杜氏利什曼原虫两个发育阶段基因表达系列分析文库的构建及HSP90在其阶段转变过程中的作用
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摘要
利什曼原虫可在人体导致多种疾病,是对人体危害严重的人兽共患寄生虫病,其中最致命的是由杜氏利什曼原虫引起黑热病。杜氏利什曼原虫生活史有前鞭毛体及无鞭毛体两个时期。利什曼原虫由前鞭毛体向无鞭毛体的转变过程是一个复杂的包括形态学及生理学的改变。这一过程中发生的改变不仅导致前鞭毛体形态上的变化,还使其逃避了宿主巨噬细胞的杀伤存活下来。目前为止,各阶段疫苗都为利什曼原虫病的防治发挥着作用,但各阶段疫苗都存在缺陷。比如说还没找到一种或几种能激发对所有原虫产生保护免疫的抗原,疫苗的构建结构也不理想。但是,利什曼原虫在实验室很容易得到,并且一些新技术的应用都为解决这些问题提供了有效的途径和希望。研究这个过程中的基因表达谱的改变有助于我们找出与利什曼原虫阶段转变有关的一些重要基因。
以前也有一些学者用双向电泳和基因芯片的方法对利什曼原虫的两个发育阶段的表达谱进行了研究。双向电泳由于技术的原因,找到的差异蛋白数量比较少;基因芯片的优点在于可以高通量的检测基因表达情况,但由于探针序列一般来自于已知基因的cDNA或EST库,只能检测已知基因,不能发现未知基因。因此,本研究以体外培养的前鞭毛体及无鞭毛体为研究对象,引入基因表达系列分析的方法,建立这两个发育阶段基因表达系列分析文库,希望为进一步的探讨其转化机制,以及转化过程与宿主的关系提供更多的线索。
在以前的研究中,研究者们报道了多种利什曼原虫前鞭毛体的培养方法,简单重复文献中的培养方法没有成功得到杜氏利什曼原虫MHOM/CN/Gansu-8801种的无鞭毛体,总结后我们发现不同种的利什曼原虫其前鞭毛体的培养步骤和培养条件均有所不同。因此,在本研究中我们首先改进了杜氏利什曼原虫MHOM/CN/Gansu-8801种无外源细胞污染的无鞭毛体体外培养的方法,得到了大量的无鞭毛体供后续研究。接下来,我们应用基因表达系列分析(Serial analysis of gene expression, SAGE)的方法建立了杜氏利什曼原虫前鞭毛体及无鞭毛体两个发育阶段基因表达文库。一共获得总标签数为40,431个,在前鞭毛体文库和无鞭毛体文库中分别为20,299和20,132个标签。大约89%的基因在两个文库中均有表达,有968个基因的表达水平在两个文库之间有显著性差异。其中,326个基因在无鞭毛体文库中是表达下调的,642个基因表达上调。我们选取了两个文库中表达差异较大的标签,BLAST查找比对找到28个标签与其对应的基因,这其中包括histone 4,elongation factor 1-alpha,alpha tubulin,acidic ribosomal protein,LACK,ubiquitin-fusion protein,40S ribosomal protein S2,40S ribosomal protein S33,60S ribosomal protein l21,60S ribosomal protein L28等基因。在这其中,延长因子1-alpha和LACK基因已经用于制备多克隆抗体,做为新的抗利什曼原虫药物和疫苗研究的候选基因。而其它表达差异显著的基因也有可能成为新的药物靶标。
我们用实时定量PCR的方法选取了7个基因对SAGE的结果进行了验证,结果表明实时定量PCR与SAGE所揭示的基因在杜氏利什曼原虫两个发育阶段表达变化是一致的。
其次,在培养无鞭毛体的过程中,我们发现有研究报道用HSP90的特异性抑制剂葛尔德霉素(Geldanamycine, GA)可以在室温和碱性条件下诱导利什曼原虫前鞭毛体转变为无鞭毛体,而仅仅在碱性条件下提高培养温度到37℃又可以诱导前鞭毛体发生凋亡。因此,我们观测了HSP90特异性抑制剂GA在利什曼原虫前鞭毛体向无鞭毛体转化过程中的作用,在温度升高的条件下,GA抑制HSP90后会诱导前鞭毛体凋亡还是发生阶段转变?期望找到更多关于HSP90在这一转化过程中的作用的证据及其发挥作用的可能途径。结果发现,用GA处理后的细胞在光学显微镜下和扫描电镜下观察可见体积变小、细胞膜完整但出现发泡现象、染色质浓缩、边缘化,核膜裂解等典型的凋亡形态。用TUNEL原位末端标记法(TdT--mediated dUTP Nick-End Labeling)处理细胞后,在激光共聚焦显微镜下可以直观看到细胞有明显的绿色荧光的阳性凋亡信号,并随时间增加而增多。用TUNEL法经流式细胞仪检测细胞发生凋亡的情况及比例,结果显示,GA诱导杜氏利什曼原虫前鞭毛体在阶段转变过程中发生凋亡这一作用呈时间和剂量依赖性。我们用PI染色后,通过流式细胞仪分析了GA对杜氏利什曼原虫细胞周期的影响,GA处理24h后细胞阻滞于G0/G1期,伴有S期减少,并出现明显的凋亡峰。用PI染色分析细胞凋亡情况,结果与TUNEL法检测凋亡结果一致。以上结果表明GA可以诱导杜氏利什曼原虫前鞭毛体在阶段转变过程中发生凋亡和细胞周期改变。检测处理前后细胞内ROS和GSH水平变化后发现,GA对杜氏利什曼原虫前鞭毛体在阶段转变过程中的存活和分化的影响与其引起的ROS含量上升和GSH含量下降有关,而提高细胞内GSH水平可抑制其毒性作用。GA诱导杜氏利什曼原虫前鞭毛体发生凋亡这一作用可能是跟GA引起细胞线粒体功能障碍有关。进一步说明HSP90在细胞阶段转变中发挥着重要作用。我们还比较了不同培养基pH值在GA诱导利什曼原虫凋亡的过程中的影响,发现酸性环境可能有利于细胞存活。同时我们还选择了5个SAGE文库结果中表达差异显著的基因,检测了在GA处理的过程中这些基因的表达水平变化情况。用实时定量PCR方法分析发现GA可以影响基因表达变化,ATPase subunit 9和Ubiquitin-fusion protein h的表达量均明显增高,而Elongation factor 1-α和H3的表达量是降低的。Ribosomal subunit protein L31的表达量却无明显变化。相关性分析结果显示,Elongation factor 1-α和H3的表达量与细胞凋亡比例之间有相关性。提示这两个基因在GA诱导的细胞凋亡过程可能发挥一定作用。
Leishmania is a protozoan parasite known to cause widespread human diseases around the world. Leishmaniasis is a major and increasingly prevalent public health problem in many regions of the world, particularly in Africa, Asia, and South America. These protozoan parasites have a life cycle characterized by the presence of a flagellated promastigote stage within the sand fly host and a nonmotile amastigote stage within the mammalian host. The promastigote-to-amastigote cytodifferentiation is a profound morphological and physiological transformation. During the process of differentiation, the parasite loses its flagellum, rounds up, changes its glycoconjugate coat, and begins to express a set of metabolic enzymes optimally active at a low pH. The transformation of Leishmania promastigotes to amastigotes during the infection of the host macrophage appears to involve a series of steps. These steps not only result in morphologic transformation, but also allow survival within the parasitophorous vacuole. The promastigote-amastigote cytodifferentiation’s significance in establishing an infection within the mammalian host has prompted us to identify molecular events involved in this process.
In this study, we examined the transcriptome of Leishmania donovani promastigotes and axenic amastigotes to identify differentially regulated mRNAs utilizing the serial analysis of gene expression (SAGE). The axenic culture of amastigotes was initiated from stationary-phase promastigotes. Transformation from promastigote to amastigote occurred when cultures in Medium 199 (pH 5.5), supplemented with 20% (v/v) FBS, were transferred from 26℃to 37℃. A total of 20,299 and 20,132 tags were generated from promastigote and amastigote libraries, respectively. The containing unique genes identified in these two SAGE libraries were 8,615 and 7,835, respectively.
Characteristics of the expressed genes’frequency distribution were remarkably similar in both libraries: the most abundant tags (frequency≥20), whose levels were equal to or > 1.3% of the identified tags, constituted > 23% of the total sequenced tags. Correspondingly, 75.72%, or 71.65% of the genes accounted for those tags present at low abundance (frequency=1), contributed only 32.13%, or 27.89%, of the total tags. A total of 968 genes (11.2% of the total genes in promastigotes and 12.4% in amastigotes) were recorded to have statistically different transcript levels between promastigotes and axenic amastigotes. Of the 968 distinct total genes, there are 326 promastigote-enriched transcripts and 642 amastigote-enriched mRNAs. Additional confirmation of the SAGE data was obtained utilizing quantitative real-time PCR.
The present study also investigates the role of geldanamycin (GA),a specific inhibitor of HSP90, during L. donovani promastigote-to- amastigote transformation stage in axenic conditions. Previous study demonstrated that promastigote-to-amastigote differentiation could be induced at a low temperature (25°C) and neutral pH by using GA. Curiously, another study has shown that heat stress triggers a process of programmed cell death in Leishmania infantum promastigotes. Hence, this prompted us to study the effects of GA and pH of media during the promastigote-to-amastigote transformation stage. Primary interest lies in knowing whether GA can induce apoptosis-like death or stage-transformation in L. donovani promastigote at a high temperature and a low pH. Moreover, finding a possible evidence of Hsp90 protection pathway is anticipated and the effects of media pH during GA treatment. In lieu of this, five selected gene expression levels between GA treated and untreated cells were also evaluated. These are selected from the previous results of serial analysis of gene expression (SAGE), which exhibited significantly different expression levels between L. donovani promastigote and amastigote stages.
Promastigotes exhibited morphologic changes, including cell shrinkage, cell rounding, and cytoplasmic blebbing after GA treatment at a high temperature. The positive apoptosis cells could be observed in situ by TdT-dUTP terminal nick-end labeling (TUNEL). Flow cytometry analysis shows a significant increase (P<0.01) in proportion to apoptotic cells with the effect in a dose- and time-dependant manner. Meanwhile, cell cycle analysis with propidium iodide stain shows a significant increase in the G1/G0 phase and a decrease in the S and G2/M phases (P<0.05). In addition, cellular glutathione level was reduced and reactive oxygen species (ROS) was increased afterwards. Pretreatment with antioxidants bring down the percentage of GA induced cell apoptosis. After treatment, cultures in pH 5.5 showed a lower percentage of apoptosis than in pH 7.4(P<0.05), indicating that acidic environments with a high temperature may play a protective role during the transformation stage. In sum, this study provides further evidence that both the Hsp90 and acidic conditions are likely to be crucial to the transformation and survival of the parasite within its human host.
以前也有一些学者用双向电泳和基因芯片的方法对利什曼原虫的两个发育阶段的表达谱进行了研究。双向电泳由于技术的原因,找到的差异蛋白数量比较少;基因芯片的优点在于可以高通量的检测基因表达情况,但由于探针序列一般来自于已知基因的cDNA或EST库,只能检测已知基因,不能发现未知基因。因此,本研究以体外培养的前鞭毛体及无鞭毛体为研究对象,引入基因表达系列分析的方法,建立这两个发育阶段基因表达系列分析文库,希望为进一步的探讨其转化机制,以及转化过程与宿主的关系提供更多的线索。
在以前的研究中,研究者们报道了多种利什曼原虫前鞭毛体的培养方法,简单重复文献中的培养方法没有成功得到杜氏利什曼原虫MHOM/CN/Gansu-8801种的无鞭毛体,总结后我们发现不同种的利什曼原虫其前鞭毛体的培养步骤和培养条件均有所不同。因此,在本研究中我们首先改进了杜氏利什曼原虫MHOM/CN/Gansu-8801种无外源细胞污染的无鞭毛体体外培养的方法,得到了大量的无鞭毛体供后续研究。接下来,我们应用基因表达系列分析(Serial analysis of gene expression, SAGE)的方法建立了杜氏利什曼原虫前鞭毛体及无鞭毛体两个发育阶段基因表达文库。一共获得总标签数为40,431个,在前鞭毛体文库和无鞭毛体文库中分别为20,299和20,132个标签。大约89%的基因在两个文库中均有表达,有968个基因的表达水平在两个文库之间有显著性差异。其中,326个基因在无鞭毛体文库中是表达下调的,642个基因表达上调。我们选取了两个文库中表达差异较大的标签,BLAST查找比对找到28个标签与其对应的基因,这其中包括histone 4,elongation factor 1-alpha,alpha tubulin,acidic ribosomal protein,LACK,ubiquitin-fusion protein,40S ribosomal protein S2,40S ribosomal protein S33,60S ribosomal protein l21,60S ribosomal protein L28等基因。在这其中,延长因子1-alpha和LACK基因已经用于制备多克隆抗体,做为新的抗利什曼原虫药物和疫苗研究的候选基因。而其它表达差异显著的基因也有可能成为新的药物靶标。
我们用实时定量PCR的方法选取了7个基因对SAGE的结果进行了验证,结果表明实时定量PCR与SAGE所揭示的基因在杜氏利什曼原虫两个发育阶段表达变化是一致的。
其次,在培养无鞭毛体的过程中,我们发现有研究报道用HSP90的特异性抑制剂葛尔德霉素(Geldanamycine, GA)可以在室温和碱性条件下诱导利什曼原虫前鞭毛体转变为无鞭毛体,而仅仅在碱性条件下提高培养温度到37℃又可以诱导前鞭毛体发生凋亡。因此,我们观测了HSP90特异性抑制剂GA在利什曼原虫前鞭毛体向无鞭毛体转化过程中的作用,在温度升高的条件下,GA抑制HSP90后会诱导前鞭毛体凋亡还是发生阶段转变?期望找到更多关于HSP90在这一转化过程中的作用的证据及其发挥作用的可能途径。结果发现,用GA处理后的细胞在光学显微镜下和扫描电镜下观察可见体积变小、细胞膜完整但出现发泡现象、染色质浓缩、边缘化,核膜裂解等典型的凋亡形态。用TUNEL原位末端标记法(TdT--mediated dUTP Nick-End Labeling)处理细胞后,在激光共聚焦显微镜下可以直观看到细胞有明显的绿色荧光的阳性凋亡信号,并随时间增加而增多。用TUNEL法经流式细胞仪检测细胞发生凋亡的情况及比例,结果显示,GA诱导杜氏利什曼原虫前鞭毛体在阶段转变过程中发生凋亡这一作用呈时间和剂量依赖性。我们用PI染色后,通过流式细胞仪分析了GA对杜氏利什曼原虫细胞周期的影响,GA处理24h后细胞阻滞于G0/G1期,伴有S期减少,并出现明显的凋亡峰。用PI染色分析细胞凋亡情况,结果与TUNEL法检测凋亡结果一致。以上结果表明GA可以诱导杜氏利什曼原虫前鞭毛体在阶段转变过程中发生凋亡和细胞周期改变。检测处理前后细胞内ROS和GSH水平变化后发现,GA对杜氏利什曼原虫前鞭毛体在阶段转变过程中的存活和分化的影响与其引起的ROS含量上升和GSH含量下降有关,而提高细胞内GSH水平可抑制其毒性作用。GA诱导杜氏利什曼原虫前鞭毛体发生凋亡这一作用可能是跟GA引起细胞线粒体功能障碍有关。进一步说明HSP90在细胞阶段转变中发挥着重要作用。我们还比较了不同培养基pH值在GA诱导利什曼原虫凋亡的过程中的影响,发现酸性环境可能有利于细胞存活。同时我们还选择了5个SAGE文库结果中表达差异显著的基因,检测了在GA处理的过程中这些基因的表达水平变化情况。用实时定量PCR方法分析发现GA可以影响基因表达变化,ATPase subunit 9和Ubiquitin-fusion protein h的表达量均明显增高,而Elongation factor 1-α和H3的表达量是降低的。Ribosomal subunit protein L31的表达量却无明显变化。相关性分析结果显示,Elongation factor 1-α和H3的表达量与细胞凋亡比例之间有相关性。提示这两个基因在GA诱导的细胞凋亡过程可能发挥一定作用。
Leishmania is a protozoan parasite known to cause widespread human diseases around the world. Leishmaniasis is a major and increasingly prevalent public health problem in many regions of the world, particularly in Africa, Asia, and South America. These protozoan parasites have a life cycle characterized by the presence of a flagellated promastigote stage within the sand fly host and a nonmotile amastigote stage within the mammalian host. The promastigote-to-amastigote cytodifferentiation is a profound morphological and physiological transformation. During the process of differentiation, the parasite loses its flagellum, rounds up, changes its glycoconjugate coat, and begins to express a set of metabolic enzymes optimally active at a low pH. The transformation of Leishmania promastigotes to amastigotes during the infection of the host macrophage appears to involve a series of steps. These steps not only result in morphologic transformation, but also allow survival within the parasitophorous vacuole. The promastigote-amastigote cytodifferentiation’s significance in establishing an infection within the mammalian host has prompted us to identify molecular events involved in this process.
In this study, we examined the transcriptome of Leishmania donovani promastigotes and axenic amastigotes to identify differentially regulated mRNAs utilizing the serial analysis of gene expression (SAGE). The axenic culture of amastigotes was initiated from stationary-phase promastigotes. Transformation from promastigote to amastigote occurred when cultures in Medium 199 (pH 5.5), supplemented with 20% (v/v) FBS, were transferred from 26℃to 37℃. A total of 20,299 and 20,132 tags were generated from promastigote and amastigote libraries, respectively. The containing unique genes identified in these two SAGE libraries were 8,615 and 7,835, respectively.
Characteristics of the expressed genes’frequency distribution were remarkably similar in both libraries: the most abundant tags (frequency≥20), whose levels were equal to or > 1.3% of the identified tags, constituted > 23% of the total sequenced tags. Correspondingly, 75.72%, or 71.65% of the genes accounted for those tags present at low abundance (frequency=1), contributed only 32.13%, or 27.89%, of the total tags. A total of 968 genes (11.2% of the total genes in promastigotes and 12.4% in amastigotes) were recorded to have statistically different transcript levels between promastigotes and axenic amastigotes. Of the 968 distinct total genes, there are 326 promastigote-enriched transcripts and 642 amastigote-enriched mRNAs. Additional confirmation of the SAGE data was obtained utilizing quantitative real-time PCR.
The present study also investigates the role of geldanamycin (GA),a specific inhibitor of HSP90, during L. donovani promastigote-to- amastigote transformation stage in axenic conditions. Previous study demonstrated that promastigote-to-amastigote differentiation could be induced at a low temperature (25°C) and neutral pH by using GA. Curiously, another study has shown that heat stress triggers a process of programmed cell death in Leishmania infantum promastigotes. Hence, this prompted us to study the effects of GA and pH of media during the promastigote-to-amastigote transformation stage. Primary interest lies in knowing whether GA can induce apoptosis-like death or stage-transformation in L. donovani promastigote at a high temperature and a low pH. Moreover, finding a possible evidence of Hsp90 protection pathway is anticipated and the effects of media pH during GA treatment. In lieu of this, five selected gene expression levels between GA treated and untreated cells were also evaluated. These are selected from the previous results of serial analysis of gene expression (SAGE), which exhibited significantly different expression levels between L. donovani promastigote and amastigote stages.
Promastigotes exhibited morphologic changes, including cell shrinkage, cell rounding, and cytoplasmic blebbing after GA treatment at a high temperature. The positive apoptosis cells could be observed in situ by TdT-dUTP terminal nick-end labeling (TUNEL). Flow cytometry analysis shows a significant increase (P<0.01) in proportion to apoptotic cells with the effect in a dose- and time-dependant manner. Meanwhile, cell cycle analysis with propidium iodide stain shows a significant increase in the G1/G0 phase and a decrease in the S and G2/M phases (P<0.05). In addition, cellular glutathione level was reduced and reactive oxygen species (ROS) was increased afterwards. Pretreatment with antioxidants bring down the percentage of GA induced cell apoptosis. After treatment, cultures in pH 5.5 showed a lower percentage of apoptosis than in pH 7.4(P<0.05), indicating that acidic environments with a high temperature may play a protective role during the transformation stage. In sum, this study provides further evidence that both the Hsp90 and acidic conditions are likely to be crucial to the transformation and survival of the parasite within its human host.
引文
1.詹希美主编,人体寄生虫学,北京,人民卫生出版社,2001,50-51
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