabetic nephropathy. MSCs therapy plays a positive role topromote diabetic nephropathy repair.
糖尿病肾病(diabetic nephropathy, DN)是终末期肾脏疾病(end-stage renal disease,ESRD)的主要原因，与ESRD较高的死亡率密切相关。目前广泛认为小管间质损害是ESRD可靠的预测因子并与肾功能障碍密切相关。微量白蛋白尿是糖尿病肾病的早期征象。现有许多证据表明对糖尿病肾病进行早期干预和精确调控可以减缓，甚至逆转糖尿病肾病的进程。因此，对早期糖尿病肾病进行及早干预、提高治疗物质在肾间质的聚集是目前医学领域亟待解决的问题。目前治疗糖尿病肾病的常用方法诸如调控血糖、胰腺和肾脏移植、透析治疗等虽然能改善肾功能，却因当前药物治疗和治疗技术的有限性，导致并发症多、生存质量不高，效果并不显著。
骨髓来源的间充质干细胞(bone marrow-derived mesenchymal stem cells, BM-MSCs)移植治疗是近年来国内外研究的重点，其易于分离提取，具有高度的扩增潜能，有良好的基因稳定性、组织相容性好、不涉及伦理道德问题，为受损组织和器官的修复开辟了一条新途径。MSCs能够分化为功能性的胰岛素生成细胞，逆转糖尿病大鼠高血糖状态，也能够分化为肾脏细胞修复受损肾脏。因而，MSCs移植治疗被认为是改善高血糖和肾功能的、具有极大吸引力的治疗手段。但骨髓中MSCs含量极少，远远小于肾脏修复所需要细胞的量，加上目前干细胞移植治疗移植后细胞存活率较低、干细胞靶向归巢能力欠佳等问题，均极大地限制了其应用。因此，如何调控骨髓干细胞的肾向性、提高干细胞靶向归巢能力和移植的有效性是干细胞移植治疗肾脏疾病的关键。
超声介导的微泡破坏(Ultrasound-mediated microbubble destruction)作为一种新的、极具前景的治疗方法应用于多个领域，例如将药物和基因传递到心血管系统，开放血脑屏障和血瘤屏障，增强毛细血管和血管通透性以及促进干细胞归巢等。肾脏是高滤过器官，对生物学效应较为敏感，对于肾脏疾病的干细胞移植治疗，超声介导的微泡破坏作用是否同样有效呢？研究显示，超声基因转染治疗仪(1.0W/cm~2)联合自制微泡能有效促进MSCs归巢至肾脏，使急性肾损伤大鼠肾功能得到明显改善。该研究还发现，超声+微泡组大鼠肾脏ICAM-1mRNA表达高于对照组、超声+微泡+MSCs组大鼠肾脏ICAM-1mRNA表达高于单纯MSCs移植组，说明超声介导的微泡破坏可以增加MSCs的靶向黏附、提高MSCs归巢至肾脏的能力，进而更有效地实现MSCs靶向移植治疗肾脏疾病的效果。
然而，关于慢性肾脏疾病的干细胞治疗，超声介导的微泡破坏作用对其影响的研究较少，本实验尝试利用超声介导的微泡靶向破坏作用，经静脉移植MSCs治疗链脲佐菌素(streptozotocin, STZ)诱导的1型糖尿病肾病大鼠，并试图探索其安全性及可能机制，期望能提供一种新的、非侵入性的方法，对糖尿病肾病患者给予系统性药物治疗的前提下选择性增强干细胞在肾脏的聚集，最大化提高糖尿病肾病的治疗效果，为提高干细胞无创、有效地靶向归巢及减缓疾病进程、或是逆转糖尿病肾病病程进展提供一种新思路和新方法。
研究目的：
1.探讨诊断超声介导的微泡破坏促进经静脉移植的同源异体MSCs归巢糖尿病肾病大鼠肾脏的可行性和安全性；
2.探讨在诊断超声介导的微泡破坏作用下，经静脉移植的MSCs对修复大鼠糖尿病肾病的有效性；
3.初步探讨诊断超声介导的微泡破坏技术对促进静脉移植的MSCs靶向归巢并修复大鼠糖尿病肾病的可能机制。
研究方法：
1.采用全骨髓培养法对SD (Sprague Dawley)大鼠骨髓MSCs进行分离、培养、及纯化，检测其生物学特性。流式细胞仪检测细胞表面标记分子，成脂、成骨诱导分化进行细胞鉴定。为便于对体内干细胞的追踪，采用携带增强型绿色荧光蛋白的慢病毒对培养的MSCs进行转染，并观察转染后MSCs表达绿色荧光情况及细胞毒性反应。
2.腹腔单次注射60mg/Kg SZT建立稳定的大鼠1型早期糖尿病肾病模型，随机血糖浓度和尿白蛋白排泄率(urinary albumin excretion rate, UAER)评价模型是否成功建立。
3.①观察eGFP标记的MSCs归巢肾脏的情况。模型大鼠被随机分为MSCs组和超声+微泡+MSCs组，两组均经尾静脉移植MSCs，72h后采用激光共聚焦显微镜观察eGFP标记的MSCs在肾脏的定植情况，观察MSCs在MSCs组与超声+微泡+MSCs组肾脏内的荧光分布并进行阳性细胞计数和统计学比较。②超声介导的微泡破坏对糖尿病肾病模型大鼠肾脏通透性的影响。模型大鼠被随机分为三个批次，每一批次均分为对照组(未处理组)、超声组、微泡组与超声+微泡组。第一批次大鼠在注射伊文思蓝(evans blue, EB)同时接受相应处理，1小时后检测EB含量、激光共聚焦显微镜及透射电镜观察各组毛细血管通透性改变，实时荧光定量PCR (Real-Time PCR)和蛋白质印迹法(Western blot)检测E选择素(E-selectin)的mRNA和蛋白表达，肾脏组织学观察超声介导的微泡破坏对组织结构的可能损害；第二批次大鼠(处理同第一批次)24小时后检测UAER评价超声介导的微泡破坏对肾脏滤过功能可能的影响；第三批次大鼠(恢复组)接受相应处理后24小时再接受EB注射，观察毛细血管通透性的恢复。
4.模型大鼠被随机分为正常非糖尿病肾病组、未治疗组、超声+微泡组、MSCs组与超声+微泡+MSCs组。给予相应处理后1、2、4、8周每周固定时间点检测各组随机血糖浓度。8周后，检测大鼠UAER和血浆胰岛素水平、透射电镜观察肾小球和肾小管超微结构、肾脏PAS染色和胰腺HE染色观察肾脏和胰腺病理结构，胰腺组织免疫荧光双标技术观察胰岛β细胞和α细胞分布及数量恢复，酶联免疫吸附测定(Enzyme-linkedimmunosorbent assay, ELISA)检测抗炎因子白细胞介素10(IL-10)的蛋白表达，免疫组织化学法(Immunohistochemistry, IHC)检测促纤维化因子TGF-β1在肾内的分布和表达，并进行半定量分析。
结果：
1.培养的MSCs贴壁生长，形态以梭形为主；细胞表面高表达CD44(99.44%)和CD90(99.37%)，几乎不表达CD34(1.17%)和CD45(7.12%)。成脂诱导培养1周后油红O染色显示胞浆红色脂滴形成，成骨诱导2周后茜素红S染色显示红色钙结节形成。选择感染复数(Multiplicity of infection, MOI)为10转染MSCs，转染后72h荧光显微镜下观察，绝大部分MSCs呈现明亮的绿色荧光，eGFP转染阳性率约90%。转染后MSCs形态仍为梭形，生长良好，未见明显毒性反应，传代后荧光表达稳定。
2. STZ注射3天后，大鼠逐渐出现多饮、多食、多尿、精神萎靡、活动减少、反应迟钝、鼠毛稀疏、晦暗无光泽等体征，连续3天监测随机血糖浓度均大于16.7mmol/L，提示1型糖尿病模型建立成功。糖尿病模型成功建立后4周，血糖值稳定在20.8-33.8mmol/L之间，大鼠出现微量白蛋白尿，UAER值从(18.64±6.43μg/mg，对照组)升高至(46.79±15.42μg/mg，糖尿病肾病组)。胰腺及肾脏病理学均提示早期糖尿病肾病模型成功复制。
3.①荧光显微镜计数并比较MSCs组与超声+微泡+MSCs组肾内eGFP阳性细胞数，分别为(5.7±0.8)个，(18.3±2.9)个，差异有统计学意义(P＜0.01)。eGFP标记的MSCs主要位于肾间质毛细血管外、管周区域，少数位于肾小球。②超声联合微泡组肾内EB蓝染面积最明显，表面与冠状面蓝染面积分别为(49.12±7.31)%和(37.99±4.36)%，超声组肾内有轻微蓝染，表面与冠状面蓝染面积分别为(9.37±3.45)%和(10.10±3.24)%，对照组和微泡组未见蓝染。③超声联合微泡组的EB含量(37.267±4.948μg/g)明显高于对照组(13.942±2.848μg/g)、超声组(14.126±2.570μg/g)、微泡组(13.969±2.076μg/g)和恢复组(14.658±3.916μg/g)。④激光共聚焦结果显示超声+微泡组肾间质毛细血管周围EB渗出较其他组为明显，似呈“渔网状”，余各组未见EB渗出。⑤透射电镜观察到超声辐照后间质毛细血管壁变薄，变得不连续、不光滑，对照组、超声组和微泡组的间质毛细血管保持完整。⑥超声+微泡组E-selectin的mRNA和蛋白表达明显高于对照组、超声组和微泡组。⑦肾脏HE染色后观察，各组肾脏组织没有出血、坏死和肾脏结构的改变。⑧各组之间UAER值没有统计学差异。⑨各组未见血尿。
4.①细胞治疗后1周，MSCs组和超声+微泡+MSCs组血糖浓度明显降低。细胞移植后第2周，血糖浓度降到最低水平并持续至整个实验阶段。细胞治疗后8周，MSCs组和超声+微泡+MSCs组，血糖浓度无明显差异(23.8±5.5mmol/L&24.0±4.6mmol/L,P=0.395)。未治疗组和超声+微泡组直至实验结束仍保持较高血糖水平，浓度分别为(28.4±6.2) mmol/L及(27.9±6.5) mmol/L。②细胞治疗后8周，与正常组(25.27±2.34mIU/L)相比，未治疗组和超声+微泡组的血浆胰岛素水平明显降低，分别为(16.42±2.17) mIU/L和(16.67±2.33) mIU/L，二者之间无明显统计学差异，而MSCs组和超声+微泡+MSCs组的血浆胰岛素水平有所上升，分别为(19.31±1.68) mIU/L和(19.53±1.59) mIU/L，二者之间也无明显统计学差异。③细胞治疗后8周，未治疗组和超声+微泡组的UAER值均保持较高水平，分别为(643.25±204.58) μg/mg和(637.29±212.24) μg/mg。该值约4倍高于同时期正常组大鼠UAER值(128.57±36.93μg/mg)。MSCs组和超声+微泡+MSCs组的UAER值明显降低，分别为(302.41±49.21) μg/mg和(252.83±39.58) μg/mg。尽管MSCs组和超声+微泡+MSCs组的UAER值均未降至正常水平，但超声+微泡+MSCs组的UAER水平明显低于MSCs组。④MSCs治疗后8周，未治疗组和超声+微泡组大鼠肾小球基底膜增厚，细胞外基质增生，系膜区增宽，足突广泛融合、增宽；肾小管上皮细胞胞浆内可见髓鞘样结构，小管上皮细胞微绒毛肿胀，线粒体肿胀并伴大量空泡变。MSCs组大鼠肾小球基底膜增厚、细胞外基质增生以及系膜区增宽情况减轻，小部分足突融合，大部分修复；肾小管上皮细胞部分线粒体肿胀伴少量空泡变性。超声+微泡+MSCs组大鼠肾小球基底膜大部分未见明显增厚，细胞外基质增生明显减轻，系膜区未见增宽，足突融合明显好转；肾小管上皮细胞线粒体轻度肿胀，未见明显空泡变性。⑤肾脏PAS染色显示，至整个实验结束，未治疗组与超声+微泡组的肾小球和肾小管在光镜下有类似改变，均表现为肾小球硬化、体积增大、基底膜增厚、系膜区增宽、系膜基质增生和肾小管扩张，肾小管上皮细胞空泡变性。MSCs组和超声+微泡+MSCs组上述情况均明显好转，MSCs组仅有一小部分肾小球硬化和肾小管扩张，超声+微泡+MSCs组光镜下可观察到几乎正常的肾小球和肾小管结构。⑥胰腺HE染色和胰腺组织免疫荧光双标检查均显示，未治疗组和超声+微泡组胰岛和胰岛β细胞大面积破坏，与正常组胰岛或胰岛β细胞相比，胰岛形态不规则、体积减小、胰岛素生成细胞数量减少并向周边分布，而胰高血糖素生成细胞数量增多并向中央分布。MSCs移植治疗后8周，MSCs组和超声+微泡+MSCs组胰岛或胰岛β细胞无论在数量、结构或是体积上均明显改善，经治疗后的胰岛素生成细胞重新向胰岛中央分布，胰高血糖素生成细胞重回周边分布。⑦免疫组织化学法检测TGF-β1在正常组中有少量表达，在未治疗组和超声+微泡组中强表达，MSCs组表达次之，超声+微泡+MSCs组弱表达，半定量分析后显示，超声+微泡+MSCs组与MSCs组相比，差异有统计学意义(P＜0.05)。⑧ELISA法检测IL-10在正常肾组织中高表达，未治疗组和超声+微泡组中表达明显降低，而MSCs组表达增加，超声+微泡+MSCs组表达进一步增加。
结论：
1.成功实现大鼠MSCs的分离、培养及纯化，骨髓贴壁法是获取、纯化、扩增MSCs简便、易行的方法。通过生长曲线、细胞周期、透射电镜等生物学特性检测，提示培养的MSCs呈低分化状态，有较强的自我更新能力。通过流式细胞仪检测细胞表面标记分子，以及体外成脂、成骨诱导分化，初步判定所获细胞为MSCs。
2.腹腔单次注射链脲霉素(60mg/Kg)可建立稳定的大鼠1型糖尿病肾病模型，随机血糖浓度和UAER可有效评价模型是否成功建立。
3. eGFP转染MSCs可有效示踪MSCs在靶器官的分布，按照MOI=10转染细胞，细胞转染后无明显毒性且荧光表达稳定，其方法简便，操作便捷，可应用于MSCs的标记。
4.诊断超声介导的微泡破坏技术可增加糖尿病肾病大鼠肾间质毛细血管通透性，为经静脉移植的MSCs归巢并修复受损肾脏提供了研究基础。其可能机制为超声介导的微泡破坏所产生的空化效应可刺激超声辐照区域肾间质毛细血管的破裂及局部炎症发生，上调E-selectin炎症因子的表达，改变微环境，进而增加移植的MSCs向受损肾脏聚集与归巢。
5.超声介导的微泡破坏技术在合适的参数下对肾脏可能是安全的，对大鼠肾脏滤过功能没有影响。肾间质毛细血管通透性的增加是可逆的，24小时后可恢复到正常水平。
6.诊断超声介导的微泡破坏产生的空化效应通过增加间质毛细血管通透性、增加炎症因子E-selectin的表达，局部改变微环境，促进更多的外源性MSCs黏附、聚集、归巢至肾间质，抑制促纤维化因子TGF-β1表达，减轻肾小管和间质损害，阻止糖尿病肾病的进程。
7. MSCs移植通过旁分泌效应产生抗炎因子IL-10，调节炎性细胞因子/抗炎细胞因子的产生，抑制机体炎症反应，从而减轻肾小球增大和肾小管扩张程度，延缓肾损害，保护肾脏。
8. MSCs移植降低血糖浓度，促进胰岛恢复，有效遏制糖尿病肾病的进程，对促进肾脏修复产生积极作用。
Background：
As one of the most detrimental long-term complications of diabetes mellitus (DM),diabetic nephropathy (DN) has evolved as a leading cause of end-stage renal disease (ESRD)around the world, which is highly associated with premature morbidity and mortality ofdiabetic patients. Traditionally DN is considered to be part of the microvascular complicationsof DM, while advanced DN is also characterized by tubulointerstitial fibrosis. It is widelyaccepted that tubulointerstitial damage correlates with the degree of renal dysfunction and thatit is a reliable predictor for ESRD. Microalbuminuria is an early sign of diabetic nephropathy.There is now strong evidence that early intervention and tight control have the potential toslow, or perhaps even reverse the progression of early diabetic nephropathy. Consequently,early prevention of DN is an urgent medical issue at present. At the moment, transplantationof pancreatic islet and kidney is the most preferred cell replacement therapy to DN. However,the scarcity of transplantable donors and the need for lifelong immunosuppression limit thewidespread use of the curative therapy. New antifibrotic therapies aimed at inhibition ofprofibrotic mediators are rapidly progressing but require a large amount of antibodies, as wellas will need to be tested on top of the existing therapy of renin-angiotensin system inhibition.The sustained hyperglycemic state accelerates the progress of early diabetic nephropathy inboth type1and type2diabetic patients. Meticulous control of blood glucose decreases therisk of developing nephropathy but is not always feasible due to limitations in the currentlyavailable drugs and therapeutic techniques.
Mesenchymal stem cells (MSCs) are the stromal component of bone marrow (BM) andeasily obtained from BM, have the potential to differentiate into several cell types, and showimmunomodulatory properties. MSCs are capable of differentiate into functional insulin- producing cells in vitro, which can reverse hyperglycemia in diabetes rats. MSCs also havethe potential to differentiate into renal cells in vivo, which can repair the destroyed kidney.Therefore, MSCs can be regarded as an attractive strategy to ameliorate hyperglycemia andimprove renal function though intracardiac injection, intra arterial or intravenous injection,and intrarenal injection. On the other hand, their utility for targeting tissue in living animalshas proved to be limited. For instance, MSCs transplantation usually resulted in aninsufficient number of engrafted MSCs in injury site. In view of the drawback, we havedeveloped a technique that applies ultrasound-mediated microbubble destruction to promotehoming of MSCs to impaired kidney.
Ultrasound microbubble contrast agent has been developed as a method to enhanceconventional ultrasound imaging in clinical ultrasonography, by destroying intravascularmicrobubbles to characterize refill kinetics. Furthermore, ultrasound-mediated microbubbledestruction is a new and promising technique applied in many fields for drug and genedelivery to cardiovascular system, blood-brain barrier and blood-tumor barrier opening,capillary and vascular permeability enhancement, and stem cells homing. Kidney, as a highefficiency filter, appears to be especially sensitive to the bioeffects due to the relatively highblood pressure in the capillaries of the glomeruli. Microbubble destruction by ultrasound genetransfection treatment (1.0W/cm2) promoted renal recovery in acute kidney injury in rats. Theresults from RT-PCR revealed that the US+MB and US+MB+MSCs groups markedlyincreased the level of inter-cellular adhesion molecule1(ICAM-1) messenger ribonucleicacid (mRNA) compared with the control group and the MSCs group (p＜0.05).
So far, no studies have been reported whether this technique provides an equalcontribution to diabetic kidney disease which acts as a complication of primary disease. Basedon these previous findings, we attempt to explore whether this technique is implemented toincrease interstitial capillary permeability of DN. It is possible to take advantage of thistechnique to selectively increase drug and gene deliver to tubulointerstitial area, to enhancetherapeutic effect, to slow down disease progression, or perhaps even reverse the progressionof DN in later work.
Objevtives：
1. To study the feasibility and safety of the improvement of targeted homing of bonemarrow derived mesenchymal stem cells by diagnostic ultrasound-mediated microbubbles destruction to the early diabetic nephropathy rats.
2. To study the efficacy of intravenous MSCs implantation by diagnostic ultrasound-mediated mirobubbles destruction on the improvement of diabetic nephropathy of rats.
3. To study the preliminary mechanisms of the improvement of diabetic nephropathy ofrats by MSCs transplantation by diagnostic ultrasound-mediated microbubbles destruction.
Methods：
1. Adherent culture method was used in the isolation and cultivation of MSCs. Themorphological and other characterics were detected. Flow cytometry (FCM) was applied todetect the expression of surface marker molecules on the stem cells. Osteogenic andadipogenesis induction of MSCs were performed.
2. All rats were received a single intraperitoneal injection of60mg/kg streptozotocin(STZ, Sigma) immediately after dissolving in0.1M citrate buffer at pH4.5. Three days afterSTZ injection, diabetes was confirmed by random blood glucose concentrations of greaterthan16.7mmol/L for3consecutive days.4weeks after onset diabetes, diabetic rats presentedmild microalbuminuria (an early sign of DN) and were considered to be diabetic nephropathy.
3. MSCs (1×106cells) were transfected with lentiviral vectors carrying eGFP to track theengrafted cells in kidneys. DN rats (n=10) were equally divided into eGFP-labeled MSCsgroup and eGFP-labeled MSCs+US+MB group. Three days after cells transplantation, ratswere killed by anesthetic overdose. Kidneys were separated and immediately snap-frozen inliquid nitrogen for frozen sections (5μm in thickness). The survival of implanted cells wasobserved and counted in frozen sections under a laser scanning confocal microscope (LSCM).Rats were randomly assigned into three batches. The first batch (88rats) was equally dividedinto four groups: control group received Evans blue (EB,2%in saline,50mg/kg) injectiononly; US group received EB injection, followed by0.5mL of PBS injection (an equivalentvolume of microbubble injection) and ultrasonic irradiation; MB group received EB injection,followed by microbubble injection (0.05mL/kg) without ultrasonic irradiation; US+MB groupreceived EB injection, followed by microbubble injection and ultrasonic irradiation. Beforeinjection, the bottle filled with microbubbles was shaken45s to form microbubblessuspension using a custom-built oscillation apparatus. Microbubbles were injected into lateraltail veins through26-gauge needles connected with a1mL syringe via a15cm length catheter,controlled by a syringe pump within30s and followed by0.5mL saline to wash the tube. A blanket covered the treated rats for1h.1h after treatment, the change of state of endothelialcells after ultrasonic irradiation was assessed by E-selectin mRNA and protein expression.Renal interstital capillary permeability was assessed by EB content assay, confocal laserscanning microscope (CLSM) and transmission electron microscope (TEM) examination. Thepossible injury in renal structure was evaluated by renal histology. To assess the impact ofultrasound-mediated microbubble destruction on kidney, urine samples of the third batch (32rats) which had the same grouping (four groups, n=8/group) and treatments as the first batchwere collected after24h post-treatment and measured for red blood cells, urinary albumin,and urinary creatinine. Urinary albumin excretion rate (UAER) was determined using urinaryalbumin-to-urinary creatinine ratio. Additionally, to assess the permeability recovery ofkidneys after ultrasound-mediated microbubble destruction, the second batch (32rats) withthe same grouping as above (four groups, n=8/group) received microbubbles injection, orultrasonic irradiation, or microbubble-ultrasound interaction, and then received EB injectionafter24h (Recovery group).
4. Random blood glucose concentrations were measured at1,2,4,8weeks after MSCstransplantation. UAER values and plasma insulin levels were detected at8weeks after MSCstransplantation. In addition, the ultrastructure of glomeruli and tubules was observed bytransmission electron microscope. The histological structure of pancreas and kidney wasobserved by HE and PAS staining, respectively. The distribution and quantity of islets β/α cellwere observed by pancreas double-label immunohistofluorescence. IL-10protein expressionin kidney was detected by ELISA. The distribution, expression, and quantitative analysis ofTGF-β1in kidney were assessed by immunohistochemistry.
Results：
1. Most of cultured MSCs were spindle shaped, attached to the culture dish tightly,proliferated in culture medium, and became more uniform after several passages. Theintracellular lipid droplets stained with oil red O staining and the calcium depositions stainedwith alizarin red S staining were observed after1week and2week of treatment, respectively.Flow cytometry analysis showed that these expanded MSCs were positive for CD44(99.44%),CD90(99.37%), and negative for the leukocyte common antigen CD34(1.17%), CD45(7.12%).
2. Three days after STZ injection, diabetes was confirmed by random blood glucose concentrations of greater than16.7mmol/L for3consecutive days.4weeks after onsetdiabetes, diabetic rats presented mild microalbuminuria (an early sign of DN) and wereconsidered to be diabetic nephropathy.
3.①The number ofeGFP-labeled MSCs in tubulointerstitial area of US+MB+MSCsgroup (18.3±2.9) was much more than that of MSCs group (5.7±0.8) under CLSM(P=0.0006). eGFP-labeled MSCs were mostly localized in outer region of interstitial capillary,the peritubular area and a small number of eGFP-MSCs located in glomeruli.②There was abit of blue staining in US group which the percentage of blue staining of renal surface andcoronal plane was (9.37±3.45%) and (10.10±3.24%), respectively, but no visible staining incontrol group and MB group. The percentage of blue staining of renal surface and coronalplane in US+MB group was (49.12±7.31%) and (37.99±4.36%), respectively.③EB contentwas significantly higher in US+MB group (37.267±4.948μg/g) than that in control group(13.942±2.848μg/g, P＜0.01), US group (14.126±2.570μg/g, P＜0.01), MB group(13.969±2.076μg/g, P＜0.01), and recovery group (14.658±3.916μg/g, P＜0.01).④Renalinterstitial capillary structure maintained intact and clear, and there was no EB extravasationin renal interstitium in control group, US group, and MB group. While the extravasation ofred EB fluorescence around interstitial capillary was evident in US+MB group, causing“fishing net” graph.⑤A part of interstitial capillary walls became thinned, discontinuous,and unsmoothed in US+MB group and they kept intact in control group, US group, and MBgroup.⑥The increase of E-selectin mRNA and protein expression were apparently morepronounced in US+MB group than in control group, US group, and MB group (P＜0.01,respectively).⑦Hematoxylin-eosin staining confirmed that there was no hemorrhage andnecrosis in renal structure in all groups.⑧There was no significant difference in UAER valuebetween US+MB group and Control group (60.12±25.67μg/mg creatinine&55.99±20.55μg/mg creatinine, P＞0.05), which was similar to that of US+MB group and US group(60.12±25.67μg/mg creatinine&57.43±18.40μg/mg creatinine, P＞0.05), US+MB groupand MB group (60.12±25.67μg/mg creatinine&53.86±22.42μg/mg creatinine, P＞0.05).⑨Hematuria was not detected in any group.
4.①Within1week after cell therapy, blood glucose concentrations significantlydecreased in MSCs group (22.5±5.3mmol/L) and US+MB+MSCs group (22.1±5.0mmol/L),reached the lowest levels at2weeks and lasted at least during the observation period. In contrast, untreated PBS group (28.4±6.2mmol/L) and US+MB group (27.9±6.5mmol/L)remained a high level of blood glucose until the end of the experiment. No obvious differencewas found in blood glucose concentration at the end of the experiment between MSCs group(23.8±5.5mmol/L) and US+MB+MSCs group (24.0±4.6mmol/L), P=0.395, which was thesame as that between PBS group (28.4±6.2mmol/L) and US+MB group (27.9±6.5mmol/L),P=0.816.②At the end of the experiment, compared with Normal group (25.27±2.34mIU/L),plasma insulin levels decreased in PBS group (16.42±2.17mIU/L) and US+MB group(16.67±2.33mIU/L), and improved in MSCs group (19.31±1.68mIU/L) and US+MB+MSCsgroup (19.53±1.59mIU/L). There was no significant difference in plasma insulin levelsbetween MSCs group and US+MB+MSCs group (P=0.574), which was the same as thatbetween PBS group and US+MB group (P=0.564).③At the end of the experiment, UAERvalues maintained a high level in PBS group (643.25±204.58μg/mg) and US+MB group(637.29±212.24μg/mg) and both of them were more than fourfold higher than that ofage-matched normal control rats (128.57±36.93μg/mg). As a comparison, UAER valuesreduced in MSCs group (302.41±49.21μg/mg) and US+MB+MSCs group (252.83±39.58μg/mg). Furthermore, UAER values were significantly milder in US+MB+MSCs group(252.83±39.58μg/mg) than that in MSCs groups (302.41±49.21μg/mg).④PBS group andUS+MB group rats showed glomerular basement membrane thickening, extracellular matrixdeposition, glomerular mesangial expansion and podocyte fusion in glomerular ultrastructureunder transmission electron microscope. There were myelin-like structures in renal tubularepithelial cells, microvilli swelling in tubule epithelial cells, and mitochondrial swelling witha large number of vacuolation in tubular ultrastructure under TEM. There was a significantimprovement in MSCs group and US+MB+MSCs gorup, especially in US+MB+MSCs group.⑤Renal histological analysis showed that there have similar changes both in PBS group andUS+MB group in glomeruli and tubule which displayed glomerular sclerosis, mesangialexpansion, and tubular dilatation observed by light microscope at the end of experiment.However, the extent of such changes of glomeruli and tubule was extenuated in MSCs groupand US+MB+MSCs group. Only a small part of glomerular hyalinosis and tubular dilatationwere observed in MSCs group. Further improvements in structure or even nearly normalstructure of glomeruli and tubule were observed in US+MB+MSCs group.⑥At the end ofthe experiment, PBS group and US+MB group showed a massive destruction of pancreatic islets/β cells observed by pancreatic pathology and double-label immunohistofluorescence.Compared to normal pancreatic islets/β cells, morphological irregularity, volume reduction,less insulin-and more glucagon-positive cells were observed in PBS group and US+MBgroup. As expected, MSCs group and US+MB+MSCs group exhibited a good recovery onamount, architecture and volume of pancreatic islets/β cells.⑦TGF-β1protein expressiondetected by immunohistochemistry was markedly increased in glomeruli and interstitialspaces in PBS group and US+MB group, compared with Normal group. MSCs therapydecreased TGF-β1protein expression in glomeruli and interstitial spaces. Furthermore,TGF-β1protein expression in glomeruli and interstitial spaces was significantly lower inUS+MB+MSCs group than that in MSCs group.⑧IL-10protein expression in renal tissuedetected by ELISA was markedly decreased in PBS group and US+MB group, compared withNormal group. MSCs therapy increased IL-10protein expression. Furthermore, IL-10proteinexpression was significantly higher in US+MB+MSCs group than that in MSCs group.
Conclusions：
1. Purified MSCs can be obtained via the adherent culture methods using morphologicalobservation, surface marker identification and inducing differentiation. It is a simple availablemethod for the isolation and culture of MSCs.
2. Three days after a single intraperitoneal injection of60mg/kg STZ injection, diabeteswas confirmed by random blood glucose concentrations of greater than16.7mmol/L for3consecutive days.4weeks after onset diabetes, diabetic rats presented mild microalbuminuria(an early sign of DN) and were considered to be diabetic nephropathy.
3. eGFP labeled MSCs could efficiently track the distribution of MSCs in organs.
4. Ultrasound-mediated microbubble destruction locally increased renal interstitialcapillary permeability in early diabetic nephropathy rats, which should be considered anenhancement therapy for drug, gene, antifibrotic agent, or stem cells delivery to kidney in thefuture. The underlying mechanism may be that ultrasound-mediated microbubble cavitationruptured interstitial capillary walls, which promoting therapeutic substances to cross throughdiscontinuous walls, enter renal interstitium, and maximize therapeutic effect. The mild injuryof interstitial capillary after cavitation effect caused the up-regulation of E-selectin, whichperhaps promoted the homing of transplanted stem cells to DN. This technology perhapsprovides a new idea for personalized cell therapy to DN by using stem cells.
5. Microbubble insonation and inertial cavitation may be safe to kidney underappropriate parameters. The increased permeability in renal interstitial capillary is reversibleand EB content returned to the control group level after24h.
6. The interstitial capillary permeability and E-selectin mRNA and protein expressionwere increased, and the microenvironment was changed by ultrasound-mediated microbubbledestruction, which facilitated the homing and gathering of MSCs to kidney, inhibited TGF-β1expression, reduce the damage to the tubules and interstitium, and prevented the process ofdiabetic nephropathy.
7. The increased expression of IL-10and inhibition of TGF-β1in US+MB+MSCs groupmay be induced by paracrine effect of the implanted MSCs, which augmented the adhension,homging and gathering ability to kidney, inhibited the inflammatory response, reduced thedegree of renal hypertrophy, and improved renal function.
8. MSCs transplantation reduced blood glucose concentrations, promoted islets recovery,and prevented the process of diabetic nephropathy. MSCs therapy plays a positive role topromote diabetic nephropathy repair.
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