Glioma is the most malignant brain cancer. The neurons, macrophages, T cells and other immune cells constitute the glioma immunosuppressive microenvironment. The accurate spatial distribution of these cells in the glioma microenvironment and its relationship with glioma metastasis is unknown. We constructed a mouse glioma cell line stably expressing the large Stokes-shifted yellow fluorescent protein and applied it to the multicolor immunofluorescence imaging. The imaging data revealed that the neurons were sparsely distributed in the glioma core and the number of neurons decreased by 90% compared with normal brain site. The spatial distribution of monocyte-macrophages and microglia is heterogeneous. The monocyte-macrophages and T cells were heavily recruited into the glioma core and metastasis. There was no significant difference in the distribution of microglia among glioma core, margin, and normal brain site. Our results provided new perspectives for targeting immune regulation cells and developing new immunotherapy strategies for glioma.Glioma is the most malignant brain cancer. The neurons, macrophages, T cells and other immune cells constitute the glioma immunosuppressive microenvironment. The accurate spatial distribution of these cells in the glioma microenvironment and its relationship with glioma metastasis is unknown. We constructed a mouse glioma cell line stably expressing the large Stokes-shifted yellow fluorescent protein and applied it to the multicolor immunofluorescence imaging. The imaging data revealed that the neurons were sparsely distributed in the glioma core and the number of neurons decreased by 90% compared with normal brain site. The spatial distribution of monocyte-macrophages and microglia is heterogeneous. The monocyte-macrophages and T cells were heavily recruited into the glioma core and metastasis. There was no significant difference in the distribution of microglia among glioma core, margin, and normal brain site. Our results provided new perspectives for targeting immune regulation cells and developing new immunotherapy strategies for glioma.

本文对荧光自相关光谱(FACS)技术作为免疫分析技术的具体实现和潜在应用进行探究。实验分别以Alexa Fluor647荧光分子和绿色荧光蛋白抗原与抗体相互作用为模型,利用一款桌面式荧光相关光谱仪分别采集荧光分子抗原与不同浓度抗体混合液的荧光自相关数据。将传统FACS技术与最大熵值法相结合,我们可以对抗原-抗体结合程度进行定量和定性评估。使用自主开发的数据分析软件对采集的荧光自相关数据进行分析以获取抗原-抗体解离常数。实验结果表明,FACS技术应用于抗原-抗体亲和力检测,具有简便、快速、灵敏的特点;检测限在皮摩尔和纳摩尔之间;模块化的数据分析软件可标准化操作流程、提高数据分析准确性和可重复性。

PAiRFP1是一种以根癌农杆菌中细菌光敏色素Agp2为基础, 经过蛋白工程手段改造后获得的近红外荧光蛋白。 与其他近红外荧光蛋白不同, PAiRFP1具有光活化行为。 这一特性可以有效改善近红外荧光蛋白在体内的成像信噪比。 然而, 关于光活化行为的影响因素却研究甚少。 主要采用荧光光谱学手段研究了PAiRFP1蛋白浓度、 pH、 金属离子、 氧化还原环境对于PAiRFP1光活化行为的影响, 结果发现: (1) PAiRFP1的最大光活化效率与该近红外荧光蛋白的浓度不呈线性关系; (2) 随着pH从6.5升高至7.8和9.0, PAiRFP1的最大光活化效率从36.8%提高至52.0%和60.8%; (3) 金属离子K+, Na+, Ca2+的加入对于PAiRFP1的最大光活化效率没有显著影响; 类似现象在H2O2和DTT的处理的情况下也被观察到。 除了上述影响因素外, 还发现当PAiRFP1中276位的缬氨酸被突变为甘氨酸产生V276G突变体后, 蛋白的最大光活化效率从~50.0%下降至19.4%。 DTT处理后导致V276G突变体最大光活化效率从19.4%提高到了27.1%。 此外, 比较研究了各种影响因素对于光活化速率的影响。 以上结果为今后更好地将此类光激活的近红外蛋白应用于活体成像中提供了更好地理论指导和优化解决方案。

近几年, 可逆光激活荧光蛋白的研制越来越受到人们的重视, 这类荧光蛋白极大地促进了活细胞超高分辨显微成像技术的发展及应用。可逆光激活荧光蛋白可被不同波长的光多次可逆地进行调制, 因而被广泛地应用于高密度数据的光存储、光致变色荧光共振能量转移的测量以及基于可逆饱和线性荧光跃迁原理的超高分辨率显微成像中。从研制这类荧光蛋白所涉及的关键氨基酸位点出发, 本文综述了近几年可逆光激活绿色荧光蛋白的研制进展, 并简要地讨论荧光蛋白结构与光学特性的关系, 从而为后续结构导向的可逆光激活荧光蛋白的研制提供参考。

The development of experimental animal models for head and neck tumors generally rely on the bioluminescence imaging to achieve the dynamic monitoring of the tumor growth and metastasis due to the complicated anatomical structures. Since the bioluminescence imaging is largely affected by the intracellular luciferase expression level and external D-luciferin concentrations, its imaging accuracy requires further confirmation. Here, a new triple fusion reporter gene, which consists of a herpes simplex virus type 1 thymidine kinase (TK) gene for radioactive imaging, a far-red fluorescent protein (mLumin) gene for fluorescent imaging, and a firefly luciferase gene for bioluminescence imaging, was introduced for in vivo observation of the head and neck tumors through multi-modality imaging. Results show that fluorescence and bioluminescence signals from mLumin and luciferase, respectively, were clearly observed in tumor cells, and TK could activate suicide pathway of the cells in the presence of nucleotide analog-ganciclovir (GCV), demonstrating the effectiveness of individual functions of each gene. Moreover, subcutaneous and metastasis animal models for head and neck tumors using the fusion reporter gene-expressing cell lines were established, allowing multi-modality imaging in vivo. Together, the established tumor models of head and neck cancer based on the newly developed triple fusion reporter gene are ideal for monitoring tumor growth, assessing the drug therapeutic efficacy and verifying the effectiveness of new treatments.

Background and aims: The spectral properties of enhanced green fluorescent protein (EGFP) used in current visualizable animal models for nasopharyngeal carcinoma (NPC) result in a limited imaging depth. Far-red fluorescent proteins have optimal spectral wavelengths that allow deep tissue penetration, thus are well-suited for the imaging of tumor growth and metastases in live animals. This study aims to establish an imageable animal model of NPC using far-red fluorescent proteins. Methods: Eukaryotic expression vectors of far-red fluorescent proteins, mLumin and Katushka S158A, were separately transfected into 5-8F NPC cells, and cell lines stably expressing the far-red fluorescent proteins were obtained. These cells were intraperitoneally or intravenously injected into mice, and their tumorigenic and metastatic potential were examined through fluorescence imaging. Finally, factors affecting their tumorigenic ability were further assessed through testing side population (SP) cells proportion by flow cytometry. Results: NPC cell line with high tumorigenicity and metastasis (5-8F-mL2) was screened out, which stably expressed far-red fluorescent protein. Intraperitoneal and intravenous injection of 5- 8F-mL2 cells resulted in an abdomen metastasis model and a lung metastasis model. In addition, NPC cell line without tumorigenicity (5-8F-Katushka S158A) was screened out. The percentage of SP cells between 5-8F-mL2 and 5-8F-Katushka S158A was found different, suggesting that the SP cell proportion may play a key role in the determination of cell tumorigenic ability. Conclusion: We successfully established animal models for NPC with high tumorigenicity and metastasis using a super-bright far-red fluorescent protein. Owing to the super-brightness and excellent wavelength parameters, these models may be applied as useful tools for intuitive and efficient monitoring of tumor growth and metastasis, as well as assessing the efficacy of nasopharyngeal cancer drugs.

绿色荧光蛋白(GFP)荧光图像提供蛋白质功能定位信息，相衬图像提供高分辨率的结构信息，二者的融合对于细胞蛋白质的功能分析和亚细胞的精细结构定位具有重要价值。基于轮廓波变换对细节信息优异的表达能力，提出一种HIS空间下基于轮廓波变换的多尺度混合分层图像融合算法，该算法利用轮廓波变换分别对GFP荧光图像和相衬图像的亮度分量进行分解，对不同子带系数采用不同的融合策略以力求融合图像在保留GFP荧光图像定位信息的同时融入相衬图像的高频细节，并引入视觉保真度(VIF)作为图像融合的评价指标。对来自约翰英纳中心的117组拟南芥细胞图像进行的融合实验表明，该算法能够有效保留源图像中的细节信息，提高了融合图像的可视性，相比传统算法更为优越。

为探究绿色荧光蛋白(GFP)对结肠直肠癌细胞遗传物质稳定性是否存在影响, 选取3种常用的结肠直肠癌细胞系HCT116, SW480, DLD-1; 采用脂质体转染法把GFP转入细胞, 统计几种细胞系微核、核芽的比例; 分别比较各种细胞系自发微核、核芽和GFP组的差异。结果发现, HCT116细胞中对照组与GFP组的微核细胞率分别为3.24 %、2.76 %, 核芽细胞率为0.24 %、0.26 %; SW480细胞中对照组与GFP组的微核细胞率分别为3.49 %、3.58 %, 核芽细胞率为1.48 %、1.33 %; DLD-1细胞中对照组与GFP组的微核细胞率分别为1.12 %、1.28 %, 核芽细胞率为0.50 %、0.56 %。三种细胞系中自发微核、核芽数目和GFP组经2×2卡方检验, P值均大于0.05, 它们之间没有显著性差异。实验结果表明, 转染GFP不影响三种细胞系的遗传物质稳定性, GFP可以作为一种安全可靠的标记物用于哺乳动物细胞核的标记。

以绿色荧光蛋白(green fluorescent protein, GFP)为报告基因, 将含TMV表达载体的质粒p35S-30B: GFP转化农杆菌EHA 105, 通过渗透法把经MMA诱导后的农杆菌悬浮液注射到本氏烟叶片内, 测定了鸦胆子素D (Bruceine D) 对烟草植株内TMV的增殖和运动的抑制作用; 通过PEG介导法把p35S-30B: GFP转化到本氏烟叶肉细胞原生质体内, 测定了Bruceine D对烟草原生质体中TMV增殖的抑制效果。结果表明, 在10 μg/mL浓度下, Bruceine D不仅可抑制烟草叶肉细胞原生质体中TMV的增殖, 还可以抑制烟草接种叶中TMV向茎部及植株上部叶片移动, 且对寄主植物不造成明显的毒害。

提取北京油鸡8种组织的总RNA, 利用RT-PCR检测purH基因mRNA的差异表达。将purH基因完整开放阅读框定向插入pEGFP-N3, 构建带有GFP报告基因重组表达载体pEGFP-purH, 利用脂质体介导法将其导入北京油鸡体外培养细胞中, 进行G418药物筛选和克隆化培养。结果表明: 北京油鸡purH基因(GenBank 登录号: EU334506) 编码区为1 782 bp, 编码593 aa的多肽, 转染后24 h、48 h和72 h, pEGFP-purH转染率在10.3 %～53.2 %之间, 绿色荧光均匀分布于细胞质与细胞核中, 随表达量的增加, 绿色荧光在细胞核中聚集成团块状或颗粒状。经药物筛选和克隆化培养, 获得表达pEGFP-purH融合蛋白的克隆细胞株, RT-PCR与Western blotting检测确认pEGFP-purH已整合到北京油鸡成纤维细胞基因组中, 在优化的条件下, 阳性细胞凋亡率、生长与增殖状况与对照组比较差异不显著(P>0.05)。