本文選題：重組蛋白 + 表皮生長(zhǎng)因子　； 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：第一部分：可激活免疫細(xì)胞靶向EGFR高表達(dá)腫瘤細(xì)胞重組蛋白的設(shè)計(jì)、表達(dá)及對(duì)腫瘤的治療作用腫瘤靶向治療是當(dāng)前癌癥治療的研究熱點(diǎn),并代表未來(lái)發(fā)展的方向。而腫瘤免疫治療是繼手術(shù)、放療和化療之后的第四種腫瘤治療模式—腫瘤生物治療,具有廣闊的應(yīng)用前景。有效地將兩者結(jié)合起來(lái)是研發(fā)新型抗腫瘤藥物的一大趨勢(shì)。目前在抗腫瘤靶向藥物的開(kāi)發(fā)中,表皮生長(zhǎng)因子受體(Epidermal growth factor receptor, EGFR)是公認(rèn)的、選用最多的靶向治療靶點(diǎn)�，F(xiàn)在臨床上主要有兩大類EGFR靶向的抗腫瘤藥物：一,小分子酪氨酸激酶抑制劑(small-molecule tyrosine kinase inhibitors, TKIs);二,單克隆抗體(monoclonal antibodies, mAbs)。但是在應(yīng)用過(guò)程中發(fā)現(xiàn)有顯著一部分患者對(duì)這兩類藥物均明顯耐藥,而最初敏感的患者在經(jīng)過(guò)治療后,轉(zhuǎn)而發(fā)展為耐藥,最終導(dǎo)致預(yù)后較差。通過(guò)分子水平的機(jī)制研究發(fā)現(xiàn)這種耐藥多是由于EGFR激酶結(jié)構(gòu)域存在突變,或其下游信號(hào)通路的持續(xù)活化造成。在這種情況下,腫瘤細(xì)胞不再依賴表皮生長(zhǎng)因子(Epidermal growth factor, EGF)結(jié)合EGFR的啟動(dòng)激活信號(hào),卻可以持續(xù)地生長(zhǎng)增殖,腫瘤細(xì)胞呈自主性生長(zhǎng)狀態(tài)。因此針對(duì)EGF-EGFR信號(hào)不敏感的腫瘤細(xì)胞,需要新的治療方法。本研究旨在開(kāi)發(fā)能夠靶向免疫治療該類腫瘤的重組蛋白類藥物,即新型的Biosimih ar。為充分調(diào)動(dòng)機(jī)體的免疫系統(tǒng),將抗腫瘤免疫治療機(jī)制運(yùn)用至新型藥物中,本研究將顯性抗原肽構(gòu)建至重組蛋白,組成一個(gè)EGFR靶向的雙功能重組免疫調(diào)節(jié)蛋白,用于高表達(dá)EGFR腫瘤的治療。本研究采用EGFR的天然配體EGF作為靶向結(jié)合載體,將其與來(lái)自李斯特菌溶細(xì)胞素O (Listeriolysin O, LLO)的3個(gè)顯性T細(xì)胞表位融合,通過(guò)序列拼接構(gòu)建了重組融合蛋白pLLO-hEGF。人EGF是一個(gè)由53個(gè)氨基酸殘基組成的單鏈多肽,分子量小,與EGFR的親和力高。而LLO是一個(gè)已經(jīng)證實(shí)的強(qiáng)大的免疫原性分子,具有豐富的CD4+和CD8+T細(xì)胞抗原表位。本研究選用了經(jīng)文獻(xiàn)證實(shí)的2個(gè)CD4+和1個(gè)CD8+T細(xì)胞表位。所構(gòu)建的重組蛋白pLLO-hEGF經(jīng)生物信息學(xué)軟件分析,理論分子量?jī)H為16 kDa,性質(zhì)穩(wěn)定,血管穿透性將優(yōu)于mAb。同時(shí)該重組蛋白應(yīng)具有2種功能：既可以通過(guò)EGF靶向結(jié)合高表達(dá)EGFR腫瘤細(xì)胞,同時(shí)其LLO的顯性抗原表位又可以充分活化機(jī)體的免疫細(xì)胞,使免疫細(xì)胞在腫瘤細(xì)胞部位聚集,從而加強(qiáng)攻擊和殺傷腫瘤細(xì)胞,發(fā)揮靶向免疫治療腫瘤的作用。我們首先采用生物工程的分子克隆技術(shù)以及蛋白表達(dá)純化技術(shù),成功克隆表達(dá)和獲得了可溶性雙功能重組蛋白pLLO-hEGF,4L重組菌液量可得(4.5-6)mL濃度為800 μg/mL左右(最大1.0 mg/m1)的純化蛋白。接下來(lái)通過(guò)Western blot篩選了高表達(dá)EGFR的人腫瘤細(xì)胞系,從不同細(xì)胞系中各挑選出2種,包括人乳腺癌(MDA-MB-231、SK-BR-3)、人肺癌(A549、NCI-H157)和人結(jié)直腸癌(HCT116、HT-29),用于雙功能重組蛋白pLLO-hEGF相關(guān)的功能研究。細(xì)胞免疫化學(xué)熒光染色實(shí)驗(yàn)結(jié)果顯示了pLLO-hEGF可以很好地靶向結(jié)合這些腫瘤細(xì)胞表面。細(xì)胞增殖實(shí)驗(yàn)結(jié)果顯示pLLO-hEGF對(duì)這些腫瘤細(xì)胞不產(chǎn)生促增殖作用。其免疫激活作用研究結(jié)果顯示：pLLO-hEGF可使體外培養(yǎng)的人外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cells, PBMCs)中 CD3+CD4+ T細(xì)胞明顯增殖。單次實(shí)驗(yàn)結(jié)果顯示pLLO-hEGF刺激14天時(shí)PBMCs中CD3+CD4+ T細(xì)胞百分比可增至54.5%,而對(duì)照組為44.8%。且刺激增殖活化的T淋巴細(xì)胞在體內(nèi)外實(shí)驗(yàn)中均顯示出顯著的殺傷腫瘤細(xì)胞作用。體外對(duì)上述幾種表達(dá)EGFR腫瘤細(xì)胞的殺傷率分別為：HCT116, (69.89 ±1.05)%; HT-29, (60.40±2.33)%; A549, (49.45±1.72)%; NCI-H157, (85.26±4.82)%；SK-BR-3,(47.90±1.39)%。體內(nèi)移植瘤模型實(shí)驗(yàn)結(jié)果顯示,將刺激增殖的T淋巴細(xì)胞注射裸小鼠體內(nèi),顯著抑制了腫瘤的生長(zhǎng),平均瘤重(0.178/0.224 g)明顯小于對(duì)照組(0.550 g)(*P0.05)。綜上所述,我們的研究提供了一種研發(fā)新型抗腫瘤藥物的新思路和新策略。此部分內(nèi)容申請(qǐng)了國(guó)家發(fā)明專利(申請(qǐng)?zhí)枺?01410152549.2),目前在實(shí)審階段,且相關(guān)內(nèi)容已發(fā)表于Hum Vaccin Immunother (Human vaccines) (IF=3.643)和CellPhysiol Biochem雜志(IF=3.55)。第二部分：可激活免疫細(xì)胞靶向CD20陽(yáng)性人淋巴瘤細(xì)胞重組蛋白的設(shè)計(jì)、表達(dá)及抗淋巴瘤作用的初步研究B細(xì)胞淋巴瘤是一種血液系統(tǒng)疾病,采用傳統(tǒng)治療手段臨床療效較差。隨著對(duì)CD20分子結(jié)構(gòu)特點(diǎn)的認(rèn)識(shí),抗CD20單克隆抗體的設(shè)計(jì)開(kāi)發(fā)在B細(xì)胞淋巴瘤的臨床治療中取得重要進(jìn)展。其中最具代表性的是1997年美國(guó)FDA批準(zhǔn)上市的人鼠嵌合抗CD20單克隆抗體一Rituximab (C2B8,美羅華)。但人鼠嵌合抗體由于其分子量大,穿透力弱,且毒副作用大,明顯限制了其臨床效果。近年來(lái),一些人源化抗體、小型或改型抗體、基因修飾抗體逐漸發(fā)展起來(lái)。其中單鏈抗體(single chain variable fragments, ScFv)由于其分子量小、穿透力強(qiáng)、能夠較好地保持抗原親和性等特點(diǎn),成為應(yīng)用生物工程方法進(jìn)行腫瘤免疫治療的一種重要手段。本研究同樣利用LLO的顯性抗原肽設(shè)計(jì)、構(gòu)建了靶向CD20陽(yáng)性B細(xì)胞淋巴瘤的雙功能重組蛋白ScFv-pLLO。首先,我們?cè)O(shè)計(jì)了針對(duì)人CD20的ScFv序列,然后將LLO顯性抗原肽與ScFv序列拼接,使構(gòu)建的重組蛋白既可靶向結(jié)合CD20陽(yáng)性B細(xì)胞淋巴瘤細(xì)胞,同時(shí)其LLO抗原表位又可增強(qiáng)腫瘤細(xì)胞的抗原性,從而充分激活機(jī)體的細(xì)胞免疫應(yīng)答,發(fā)揮更長(zhǎng)效的抗腫瘤免疫,達(dá)到靶向免疫治療的效果。雙功能重組蛋白ScFv-pLLO經(jīng)原核克隆、表達(dá)和純化,并初步分析了其抗B細(xì)胞淋巴瘤作用。生物信息學(xué)軟件分析顯示重組蛋白理論分子量為32 kDa,化學(xué)性質(zhì)穩(wěn)定。流式細(xì)胞術(shù)定量分析幾種人淋巴瘤細(xì)胞系細(xì)胞表面CD20表達(dá)情況,結(jié)果顯示Daudi細(xì)胞CD20陽(yáng)性率在98%以上,Raji和NCI-BL2009細(xì)胞CD20陽(yáng)性率分別為89.7%和92.2%,而RAMOS細(xì)胞CD20陽(yáng)性率是52.3%。流式細(xì)胞術(shù)分析ScFv-pLLO靶向結(jié)合人淋巴瘤細(xì)胞的能力,結(jié)果顯示ScFv-pLLO可不同程度的靶向結(jié)合CD20表達(dá)水平不同的人淋巴瘤細(xì)胞,其濃度為10μg/mL時(shí),對(duì)Daudi和NCI-BL2009細(xì)胞的平均結(jié)合率分別為(93.25±3.45)%和(73.85±3.95)%。免疫共沉淀實(shí)驗(yàn)結(jié)果進(jìn)一步證明ScFv-pLLO可特異靶向結(jié)合人淋巴瘤細(xì)胞表面的CD20分子。細(xì)胞增殖實(shí)驗(yàn)結(jié)果顯示ScFv-pLLO可抑制Raji細(xì)胞的生長(zhǎng),而對(duì)Daudi、RAMOS和NCI-BL2009淋巴瘤細(xì)胞的生長(zhǎng)無(wú)影響。凋亡實(shí)驗(yàn)結(jié)果顯示ScFv-pLLO可直接誘導(dǎo)Raji細(xì)胞凋亡,20μg/mL的ScFv-pLLO作用24 h后,可誘導(dǎo)24.6%的Raji細(xì)胞凋亡(早期+晚期),作用48 h后,Raji細(xì)胞總凋亡率可達(dá)47.0%。本研究的初步結(jié)果顯示,雙功能重組蛋白ScFv-pLLO可特異靶向結(jié)合CD20陽(yáng)性B細(xì)胞淋巴瘤,對(duì)某些人淋巴瘤細(xì)胞系,如Raji細(xì)胞,可直接誘導(dǎo)其凋亡,結(jié)構(gòu)和功能上類似“小型抗體”。同時(shí),雙功能重組蛋白ScFv-pLLO由于攜帶顯性抗原肽,不同于純粹的單克隆抗體,其靶向結(jié)合淋巴瘤細(xì)胞表面CD20分子后可增強(qiáng)腫瘤細(xì)胞激活免疫系統(tǒng)的能力,從而為我們下一步研究其對(duì)免疫細(xì)胞的激活作用以及激發(fā)長(zhǎng)效抗腫瘤細(xì)胞免疫的功能奠定了基礎(chǔ)。此部分內(nèi)容已發(fā)表于《中國(guó)免疫學(xué)雜志》。第三部分：Leptin在結(jié)直腸癌細(xì)胞侵襲轉(zhuǎn)移中的作用研究隨著腫瘤分子生物學(xué)研究的深入,在腫瘤靶向治療中,不斷有新的候選抗腫瘤靶點(diǎn)出現(xiàn)。近幾年,越來(lái)越多的研究發(fā)現(xiàn)Leptin介導(dǎo)的信號(hào)在腫瘤細(xì)胞的增殖、侵襲和腫瘤干細(xì)胞的誘導(dǎo)生成中發(fā)揮了重要作用,并被認(rèn)為是潛在的有效抗腫瘤靶向治療靶點(diǎn)。Leptin的中文名稱為瘦素,是一種由167個(gè)氨基酸組成的非糖基化蛋白質(zhì)類激素,主要由脂肪細(xì)胞分泌,參與機(jī)體的能量代謝。研究表明腫瘤組織也可表達(dá)Leptin及其受體(LEPR/Ob-R),其中較多數(shù)據(jù)顯示結(jié)直腸癌的惡性程度與Leptin的表達(dá)量密切相關(guān)。一些實(shí)驗(yàn)結(jié)果顯示高濃度的外源性Leptin可以促進(jìn)人結(jié)直腸癌細(xì)胞的增殖,并增強(qiáng)其運(yùn)動(dòng)和侵襲能力。但目前較少關(guān)于腫瘤細(xì)胞產(chǎn)生的內(nèi)源性Leptin在腫瘤細(xì)胞生長(zhǎng)和侵襲力方面確切生物學(xué)作用的研究,而明確Leptin介導(dǎo)的信號(hào)通路對(duì)腫瘤細(xì)胞生物學(xué)行為的影響,有助于開(kāi)發(fā)新型抗腫瘤靶向治療藥物。在研究中,我們首先分析了幾種人結(jié)直腸癌細(xì)胞系中Leptin及其受體LEPR的表達(dá)情況,包括mRNA和蛋白水平。結(jié)果顯示人結(jié)直腸癌細(xì)胞系均普遍表達(dá)Leptin和LEPR,包括細(xì)胞系HCT116、HT-29、COLO201、COLO205和SW480。以人結(jié)直腸癌細(xì)胞系HCT116和HT-29細(xì)胞為例,我們用siRNA有效敲低了腫瘤細(xì)胞中內(nèi)源性Leptin的表達(dá)。細(xì)胞增殖實(shí)驗(yàn)結(jié)果顯示,抑制內(nèi)源性Leptin的表達(dá)對(duì)人結(jié)直腸癌細(xì)胞系HCT116和HT-29細(xì)胞的增殖無(wú)影響。然后我們通過(guò)Real-time PCR檢測(cè)了一些與腫瘤細(xì)胞侵襲轉(zhuǎn)移能力密切相關(guān)基因的表達(dá)情況,包括MMP1、MMP9、 TIMP-1、E-cadherin、Twist等。結(jié)果顯示,抑制內(nèi)源性Leptin表達(dá),HCT116細(xì)胞中MMP1和MMP9的表達(dá)明顯上調(diào)(*P0.05),TIMP-1、TIMP-2 和 E-cadherin的表達(dá)明顯下調(diào)(*P0.05)；HT-29細(xì)胞中,MMP1和Vimentin的表達(dá)明顯上調(diào)(*P0.05),TIMP-2的表達(dá)明顯下調(diào)(*P0.05)。以HCT116為例,Western blot進(jìn)一步檢測(cè)蛋白表達(dá)水平變化,其結(jié)果與mRNA水平變化一致。此外,明膠酶譜實(shí)驗(yàn)結(jié)果顯示,抑制內(nèi)源性Leptin表達(dá),HCT116細(xì)胞中MMP9的蛋白裂解活性增強(qiáng)。Transwell體外侵襲實(shí)驗(yàn)結(jié)果顯示,與對(duì)照組穿膜細(xì)胞數(shù)(148±17)相比,HCT116細(xì)胞Leptin RNAi組穿出小室微孔膜的細(xì)胞數(shù)明顯增多(512±23)(*P0.05),表明HCT116細(xì)胞的侵襲能力明顯增強(qiáng)。本研究不同于以往的研究,重點(diǎn)分析了抑制人結(jié)直腸癌細(xì)胞內(nèi)源性Leptin的表達(dá),對(duì)腫瘤細(xì)胞增殖和侵襲能力的影響。研究發(fā)現(xiàn),降低Leptin的表達(dá)對(duì)細(xì)胞增殖無(wú)影響,但卻明顯增強(qiáng)了細(xì)胞的侵襲能力。而以往的研究結(jié)果是提高Leptin的濃度會(huì)促進(jìn)腫瘤細(xì)胞的增殖和侵襲。因此,進(jìn)一步探明不同條件下Leptin信號(hào)通路在結(jié)直腸癌細(xì)胞中的具體生物學(xué)作用機(jī)制,可為確定此通路能否用于腫瘤靶向治療靶點(diǎn)選擇奠定基礎(chǔ)。
[Abstract]:The first part: the design, expression, and therapeutic effect of the tumor targeting EGFR high expression of tumor cell recombinant protein, which is the hot spot of cancer therapy and the direction of the future development. Tumor immunotherapy is the fourth mode of tumor treatment following surgery, radiotherapy and chemotherapy. Epidermal growth factor receptor (EGFR) is the most widely accepted target for target therapy in the development of anti tumor targeting drugs. Two major EGFR targeting antitumor drugs: one, small molecule tyrosine kinase inhibitor (small-molecule tyrosine kinase inhibitors, TKIs); two, monoclonal antibodies (monoclonal antibodies, mAbs). But a significant number of patients were found to be significantly resistant to these two drugs during the application process, and the first sensitive patients were passing through After treatment, it turns to drug resistance and eventually leads to poor prognosis. Through molecular level mechanisms, it is found that the resistance is due to mutations in the EGFR kinase domain or the continuous activation of its downstream signal pathway. In this case, the tumor cells are no longer dependent on the epidermal growth factor (Epidermal growth factor, EGF) binding EGFR. The activation signal can continue to grow and proliferate, and the tumor cells are autonomous growth. Therefore, new therapies are needed for tumor cells that are not sensitive to EGF-EGFR signals. This study aims to develop recombinant protein drugs that can target the immunotherapy of this type of tumor, that is, the new type of Biosimih ar. to fully mobilize the body. The Phytophthora system, which applies the antitumor immunotherapy mechanism to the new drug, constructs the dominant antigen peptide to the recombinant protein to form a EGFR targeted double functional recombinant immunoregulatory protein for the treatment of high expression of EGFR tumor. This study uses the natural ligand EGF of EGFR as a targeting carrier, and it is from Lester bacteria. The fusion of 3 dominant T cell epitopes of Listeriolysin O (LLO) O, the recombinant fusion protein pLLO-hEGF. human EGF is a single strand polypeptide composed of 53 amino acid residues. The molecular weight is small and the affinity to EGFR is high. LLO is a powerful immunogenic molecule that has been proved to be rich in CD4+. This study selected 2 CD4+ and 1 CD8+T cell epitopes confirmed by the literature. The constructed recombinant protein pLLO-hEGF was analyzed by bioinformatics software. The molecular weight of the recombinant protein was only 16 kDa, the properties were stable, the blood vessel penetration would be better than mAb. and the recombinant egg white should have the function of the EGF target node. At the same time, the high expression of EGFR tumor cells, and the dominant antigen epitopes of LLO can fully activate the immune cells in the body, so that the immune cells are gathered in the tumor cells, thus strengthening the attack and killing the tumor cells and playing the role of targeting the tumor in the target immunotherapy. The purified technique was successfully cloned and expressed and obtained the soluble bifunctional recombinant protein pLLO-hEGF, and the recombinant protein of 4L was obtained (4.5-6) with a concentration of about 800 mu g/mL (maximum 1 mg/m1). Then, the human tumor cell lines with high expression of EGFR were screened by Western blot, and 2 species were selected from different cell lines, including human breast. Cancer (MDA-MB-231, SK-BR-3), human lung cancer (A549, NCI-H157) and human colorectal cancer (HCT116, HT-29), used for the functional study of the bifunctional recombinant protein pLLO-hEGF related functions. The results of cell immunofluorescence staining showed that pLLO-hEGF could target the surface of these tumor cells well. The results of cell proliferation experiment showed that pLLO-hEGF against these cells. The tumor cells did not promote proliferation. The results of immunological activation showed that pLLO-hEGF could significantly increase the proliferation of CD3+CD4+ T cells in human peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMCs) cultured in vitro. The results of single experiment showed that the percentage of CD3+CD4+ T cells in PBMCs was increased at 14 days when pLLO-hEGF spiny was stimulated. To 54.5%, the control group was 44.8%. and stimulated the proliferation and activation of T lymphocytes to play a significant role in killing tumor cells in vitro and in vivo. In vitro, the killing rates of EGFR tumor cells were HCT116, (69.89 + 1.05)%; HT-29, (60.40 + 2.33)%; A549, (49.45 + 1.72)%; NCI-H157, (85.26 + 4.82)%; S K-BR-3, (47.90 + 1.39)%. In vivo transplantation tumor model experimental results showed that the proliferation of T lymphocytes injected into nude mice significantly inhibited the growth of tumor, and the average tumor weight (0.178/0.224 g) was significantly smaller than that of the control group (0.550 g) (*P0.05). In summary, our research provides a new idea for developing new antitumor drugs. New strategy. This part applies to the national invention patent (application number: 201410152549.2), currently in the actual trial stage, and the relevant content has been published in Hum Vaccin Immunother (Human vaccines) (IF=3.643) and CellPhysiol Biochem magazine (IF=3.55). The second part: can activate immune cells to target CD20 positive human lymphoma cell recombinant protein Preliminary study on the design, expression and anti lymphoma effect of B cell lymphoma is a blood system disease. The clinical effect of traditional treatment is poor. With the understanding of the structural characteristics of the CD20 molecular structure, the design and development of anti CD20 monoclonal antibodies have made important progress in the clinical treatment of B cell lymphoma. In 1997, the human mice approved by FDA in the United States were chimed with anti CD20 monoclonal antibody 1 Rituximab (C2B8). But the human chimeric antibody, because of its large molecular weight, weak penetration and toxic side effects, obviously restricts its clinical effect. In recent years, some people have derived antibodies, small or modified antibodies, and the gene modified antibodies have gradually developed. Single chain variable fragments (ScFv) is an important means for the application of bioengineering method for tumor immunotherapy due to its small molecular weight, strong penetration and good ability to maintain antigen affinity. This study also used the explicit antigen peptide of LLO to construct the targeted CD20 positive B cell lymph. The tumor's bifunctional recombinant protein ScFv-pLLO. first, we designed the ScFv sequence for human CD20, and then spliced the LLO dominant antigen peptide with the ScFv sequence, so that the recombinant protein can target both CD20 positive B cell lymphoma cells and the LLO antigen epitopes can enhance the antigenicity of the tumor cells, thus fully activating the body's fines. Cellular immune response, exerting a more effective anti-tumor immunity to achieve the effect of targeted immunotherapy. Double functional recombinant protein ScFv-pLLO was expressed and purified by prokaryotic cloning, and its anti B cell lymphoma was preliminarily analyzed. Bioinformatics software analysis showed that the theory of recombinant protein was 32 kDa, chemical properties were stable. Flow cytometry was used. The quantitative analysis of the expression of CD20 on the cell surface of several human lymphoma cell lines showed that the positive rate of CD20 in Daudi cells was above 98%, and the positive rates of CD20 in Raji and NCI-BL2009 cells were 89.7% and 92.2% respectively, while the positive rate of RAMOS cell CD20 was the ability to analyze ScFv-pLLO targeted lymphoma cells by 52.3%. flow cytometry, and the results showed ScFv-. PLLO can target different levels of CD20 expression in human lymphoma cells with a concentration of 10 mu g/mL, the average binding rate of Daudi and NCI-BL2009 cells is (93.25 + 3.45)% and (73.85 + 3.95)%, respectively. The results of immunoprecipitation test further demonstrate that ScFv-pLLO can specifically target the CD20 molecules on the surface of human lymphoma cells. The results of cell proliferation test showed that ScFv-pLLO could inhibit the growth of Raji cells, but had no effect on the growth of Daudi, RAMOS and NCI-BL2009 lymphoma cells. Apoptosis experiment showed that ScFv-pLLO could induce apoptosis of Raji cells directly. After 24 h of ScFv-pLLO action of 20 u g/mL, apoptosis of 24.6% Raji cells (early + late) could be induced. After the action of 48 h The initial results of the total apoptosis rate of the cell up to 47.0%. show that the bifunctional recombinant protein ScFv-pLLO can specifically target the binding of CD20 positive B cell lymphoma. For some human lymphoma cell lines, such as Raji cells, the apoptosis can be directly induced and the structure and function are similar to "small anti body". At the same time, the dual function recombinant protein ScFv-pLLO is carried out. The dominant antigen peptide, which is different from the pure monoclonal antibody, can enhance the ability of the tumor cells to activate the immune system after binding to the CD20 molecules on the surface of the lymphoma cells. This provides a basis for our next step to study the activation of the immune cells and to stimulate the function of the long effective anti-tumor cell immunity. The third part of China's Journal of Immunology >. The third part: the role of Leptin in the invasion and metastasis of colorectal cancer cells, with the development of tumor molecular biology, new candidate anti-tumor targets are constantly emerging in tumor targeting therapy. In recent years, more and more studies have been conducted on the proliferation of Leptin mediated signals in tumor cells and the invasion of tumor cells in recent years. It has played an important role in the induction and formation of tumor stem cells. The Chinese name is leptin, which is considered as a potential target of effective antitumor targeting therapy.Leptin. It is a non glycosylated protein hormone composed of 167 amino acids, which is secreted mainly by fat cells and participates in the body's energy metabolism. Leptin and its receptor (LEPR/Ob-R) were also expressed, which showed that the malignancy of colorectal cancer was closely related to the expression of Leptin. Some experimental results showed that high concentrations of exogenous Leptin could promote the proliferation of human colorectal cancer cells and enhance their movement and emplacement ability. The exact biological role of endogenous Leptin in the growth and invasion of tumor cells, and the effect of Leptin mediated signaling pathway on the biological behavior of tumor cells is helpful for the development of new antitumor targeting drugs. In the study, we first analyzed the Leptin and its receptor LEPR in several human colorectal cancer cell lines. The expression, including mRNA and protein levels, showed that Leptin and LEPR were generally expressed in human colorectal cancer cell lines, including cell line HCT116, HT-29, COLO201, COLO205 and SW480. in colorectal cancer cell lines HCT116 and HT-29 cells, and we used siRNA to effectively reduce the expression of endogenous Leptin in tumor cells. Cell proliferation was true. The results showed that inhibition of the expression of endogenous Leptin had no effect on the proliferation of HCT116 and HT-29 cells in human colorectal cancer cell lines. Then we detected some genes closely related to the invasion and metastasis of tumor cells by Real-time PCR, including MMP1, MMP9, TIMP-1, E-cadherin, Twist and so on. The results showed that the inhibition of endogenous Le was inhibited. The expression of MMP1 and MMP9 in HCT116 cells was obviously up-regulated (*P0.05), and the expression of TIMP-1, TIMP-2 and E-cadherin was obviously down regulated (*P0.05), and the expression of MMP1 and Vimentin was obviously up regulated in HT-29 cells. In addition, the results of the gelatinase spectrum test showed that the expression of endogenous Leptin was inhibited and the protein lysis activity of MMP9 in HCT116 cells increased in HCT116 cells. The results of the invasion of.Transwell in vitro showed that the number of cells in the Leptin RNAi group of HCT116 cells increased significantly (51) compared with the control group (148 + 17) (51 2 + 23) (*P0.05) showed that the invasiveness of HCT116 cells was significantly enhanced. Different from previous studies, this study focused on the effects of inhibiting the expression of endogenous Leptin in human colorectal cancer cells and on the proliferation and invasiveness of tumor cells. Invasiveness. Previous studies have shown that increasing the concentration of Leptin can promote the proliferation and invasion of tumor cells.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R730.51

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