超声联合微泡促HGF修饰BMSCs靶向归巢抗肾纤维化的实验研究
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摘要
研究背景：
肾小管间质纤维化是大多数慢性肾病的终末期表现，因此及早控制和逆转肾脏纤维化的进程是慢性肾脏疾病治疗中非常重要的治疗途径。
研究表明骨髓间充质干细胞(BMSCs)由于其高可塑，易分化等特性，在组织修复和器官再生的细胞治疗和基因治疗等领域表现出特有的优势。对于肾脏而言，骨髓干细胞可以分化为肾脏细胞包括系膜细胞、内皮细胞、足细胞和肾小管上皮细胞等，这就使得BMSCs成为修复受损肾脏最具吸引力的细胞。BMSCs还可以通过旁分泌的形式对局部缺血或再灌流造成的肾损伤进行保护,明显减少邻近细胞的坏死。然而，也有研究报道单纯BMSCs移植治疗受损的纤维化改变的肾脏效果不佳，究其原因可能与BMSCs的移植途径，靶向归巢数量少以及缺乏能够明显改善肾间质纤维化的肝细胞生长因子(HGF)等多方面因素有关。
近年来，超声造影剂(微泡)的应用已从诊断领域向治疗领域拓展。微泡借助其在超声作用下的空化作用可以作为一种基因治疗的非病毒载体。超声靶向击破微泡(Ultrasound-targeted microbubble destruction，UTMD)介导药物靶向传输和基因转染是近年发展起来并被公认为有效而且很有潜力的方法。该研究设想以此特性用HGF修饰BMSCs，通过对基因转染后的BMSCs生物学功能评价优选声学参数，获得活性显著提高的目的细胞对肾纤维化动物模型进行移植。另外，UTMD还可能改变靶器官的微环境，增强靶器官微血管通透性，使内皮细胞间隙增加，促使细胞因子分泌的改变，为BMSCs“着床”于靶组织创造条件。通过超声联合微泡介导HGF修饰BMSCs移植，促进HGF-BMSCs向损伤肾脏选择性归巢与聚集，进而起到提高疗效和改善肾纤维化的作用。将基因治疗和细胞治疗相结合，提高移植效率，对于抗肾纤维化的可行性、安全性及靶向性进行研究，并对其机制进行探讨。
研究目的：
1.探讨微泡介导治疗超声联合聚乙烯亚胺(PEI)实现HGF修饰BMSCs的有效性，可行性及其机制。试图探索一种非病毒的方法来实现BMSCs的高效转染从而避免病毒转染将会引发的安全问题；
2.探讨一定参数配比条件下的诊断超声联合微泡对于大鼠肾间质毛细血管通透性的改变情况以及对于肾脏形态结构和功能的影响；
3.探讨诊断超声联合微泡作用下静脉移植BMSCs促进其归巢单侧输尿管梗阻模型受损肾脏的可行性及其机制；
4.探讨诊断超声联合微泡介导BMSCs和HGF修饰的BMSCs移植应用于延缓大鼠肾纤维化进程的有效性、可行性、安全性及可能机制，为探讨肾纤维化新的治疗手段提供实验基础。
研究方法：
1.微泡介导治疗超声联合PEI转染大鼠骨髓间充质干细胞的体外研究
采用贴壁培养法对大鼠BMSCs进行分离培养。并通过生长曲线、细胞周期、透射电镜等检测其生物学特性。用流式细胞仪检测细胞表面标记分子并成脂、成骨诱导分化进行细胞鉴定。构建质粒DNA:pEGFP-HGF。按照均匀实验设计，对超声强度，微泡浓度，超声辐照时间三个主要影响因素进行考察，通过对细胞密度，转染效率，细胞存活率的检测，最终优选出最佳的转染参数配比。对转染后BMSCs的细胞周期，分化能力进行检测。运用激光共聚焦荧光显微镜对转染后48h，7d的BMSCs荧光表达进行观察。扫描电镜观察超声联合微泡作用对于细胞造成的损伤以及细胞的自行恢复情况。
2.微泡介导诊断超声对健康大鼠肾脏通透性的影响及安全性研究
诊断超声联合微泡作用采用不同的超声发射方式(连续式超声辐照，间歇式超声辐照)于健康大鼠肾脏。采用Evans blue (EB)定量分析，HE染色，透射电镜对靶器官肾脏间质区毛细血管通透性的改变进行观察检测；通过激光共聚焦荧光显微镜对DIO标记微泡的靶器官释放的定位及相对定量进行观察分析；对尿蛋白进行检测，评价该方法对肾小球滤过、肾小管重吸收的影响。
3.诊断超声联合微泡促进BMSCs归巢模型大鼠肾脏的研究
构建单侧输尿管梗阻(UUO)模型，采用影像学和病理学方法评价模型的建立。于建模后7d，采用超声联合微泡介导HGF-BMSCs静脉移植。分组：模型大鼠移植HGF-BMSCs组(UM组)；模型大鼠+超声+微泡+HGF-BMSCs组(UUM组)，健康大鼠移植HGF-BMSCs组(CM组)；健康大鼠+超声+微泡+HGF-BMSCs组(CUM组)。激光共聚焦观察示踪各组肾脏的绿色荧光阳性细胞计数和统计学分析。HE染色和透射电镜观察各组移植后靶组织形态结构和间质毛细血管超微结构。免疫组化对受损肾脏归巢相关因子SDF-1表达进行定量分析。
4.诊断超声联合微泡经静脉移植BMSCs改善大鼠肾纤维化的研究
构建单侧输尿管梗阻再通(RUUO)模型，采用影像学和病理学方法评价模型的建立。建模后7d进行干细胞移植治疗，于14d解除输尿管梗阻。在建模后7d，14d，21d，28d四个时相点分别对RUUO组， BMSCs组， US+MB+BMSCs组和US+MB+HGF-BMSCs组进行观察。采用影像学方法对肾脏形态进行观察；HE染色，Masson染色对肾小管间质损害进行观察和病理评分；Real time PCR检测肝细胞生长因子(HGF),转化生长因子-β(TGF-β)和α-平滑肌肌动蛋白(α-SMA)表达；免疫组织化学检测α-SMA表达；western blot检测SDF-1，α-SMA表达。生化检测血清肌酐，血清尿素氮和内生肌酐清除率评价肾脏功能改变。
结果：
1.微泡介导治疗超声联合PEI转染大鼠骨髓间充质干细胞的体外研究
成功分离培养BMSCs，检测结果显示绝大部分细胞表达CD44，CD29和CD90，几乎不表达CD34，CD45和CD11b。成脂诱导培养后油红O染色胞浆脂滴形成，成骨诱导后茜素红染色钙结节形成。超声联合微泡协同PEI转染BMSCs最佳参数：声强(Q)=0.6W/cm2，微泡(MB)=106/ml，辐照时间(T)=30s时，pEGFP-HGF转染效率38.81±2.58%和BMSCs细胞存活率82.10±1.77％达到一个相对比较匹配的数值。不同影响因素比较PEI:DNA+MB+US组同其他各组有统计学差异(P<0.05),激光共聚焦观察发现转染后直到7d仍可见绿色荧光蛋白表达。转染后7d，PEI:DNA+MB+US组的转染效率为57.43±1.56%(图12F)，而细胞活性下降到78.98±3.11%。转染后的BMSCs细胞周期及分化能力检测G1期的细胞占82.93%，成脂诱导培养后油红O染色胞浆脂滴形成，成骨诱导后茜素红染色钙结节形成。Westernblot检测细胞中HGF蛋白的表达:转染pEGFP-HGF成功表达HGF蛋白，而对应的空白对照组和转染pEGFP-N1组未检测到目的蛋白。扫描电镜观察到超声联合微泡最佳条件作用后，BMSCs的细胞膜上可以看到微小的孔,大小不一，形态各异。7天后，BMSCs基本恢复，大多数的微孔已经消失，细胞表面基本光滑。
2.微泡介导诊断对大鼠肾脏通透性的影响及安全性
Evans blue定量分析连续式超声辐照组(CUS)和间歇式超声辐照组(IUS)同对照组比较没有明显差异(P<0.05)。超声联合微泡作用(UTMD)后，无论CUS+MB组还是IUS+MB组的EB含量同其它三组比较都呈现显著增加，组间差异显著(P<0.05)。UTMD后6小时，在IUS+MB6h组EB含量达到3.15±2.20μg/g，同IUS+MB组相比较，下降显著。UTMD后24小时，IUS+MB24h组EB含量达到0.690±0.540μg/g，这与对照组差异无统计学意义(P>0.05)。病理学检测提示UTMD后间质受损，可见红细胞的漏出，肾间质内毛细血管连续性中断，肾小球未受到明显损伤。而肾间质的损伤在24h内能够自身恢复。激光共聚焦扫描荧光显微镜观察到DIO标记的MB在肾间质靶向释放，同其它脏器比较具有显著差异。其中IUS+MB组(IOD=2092.17±342.36)和CUS+MB组(IOD=943.48±179.07)同对照组和右肾比较显著增加，有统计学差异(P<0.05)。IUS+MB组同CUS+MB组相比较，呈现出显著的增加(P<0.05)。尿检结果显示各组的尿蛋白水平在正常范围内(-)到(++)，均未观察到蛋白尿的显著变化。
3.诊断超声联合微泡促进BMSCs归巢模型大鼠肾脏的研究
成功构建UUO模型。激光共聚焦观察示踪静脉移植BMSCs归巢梗阻侧肾脏，在健康大鼠移植HGF-BMSCs组(CM组)和健康大鼠+超声+微泡+HGF-BMSCs组(CUM组)的肾脏组织切片上没有观察到绿色荧光表达；而在模型大鼠移植HGF-BMSCs组(UM组)和模型大鼠+超声+微泡+HGF-BMSCs组(UUM组)肾小管间质区内观察到胞浆内表达绿色荧光的细胞，分别为(16.41±5.37)个、(86.39±8.49)个，差异有统计学意义(P<0.01)。HE染色光镜下观察发现UTMD作用后的模型大鼠肾间质的毛细血管破裂，大量红细胞漏出，间质结构疏松，水肿，肾间质通透性显著增加。透射电镜显示了类似的结果。免疫组织化学检测归巢相关因子SDF-1，CM组和CUM组SDF-1表达定位远曲小管和极少近曲小管的胞浆中，UM组和UUM组定位在大部分的肾小管上皮细胞胞浆中，甚至在个别细胞的膜上也有表达。UUM组(22146.49±1920.73)高于UM组(18643.94±2046.29)，差异不显著，P>0.05，与CUM组(5528.36±1021.81)，CM组(5162.38±1376.54)的组间差异有统计学意义，P<0.05。
4.诊断超声联合微泡经静脉移植BMSCs改善大鼠肾纤维化的研究
成功构建RUUO模型。超声观察各组肾脏在不同时相点(建模后7d,14d，21d，28d)的形态变化，US+MB+BMSCs组和US+MB+HGF-BMSCs组同RUUO组和BMSCs组比较差异显著。肾小管间质病变的病理积分发现在术后21d和28dUS+MB+BMSCs组和US+MB+HGF-BMSC组的病理积分开始明显低于BMSCs组和RUUO组(P<0.05)。其中28d时US+MB+HGF-BMSC组较其他三组差异显著(P<0.05)。Real-time PCR检测不同时相点各组肾脏组织致纤维化转化生长因子TGF-β的基因表达：14d后移植BMSCs的三个治疗组与RUUO组比较，差异显著(P<0.05)；诊断超声联合微泡作用的两个BMSCs移植治疗组表达减低，较单纯BMSCs移植组差异显著(P<0.05)；在14d和21d两时相点，US+MB+HGF-BMSCs组与US+MB+BMSCs组比较，差异显著(P<0.05)。肝细胞生长因子(HGF)的基因表达：在14d和28d两个时相点，US+MB+BMSCs组和US+MB+HGF-BMSCs组的HGF表达较RUUO组增高且差异显著(P<0.05)；而14d后的各时相点，US+MB+HGF-BMSCs组的HGF表达均高于其余各组，差异显著(P<0.05)。从基因水平，蛋白水平(Western blot检测，免疫组化检测)分别检测α-平滑肌肌动蛋白(α-SMA)的表达(α-SMA被认为是成纤维细胞的标志，参与间质纤维化)：主要定位在间质，7d时各组肾组织内α-SMA的表达差异不显著。在14d后时，US+MB+BMSCs组和US+MB+HGF-BMSCs组的α-SMA表达较RUUO组和BMSCs组减低，差异显著(P<0.05)。SDF-1表达：14d时各组达到峰值。而US+MB+BMSCs组和US+MB+HGF-BMSCs组的增加程度较RUUO组和BMSCs组更为显著(P<0.05)。随着时间的延长，表达逐渐下降，但US+MB+BMSCs组和US+MB+HGF-BMSCs组在相同时相点SDF-1的表达均显著强于RUUO组和BMSCs组(P<0.05)。各时相点各组血清尿素氮，血清肌酐以及肾小球滤过率(内生肌酐清除率Ccr)水平无显著性差异。
结论：
1.成功实现大鼠骨髓源性BMSCs的分离、纯化及培养。
2.通过均匀实验设计进行实验，对超声强度，微泡浓度，超声辐照时间优选出最佳的转染参数配比。成功运用微泡介导超声联合PEI对大鼠BMSCs进行了转染，初步确定该方法未改变BMSCs的高增殖特性和多向分化能力，本方法对于细胞的损伤具有可恢复性且获得一段时间的持续荧光表达。
3.对微泡介导超声联合PEI转染BMSCs的机理进行了初步探讨，空化效应是其中一个因素，但协同作用可能是造成转染效率提高的重要原因。
4.通过诊断超声联合微泡作用成功实现对于健康大鼠肾脏间质通透性的一过性靶向改变，为肾间质的靶向释放提供了一个新的途径。另外对机制进行了初步探讨，认为超声联合微泡产生的空化效应可能是造成间质毛细血管通透性增强的主要原因。
5.通过超声检查，HE染色，Masson染色等多种检查途径证实大鼠UUO模型和RUUO模型建立成功。
6.运用诊断超声联合微泡介导外源性BMSCs移植，成功实现BMSCs向UUO模型大鼠肾脏间质的靶向归巢和聚集。其作用机理一方面可能是UTMD作用产生的生物学效应为提高BMSCs细胞在靶区的停留，粘附，迁移提供了更多的可能；另一方面移植到靶区的BMSCs所产生旁分泌作用分泌SDF-1，趋化更多的内外源性BMSCs黏附、归巢到达靶区肾组织。
7.诊断超声联合微泡方式介导的BMSCs或HGF-BMSCs移植能够有效地延缓肾间质纤维化的发展进程，较单纯的BMSCs移植对于梗阻再通损伤肾脏的修复具有明显促进作用。诊断超声联合微泡产生的空化效应通过改变肾间质微环境，提高BMSCs在靶区的浓度成功实现延缓肾间质纤维化的进程和肾组织损伤的修复。
8.诊断超声联合微泡方式介导的表达HGF的BMSCs，使HGF浓度在局部升高，抑制致纤维化转化生长因子TGF-β的表达，调控α-SMA，有效抑制肾间质胶原纤维形成，进一步促进肾脏间质纤维化的改善。
Background:
Tubulointerstitial fibrosis is the final stage of most chronic nephropathy, andtubulointerstitial fibrosis control or reversing is essential in the therapy of chronic kidneydiseases.
Bone marrow stromal cells (BMSCs) have numerous advantages for cell therapy andgene therapy in tissue repair and organ regeneration, including their high plasticity and easeof differentiation. For kidney, BMSCs can differentiate into variety of renal cell, includingmesangial cells, endothelial cells, podocytes and tubular epithelial cells, which suggestBMSCs to be the most attractive cells for the repair of injury renal tissue. BMSCs can alsoprotect the injury kidney which caused by ischemia or reperfusion in the way of theparacrine which can significantly reduced the necrosis of the adjacent cells. However, somestudies showed the poor therapy effect of the simple BMSCs transplantation for thetreatment of injury fibrosis renal tissue. The reason may be related to the way of BMSCstransplantation, the small number of homing cells, the lack of hepatocyte growth factor(HGF) which can significantly improve renal interstitial fibrosis and etc.
Recently the application of ultrasound contrast agents (microbubbles) has beenexpanded from diagnosis to treatment. Studies have showed microbubbles (MB) mediatedultrasound (US) engender the ultrasonic cavitation can be used as a gene therapy non-viralvector. Ultrasound-targeted microbubble destruction (UTMD) mediated drug targeteddelivery and gene transfection is recognized as an effective and promising method in recentyears. This study tried to use the characteristic of MB combined with US to modify BMSCs*The study was fund by the National Natural Science Foundation (NO.81071160) with HGF in the optimized acoustic parameters and use the method of UTMD to transplantthe cells in the animal models of renal fibrosis for the gene and cell combination therapy. Inaddition, UTMD can change the microenvironment of the target organ, enhance themicrovascular permeability in the target organ, increase endothelial cell gap, promote thesecretion of cytokine. So all of these changes can create the conditions for BMSCs“homing” the target tissue.So US combined with MB induced the HGF modified BMSCs(HGF-BMSCs) transplantation may promote HGF-BMSCs targeted homing andaggregation to the damaged kidney. In addition this method may result to improve thetherapy efficacy and the improvement of renal fibrosis. The study tested the feasibility,safety and targeting of this medthod and trid to explore its mechanism.
Objective：
1. To study the effectiveness, feasibility and mechanisms of the MB mediatedtreatment ultrasound combined polyethyleneimine (PEI) increased the transfectionefficiency of BMSCs modified by HGF. An attempt was made to find a non-viral method toachieve high-transfected BMSCs in order to avoid the high toxicity and immunogenicity ofviral vectors.
2. To stuy the changes of renal interstitial capillary permeability induced by MBmediated the certain parameters diagnostic ultrasound(DUS) as well as renal morphologyand function.
3. To study the feasibility of intravenous BMSCs transplantation which was inducedby MB mediated DUS and was promoted its targeted homing to the impaired kidney ofunilateral ureteral obstruction model.
4. To study the effectiveness, feasibility, safety and possible mechanisms of theimprovement of the renal fibrosis treated by BMSCs transplantation and HGF-BMSCstransplantation non-invasively mediated by DUS and MB in order to explore experimentalfoundation for the renal fibrosis therapy.
Methods:
1. A vitro study of DNA transfection of BMSCs using MB mediated treatedultrasound and PEI
The adherent culture method was used in the isolation and cultivation of BMSCs. Thegrowth curve, cell cycle, transmission electron microscopy and etc. were detected for the biological characteristics. Flow cytometry was applied to detect the expression of cellsurface marker molecules on the BMSCs.Osteogenic and adipogenes is induction ofBMSCs were performed.The plasmid DNA: pEGFP-HGF was constructed. Eachexperiment was arranged based on the uniform design. After tested the cell densities, thetransfection efficiency and the cell viability, the results of the optimized transfectionparameters (the acoustic intensity, the dosage of MBs, and the exposure time) wereanalyzed by regression analysis ultimately.The cell cycle and differentiation capacity of thetransfected BMSCs were detected.The BMSCs fluorescence expression at the time point of48h and7d after transfection were observed using laser scanning confocal fluorescencemicroscopy.The cell damage caused by UTMD and the cell self-recovery were observedusing scanning electron microscopy (SEM).
2. A study on the healthy rat renal interstitial permeability changes induced by MBmediated DUS and its safety
The left kidney of healthy Sprague–Dawley (SD) rat was insonated by UTMD witheither continuous or intermittent mode for5min.The target organ kidney interstitiumcapillary permeability changes were observed by Evans blue (EB) quantification,hematoxylin and eosin (HE) staining and transmission electron microscopy (TEM).Thepositioning of the DIO labed MB targeted release which was observed by the confocal laserfluorescence microscope. Urinalysis was performed to evaluate the glomerular filtration andthe tubular reabsorption.
3. MB mediated DUS enhanced BMSCs targeted homing to the kidney in the ratmodel
The mode of unilateral ureteral obstruction (UUO) was built and evaluated by imagingtests and pathology. MB mediated DUS improved the HGF-BMSCs vein transplantation onthe7d after modeling. Grouping as follow: HGF-BMSCs transplantation in UUO rat modelgroup (UM group), US+MB+HGF-BMSCs transplantation in UUO rat model group (UUMgroup), HGF-BMSCs transplantation in healthy rat group (CM group), US+MB+HGF-BMSCs transplantation in healthy rat group (CUM group). After48hours of celltransplantation，the green fluorescence-positive cells were observed using the confocal laserfluorescence microscope. The survival implanted cells were counted and statisticalanalysised. The target tissue morphology and the ultrastructure of interstitial capillaries after transplantation were observed by HE staining and TEM. The homing factor SDF-1expression was observed and quantitative analysised using immunohistochemical fordamaged kidney.
4. MB mediated DUS enhanced BMSCs vein transplantation to improve renal fibrosisin rats
The mode of unilateral ureteral obstruction recanalization (RUUO) was built andevaluated by imaging tests and pathology. Stem cell vein transplantation was performed onthe7d after modeling. The unilateral ureteral obstruction was relieved on the14d aftermodeling. Grouping as follow: RUUO group, BMSCs group, US+MB+BMSCs group andUS+MB+HGF-BMSCs group. The four observing time points were7d,14d,21d and28dafter modeling. At each time point, items were detected as follows: Imaging tests was usedto observe the renal morphology. HE staining and Masson staining were used to observe thetubulointerstitial damage and evaluate ratings. The gene expression of hepatocyte growthfactor (HGF), transforming growth factor-β(TGF-β) andα-smooth muscle actin (α-SMA) were detected using real time PCR method. The expression of α-SMA wasdetected by immunohistochemistry method. The protein expression of SDF-1, α-SMAwere detected by western blot method. The blood and urine were collected for the renalfunction detection followed as serum creatinine, blood urea nitrogen and creatinineclearance.
Results:
1. A vitro study of DNA transfection of BMSCs using MB mediated treatedultrasound and PEIThe identification results showed that specific antigens CD44, CD29, and CD90werepositive expressed and the specific antigens CD34, CD45, and CD11b of other cell lineagewere not expressed in the BMSC cultures. Adipogenic and osteocyte differentiation weresuccessfully induced in BMSCs. The best match of parameters are as follow, Q=0.6W/cm2, MB=106/ml, T=30s. The transfection efficiency (38.81±2.58)%and cellviability (82.10±1.77)%both reached a high level in the optimal parameter. Theexpression of GFP continued for7days after transfection. Comparing the differentinfluencing factors, PEI:DNA+MB+US group was significantly different (p <0.05). By day7after transfection,57.43±1.56%of BMSCs were transfected with MB-mediated US combined with PEI:DNA; the BMSC viability on7days after transfection was78.98±3.11%. The transfected BMSCs still maintained their differentiation capability (G1phase82.93%). With osteogenic induction of transfected BMSCs, there was a significant increasein calcium nodule formation, with red nodules staining positive with alizarin red. Westernblot analysis showed that interest protein HGF was successfully expressed in thepEGFP-HGF group, compared with no expression in the control and pEGFP-N1groups.Tiny holes were seen on the cell membranes. The sonoporation pore sizes were not uniform,and there was distortion on the cell surface. By day7after sonoporation, the BMSCs hadrecovered; most of the tiny ‘‘holes’’ had disappeared, and the cell surface was smooth.
2. A study on the healthy rat renal interstitial permeability changes induced by MBmediated DUS and its safety
The Continuous DUS without MB (CUS) group or Intermittent DUS without MB (IUS)group showed no significant difference compared with control group (P>0.05) using EBquantification. After UTMD, EB content of either continuous DUS with MB group (CUS+MB) group orIntermittent DUS with MB (IUS+MB) group showed significant increasecompared with three controls respectively (P<0.05). The IUStMB post6h group, the EBcontent reached3.15±2.20μg/g which decreased significantly compared with theIUStMB group.The EB content reached0.690±0.540μg/g in the IUStMB post-24hgroup, which was no statistically significant difference between the control group (P>0.05).Pathology detection results suggested the interstitium was damaged and the microvesselwall was broken after UTMD, while the glomerular basement membrane was intact.Theinterstitial capillary injury can self-recovery in24h after UTMD. Dio-labeled MB followedby DUS and observed under fluorescent microscope suggested the MBs are predominantlylocalized in the exposed kidney over other unexposed organs. However, IOD in IUS+MB(IOD=2092.17±342.36) and CUS+MB (IOD=943.48±179.07) showed significantincrease over control group and the right kidney (P<0.05). Furthermore, IOD in IUS+MBgroup showed significant increase compared to CUS+MB group (P<0.05). The urine resultshowed the protein level from (-) up to (++) in normal rats ranges without significantchange.
3. MB mediated DUS enhanced BMSCs targeted homing to the kidney in the ratmodel
The green fluorescence positive BMSCs were observed neither in CM group nor inCUM group in healthy rats. However, in UM group and UUM group the green fluorescencepositive BMSCs were observed in model rats. The number of positive cells wererespectively16.41±5.37and86.39±8.49, with significantly difference (P<0.05). HEstaining showed that after UTMD, the interstitium was damaged and the microvessel wallwas broken, lots of red blood cells were leaked, interstitial structure was loose and edema,the renal interstitial permeability increased significantly. The similar results were foundusing TEM. The expression of homing factor SDF-1located in the cytoplasm of the distalconvoluted tubule and the rarely proximal tubule which were detected byimmunohistochemistry. The UUM group (22146.49±1920.73) is not difference highercompared with the UM group (18643.94±2046.29), P>0.05. The CUM group (5528.36±1021.81) was statistically significant difference with the CM group (5162.38±1376.54), P <0.05.
4. MB mediated DUS enhanced BMSCs vein transplantation to improve renal fibrosisin rats
The renal morphology of RUUO rat model in each group was observed usingultrasound imaging teast in different time points (7d,14d，21d，28d after modeling). TheUS+MB+BMSCs group and the US+MB+HGF-BMSCs group were significant differentbetween the RUUO group and the BMSCs group. The renal interstitium pathological scoresshowed that the US+MB+BMSCs group and US+MB+HGF-BMSCs group weresignificantly lower compared with the BMSCs group and the RUUO group (P<0.05) on the21d and28d.In addition,the US+MB+HGF-BMSCs group was significantly between groupson28d (P<0.05). Fibrogenic factor TGF-β in the renal tissue was detected by Real-timePCR on the four time points which suggested that the BMSCs transplantation treatmentgroups were significant different between the RUUO group (P<0.05) after14d. However,the US+MB+BMSCs group and US+MB+HGF-BMSCs group were significantly lowerthan the BMSCs group (P<0.05). On the time points of14d and21d, the US+MB+BMSCsgroup and US+MB+HGF-BMSC group were statistic significant difference (P<0.05). HGFin the renal tissue was detected by Real-time PCR on the four time points which suggestedthat on the time points of14d and28d, the US+MB+BMSCs group andUS+MB+HGF-BMSC group were significantly higher than the BMSCs group (P<0.05). However, the US+MB+HGF-BMSC group was significant different between groups after14d (P<0.05). Then theα-SMA was observed on the gene level and protein level (Westernblot detection, Immunohistochemical detection). The α-smooth muscle actin wasconsidered to be the sign of fibroblasts involved in interstitial fibrosis. The expression ofα-SMA mainly located in the interstitium and the difference was not significant on7d. After14d, the US+MB+BMSCs group and US+MB+HGF-BMSC group were significantly lowerthan the RUUO group and the BMSCs group (P<0.05).The expression of SDF-1was asfollow: The expression reached the peak on14d and the increasing levels in theUS+MB+BMSCs group and US+MB+HGF-BMSC group were significant higher than theRUUO group and the BMSCs group (P<0.05). With the time increased, the SDF-1expression gradually decreased. However, at the same time points, the US+MB+BMSCsgroup and US+MB+HGF-BMSC group were significant higher than the RUUO group andthe BMSCs group (P<0.05). At each time points, there were no significant differencebetween groups about the serum urea nitrogen, serum creatinine and glomerular filtrationrate (endogenous creatinine clearance Ccr).
Conclusions:
1. Successfully isolated, purified and cultured rat bone marrow derived BMSCs.
2. Each experiment was arranged based on the uniform design. The optimaltransfection parameters of the acoustic intensity, the dosage of MBs, and the exposure timewere detected. Rat BMSCs were successfully transfected using MB mediated US combinedwith PEI. Iinitially identified this method does not change the BMSCs high proliferativecharacteristics and differentiation ability. The cell damage caused by UTMD canself-recovery and the fluorescence can continue expression.
3. Preliminary study on the mechanism of BMSCs transfection induced by MBmediated US combined with PEI suggested one of the factors is cavitation, but thesynergistic effect may be the important reason.
4. The healthy rat renal interstitial permeability was temporarily changed induced byMB mediated DUS which provids a new way for renal interstitial targeted delivery. Inaddition, preliminary study the mechanism of this method which suggested the cavitationmay be the major reason for the enhancement of renal interstitial capillary permeability.
5. The rat UUO model and the rat RUUO model were successfully established which were confirmed by ultrasound imaging test, HE staining and Masson staining.
6. MB mediated DUS can improve the exogenous BMSCs transplantation whichtargeted homing and aggregated to the renal interstitium of the UUO model.One of thepossible mechanism is the biological effects caused by UTMD can improve the BMSCssettlement, adhesion and migration towards the targeted area.The other possible mechanismis the paracrine action of the BMSCs. The paracrine action can improve SDF-1secretionand then tend more endogenous and exogenous BMSCs adhesion and homing to thetargeted renal tissue.
7. The BMSCs transplantation and the HGF-BMSCs transplantation induced by MBmediated DUS can effectively delay the process of the development of renal interstitialfibrosis which significantly improved the damage kidney repairation comparing with thesimple BMSCs vein transplantation method.The cavatition erosion caused by MB mediatedDUS can change the renal interstitial micro-environment, improve the BMSCsconcentration in the target region and then delay the process of the development of renalinterstitial fibrosis and improve the renal tissue repairation.
8. The HGF-BMSCs transplantation induced by MB mediated DUS can improve theHGF concentration in local area, inhibit the expression of the fibrogenic factor TGF-β,regulate and controlα-SMA, effectively inhibit renal interstitial collagen fiber formation,further promote the improvement of renal interstitial fibrosis.
肾小管间质纤维化是大多数慢性肾病的终末期表现，因此及早控制和逆转肾脏纤维化的进程是慢性肾脏疾病治疗中非常重要的治疗途径。
研究表明骨髓间充质干细胞(BMSCs)由于其高可塑，易分化等特性，在组织修复和器官再生的细胞治疗和基因治疗等领域表现出特有的优势。对于肾脏而言，骨髓干细胞可以分化为肾脏细胞包括系膜细胞、内皮细胞、足细胞和肾小管上皮细胞等，这就使得BMSCs成为修复受损肾脏最具吸引力的细胞。BMSCs还可以通过旁分泌的形式对局部缺血或再灌流造成的肾损伤进行保护,明显减少邻近细胞的坏死。然而，也有研究报道单纯BMSCs移植治疗受损的纤维化改变的肾脏效果不佳，究其原因可能与BMSCs的移植途径，靶向归巢数量少以及缺乏能够明显改善肾间质纤维化的肝细胞生长因子(HGF)等多方面因素有关。
近年来，超声造影剂(微泡)的应用已从诊断领域向治疗领域拓展。微泡借助其在超声作用下的空化作用可以作为一种基因治疗的非病毒载体。超声靶向击破微泡(Ultrasound-targeted microbubble destruction，UTMD)介导药物靶向传输和基因转染是近年发展起来并被公认为有效而且很有潜力的方法。该研究设想以此特性用HGF修饰BMSCs，通过对基因转染后的BMSCs生物学功能评价优选声学参数，获得活性显著提高的目的细胞对肾纤维化动物模型进行移植。另外，UTMD还可能改变靶器官的微环境，增强靶器官微血管通透性，使内皮细胞间隙增加，促使细胞因子分泌的改变，为BMSCs“着床”于靶组织创造条件。通过超声联合微泡介导HGF修饰BMSCs移植，促进HGF-BMSCs向损伤肾脏选择性归巢与聚集，进而起到提高疗效和改善肾纤维化的作用。将基因治疗和细胞治疗相结合，提高移植效率，对于抗肾纤维化的可行性、安全性及靶向性进行研究，并对其机制进行探讨。
研究目的：
1.探讨微泡介导治疗超声联合聚乙烯亚胺(PEI)实现HGF修饰BMSCs的有效性，可行性及其机制。试图探索一种非病毒的方法来实现BMSCs的高效转染从而避免病毒转染将会引发的安全问题；
2.探讨一定参数配比条件下的诊断超声联合微泡对于大鼠肾间质毛细血管通透性的改变情况以及对于肾脏形态结构和功能的影响；
3.探讨诊断超声联合微泡作用下静脉移植BMSCs促进其归巢单侧输尿管梗阻模型受损肾脏的可行性及其机制；
4.探讨诊断超声联合微泡介导BMSCs和HGF修饰的BMSCs移植应用于延缓大鼠肾纤维化进程的有效性、可行性、安全性及可能机制，为探讨肾纤维化新的治疗手段提供实验基础。
研究方法：
1.微泡介导治疗超声联合PEI转染大鼠骨髓间充质干细胞的体外研究
采用贴壁培养法对大鼠BMSCs进行分离培养。并通过生长曲线、细胞周期、透射电镜等检测其生物学特性。用流式细胞仪检测细胞表面标记分子并成脂、成骨诱导分化进行细胞鉴定。构建质粒DNA:pEGFP-HGF。按照均匀实验设计，对超声强度，微泡浓度，超声辐照时间三个主要影响因素进行考察，通过对细胞密度，转染效率，细胞存活率的检测，最终优选出最佳的转染参数配比。对转染后BMSCs的细胞周期，分化能力进行检测。运用激光共聚焦荧光显微镜对转染后48h，7d的BMSCs荧光表达进行观察。扫描电镜观察超声联合微泡作用对于细胞造成的损伤以及细胞的自行恢复情况。
2.微泡介导诊断超声对健康大鼠肾脏通透性的影响及安全性研究
诊断超声联合微泡作用采用不同的超声发射方式(连续式超声辐照，间歇式超声辐照)于健康大鼠肾脏。采用Evans blue (EB)定量分析，HE染色，透射电镜对靶器官肾脏间质区毛细血管通透性的改变进行观察检测；通过激光共聚焦荧光显微镜对DIO标记微泡的靶器官释放的定位及相对定量进行观察分析；对尿蛋白进行检测，评价该方法对肾小球滤过、肾小管重吸收的影响。
3.诊断超声联合微泡促进BMSCs归巢模型大鼠肾脏的研究
构建单侧输尿管梗阻(UUO)模型，采用影像学和病理学方法评价模型的建立。于建模后7d，采用超声联合微泡介导HGF-BMSCs静脉移植。分组：模型大鼠移植HGF-BMSCs组(UM组)；模型大鼠+超声+微泡+HGF-BMSCs组(UUM组)，健康大鼠移植HGF-BMSCs组(CM组)；健康大鼠+超声+微泡+HGF-BMSCs组(CUM组)。激光共聚焦观察示踪各组肾脏的绿色荧光阳性细胞计数和统计学分析。HE染色和透射电镜观察各组移植后靶组织形态结构和间质毛细血管超微结构。免疫组化对受损肾脏归巢相关因子SDF-1表达进行定量分析。
4.诊断超声联合微泡经静脉移植BMSCs改善大鼠肾纤维化的研究
构建单侧输尿管梗阻再通(RUUO)模型，采用影像学和病理学方法评价模型的建立。建模后7d进行干细胞移植治疗，于14d解除输尿管梗阻。在建模后7d，14d，21d，28d四个时相点分别对RUUO组， BMSCs组， US+MB+BMSCs组和US+MB+HGF-BMSCs组进行观察。采用影像学方法对肾脏形态进行观察；HE染色，Masson染色对肾小管间质损害进行观察和病理评分；Real time PCR检测肝细胞生长因子(HGF),转化生长因子-β(TGF-β)和α-平滑肌肌动蛋白(α-SMA)表达；免疫组织化学检测α-SMA表达；western blot检测SDF-1，α-SMA表达。生化检测血清肌酐，血清尿素氮和内生肌酐清除率评价肾脏功能改变。
结果：
1.微泡介导治疗超声联合PEI转染大鼠骨髓间充质干细胞的体外研究
成功分离培养BMSCs，检测结果显示绝大部分细胞表达CD44，CD29和CD90，几乎不表达CD34，CD45和CD11b。成脂诱导培养后油红O染色胞浆脂滴形成，成骨诱导后茜素红染色钙结节形成。超声联合微泡协同PEI转染BMSCs最佳参数：声强(Q)=0.6W/cm2，微泡(MB)=106/ml，辐照时间(T)=30s时，pEGFP-HGF转染效率38.81±2.58%和BMSCs细胞存活率82.10±1.77％达到一个相对比较匹配的数值。不同影响因素比较PEI:DNA+MB+US组同其他各组有统计学差异(P<0.05),激光共聚焦观察发现转染后直到7d仍可见绿色荧光蛋白表达。转染后7d，PEI:DNA+MB+US组的转染效率为57.43±1.56%(图12F)，而细胞活性下降到78.98±3.11%。转染后的BMSCs细胞周期及分化能力检测G1期的细胞占82.93%，成脂诱导培养后油红O染色胞浆脂滴形成，成骨诱导后茜素红染色钙结节形成。Westernblot检测细胞中HGF蛋白的表达:转染pEGFP-HGF成功表达HGF蛋白，而对应的空白对照组和转染pEGFP-N1组未检测到目的蛋白。扫描电镜观察到超声联合微泡最佳条件作用后，BMSCs的细胞膜上可以看到微小的孔,大小不一，形态各异。7天后，BMSCs基本恢复，大多数的微孔已经消失，细胞表面基本光滑。
2.微泡介导诊断对大鼠肾脏通透性的影响及安全性
Evans blue定量分析连续式超声辐照组(CUS)和间歇式超声辐照组(IUS)同对照组比较没有明显差异(P<0.05)。超声联合微泡作用(UTMD)后，无论CUS+MB组还是IUS+MB组的EB含量同其它三组比较都呈现显著增加，组间差异显著(P<0.05)。UTMD后6小时，在IUS+MB6h组EB含量达到3.15±2.20μg/g，同IUS+MB组相比较，下降显著。UTMD后24小时，IUS+MB24h组EB含量达到0.690±0.540μg/g，这与对照组差异无统计学意义(P>0.05)。病理学检测提示UTMD后间质受损，可见红细胞的漏出，肾间质内毛细血管连续性中断，肾小球未受到明显损伤。而肾间质的损伤在24h内能够自身恢复。激光共聚焦扫描荧光显微镜观察到DIO标记的MB在肾间质靶向释放，同其它脏器比较具有显著差异。其中IUS+MB组(IOD=2092.17±342.36)和CUS+MB组(IOD=943.48±179.07)同对照组和右肾比较显著增加，有统计学差异(P<0.05)。IUS+MB组同CUS+MB组相比较，呈现出显著的增加(P<0.05)。尿检结果显示各组的尿蛋白水平在正常范围内(-)到(++)，均未观察到蛋白尿的显著变化。
3.诊断超声联合微泡促进BMSCs归巢模型大鼠肾脏的研究
成功构建UUO模型。激光共聚焦观察示踪静脉移植BMSCs归巢梗阻侧肾脏，在健康大鼠移植HGF-BMSCs组(CM组)和健康大鼠+超声+微泡+HGF-BMSCs组(CUM组)的肾脏组织切片上没有观察到绿色荧光表达；而在模型大鼠移植HGF-BMSCs组(UM组)和模型大鼠+超声+微泡+HGF-BMSCs组(UUM组)肾小管间质区内观察到胞浆内表达绿色荧光的细胞，分别为(16.41±5.37)个、(86.39±8.49)个，差异有统计学意义(P<0.01)。HE染色光镜下观察发现UTMD作用后的模型大鼠肾间质的毛细血管破裂，大量红细胞漏出，间质结构疏松，水肿，肾间质通透性显著增加。透射电镜显示了类似的结果。免疫组织化学检测归巢相关因子SDF-1，CM组和CUM组SDF-1表达定位远曲小管和极少近曲小管的胞浆中，UM组和UUM组定位在大部分的肾小管上皮细胞胞浆中，甚至在个别细胞的膜上也有表达。UUM组(22146.49±1920.73)高于UM组(18643.94±2046.29)，差异不显著，P>0.05，与CUM组(5528.36±1021.81)，CM组(5162.38±1376.54)的组间差异有统计学意义，P<0.05。
4.诊断超声联合微泡经静脉移植BMSCs改善大鼠肾纤维化的研究
成功构建RUUO模型。超声观察各组肾脏在不同时相点(建模后7d,14d，21d，28d)的形态变化，US+MB+BMSCs组和US+MB+HGF-BMSCs组同RUUO组和BMSCs组比较差异显著。肾小管间质病变的病理积分发现在术后21d和28dUS+MB+BMSCs组和US+MB+HGF-BMSC组的病理积分开始明显低于BMSCs组和RUUO组(P<0.05)。其中28d时US+MB+HGF-BMSC组较其他三组差异显著(P<0.05)。Real-time PCR检测不同时相点各组肾脏组织致纤维化转化生长因子TGF-β的基因表达：14d后移植BMSCs的三个治疗组与RUUO组比较，差异显著(P<0.05)；诊断超声联合微泡作用的两个BMSCs移植治疗组表达减低，较单纯BMSCs移植组差异显著(P<0.05)；在14d和21d两时相点，US+MB+HGF-BMSCs组与US+MB+BMSCs组比较，差异显著(P<0.05)。肝细胞生长因子(HGF)的基因表达：在14d和28d两个时相点，US+MB+BMSCs组和US+MB+HGF-BMSCs组的HGF表达较RUUO组增高且差异显著(P<0.05)；而14d后的各时相点，US+MB+HGF-BMSCs组的HGF表达均高于其余各组，差异显著(P<0.05)。从基因水平，蛋白水平(Western blot检测，免疫组化检测)分别检测α-平滑肌肌动蛋白(α-SMA)的表达(α-SMA被认为是成纤维细胞的标志，参与间质纤维化)：主要定位在间质，7d时各组肾组织内α-SMA的表达差异不显著。在14d后时，US+MB+BMSCs组和US+MB+HGF-BMSCs组的α-SMA表达较RUUO组和BMSCs组减低，差异显著(P<0.05)。SDF-1表达：14d时各组达到峰值。而US+MB+BMSCs组和US+MB+HGF-BMSCs组的增加程度较RUUO组和BMSCs组更为显著(P<0.05)。随着时间的延长，表达逐渐下降，但US+MB+BMSCs组和US+MB+HGF-BMSCs组在相同时相点SDF-1的表达均显著强于RUUO组和BMSCs组(P<0.05)。各时相点各组血清尿素氮，血清肌酐以及肾小球滤过率(内生肌酐清除率Ccr)水平无显著性差异。
结论：
1.成功实现大鼠骨髓源性BMSCs的分离、纯化及培养。
2.通过均匀实验设计进行实验，对超声强度，微泡浓度，超声辐照时间优选出最佳的转染参数配比。成功运用微泡介导超声联合PEI对大鼠BMSCs进行了转染，初步确定该方法未改变BMSCs的高增殖特性和多向分化能力，本方法对于细胞的损伤具有可恢复性且获得一段时间的持续荧光表达。
3.对微泡介导超声联合PEI转染BMSCs的机理进行了初步探讨，空化效应是其中一个因素，但协同作用可能是造成转染效率提高的重要原因。
4.通过诊断超声联合微泡作用成功实现对于健康大鼠肾脏间质通透性的一过性靶向改变，为肾间质的靶向释放提供了一个新的途径。另外对机制进行了初步探讨，认为超声联合微泡产生的空化效应可能是造成间质毛细血管通透性增强的主要原因。
5.通过超声检查，HE染色，Masson染色等多种检查途径证实大鼠UUO模型和RUUO模型建立成功。
6.运用诊断超声联合微泡介导外源性BMSCs移植，成功实现BMSCs向UUO模型大鼠肾脏间质的靶向归巢和聚集。其作用机理一方面可能是UTMD作用产生的生物学效应为提高BMSCs细胞在靶区的停留，粘附，迁移提供了更多的可能；另一方面移植到靶区的BMSCs所产生旁分泌作用分泌SDF-1，趋化更多的内外源性BMSCs黏附、归巢到达靶区肾组织。
7.诊断超声联合微泡方式介导的BMSCs或HGF-BMSCs移植能够有效地延缓肾间质纤维化的发展进程，较单纯的BMSCs移植对于梗阻再通损伤肾脏的修复具有明显促进作用。诊断超声联合微泡产生的空化效应通过改变肾间质微环境，提高BMSCs在靶区的浓度成功实现延缓肾间质纤维化的进程和肾组织损伤的修复。
8.诊断超声联合微泡方式介导的表达HGF的BMSCs，使HGF浓度在局部升高，抑制致纤维化转化生长因子TGF-β的表达，调控α-SMA，有效抑制肾间质胶原纤维形成，进一步促进肾脏间质纤维化的改善。
Background:
Tubulointerstitial fibrosis is the final stage of most chronic nephropathy, andtubulointerstitial fibrosis control or reversing is essential in the therapy of chronic kidneydiseases.
Bone marrow stromal cells (BMSCs) have numerous advantages for cell therapy andgene therapy in tissue repair and organ regeneration, including their high plasticity and easeof differentiation. For kidney, BMSCs can differentiate into variety of renal cell, includingmesangial cells, endothelial cells, podocytes and tubular epithelial cells, which suggestBMSCs to be the most attractive cells for the repair of injury renal tissue. BMSCs can alsoprotect the injury kidney which caused by ischemia or reperfusion in the way of theparacrine which can significantly reduced the necrosis of the adjacent cells. However, somestudies showed the poor therapy effect of the simple BMSCs transplantation for thetreatment of injury fibrosis renal tissue. The reason may be related to the way of BMSCstransplantation, the small number of homing cells, the lack of hepatocyte growth factor(HGF) which can significantly improve renal interstitial fibrosis and etc.
Recently the application of ultrasound contrast agents (microbubbles) has beenexpanded from diagnosis to treatment. Studies have showed microbubbles (MB) mediatedultrasound (US) engender the ultrasonic cavitation can be used as a gene therapy non-viralvector. Ultrasound-targeted microbubble destruction (UTMD) mediated drug targeteddelivery and gene transfection is recognized as an effective and promising method in recentyears. This study tried to use the characteristic of MB combined with US to modify BMSCs*The study was fund by the National Natural Science Foundation (NO.81071160) with HGF in the optimized acoustic parameters and use the method of UTMD to transplantthe cells in the animal models of renal fibrosis for the gene and cell combination therapy. Inaddition, UTMD can change the microenvironment of the target organ, enhance themicrovascular permeability in the target organ, increase endothelial cell gap, promote thesecretion of cytokine. So all of these changes can create the conditions for BMSCs“homing” the target tissue.So US combined with MB induced the HGF modified BMSCs(HGF-BMSCs) transplantation may promote HGF-BMSCs targeted homing andaggregation to the damaged kidney. In addition this method may result to improve thetherapy efficacy and the improvement of renal fibrosis. The study tested the feasibility,safety and targeting of this medthod and trid to explore its mechanism.
Objective：
1. To study the effectiveness, feasibility and mechanisms of the MB mediatedtreatment ultrasound combined polyethyleneimine (PEI) increased the transfectionefficiency of BMSCs modified by HGF. An attempt was made to find a non-viral method toachieve high-transfected BMSCs in order to avoid the high toxicity and immunogenicity ofviral vectors.
2. To stuy the changes of renal interstitial capillary permeability induced by MBmediated the certain parameters diagnostic ultrasound(DUS) as well as renal morphologyand function.
3. To study the feasibility of intravenous BMSCs transplantation which was inducedby MB mediated DUS and was promoted its targeted homing to the impaired kidney ofunilateral ureteral obstruction model.
4. To study the effectiveness, feasibility, safety and possible mechanisms of theimprovement of the renal fibrosis treated by BMSCs transplantation and HGF-BMSCstransplantation non-invasively mediated by DUS and MB in order to explore experimentalfoundation for the renal fibrosis therapy.
Methods:
1. A vitro study of DNA transfection of BMSCs using MB mediated treatedultrasound and PEI
The adherent culture method was used in the isolation and cultivation of BMSCs. Thegrowth curve, cell cycle, transmission electron microscopy and etc. were detected for the biological characteristics. Flow cytometry was applied to detect the expression of cellsurface marker molecules on the BMSCs.Osteogenic and adipogenes is induction ofBMSCs were performed.The plasmid DNA: pEGFP-HGF was constructed. Eachexperiment was arranged based on the uniform design. After tested the cell densities, thetransfection efficiency and the cell viability, the results of the optimized transfectionparameters (the acoustic intensity, the dosage of MBs, and the exposure time) wereanalyzed by regression analysis ultimately.The cell cycle and differentiation capacity of thetransfected BMSCs were detected.The BMSCs fluorescence expression at the time point of48h and7d after transfection were observed using laser scanning confocal fluorescencemicroscopy.The cell damage caused by UTMD and the cell self-recovery were observedusing scanning electron microscopy (SEM).
2. A study on the healthy rat renal interstitial permeability changes induced by MBmediated DUS and its safety
The left kidney of healthy Sprague–Dawley (SD) rat was insonated by UTMD witheither continuous or intermittent mode for5min.The target organ kidney interstitiumcapillary permeability changes were observed by Evans blue (EB) quantification,hematoxylin and eosin (HE) staining and transmission electron microscopy (TEM).Thepositioning of the DIO labed MB targeted release which was observed by the confocal laserfluorescence microscope. Urinalysis was performed to evaluate the glomerular filtration andthe tubular reabsorption.
3. MB mediated DUS enhanced BMSCs targeted homing to the kidney in the ratmodel
The mode of unilateral ureteral obstruction (UUO) was built and evaluated by imagingtests and pathology. MB mediated DUS improved the HGF-BMSCs vein transplantation onthe7d after modeling. Grouping as follow: HGF-BMSCs transplantation in UUO rat modelgroup (UM group), US+MB+HGF-BMSCs transplantation in UUO rat model group (UUMgroup), HGF-BMSCs transplantation in healthy rat group (CM group), US+MB+HGF-BMSCs transplantation in healthy rat group (CUM group). After48hours of celltransplantation，the green fluorescence-positive cells were observed using the confocal laserfluorescence microscope. The survival implanted cells were counted and statisticalanalysised. The target tissue morphology and the ultrastructure of interstitial capillaries after transplantation were observed by HE staining and TEM. The homing factor SDF-1expression was observed and quantitative analysised using immunohistochemical fordamaged kidney.
4. MB mediated DUS enhanced BMSCs vein transplantation to improve renal fibrosisin rats
The mode of unilateral ureteral obstruction recanalization (RUUO) was built andevaluated by imaging tests and pathology. Stem cell vein transplantation was performed onthe7d after modeling. The unilateral ureteral obstruction was relieved on the14d aftermodeling. Grouping as follow: RUUO group, BMSCs group, US+MB+BMSCs group andUS+MB+HGF-BMSCs group. The four observing time points were7d,14d,21d and28dafter modeling. At each time point, items were detected as follows: Imaging tests was usedto observe the renal morphology. HE staining and Masson staining were used to observe thetubulointerstitial damage and evaluate ratings. The gene expression of hepatocyte growthfactor (HGF), transforming growth factor-β(TGF-β) andα-smooth muscle actin (α-SMA) were detected using real time PCR method. The expression of α-SMA wasdetected by immunohistochemistry method. The protein expression of SDF-1, α-SMAwere detected by western blot method. The blood and urine were collected for the renalfunction detection followed as serum creatinine, blood urea nitrogen and creatinineclearance.
Results:
1. A vitro study of DNA transfection of BMSCs using MB mediated treatedultrasound and PEIThe identification results showed that specific antigens CD44, CD29, and CD90werepositive expressed and the specific antigens CD34, CD45, and CD11b of other cell lineagewere not expressed in the BMSC cultures. Adipogenic and osteocyte differentiation weresuccessfully induced in BMSCs. The best match of parameters are as follow, Q=0.6W/cm2, MB=106/ml, T=30s. The transfection efficiency (38.81±2.58)%and cellviability (82.10±1.77)%both reached a high level in the optimal parameter. Theexpression of GFP continued for7days after transfection. Comparing the differentinfluencing factors, PEI:DNA+MB+US group was significantly different (p <0.05). By day7after transfection,57.43±1.56%of BMSCs were transfected with MB-mediated US combined with PEI:DNA; the BMSC viability on7days after transfection was78.98±3.11%. The transfected BMSCs still maintained their differentiation capability (G1phase82.93%). With osteogenic induction of transfected BMSCs, there was a significant increasein calcium nodule formation, with red nodules staining positive with alizarin red. Westernblot analysis showed that interest protein HGF was successfully expressed in thepEGFP-HGF group, compared with no expression in the control and pEGFP-N1groups.Tiny holes were seen on the cell membranes. The sonoporation pore sizes were not uniform,and there was distortion on the cell surface. By day7after sonoporation, the BMSCs hadrecovered; most of the tiny ‘‘holes’’ had disappeared, and the cell surface was smooth.
2. A study on the healthy rat renal interstitial permeability changes induced by MBmediated DUS and its safety
The Continuous DUS without MB (CUS) group or Intermittent DUS without MB (IUS)group showed no significant difference compared with control group (P>0.05) using EBquantification. After UTMD, EB content of either continuous DUS with MB group (CUS+MB) group orIntermittent DUS with MB (IUS+MB) group showed significant increasecompared with three controls respectively (P<0.05). The IUStMB post6h group, the EBcontent reached3.15±2.20μg/g which decreased significantly compared with theIUStMB group.The EB content reached0.690±0.540μg/g in the IUStMB post-24hgroup, which was no statistically significant difference between the control group (P>0.05).Pathology detection results suggested the interstitium was damaged and the microvesselwall was broken after UTMD, while the glomerular basement membrane was intact.Theinterstitial capillary injury can self-recovery in24h after UTMD. Dio-labeled MB followedby DUS and observed under fluorescent microscope suggested the MBs are predominantlylocalized in the exposed kidney over other unexposed organs. However, IOD in IUS+MB(IOD=2092.17±342.36) and CUS+MB (IOD=943.48±179.07) showed significantincrease over control group and the right kidney (P<0.05). Furthermore, IOD in IUS+MBgroup showed significant increase compared to CUS+MB group (P<0.05). The urine resultshowed the protein level from (-) up to (++) in normal rats ranges without significantchange.
3. MB mediated DUS enhanced BMSCs targeted homing to the kidney in the ratmodel
The green fluorescence positive BMSCs were observed neither in CM group nor inCUM group in healthy rats. However, in UM group and UUM group the green fluorescencepositive BMSCs were observed in model rats. The number of positive cells wererespectively16.41±5.37and86.39±8.49, with significantly difference (P<0.05). HEstaining showed that after UTMD, the interstitium was damaged and the microvessel wallwas broken, lots of red blood cells were leaked, interstitial structure was loose and edema,the renal interstitial permeability increased significantly. The similar results were foundusing TEM. The expression of homing factor SDF-1located in the cytoplasm of the distalconvoluted tubule and the rarely proximal tubule which were detected byimmunohistochemistry. The UUM group (22146.49±1920.73) is not difference highercompared with the UM group (18643.94±2046.29), P>0.05. The CUM group (5528.36±1021.81) was statistically significant difference with the CM group (5162.38±1376.54), P <0.05.
4. MB mediated DUS enhanced BMSCs vein transplantation to improve renal fibrosisin rats
The renal morphology of RUUO rat model in each group was observed usingultrasound imaging teast in different time points (7d,14d，21d，28d after modeling). TheUS+MB+BMSCs group and the US+MB+HGF-BMSCs group were significant differentbetween the RUUO group and the BMSCs group. The renal interstitium pathological scoresshowed that the US+MB+BMSCs group and US+MB+HGF-BMSCs group weresignificantly lower compared with the BMSCs group and the RUUO group (P<0.05) on the21d and28d.In addition,the US+MB+HGF-BMSCs group was significantly between groupson28d (P<0.05). Fibrogenic factor TGF-β in the renal tissue was detected by Real-timePCR on the four time points which suggested that the BMSCs transplantation treatmentgroups were significant different between the RUUO group (P<0.05) after14d. However,the US+MB+BMSCs group and US+MB+HGF-BMSCs group were significantly lowerthan the BMSCs group (P<0.05). On the time points of14d and21d, the US+MB+BMSCsgroup and US+MB+HGF-BMSC group were statistic significant difference (P<0.05). HGFin the renal tissue was detected by Real-time PCR on the four time points which suggestedthat on the time points of14d and28d, the US+MB+BMSCs group andUS+MB+HGF-BMSC group were significantly higher than the BMSCs group (P<0.05). However, the US+MB+HGF-BMSC group was significant different between groups after14d (P<0.05). Then theα-SMA was observed on the gene level and protein level (Westernblot detection, Immunohistochemical detection). The α-smooth muscle actin wasconsidered to be the sign of fibroblasts involved in interstitial fibrosis. The expression ofα-SMA mainly located in the interstitium and the difference was not significant on7d. After14d, the US+MB+BMSCs group and US+MB+HGF-BMSC group were significantly lowerthan the RUUO group and the BMSCs group (P<0.05).The expression of SDF-1was asfollow: The expression reached the peak on14d and the increasing levels in theUS+MB+BMSCs group and US+MB+HGF-BMSC group were significant higher than theRUUO group and the BMSCs group (P<0.05). With the time increased, the SDF-1expression gradually decreased. However, at the same time points, the US+MB+BMSCsgroup and US+MB+HGF-BMSC group were significant higher than the RUUO group andthe BMSCs group (P<0.05). At each time points, there were no significant differencebetween groups about the serum urea nitrogen, serum creatinine and glomerular filtrationrate (endogenous creatinine clearance Ccr).
Conclusions:
1. Successfully isolated, purified and cultured rat bone marrow derived BMSCs.
2. Each experiment was arranged based on the uniform design. The optimaltransfection parameters of the acoustic intensity, the dosage of MBs, and the exposure timewere detected. Rat BMSCs were successfully transfected using MB mediated US combinedwith PEI. Iinitially identified this method does not change the BMSCs high proliferativecharacteristics and differentiation ability. The cell damage caused by UTMD canself-recovery and the fluorescence can continue expression.
3. Preliminary study on the mechanism of BMSCs transfection induced by MBmediated US combined with PEI suggested one of the factors is cavitation, but thesynergistic effect may be the important reason.
4. The healthy rat renal interstitial permeability was temporarily changed induced byMB mediated DUS which provids a new way for renal interstitial targeted delivery. Inaddition, preliminary study the mechanism of this method which suggested the cavitationmay be the major reason for the enhancement of renal interstitial capillary permeability.
5. The rat UUO model and the rat RUUO model were successfully established which were confirmed by ultrasound imaging test, HE staining and Masson staining.
6. MB mediated DUS can improve the exogenous BMSCs transplantation whichtargeted homing and aggregated to the renal interstitium of the UUO model.One of thepossible mechanism is the biological effects caused by UTMD can improve the BMSCssettlement, adhesion and migration towards the targeted area.The other possible mechanismis the paracrine action of the BMSCs. The paracrine action can improve SDF-1secretionand then tend more endogenous and exogenous BMSCs adhesion and homing to thetargeted renal tissue.
7. The BMSCs transplantation and the HGF-BMSCs transplantation induced by MBmediated DUS can effectively delay the process of the development of renal interstitialfibrosis which significantly improved the damage kidney repairation comparing with thesimple BMSCs vein transplantation method.The cavatition erosion caused by MB mediatedDUS can change the renal interstitial micro-environment, improve the BMSCsconcentration in the target region and then delay the process of the development of renalinterstitial fibrosis and improve the renal tissue repairation.
8. The HGF-BMSCs transplantation induced by MB mediated DUS can improve theHGF concentration in local area, inhibit the expression of the fibrogenic factor TGF-β,regulate and controlα-SMA, effectively inhibit renal interstitial collagen fiber formation,further promote the improvement of renal interstitial fibrosis.
引文
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