光学断层分子成像定量分析及其应用
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摘要
近十年来，光学断层分子成像（Optical Molecular Tomography, OMT）因其在预临床的重大疾病研究应用中所表现出的良好性能，受到了学者们的广泛关注。光学断层分子成像由于具有灵敏度高、成本相对低廉、可能实现绝对定量等诸多优点，因而在预临床和临床的恶性肿瘤、心血管疾病、神经系统病变等重大疾病研究中得到了广泛的应用。作为光学断层分子成像的典型代表，生物发光断层成像及超高分辨率micro-CT混合成像系统以其高灵敏性、高特异性及高分辨的解剖结构等优点，在生物医学研究中具有广阔的应用前景。本文对光学断层分子成像的绝对定量分析方法进行了深入研究，并就生物发光断层分子成像和超高分辨率micro-CT混合成像系统对胃癌和干细胞移植治疗缺血性疾病的生物医学应用进行了系统的成像研究，本论文的主要研究内容包括：
1.构建了生物发光断层分子成像和超高分辨率micro-CT混合成像系统，并对生物发光断层分子成像系统进行了定量校准。针对预临床小动物研究的需要，构建了生物发光断层分子成像系统，并对成像系统CCD相机进行了像元响应的非均匀性校准和绝对强度的定量校准。针对光学断层分子成像空间位置的差异构建了视场函数，对进入光学成像系统的光通量进行了定量分析。构建了超高分辨率micro-CT成像系统，为本论文的下肢缺血模型的血管新生定量研究奠定了基础。
2.对生物发光断层分子成像定量重建方法进行了研究。基于有限元网格剖分方法，提出了总能量定量重建的策略。通过已知光源移植到小动物体内，验证了总能量重建的可行性。建立了基于报告基因系统的光学探针所反映的生物学参数与成像系统中总能量重建的物理参数之间的内在联系，实现了生物发光断层分子成像系统对肿瘤细胞和干细胞的细胞数量的定量分析。通过PC-3M-luc-C6前列腺癌和A549-luc-C8非小细胞肺癌细胞系以及脂肪来源的间充质干细胞的在体定量研究，证实了细胞水平定量分析的可行性和适用性。
3.研究了光学断层分子成像在光学探针峰值波长偏移及解剖结构模型偏差等情况下引入的重建误差及其贡献率的大小。以生物发光断层成像为例分析了光学断层分子成像的重建误差的主要来源。（1）在假定光学参数准确的前提下，研究了动物模型解剖结构偏差带来的光学重建在定位和定量上的偏差。（2）在假定解剖结构准确的情况下，研究了光学探针峰值波长偏移对重建误差在定位和定量方面的影响。通过对解剖结构偏差以及峰值波长偏移的实验数据的系统测试、重建及数据分析，表明二者均会对重建的定位和定量带来较大的影响；其中，当光学探针峰值波长在640nm以下时，解剖结构的偏差对重建在定位和定量有更大的影响。实验数据也揭示了光学探针峰值波长漂移量在±10nm范围内、动物模型的解剖结构偏差在亚毫米级别以内时都可以得到较理想的定位和定量重建结果。
4.利用自主研发的混合成像系统对胃癌的生物学行为进行定量成像研究。利用生物发光断层分子成像对胃癌原位动物模型实现在体定量分析以及肿瘤模型的长程动态观测，为胃癌的预临床研究提供了新的成像方法和成像工具。通过micro-CT造影成像精确计算出肿瘤的实际体积大小，同时在研究中提出了新颖的低成本欧乃派克时间累积对比增强造影方法，可以实现微小胃癌转移灶的在体定位和定量分析。
5.对干细胞移植治疗缺血性疾病促进血管新生开展了混合成像系统的定量示踪和基于空间血管体积因数SVVF定量评估的成像研究。（1）采用生物发光断层分子成像及超高分辨率micro-CT混合成像系统在三维空间实现干细胞的在体定量示踪以及血管新生的血管灌注分析，实现对干细胞在体空间分布和细胞数量的定量示踪；并根据干细胞的迁移、存活数量和空间分布，进一步交互式实施高分辨的微血管网络密度的定量评估。（2）提出了空间血管体积因数（Spatial VascularVolume Fraction, SVVF）的概念，并将其用于小鼠下肢缺血模型血管新生的定量评估。（3）通过血管体积因数SVVF的系列成像研究发现在时间和空间上血管新生的量化指标，进而探讨性地分析了干细胞移植后旁分泌的作用效果和作用半径。（4）通过多种传统的方法对干细胞移植后的血管新生进行了交叉验证。利用血管铸型验证血管新生的宏观变化情况；通过扫描电镜成像证实了在干细胞移植后促进血管新生作用的不同时间点血管新生的微观结构及发芽情况；通过共聚焦荧光显微成像证实了eGFP转基因小鼠来源的脂肪间充质干细胞在缺血组织的存在性和血管新生的情况；通过激光多普勒成像观测血流灌注的恢复情况。本文提出的SVVF方法以及交叉验证的结果一致发现，脂肪来源的间充质干细胞移植与对照组相比可以明显地增大了血管密度，进而证实脂肪来源的间充质干细胞移植促进了血管新生。综上，在三维空间的血管体积因数成像以干细胞在三维空间的在体示踪存活的数量和空间分布作为反馈信息，进而对血管新生进行定量成像研究。生物发光断层成像及超高分辨率micro-CT混合成像系统在三维空间为基于细胞移植治疗的机制研究和长程监测研究提供了新颖的成像工具和功能评估手段。
本论文的研究涉及到的动物实验均按照动物操作规程执行，并得到了本单位实验动物保护委员会的批准。
In recent years, Optical Molecular Tomography technologies and the applicationsin serious diseases have brought into focus owing to good performances and effects.Due to optical molecular tomography possesses their several advantages such as highsenstivity, cost-effectiveness and potential absolute quantification characteristics. Untilnowadays, optical molecular tomography has been widely used in tumors,cardiovascular diseases, neuroscience etc. As one of the optical tomography,Bioluminescence Tomography also has the advantages of high-senstivity,high-sepcificity, and high-resolution for anatomical structure etc. It has abroad potentialfor biomedical applications, realization of absolute quantification analysis. The gastriccancer and stem cell transplantation for the therapy of ischemic diseases have beensystematic researched. The author’s major researches are outlined as follows:
1. Hybrid system of high-resilution micro-CT and Bioluminescence Tomography(BLT) was developed for application research such as tumor and hindlimb ischemiadeseases. Quantitative calibrations were performed for BLT system, the calibrationsinclude non-uniformity calibration for the pixel response and absolute intensitycalibration for quantitative imaging. Moreover, a view-field function was constructedfor consideration of the spatial location of the tomographic optical imaging system, thus,the luminous flux could be quantified by passing through the optical lens. All aboveworks would promote the research for quantitative angiogenesis of hindlimb ischemia.
2. Quantitative reconstruction method was studied for BioluminescenceTomography. A total power reconstruction strategy was provided based on finiteelement mesh generation method. We constructed the relationship between thebiological parameters and optical reporter gene characteristics and confirmed thefeasibility of total power reconstruction through transplanting a given source into mousebody. Cell number level quantification analysis was set up for tumor cell line and stemcell through PC-3M-luc-C6prostate cancer line, A549-luc-C8non-small cell lungcancer line and adipose-derived mesenchymal stem cells (AD-MSCs).
3. We concentrated on the analysis of quantitative reconstruction deviation anddeviation contribution rate from peak wavelength shift of luminescent source and theanatomical structure deviation of animal models. Firstly, reconstruction deviations inlocalization and quantification were studied based on the hypothesis of the accurateoptical parameters of the emission peak wavelength. Secondly, reconstruction deviationwith peak wavelength shift was investigated based on the hppothesis of the accurate anatomical structure of animal model. Experimantal data suggested both the emissionpeak wavelength shift of luminescent light and deviation of anatomical structure modellead to errors in localization and quantification for BLT reconstruction. We find thatsub-millimeter accuracy for anntomical structure animal model and less than±10nmpeak wavelength shift are expected to be used in BLT reconstruction. Futhermore, theexperiments suggested that the deviation of the annotimcal structure model has muchmore influence on the reconstruction precision than the emission peak wavelength shiftdoes.
4. Noninvasive monitoring and quantitative imaging research have been studied forthe multistep process of gastric cancer. Bioluminescence Tomography techniques wereused for longitudinal observation of orthotopic gastric cancer model and some signaldevelopments during tumor progression in the first time. We also evaluated thefeasibility of the detection for micro-metastasis of gastric carcinoma by high-resolutionmicro-CT system with omnipaque accumulative contrast enhancement method in theorgans. Moreover, this method not only privides high-quality anatomical information ofthe metastasis in vivo, but also reveals the accurate solid tumors volume of the etastasis.Hybrid imaging system of high-resolution micro-CT and Bioluminescence Tomographybecomes a useful tool for preclinical gastric cancer research.
5. Quantitative assessment of angiogenesis has been studied through SpatialVascular Volume Fraction (SVVF) accompanying tracking AD-MSCs using hybridimaging system in vivo. Firstly, Noninvasive imaging of the AD-MSCs distribution andsurvival cells allows three-dimensional spatial visualization of the angiogenic perfusionand stem cells in vivo quantitative tracking in mice. In addition, angiogenesisassessment and quantification of stem cells with interactive imaging were performedusing hybrid system. Secondly, we reported a novel serial imaging of SVVF to exploreangiogenesis and this method could be used for quantitative assessment theangiogenesis in hindlimb mouse model. Thirdly, we discussed the strong active radii ofAD-MSCs through serial SVVF imaging analysis in spatiotemporal distribution.Fourthly, cross-validation was performed by several traditional methods to comfirm theangiogenesis after AD-MSCs transportation. a) Vascular corrosion casting validated thevascular density enhancement after AD-MSCs treatments. b) Scanning electronmicroscopic imaging performed visualization of the remarkably detailed narrativesabout some vascular sprouting. c) Confocal photomicrographs revealed the vasculardensity at the ischemic region and AD-MSCs presence in the ischemic tissue. d) Laser Doppler perfusion imaging (LDPI) was performed to assess tissue perfusion in theischemic hindlimb. Experimental data suggested significant enhancements of vasculardensity comparing between the treatment and control groups. In a word, this studyprovides an interactive SVVF quantification method for angiogenesis in3D space,which combined with BLT imaging analysis of the survival numbers and spatialdistribution of stem cells as a feedback. This combination of hybrid imagingmethodology and functional evaluation is suitable for the longitudinal mechanisticinvestigations of cellular therapy in vivo in3D space.
Animal protocols were approved by the Institutional Administrative Panel onLaboratory Animal Care.
1.构建了生物发光断层分子成像和超高分辨率micro-CT混合成像系统，并对生物发光断层分子成像系统进行了定量校准。针对预临床小动物研究的需要，构建了生物发光断层分子成像系统，并对成像系统CCD相机进行了像元响应的非均匀性校准和绝对强度的定量校准。针对光学断层分子成像空间位置的差异构建了视场函数，对进入光学成像系统的光通量进行了定量分析。构建了超高分辨率micro-CT成像系统，为本论文的下肢缺血模型的血管新生定量研究奠定了基础。
2.对生物发光断层分子成像定量重建方法进行了研究。基于有限元网格剖分方法，提出了总能量定量重建的策略。通过已知光源移植到小动物体内，验证了总能量重建的可行性。建立了基于报告基因系统的光学探针所反映的生物学参数与成像系统中总能量重建的物理参数之间的内在联系，实现了生物发光断层分子成像系统对肿瘤细胞和干细胞的细胞数量的定量分析。通过PC-3M-luc-C6前列腺癌和A549-luc-C8非小细胞肺癌细胞系以及脂肪来源的间充质干细胞的在体定量研究，证实了细胞水平定量分析的可行性和适用性。
3.研究了光学断层分子成像在光学探针峰值波长偏移及解剖结构模型偏差等情况下引入的重建误差及其贡献率的大小。以生物发光断层成像为例分析了光学断层分子成像的重建误差的主要来源。（1）在假定光学参数准确的前提下，研究了动物模型解剖结构偏差带来的光学重建在定位和定量上的偏差。（2）在假定解剖结构准确的情况下，研究了光学探针峰值波长偏移对重建误差在定位和定量方面的影响。通过对解剖结构偏差以及峰值波长偏移的实验数据的系统测试、重建及数据分析，表明二者均会对重建的定位和定量带来较大的影响；其中，当光学探针峰值波长在640nm以下时，解剖结构的偏差对重建在定位和定量有更大的影响。实验数据也揭示了光学探针峰值波长漂移量在±10nm范围内、动物模型的解剖结构偏差在亚毫米级别以内时都可以得到较理想的定位和定量重建结果。
4.利用自主研发的混合成像系统对胃癌的生物学行为进行定量成像研究。利用生物发光断层分子成像对胃癌原位动物模型实现在体定量分析以及肿瘤模型的长程动态观测，为胃癌的预临床研究提供了新的成像方法和成像工具。通过micro-CT造影成像精确计算出肿瘤的实际体积大小，同时在研究中提出了新颖的低成本欧乃派克时间累积对比增强造影方法，可以实现微小胃癌转移灶的在体定位和定量分析。
5.对干细胞移植治疗缺血性疾病促进血管新生开展了混合成像系统的定量示踪和基于空间血管体积因数SVVF定量评估的成像研究。（1）采用生物发光断层分子成像及超高分辨率micro-CT混合成像系统在三维空间实现干细胞的在体定量示踪以及血管新生的血管灌注分析，实现对干细胞在体空间分布和细胞数量的定量示踪；并根据干细胞的迁移、存活数量和空间分布，进一步交互式实施高分辨的微血管网络密度的定量评估。（2）提出了空间血管体积因数（Spatial VascularVolume Fraction, SVVF）的概念，并将其用于小鼠下肢缺血模型血管新生的定量评估。（3）通过血管体积因数SVVF的系列成像研究发现在时间和空间上血管新生的量化指标，进而探讨性地分析了干细胞移植后旁分泌的作用效果和作用半径。（4）通过多种传统的方法对干细胞移植后的血管新生进行了交叉验证。利用血管铸型验证血管新生的宏观变化情况；通过扫描电镜成像证实了在干细胞移植后促进血管新生作用的不同时间点血管新生的微观结构及发芽情况；通过共聚焦荧光显微成像证实了eGFP转基因小鼠来源的脂肪间充质干细胞在缺血组织的存在性和血管新生的情况；通过激光多普勒成像观测血流灌注的恢复情况。本文提出的SVVF方法以及交叉验证的结果一致发现，脂肪来源的间充质干细胞移植与对照组相比可以明显地增大了血管密度，进而证实脂肪来源的间充质干细胞移植促进了血管新生。综上，在三维空间的血管体积因数成像以干细胞在三维空间的在体示踪存活的数量和空间分布作为反馈信息，进而对血管新生进行定量成像研究。生物发光断层成像及超高分辨率micro-CT混合成像系统在三维空间为基于细胞移植治疗的机制研究和长程监测研究提供了新颖的成像工具和功能评估手段。
本论文的研究涉及到的动物实验均按照动物操作规程执行，并得到了本单位实验动物保护委员会的批准。
In recent years, Optical Molecular Tomography technologies and the applicationsin serious diseases have brought into focus owing to good performances and effects.Due to optical molecular tomography possesses their several advantages such as highsenstivity, cost-effectiveness and potential absolute quantification characteristics. Untilnowadays, optical molecular tomography has been widely used in tumors,cardiovascular diseases, neuroscience etc. As one of the optical tomography,Bioluminescence Tomography also has the advantages of high-senstivity,high-sepcificity, and high-resolution for anatomical structure etc. It has abroad potentialfor biomedical applications, realization of absolute quantification analysis. The gastriccancer and stem cell transplantation for the therapy of ischemic diseases have beensystematic researched. The author’s major researches are outlined as follows:
1. Hybrid system of high-resilution micro-CT and Bioluminescence Tomography(BLT) was developed for application research such as tumor and hindlimb ischemiadeseases. Quantitative calibrations were performed for BLT system, the calibrationsinclude non-uniformity calibration for the pixel response and absolute intensitycalibration for quantitative imaging. Moreover, a view-field function was constructedfor consideration of the spatial location of the tomographic optical imaging system, thus,the luminous flux could be quantified by passing through the optical lens. All aboveworks would promote the research for quantitative angiogenesis of hindlimb ischemia.
2. Quantitative reconstruction method was studied for BioluminescenceTomography. A total power reconstruction strategy was provided based on finiteelement mesh generation method. We constructed the relationship between thebiological parameters and optical reporter gene characteristics and confirmed thefeasibility of total power reconstruction through transplanting a given source into mousebody. Cell number level quantification analysis was set up for tumor cell line and stemcell through PC-3M-luc-C6prostate cancer line, A549-luc-C8non-small cell lungcancer line and adipose-derived mesenchymal stem cells (AD-MSCs).
3. We concentrated on the analysis of quantitative reconstruction deviation anddeviation contribution rate from peak wavelength shift of luminescent source and theanatomical structure deviation of animal models. Firstly, reconstruction deviations inlocalization and quantification were studied based on the hypothesis of the accurateoptical parameters of the emission peak wavelength. Secondly, reconstruction deviationwith peak wavelength shift was investigated based on the hppothesis of the accurate anatomical structure of animal model. Experimantal data suggested both the emissionpeak wavelength shift of luminescent light and deviation of anatomical structure modellead to errors in localization and quantification for BLT reconstruction. We find thatsub-millimeter accuracy for anntomical structure animal model and less than±10nmpeak wavelength shift are expected to be used in BLT reconstruction. Futhermore, theexperiments suggested that the deviation of the annotimcal structure model has muchmore influence on the reconstruction precision than the emission peak wavelength shiftdoes.
4. Noninvasive monitoring and quantitative imaging research have been studied forthe multistep process of gastric cancer. Bioluminescence Tomography techniques wereused for longitudinal observation of orthotopic gastric cancer model and some signaldevelopments during tumor progression in the first time. We also evaluated thefeasibility of the detection for micro-metastasis of gastric carcinoma by high-resolutionmicro-CT system with omnipaque accumulative contrast enhancement method in theorgans. Moreover, this method not only privides high-quality anatomical information ofthe metastasis in vivo, but also reveals the accurate solid tumors volume of the etastasis.Hybrid imaging system of high-resolution micro-CT and Bioluminescence Tomographybecomes a useful tool for preclinical gastric cancer research.
5. Quantitative assessment of angiogenesis has been studied through SpatialVascular Volume Fraction (SVVF) accompanying tracking AD-MSCs using hybridimaging system in vivo. Firstly, Noninvasive imaging of the AD-MSCs distribution andsurvival cells allows three-dimensional spatial visualization of the angiogenic perfusionand stem cells in vivo quantitative tracking in mice. In addition, angiogenesisassessment and quantification of stem cells with interactive imaging were performedusing hybrid system. Secondly, we reported a novel serial imaging of SVVF to exploreangiogenesis and this method could be used for quantitative assessment theangiogenesis in hindlimb mouse model. Thirdly, we discussed the strong active radii ofAD-MSCs through serial SVVF imaging analysis in spatiotemporal distribution.Fourthly, cross-validation was performed by several traditional methods to comfirm theangiogenesis after AD-MSCs transportation. a) Vascular corrosion casting validated thevascular density enhancement after AD-MSCs treatments. b) Scanning electronmicroscopic imaging performed visualization of the remarkably detailed narrativesabout some vascular sprouting. c) Confocal photomicrographs revealed the vasculardensity at the ischemic region and AD-MSCs presence in the ischemic tissue. d) Laser Doppler perfusion imaging (LDPI) was performed to assess tissue perfusion in theischemic hindlimb. Experimental data suggested significant enhancements of vasculardensity comparing between the treatment and control groups. In a word, this studyprovides an interactive SVVF quantification method for angiogenesis in3D space,which combined with BLT imaging analysis of the survival numbers and spatialdistribution of stem cells as a feedback. This combination of hybrid imagingmethodology and functional evaluation is suitable for the longitudinal mechanisticinvestigations of cellular therapy in vivo in3D space.
Animal protocols were approved by the Institutional Administrative Panel onLaboratory Animal Care.
引文
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