【作者】 唐海林；

【导师】 王志刚；

【作者基本信息】 重庆医科大学 ， 影像医学与核医学， 2012， 博士

【摘要】 急性肾功能衰竭（ARF）在临床上是常见的急危重症，尤其在重症监护室。大部分缺血和中毒所致的急性肾功能衰竭是可治的，但是，往往是伴有多器官衰竭的因素。实际上，尽管肾脏治疗有了很大的发展，医院内获得性急性肾功能衰竭的病死率并没有明显减少，仍高达30-80%。急性肾小管坏死（ATN）是缺血和中毒所致的急性肾功能衰竭的主要原因。因此，怎么能够促进ATN后对其修复与再生，从而到达使ATN的死亡率降低。研究表明，生长因子在ATN肾小管皮细胞再生中起积极的作用。在动物ATN的模型上，用如下的生长因子治疗：上皮生长因子(EGF)、胰岛素样生长因子1(IGF-1)及肝细胞生长因子（HGF），可以改善其增殖再生，从而到达修复的目的。虽然在实验动物中，发现这些细胞因子促进ATN的再生和修复，但在人患有急性肾脏损伤后，用胰岛素样生长因子1对其进行治疗，疗效不佳。有学者推测，人体ATN的修复有着十分复杂的病理生理过程，其修复可能涉及到肾微环境的网络蛋白综合作用，用单个基因产物来治疗作用非常有限。骨髓间充质干细胞是近年来研究的热点，越来越多应用于各种组织器官损伤的再生治疗，为组织器官的修复开辟了一条新的途径。骨髓干细胞移植是治疗ATN的一种新方法。骨髓干细胞在病理条件下会归巢至肾小管损伤部位，并分化为肾小管皮细胞，促进肾组织形态和功能改变。但是有一些学者持怀疑态度，认为骨髓源性的间充质干细胞不能转化为肾小管上皮细胞。在参与肾损伤修复过程的间充质干细胞中，骨髓源性的间充质干细胞所占比例很少。用干细胞移植来治疗ATN并希望干细胞分化成肾小管上皮细胞的设想很难实现。因此，能改善骨髓干细胞移植治疗ATN的方法吗？干细胞植入的有效性及靶向性是干细胞移植治疗ATN的关键。而细胞间黏附分子表达有利于干细胞的归巢，提高干细胞移植的疗效，为骨髓干细胞治疗急性肾小管坏死提供了新的思路。近年人们在尝试药物及基因靶向释放的新方法（超声靶向破坏微泡）。超声介导微泡破裂后产生有如下的生物效应：内皮细胞间隙增宽，细胞因子表达改变，中性粒细胞聚集，炎性反应等。感兴趣的是，超声辐照微泡后骨骼肌毛细血管内皮细胞粘附分子-1（ICAM-l）及血小板源生长因子PDF表达增加。在后来实验中也发现超声介导微泡后心肌组织毛细血管内皮细胞粘附分子-1（VCAM-1及ICAM-1）表达增加。而且应用超声介导微泡后产生的生物学效应，转导骨髓干细胞到下肢缺血模型中及转导内皮祖细胞到非缺血性心肌病，都取得了一定疗效。后来应用聚焦超声及应用诊断超声介导微泡转导骨髓干细胞到缺血性心脏病模型，也有同样的效果。而未见报道将超声介导微泡的这种生物学效应应用于骨髓干细胞移植到肾脏模型是否也是一种可行的方法。因此，我们尝试将超声介导微泡转导骨髓干细胞到大鼠ATN肾组织并促进ATN修复研究。超声介导微泡的生物学效应被广泛的关注及研究，微泡的空化效应可对周边的毛细血管壁造成一些微观的生物学反应，包括微血管的渗漏、毛细血管破裂、诱导周边细胞的凋亡及引起局部炎症反应等。尽管这些现象科发生在多个器官，但是肾脏似乎对上述的生物学效应更加敏感，这可能与肾小球毛细血管内血压较高有关，严重者可以引起持续出血，导致肾单位的损伤，甚至肾功能不全。因此，将超声介导微泡的这种生物学效应应用于骨髓干细胞移植到肾脏模型，超声辐照微泡的条件需要优化。针对上述理论及存在的问题，本研究以二氯化汞（HgC12）致大鼠急性肾小管坏死为模型，观察不同强度超声介导微泡后对肾组织微环境的影响，探寻合适超声辐照条件以达到干细胞移植治疗的目的。并在超声辐照介导改善肾微环境后进行4’,6-二脒基-2-苯基吲(DAPI)标记的干细胞移植治疗，采用荧光显微镜观察MSCs在肾组织的分布情况，荧光定量PCR测定基因表达水平， Western blot测定肾组织中蛋白质的表达情况，HE染色观察肾小管坏死后修复情况。共包括以下三个部分。第一部分：大鼠骨髓间充质干细胞的体外分离、培养及鉴定目的：探讨一种体外分离、培养的高纯度大鼠间充质干细胞（MSCs）方法并对其鉴定。方法：为获得高纯度的间充质干细胞，采用如下方法：取大鼠胫骨及股骨骨髓，通过密度梯度离心法分离出单一核细胞，再采用贴壁培养方法。采用免疫组化对CD34、CD31、CD44和CD45的细胞膜表面抗原进行检测，并用流式细胞仪检测细胞表面特异性抗原CD29及对大鼠MSCs进行周期测定。结果：细胞培养结果是分离出骨髓单个核细胞并贴壁。细胞鉴定结果是大鼠干细胞膜表面抗原阳性的是CD44，表达阴性的是CD45、CD31和CD34；细胞周期检测90.36%的细胞处在G0/G1期；流式检测CD29阳性率是91.54%。结论：通过Percoll分离液筛选，经贴壁培养后获得高纯度的间充质干细胞，可为治疗大鼠肾小管坏死提供充足的细胞来源。第二部分：超声辐照微泡对急性肾小管坏死肾组织微环境的影响目的：探讨不同条件超声介导微泡对急性肾小管坏死肾组织的生物学效应，选择合适的超声参数以促进干细胞治疗ANT。方法：急性肾小管坏死模型的建立：腹腔注射HgCl2：(0．2％)的方法，剂量为0.75mg／kg。将HgC12致急性肾小管坏死为模型的28只SD大鼠随机分为4组：不采用超声辐照第1组大鼠，作为对照组；第2、3、4组大鼠在肾小管坏死模型第1天及第3天后通过静脉输入微泡，采用频率为1MHz，强度分别为0.5W/cm~2、1.0W/cm~2、2.0W/cm~2的脉冲超声经肾区辐照大鼠肾组织，每次辐照1分钟。第2次辐照后4天将大鼠处死，取肾组织，定量PCR及免疫组织化学法检测细胞间黏附分子1(ICAM-1)及肿瘤坏死因子α（TNFα）基因及蛋白质的表达水平。结果：荧光PCR结果显示：2.0W/cm~2组肾组织表达ICAM-1及TNFα较对照组分别增加约3.1倍及6.3倍；1.0W/cm~2组表达ICAM-1及TNFα较对照组分别增加约2.9倍及3.5倍；2.0W/cm~2组的超声辐照肾组织表达TNFα较1.0W/cm~2组明显增加(P<0.05)；免疫组织化学法检测结果表明强度为1.0W/cm~2及2.0W/cm2的超声辐照肾组织表达ICAM-1及TNFα较对照组明显增加(P<0.05)；结论：强度为1.0W/cm~2的超声介导破裂微泡作用于大鼠急性肾小管坏死模型，上调肾组织ICAM-1的表达水平及轻度炎性反应，有利于干细胞的归巢。第三部分：超声靶向破坏微泡诱导肾组织微环境改变联合骨髓间充质干细胞促进急性肾小管坏死修复实验研究目的：探讨超声靶向破坏微泡是否使骨髓间充质干细胞归巢于肾组织并促进大鼠急性肾小管坏死修复。方法：急性肾小管坏死模型的建立：腹腔注射HgCl2：(0．2％)的方法，剂量为0.75mg／kg。采用UGT1025型超声辐照仪，强度为1.0W/cm2及频率为1MHz超声照射肾脏组织，并尾静脉输入0.5ml造影剂，浓度为1.0×108个/ml左右，辐照时间5s，停5s，总共1min。在急性肾小管坏死模型的建立后第1d及3d分别辐照1次。将制备成功的40只肾小管坏死模型大鼠随机分为4组（10只），即(1)单纯模型组（对照组）；(2)1.0W/cm2超声+微泡组（1.0US+MB）；(3)干细胞组（MSCs）；(4)1.0W/cm2超声+微泡+干细胞组（1.0US+MB+MSCs）。最后一次辐照完毕后1min内经股静脉注入DAPI标记的骨髓间充质干细胞1ml（2.0×106个/ml）。7d后将各组大鼠处死，取材用于荧光显微镜观察MSCs在肾组织的分布情况，采用荧光定量PCR测定细胞间黏附分子（1ICAM-1）、表皮细胞生长因子(EGF)及肝细胞生长因子(HGF)基因表达水平。采用Western blot测定肾组织中HGF及EGF蛋白表达水平，采用HE染色观察肾小管坏死后修复情况。结果：采用荧光显微镜观察，DAPI标记的MSCs细胞核（细胞核带蓝色荧光），1.0US+MB+MSCs组肾组织骨髓干细胞数量明显多于MSCs组(P<0.05)。荧光定量PCR结果表明1.0US+MB组及1.0US+MB+MSCs组ICAM-1的基因水平明显高于对照组及MSCs组(P<0.05)。1.0US+MB+MSCs组HGF及EGF基因水平明显高于其他各组(P<0.05)。Western blot检测结果表明1.0US+MB+MSCs组HGF及EGF蛋白表达水平明显高于其他各组(P<0.05)。HE染色结果表明1.0US+MB+MSCs组肾小管坏死修复情况明显好于其他各组(P<0.05)。结论：1.0W/cm2超声介导微泡促进骨髓间充质干细胞归巢于肾脏，并通过旁分泌或自分泌形式分泌HGF及EGF，促进急性肾小管坏死修复。更多还原

【Abstract】 Acute renal failure (ARF) is clinically common acute and critically illin hospitalized patients，especially in the intensive care unit. ARF mostfrequently ensues upon a toxic or ischemic insult to the kidney and ispotentially reversible, however, being often just one element of multipleorgan damage. The mortality rate in hospital acquired ARF still ranges from30to80%actually. Modern dialysis techniques had no significant impact onoverall mortality. Acute tubular necrosis (ATN) plays central roles in theprocess underlying the failure of the kidney after toxic or ischemicchallenge.In animal models, the rate of recovery depends strictly on thereplacement of damaged and/or dead epithelium with a new functioning one.Growth factors, such as insulin-like growth factor-1(IGF-1), hepatocytegrowth factor（HGF）, and epidermal growth factor(EGF), have been usedconsistently to potentiate tubular regeneration in experimenta ARF andaccelerated the proliferation and migration of resident cells. However, a therapeutic trial of recombinant human IGF-1in patients with ATN failed todemonstrate efficacy. The pathophysiology of human renal tubular necrosisand repair is very complex. Regenerative factors of renal tubular repair maybe involved with the combined effects of the protein network and a singlegene product is difficult to achieve therapeutic purposes.Tissue-based stem cells have traditionally been viewed asmultipotential precursor cells that are capable of generating tissue-specificdifferentiated cells. Intravascular transplantation of BM-MSCs is apromising therapeutic approach after ATN. Some scholars reported thatMSCs homed to the kidneys with ATN, differentiated into tubular epithelialcells and promoted the recovery of morphological and functional alterations.However, some studies argue against the direct transdifferentiation of bonemarrow–derived stem cells or MSCs into the kidney. The engraftmentfrequency of bone marrow–derived stem cells was reported to be relativelysmall. The present studies demonstrate that in vivo differentiation of MSCsinto renal tubular cells may not occur at all or that it is, at most, a minorcomponent of the repair process after ischemic injury. Is there a method toimprove cell transplantation? The efficacy and targeting of MSCsengraftment should now be crucial variables determining the therapeuticvalue of MSCs transplantation. Strategies involving adhesion moleculeshave been proposed to augment the attraction and invasion of stem cells andincrease their integration into pre-treated tissue. Ultrasound targeted microbubble destruction (UTMD) has evolved as apromising tool for organ specific gene and drug delivery.Ultrasound-mediated microbubble destruction resulting from the followingbiological effects: broaden the endothelial cell gap，cytokine expressionchange, neutrophil aggregation, and inflammatory response. Interestingly,platelet-derived proinflammatory factors activated by acoustic cavitationinduce the expression of adhesion molecules (P-selectin and ICAM-1),leading to the attachment of transplanted BM-MSCs onto the endothelium.Acoustic cavitation induces the expression of adhesion molecules (VCAM-1and ICAM-1) in capillaries. Moreover, the targeting of transplanted bonemarrow-derived stem cells has been facilitated by acoustic cavitation inischemic skeletal muscle non-ischemic cardiomyopathy and acutemyocardial infarction tissue. However, no studies have been reported thatdetermine whether this method is feasible for delivering MSCs to kidneytissue in ATN. Based on these previous findings, we attempted to explorewhether acoustic cavitation augments the site-targeted engraftment of MSCsto the kidney and promotes kidney recovery in ATN in rats.The bioeffects of ultrasound in combination with microbubbles havebeen extensively studied. These studies demonstrated that inertial cavitationof microbubbles in close proximity of the capillary wall can causemicroscopic bioeffects, including microvascular leakage, capillary rupture,and local induction of cell death and inflammation. Although these phenomena can occur in several organs, the kidney appears to be especiallysensitive to the adverse side-effects of ultrasound and microbubbles. Thismay be related to the relatively high blood pressure within the capillaries ofthe glomerulus, resulting in substantial bleeding into the Bowman’s spacewith subsequent loss of function of the whole nephron. Therefore, theparameters of ultrasound need to be optimized.Based on the above mentioned theoretical basis and problems, ATNwas induced by the subcutaneous injection of mercuric chloride (HgCl2) inrats and microenvironment of renal tissue induced by different intensityultrasound-mediated microbubble will be investigated, and to explore thesuitable ultrasonic irradiation condition which is suitable for MSCstransplantation will be determined in this study.4’,6-diamidino-2-phenylindole (DAPI) labeled MSCs transplantation willbe performed after environment changes of renal tissue induced byacoustic cavitation. The number of MSCs was evaluated by fluorescencemicroscopy, and real-time polymerase chain reaction (RT-PCR) and westernblotting and histological examination were performed7days after MSCstransplantation.Overall，The study was composed of the following three parts. PART I: SOLATION, CULTIVATION ANDIDENTIFICATION OF RATS BONE MARROW-DERIVEDMESENCHYMAL STEM CELLS IN VITROObjective: To investigate a stable method for the isolation, purification,cultivation and phenotype analysis of rats bone marrow-derivedmesenchymal stem cells in vitro. Methods：BM-MSCs were obtained from4-week-old rats. Bone marrow mononuclearcells were extracted from ratsfemurs and tibias bone marrow and purified via density gradientcentrifugationand then adherent culture. The expression of CD31，CD34，CD44and CD45were measured by immunohistochemistry. Flow cytometrywas used to determine the expression of CD29. Flow cytometry was used todetermine the cycle of MSCs by surface maker protein. Results:Immunohistochemical detection indicated positive expression of CD44.The expression of CD45, CD31and CD34was negative.The cellscomprised a unique phenotypic population, as visualised by flow cytometricanalysis of expressed surface antigens. These expanded MSCs wereuniformly positive for CD29(91.54%). The results from generation cycledetection showed90.36%cells in the period of G0/G1by flow cytometricanalysis.Conclusion: MSCs can be obtained by isolating rat femurs and tibiasbone marrow and purifying with adherent culture, providing a sufficient source of cells for the treatment of rat renal tubular necrosis. PART II: EFFECTS ON KIDNEYS TISSUEMICROENVIROMENT INDUCED BY ULTRASOUNDDESTRUCTION OF MICROBUBBLES IN ACUTETUBULAR NECROSIS MODELObjective: To explore the effects on kidneys tissue microenviromentinduced by acoustic cavitation with different ultrasound intensities in ratsand to determine the proper ultrasound intensity which is suitable for stemcells transplantation. Methods: ATN was induced by the subcutaneousinjection of mercuric chloride (HgCl2). Tweny eight Sprague-Dawley (SD)rats were randomly divided into the4group after the establishment of animalmodels of acute tubular necrosis. No acoustic cavitation in group1(controlgroup). One days and three days after ATN, rats in group2、3and4groupwere received pulse ultrasound (insonate for5s at1MHz and with a5s pause,totalling60s) targeted microbubble destruction focused on the lower backregion of rats with intensity of0.5W/cm2US，1.0W/cm2US and2.0W/cm2US respectively. All rats were sacrificed and the kidenys tissue washarvested4days after the last acoustic cavitation in experimental group. Real-time polymerase chain reaction (RT-PCR) and histologicalexamination were performed. Results: The results from RT-PCR revealedthat2.0W/cm2US+MB group markedly increased the level of ICAM-1andTNFα (P<0.05,3.1fold and6.3fold, respectively) compared with thecontrol group.1.0W/cm2US+MB treatment markedly increased the level ofICAM-1and TNFα (P<0.05,2.9fold and3.5fold, respectively) comparedwith the control group. The level of TNFα in2.0W/cm2US+MB group wasobviously higher than those with1.0W/cm2US+MB group (P<0.05). Theresults from Immunohistochemical examination revealed that the level ofICAM-1and TNFα in2.0W/cm2US+MB and1.0W/cm2US+MB groupwere marked increased than0.5W/cm2US+MB and the control group(P<0.05). Conclusion: Microbubble destruction by1.0W/cm2ultrasoundcan increase the level of ICAM-1of the kidney in ATN in rats and resultingin favor of the homing of stem cells with mild inflammatory response. PART Ⅲ: DELIVERY OF MSCS AFTERMICROENVIRONMENT CHANGES INDUCED BYULTRASOUND TARGETED MICROBUBBLEDESTRUCTION PROMOTE KIDNEY RECOVERY INACUTE TUBULAR NECROSIS MODELObjective: The aim of the present study was to explore whether ultrasound microbubble destruction augments site-targeted engraftment ofBM-MSCs to kidney tissue and promotes recovery of the kidney in acutetubular necrosis in rats. Methods: ATN was induced by the subcutaneousinjection of mercuric chloride (HgCl2).We performed the0.5ml infusion ofmicrobubbles via the contralateral femoral vein at days1and3after ATN.Simultaneous insonation was started at the lower back region of rats, usingan ultrasound gene transfection treatment meter [UGT]1025with thefollowing settings: insonate for5s at1MHz and1.0W/cm2with a5s pause,totalling60s. Forty Sprague-Dawley (SD) rats were randomly divided intothe following groups after the establishment of animal models of ATN injury(n=10):(1) Model group alone (control group);(2)1.0W/cm2US+MB(US/MB group), Bubble (0.5ml)+1.0W/cm2US;(3) MSCs group (ratMSCs at a concentration of2×106in1ml saline were infused via thecontralateral femoral vein at3day after ATN);(4)1.0W/cm2US+MB+MSCs group (US/MB+MSCs group), which received an infusion of ratMSCs (2×106in1ml saline) via the contralateral femoral vein1minuteafter US/MB at3day after ATN. The number of4’,6-diamidino-2-phenylindole (DAPI) labelled MSCs was evaluated byfluorescence microscopy, and RT-PCR and western blotting and histologicalexamination were performed7days after MSCs transplantation. Results: Itwas observed via fluorescence microscopy that the number of DAPI labelled MSCs in the kidney for the US/MB+MSCs group was significantly morethan the MSCs group (P<0.05). The results from RT-PCR revealed that theUS/MB and US/MB+MSCs groups markedly increased the level ofICAM-1messenger ribonucleic acid (mRNA) compared with the controlgroup and the MSCs group (P<0.05). RT-PCR and Western blot analysisshowed that the expression of hepatocyte growth factor (HGF) andepidermal growth factor (EGF) in the US/MB+MSCs group were markedlyincreased compared with the all other groups (P<0.01). The extent of tubularnecrosis and dilation was significantly milder in the US/MB+MSCs group(acoustic exposure conditions:5s at1MHz and1.0W/cm2with a5s pause,totalling60s) than the all other groups (P<0.05). Conclusion: Microbubbledestruction by1.0W/cm2ultrasound can promote both the homing ofBM-MSCs to kidney tissue and the recovery of the kidney in ATN in ratswith the secretion of paracrine or autocrine of HGF and EGF.更多还原