近江牡蛎天然免疫相关蛋白的分子生物学及功能研究
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摘要
牡蛎等无脊椎动物的天然免疫相关蛋白及其与病原的相互作用是国际病害与分子免疫学研究的热点问题。类立克次体是一类专性细胞内寄生的革兰氏阴性细菌,可引起牡蛎的大规模死亡,但目前在贝类抗RLO感染机理方面所知甚少。本博士学位论文在对近江牡蛎天然免疫相关蛋白的基因(sTRAIL,Tolloid-like,MAK3和p38)和／或蛋白质结构的分子生物学进行研究的基础上,对它们的功能进行了研究,即：一方面采用类立克次体对近江牡蛎宿主进行诱导刺激,系统地分析了宿主与类立克次体病原的相互作用：即牡蛎sTRAIL,Tolloid-like,MAK3和p38在类立克次体诱导的宿主免疫反应中的功能、作用机制,以及sTRAIL在免疫反应中的信号传导通路。另一方面本研究系统地探索研究了sTRAIL对牡蛎正常血淋巴细胞和肿瘤细胞的细胞毒作用及其作用机制和信号传导通路。
一、关于近江牡蛎sTRAIL促细胞凋亡及抗类立克次体感染免疫的研究
取近江牡蛎血淋巴细胞,采用TRIzol法提取总RNA,制备cDNA模板。根据人和不同动物TRAIL基因序列设计引物,采用RT-PCR技术首次在双壳贝类中获得了CasTRAIL的编码序列。该cDNA序列含有一个501 bp的读码框,编码一个18.4kDa的蛋白,该蛋白含有一个典型的TNF结构域。CasTRAIL与人的TRAIL基因同源性较高,两者的cDNA序列同源性为99%,氨基酸序列的同源性为98%；与草鱼的同源性则较低,为42%。各组织原位杂交和RT-PCR结果表明：CasTRAIL在牡蛎各正常组织中均有表达,在外套膜、血淋巴和鳃中表达量较高,性腺中表达量很低,提示该基因可能与免疫调节有关。通过构建重组表达质粒pET-CasTRAIL,在大肠杆菌中对CasTRAIL进行了诱导表达,并纯化蛋白制备了多克隆抗体；同时,利用该抗体对牡蛎TRAIL的亚细胞定位进行了研究：结果表明CasTRAIL在大肠杆菌中得到了高效表达,所制备的兔多克隆抗体的效价为1：4000；牡蛎TRAIL蛋白主要表达于血淋巴细胞的细胞膜上,是一个跨膜蛋白。随后利用Real time RT-PCR、流式细胞仪、DNA电泳、Western blotting和抗体技术等探索研究了CasTRAIL对牡蛎正常血淋巴细胞和肿瘤细胞的细胞毒作用及其作用机制和信号传导通路。结果证明：CasTRAIL可通过激活死亡受体(DR4和DR5)途径引起人类肿瘤细胞HL-60的凋亡；对正常牡蛎血淋巴细胞无毒性,但可引起p38和ERK等磷酸化水平的提高,表明MAPKs与CasTRAIL信号传导的和功能有关。接着,利用原位杂交、Real time RT-PCR技术探索研究了CasTRAIL在类立克次体诱导刺激条件下的激活机理及其功能。结果显示：CasTRAIL在外套膜、血淋巴和肌肉中明显增强,在鳃中显著降低,而在性腺中则未见明显变化,表明CasTRAIL在类立克次体诱导的宿主免疫反应中具有功能。
二、类立克次体诱导刺激后近江牡蛎Tolloid-like、MAK3和p38MAPK基因的克隆与鉴定
我们进一步克隆鉴定了近江牡蛎的Tolloid-like,MAK3和p38基因,并对它们的功能进行了初步研究。①CaTLL全长3492 bp,具有一个2811 bp的读码框,编码含936个氨基酸、分子量大小约103 kDa的蛋白,该蛋白含有虾红素超家族成员几个特征性结构域或基序：astacin-like结构域、按CUB-CUB-EGF-CUB-EGF-CUB-CUB排列的CUBs和EGFs结构域、RTRR基序、zn2+结合基序：HELGHVIGFWHEH和Met-turn:SIMHY。其中astacin-like结构域与其它动物该结构域的相似度在30%—97%之间,提示其催化功能的保守性。RT-PCR结果显示：CaTLL在各正常组织中均有表达,在鳃中的表达量较高,在血淋巴细胞中的表达量显著偏低；而实时定量PCR结果显示：类立克次体诱导刺激后血淋巴细胞中CaTLL的表达明显增强,表明CaTLL与RLO诱导的宿主免疫反应有关。②CaMAK3基因的编码序列长510 bp,具有一个510 bp的完整读码框,编码169个氨基酸、分子量大小约18.6 kDa的蛋白,该蛋白的特征性结构域Acetyltransf_1与酵母菌的相似度为27%；与各种动物的相似度在53%--100%之间,是一个非常保守的结构域。RT-PCR结果显示：CaMAK3在各正常组织中均有表达,其中鳃和血淋巴细胞中表达量较高,外套膜和闭壳肌中的表达量较低；而在类立克次体诱导刺激后血淋巴细胞中的表达明显增强,表明CaMAK3与RLO诱导的宿主免疫反应有关。③Cap38全长1244 bp,含有一个1065 bp的完整读码框,编码355个氨基酸的蛋白质,其分子量近39 kDa。该蛋白含有一个保守的丝氨酸／苏氨酸蛋白激酶催化结构域S_TKc,该结构域和其它动物的相似度在62%和77%之间,说明p38蛋白是一个较保守的激酶。RT-PCR结果显示：p38在各组织中均有表达,其中鳃、血淋巴细胞和外套膜中表达量较高；而在类立克次体诱导刺激后血淋巴细胞中Cap38的表达水平在15min后即迅速增至峰值,表明Cap38在RLO诱导的宿主免疫反应中具有一定作用。
综合上述结果得到如下结论：CasTRAIL在类立克次体诱导的免疫反应中,类立克次体可以诱导增强CasTRAIL的表达水平,证明了CasTRAIL具有免疫功能,且CasTRAIL重组蛋白诱导试验证明CasTRAIL可从mRNA和蛋白两个水平上同时激活p38-MAPK的表达,表明在CasTRAIL介导的抗类立克次体感染的免疫反应中,可能和p38-MAPK介导的信号通路有关；而Tolloid-like,MAK3和p38等基因在类立克次体诱导的宿主免疫反应中都具有一定的功能。鉴于这些结果,病原/TRAIL/MAPKs/信号途径可能存在于牡蛎天然免疫系统中,而Tolloid-like,MAK3可能在对该信号途径进行调节、修饰等方面发挥一定作用,从而共同抵御外来病原的入侵。另外,通过检测p38, ERK, JNK等MAPks激酶磷酸化水平的变化,证明CasTRAIL蛋白对正常血淋巴细胞无毒性作用,但对人类的肿瘤细胞具有细胞毒作用,可引起肿瘤细胞HL60的凋亡,并且该作用是通过死亡受体(DR4和DR5)介导的。
The studies on molecular biology and function of innate immune-related proteins in inverteberate species including oysters are currently a key topic in the frontier of pathogen and molecular immunology in the world. Rickettsia-like organisms (RLOs) are obligate intracellular Gram-negative bacteria which caused mass mortality of oysters. However, little is known about the mechanisms of anti-RLO infection in oyster. The studies on molecular biology and function of innate immune-related proteins were carried out in this scientific dissertation which included the immune function and mechanism of genes or proteins including sTRAIL,Tolloid-like,MAK3 and p38, and signal pathways of sTRAIL involved in the host defense under the induction of RLO. On the other hand, studies on the cytotoxicity and its mechanims and signal pathway of CasTRAIL on oyster hemocytes and HL-60 tumor cells were also carried out in this dissertation
1.Study on the funtion of anti-tumor and anti-RLO immune response of CasTRAIL in oyster Crassostrea ariakensis
Total RNAwas extracted from hemocytes of RLO-challenged and unchallenged oysters using TRIzol method and was reverse transcribed into cDNA. Degenerate oligonucleotide primers were designed according to the sequences of TRAIL genes publisded in NCBI. Here, we first report that a homologue of human sTRAIL, CasTRAIL, was cloned and identified by RT-PCR in bivalves. The sequence of TRAIL cDNA includes an open reading frame of 501 nucleotide encoding a hypothetical protein of 167 amino acids with a molecular mass about 18.4kDa. Amino acid sequence analysis revealed that CasTRAIL shares conserved signature motifs with other TNF proteins and is highly homologous to sTRAIL of human (98%), it has a 99% similarity over the cDNA sequence and 98% over the amino acid sequence between them. On the contrary, it was only 42% of similarity compared to grass Carp (Ctenopharyngodon idella).
The expression level of CasTRAIL in various tissues was investigated by RT-PCR and in situ hybridization(ISH).The results showed that CasTRAIL was ubiquitously expressed in all examined tissues, and expression levels in mantle, hemocytes and gills were higher than the others, while the lowlest level was detected in gonad, suggesting that CasTRAIL may be involved in immune responses. To investigate the biological functions of CasTRAIL, the recombinant plasmids (pET-CasTRAIL) were constructed and transformed into competent cells for expression. The recombinant fusion proteins were purified by affinity chromatography, then the antiserum against New Zealand white rabbits was prepared and used to study the subcellular location of oyster TRAIL protein. The results showed that: recombinant protein CasTRAIL was successfully expressed in E. coli, the valence of antibody raised with recombinant CasTRAIL was more than 1:4000.Oyster TRAIL protein was primarily located in the cell membrane, and it is a transmember protein. The results of SDS-PAGE, Flow cytometric analysis, DNA fragmentation ,real time RT-PCR and Western blotting showed that: CasTRAIL could induce apoptosis in HL-60 tumor cells, by binding to death receptors (DR4 and DR5) which were up-regulated by CasTRAIL induction. However, it showed no cytotoxicity to normal hemocytes of oyster,although the rapid increase in the phospho-ERK and phospho-p38 levels were observed, indicated that the MAPK pathway may be involved in CasTRAIL-mediated signaling.Then the results of ISH and real time RT-PCR showed that: obviously increased expression level were detected in mantle, hemolymph and muscle, while decreased in gill, and no obvious changes were found in gonad, suggesting that CasTRAIL is involved in anti-RLO defense mechanisms.
2.Molecular cloning and identification of Tolloid-like、MAK3 and p38MAPK genes in oyster Crassostrea ariakensis after RLO chanllenge
Based on our previous studies, other host genes, including Tolloid-like,MAK3 and p38 of oyster Crassostrea ariakensis were cloned, and their functions in the immune response against RLO were also investigated.①The full-length cDNA of CaTLL spans 3492 nucleotides including an open reading frame of 2811 nucleotides which encodes a hypothetical protein of 936 amino acids, with a molecular mass of approximately 103 kDa. The CaTLL molecule possessed structural features of several motifs of astacin family including an N-terminal signal peptide sequence, a prodomain with a RTRR motif, an astacin-like domain that contains a conserved zinc-binding motif HELGHVIGFWHEH, five CUBs and two EGF domains with the arrangement CUB-CUB-EGF-CUB-EGF-CUB-CUB. The proteolytic domain of CaTLL shares more than 30% identity with other astacins of various animals from squail to mammals, indicating its conserved catalytic ability. RT-PCR and qantitative real-time PCR analyses revealed that CaTLL showed the lowest expression level in hemocytes of normal groups, but was affected significantly by RLO-challenge, suggesting that CaTLL is involved in the molluscan immune response.②The ORF of CaMAK3 consists 510bp encoding a protein of 169 amino acids, with a molecular mass of approximately 18.6kDa. The Acetyltransf_1 domain of CaMAK3 shares about 30% identity with yeast Saccharomyces cerevisiae, and 53% to 100% identity with animals selected, indicating that it is a very conserved domain. The results of RT-PCR showed that CaMAK3 was expressed in all examined tissues, and expression levels in gills and hemocytes were higher than the others. Meanwhile, obvious changes in the expression level of CaMAK3 were found after RLO challenge by RT-PCR, suggesting that it is involved in immune responses.③The full-length cDNA of Cap38 spans 1244bp,which contains an ORF of 1065 bp encoding a protein of 169 amino acids about 39kDa. Structure analysis founded that Cap38 shares conserved signature motifs--S_TKc with other p38 proteins, which shares 56% to 70% identity with other animals, indicating that it is a very conserved kinase. As RT-PCR indicated, Cap38 was ubiquitously expressed in all examined tissues, and expression levels in gills,mantle and hemocytes were higher than that in others.And obvious changes in the expression level of Cap38 were found in hemocytew after RLO challenge for 15min, suggesting that it play certain roles in immune responses.
All together, we could draw the following conclusions:The expression level of CasTRAIL could be significantly up-regulated by RLO stimulation, indicating that CasTRAIL was involved in anti-RLO defense mechanisms. Moreover, CasTRAIL could up- regulate the expression levels p38 both from mRNA level and protein level, which suggesting that p38 may play a role in CasTRAIL-mediated anti-RLO signaling pathway, In addition, Tolloid-like,MAK3 and p38 are all involved in anti-RLO immune responses. These results support the hypothesis that the Pathogen/TRAIL/MAPKs/NFκB pathway may exist in the immune system of C. ariakensis and play an immportant role in the immune response against RLO infection, and Tolloid-like and MAK3 might also play some roles in regulation and acetylation modification of molecules involved in this pathway.In addition, CasTRAIL also showed an ability to induce apoptosis in HL-60 tumor cells through death receptors (DR4 and DR5) pathway but has no cytotoxicity to normal hemocytes of oyster.
一、关于近江牡蛎sTRAIL促细胞凋亡及抗类立克次体感染免疫的研究
取近江牡蛎血淋巴细胞,采用TRIzol法提取总RNA,制备cDNA模板。根据人和不同动物TRAIL基因序列设计引物,采用RT-PCR技术首次在双壳贝类中获得了CasTRAIL的编码序列。该cDNA序列含有一个501 bp的读码框,编码一个18.4kDa的蛋白,该蛋白含有一个典型的TNF结构域。CasTRAIL与人的TRAIL基因同源性较高,两者的cDNA序列同源性为99%,氨基酸序列的同源性为98%；与草鱼的同源性则较低,为42%。各组织原位杂交和RT-PCR结果表明：CasTRAIL在牡蛎各正常组织中均有表达,在外套膜、血淋巴和鳃中表达量较高,性腺中表达量很低,提示该基因可能与免疫调节有关。通过构建重组表达质粒pET-CasTRAIL,在大肠杆菌中对CasTRAIL进行了诱导表达,并纯化蛋白制备了多克隆抗体；同时,利用该抗体对牡蛎TRAIL的亚细胞定位进行了研究：结果表明CasTRAIL在大肠杆菌中得到了高效表达,所制备的兔多克隆抗体的效价为1：4000；牡蛎TRAIL蛋白主要表达于血淋巴细胞的细胞膜上,是一个跨膜蛋白。随后利用Real time RT-PCR、流式细胞仪、DNA电泳、Western blotting和抗体技术等探索研究了CasTRAIL对牡蛎正常血淋巴细胞和肿瘤细胞的细胞毒作用及其作用机制和信号传导通路。结果证明：CasTRAIL可通过激活死亡受体(DR4和DR5)途径引起人类肿瘤细胞HL-60的凋亡；对正常牡蛎血淋巴细胞无毒性,但可引起p38和ERK等磷酸化水平的提高,表明MAPKs与CasTRAIL信号传导的和功能有关。接着,利用原位杂交、Real time RT-PCR技术探索研究了CasTRAIL在类立克次体诱导刺激条件下的激活机理及其功能。结果显示：CasTRAIL在外套膜、血淋巴和肌肉中明显增强,在鳃中显著降低,而在性腺中则未见明显变化,表明CasTRAIL在类立克次体诱导的宿主免疫反应中具有功能。
二、类立克次体诱导刺激后近江牡蛎Tolloid-like、MAK3和p38MAPK基因的克隆与鉴定
我们进一步克隆鉴定了近江牡蛎的Tolloid-like,MAK3和p38基因,并对它们的功能进行了初步研究。①CaTLL全长3492 bp,具有一个2811 bp的读码框,编码含936个氨基酸、分子量大小约103 kDa的蛋白,该蛋白含有虾红素超家族成员几个特征性结构域或基序：astacin-like结构域、按CUB-CUB-EGF-CUB-EGF-CUB-CUB排列的CUBs和EGFs结构域、RTRR基序、zn2+结合基序：HELGHVIGFWHEH和Met-turn:SIMHY。其中astacin-like结构域与其它动物该结构域的相似度在30%—97%之间,提示其催化功能的保守性。RT-PCR结果显示：CaTLL在各正常组织中均有表达,在鳃中的表达量较高,在血淋巴细胞中的表达量显著偏低；而实时定量PCR结果显示：类立克次体诱导刺激后血淋巴细胞中CaTLL的表达明显增强,表明CaTLL与RLO诱导的宿主免疫反应有关。②CaMAK3基因的编码序列长510 bp,具有一个510 bp的完整读码框,编码169个氨基酸、分子量大小约18.6 kDa的蛋白,该蛋白的特征性结构域Acetyltransf_1与酵母菌的相似度为27%；与各种动物的相似度在53%--100%之间,是一个非常保守的结构域。RT-PCR结果显示：CaMAK3在各正常组织中均有表达,其中鳃和血淋巴细胞中表达量较高,外套膜和闭壳肌中的表达量较低；而在类立克次体诱导刺激后血淋巴细胞中的表达明显增强,表明CaMAK3与RLO诱导的宿主免疫反应有关。③Cap38全长1244 bp,含有一个1065 bp的完整读码框,编码355个氨基酸的蛋白质,其分子量近39 kDa。该蛋白含有一个保守的丝氨酸／苏氨酸蛋白激酶催化结构域S_TKc,该结构域和其它动物的相似度在62%和77%之间,说明p38蛋白是一个较保守的激酶。RT-PCR结果显示：p38在各组织中均有表达,其中鳃、血淋巴细胞和外套膜中表达量较高；而在类立克次体诱导刺激后血淋巴细胞中Cap38的表达水平在15min后即迅速增至峰值,表明Cap38在RLO诱导的宿主免疫反应中具有一定作用。
综合上述结果得到如下结论：CasTRAIL在类立克次体诱导的免疫反应中,类立克次体可以诱导增强CasTRAIL的表达水平,证明了CasTRAIL具有免疫功能,且CasTRAIL重组蛋白诱导试验证明CasTRAIL可从mRNA和蛋白两个水平上同时激活p38-MAPK的表达,表明在CasTRAIL介导的抗类立克次体感染的免疫反应中,可能和p38-MAPK介导的信号通路有关；而Tolloid-like,MAK3和p38等基因在类立克次体诱导的宿主免疫反应中都具有一定的功能。鉴于这些结果,病原/TRAIL/MAPKs/信号途径可能存在于牡蛎天然免疫系统中,而Tolloid-like,MAK3可能在对该信号途径进行调节、修饰等方面发挥一定作用,从而共同抵御外来病原的入侵。另外,通过检测p38, ERK, JNK等MAPks激酶磷酸化水平的变化,证明CasTRAIL蛋白对正常血淋巴细胞无毒性作用,但对人类的肿瘤细胞具有细胞毒作用,可引起肿瘤细胞HL60的凋亡,并且该作用是通过死亡受体(DR4和DR5)介导的。
The studies on molecular biology and function of innate immune-related proteins in inverteberate species including oysters are currently a key topic in the frontier of pathogen and molecular immunology in the world. Rickettsia-like organisms (RLOs) are obligate intracellular Gram-negative bacteria which caused mass mortality of oysters. However, little is known about the mechanisms of anti-RLO infection in oyster. The studies on molecular biology and function of innate immune-related proteins were carried out in this scientific dissertation which included the immune function and mechanism of genes or proteins including sTRAIL,Tolloid-like,MAK3 and p38, and signal pathways of sTRAIL involved in the host defense under the induction of RLO. On the other hand, studies on the cytotoxicity and its mechanims and signal pathway of CasTRAIL on oyster hemocytes and HL-60 tumor cells were also carried out in this dissertation
1.Study on the funtion of anti-tumor and anti-RLO immune response of CasTRAIL in oyster Crassostrea ariakensis
Total RNAwas extracted from hemocytes of RLO-challenged and unchallenged oysters using TRIzol method and was reverse transcribed into cDNA. Degenerate oligonucleotide primers were designed according to the sequences of TRAIL genes publisded in NCBI. Here, we first report that a homologue of human sTRAIL, CasTRAIL, was cloned and identified by RT-PCR in bivalves. The sequence of TRAIL cDNA includes an open reading frame of 501 nucleotide encoding a hypothetical protein of 167 amino acids with a molecular mass about 18.4kDa. Amino acid sequence analysis revealed that CasTRAIL shares conserved signature motifs with other TNF proteins and is highly homologous to sTRAIL of human (98%), it has a 99% similarity over the cDNA sequence and 98% over the amino acid sequence between them. On the contrary, it was only 42% of similarity compared to grass Carp (Ctenopharyngodon idella).
The expression level of CasTRAIL in various tissues was investigated by RT-PCR and in situ hybridization(ISH).The results showed that CasTRAIL was ubiquitously expressed in all examined tissues, and expression levels in mantle, hemocytes and gills were higher than the others, while the lowlest level was detected in gonad, suggesting that CasTRAIL may be involved in immune responses. To investigate the biological functions of CasTRAIL, the recombinant plasmids (pET-CasTRAIL) were constructed and transformed into competent cells for expression. The recombinant fusion proteins were purified by affinity chromatography, then the antiserum against New Zealand white rabbits was prepared and used to study the subcellular location of oyster TRAIL protein. The results showed that: recombinant protein CasTRAIL was successfully expressed in E. coli, the valence of antibody raised with recombinant CasTRAIL was more than 1:4000.Oyster TRAIL protein was primarily located in the cell membrane, and it is a transmember protein. The results of SDS-PAGE, Flow cytometric analysis, DNA fragmentation ,real time RT-PCR and Western blotting showed that: CasTRAIL could induce apoptosis in HL-60 tumor cells, by binding to death receptors (DR4 and DR5) which were up-regulated by CasTRAIL induction. However, it showed no cytotoxicity to normal hemocytes of oyster,although the rapid increase in the phospho-ERK and phospho-p38 levels were observed, indicated that the MAPK pathway may be involved in CasTRAIL-mediated signaling.Then the results of ISH and real time RT-PCR showed that: obviously increased expression level were detected in mantle, hemolymph and muscle, while decreased in gill, and no obvious changes were found in gonad, suggesting that CasTRAIL is involved in anti-RLO defense mechanisms.
2.Molecular cloning and identification of Tolloid-like、MAK3 and p38MAPK genes in oyster Crassostrea ariakensis after RLO chanllenge
Based on our previous studies, other host genes, including Tolloid-like,MAK3 and p38 of oyster Crassostrea ariakensis were cloned, and their functions in the immune response against RLO were also investigated.①The full-length cDNA of CaTLL spans 3492 nucleotides including an open reading frame of 2811 nucleotides which encodes a hypothetical protein of 936 amino acids, with a molecular mass of approximately 103 kDa. The CaTLL molecule possessed structural features of several motifs of astacin family including an N-terminal signal peptide sequence, a prodomain with a RTRR motif, an astacin-like domain that contains a conserved zinc-binding motif HELGHVIGFWHEH, five CUBs and two EGF domains with the arrangement CUB-CUB-EGF-CUB-EGF-CUB-CUB. The proteolytic domain of CaTLL shares more than 30% identity with other astacins of various animals from squail to mammals, indicating its conserved catalytic ability. RT-PCR and qantitative real-time PCR analyses revealed that CaTLL showed the lowest expression level in hemocytes of normal groups, but was affected significantly by RLO-challenge, suggesting that CaTLL is involved in the molluscan immune response.②The ORF of CaMAK3 consists 510bp encoding a protein of 169 amino acids, with a molecular mass of approximately 18.6kDa. The Acetyltransf_1 domain of CaMAK3 shares about 30% identity with yeast Saccharomyces cerevisiae, and 53% to 100% identity with animals selected, indicating that it is a very conserved domain. The results of RT-PCR showed that CaMAK3 was expressed in all examined tissues, and expression levels in gills and hemocytes were higher than the others. Meanwhile, obvious changes in the expression level of CaMAK3 were found after RLO challenge by RT-PCR, suggesting that it is involved in immune responses.③The full-length cDNA of Cap38 spans 1244bp,which contains an ORF of 1065 bp encoding a protein of 169 amino acids about 39kDa. Structure analysis founded that Cap38 shares conserved signature motifs--S_TKc with other p38 proteins, which shares 56% to 70% identity with other animals, indicating that it is a very conserved kinase. As RT-PCR indicated, Cap38 was ubiquitously expressed in all examined tissues, and expression levels in gills,mantle and hemocytes were higher than that in others.And obvious changes in the expression level of Cap38 were found in hemocytew after RLO challenge for 15min, suggesting that it play certain roles in immune responses.
All together, we could draw the following conclusions:The expression level of CasTRAIL could be significantly up-regulated by RLO stimulation, indicating that CasTRAIL was involved in anti-RLO defense mechanisms. Moreover, CasTRAIL could up- regulate the expression levels p38 both from mRNA level and protein level, which suggesting that p38 may play a role in CasTRAIL-mediated anti-RLO signaling pathway, In addition, Tolloid-like,MAK3 and p38 are all involved in anti-RLO immune responses. These results support the hypothesis that the Pathogen/TRAIL/MAPKs/NFκB pathway may exist in the immune system of C. ariakensis and play an immportant role in the immune response against RLO infection, and Tolloid-like and MAK3 might also play some roles in regulation and acetylation modification of molecules involved in this pathway.In addition, CasTRAIL also showed an ability to induce apoptosis in HL-60 tumor cells through death receptors (DR4 and DR5) pathway but has no cytotoxicity to normal hemocytes of oyster.
引文
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