摘要
糖尿病是一种常见的、严重威胁人类健康的疾病,其发病率约为2～6%。近年来,我国糖尿病的发病率不断上升,目前糖尿病患者约3000万,预计到2025年将达5000万。每年我国用于糖尿病的保健费用高达2500亿,而且逐年提高。胰岛移植被认为是目前治疗胰岛素依赖型糖尿病的最有效手段,但由于缺乏有效的、无创伤的胰岛移植物监测技术,不能早期预防、干预原发性移植物失功能和免疫排斥反应发生,导致目前移植效率及疗效不太理想。近几年分子影像学无创监测活体胰岛移植出现新的突破,为存活β细胞数量(β-cell mass,BCM)监测技术发展奠定良好基础。研制理想的示踪剂,建立胰岛活体功能分子影像学示踪技术,无创伤性的识别、跟踪移植后的胰岛细胞,动态监测胰岛移植后不同阶段胰岛功能活力维持与凋亡发生、发展,预测胰岛移植物的功能变化,评价胰岛移植成败及处理措施的有效性,对临床上推动胰岛移植的发展和糖尿病的防治具有非同寻常的意义。
分子影像学是将活体动物及人类复杂的生理病理过程在细胞或分子水平上变成直观的图像,从而向我们提供传统影像学所无法得到的细胞或分子水平的信息。根据成像方法不同,分子影像学主要包括生物光学成像(bioluminescence imaging,BLI)、核素分子成像(positron emission tomography,PET),磁共振成像(magneticresonance imaging,MRI)等,其中MRI因其优异的空间分辨率而在细胞活体示踪的研究中备受瞩目。有关磁标记细胞及MRI活体示踪的研究,国内外部分学者已进行了有益的尝试并取得初步成功。早期研究多采用高场强MRI系统进行单个细胞成像,近年来,随着标记方法的不断改进和标记效率的明显提高,也有研究应用1.5-Tesla(T)MRI成功地进行了中枢神经系统和心脏等部位磁标记干细胞的体外成像及活体示踪,这为活体移植胰岛无创示踪提出可能。用MRI示踪移植胰岛细胞,需要对细胞进行磁性标记。一般的细胞磁性标记技术是在细胞表面吸附几百纳米或微米大小的磁珠,这种方法用于体外细胞分选非常有效,但不适用于细胞的活体示踪,因为表面被磁珠标记的细胞在体内会很快被网状内皮系统识别并清除。所以,MRI活体移植胰岛示踪技术需要寻找一种安全、有效、稳定的细胞内磁性标记物。超顺磁性氧化铁纳米颗粒(superparamagnetic iron oxidenanoparticles,SPIO)的生物相容性好,毒性小,进入靶细胞后完全呈惰性,在子代细胞中呈线性分布,是移植细胞较为理想的标记物。2007年Vescovi和Gritti报道用商品化的SPIO(Sinerem和Endorem)标记人神经干细胞,成功进行小鼠脑内移植MRI活体示踪。
目前常用的SPIO都为国外产品,国内缺少正式上市的相应产品。本研究旨在首次探讨自行制备聚乙烯吡咯烷酮表面修饰的超顺磁性氧化铁纳米颗粒(polyvinylpyrrolidone coated superparamagnetic iron oxide nanoparticles,PVP-SPIO)标记小鼠胰岛,同种异体移植后3.0-T临床磁共振成像系统动物线圈活体示踪的可行性、安全性和有效性。初步探索用自行制备的PVP-SPIO标记小鼠胰岛细胞,进行MRI活体功能示踪。为胰岛移植治疗糖尿病的基础研究和临床应用的开展与推广,寻找一种无创、有效、可重复性强的活体示踪技术。
目的:
自行研制PVP表面修饰的SPIO,并对制备的磁纳米颗粒的结构、形貌和理化性状进行表征。
方法:
采用高温水解金属醇盐螯合物过程制备PVP表面修饰的SPIO。并进一步对制备的PVP-SPIO通过等离子发射光谱(ICP)、透射电子显微镜(TEM)、高分辨透射电镜(HRTEM)、选区电子衍射(SAED)、X射线衍射(XRD)、傅立叶红外吸收光谱(FTIR)、纳米激光粒度仪(DLS)、振动样品磁强计(VSM)和磁共振成像系统(MR scanner)等方法对其理化性状进行研究。
结果:
采用高温水解金属醇盐螯合物过程制备的PVP-SPIO呈黄褐色,长时间放置不产生团聚。TEM扫描提示PVP-SPIO颗粒核心直径约为5～10nm,分布集中,在水溶液中分散性良好。从HRTEM照片中可以看到两组高分辨条纹,一组条纹之间的间距为0.25nm,对应立方相Fe_3O_4的{220}晶面;一组条纹之间的间距为0.29nm,对应立方相Fe_3O_4的{311}晶面。从SAED图中可以观察到很清晰的衍射环,对衍射花样进行标定可以知道颗粒为立方相的Fe_3O_4。XRD图谱显示纳米颗粒的所有衍射峰位都与立方相的Fe_3O_4标准图谱(JCPDS 85-1436)一致,而且没有观察到其他杂质的衍射峰。FTIR图中可以明显看到PVP的特征峰。纳米激光粒度仪分析结果提示粒径平均尺寸为45nm。制备的PVP-SPIO在室温(300K)下,外磁场为2T时的磁滞回线提示制备的PVP-SPIO的饱和磁化强度达到63emu/g,明显高于Feridex。MRI显示PVP-SPIO可以明显缩短T_1和T_2弛豫时间,T_2信号强度与样品的Fe浓度正相关,而在相同Fe浓度下PVP-SPIO磁信号明显强于Feridex。
结论:
采用高温水解金属醇盐螯合物过程成功制备出具有高分散性、强磁响应性、粒径均匀的PVP-SPIO,3.0-T MRI显示PVP-SPIO具备良好的超顺磁性。
第二部分PVP-SPIO标记小鼠胰岛细胞系Beta-TC-6的细胞生物学研究
目的:
研究自制的PVP-SPIO体外磁性标记小鼠胰岛细胞的可行性、标记效率、以及对细胞活力和功能的影响,为胰岛移植MRI活体示踪奠定基础。
方法:
培养小鼠胰岛细胞系Beta-TC-6,使用PVP-SPIO标记Beta-TC-6细胞。采用普鲁士兰染色和透射电镜观察被标记细胞内铁颗粒情况。利用梯度浓度PVP-SPIO标记Beta-TC-6细胞,普鲁士兰染色和体外MRI扫描检测其标记效率。采用四唑盐(MTT)比色试验检测PVP-SPIO标记的Beta-TC-6细胞活力。通过生长曲线观察PVP-SPIO对Beta-TC-6细胞增殖的影响。应用流式细胞仪、活性氧簇(ROS)荧光检测标记后细胞的凋亡和氧化损伤水平。采用葡萄糖刺激胰岛素释放试验对标记后Beta-TC-6细胞的胰岛素分泌功能进行评估。
结果:
细胞内铁染色:普鲁士蓝染色显示PVP-SPIO标记的Beta-TC-6细胞胞质内出现细小的蓝色铁颗粒。电镜结果进一步证实PVP-SPIO标记的Beta-TC-6细胞胞质内含有较多包裹纳米铁颗粒的囊泡。标记效率的检测:体外MRI扫描和普鲁士蓝染色提示,不同铁浓度PVP-SPIO和Feridex分别标记2×10~5Beta-TC-6细胞24h,MRI扫描T_2信号随标记铁浓度升高而降低,细胞质内的蓝色铁颗粒也相应增加。在相同铁浓度(100μg Fe/mL)下作用24h,PVP-SPIO较Feridex对Beta-TC-6细胞的标记效率更高,所标记的细胞MRI体外扫描T_2弛豫时间更短。细胞形态学观察:倒置相差显微镜下PVP-SPIO标记组、Feridex标记组和未标记组Beta-TC-6细胞之间,细胞形态无明显差异。PVP-SPIO对细胞活力的影响:MTT结果显示,PVP-SPIO和Feridex在400μg Fe/mL浓度以下标记Beta-TC-6细胞24h,对细胞活力无显著性影响。PVP-SPIO对细胞增殖的影响:PVP-SPIO和Feridex以100μgFe/mL浓度标记Beta-TC-6细胞24h,细胞生长曲线无显著改变。PVP-SPIO对细胞早期凋亡水平的影响:流式细胞仪检测显示,PVP-SPIO和Feridex以100μg Fe/mL浓度标记Beta-TC-6细胞24h,细胞凋亡无显著性增加。PVP-SPIO对细胞发生氧化损伤的影响:流式细胞仪检测显示,PVP-SPIO和Feridex以100μg Fe/mL浓度标记Beta-TC-6细胞24h,细胞内ROS的水平无显著性增加。PVP-SPIO对细胞功能的影响:葡萄糖刺激胰岛素释放试验结果显示,PVP-SPIO和Feridex以100μgFe/mL浓度标记24h,对Beta-TC-6细胞胰岛素释放功能无显著性影响。
结论:
制备的PVP-SPIO具有良好的生物相容性和超顺磁性,能够有效标记小鼠Beta-TC-6细胞,对细胞的活性和功能无显著性影响,在PVP-SPIO标记的Beta-TC-6细胞MRI体外扫描中表现出理想的磁信号。
第三部分PVP-SPIO标记小鼠胰岛细胞MRI活体示踪研究
目的:
建立PVP-SPIO标记小鼠胰岛细胞同种异体肾包膜下移植模型,探讨3.0-T临床磁共振成像系统动物线圈,活体示踪PVP-SPIO标记小鼠胰岛细胞的可行性。了解PVP-SPIO标记胰岛细胞的MRI成像规律,寻找MRI成像的最佳序列和参数,为进一步探索胰岛移植MRI活体功能示踪奠定基础。
方法:
将制备的PVP-SPIO,加PLL(其质量浓度与Fe之比为100:1),以100μg Fe/mL浓度与Beta-TC-6细胞共孵育24h。分别取0.5×10~5,1×10~5,2×10~5,4×10~,8×10~5,1.6×10~6,3.2×10~6,6.4×10~6个标记的细胞,进行MRI体外扫描,观察信号变化。取2×10~5PVP-SPIO标记Beta-TC-6细胞,进行C57BL/6小鼠同种异体肾包膜下移植,术后对移植物进行MRI活体示踪。将移植模型肾脏进行石蜡切片,胰岛素免疫组织化学染色和普鲁士蓝染色观察。分离纯化100个C57BL/6小鼠胰岛,用制备的PVP-SPIO标记后,进行C57BL/6小鼠同种异体肾包膜下移植,术后第1,8,15d对移植物进行MRI活体示踪。
结果:
通过PVP-SPIO以100μg Fe/mL浓度标记数量梯度的Beta-TC-6细胞MRI体外扫描,确定MRI扫描的有效细胞数量范围。PVP-SPIO以100μg Fe/mL浓度标记的2×10~5个Beta-TC-6细胞小鼠肾包膜下移植,3.0-T MRI活体扫描移植物表现为明显的T_2低信号。移植模型肾石蜡切片,胰岛素免疫组化/普鲁士蓝双染色,证实肾包膜下PVP-SPIO标记的胰岛移植物存在。PVP-SPIO以100μg Fe/mL浓度标记的100个C57BL/6小鼠原代胰岛同种异体肾包膜下移植,MRI活体示踪可以观测到理想的磁信号,时间可以持续15d,MRI信号强度随移植后时间延长而逐渐减弱。
结论:
制备的PVP-SPIO可以有效标记小鼠胰岛细胞系Beta-TC-6和原代胰岛,并通过3.0-T临床磁共振成像系统动物线圈进行同种异体肾包膜下移植MRI活体示踪。
目的:
建立小鼠胰岛Beta-TC-6细胞氧化损伤肾包膜下移植模型,初步探讨PVP-SPIO标记不同功能状态小鼠胰岛细胞进行MR/活体功能示踪。
方法:
H_2O_2以不同浓度,短时间作用小鼠胰岛Beta-TC-6细胞后,检测细胞活性、早期凋亡和细胞内ROS水平来反映细胞损伤程度。以制备的PVP-SPIO分别标记正常和H_2O_2氧化损伤组小鼠Beta-TC-6细胞,进行C57BL/6小鼠肾包膜下移植。移植后1d,用3.0-T临床磁共振成像系统动物线圈扫描,测量移植物和正常肾皮质的T_2map序列的T_2和R_2值,计算每个受体的移植物和正常肾皮质的R_2值之比,对正常组和损伤组的R_2比值进行统计学分析。对正常组和损伤组的移植模型肾脏,进行石蜡切片和原位脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)染色,观察移植物的凋亡发生程度。
结果:
通过MTT比色法、流式细胞仪对检测细胞凋亡和细胞内ROS水平的检测,证明H_2O_2 500μM浓度作用1h造成小鼠胰岛Beta-TC-6细胞氧化损伤模型成立。制备的PVP-SPIO标记正常组和氧化损伤组的小鼠Beta-TC-6细胞,移植后MRI活体示踪图像显示,正常组T_2弛豫时间明显较损伤组更短。计算T_2map序列的R_2比值,提示正常组R_2比值较损伤组高,并且具有统计学意义(P<0.05)。移植小鼠肾脏TUNEL染色证实氧化损伤组移植物凋亡程度明显高于正常组,其结果与MR/扫描结果一致。
结论:
制备的PVP-SPIO标记不同活力状态的小鼠胰岛Beta-TC-6细胞,进行肾包膜下移植,可以通过3.0-T临床磁共振成像系统动物线圈扫描观测移植物图像和信号的差异,初步反映移植胰岛细胞的活力状态、凋亡和氧化损伤程度。
Diabetes mellitus(DM),which affects millions of people worldwide,is characterized by abnormally high levels of glucose in blood caused by either absolute (type 1 DM) or relative(type 2 DM) insulin deficiency due to the destruction of pancreatic P-cells by T cells of the immune system or decreased insulin sensitivity respectively.By 2010 the number of people with diabetes is expected to exceed 350 million.Late diabetic complications will cause considerable morbidity in 5-10%of these patients and place an enormous burden on society.Islet cell replacement is considered as the optimal treatment recently.However the success of this approach is hampered by the absence of reliable methods to follow the fate of transplanted islets non-invasively.In vivo molecular imaging seems to be the most appropriate technique to achieve this goal in small animals and eventually in humans.Such measures would potentially allow the assessment of islet engraftment and the early recognition of graft loss,leading to greater improvements in islet graft survival and function.
The term molecular imaging can be broadly defined as the in vivo characterization and measurement of biologic processes at the cellular and molecular level.Three modalities have demonstrated applicability in the near future:bioluminescence imaging (BLI),magnetic resonance imaging(MRI) and positron emission tomography(PET).A main advantage of using MRI is its high spatial resolution and the ability to extract more than one measurement parameter at a given imaging session.Superparamagnetic iron oxide nanoparticles(SPIO) have demonstrated their utility as an important tool for enhancing magnetic resonance contrast,allowing researchers to monitor not only anatomical changes,but physiological and molecular changes as well.Recently,stem and progenitor cells of neural and non-neural origin have been magnetically labeled using SPIO in order to track them by MRI in cell therapy approaches for CNS and cardiovascular disorders.
In this study,we have developed method of high temperature hydrolysis of chelate metal alkoxide complexes to coat iron oxide nanoparticles with biocompatible polymer, polyvinylpyrrolidone(PVP).Synthesized PVP-SPIO nanoparticles have demonstrated excellent biocompatibility and superparamagnetic behavior.The imaging of 100 PVP-SPIO labeled islets in mice renal subcapsular model of transplantation under a clinical 3.0-Tesla MR scanner showed high spatial resolution in vivo.These results indicated the great potential applications of PVP-SPIO as MRI contrast agent might be possible for monitoring the transplanted islet grafts in the clinical management of diabetes in the near future.
Part 1 Synthesis and Characterization of PVP-SPIO
Objective:
Preparation and research of the physicochemical characteristics of PVP-SPIO.
Methods:
PVP-SPIO was synthesized by high-temperature hydrolysis of chelate metal alkoxide complexes in liquid polyol.The morphology and structure of PVP-SPIO was characterized by transmission electron microscopy(TEM),high-resolution transmission electron microscopy(HRTEM) and X-ray powder diffraction(XRD).The size of PVP-SPIO was measured by dynamic light scattering(DLS).Fourier transmission infrared spectra(FTIR) were performed by FTIR spectrometer.The magnetic properties of PVP-SPIO were examined by vibrating sample magnetometer(VSM).
Results:PVP-SPIO was fabricated by high-temperature polyol approach.TEM shows the core sizes of nanoparticles are 5～10 nm.The HRTEM image reveals that there are two kinds of the lattice fringes with lattice spacing of about 0.25 and 0.29 nm corresponding to the {220} and the {311} plane of SPIO,respectively.The DLS measurement indicates that the average hydrodynamic size of PVP-SPIO is about 45 nm. The XRD pattern and FTIR analysis further confirm the formation of PVP-SPIO.The saturation magnetization of PVP-SPIO examined by VSM is 63emu/g at 2T.PVP-SPIO exhibits the typical property of superparamagnetic iron oxide in shortening T_1 and T_2 relaxation time with a gradually increasing superparamagnetic effect according to the increasing Fe concentration.
Conclusions:Successfully synthesized PVP-SPIO demonstrates high stability, crystalline and magnetization.
Part 2 Cytobiology study of Beta-TC-6 cells labeling with PVP-SPIO
Objective:
Study on the labeling effiency and biocompatibility of synthesized PVP-SPIO.
Methods:
Prussian blue staining and TEM were performed to detect uptaking of PVP-SPIO by Beta-TC-6 cells.In vitro MRI and prussian blue staining of gradient PVP-SPIO labeled cells was investigated to measure the label efficiency.MTT assays,growth curves and flow cytometry were performed to evaluate the cytotoxicity corresponding to the biocompatibility of PVP-SPIO on Beta-TC-6 cells.The labeled Beta-TC-6 cells function was evaluated by Glucose-stimulated insulin release assays.
Results:
Prussian blue method and TEM reveal intracellular iron accumulation.In vitro MRI show higher lebeling efficiency of PVP-SPIO than Feridex.No significant difference in morphology was observed between PVP-SPIO labeled,Feridex labeled and control cells.MTT assay demonstrate no significant difference in viability was evident with PVP-SPIO labeled and untreated cells.The flow cytometry assay suggests that there was no significant difference in cellular apoptosis detection and ROS level. There is no difference in glucose-stimulated insulin release between PVP-SPIO labeled group and Feridex labeled group or between PVP-SPIO labeled and control group.
Conslusions:
The newly synthesized PVP-SPIO demonstrates excellent biocompatibility and superparamagnetic behavior.PVP-SPIO could be efficiently internalized into Beta-TC-6 cells,without affecting cell viability and function.
Part 3 In vivo MRI tracking and morphological study of PVP-SPIO labeled mice islets transplantation
Objective:
To investigate the possibility of in vivo detection of PVP-SPIO labeled mice islets in a clinical 3.0-T MR scanner with 30-mm mouse coil.To find an optimal sequence and parameter for MRI of PVP-SPIO labeled islets for the following in vivo study.
Methods:
0.5×10~5,1×10~5,2×10~5,4×10~5,8×10~5,1.6×10~6,3.2×10~6,6.4×10~6 Beta-TC-6 cells were respectively collected after labeling with PVP-SPIO(100μg Fe/mL) for 24h.In vitro MRI of cell pellets was performed at a clinical 3.0-T MR scanner.2×10~5 Beta-TC-6 cells labeled with PVP-SPIO were transplanted beneath the renal capsule of C57BL/6 mice tracked with in vivo MRI.The transplanted tissue samples were sectioned to investigate with immunohistochemistry and prussian blue staining immediately after MRI.100 islets labeled with PVP-SPIO were transplanted beneath the renal capsule of C57BL/6 mice.In vivo MRI was performed 1d,8d,15d after syngenetic transplantation respectively.
Results:
Beta-TC-6 cells labeled with PVP-SPIO demonstrate hypointense spot in T_2-weighted image at the transplantation area,which can be detected by the clinical 3.0-T MR scanner.Immunohistochemistry and prussian blue staining confirm the transplanted location of PVP-SPIO labeled islet cells in the renal subcapsular area. Within 15 days after transplantation,100 PVP-SPIO labeled islets can be detected by in vivo MRI,with signal faded over time.
Conslusions:
The newly synthesized PVP-SPIO can be used to efficiently label mice islets transplanted beneath the renal capsule of C57BL/6 mice.The labeled islet grafts can be tracked in vivo by the clinical 3.0-T MR scanner with mouse coil.
Part 4 Functional graft imaging of PVP-SPIO labeled Beta-TC-6 cells with in vivo MRI
Objective:
To establish the oxidative injury transplantation model of Beta-TC-6 cells.We have adapted for monitoring the function of grafts noninvasively with MRI.
Methods:
To determine the presence of apoptosis and oxidative stress,MTT assays and flow cytometry were performed after Beta-TC-6 cells treated with H_2O_2.Normal group and oxidative injury group of Beta-TC-6 cells labeled with PVP-SPIO were transplanted beneath the renal capsule of C57BL/6 mice.In vivo MRI was performed days 1 after transplantation respectively.The signal changing on T_amap weight image was analyzed according to the R_2 Ratio=(R_(2 Graft)/R_(2 Kidney))×100%.The transplanted tissue samples were sectioned to investigate with TUNEL assay.
Results:
MTT assays and flow cytometry demonstrate that H_2O_2 induced apoptosis of Beta-TC-6 cells and reduced the cell viability in a dose-dependent manner.The R_2 Ratio on in vivo MRI for graft in normal group is significantly higher than oxidative injury group induced by H_2O_2(P<0.05).The number of apoptosis of TUNEL positive cells is obviously lower in normal group than in oxidative injury group.
Conslusions:
The newly synthesized PVP-SPIO in combination with our model of mice islet transplantation,can be used to monitoring the function of grafts noninvasively with the clinical 3.0-T MR scanner with mouse coil.
引文
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