本文關(guān)鍵詞： 乙肝病毒 原核表達(dá) 樹(shù)突狀細(xì)胞 B7-DC RNA干擾　出處：《華東師范大學(xué)》2009年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 乙型肝炎病毒是一種非細(xì)胞裂解性病毒,它是引起急、慢性肝炎的主要病原。研究發(fā)現(xiàn),在慢性乙肝患者體內(nèi)的CD8+ T細(xì)胞的增殖和效應(yīng)能力很弱,提示了增強(qiáng)病毒特異性T細(xì)胞的反應(yīng)能力可能是控制慢性乙肝病毒持續(xù)感染的一條途徑。目前,樹(shù)突狀細(xì)胞疫苗被公認(rèn)為是抗腫瘤和抗慢性病毒感染的有效方法之一。因此,基于樹(shù)突狀細(xì)胞的治療性疫苗對(duì)于乙肝的免疫治療是可行的。 許多研究表明,B7家族提供的共刺激信號(hào)對(duì)促進(jìn)和抑制T細(xì)胞活化具有至關(guān)重要的作用。B7-DC是B7家族的第五個(gè)成員,被認(rèn)為是PD-1的第二個(gè)配體,可以負(fù)向調(diào)節(jié)T細(xì)胞的活化。B7-DC選擇性表達(dá)于樹(shù)突狀細(xì)胞上,因此,如果阻斷樹(shù)突狀細(xì)胞與T細(xì)胞B7-DC/PD-1間的相互作用,有可能使樹(shù)突狀細(xì)胞誘導(dǎo)更強(qiáng)的T細(xì)胞反應(yīng)。 RNA干擾是一種新穎的基因調(diào)節(jié)機(jī)制,是通過(guò)雙鏈RNA介導(dǎo)的轉(zhuǎn)錄后基因沉默技術(shù),它是目前封閉基因表達(dá)行之有效的方法之一。 本研究中,我們從小鼠骨髓來(lái)源的未成熟樹(shù)突狀細(xì)胞中提取總RNA,經(jīng)RT-PCR擴(kuò)增B7-DC胞外段,并將其克隆至原核表達(dá)載體pET32a(+)中,構(gòu)建重組表達(dá)質(zhì)粒pET32a(+)-B7-DC_(ECD)。重組質(zhì)粒經(jīng)雙酶切鑒定及序列測(cè)定后,轉(zhuǎn)化E.coliBL21,經(jīng)IPTG誘導(dǎo)表達(dá)目的蛋白,并用SDS-PAGE和Western blot進(jìn)行檢測(cè)。實(shí)驗(yàn)結(jié)果表明,成功獲得全長(zhǎng)為582 bp的小鼠B7-DC胞外段基因,經(jīng)測(cè)序證實(shí)其序列正確。SDS-PAGE和Western blot分析證實(shí)重組質(zhì)�？杀磉_(dá)出Mr約為41 000的B7-DC胞外段蛋白。同時(shí)我們對(duì)攜帶有針對(duì)目標(biāo)基因的shRNA在抑制樹(shù)突狀細(xì)胞上B7-DC基因的表達(dá)水平,以及基因修飾的樹(shù)突狀細(xì)胞的功能方面作了初步的研究。成功構(gòu)建兩個(gè)siRNA真核表達(dá)載體并在體外轉(zhuǎn)染樹(shù)突狀細(xì)胞,檢測(cè)結(jié)果表明兩個(gè)siRNA真核表達(dá)載體均可抑制樹(shù)突狀細(xì)胞上B7-DC基因的表達(dá),重組干擾質(zhì)粒shRNA1-B7-DC和shRNA2-B7-DC抑制B7-DC基因的效率分別為61.4%、45.9%�；旌狭馨图�(xì)胞反應(yīng)中B7-DC基因沉默的樹(shù)突狀細(xì)胞可顯著刺激同種異體T淋巴細(xì)胞增殖。將基因修飾的樹(shù)突狀細(xì)胞免疫HBV轉(zhuǎn)基因小鼠,檢測(cè)結(jié)果表明在效:靶比為50:1時(shí),經(jīng)shRNA1-B7-DC轉(zhuǎn)染后致敏的DCs細(xì)胞免疫組小鼠脾細(xì)胞CTL殺傷活性顯著高于空載體pAS轉(zhuǎn)染組和非轉(zhuǎn)染組,提示樹(shù)突狀細(xì)胞中B7-DC基因沉默后誘導(dǎo)更強(qiáng)的CTL殺傷活性。此外,我們檢測(cè)了免疫小鼠血清中乙肝表面抗原的水平和血清中HBV DNA的濃度。結(jié)果顯示,乙肝病毒特異性表位肽脈沖的樹(shù)突狀細(xì)胞可誘導(dǎo)特異性的抗乙肝病毒免疫,顯著降低了血清中乙肝表面抗原和HBV DNA的濃度,提示了B7-DC基因沉默在樹(shù)突狀細(xì)胞疫苗治療乙肝上的潛在應(yīng)用價(jià)值。
[Abstract]:Hepatitis B virus is a non-cellular lytic virus, it is the main cause of acute and chronic hepatitis. The study found that the proliferation and effect of CD8 T cells in patients with chronic hepatitis B is very weak. It is suggested that enhancing the response ability of viral specific T cells may be a way to control the persistent infection of chronic hepatitis B virus. Dendritic cell vaccine is recognized as one of the effective methods of anti-tumor and anti-chronic virus infection. Therefore, the therapeutic vaccine based on dendritic cells is feasible for hepatitis B immunotherapy. Many studies have shown that the costimulatory signal provided by the B7 family plays an important role in promoting and inhibiting the activation of T cells. B7-DC is the fifth member of the B7 family. It is considered to be the second ligand of PD-1, which can negatively regulate the activation of T cells. B7-DC is selectively expressed on dendritic cells. If the interaction between dendritic cells and T cells B7-DC-PD-1 is blocked, it is possible that dendritic cells induce stronger T cell responses. RNA interference is a novel gene regulation mechanism, which is a post-transcriptional gene silencing technique mediated by double-stranded RNA. It is one of the effective methods for blocking gene expression. In this study, we extracted total RNAs from immature dendritic cells from mouse bone marrow and amplified the extracellular segments of B7-DC by RT-PCR. The recombinant plasmid pET32a was cloned into prokaryotic expression vector pET32a (pET32a), and the recombinant plasmid was identified by double enzyme digestion and sequenced. E. coli BL21 was transformed into E. coli BL21. The target protein was induced by IPTG and detected by SDS-PAGE and Western blot. A 582 BP mouse B7-DC extracellular segment gene was successfully obtained. Sequencing confirmed that its sequence was correct. SDS-PAGE and Western blot analysis confirmed that the recombinant plasmid could express Mr 41. At the same time, we inhibited the expression of B7-DC gene in dendritic cells by shRNA carrying target gene. Two siRNA eukaryotic expression vectors were successfully constructed and transfected into dendritic cells in vitro. The results showed that two siRNA eukaryotic expression vectors could inhibit the expression of B7-DC gene in dendritic cells. The efficiency of inhibition of B7-DC gene by recombinant interference plasmid shRNA1-B7-DC and shRNA2-B7-DC was 61.4%, respectively. The dendritic cells silenced by B7-DC gene could significantly stimulate the proliferation of allogeneic T lymphocytes. The genetically modified dendritic cells were immunized with HBV transgenic mice. The results show that the target ratio is 50: 1. The CTL killing activity of spleen cells in DCs cells immunized with shRNA1-B7-DC was significantly higher than that in empty vector pAS transfection and non-transfection groups. These results suggest that B7-DC gene silencing induces stronger CTL killing activity in dendritic cells. The serum level of hepatitis B surface antigen and the concentration of HBV DNA in serum of immunized mice were determined. Dendritic cells with HBV-specific epitope peptide pulse could induce specific anti-HBV immunity and significantly reduce the concentration of HBV surface antigen and HBV DNA in serum. The potential value of B 7-DC gene silencing in the treatment of hepatitis B with dendritic cell vaccine was suggested.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 吳海霞;張維銘;李曉眠;;基因轉(zhuǎn)染技術(shù)的發(fā)展與現(xiàn)狀[J];國(guó)際生物醫(yī)學(xué)工程雜志;2007年06期

2 黃神安;鄧歡;張吉翔;;共價(jià)閉合環(huán)狀DNA在肝病發(fā)展中的作用[J];國(guó)際消化病雜志;2007年02期

3 唐國(guó)全;;樹(shù)突狀細(xì)胞疫苗的研究與腫瘤的免疫治療[J];廣西醫(yī)學(xué);2006年12期

4 任穎,銀平章,孔令非,唐華;樹(shù)突狀細(xì)胞的制備及生物學(xué)特性的研究[J];實(shí)用診斷與治療雜志;2004年03期

5 王曉軍,張榮珍,胡苑笙,梁曉峰;我國(guó)病毒性肝炎流行現(xiàn)狀研究[J];疾病監(jiān)測(cè);2004年08期

6 徐寬楓;B7家族新成員PD-L1、PD-L2的研究進(jìn)展[J];細(xì)胞與分子免疫學(xué)雜志;2003年05期

7 李珊;潘全;李云龍;葉昕;;人Cyclin E原核表達(dá)載體的構(gòu)建及表達(dá)[J];細(xì)胞與分子免疫學(xué)雜志;2007年01期

8 王全楚;聶青和;;治療性疫苗-慢性乙型肝炎患者的希望[J];世界華人消化雜志;2003年06期

9 戎晶晶;刁振宇;周?chē)?guó)華;;大腸桿菌表達(dá)系統(tǒng)的研究進(jìn)展[J];藥物生物技術(shù);2005年06期

10 王強(qiáng),彭毅志;小鼠骨髓未成熟樹(shù)突狀細(xì)胞體外擴(kuò)增及鑒定[J];中華燒傷雜志;2003年06期

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