The Applications of High Sensitivity Flow Cytometry in the Detection of Bacteria

自然界中存在着许多微纳米尺寸的生物颗粒，比如细菌、病毒、分子组装体和生物大分子等。其中细菌具有体形微小、繁殖迅速、容易变异及环境适应能力强等特点，对人们的日常生活与生产具有诸多影响。一方面经过基因改造的工程菌在生物、医学、医药、工农业生产、环境治理等众多领域发挥着重要的应用。另一方面，各种致病菌的存在严重威胁着人类的生存和健康：不仅影响食品的质量和安全，而且带有耐药性的致病菌往往容易导致疾病治疗的失败。然而，传统的细菌检测方法操作复杂、检测周期长，难以满足实际需求。因此，发展快速、灵敏、特异的细菌检测分析方法对食品安全、环境监测、疾病机理及诊断治疗和生物恐怖袭击的防范等方面具有重要意义。 流...There are a wide variety of biological particles which fall into the nanometer to sub-micron scale such as bacteria, viruses, molecular assemblies and biomacromolecules. Having the characteristics like small size, propagation rapidly, variation easily and strong adaptability of environmental, bacteria could affect the people's daily lives and production. The engineered bacteria play an important ...学位：理学博士院系专业：化学化工学院_化学生物学学号：2052012015347

柯里拉京对宫颈癌Hela细胞增殖与凋亡的影响

为了探讨柯里拉京对宫颈癌Hela细胞增殖与凋亡的影响,不同浓度柯里拉京作用Hela细胞后,利用MTT法和克隆形成实验检测柯里拉京对Hela细胞的增殖抑制作用,用倒置显微镜观察Hela细胞的形态变化,用流式细胞仪分析细胞周期与细胞死亡率,用Western blot法检测细胞PCNA、Caspase-3、Caspase-8、Caspase-9、PARP蛋白的表达。结果:柯里拉京能显著抑制宫颈癌Hela细胞增殖,并呈浓度依赖效应;流式细胞仪测定结果表明柯里拉京能使Hela细胞发生S期周期阻滞;Western blot结果显示加药组细胞中与增殖有关蛋白PCNA的表达量显著下调,与凋亡有关蛋白Caspase-3、Caspase-8、Caspase-9、PARP被剪切激活。结论:柯里拉京对宫颈癌Hela细胞有明显的抑制作用,其抑制作用与柯里拉京诱导细胞周期阻滞及凋亡有关。福建省高校产学合作重大项目(2016Y4011

Flow Cytometric Sorting Based Molecular Ecological Study of Prochlorococcus in the South China Sea

海洋原绿球藻(Prochlorococcus)是贫营养大洋的优势自养微型生物，可划分为光生理以及16SrRNA基因系统发育显著差异的两个基本生态型，即高光生态型(Highlightecotype，HL)和低光生态型(Lowlightecotype，LL)。在高光生态型内部有两个不同的亚类，低光生态型有四个亚类，但近年来研究发现还存在着未知的亚类，所以寻找未知亚类是改善原绿球藻定量研究以及加深了解其环境适应机制的必要途径。 流式细胞仪是研究超微型生物的重要工具，本论文与传统的膜过滤采样方法不同，利用流式细胞仪的分选功能获得原绿球藻浓缩样品，并采用简洁的冻融法提取DNA，通过克隆文库以及变性梯度...Prochlorococcus is the most abundant photosynthetic organism in the oligotrophic ocean. It can be divided into two basical ecotypes with distinct photophysiology and phylogenetically distinct 16S rRNA markers, the high light (HL) and low light (LL) ecotypes. Two ecologically distinct subclusters have been identified within the HL ecotype and at least four distinct genotypes within the LL. Recently...学位：理学硕士院系专业：海洋与环境学院_环境科学学号：2005130222

The Systemic Evaluation and Clinical Significance of Immunological Function for Advanced Lung Cancer Patients

Background and objective The actual evaluation of immunological function is significant for studing the tumor development and devising a treatment in time. The aim of this study is to evaluate the immunological function of advanced lung cancer patients systematically, and to discuss the clinical significance. Methods The nucleated cell amounts of advanced lung cancer patients and the healthy individuals were counted. The immune cell subsets and the levels of IL-4, INF-γ, perforin and granzyme in CD8+T cells by the flow cytometry were measured. The proliferation activity and the inhibition ratio of immune cells to several tumor cell lines were evaluated by MTT assay. Results The absolute amounts and subsets of T, B, NK cells of advanced lung cancer patients were lower than the healthy individuals (P < 0.05); However, the proportion of regulatory T cells of advanced lung cancer patients (4.00±1.84)% was lower than the healthy individuals (1.27±0.78)% (P < 0.05). The positive rates of IFN-γ perforin, granzyme in CD8+T cells decreased while them in IL-4 did not in the advanced lung cancer patients compared to the healthy control group (P < 0.05). The proliferation activity of immune cells, the positive rate of PPD masculine and the inhibition ratio to tumor cells in the advanced lung cancer patients was lower than the healthy subsets obviously (P < 0.05). Conclusion There was a significant immune depression in the advanced lung cancer patients compared to the healthy individuals

Curcumin Promoted the Apoptosis of Cisplain-resistant Human Lung Carcinoma Cells A549/DDP through Down-regulating miR-186*

Background and objective Curcumin, a natural compound, is derived from the rthizom of Curcuma longa. In vitro and in vivo preclinical studies have shown its anti-inflammatory, antioxidant, anticancer activities and so on. miR-186*, which was found by microarray technology, was highly expressed in lung carcinoma cells A549/DDP. The aim of this study is to illustrate whether Curcumin could promote the apoptosis of A549/DDP cells through regulating the expression of miR-186*. Methods An oligonucleotide microarray chip was used to profile microRNA (miRNA) expressions in A549/DDP cells treated with and without Curcumin. The significantly differentially expressed miRNA, which was selected from microarray chip, validated by quantitative real-time PCR. Ultimately, the remarkably expressed miRNA modulated the apoptosis assaying by flow cytometry expriments and the survival rate was measured by MTT method. Results The microarray chip results demonstrated: Curcumin altered the expression level of miRNAs compared with untreated control in A549/DDP cell line, miR-186* was significantly down-regulated after Curcumin treatment, which confirmed by quantitative real-time PCR. Downregulation of miR-186* expression by curcumin elevated the apoptosis, and the survival rate of A549/DDP cells decreased; but up-regulation of miR-186* expression by transfection its mimics restrained the apoptosis, the survival rate of A549/DDP cells increased, which were assayed by flow cytometry expriments and MTT method. Conclusion Modulation of miRNAs expression may be an important mechanism underlying the biological roles of Curcumin

Establishment of MDCK cell lines which stably express visualable human neonatal Fc receptor

[目的]建立稳定表达融合EGFP的人新生儿Fc受体（h FcRn）的MDCK细胞株。[方法]构建重组慢病毒质粒p EGFP-h FcRn,采用四质粒包装系统共转染HEK 293T细胞生产重组慢病毒,感染MDCK细胞后对EGFP阳性细胞进行流式单细胞分选;通过Western Blot及EGFP-β2m荧光共定位验证h FcRn的完整性,并用流式细胞仪检测h FcRn与人Ig G的结合活性。[结果]测序结果表明成功构建p EGFP-FcRn慢病毒表达载体;感染后EGFP阳性MDCK细胞比例约为26.5%,流式单细胞分选后得到纯阳性细胞;荧光共定位及Western Blot均检测到h FcRn的完整表达;流式分析表明细胞株上的h FcRn与Ig G存在p H依赖性结合。[结论]成功获得稳定表达具有生物活性的可视化h FcRn的MDCK细胞株。[ Objective] To establish MDCK cell line stably expressing EGFP- human neonatal Fc receptor（hFcRn） fusion protein. [ Methods ] The lentiviral expression vector for EGFP - hFcRn fusion protein was constructed. Generating by co - transfection of four -plasmids into HEK 293T cells ,the lentivirus particles were used to infect MDCK cell line. EGFP positive single cell was obtained by FACS, and then FcRn expression was identified by fluorescence co -location with EGFP - β2m and confirmed by Western Blot. Flow cytometry was used to detect binding activity of hFcRn and human IgG. [ Results ] DNA se- quencing demonstrated that the lentivirus vector pEGFP - FcRn was constructed successfully. The percentage of EGFP - posi- tive ceils was about 26.5% after infection. Expression of the complete protein was detected through fluorescence co - location and Western Blot, respectively. Flow cytometry analysis showed that the cell lines could pH - dependently capture human IgG. [ Conclusion] MDCK cell line stably expressing functional visualable hFcRn was established.基金项目：国家自然科学基金项目（“结构生物学指导的HBV治疗性抗体人源化及其关键技术研究”,No.31600748;“抗呼吸道合胞病毒高中和活性抗体的保护机制研究”,No.81401668;“基于广谱中和单抗的通用型流感疫苗设计及其结构基础研究”,No.31670934

基于遗传算法的多组分光谱解析方法研究

光谱解析方法是一种常见的光谱分析方法,广泛用于各种化学计量学领域。现有的解析方法无法处理纯组分未知的应用体系,本文针对该情况,提出了基于遗传算法和最小二乘法的多元组分光谱解析定量分析方法。该方法首先通过遗传算法在混合光谱上寻找未知组分的最优峰位置和最优峰形,得到一组的最优纯组分光谱矩阵,再利用最小二乘拟合曲线,能够快速有效地解析混合光谱。在实验中,对纯组分光谱全未知、纯组分光谱部分未知及不同参数设置下算法的表现进行了讨论,分析其对算法收敛速度及计算结果精确性和稳定性的影响。利用该方法对流式细胞仪光谱数据进行处理,解析效果良好,谱线的契合程度高,验证其用于多组分流式细胞仪光谱数据分析的可行性、有效性和精确性。国家自然科学基金(NO.21503171);;国家重大科研仪器研制项目(NO.21627811

Research on the rapid inactivation of typical algae blooms by hydroxyl radical

以典型水华藻铜绿微囊藻、针杆藻和四尾栅藻为研究对象,利用大气压强电离放电高效生成的羟基自由基(·OH)对3种藻进行杀灭.采用荧光染色、流式细胞仪; 和光合活性等生物学方法,确定·OH杀灭的阈值浓度和时间,并观察细胞形态变化.结果表明,当混合藻中铜绿微囊藻、针杆藻和四尾栅藻的初始藻密度分别为1; 9.5*10~4、21.8*10~4和4.90*10~4cells/mL时,; ·OH杀灭的阈值浓度为1.07mg/L,致死时间为4.5s;形态观察结果表明,处理后各种藻的形态是完整的,无内溶质溢出.因此,采用·OH可实现高; 效快速杀灭水华藻,有效保障饮用水安全.Algae blooming in water sources breaks out increasingly and seriously; threatened the water supply safety. Bench scale tests were conducted to; study the effects of ·OH generated from strong ionization discharge and; high pressure water jet cavitation on cell density, cell integrity and; photosynthetic capacity of 3kinds of typical freshwater algae. Algae; species including Microcystis aeruginosa, Synedra sp., and Scenedesmus; quadricuauda were respectively prepared at concentrations of 19.5*10~4,; 21.8*10~4 and 4.90*10~4 cells/mL, and the cell integrity was assessed by; flow cytometry. Results suggested that the ·OH lethal threshold of the; algae was 1.07mg/L within the exposure time of 4.5s. The cell; morphological observation results showed that all the cells were; integral and no cytoplasm composition spilled. Hence, large-scale; production of ·OH is a novel method to inactive typical algae species; efficiently and to protect drinking water safety simultaneously.国家科技支撑计划资助项目; 国家重大科研仪器研制项

荧光原位杂交技术和流式细胞仪用于环境样品中产毒及非产毒微囊藻的定量监测

全球范围内,高频次、大范围暴发的蓝藻水华对淡水水体环境造成严重影响.微囊藻因其在生长特别是衰亡过程中向水体释放微囊藻毒素而威胁人类健康.因此,分析其产毒株及非产毒株在环境样品中的组成,建立产毒蓝藻的预报及评价体系显得极为重要.本文采用荧光原位杂交技术结合流式细胞技术实现对环境样品中产毒藻株的鉴别与定量.针对目标基因mcyA设计的、以地高辛标记的双链DNA探针可有效应用于产毒微囊藻FACHB905和PCC7806的鉴别.分别对来自滇池、太湖和关桥的11个样品进行分析显示,该方法与传统的形态学鉴定及PCR方

抗人DR5单克隆抗体的制备、鉴定及活性分析

目的制备抗人DR5单克隆抗体(mAb),鉴定其特性,并进行生物学活性分析。方法以纯化的可溶性DR5(sDR5)免疫Balb/c小鼠,杂交瘤技术制备抗人DR5mAb;运用ELISA、SDS-PAGE电泳方法测定抗DR5mAb与sDR5结合的特性;Ig亚类ELISA试剂盒鉴定抗DR5mAb亚类;间接ELISA法检测腹水mAb效价;流式细胞仪检测肿瘤细胞表面DR5的表达水平;流式细胞仪检测抗DR5单克隆抗体(mAb)诱导肿瘤细胞凋亡的功能。结果获得1株可分泌抗DR5mAb的杂交瘤细胞系R150。SDS-PAGE电泳检测证实,获得的R150可特异性地识别DR5;R150的Ig亚类为IgGI(λ型);腹水效价为1×106;通过流式细胞仪可敏感地检测到肿瘤细胞表面DR5的表达水平及R150诱导肿瘤细胞凋亡情况。结论获得1株可分泌抗DR5mAb的细胞系R150,抗体具有效价高、特异性强等特点并能有效诱导肿瘤细胞凋亡,具有较好的应用价值