入核型IL-1α propiece分子调控机制研究

发布时间：2018-05-04 18:21

本文选题：分子克隆 + 慢病毒　；参考：《苏州大学》2016年博士论文

【摘要】：第一部分IL-1α表达系统的构建目的:IL-1α基因的N端含有核定位序列,能够进入细胞核,但入核型IL-1α具体功能机制并不清楚。研究表明白血病细胞中IL-1α水平异常升高。为了探寻入核型IL-1αpropiece在白血病细胞中的生物学功能以及可能的调控机制,为IL-1α的入核功能的研究提供实验基础。方法:利用PCR方法克隆IL-1αpropiece基因,经过测序验证之后连接到慢病毒载体。其中包含带有FLAG标签与strep标签的IL-1αpropiece,用于对propiece进行定位与纯化。非标签IL-1αpropiece用于减少标签蛋白对蛋白功能最少程的影响。度包装慢病毒颗粒对jurkat细胞进行感染,利用流式细胞仪技术分选出高表达IL-1αpropiece的细胞。分离细胞浆蛋白与细胞核蛋白,使用免疫印迹的方法对不同组分中标签蛋白进行检测,从而对IL-1αpropiece的表达空间进行质控。免疫荧光也被用来对IL-1αpropiece的核定位进行质控。细胞核被分离出来,经过免疫荧光染色使用流式细胞仪进行检测。对整个细胞进行免疫荧光染色后,共聚焦检测标签蛋白在细胞中的定位。结果:入核型IL-1α克隆至慢病毒表达载体,成功包装出病毒颗粒,流式分选感染慢病毒后高表达GFP的细胞。细胞胞浆蛋白与核蛋白中对标签蛋白的免疫印迹表明IL-1αpropiece存在于细胞核组份中。共聚焦检测发现IL-1αpropiece与DAPI的信号出现共定位。而在对染色后的细胞核流式染色中,发现IL-1αpropiece组的平均荧光强度高于对照组。结论:成功制备高表达IL-1αpropiece的jurkat细胞。免疫印迹实验、共聚焦显微镜及细胞核的流式染色均表明propiece定位表达于细胞核中。IL-1αpropiece真核过表达平台构建成功。第二部分IL-1α在急性淋巴细胞白血病细胞系中的生物学功能目的:研究在急性淋巴细胞白血病细胞系过表达IL-1αpropiece对细胞增殖、细胞周期、细胞凋亡的影响。研究低血清生存压力胁迫下及抗肿瘤药物顺铂毒性下,过表达IL-1αpropiece在急性淋巴白血病细胞中的生物学作用。方法:在过表达IL-1αpropiece的急性淋巴白血病细胞系中MTT法检测24、48,72小时细胞增殖情况,并检测低血清生存胁迫与顺铂药物毒性下的细胞增殖情况。Annexin V与7AAD双染检测IL-1αpropiece过表达对细胞凋亡的影响。PI染色检测低血清生存压力胁迫与顺铂药物毒性下过表达IL-1αpropiece对细胞周期的影响。结果:IL-1αpropiece过表达能够促进jurkat细胞与P388细胞增殖,增加G2/M期细胞百分比,并在顺铂毒性及低血清胁迫实验中降低细胞凋亡水平。结论:IL-1αpropiece过表达能够促进急性淋巴白血病细胞增殖,抑制细胞凋亡以及增加G2/M期细胞。第三部分IL-1αpropiece过表达生物芯片检测与数据挖掘目的:探寻IL-1αpropiece在急性淋巴白血病细胞中的作用机制,生物芯片检测过表达IL-1αpropiece引起的差异表达基因,并对信号通路、信号网络、分子功能的影响进行数据挖掘。方法:在过表达IL-1αpropiece急性淋巴细胞白血病细胞中使用Agilent Human4x44K gene expression array对基因表达谱的变化进行检测。聚类分析差异表达基因。选取Gene Go与GO(Gene Ontology)数据库找出与差异基因相关的通路与生物学过程。通过I-TASSER对propiece的空间结构进行预测,结合DBD-Hunter数据库,Bind N数据库,DISPLAR数据库与i DBPs server数据库的结果对propiece与DNA的结合能力进行分析。利用PRIMA算法富集了差异表达基因的共同启动子。结果:芯片信息通过聚类分析可以发现MT家族基因与NF-κB靶基因均上调。基于Gene Go数据库的分析中,通过对比110个已经注释过的信号动态网络,推测propiece可能通过从血管新生,炎症,抗凋亡,神经新生等方面对疾病进行调控。I-TASSER数据库分析出的空间结构也具有与DNA结合的可能性,并且PRIMA算法富集得到Sp1启动子为下调基因的共同启动子区域。结论:IL-1αpropiece能够对动态神经网络的进行调节,并且预测具有核酸结合的结合区域。可能通过调控Sp1启动子区域达到生物学功能。第四部分IL-1αpropiece对信号通路的调控及其核酸结合片段的筛选目的:对本课题前期通过生物信息取得的预测结果进行验证。构建Sp1 mini promoter文库寻找能与IL-1αpropiece发生相互作用的DNA序列信息。方法:使用免疫印迹对信号通路中发生改变的关键节点基因检测,对利用差异表达基因数据挖掘产生的信号网络进行验证。获得重组IL-1αpropiece蛋白,与Sp1 promoter deletion assay混合,经过SELEX方法筛选,经过四轮进化筛选。最终亲和力高的文库片段被连入T载体。测序后根据结果对每个区域计数统计,获得IL-1αpropiece的高亲和力区域。并且分析其结合DNA motifs。使用丰度更高的商品化文库与IL-1αpropiece进行SELEX实验,提高IL-1αpropiece结合DNA序列的分辨率。结果:IL-1αpropiece能激活NF-κB,AKT与p38 MAPK通路,降低外源性凋亡,上调抗凋亡基因。IL-1αpropiece能与Sp1启动子部分DNA发生相互作用。结论:IL-1αpropiece通过调控NF-κB通路,AKT通路与p38 MAPK通路协同作用发挥细胞促进增殖,抑制凋亡的功能。SELEX的方法筛选与IL-1αpropiece结合的DNA序列,DNA序列具有CG含量高的特点。
[Abstract]:The first part of the construction of the IL-1 alpha expression system: the N end of the IL-1 alpha gene contains a nuclear location sequence, which can enter the nucleus, but the specific functional mechanism of the nuclear type IL-1 alpha is not clear. The study shows that the level of IL-1 alpha in the leukemia cells is abnormal. In order to explore the biological function and possibility of the nucleate IL-1 alpha propiece in leukemia cells. The regulatory mechanism provides an experimental basis for the study of IL-1 alpha nucleation function. Methods: the IL-1 alpha propiece gene was cloned by PCR method. After sequencing, it was connected to the lentivirus vector, including the IL-1 alpha propiece with the FLAG tag and strep label, which was used to locate and purify propiece. The non label IL-1 alpha propiece was used to reduce it. The effect of the less labelling protein on the function of the protein function at least. The Jurkat cells were infected by the lentivirus, and the cells with high expression of IL-1 alpha propiece were separated by flow cytometry. The cytoplasmic protein and nuclear protein were separated and the labelling proteins in different components were detected by immunoblotting, thus the IL-1 alpha prop was detected. The expression space of iece is controlled. Immunofluorescence is also used to control the nuclear location of IL-1 alpha propiece. The nucleus is separated and detected by immunofluorescence staining with flow cytometry. After immunofluorescence, the location of the confocal labeling protein in the cell is detected. Results: nucleate IL-1 alpha grams The virus particles were successfully packaged, and the cells with high expression of GFP after the infection of the lentivirus were successfully packed. The immunoblotting of cytoplasmic protein and the nuclear protein showed that the IL-1 alpha propiece existed in the nuclear components. Confocal detection found that the signals of IL-1 alpha propiece and DAPI were Co located. In the post color nuclear flow staining, the average fluorescence intensity of IL-1 alpha propiece group was higher than that of the control group. Conclusion: the Jurkat cells with high expression of IL-1 alpha propiece were successfully prepared. The immunoblot test, confocal microscopy and the flow staining of the nucleus showed that the propiece localization was expressed in the eukaryotic expression platform of.IL-1 alpha propiece in the nucleus. Second part of the biological function of IL-1 alpha in acute lymphoblastic leukemia cell lines: To study the effect of IL-1 alpha propiece on cell proliferation, cell cycle and apoptosis in acute lymphoblastic leukemia cell lines. Study the expression of IL-1 alpha under the low serum pressure stress and the toxicity of antitumor drug cisplatin. Biological effects of propiece in acute lymphoblastic leukemia cells. Methods: in the acute lymphoblastic leukemia cell lines that overexpressed IL-1 alpha propiece, MTT assay was used to detect the proliferation of 24,48,72 hours cells, and the cell proliferation under low serum stress and cisplatin toxicity was detected by.Annexin V and 7AAD double staining for the detection of IL-1 alpha propiece The effect of.PI staining on the survival pressure of low serum and the overexpression of IL-1 alpha propiece on the cell cycle of cisplatin. Results: the overexpression of IL-1 alpha propiece can promote the proliferation of Jurkat cells and P388 cells, increase the percentage of G2/M phase cells, and reduce the detail in the toxicity of cisplatin and the low serum stress test. Conclusion: IL-1 alpha propiece overexpression can promote the proliferation of acute lymphoblastic leukemia cells, inhibit cell apoptosis and increase G2/M phase cells. Third part IL-1 alpha propiece overexpression biochip detection and data mining aim to explore the mechanism of IL-1 alpha propiece in acute lymphoblastic leukemia cells, biochip detection Overexpression of differentially expressed genes caused by IL-1 alpha propiece and data mining on the effects of signal pathways, signal networks, and molecular functions. Methods: detection of the changes in gene expression profiles using Agilent Human4x44K gene expression array in IL-1 alpha propiece acute lymphoblastic leukemia cells. Cluster analysis differential table Gene Go and GO (Gene Ontology) database were selected to identify the pathways and biological processes related to differential genes. The spatial structure of propiece was predicted by I-TASSER, and the combining ability of DBD-Hunter database, Bind N database, DISPLAR database and I database were analyzed. PRIMA algorithm is used to enrich the common promoter of differentially expressed genes. Results: the MT family gene and the target gene of NF- kappa B can be found by cluster analysis. Based on the analysis of the Gene Go database, by comparing 110 signal dynamic networks that have already been annotated, the propiece may be measured from the angiogenesis, inflammation, and resistance to withering. The spatial structure of the.I-TASSER database analyzed by the.I-TASSER database also has the possibility of combining with DNA, and the PRIMA algorithm enriching the Sp1 promoter as the co promoter region of the down regulated gene. Conclusion: IL-1 alpha propiece can regulate the dynamic neural network and predict the binding of nucleic acid. The combination area. Maybe by regulating the Sp1 promoter region to achieve biological function. Fourth part IL-1 alpha propiece regulation of the signal pathway and the screening of nucleic acid binding fragments: to verify the prediction results obtained by biological information in the early stage of this project. The construction of Sp1 Mini promoter library can be found to occur with IL-1 alpha propiece. DNA sequence information of interaction. Method: using immunoblotting to detect the key node genes that change in the signal pathway, verify the signal network produced by the differential expression gene data mining. The recombinant IL-1 alpha propiece protein is obtained and mixed with Sp1 promoter deletion assay. Through SELEX method, four rounds of evolution sieve are screened by SELEX method. Selection. The final affinity high library fragments were connected to the T carrier. After sequencing, the high affinity regions of IL-1 alpha propiece were obtained according to the results. And the higher abundance commercial library of DNA motifs. and IL-1 alpha propiece were used to carry out SELEX experiments, and the resolution of IL-1 alpha propiece combined with DNA sequence was raised. Fruit: IL-1 alpha propiece activates NF- kappa B, AKT and p38 MAPK pathway, reduces exogenous apoptosis and up-regulates the DNA interaction of the anti apoptotic gene.IL-1 alpha propiece and Sp1 promoter part. Methods DNA sequences were screened with IL-1 alpha propiece, and the DNA sequence was characterized by high CG content.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392

【相似文献】