【作者】 胡晓鹏；

【导师】 吴亚群；

【作者基本信息】 华中科技大学 ， 普通外科学， 2013， 博士

【摘要】 目的探讨细胞培养中支原体污染的种类及清除方法。方法检测实验室不同培养者培养的各细胞系污染支原体情况。首先设计支原体种属特异性的简并引物，针对其16s-23s基因间隔区序列，用来检测支原体阳性片段条带。根据简并引物扩增的片段测序明确为何种支原体污染。然后针对污染支原体的种类和滴度，采用方差分析方法，经过统计检验，探索一种合理的配方药物用于细胞培养，能够短期杀灭细胞培养中污染的支原体。对于药物对支原体的杀灭作用采用定量PCR的方法检测，对于药物对细胞的副作用，采用流式细胞数测细胞周期的方法作为指标来评估。对于细胞内支原体的污染，采用免疫荧光后激光共聚焦显微镜观察的方法来明确细胞内变化。结果1）设计的支原体属及莱氏无胆甾原体属简并引物及特异性引物可以快速的检测出有无支原体污染，根据条带对比可以初步明确为何种支原体污染，进一步明确还可以通过测序的金标准来明确。通过该方法可以快速的检测且准确性可靠。2）经方差分析各种药物浓度组合，筛选出具有抗支原体作用显著的几种组合。药物选自三种类型，分别为：截短侧耳素类，第三代喹诺酮类及以上，四环素类。分别配成A,B,C液。所述试剂由A液、B液等体积混合或A液、B液、C液等体积混合而成，其中，A液由半合成截短侧耳素衍生物和0.01M PBS混合而成，混合液中半合成截短侧耳素衍生物的质量-体积百分比浓度为0.5%—1.5%；B液由喹诺酮类衍生物和0.01M PBS混合而成，混合液中喹诺酮类衍生物质量-体积百分比浓度为0.5%—1.5%；C液由四环素类衍生物和0.01M PBS混合而成，混合后四环素类衍生物的质量-体积百分比浓度为0.25%—0.75%；3）该种配方组合不仅可以杀灭细胞外支原体污染，还可以杀灭潜藏于宿主细胞内的支原体。使用之后细胞内部微生物感染引起的自噬反应消失。4）该种配方组合在正常使用浓度下对实验用肿瘤细胞系无明显毒副作用。使用治疗浓度10倍以上的浓度处理细胞未见明显细胞周期变化。结论该发明的目的是提供一种用于清除细胞培养中支原体污染的试剂及其使用方法，能够系统的检测出支原体的种类并将清除，可以有效的清除支原体污染，并不会对细胞的本身代谢带来影响。目的探讨支原体对宿主肿瘤细胞的影响，鉴别一种毒力因子SGLP（smallGTPase-like protein fragment），并探索其与宿主细胞内分子相互作用的机制。方法首先通过生物信息学的方法，分析支原体基因组序列，寻找一些具有保守性的共有序列。在候选的序列中，通过查阅相关文献，确定SGLP为可能的突破口，即可能和Rac1以及Stat3相互作用的支原体蛋白。然后将目的片段的编码序列分别克隆至原核和真核表达载体。通过GST融合蛋白和GFP融合蛋白在原核和真核实验系统中的表达来研究目的蛋白片段和宿主分子Rac1和Stat3的相互作用。通过激光共聚焦显微镜观察目的蛋白分子和宿主细胞Rac1和Stat3在细胞内的共定位。通过Western blot研究该支原体蛋白片段对Rac1和Stat3活性的影响。通过转染突变型的Rac1质粒，改变宿主细胞内Rac1的活性，研究支原体蛋白片段对Stat3的激活是否依赖于Rac1的活性。通过Transwell实验和BrdU掺入实验，研究该支原体蛋白片段对宿主肿瘤细胞体外的迁移和增殖能力的影响。用流式检测支原体蛋白片段对细胞内活性氧生成水平的影响，并研究其效应是否依赖于Rac1。用染色质免疫共沉淀证实Stat3的可能靶基因是否包含Rab7。结果本实验证实所克隆的支原体保守蛋白Chromosomepartition protein Smc的N端240个氨基酸残基具有类似小GTP结合蛋白的序列特征（SGLP）。结果显示，SGLP是分子量约27KDa的蛋白片段，可以和宿主细胞Rac1以及Stat3相互作用。SGLP可以上调Rac1的活性和Stat3的磷酸化水平，且促进Stat3磷酸化的作用依赖于Rac1的活性。本实验还发现，SGLP可以促进肿瘤细胞体外的迁移和增殖能力。本实验还探索了SGLP激活Rac1和Stat3的下游效应。为进一步研究打下基础。结论支原体保守蛋白片段SGLP可以通过和Rac1以及Stat3的相互作用促进Rac1和Stat3的激活，从而促进宿主肿瘤细胞的迁移和增殖，以及产生一系列其他的影响。更多还原

【Abstract】 Objective To evaluate the situation of mycoplasma contamination in cellcultures for laboratory research use, and to find a method to eliminate the mycoplasmacontamination in a short time. Method First, we designed the degenerate primer pairs foramplification of the16s-23s gene spacer region from mycoplasma genera. According to thelength of the amplified product, we preliminarily confirm which species the contaminant isresulted from. Then, based on the identified mycoplasma species and titers of the infection,we utilize ANOVA analysis to examine an appropriate formulation of three classes ofantimicrobials to eliminate the infection in a short time. The efficiency of theanti-mycoplasma effect was assessed by real-time PCR using a method based on SybrGreen. The side effects of the antimicrobials on host cell were indicated by alterations incell cycle measured by Flow cytometry. The alterations caused by intracellular mycoplasmainfection was visualized by fluorescence immunostaining followed by confocal microscopy.Results1) The designed degenerate primers for Mycoplasma and Acholeplasma canamplify these two genera of mycoplasma in2hours of amplification reaction. The speciesof the contaminants can be assessed preliminarily according to the length of the productfragment. The golden standard for identification can also be conducted after theamplification by sequencing.2) Based on the analysis of ANOVA, we figured out threetypes of antimicrobial dr gs that can be used to eliminate mycoplasma infection in cellscultures. The dr gs used were…, which are designated as A, B and C, respectively. Theformulation of the regime are: solution A was0.5%-1.5%m/v resolved with0.01M PBS;solution B was0.5%-1.5%m/v resolved with0.01M PBS; solution C was0.25%-0.75%m/v resolved with0.01M PBS.3) After administration of this formulation of antimicrobialdr gs, the intracellular mycoplasma as well as the extracellular mycoplasma can both be eliminated. The autophagy flux which are characterized by the LC3-II puncta recovered tonormal state after elimination of mycoplasma infection.4) This formulation did not causemuch prominent side effect to host cells. The cell cycle did not change significantlydetermined by Flow cytometry. Conclusion This innovation of a method for detection ofmycoplasma followed by specifically eliminating both extracellular and intracellularmycoplasma can be used in cell culture laboratory. Objective The author so ght to indentify a mycoplasmal virulence factor thatcan interact with host cellular molecules, by which mycoplasma may affect host tumor cellfunctions. And the author also endeavored to explore the molecular basis for thepathogenesis caused by mycoplasmal virulence factors. Method First, the author utilizedthe method based on bioinformatics to scrutinize mycoplasma genomes in searching forsome highly conserved sequences among mycoplasma species. Based on ample evidencesfrom papers about prokaryotic virulence factors, the author chose a small GTPase-likeprotein fragment (SGLP) of mycoplasma protein, chromosome partition protein Smc, as acandidate for research. The author cloned the SGLP sequence and inserted it into vectorsfor expression in E coli. and HeLa cells separately. The protein interactions between SGLPand Rac1/Stat3were assessed by GST pull-down assay and co-immunoprecipitation assay.The intracellular colocalization of SGLP with Rac1and Stat3was determined by confocalmicroscopy. Rac1activation was detected using Western blot analysis based on aGST-Pak1pull-down assay. Stat3phosphorylation was also detected using Western blotanalysis. The author transfected HeLa cells with dominant negative or constitutively activeRac1constructs to manipulate the cellular Rac1activity to investigate whetherSGLP-induced Stat3phosphorylation was dependent on Rac1activity. The effect of SGLPon HeLa cell migration was studied thro gh a Transwell assay, and the effect of SGLP onHeLa cell proliferation was also studied thro gh a BrdU incorporation assay determined byFlow cytometry. The ROS generation was tested by Flow cytometry after depletion of Rac1using siRNA. Chromatin immunoprecipitation assay was conducted to investigate whetherRab7was a Stat3target gene. Results1) SGLP can interacts with Rac1and Stat3;2)SGLP can upregulate Rac1activity and Stat3phosphorylation at705-tyrosine residues.3)SGLP-induced Stat3phosphorylation is dependent on Rac1activity.4) SGLP can promote tumor cell migration and has a pro-proliferative effect on tumor cells.4) studies aboutdownstream effects of SGLP imposed on host cells is preliminarily explored. ConclusionThe highly conserve mycoplasma protein fragment, SGLP, may induce activation of Rac1and Stat3thro gh interaction with Rac1and Stat3, which may be responsible for theobserve increase in tumor cell migration and proliferation. Studies about its ripples effectspave road for future studies in this field.更多还原