家蚕血细胞特异性分子标记鉴定及造血调控机制研究

发布时间：2018-06-23 08:49

  本文选题：家蚕 + 血细胞　； 参考：《西南大学》2017年博士论文

【摘要】：昆虫整个体腔均浸泡在循环血系统中,血淋巴不仅是滋养、代谢的中心场所,也是清除组织和细胞碎片、抗击外源物入侵的主要发生地之一。家蚕作为一种经典的鳞翅目昆虫和重要的经济昆虫,是研究昆虫造血及血细胞功能的良好模型。根据传统的形态学分类方法,家蚕血细胞主要包括五种不同的类型,即原血细胞、颗粒细胞、浆细胞、拟绛色细胞和小球细胞。由于研究工具的匮乏,目前对于家蚕血细胞的发育及其调控机制的研究仍然处于初始阶段。本研究尝试利用转录组测序分析方法对幼虫不同的造血谱系间基因表达模式的差异进行研究,从分子水平揭示颗粒细胞和浆细胞这两种最主要的血细胞类型的发育和功能上的差异。筛选血细胞特异性分子标记,利用其特异性追踪家蚕血细胞的来源及其在免疫中的功能。利用CRISP/Cas9基因编辑技术对浆细胞特异性标记Integrinβ2和Integrinβ3分别进行基因敲除,探索其功能。克隆鉴定血细胞特异性启动子,利用转基因手段探讨体内活性。另外,利用特异性信号通路激活剂和抑制剂,研究MAPK和PI3K信号通路在家蚕造血中的作用。主要研究结果如下:1、家蚕幼虫颗粒细胞和浆细胞转录组分析成功分选出颗粒细胞和浆细胞,利用转录组测序对基因的表达情况进行分析。RNA-Seq共鉴定得到10126个Unigene,颗粒细胞与浆细胞之间共有差异表达基因831个,其中颗粒细胞上调表达基因420个,浆细胞上调表达基因411个。GO分析结果显示这些基因主要涉及结合、催化活性、代谢进程、单一生物过程、细胞进程、膜、膜组分、对刺激的反应和生物调节等生物学过程;KEGG分析结果显示,差异表达基因主要涉及到背腹轴的形成、α-亚麻酸代谢、细胞外基质与受体相互作用、甘油酯代谢和烟酸和烟酰胺代谢等。对代谢相关通路进行分析,结果显示颗粒细胞和浆细胞具有不同的代谢模式,颗粒细胞氨基酸和脂肪代谢活动更为旺盛,而浆细胞核酸和碳水化合物代谢更强。对免疫相关基因进行分析,结果显示浆细胞在抗菌肽、丝氨酸蛋白酶级联的黑化反应、过氧化物酶、几丁质酶、超氧化物歧化酶等方面具有更高的活性,而颗粒细胞中半胱氨酸蛋白酶和清道夫受体等因子表达水平更高,这些结果暗示浆细胞和颗粒细胞在免疫反应中具有不同的角色分工。2、血细胞特异性分子标记筛选与鉴定通过转录组分析,筛选获得139个颗粒细胞和141个浆细胞特异性表达候选基因。利用荧光定量pcr,对转录组分析结果进行验证。结果显示integrinβ2和integrinβ3高表达于浆细胞,bmscrb8和bmsr-c高表达于颗粒细胞,而integrinβ1在两种细胞中均有表达,但在浆细胞中的表达水平要高于颗粒细胞。随后,成功鉴定克隆了这些候选基因,经原核表达和蛋白纯化获得相应抗原后免疫动物制备抗体。免疫荧光结果表明integrinβ1可以标记所有的血细胞类型,integrinβ2和integrinβ3特异标记浆细胞,bmscrb8可以特异性标记颗粒细胞和拟浆色细胞,bmsr-c可以标记拟浆色细胞和部分颗粒细胞。3、浆细胞特异性分子标记的鉴定及浆细胞起源研究(1)integrinβ2和integrinβ3序列分析通过pcr和race技术获得家蚕integrinβ2和integrinβ3基因全长序列,这两个基因均定位于4号染色体nscaf2847上,转录方向相反。integrinβ2基因全长6311bp,由8个外显子和7个内含子构成,其cdna全长2434bp,5’和3’utr分别为118bp和72bp。其cds为2244bp,编码747aa的蛋白,预测蛋白质分子量为84.42kda,等电点为5.349。integrinβ3基因全长10086bp,由7个外显子和6个内含子构成,其cdna全长2653bp,5’和3’utr分别为122bp和410bp。其cds为2172bp,编码723aa的蛋白,预测蛋白质分子量为81.79kda,等电点为5.22。这两个β亚基均含有整合素家族保守结构特征,均由一段较长的胞外域、一个单次跨膜结构域和一个较短的胞内域构成。(2)integrinβ2和β3表达分析利用qrt-pcr和westernblot分别在rna水平和蛋白水平检测了integrinβ2和integrinβ3在家蚕5龄3天各组织中的表达情况,发现这两个基因及其编码的蛋白均特异表达于造血系统。利用qrt-pcr检测幼虫血细胞各时期integrinβ2和integrinβ3的表达情况,发现均在血细胞中持续高表达,在5龄6天时达到峰值。原核表达获得重组蛋白,经蛋白纯化后免疫大鼠或小鼠,制备多克隆抗体。westernblot检测结果显示免疫血清能够识别重组蛋白。免疫荧光结果显示,anti-integrinβ2和anti-integrinβ3抗体均可以特异性识别循环系统中的浆细胞,并且定位于细胞膜上。利用a3-egfp转基因蚕血细胞对anti-integrinβ2抗体进行免疫染色,发现integrinβ2信号与egfp信号高度吻合。这些结果显示integrinβ2和integrinβ3特异表达于浆细胞,可以作为浆细胞特异性分子标记。(3)家蚕浆细胞起源利用qrt-pcr和westernblot检测integrinβ2和integrinβ3在家蚕胚胎时期的表达情况,结果显示这两个浆细胞标记分子从胚胎七天开始表达,此后一直维持比较高的表达水平,说明浆细胞从胚胎7天开始产生,之后数量逐渐增多并维持在一定水平。摘除幼虫造血器官后,循环浆细胞数量和其增殖能力显著降低,暗示造血器官可能是循环浆细胞的重要来源。造血器官体外新生的血细胞可以被浆细胞特异性分子标记染色,且具有较高的增殖活性,但随着培养时间的延长,增殖能力迅速降低。edu标记滞留实验结果显示浆细胞可以长期被edu标记,而其他的血细胞类型edu标记信号迅速降低直至消失。综合上述数据,推测循环浆细胞主要来源于造血器官,而非循环细胞的增殖。新生浆细胞具有强增殖特性,但释放至循环系统后增殖能力迅速降低。4、integrinβ2和β3在血细胞中的功能研究对家蚕幼虫注射不同种类的病原微生物和病原相关模式分子后,integrinβ2和integrinβ3基因均出现不同程度的表达上调。细菌凝集实验发现integrinβ2和integrinβ3这两种重组蛋白在体外均对金黄色葡萄球菌具有显著的凝集作用。细菌结合实验结果表明,这两种重组蛋白均能够与金黄色葡萄球菌,而非大肠杆菌和绿脓杆菌结合。为了检测这两种蛋白与病原相关模式分子lps和pgn结合情况而进行的elisa检测结果表明,重组蛋白能与pgn和lps结合,但与pgn的结合能力要强于lps。上述实验结果表明,integrinβ2和integrinβ3可能参与病原微生物,特别是金黄色葡萄球菌的识别与免疫反应。利用crisp/cas9技术,成功构建了分别表达cas9蛋白和grna的转基因个体。通过杂交和子代自交筛选和测序检测,获得了integrinβ2基因突变纯合个体,westernblot和免疫荧光结果证实integrinβ2蛋白不能正常表达。在个体水平和细胞水平均未观察和检测到明显的表型变化,qrt-pcr、westernblot和免疫荧光结果发现随着integrinβ2的功能的缺失,其同源基因integrinβ3的表达却显著上调,推测integrinβ3可能在integrinβ2缺失时通过上调表达以维持血细胞正常功能。5、血细胞特异性启动子的筛选、鉴定及获得(1)血细胞特异性基因的筛选与鉴定分析家蚕组织表达芯片数据库,筛选血细胞特异表达基因,并利用qrt-pcr进行验证,最终获得4个血细胞特异表达基因,即integrinβ2、integrinβ3、cathpsino和一个探针号为sw04862的新基因。integrinβ2和integrinβ3属整合素家族,cathpsino属于半胱氨酸蛋白酶家族,而sw04862信息未知。(2)启动子克隆及活性分析将候选基因启动子成功克隆至经过改装的重组杆状病毒系统,转染家蚕BmE细胞后获得重组病毒。病毒感染家蚕幼虫后检测血细胞、丝腺和脂肪体中报告基因的表达,发现Integrinβ2、Integrinβ3和Cathpsin O的启动子均能够驱动报告基因特异表达于血细胞,而sw04862的启动子无组织特异性。(3)转基因载体构建及阳性个体的获得将Integrinβ2和Integrinβ3基因的启动子分别构建至转基因表达系统中,胚胎显微注射后成功获得转基因阳性个体。实时荧光定量PCR和western blot检测结果表明报告基因EGFP特异性高表达于血细胞。免疫荧光观察结果发现EGFP在造血器官中也有一定的表达,而在丝腺和脂肪体中未检测到荧光信号。这些结果说明Integrinβ2和Integrinβ3启动子在体内具有非常高的血细胞特异性。进一步检测EGFP在血细胞中的表达情况,发现在这两种转基因品系中,报告基因EGFP信号均定为于浆细胞,而非其他类型的血细胞,表明Integrinβ2和Integrinβ3启动子具有高度的浆细胞特异性。6、家蚕造血调控机制解析牛胰岛素能够显著促进造血器官体外血细胞的释放,并且呈现一定的浓度梯度依赖效应,western blot检测结果表明牛胰岛素可以显著激活Akt和Erk的磷酸化。利用信号通路抑制剂,检测MAPK和PI3K信号通路对家蚕造血的影响,发现抑制PI3K通路活性会显著抑制造血作用,而抑制MAPK通路活性则显著促进血细胞的释放。利用牛胰岛素激活胰岛素及其下游通路时,添加U0126抑制MAPK通路活性可以进一步促进造血,而添加LY294002抑制PI3K通路活性则会显著抑制造血。这些结果暗示胰岛素下游的MAPK和PI3K通路可能扮演着不同的角色。在高浓度U0126作用的同时,加入不同浓度的LY294002可以显著抑制U0126对造血的促进作用,且呈现浓度依赖效应;而在高浓度LY294002作用的同时,加入不同浓度的U0126,可以挽救LY294002对造血的抑制作用,并且也呈现浓度依赖效应。胰岛素是家蚕造血调控的一个重要因子,推测可能通过激活其下游的PI3K和MAPK通路来调控造血,其中PI3K促进血细胞的释放,以增加循环系统血细胞的数量以应对正常发育和各种免疫反应需求,而与此同时胰岛素也通过激活MAPK信号通路抑制血细胞的释放,从而严格控制血细胞的数量。我们的结果表明PI3K和MAPK信号通路通过相互拮抗,从而使得机体能够根据自身需求动态维持血细胞数量的稳定。
[Abstract]:The whole body cavity of insects is soaked in the circulatory blood system. Hemolymph is not only the center of nourishing and metabolism, but also one of the main sites for scavenging tissue and cell debris and resisting exogenous invasion. As a classic Lepidoptera and important economic insect, the silkworm is a good model for the study of the hematopoiesis and blood cell function of insects. According to the traditional morphological classification, the blood cells of silkworm mainly include five different types, namely, primary blood cells, granulosa cells, plasma cells, pseudo crimson cells and small ball cells. Because of the lack of research tools, the research on the development and regulation mechanism of silkworm blood cells is still in the initial stage. This study attempts to use transcription. Group sequencing analysis method was used to study the difference of gene expression patterns between different hematopoietic lineages in the larva. The development and functional differences of the two main types of blood cells were revealed from the molecular level. The specific molecular markers of blood cells were screened and the origin of the blood cells of the silkworm was traced by their specificity and the specific trace of the blood cells. The function of CRISP/Cas9 gene editing technique was used to detect the function of plasma cell specific marker Integrin beta 2 and Integrin beta 3 respectively. The specific promoter of blood cells was cloned and identified, and the activity of the body was explored by transgenic means. In addition, the specific signaling pathway activator and inhibitor were used to study MAPK and P. The main research results of I3K signaling pathway in silkworm hematopoiesis are as follows: 1, granulosa cells and plasma cells were successfully identified by the analysis of the silkworm larvae and plasma cell transcriptional groups. The analysis of gene expression by the sequence of transcriptional sequences was used to identify 10126 Unigene and the difference between the granulosa cells and the plasma cells. 831 genes are expressed, 420 of which are up-regulated by granulosa cells, and 411.GO analysis results for up regulation of plasma cells show that these genes are mainly involved in the biological processes such as binding, catalytic activity, metabolic process, single biological process, cell process, membrane, membrane components, response to stimulation and biological regulation, and the results of KEGG analysis show that the difference is poor. The abnormal expression genes mainly involve the formation of the dorsal ventral axis, the metabolism of alpha linolenic acid, the interaction of extracellular matrix and receptor, glyceryl ester metabolism and nicotinic acid and nicotinamide metabolism. The metabolic pathways are analyzed. The results show that the granular cells and plasma cells have different metabolic modes, and the amino acid and fat metabolism of granular cells are more active. The plasma cell nucleic acid and carbohydrate metabolism are stronger. The immune related genes are analyzed. The results show that the pulp cells have higher activity in the antibacterial peptide, the serine protease cascade blackening reaction, the peroxidase, chitinase, superoxide dismutase and so on, and the cysteine protease and the scavenger in granulosa cells are affected. The expression level of body and other factors is higher. These results suggest that plasma cells and granulosa cells have different roles of division of labor in the immune response. Screening and identification of specific molecular markers in blood cells are screened and identified by transcriptional analysis. 139 granular cells and 141 plasma cell specific tables are screened for candidate genes. Using fluorescence quantitative PCR, the transcriptional group is applied to the transcriptional group. The results were verified. The results showed that integrin beta 2 and integrin beta 3 were highly expressed in plasma cells, bmscrb8 and bmsr-c were highly expressed in granulosa cells, and integrin beta 1 expressed in two cells, but the expression level in plasma cells was higher than that of granular cells. Immunofluorescence results showed that integrin beta 1 could mark all types of blood cells, integrin beta 2 and integrin beta 3 specifically labeled plasma cells, bmscrb8 could specifically label granulosa cells and plasma coloured cells, bmsr-c could mark plasma color cells and partial granular cells.3, plasma cell specificity. Identification of molecular markers and plasma cell origin (1) integrin beta 2 and integrin beta 3 sequence analysis by PCR and race technology to obtain the whole long sequence of integrin beta 2 and integrin beta 3 gene of silkworm, the two genes are located on the 4 chromosome nscaf2847, and the transcriptional direction is contrary to the.Integrin beta 2 gene in 6311bp, 8 exons and 7 inclusions. The cDNA full length 2434bp, 5 'and 3' UTR are 118bp and 72bp., respectively, the CDs is 2244bp, the protein of 747aa is encoded, the protein molecular weight is 84.42kda, the isoelectric point is 5.349.integrin beta 3 gene full length 10086bp, 7 exons and 6 introns, 5 'and 3' respectively. The protein encoded 723aa, the predicted protein molecular weight is 81.79kda, and the isoelectric point is 5.22., the two beta subunits all contain the integrin family conserved structure, which are composed of a long period of extracellular domain, a single transmembrane domain and a shorter intracellular domain. (2) integrin beta 2 and beta 3 expression analysis using qRT-PCR and Westernblot in RNA, respectively The expression of integrin beta 2 and integrin beta 3 in the tissues of the silkworm 5 days and 3 days was detected by the level and protein level. The two genes and their encoded proteins were expressed specifically in the hematopoietic system. The expression of integrin beta 2 and integrin beta 3 at various stages of the larval blood cells was detected by qRT-PCR, and they were found to be highly expressed in the blood cells. The recombinant protein was obtained at 5 years of age and 6 days. The recombinant protein was obtained by prokaryotic expression and immunized in rats or mice after protein purification. The results of the preparation of polyclonal antibody.Westernblot showed that the immune sera could identify the recombinant protein. The immunofluorescence results showed that both anti-integrin beta 2 and anti-integrin beta 3 could identify the pulp in the circulatory system specifically. The cells were located on the cell membrane. The a3-egfp transgenic silkworm blood cells were used to immunize the anti-integrin beta 2 antibody. It was found that the integrin beta 2 signal was highly consistent with the EGFP signal. These results showed that integrin beta 2 and integrin beta 3 were specifically expressed in plasma cells and could be used as plasma cell specific molecular markers. (3) the origin of silkworm cells QRT-PCR and Westernblot were used to detect the expression of integrin beta 2 and integrin beta 3 in the embryo of the silkworm. The results showed that the two plasma cell markers were expressed from the embryo seven days. After that, the high expression level was maintained, indicating that the plasma cells began to produce from 7 days after the embryo, and the number of plasma cells gradually increased and maintained at a certain level. After removal of the larval hematopoietic organs, the number of circulating plasma cells and its proliferation ability significantly decreased, suggesting that the hematopoietic organs may be an important source of circulating plasma cells. The hematopoietic cells in the hematopoietic organs can be stained by the plasma cell specific molecules and have high proliferative activity, but with the prolongation of the culture time, the proliferation ability is fast. The results of the rapid reduction of.Edu markers showed that the plasma cells could be labeled by edu for a long time, while the other edu markers of blood cells decreased rapidly until it disappeared. It was concluded that the circulating plasma cells were mainly derived from the hematopoietic organs, but not the proliferation of circulating cells. The proliferation of plasma cells was strong, but released to the circulatory system. The function of.4, integrin beta 2 and beta 3 in the blood cells was rapidly reduced, and the expression of integrin beta 2 and integrin beta 3 were up-regulated in different degrees after injecting different kinds of pathogenic microbes and pathogenic factors related to the silkworm larvae. The bacterial agglutination experiments showed two recombinant eggs of integrin beta 2 and integrin beta 3. The results of bacterial binding assay showed that all two recombinant proteins could be combined with Staphylococcus aureus, not Escherichia coli and Pseudomonas aeruginosa. In order to detect the binding of the two proteins to the pathogen associated model molecules LPS and PGN, the results of ELISA detection were detected. The recombinant protein can be combined with PGN and LPS, but the binding ability with PGN is stronger than that of lps.. Integrin beta 2 and integrin beta 3 may be involved in the pathogenic microorganism, especially the identification and immune response of Staphylococcus aureus. By using crisp/cas9 technology, the transgenic individuals expressing cas9 protein and gRNA respectively are constructed. Integrin beta 2 gene mutant homozygous individuals were obtained by hybridization and progeny self screening and sequencing. Westernblot and immunofluorescence results confirmed that integrin beta 2 protein could not be expressed normally. The obvious phenotypic changes were not observed and detected at the individual level and cell level, and the results of qRT-PCR, Westernblot and immunofluorescence were found along with int The deletion of the function of egrin beta 2 and the expression of its homologous gene integrin beta 3 are significantly up-regulated. It is presumed that integrin beta 3 may be expressed by up regulation of integrin beta 2 to maintain normal function.5 of blood cells, screening and identification of specific promoters of blood cells, identification and identification and identification of the expression core of silkworm tissue by screening and identification of the specific genes of blood cells (1) By screening the specific expression genes of blood cells and using qRT-PCR, 4 specific genes were obtained, namely, integrin beta 2, integrin beta 3, cathpsino and a new gene sw04862 for.Integrin beta 2 and integrin beta 3 integrin family, cathpsino belongs to the cysteine protease family, and sw04862 letter. (2) the Promoter Cloning and activity analysis successfully cloned the candidate gene promoter to the modified recombinant baculovirus system and transfected the Bombyx mori BmE cells to obtain the recombinant virus. The virus infected the silkworm larvae and detected the expression of the reporter gene in the blood cells, the silk gland and the fat body, and found the initiation of Integrin beta 2, Integrin beta 3 and Cathpsin O. The promoter could drive the reporter gene specifically expressed in the blood cells, and the promoter of sw04862 was no tissue specific. (3) the promoter of the transgenic vector and the positive individual were constructed to the transgenic expression system, respectively. The promoter of Integrin beta 2 and Integrin beta 3 gene was successfully obtained after the microinjection of the embryos. The results of light quantitative PCR and Western blot showed that the specificity of the reporter gene EGFP was highly expressed in the blood cells. The results of immunofluorescence showed that EGFP was also expressed in the hematopoietic organs, but the fluorescence signals were not detected in the silk gland and the fat body. These results indicate that the Integrin beta 2 and the Integrin beta 3 promoter are very high in the body. Blood cell specificity. Further detecting the expression of EGFP in blood cells, it is found that in these two transgenic lines, the EGFP signal of the reported gene is determined to be in plasma cells, not other types of blood cells, indicating that Integrin beta 2 and Integrin beta 3 promoter have a high plasma cell specific.6, and the regulation mechanism of silkworm haematopoiesis analysis bovine insulin It can significantly promote the release of hematopoietic cells in hematopoietic organs in vitro, and present a certain concentration gradient dependence. Western blot detection results show that bovine insulin can significantly activate the phosphorylation of Akt and Erk. Using signal pathway inhibitors, the effects of MAPK and PI3K signaling pathways on the hematopoiesis of silkworm were detected, and the inhibition of PI3K pathway activity was found to be significant. Inhibition of hematopoiesis, while inhibiting the activity of MAPK pathway significantly promotes the release of blood cells. When using bovine insulin to activate insulin and its downstream pathway, adding U0126 to inhibit MAPK pathway activity can further promote hematopoiesis, while adding LY294002 to inhibit the activity of PI3K pathway can significantly inhibit hematopoiesis. These results suggest the MA downstream of insulin. PK and PI3K pathways may play different roles. At the same time of high concentration of U0126, adding different concentrations of LY294002 can significantly inhibit the promoting effect of U0126 on hematopoiesis, and present a concentration dependent effect, while adding different concentrations of U0126 can save the inhibitory effect of LY294002 on hematopoiesis at the same time of high concentration of LY294002. Insulin is an important factor of concentration dependence. Insulin is an important factor in the regulation of hematopoiesis in silkworm. It is presumed that hematopoiesis may be regulated by activating its downstream PI3K and MAPK pathways, in which PI3K promotes the release of blood cells in order to increase the number of blood cells in the circulatory system in response to normal development and various immune response needs, while at the same time insulin We also controlled the release of blood cells by activating the MAPK signaling pathway, thus strictly controlling the number of blood cells. Our results indicate that PI3K and MAPK signals are available.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q963
，

本文编号：2056564