WO2013185487A1 - 基于金纳米簇的肿瘤靶向活体多模态成像方法 - Google Patents

基于金纳米簇的肿瘤靶向活体多模态成像方法 Download PDF

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WO2013185487A1
WO2013185487A1 PCT/CN2013/070928 CN2013070928W WO2013185487A1 WO 2013185487 A1 WO2013185487 A1 WO 2013185487A1 CN 2013070928 W CN2013070928 W CN 2013070928W WO 2013185487 A1 WO2013185487 A1 WO 2013185487A1
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imaging
tumor
raman
fluorescence
cells
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WO2013185487A8 (zh
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王雪梅
王建玲
李奇维
姜晖
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东南大学
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    • A—HUMAN NECESSITIES
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    • A61K49/0065—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle
    • A—HUMAN NECESSITIES
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    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A—HUMAN NECESSITIES
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    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
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    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • A—HUMAN NECESSITIES
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    • A61B8/08—Detecting organic movements or changes, e.g. tumours, cysts, swellings
    • A61B8/0833—Detecting organic movements or changes, e.g. tumours, cysts, swellings involving detecting or locating foreign bodies or organic structures
    • A61B8/085—Detecting organic movements or changes, e.g. tumours, cysts, swellings involving detecting or locating foreign bodies or organic structures for locating body or organic structures, e.g. tumours, calculi, blood vessels, nodules
    • A—HUMAN NECESSITIES
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    • A61B8/481—Diagnostic techniques involving the use of contrast agent, e.g. microbubbles introduced into the bloodstream
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    • G01N21/64—Fluorescence; Phosphorescence
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    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering

Definitions

  • the present invention relates to the field of tumor detection technology, and more particularly to a method for preparing a tumor nano-probe, which grows a nano-bioprobe such as a gold nanocluster with tumor-targeted molecular imaging in situ in a living body lesion.
  • Functional nanoprobes combine tumor targeting, fluorescence imaging, Raman imaging, and ultrasound imaging to enable multimodal simultaneous monitoring, and this in situ bioimaging method can not only perform early tumor diagnosis but also Timely monitoring during cancer treatment.
  • Tumors especially malignant tumors - cancer is the leading cause of human death.
  • tumor biology and oncology such as the discovery of tumor biomarkers, the ease of surgical procedures, the development of radiotherapy and chemotherapy, the overall cancer survival rate has not improved significantly over the years.
  • fluorescence imaging and Raman imaging are particularly concerned with medical diagnosis because of their high sensitivity and ability to provide quantitative and dynamic biological information.
  • fluorescence imaging and Raman imaging technology can quickly measure the growth of tumors in various cancer models, and can timely evaluate the changes of cancer cells in cancer treatment; Quantitative detection of in situ tumors, metastases, and spontaneous tumors in mice as a whole.
  • Hollingshead U251-HRE cells were constructed using the human glioma cell line U251, and the tumor cells were implanted in nude mice. They found that when the tumor reached 300-500. At the time of mg, local tissue showed hypoxia, and significant expression of luciferase was detected at this time.
  • This method not only monitors the tumor itself, More importantly, the microenvironment in which the tumor cells are located can be monitored.
  • fluorescence imaging and Raman imaging can accurately provide information on changes in molecular characteristics in cells and tissues, revealing tissue morphology and anatomical details through spectral fingerprints of molecular structures and locations, enabling clinical diagnostic tools. To help medical staff detect disease and treatment response at the molecular level.
  • Nanomaterials not only provide highly sensitive and specific imaging information for cancer patients, but also transport anticancer drugs to tumor sites. In addition, it is more interesting and important that some nanomaterials can be used as wonderful drugs to treat cancer. .
  • Today, our understanding of these aspects is still limited: one is the biomarker suitable for imaging; the other is the choice of imaging target and contrast enhancing material; the third is the chemical method used for imaging probe biochemistry.
  • the object of the present invention is to provide a nano biological probe such as a gold nanocluster with in-situ growth and integrated in vivo or ex vivo tumor targeting, fluorescence, enhanced Raman information and ultrasound imaging. And can be used for tumor-targeted fluorescence imaging, Raman imaging and ultrasound imaging of living cells or living tumors. Ultrasound imaging can determine the size and location of tumors, monitor the treatment process and the recovery process after treatment. In addition, according to the fluorescence distribution of fluorescence imaging and its fluorescence intensity and the wave number position and intensity of the Raman signal in the Raman imaging map, the distribution and quantity of the biochemical components of the tumor site are qualitatively and quantitatively analyzed to realize the tumor. Early diagnosis and timely monitoring of the tumor treatment process.
  • the gold nanocluster-based tumor-targeting living body multimodal imaging method of the present invention is: co-cultivating chloroauric acid or a salt solution thereof with tumor cells, using a large number of gold nanoclusters specifically generated in tumor cells, using a fluorescence microscope High-resolution microscopy imaging of tumor cells by Raman microscopy or ultrasound imaging.
  • Qualitative or quantitative analysis of cellular structure or chemical composition is: co-cultivating chloroauric acid or a salt solution thereof with tumor cells, using a large number of gold nanoclusters specifically generated in tumor cells, using a fluorescence microscope High-resolution microscopy imaging of tumor cells by Raman microscopy or ultrasound imaging.
  • the imaging is used to inject chloroauric acid or a salt solution thereof into tumor tissue or tumor tissue during body tumor imaging, using a large number of gold nanoclusters specifically generated in tumor cells, using Raman imager and living fluorescence
  • the imager performs Raman imaging or fluorescence imaging on the tumor site, determines the size and position of the tumor by ultrasound imaging, monitors the treatment process and the recovery process after the treatment, and, according to the intensity and distribution of the Raman and fluorescence signals, the tumor site Qualitative and quantitative analysis of the distribution and quantity of biochemical components;
  • the invention Compared with the prior art method, the invention has the following advantages and effects:
  • the method for imaging the isolated tumor cells is to co-culture chloroauric acid or a salt solution thereof with the tumor cells, and utilize a large number of gold nanoclusters specifically generated in the tumor cells, using fluorescence imaging, Raman imaging or super generation.
  • the cell structure or chemical composition of tumor cells can be analyzed.
  • the method for imaging in vivo tumors is to inject chloroauric acid or a salt solution thereof around tumor tissue or tumor tissue, and utilize a large number of gold nanoclusters specifically formed in tumor cells, using Raman imaging and living fluorescence. Imaging technology can analyze the distribution and quantity of biochemical components of tumor cells.
  • the present invention adopts the following technical measures:
  • Raman imaging or fluorescence imaging is used to perform Raman imaging or fluorescence imaging of the tumor site, the size and location of the tumor are determined by ultrasound imaging, and the treatment process and the recovery process after treatment are monitored.
  • the distribution and quantity of the biochemical components of the tumor site are qualitatively and quantitatively analyzed to achieve early diagnosis of the tumor and timely monitoring of the tumor treatment process.
  • Example 1 Imaging method based on intracellular biosynthesis of gold nanoclusters
  • a sterile concentration of 1 to 1000 ⁇ mol/L of chloroauric acid or a salt solution thereof is incubated with HepG2 cells for 16 to 48 hours (37 ° C, 5% CO 2 , RH 95%) to obtain an in vivo body.
  • In situ biosynthesis of gold nanoclusters The medium in the culture dish was carefully removed, and the cells were gently rinsed with sterile PBS of pH 7.2, placed under a laser focused fluorescence microscope, and the green fluorescence of the cells was collected by excitation with a wavelength of 488 nm blue light. The image, through the fluorescence tomography technique, can clearly observe that the gold nanoclusters are mainly concentrated in the nuclear region.
  • Example 2 Tumor-targeted imaging method based on in situ growth of gold nanoclusters in living lesions
  • subcutaneous injection of 0.1 ⁇ 0.5 mL of sterile concentration in the vicinity of the tumor of the nude mouse that has been implanted into the liver cancer tumor model is 1 ⁇ 1000.
  • Methyl/L chloroauric acid or its salt solution after 12 to 36 hours of incubation, the experimental nude mice were anesthetized with 5% isoflurane gas, and then placed on a small animal living imager operating platform, An image of the tumor area can be acquired by selecting blue light excitation.

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Abstract

一种基于金纳米簇的肿瘤靶向活体多模态成像分析方法，包括：通过包括不同种类肿瘤细胞及正常细胞的体外细胞实验，将相关细胞与一定浓度的氯金酸及其盐溶液在生理条件下孵育产生金纳米簇，实现了实时非侵入性的高分辨肿瘤细胞荧光成像、拉曼成像及超声成像。在此基础上，进一步采用局部皮下注射方法，在移植肿瘤裸鼠模型上实现了实时原位活体肿瘤靶向荧光、拉曼和超声成像。该功能性纳米探针将肿瘤靶向、荧光成像、拉曼成像以及超声成像多功能融为一体，能够实现多模态的同步监测；同时，这种基于金纳米簇的原位活体成像方法能够进行准确定位与肿瘤靶向成像分析。

Description

基于金纳米簇的肿瘤靶向活体多模态成像方法 技术领域
本发明涉及肿瘤检测技术领域，更具体涉及一种靶向肿瘤纳米探针的制备方法，通过在生命体病灶原位生长出具有肿瘤靶向性分子成像的金纳米簇等纳米生物探针，该功能纳米探针将肿瘤靶向、荧光成像、拉曼成像、超声成像多功能融为一体，能够实现多模态的同步监测，而且这种原位活体成像方法不仅能够进行肿瘤早期诊断而且能够在癌症治疗过程中进行适时监控。
 背景技术
肿瘤，尤其是恶性肿瘤——癌症是人类死亡的主要原因。尽管肿瘤生物学和肿瘤医学有了很大的发展，比如：肿瘤生物标志物的发现，简便的手术流程，放疗和化疗的发展，但是总癌症存活率多年来一直都没有显著的提高。为了提高肿瘤患者的存活率和生活质量，我们继续发展新的肿瘤早期诊断的方法和治疗方法。
当今，肿瘤靶向性纳米探针的发展存在很大挑战，与肿瘤学领域最常规的方法相比，分子成像有可能更早地检测疾病的进展或治疗效力。其中荧光成像和拉曼成像因为其具有高灵敏和能够提供定量和动态的生物信息而受到医学诊断的特别关注。随着研究的不断深入和系统的不断改进，荧光成像和拉曼成像技术可以快速的测量各种癌症模型中肿瘤的生长，并可对癌症治疗中癌细胞的变化进行适时观测评估；可以无创伤地定量检测小鼠整体的原位瘤、转移瘤及自发瘤。如Hollingshead 等利用人类胶质瘤细胞系U251构建U251-HRE细胞，将此肿瘤细胞植于裸鼠体内，他们发现当肿瘤达到了300-500 mg时，局部组织出现低氧状态，此时可监测到荧光素酶显著表达。这种方法不仅仅监测肿瘤本身, 更重要的是可以监测肿瘤细胞所处的微环境。另外，荧光成像和拉曼成像技术可以精确地提供细腻的细胞和组织中分子特征变化信息，能够通过分子的结构和位置的光谱指纹揭示组织形态和解剖学上的细节，从而能够成为临床诊断工具，帮助医务人员检测出分子水平上的疾病和治疗反应情况。
纳米科学和技术的发展带动了用于分子细胞成像和癌症治疗的纳米材料的发展，也带动了用于癌症检测和筛查的纳米器件的开发。纳米材料不仅能提供癌症病人的高灵敏和特异性成像信息，而且还能运输抗癌药物到达肿瘤的位置，另外，更有意思同时也很重要的是有些纳米材料本身可以作为奇妙的药物来治疗癌症。当今，我们在以下这些方面的认识仍然是有限的：一是适合用来成像的生物标志物；二是成像靶标和反差增强材料的选择；三是用来是成像探针生物化的化学方法。我们在发展癌症特异性成像试剂上同样遇到很多困难，包括：靶向组织或肿瘤的探针的运输不佳；生物毒性大；探针的稳定性不佳；体内信号增强强度低等。本发明已解决了这些问题。
 技术问题
本发明的目的是在于提供了一种原位生长并且集在体或离体肿瘤靶向、荧光、增强拉曼信息及超声成像等多功能生物相容性好的金纳米簇等纳米生物探针，并能够用于肿瘤的活细胞或活体的肿瘤靶向的荧光成像、拉曼成像及超声成像的新方法，通过超声成像可确定肿瘤的尺寸和位置，监控治疗过程以及治疗后的恢复过程，另外，根据荧光成像的荧光分布情况和其荧光强度以及拉曼成像的图谱中拉曼信号的波数位置及其强度对肿瘤部位的生物化学组分的分布以及数量进行定性和定量分析，以便实现肿瘤的早期诊断以及肿瘤治疗过程适时监控。
技术解决方案
本发明的基于金纳米簇的肿瘤靶向活体多模态成像方法为：将氯金酸或其盐溶液与肿瘤细胞共育，利用肿瘤细胞中特异性生成的大量的金纳米簇，使用荧光显微镜、拉曼显微镜或超声成像技术对肿瘤细胞进行高分辨显微成像，通过超声成像、荧光成像的荧光分布情况和其荧光强度以及拉曼成像的图谱中拉曼信号的波数位置及其强度来对细胞结构或化学组分进行定性或定量分析。
具体措施如下：
首先在细胞水平进行研究，其具体步骤是：
1）选择肝癌HepG2和白血病K562细胞作为研究对象，将这两种细胞分别与1~1000 μmol/L的氯金酸或其盐溶液进行16~48小时细胞培养箱中孵育以得到原位生物合成的金纳米簇；
2）与1~1000 μmol/L的无菌氯金酸或其盐溶液进行16~48小时孵育的HepG2（K562）细胞，用荧光显微镜表征金纳米簇在细胞中的分布情况，用荧光显微镜或拉曼显微镜来探究肿瘤细胞内原位生长的金纳米簇用于肿瘤的活细胞的肿瘤靶向的荧光成像和拉曼成像；通过超声成像、荧光成像的荧光分布情况和其荧光强度以及拉曼成像的图谱中拉曼信号的波数位置及其强度来对细胞结构或化学组分进行定性或定量分析。
该成像用于在体肿瘤成像时，将氯金酸或其盐溶液注射到肿瘤组织周围或肿瘤组织中，利用肿瘤细胞中特异性生成的大量的金纳米簇，使用拉曼成像仪及活体荧光成像仪对肿瘤部位进行拉曼成像或荧光成像，通过超声成像确定肿瘤的尺寸和位置，监控治疗过程以及治疗后的恢复过程，另外，根据拉曼及荧光信号的强度及分布情况从而对肿瘤部位的生物化学组分的分布以及数量进行定性和定量分析；
然后在动物活体模型层面进行研究，其具体步骤是：
1）构建移植肝癌肿瘤的裸鼠模型；
2）采用于肿瘤附近局部皮下注射0.1~0.5 mL无菌的浓度为1~1000 mmol/L的氯金酸或其盐溶液，并且经过12~36小时的孵育，在肿瘤组织内原位生成集肿瘤靶向、荧光、拉曼以及超声成像的多功能金纳米簇；
3）利用过程2）中所生成的金纳米簇，使用拉曼成像或荧光成像技术对肿瘤部位进行拉曼成像或荧光成像，通过超声成像确定肿瘤的尺寸和位置，监控治疗过程以及治疗后的恢复过程，另外，根据荧光或拉曼信号的强度及分布情况从而对肿瘤部位的生物化学组分的分布以及数量进行定性和定量分析。
 有益效果
本发明与现有技术方法相比，具有以下优点和效果：
本研究采用生命体内原位生长多功能分子成像的金纳米簇等纳米生物探针的方法，这种方法既避免了传统纳米材料合成过程中引入的化学试剂以及纳米材料稳定剂对有机体造成的生物毒性，也避免了传统纳米材料易被网状内皮系统捕获从而被清除而不能到达病灶组织的缺点，以便实现活体肿瘤靶向性荧光、拉曼及超声成像。该功能性纳米探针将肿瘤靶向、荧光成像、拉曼成像及超声成像多功能融为一体，能够实现多模态的同步监测，这种原位活体成像方法可望应用于肿瘤临床诊断的多功能适时成像以便实现肿瘤早期诊断以及肿瘤治疗过程中的适时跟踪。
 本发明的实施方式
所述的离体肿瘤细胞成像的方法为，将氯金酸或其盐溶液与肿瘤细胞共育，利用肿瘤细胞中特异性生成的大量的金纳米簇，使用荧光成像、拉曼成像或超生成像技术便可对肿瘤细胞的细胞结构或化学组分进行分析。
所述的在体肿瘤成像的方法为，将氯金酸或其盐溶液注射到肿瘤组织周围或肿瘤组织中，利用肿瘤细胞中特异性生成的大量的金纳米簇，采用拉曼成像及活体荧光成像技术便可对肿瘤细胞的生物化学组分的分布及其数量进行分析。
为了实现上述的目的，本发明采用以下技术措施：
首先在细胞水平进行研究，其具体步骤是：
1）选择肝癌HepG2和白血病K562细胞作为研究对象，将这两种细胞分别与1~1000 μmol/L的氯金酸或其盐溶液进行16~48小时细胞培养箱中孵育以得到原位生物合成的金纳米簇；
2）与1~1000 μmol/L的无菌氯金酸或其盐溶液进行16~48小时孵育的HepG2（K562）细胞，用荧光显微镜表征金纳米簇在细胞中的分布情况，用荧光显微镜或拉曼显微镜来探究肿瘤细胞内原位生长的金纳米簇用于肿瘤的活细胞的肿瘤靶向的荧光成像和拉曼成像；通过超声成像、荧光成像的荧光分布情况和其荧光强度以及拉曼成像的图谱中拉曼信号的波数位置及其强度来对细胞结构或化学组分进行定性或定量分析。
然后在动物活体模型进行研究，其具体步骤是：
1） 构建移植肝癌肿瘤的裸鼠模型。
2） 采用于肿瘤附近局部皮下注射0.1~0.5 mL无菌的浓度为1~1000 mmol/L的氯金酸或其盐溶液，并且经过12~36小时的孵育，在肿瘤组织内原位生成集肿瘤靶向、荧光、拉曼以及超声成像的多功能金纳米簇。
3） 利用过程2）中所生成的金纳米簇，使用拉曼成像或荧光成像技术对肿瘤部位进行拉曼成像或荧光成像，通过超声成像确定肿瘤的尺寸和位置，监控治疗过程以及治疗后的恢复过程，另外，根据荧光或拉曼信号的强度及分布情况从而对肿瘤部位的生物化学组分的分布以及数量进行定性和定量分析，以实现肿瘤的早期诊断以及肿瘤治疗过程适时监控。
实例1 基于细胞内原位生物合成金纳米簇的成像方法
首先将无菌的浓度为1~1000 μmol/L的氯金酸或其盐溶液与HepG2细胞共孵育16~48小时（37 °C，5 % CO2，RH 95%），即可得到有体内原位生物合成的金纳米簇。然后将培养皿中的培养基小心移出，并用pH 7.2的无菌PBS轻轻淋洗细胞，将其置于激光聚焦荧光显微镜下，采用波长为488 nm蓝光进行激发即可采集到细胞的绿色荧光图像，通过荧光断层扫描技术可以清晰的观察到这种金纳米簇主要集中在细胞核区。
实例2 基于活体病灶原位生长金纳米簇的肿瘤靶向成像方法
首先在已经植入肝癌肿瘤模型裸鼠的肿瘤附近局部皮下注射0.1~0.5 mL无菌的浓度为1~1000 mmol/L的氯金酸或其盐溶液，经过12~36小时的孵育后，将该实验裸鼠用5%异氟烷进行气体麻醉，然后将其置于小动物活体成像仪操作平台上，选择蓝光激发即可采集到肿瘤区域的图像。

Claims (2)

  1. 一种基于金纳米簇的肿瘤靶向活体多模态成像方法，其特征在于该成像的方法为：将氯金酸或其盐溶液与肿瘤细胞共育，利用肿瘤细胞中特异性生成的大量的金纳米簇，使用荧光显微镜、拉曼显微镜或超声成像技术对肿瘤细胞进行高分辨显微成像，通过超声成像、荧光成像的荧光分布情况和其荧光强度以及拉曼成像的图谱中拉曼信号的波数位置及其强度来对细胞结构或化学组分进行定性或定量分析；
    具体措施如下：
    首先在细胞水平进行研究，其具体步骤是：
    1）选择肝癌HepG2和白血病K562细胞作为研究对象，将这两种细胞分别与1~1000 μmol/L的氯金酸或其盐溶液进行16~48小时细胞培养箱中孵育以得到原位生物合成的金纳米簇；
    2）与1~1000 μmol/L的无菌氯金酸或其盐溶液进行16~48小时孵育的HepG2（K562）细胞，用荧光显微镜表征金纳米簇在细胞中的分布情况，用荧光显微镜或拉曼显微镜来探究肿瘤细胞内原位生长的金纳米簇用于肿瘤的活细胞的肿瘤靶向的荧光成像和拉曼成像；通过超声成像、荧光成像的荧光分布情况和其荧光强度以及拉曼成像的图谱中拉曼信号的波数位置及其强度来对细胞结构或化学组分进行定性或定量分析。
  2. 一种基于金纳米簇的肿瘤靶向活体多模态成像分析方法，其特征在于该成像用于在体肿瘤成像时，将氯金酸或其盐溶液注射到肿瘤组织周围或肿瘤组织中，利用肿瘤细胞中特异性生成的大量的金纳米簇，使用拉曼成像仪及活体荧光成像仪对肿瘤部位进行拉曼成像或荧光成像，通过超声成像确定肿瘤的尺寸和位置，监控治疗过程以及治疗后的恢复过程，另外，根据拉曼及荧光信号的强度及分布情况从而对肿瘤部位的生物化学组分的分布以及数量进行定性和定量分析；
    然后在动物活体模型层面进行研究，其具体步骤是：
    1）构建移植肝癌肿瘤的裸鼠模型；
    2）采用于肿瘤附近局部皮下注射0.1~0.5 mL无菌的浓度为1~1000 mmol/L的氯金酸或其盐溶液，并且经过12~36小时的孵育，在肿瘤组织内原位生成集肿瘤靶向、荧光、拉曼以及超声成像的多功能金纳米簇；
    3）利用过程2）中所生成的金纳米簇，使用拉曼成像或荧光成像技术对肿瘤部位进行拉曼成像或荧光成像，通过超声成像确定肿瘤的尺寸和位置，监控治疗过程以及治疗后的恢复过程，另外，根据荧光或拉曼信号的强度及分布情况从而对肿瘤部位的生物化学组分的分布以及数量进行定性和定量分析。
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