本文選題：家蠶 + 血細(xì)胞��； 參考：《西南大學(xué)》2017年博士論文

【摘要】：昆蟲整個(gè)體腔均浸泡在循環(huán)血系統(tǒng)中,血淋巴不僅是滋養(yǎng)、代謝的中心場(chǎng)所,也是清除組織和細(xì)胞碎片、抗擊外源物入侵的主要發(fā)生地之一。家蠶作為一種經(jīng)典的鱗翅目昆蟲和重要的經(jīng)濟(jì)昆蟲,是研究昆蟲造血及血細(xì)胞功能的良好模型。根據(jù)傳統(tǒng)的形態(tài)學(xué)分類方法,家蠶血細(xì)胞主要包括五種不同的類型,即原血細(xì)胞、顆粒細(xì)胞、漿細(xì)胞、擬絳色細(xì)胞和小球細(xì)胞。由于研究工具的匱乏,目前對(duì)于家蠶血細(xì)胞的發(fā)育及其調(diào)控機(jī)制的研究仍然處于初始階段。本研究嘗試?yán)棉D(zhuǎn)錄組測(cè)序分析方法對(duì)幼蟲不同的造血譜系間基因表達(dá)模式的差異進(jìn)行研究,從分子水平揭示顆粒細(xì)胞和漿細(xì)胞這兩種最主要的血細(xì)胞類型的發(fā)育和功能上的差異。篩選血細(xì)胞特異性分子標(biāo)記,利用其特異性追蹤家蠶血細(xì)胞的來源及其在免疫中的功能。利用CRISP/Cas9基因編輯技術(shù)對(duì)漿細(xì)胞特異性標(biāo)記Integrinβ2和Integrinβ3分別進(jìn)行基因敲除,探索其功能�？寺¤b定血細(xì)胞特異性啟動(dòng)子,利用轉(zhuǎn)基因手段探討體內(nèi)活性。另外,利用特異性信號(hào)通路激活劑和抑制劑,研究MAPK和PI3K信號(hào)通路在家蠶造血中的作用。主要研究結(jié)果如下:1、家蠶幼蟲顆粒細(xì)胞和漿細(xì)胞轉(zhuǎn)錄組分析成功分選出顆粒細(xì)胞和漿細(xì)胞,利用轉(zhuǎn)錄組測(cè)序?qū)虻谋磉_(dá)情況進(jìn)行分析。RNA-Seq共鑒定得到10126個(gè)Unigene,顆粒細(xì)胞與漿細(xì)胞之間共有差異表達(dá)基因831個(gè),其中顆粒細(xì)胞上調(diào)表達(dá)基因420個(gè),漿細(xì)胞上調(diào)表達(dá)基因411個(gè)。GO分析結(jié)果顯示這些基因主要涉及結(jié)合、催化活性、代謝進(jìn)程、單一生物過程、細(xì)胞進(jìn)程、膜、膜組分、對(duì)刺激的反應(yīng)和生物調(diào)節(jié)等生物學(xué)過程;KEGG分析結(jié)果顯示,差異表達(dá)基因主要涉及到背腹軸的形成、α-亞麻酸代謝、細(xì)胞外基質(zhì)與受體相互作用、甘油酯代謝和煙酸和煙酰胺代謝等。對(duì)代謝相關(guān)通路進(jìn)行分析,結(jié)果顯示顆粒細(xì)胞和漿細(xì)胞具有不同的代謝模式,顆粒細(xì)胞氨基酸和脂肪代謝活動(dòng)更為旺盛,而漿細(xì)胞核酸和碳水化合物代謝更強(qiáng)。對(duì)免疫相關(guān)基因進(jìn)行分析,結(jié)果顯示漿細(xì)胞在抗菌肽、絲氨酸蛋白酶級(jí)聯(lián)的黑化反應(yīng)、過氧化物酶、幾丁質(zhì)酶、超氧化物歧化酶等方面具有更高的活性,而顆粒細(xì)胞中半胱氨酸蛋白酶和清道夫受體等因子表達(dá)水平更高,這些結(jié)果暗示漿細(xì)胞和顆粒細(xì)胞在免疫反應(yīng)中具有不同的角色分工。2、血細(xì)胞特異性分子標(biāo)記篩選與鑒定通過轉(zhuǎn)錄組分析,篩選獲得139個(gè)顆粒細(xì)胞和141個(gè)漿細(xì)胞特異性表達(dá)候選基因。利用熒光定量pcr,對(duì)轉(zhuǎn)錄組分析結(jié)果進(jìn)行驗(yàn)證。結(jié)果顯示integrinβ2和integrinβ3高表達(dá)于漿細(xì)胞,bmscrb8和bmsr-c高表達(dá)于顆粒細(xì)胞,而integrinβ1在兩種細(xì)胞中均有表達(dá),但在漿細(xì)胞中的表達(dá)水平要高于顆粒細(xì)胞。隨后,成功鑒定克隆了這些候選基因,經(jīng)原核表達(dá)和蛋白純化獲得相應(yīng)抗原后免疫動(dòng)物制備抗體。免疫熒光結(jié)果表明integrinβ1可以標(biāo)記所有的血細(xì)胞類型,integrinβ2和integrinβ3特異標(biāo)記漿細(xì)胞,bmscrb8可以特異性標(biāo)記顆粒細(xì)胞和擬漿色細(xì)胞,bmsr-c可以標(biāo)記擬漿色細(xì)胞和部分顆粒細(xì)胞。3、漿細(xì)胞特異性分子標(biāo)記的鑒定及漿細(xì)胞起源研究(1)integrinβ2和integrinβ3序列分析通過pcr和race技術(shù)獲得家蠶integrinβ2和integrinβ3基因全長(zhǎng)序列,這兩個(gè)基因均定位于4號(hào)染色體nscaf2847上,轉(zhuǎn)錄方向相反。integrinβ2基因全長(zhǎng)6311bp,由8個(gè)外顯子和7個(gè)內(nèi)含子構(gòu)成,其cdna全長(zhǎng)2434bp,5’和3’utr分別為118bp和72bp。其cds為2244bp,編碼747aa的蛋白,預(yù)測(cè)蛋白質(zhì)分子量為84.42kda,等電點(diǎn)為5.349。integrinβ3基因全長(zhǎng)10086bp,由7個(gè)外顯子和6個(gè)內(nèi)含子構(gòu)成,其cdna全長(zhǎng)2653bp,5’和3’utr分別為122bp和410bp。其cds為2172bp,編碼723aa的蛋白,預(yù)測(cè)蛋白質(zhì)分子量為81.79kda,等電點(diǎn)為5.22。這兩個(gè)β亞基均含有整合素家族保守結(jié)構(gòu)特征,均由一段較長(zhǎng)的胞外域、一個(gè)單次跨膜結(jié)構(gòu)域和一個(gè)較短的胞內(nèi)域構(gòu)成。(2)integrinβ2和β3表達(dá)分析利用qrt-pcr和westernblot分別在rna水平和蛋白水平檢測(cè)了integrinβ2和integrinβ3在家蠶5齡3天各組織中的表達(dá)情況,發(fā)現(xiàn)這兩個(gè)基因及其編碼的蛋白均特異表達(dá)于造血系統(tǒng)。利用qrt-pcr檢測(cè)幼蟲血細(xì)胞各時(shí)期integrinβ2和integrinβ3的表達(dá)情況,發(fā)現(xiàn)均在血細(xì)胞中持續(xù)高表達(dá),在5齡6天時(shí)達(dá)到峰值。原核表達(dá)獲得重組蛋白,經(jīng)蛋白純化后免疫大鼠或小鼠,制備多克隆抗體。westernblot檢測(cè)結(jié)果顯示免疫血清能夠識(shí)別重組蛋白。免疫熒光結(jié)果顯示,anti-integrinβ2和anti-integrinβ3抗體均可以特異性識(shí)別循環(huán)系統(tǒng)中的漿細(xì)胞,并且定位于細(xì)胞膜上。利用a3-egfp轉(zhuǎn)基因蠶血細(xì)胞對(duì)anti-integrinβ2抗體進(jìn)行免疫染色,發(fā)現(xiàn)integrinβ2信號(hào)與egfp信號(hào)高度吻合。這些結(jié)果顯示integrinβ2和integrinβ3特異表達(dá)于漿細(xì)胞,可以作為漿細(xì)胞特異性分子標(biāo)記。(3)家蠶漿細(xì)胞起源利用qrt-pcr和westernblot檢測(cè)integrinβ2和integrinβ3在家蠶胚胎時(shí)期的表達(dá)情況,結(jié)果顯示這兩個(gè)漿細(xì)胞標(biāo)記分子從胚胎七天開始表達(dá),此后一直維持比較高的表達(dá)水平,說明漿細(xì)胞從胚胎7天開始產(chǎn)生,之后數(shù)量逐漸增多并維持在一定水平。摘除幼蟲造血器官后,循環(huán)漿細(xì)胞數(shù)量和其增殖能力顯著降低,暗示造血器官可能是循環(huán)漿細(xì)胞的重要來源。造血器官體外新生的血細(xì)胞可以被漿細(xì)胞特異性分子標(biāo)記染色,且具有較高的增殖活性,但隨著培養(yǎng)時(shí)間的延長(zhǎng),增殖能力迅速降低。edu標(biāo)記滯留實(shí)驗(yàn)結(jié)果顯示漿細(xì)胞可以長(zhǎng)期被edu標(biāo)記,而其他的血細(xì)胞類型edu標(biāo)記信號(hào)迅速降低直至消失。綜合上述數(shù)據(jù),推測(cè)循環(huán)漿細(xì)胞主要來源于造血器官,而非循環(huán)細(xì)胞的增殖。新生漿細(xì)胞具有強(qiáng)增殖特性,但釋放至循環(huán)系統(tǒng)后增殖能力迅速降低。4、integrinβ2和β3在血細(xì)胞中的功能研究對(duì)家蠶幼蟲注射不同種類的病原微生物和病原相關(guān)模式分子后,integrinβ2和integrinβ3基因均出現(xiàn)不同程度的表達(dá)上調(diào)。細(xì)菌凝集實(shí)驗(yàn)發(fā)現(xiàn)integrinβ2和integrinβ3這兩種重組蛋白在體外均對(duì)金黃色葡萄球菌具有顯著的凝集作用。細(xì)菌結(jié)合實(shí)驗(yàn)結(jié)果表明,這兩種重組蛋白均能夠與金黃色葡萄球菌,而非大腸桿菌和綠膿桿菌結(jié)合。為了檢測(cè)這兩種蛋白與病原相關(guān)模式分子lps和pgn結(jié)合情況而進(jìn)行的elisa檢測(cè)結(jié)果表明,重組蛋白能與pgn和lps結(jié)合,但與pgn的結(jié)合能力要強(qiáng)于lps。上述實(shí)驗(yàn)結(jié)果表明,integrinβ2和integrinβ3可能參與病原微生物,特別是金黃色葡萄球菌的識(shí)別與免疫反應(yīng)。利用crisp/cas9技術(shù),成功構(gòu)建了分別表達(dá)cas9蛋白和grna的轉(zhuǎn)基因個(gè)體。通過雜交和子代自交篩選和測(cè)序檢測(cè),獲得了integrinβ2基因突變純合個(gè)體,westernblot和免疫熒光結(jié)果證實(shí)integrinβ2蛋白不能正常表達(dá)。在個(gè)體水平和細(xì)胞水平均未觀察和檢測(cè)到明顯的表型變化,qrt-pcr、westernblot和免疫熒光結(jié)果發(fā)現(xiàn)隨著integrinβ2的功能的缺失,其同源基因integrinβ3的表達(dá)卻顯著上調(diào),推測(cè)integrinβ3可能在integrinβ2缺失時(shí)通過上調(diào)表達(dá)以維持血細(xì)胞正常功能。5、血細(xì)胞特異性啟動(dòng)子的篩選、鑒定及獲得(1)血細(xì)胞特異性基因的篩選與鑒定分析家蠶組織表達(dá)芯片數(shù)據(jù)庫(kù),篩選血細(xì)胞特異表達(dá)基因,并利用qrt-pcr進(jìn)行驗(yàn)證,最終獲得4個(gè)血細(xì)胞特異表達(dá)基因,即integrinβ2、integrinβ3、cathpsino和一個(gè)探針號(hào)為sw04862的新基因。integrinβ2和integrinβ3屬整合素家族,cathpsino屬于半胱氨酸蛋白酶家族,而sw04862信息未知。(2)啟動(dòng)子克隆及活性分析將候選基因啟動(dòng)子成功克隆至經(jīng)過改裝的重組桿狀病毒系統(tǒng),轉(zhuǎn)染家蠶BmE細(xì)胞后獲得重組病毒。病毒感染家蠶幼蟲后檢測(cè)血細(xì)胞、絲腺和脂肪體中報(bào)告基因的表達(dá),發(fā)現(xiàn)Integrinβ2、Integrinβ3和Cathpsin O的啟動(dòng)子均能夠驅(qū)動(dòng)報(bào)告基因特異表達(dá)于血細(xì)胞,而sw04862的啟動(dòng)子無(wú)組織特異性。(3)轉(zhuǎn)基因載體構(gòu)建及陽(yáng)性個(gè)體的獲得將Integrinβ2和Integrinβ3基因的啟動(dòng)子分別構(gòu)建至轉(zhuǎn)基因表達(dá)系統(tǒng)中,胚胎顯微注射后成功獲得轉(zhuǎn)基因陽(yáng)性個(gè)體。實(shí)時(shí)熒光定量PCR和western blot檢測(cè)結(jié)果表明報(bào)告基因EGFP特異性高表達(dá)于血細(xì)胞。免疫熒光觀察結(jié)果發(fā)現(xiàn)EGFP在造血器官中也有一定的表達(dá),而在絲腺和脂肪體中未檢測(cè)到熒光信號(hào)。這些結(jié)果說明Integrinβ2和Integrinβ3啟動(dòng)子在體內(nèi)具有非常高的血細(xì)胞特異性。進(jìn)一步檢測(cè)EGFP在血細(xì)胞中的表達(dá)情況,發(fā)現(xiàn)在這兩種轉(zhuǎn)基因品系中,報(bào)告基因EGFP信號(hào)均定為于漿細(xì)胞,而非其他類型的血細(xì)胞,表明Integrinβ2和Integrinβ3啟動(dòng)子具有高度的漿細(xì)胞特異性。6、家蠶造血調(diào)控機(jī)制解析牛胰島素能夠顯著促進(jìn)造血器官體外血細(xì)胞的釋放,并且呈現(xiàn)一定的濃度梯度依賴效應(yīng),western blot檢測(cè)結(jié)果表明牛胰島素可以顯著激活A(yù)kt和Erk的磷酸化。利用信號(hào)通路抑制劑,檢測(cè)MAPK和PI3K信號(hào)通路對(duì)家蠶造血的影響,發(fā)現(xiàn)抑制PI3K通路活性會(huì)顯著抑制造血作用,而抑制MAPK通路活性則顯著促進(jìn)血細(xì)胞的釋放。利用牛胰島素激活胰島素及其下游通路時(shí),添加U0126抑制MAPK通路活性可以進(jìn)一步促進(jìn)造血,而添加LY294002抑制PI3K通路活性則會(huì)顯著抑制造血。這些結(jié)果暗示胰島素下游的MAPK和PI3K通路可能扮演著不同的角色。在高濃度U0126作用的同時(shí),加入不同濃度的LY294002可以顯著抑制U0126對(duì)造血的促進(jìn)作用,且呈現(xiàn)濃度依賴效應(yīng);而在高濃度LY294002作用的同時(shí),加入不同濃度的U0126,可以挽救LY294002對(duì)造血的抑制作用,并且也呈現(xiàn)濃度依賴效應(yīng)。胰島素是家蠶造血調(diào)控的一個(gè)重要因子,推測(cè)可能通過激活其下游的PI3K和MAPK通路來調(diào)控造血,其中PI3K促進(jìn)血細(xì)胞的釋放,以增加循環(huán)系統(tǒng)血細(xì)胞的數(shù)量以應(yīng)對(duì)正常發(fā)育和各種免疫反應(yīng)需求,而與此同時(shí)胰島素也通過激活MAPK信號(hào)通路抑制血細(xì)胞的釋放,從而嚴(yán)格控制血細(xì)胞的數(shù)量。我們的結(jié)果表明PI3K和MAPK信號(hào)通路通過相互拮抗,從而使得機(jī)體能夠根據(jù)自身需求動(dòng)態(tài)維持血細(xì)胞數(shù)量的穩(wěn)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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q963
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本文編號(hào)：2056564