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

【摘要】：第一部分：可激活免疫細胞靶向EGFR高表達腫瘤細胞重組蛋白的設計、表達及對腫瘤的治療作用腫瘤靶向治療是當前癌癥治療的研究熱點,并代表未來發(fā)展的方向。而腫瘤免疫治療是繼手術、放療和化療之后的第四種腫瘤治療模式—腫瘤生物治療,具有廣闊的應用前景。有效地將兩者結(jié)合起來是研發(fā)新型抗腫瘤藥物的一大趨勢。目前在抗腫瘤靶向藥物的開發(fā)中,表皮生長因子受體(Epidermal growth factor receptor, EGFR)是公認的、選用最多的靶向治療靶點�，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)。但是在應用過程中發(fā)現(xiàn)有顯著一部分患者對這兩類藥物均明顯耐藥,而最初敏感的患者在經(jīng)過治療后,轉(zhuǎn)而發(fā)展為耐藥,最終導致預后較差。通過分子水平的機制研究發(fā)現(xiàn)這種耐藥多是由于EGFR激酶結(jié)構(gòu)域存在突變,或其下游信號通路的持續(xù)活化造成。在這種情況下,腫瘤細胞不再依賴表皮生長因子(Epidermal growth factor, EGF)結(jié)合EGFR的啟動激活信號,卻可以持續(xù)地生長增殖,腫瘤細胞呈自主性生長狀態(tài)。因此針對EGF-EGFR信號不敏感的腫瘤細胞,需要新的治療方法。本研究旨在開發(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)動機體的免疫系統(tǒng),將抗腫瘤免疫治療機制運用至新型藥物中,本研究將顯性抗原肽構(gòu)建至重組蛋白,組成一個EGFR靶向的雙功能重組免疫調(diào)節(jié)蛋白,用于高表達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,將其與來自李斯特菌溶細胞素O (Listeriolysin O, LLO)的3個顯性T細胞表位融合,通過序列拼接構(gòu)建了重組融合蛋白pLLO-hEGF。人EGF是一個由53個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是一個已經(jīng)證實的強大的免疫原性分子,具有豐富的CD4+和CD8+T細胞抗原表位。本研究選用了經(jīng)文獻證實的2個CD4+和1個CD8+T細胞表位。所構(gòu)建的重組蛋白pLLO-hEGF經(jīng)生物信息學軟件分析,理論分子量僅為16 kDa,性質(zhì)穩(wěn)定,血管穿透性將優(yōu)于mAb。同時該重組蛋白應具有2種功能：既可以通過EGF靶向結(jié)合高表達EGFR腫瘤細胞,同時其LLO的顯性抗原表位又可以充分活化機體的免疫細胞,使免疫細胞在腫瘤細胞部位聚集,從而加強攻擊和殺傷腫瘤細胞,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以及蛋白表達純化技術,成功克隆表達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。接下來通過Western blot篩選了高表達EGFR的人腫瘤細胞系,從不同細胞系中各挑選出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相關的功能研究。細胞免疫化學熒光染色實驗結(jié)果顯示了pLLO-hEGF可以很好地靶向結(jié)合這些腫瘤細胞表面。細胞增殖實驗結(jié)果顯示pLLO-hEGF對這些腫瘤細胞不產(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)的人外周血單個核細胞(peripheral blood mononuclear cells, PBMCs)中 CD3+CD4+ T細胞明顯增殖。單次實驗結(jié)果顯示pLLO-hEGF刺激14天時PBMCs中CD3+CD4+ T細胞百分比可增至54.5%,而對照組為44.8%。且刺激增殖活化的T淋巴細胞在體內(nèi)外實驗中均顯示出顯著的殺傷腫瘤細胞作用。體外對上述幾種表達EGFR腫瘤細胞的殺傷率分別為：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)移植瘤模型實驗結(jié)果顯示,將刺激增殖的T淋巴細胞注射裸小鼠體內(nèi),顯著抑制了腫瘤的生長,平均瘤重(0.178/0.224 g)明顯小于對照組(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)容申請了國家發(fā)明專利(申請?zhí)枺?01410152549.2),目前在實審階段,且相關內(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：可激活免疫細胞靶向CD20陽性人淋巴瘤細胞重組蛋白的設計、表達及抗淋巴瘤作用的初步研究B細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CD20分子結(jié)構(gòu)特點的認識,抗CD20單克隆抗體的設計開發(fā)在B細胞淋巴瘤的臨床治療中取得重要進展。其中最具代表性的是1997年美國FDA批準上市的人鼠嵌合抗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,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。其中單鏈抗體(single chain variable fragments, ScFv)由于其分子量小、穿透力強、能夠較好地保持抗原親和性等特點,成為應用生物工程方法進行腫瘤免疫治療的一種重要手段。本研究同樣利用LLO的顯性抗原肽設計、構(gòu)建了靶向CD20陽性B細胞淋巴瘤的雙功能重組蛋白ScFv-pLLO。首先,我們設計了針對人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陽性B細胞淋巴瘤細胞,同時其LLO抗原表位又可增強腫瘤細胞的抗原性,從而充分激活機體的細胞免疫應答,發(fā)揮更長效的抗腫瘤免疫,達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、表達和純化,并初步分析了其抗B細胞淋巴瘤作用。生物信息學軟件分析顯示重組蛋白理論分子量為32 kDa,化學性質(zhì)穩(wěn)定。流式細胞術定量分析幾種人淋巴瘤細胞系細胞表面CD20表達情況,結(jié)果顯示Daudi細胞CD20陽性率在98%以上,Raji和NCI-BL2009細胞CD20陽性率分別為89.7%和92.2%,而RAMOS細胞CD20陽性率是52.3%。流式細胞術分析ScFv-pLLO靶向結(jié)合人淋巴瘤細胞的能力,結(jié)果顯示ScFv-pLLO可不同程度的靶向結(jié)合CD20表達水平不同的人淋巴瘤細胞,其濃度為10μg/mL時,對Daudi和NCI-BL2009細胞的平均結(jié)合率分別為(93.25±3.45)%和(73.85±3.95)%。免疫共沉淀實驗結(jié)果進一步證明ScFv-pLLO可特異靶向結(jié)合人淋巴瘤細胞表面的CD20分子。細胞增殖實驗結(jié)果顯示ScFv-pLLO可抑制Raji細胞的生長,而對Daudi、RAMOS和NCI-BL2009淋巴瘤細胞的生長無影響。凋亡實驗結(jié)果顯示ScFv-pLLO可直接誘導Raji細胞凋亡,20μg/mL的ScFv-pLLO作用24 h后,可誘導24.6%的Raji細胞凋亡(早期+晚期),作用48 h后,Raji細胞總凋亡率可達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陽性B細胞淋巴瘤,對某些人淋巴瘤細胞系,如Raji細胞,可直接誘導其凋亡,結(jié)構(gòu)和功能上類似“小型抗體”。同時,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)合淋巴瘤細胞表面CD20分子后可增強腫瘤細胞激活免疫系統(tǒng)的能力,從而為我們下一步研究其對免疫細胞的激活作用以及激發(fā)長效抗腫瘤細胞免疫的功能奠定了基礎。此部分內(nèi)容已發(fā)表于《中國免疫學雜志》。第三部分：Leptin在結(jié)直腸癌細胞侵襲轉(zhuǎn)移中的作用研究隨著腫瘤分子生物學研究的深入,在腫瘤靶向治療中,不斷有新的候選抗腫瘤靶點出現(xiàn)。近幾年,越來越多的研究發(fā)現(xiàn)Leptin介導的信號在腫瘤細胞的增殖、侵襲和腫瘤干細胞的誘導生成中發(fā)揮了重要作用,并被認為是潛在的有效抗腫瘤靶向治療靶點。Leptin的中文名稱為瘦素,是一種由167個氨基酸組成的非糖基化蛋白質(zhì)類激素,主要由脂肪細胞分泌,參與機體的能量代謝。研究表明腫瘤組織也可表達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的表達量密切相關。一些實驗結(jié)果顯示高濃度的外源性Leptin可以促進人結(jié)直腸癌細胞的增殖,并增強其運動和侵襲能力。但目前較少關于腫瘤細胞產(chǎn)生的內(nèi)源性Leptin在腫瘤細胞生長和侵襲力方面確切生物學作用的研究,而明確Leptin介導的信號通路對腫瘤細胞生物學行為的影響,有助于開發(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)直腸癌細胞系中Leptin及其受體LEPR的表達情況,包括mRNA和蛋白水平。結(jié)果顯示人結(jié)直腸癌細胞系均普遍表達Leptin和LEPR,包括細胞系HCT116、HT-29、COLO201、COLO205和SW480。以人結(jié)直腸癌細胞系HCT116和HT-29細胞為例,我們用siRNA有效敲低了腫瘤細胞中內(nèi)源性Leptin的表達。細胞增殖實驗結(jié)果顯示,抑制內(nèi)源性Leptin的表達對人結(jié)直腸癌細胞系HCT116和HT-29細胞的增殖無影響。然后我們通過Real-time PCR檢測了一些與腫瘤細胞侵襲轉(zhuǎn)移能力密切相關基因的表達情況,包括MMP1、MMP9、 TIMP-1、E-cadherin、Twist等。結(jié)果顯示,抑制內(nèi)源性Leptin表達,HCT116細胞中MMP1和MMP9的表達明顯上調(diào)(*P0.05),TIMP-1、TIMP-2 和 E-cadherin的表達明顯下調(diào)(*P0.05)；HT-29細胞中,MMP1和Vimentin的表達明顯上調(diào)(*P0.05),TIMP-2的表達明顯下調(diào)(*P0.05)。以HCT116為例,Western blot進一步檢測蛋白表達水平變化,其結(jié)果與mRNA水平變化一致。此外,明膠酶譜實驗結(jié)果顯示,抑制內(nèi)源性Leptin表達,HCT116細胞中MMP9的蛋白裂解活性增強。Transwell體外侵襲實驗結(jié)果顯示,與對照組穿膜細胞數(shù)(148±17)相比,HCT116細胞Leptin RNAi組穿出小室微孔膜的細胞數(shù)明顯增多(512±23)(*P0.05),表明HCT116細胞的侵襲能力明顯增強。本研究不同于以往的研究,重點分析了抑制人結(jié)直腸癌細胞內(nèi)源性Leptin的表達,對腫瘤細胞增殖和侵襲能力的影響。研究發(fā)現(xiàn),降低Leptin的表達對細胞增殖無影響,但卻明顯增強了細胞的侵襲能力。而以往的研究結(jié)果是提高Leptin的濃度會促進腫瘤細胞的增殖和侵襲。因此,進一步探明不同條件下Leptin信號通路在結(jié)直腸癌細胞中的具體生物學作用機制,可為確定此通路能否用于腫瘤靶向治療靶點選擇奠定基礎。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R730.51

【參考文獻】

相關期刊論文 前5條

1 German G.Gomez;Jill Wykosky;Ciro Zanca;Frank B.Furnari;Webster K.Cavenee;;Therapeutic resistance in cancer: microRNA regulation of EGFR signaling networks[J];Cancer Biology & Medicine;2013年04期

2 甘紹舉;王青;朱麗敏;謝浩;丁先鋒;;靶向治療藥物在乳腺癌中的研究進展[J];基礎醫(yī)學與臨床;2015年01期

3 張久丁;張冠一;師明磊;胡顯文;;CD20生物學功能的研究進展[J];生物技術通訊;2009年02期

4 ;Leptin: a multifunctional hormone[J];Cell Research;2000年02期

5 Kevin Becker;Yiqing Xu;;Management of tyrosine kinase inhibitor resistance in lung cancer with EGFR mutation[J];World Journal of Clinical Oncology;2014年04期

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