磁性纳米粒子高效纯化及双色荧光量子点探针筛查外周血肺癌微转移方法的研究
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摘要
肺癌现已成为全球范围内对人类健康威胁最大的恶性肿瘤之一,尽管手术能够完全切除局部病灶,但仍有患者在5年内复发和转移,因此科学家提出微转移的概念,认为如果能及时诊断出微转移,患者将有可能得到更及时、系统和有针对性的治疗。磁性纳米粒子以其优越的物理特性在生物分离领域显示出独特的优势,荧光量子点与有机荧光染料相比具有独特的光学特性,在荧光标记领域的应用也突显优势。本文通过将广谱角蛋白pan-ck抗体偶联到Fe3O4磁性纳米粒子表面,肺组织特异性Lunx抗体及SP-A抗体偶联到水溶性CdTe荧光量子点表面,然后利用已标识有pan-ck抗体的磁性纳米粒子进行外周血癌细胞的富集与分选,最后应用连接有Lunx和SP-A抗体的荧光量子点对富集到的细胞进行肺组织特异性鉴定,以实现对肺癌微转移的诊断。本研究成功地将pan-ck抗体偶联到磁性纳米粒子表面,Lunx和SP-A抗体偶联到荧光量子点表面,并且能够从外周血中富集与分选到癌细胞,通过双色荧光量子点探针成功地对细胞内两种蛋白的表达进行了同步鉴定,明显提高了鉴定的效率与准确性,为进一步建立外周血肺癌微转移方法奠定了较好的研究基础。
Pulmonary carcinoma has become one of the biggest malignant tumors threatening to human all over the world. Its incidence rate and death rate have a incremental tendency year by year. Pulmonary carcinoma is divided into small cell lung cancer and non small cell lung cancer(NSCLC) according international standard. NSCLC occupy 85 percent of pulmonary carcinoma. To the NSCLC patients, operation is the preferred treatment prescription. Although operation can completely resect local lesion,there still have patients occur recidivation and metabasis,and routine pathobiology and imageology method have difficultity to find the focus. So scientists propose the concept of occult micrometastasis. They consider that if we can diagnose occult micrometastasis in time,then the patients have the chance to obtain directed and systematical treatment promptly. Thus people put the expection to the new detect method that screening peripheral blood caner cells for the people that has lung carcinoma risk factor or the patient that final diagnosis is lung carcinoma,thereby achieve the aims of early diagnosis and preventing recurrence.
At present, the main methods of screening cancer cells in peripheral blood are RT-PCR detection、flow cytometry and immunomagnetic beads technique et al. RT-PCR detection is that designing specific primer of tumor marked gene or target mRNA and useing RT-PCR method to detect peripheral blood sample whether contain tumor cells marked gene or mRNA.Because this method suffer from experiment contamination、illegitimate transcription et al.,so this method has high false positive rate.Thus it limit the usage for screening. Flow cytometry can carry out multiparameter and volant quantitate analysis and sorting to the cells of quick streamline flow state. Even though flow cytometry can gain precise cells quantitation,it cann’t provide cell morphology and the instrument price is so high that restrict its generalization. Immunomagnetic beads is synthetical ferruginated microparticles. Outer layer is coated by functional layer. Functional layer can bind biomolecule and be drawen by magnetic force.So under the magnetic force, compound particles happen mechanic movement and achieve be separated from other materials. In spite of immunomagnetic beads technique achieve determinate result, because of bigger diameter of magnetic beads, magnetic beads is easy to aggregate and sedimentate,so cause enriching efficiency to decrease, magnetic beads have difficulty to desorpt from cell surface.These defects confine the application of magnetic beads.
Magnetic nanoparticles is a new type nanometer magnetic.Comparing with common magnetic particle, the diameter of magnetic nanoparticles is small,it has favourable surface effection, functional group density of nanoparticle is increased,the time of dsorption equilibrium is decurtated. Secondly, magnetic nanoparticles has fine magnetic response,it has superparamagnetism. Thirdly, physical and chemistry property of magnetic nanoparticles are stable.it has enough mechanical strength to tolerance acid and alkali fusion and degradation of microbe.Finally,magnetic nanoparticle surface has functional group,so it can connect biomolecule.under the magnetic force, compound happen mechanic movement and achieve be separated from other material.
Quantum dots are new type of fluorophores with unique photophysical properties that make them attractive to biologists for use in fluorescence assays. Comparing with organic fluorescent dyes, quantum dots has broad absorption spectra coupled to narrow size-tunable photoluminescent emission spectra. Distinct populations of quantum dots can be simultaneously excited by a single excitation wavelength far removed from their respective emissions, the properties suggests they could be especially suited for multiplexing assays via simultaneous detection of multiple signals. Quantum dots have the ability to resist photobleaching.when using optical excitation, organic fluorescent dyes have faded,quantum still has strong fluorescence.
In my experiments, We coupled pan-ck antibody to the surface of magnetic nanoparticles, Lunx and SP-A antibody to the surface of quantum dots, then we enriched tumor cells by magnetic nanoparticle probe, and identified lung tissue specificity by quantum dots labelled antibody. Thus we could realize diagnose of earlier period micrometastasis. Experiments were divided four parts: 1. Preparation and modification of Fe3O4 magnetic nanoparticles and aqueous soluble CdTe quantum dots. 2. Coupling antibody to the surface Fe3O4 magnetic nanoparticles and aqueous soluble CdTe quantum dots. 3. Constructing a method on enriching and separating cancer cells in peripheral blood by immunomagnetic nanoparticles. 4. Identifing pulmonary carcinoma by quantum dots labelled Lunx and SP-A antibody. The results are as follows.
1. Preparation and modification of Fe3O4 magnetic nanoparticles
Fe3O4/poly(St-co-MPS)/SiO2 composite particles were prepared by combinating miniemulsion polymerization and St?ber coating methods.(patent delegation NO. CN100425627C ).Carboxyl-functionalized Fe3O4/Polystyrene composite particles were also prepared by copolymerization of acrylic acid and styrene, where could also prepare magnetite composite particles with different functionalized groups which could couple biomolecules.the small diameter of magnetic nanoparticles had fine biocompatibility, magnetic nanoparticles couple to biomolecule,then be used in the field of biological tag and bioseparation.
2. Preparation and modification of aqueous soluble CdTe quantum dots
Aqueous soluble CdTe quantum dots were prepared in aqueous solution by using thioglycolic acid as the stabilizer and then characterized by fluorescence emission spectrum, absorption band, and TEM, respectively. The results show that these quantum dots had good optical properties, such as narrow spectral line widths and continuous absorption profiles, peak width at half is 50 nm, TEM picture show that quantum dots dispersibility was fine,the diameter is uniform, which provide a powerful foundation for further applications.
3. Coupling pan-ck antibody to the surface Fe3O4 magnetic nanoparticles
Carbodiimide-mediated condensation method was used to couple pan-ck antibody to the surface of magnetic nanoparticles. In order to obtain the best coupling buffer system,we changed PH value, antibody addition and cross linking agent addition of MES coupling buffer and bangslabs coupling buffer. The result show that when PH value was 5.5, pan-ck antibody addition was 50μg,EDC addition was 0.25mg,the bangslabs coupling buffer system achieve the biggest coupling rate.
4. Coupling Lunx or SP-A antibody to the surface aqueous soluble CdTe quantum dots
Carbodiimide-mediated condensation method was used to couple Lunx and SP-A antibody to the surface aqueous soluble CdTe quantum dots. In order to obtain the best coupling buffer system,we changed the concentration and PH value of borate buffer.The result show that when PH value was 7.5, concentration was 0.05M, emission spectrum of CdTe quantum dots was the highest. After coupling finished, using dislysis method to eliminate quantum dots that didn’t couple antibody. We detected fluorescence emission spectrum after dislysis.the result show that the biggest emission peak had slight red shift,and peak shape didn’t change.these results hinted us that quantum dots didn’t happen gather and its dispersibility is fine, which provide a powerful foundation for further applications.
5. Constructing a method on enriching and separating cancer cells in peripheral blood by immunomagnetic nanoparticles.
We cultured A549 cells line,SPC-A-1 cell line,HepG2 cell line,HCT-8 cell line and identified expression of pan-ck protein in four epithelial cell lines. Four epithelial cell lines had high expression of pan-ck protein. This hint us that pan-ck can be served as capture antibody to screen epithelial cells. We investigated enriching efficiency by change the proportion of cancer epithelial line and PBMC,then enriching cancer cell by immunomagnetic nanoparticles.The result show that enriching efficiency is 5×106 ,comparing with the work of Zigeuner that use immunomagnetic beads to enrich,the enriching efficiency raised 5 times.along with proportion of cancer cells elevating,enriching cells means was increasing. After enriching,we used HE stain to initially identify cancer cells.HE stain show us that nucleus body of cells enriched by immunomagnetic nanoparticles was enlarged and nucleus dye deep,while main part of PBMC was lymph cells,comparing with cancer cells, lymph cells body was small. We collected 6 case peripheral blood of pulmonary carcinoma patients,then used immunomagnetic nanoparticles enriching epithelial cancer cells.5 cases peripheral blood could enrich cancer cells,while 1 case couldn’t enrich to obtain cancer cells. The cause was owe to that the extent of enriching efficiency was limited. So we should improve enriching efficiency.
6. Identifing pulmonary carcinoma by quantum dots labelled Lunx and SP-A antibodies
After enriching,we needed identify cells character using further detect method. Firstly, detecting expression of Lunx and SP-A in four epithelial cancer cells by immunocytochemical method. Then we investigated accuracy of quantum dots labeling by QDs-Lunx and QDs-SP-A to label four epithelial cancer cells. The result show that positive cell rate of the two methods of Immunocytochemical stain and quantum dots single labeling method was coincident.Quantum dots double labeling method can elevate positive cell rate. After ascertaining accuracy and superiority,we identified 5 case cells sample of enriching from peripheral blood by QDs-Lunx and QDs-SP-A probe. The result show that 2 cases expressed Lunx and SP-A at the same time.1 case only expressed SP-A. 2 cases only express Lunx. These results suggest us that using Lunx and SP-A antibody can effectively identify lung tissue specificity and quantum dots double labeling method can detect multiprotein expression at one sample simultaneously and effectively.
Pulmonary carcinoma has become one of the biggest malignant tumors threatening to human all over the world. Its incidence rate and death rate have a incremental tendency year by year. Pulmonary carcinoma is divided into small cell lung cancer and non small cell lung cancer(NSCLC) according international standard. NSCLC occupy 85 percent of pulmonary carcinoma. To the NSCLC patients, operation is the preferred treatment prescription. Although operation can completely resect local lesion,there still have patients occur recidivation and metabasis,and routine pathobiology and imageology method have difficultity to find the focus. So scientists propose the concept of occult micrometastasis. They consider that if we can diagnose occult micrometastasis in time,then the patients have the chance to obtain directed and systematical treatment promptly. Thus people put the expection to the new detect method that screening peripheral blood caner cells for the people that has lung carcinoma risk factor or the patient that final diagnosis is lung carcinoma,thereby achieve the aims of early diagnosis and preventing recurrence.
At present, the main methods of screening cancer cells in peripheral blood are RT-PCR detection、flow cytometry and immunomagnetic beads technique et al. RT-PCR detection is that designing specific primer of tumor marked gene or target mRNA and useing RT-PCR method to detect peripheral blood sample whether contain tumor cells marked gene or mRNA.Because this method suffer from experiment contamination、illegitimate transcription et al.,so this method has high false positive rate.Thus it limit the usage for screening. Flow cytometry can carry out multiparameter and volant quantitate analysis and sorting to the cells of quick streamline flow state. Even though flow cytometry can gain precise cells quantitation,it cann’t provide cell morphology and the instrument price is so high that restrict its generalization. Immunomagnetic beads is synthetical ferruginated microparticles. Outer layer is coated by functional layer. Functional layer can bind biomolecule and be drawen by magnetic force.So under the magnetic force, compound particles happen mechanic movement and achieve be separated from other materials. In spite of immunomagnetic beads technique achieve determinate result, because of bigger diameter of magnetic beads, magnetic beads is easy to aggregate and sedimentate,so cause enriching efficiency to decrease, magnetic beads have difficulty to desorpt from cell surface.These defects confine the application of magnetic beads.
Magnetic nanoparticles is a new type nanometer magnetic.Comparing with common magnetic particle, the diameter of magnetic nanoparticles is small,it has favourable surface effection, functional group density of nanoparticle is increased,the time of dsorption equilibrium is decurtated. Secondly, magnetic nanoparticles has fine magnetic response,it has superparamagnetism. Thirdly, physical and chemistry property of magnetic nanoparticles are stable.it has enough mechanical strength to tolerance acid and alkali fusion and degradation of microbe.Finally,magnetic nanoparticle surface has functional group,so it can connect biomolecule.under the magnetic force, compound happen mechanic movement and achieve be separated from other material.
Quantum dots are new type of fluorophores with unique photophysical properties that make them attractive to biologists for use in fluorescence assays. Comparing with organic fluorescent dyes, quantum dots has broad absorption spectra coupled to narrow size-tunable photoluminescent emission spectra. Distinct populations of quantum dots can be simultaneously excited by a single excitation wavelength far removed from their respective emissions, the properties suggests they could be especially suited for multiplexing assays via simultaneous detection of multiple signals. Quantum dots have the ability to resist photobleaching.when using optical excitation, organic fluorescent dyes have faded,quantum still has strong fluorescence.
In my experiments, We coupled pan-ck antibody to the surface of magnetic nanoparticles, Lunx and SP-A antibody to the surface of quantum dots, then we enriched tumor cells by magnetic nanoparticle probe, and identified lung tissue specificity by quantum dots labelled antibody. Thus we could realize diagnose of earlier period micrometastasis. Experiments were divided four parts: 1. Preparation and modification of Fe3O4 magnetic nanoparticles and aqueous soluble CdTe quantum dots. 2. Coupling antibody to the surface Fe3O4 magnetic nanoparticles and aqueous soluble CdTe quantum dots. 3. Constructing a method on enriching and separating cancer cells in peripheral blood by immunomagnetic nanoparticles. 4. Identifing pulmonary carcinoma by quantum dots labelled Lunx and SP-A antibody. The results are as follows.
1. Preparation and modification of Fe3O4 magnetic nanoparticles
Fe3O4/poly(St-co-MPS)/SiO2 composite particles were prepared by combinating miniemulsion polymerization and St?ber coating methods.(patent delegation NO. CN100425627C ).Carboxyl-functionalized Fe3O4/Polystyrene composite particles were also prepared by copolymerization of acrylic acid and styrene, where could also prepare magnetite composite particles with different functionalized groups which could couple biomolecules.the small diameter of magnetic nanoparticles had fine biocompatibility, magnetic nanoparticles couple to biomolecule,then be used in the field of biological tag and bioseparation.
2. Preparation and modification of aqueous soluble CdTe quantum dots
Aqueous soluble CdTe quantum dots were prepared in aqueous solution by using thioglycolic acid as the stabilizer and then characterized by fluorescence emission spectrum, absorption band, and TEM, respectively. The results show that these quantum dots had good optical properties, such as narrow spectral line widths and continuous absorption profiles, peak width at half is 50 nm, TEM picture show that quantum dots dispersibility was fine,the diameter is uniform, which provide a powerful foundation for further applications.
3. Coupling pan-ck antibody to the surface Fe3O4 magnetic nanoparticles
Carbodiimide-mediated condensation method was used to couple pan-ck antibody to the surface of magnetic nanoparticles. In order to obtain the best coupling buffer system,we changed PH value, antibody addition and cross linking agent addition of MES coupling buffer and bangslabs coupling buffer. The result show that when PH value was 5.5, pan-ck antibody addition was 50μg,EDC addition was 0.25mg,the bangslabs coupling buffer system achieve the biggest coupling rate.
4. Coupling Lunx or SP-A antibody to the surface aqueous soluble CdTe quantum dots
Carbodiimide-mediated condensation method was used to couple Lunx and SP-A antibody to the surface aqueous soluble CdTe quantum dots. In order to obtain the best coupling buffer system,we changed the concentration and PH value of borate buffer.The result show that when PH value was 7.5, concentration was 0.05M, emission spectrum of CdTe quantum dots was the highest. After coupling finished, using dislysis method to eliminate quantum dots that didn’t couple antibody. We detected fluorescence emission spectrum after dislysis.the result show that the biggest emission peak had slight red shift,and peak shape didn’t change.these results hinted us that quantum dots didn’t happen gather and its dispersibility is fine, which provide a powerful foundation for further applications.
5. Constructing a method on enriching and separating cancer cells in peripheral blood by immunomagnetic nanoparticles.
We cultured A549 cells line,SPC-A-1 cell line,HepG2 cell line,HCT-8 cell line and identified expression of pan-ck protein in four epithelial cell lines. Four epithelial cell lines had high expression of pan-ck protein. This hint us that pan-ck can be served as capture antibody to screen epithelial cells. We investigated enriching efficiency by change the proportion of cancer epithelial line and PBMC,then enriching cancer cell by immunomagnetic nanoparticles.The result show that enriching efficiency is 5×106 ,comparing with the work of Zigeuner that use immunomagnetic beads to enrich,the enriching efficiency raised 5 times.along with proportion of cancer cells elevating,enriching cells means was increasing. After enriching,we used HE stain to initially identify cancer cells.HE stain show us that nucleus body of cells enriched by immunomagnetic nanoparticles was enlarged and nucleus dye deep,while main part of PBMC was lymph cells,comparing with cancer cells, lymph cells body was small. We collected 6 case peripheral blood of pulmonary carcinoma patients,then used immunomagnetic nanoparticles enriching epithelial cancer cells.5 cases peripheral blood could enrich cancer cells,while 1 case couldn’t enrich to obtain cancer cells. The cause was owe to that the extent of enriching efficiency was limited. So we should improve enriching efficiency.
6. Identifing pulmonary carcinoma by quantum dots labelled Lunx and SP-A antibodies
After enriching,we needed identify cells character using further detect method. Firstly, detecting expression of Lunx and SP-A in four epithelial cancer cells by immunocytochemical method. Then we investigated accuracy of quantum dots labeling by QDs-Lunx and QDs-SP-A to label four epithelial cancer cells. The result show that positive cell rate of the two methods of Immunocytochemical stain and quantum dots single labeling method was coincident.Quantum dots double labeling method can elevate positive cell rate. After ascertaining accuracy and superiority,we identified 5 case cells sample of enriching from peripheral blood by QDs-Lunx and QDs-SP-A probe. The result show that 2 cases expressed Lunx and SP-A at the same time.1 case only expressed SP-A. 2 cases only express Lunx. These results suggest us that using Lunx and SP-A antibody can effectively identify lung tissue specificity and quantum dots double labeling method can detect multiprotein expression at one sample simultaneously and effectively.
引文
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