【作者】 郭芝刚；

【导师】 蔡绍皙；

【作者基本信息】 重庆大学 ， 生物医学工程， 2007， 博士

【摘要】 趋化因子受体CXCR4(CXC chemokine receptor 4),属于七次跨膜G蛋白偶联受体家族的细胞膜蛋白,目前已知唯一能与其结合并能激活它的趋化因子是基质细胞衍生因子1(Stromal cell-derived factor-1,SDF-1),又称CXCL12。高水平表达于肿瘤细胞表面的CXCR4作为卵巢癌发生或者转移的一个生物学表型,可以通过与SDF-1的相互作用直接参与卵巢癌等肿瘤疾病的转移。表皮生长因子受体EGFR (Epidermal Growth Factor Receptor)是存在于细胞表面的跨膜糖蛋白分子,在生理状态下是细胞的生长调节因子,EGFR的过度表达干扰细胞分化的正常调控状态,引起细胞持续增殖,发生恶性转化。近年的研究证实:在共表达CXCR4和EGFR的癌细胞中,两个受体间存在一个复杂的网络,通过受体间的“cross-talk”,肿瘤的恶性程度增加,但其详细的调控机理仍不清楚。本研究用一株共表达CXCR4和EGFR的卵巢癌细胞SKOV3作为研究材料,初步研究了CXCR4和EGFR作用下的癌细胞侵袭与转移机制。研究发现:①在EGF的作用下,PI-3K/AKT通路激活,表达趋化因子受体CXCR4的细胞阳性率增加,细胞的趋化能力增强;②PI-3K/AKT通路参与了EGF和SDF-1作用下的与肿瘤细胞侵袭能力密切相关的MMP9表达水平增高。结合同行的研究结果,我们可知在CXCR4和EGFR的作用下,癌细胞的增殖、侵袭、转移等能力增加,肿瘤细胞恶性转化。研究结果与新近报道的共表达CXCR4和EGFR的肿瘤患者预后差,综合生存率低的临床结果相吻合。肿瘤的恶性转变是一个复杂的过程,单纯地抑制某个靶点并不能完全消除肿瘤,而同时针对两个或多个靶点进行治疗则是更有效的治疗方法。以该机理为理论基础,我们对野生型hSDF-1进行定向遗传改造,获得对CXCR4具高拮抗活性效应的hSDF-1突变体SDF-1/54。在此基础上,借助抗体重链铰链片段Hinge将重组Decorin成熟肽与SDF-1/54突变体连接,构建了双重靶效应的Decorin与SDF-1/54突变体基因重组嵌合体SDF-1/54-DCN。该嵌合体具有以下特点:①因能同步识别CXCR4和EGFR高表达的卵巢肿瘤细胞,可更有选择性地大量聚集在卵巢原发肿瘤及其转移灶组织;②利用SDF-1/54突变体结构中CXCR4拮抗性配体部分与CXCR4特异性结合,封闭CXCR4,破坏了在原发灶与远程特定转移器官间存在的SDF-1梯度,切断卵巢癌转移的潜在通路;③利用其结构中Decorin,特异性阻断EGFR高表达所产生的卵巢癌浸润性生长和转移产生的效应。将嵌合重组体基因SDF-1/54-DCN装入原核表达载体pET30a(+),在大肠杆菌BL21(DE3)中成功地以包涵体形式表达,通过包涵体复性和蛋白纯化,获得了纯度在85%以上的活性蛋白。在体外,选用高表达CXCR4的MOLT-4细胞,高表达EGFR的A431细胞和共表达CXCR4和EGFR的SKOV3细胞,对SDF-1/54-DCN嵌合体蛋白细胞活性进行检测。结果显示该嵌合体蛋白具有以下活性:①诱导CXCR4和EGFR的内在化;②抑制表达CXCR4的癌细胞在SDF-1作用下的趋化;③对表达EGFR癌细胞具有增殖抑制的活性。研究结果表明我们成功获得了具有SDF-1/54突变体和Decorin双重活性的SDF-1/54-DCN基因重组嵌合体蛋白,它能同时针对CXCR4和EGFR发挥相应的生物活性,将为探究以CXCR4和EGFR为靶点的双靶向肿瘤治疗提供直接的实验依据。更多还原

【Abstract】 Stromal cell-derived factor-1(SDF-1), also named SDF-1 is known as the only chemokine until now that could combine and activate the chemokine receptor 4(CXCR 4), a membrane protein as one member of G protein-coupled receptor family. CXCR4 highly expressing on the surface of tumor cell plays a role as a biological phenotype. It functioned in cancer metastasis by the interaction with SDF-1. Epidermal growth factor receptor is one kind of transmembrane glycoprotein which serves as a growth regulation factor in Physiological status. However overexpression of EGFR break cell’s normal growth regulation status with a consequence of continuous proliferation and malignant transformation. Recent researches have discovered in tumor cells expressing both EGFR and CXCR4 that a complex network between these two receptors deepens the malignant degree by their“cross-talk”with the accurate mechanism still unknown by now.After screening, we choose ovarian cancer cell SKOV3 which coexpresses CXCR4 and EGFR to investigate the synergistic effect of these two recptors on cancer invasion and metastasis. Our research suggested that:①PI-3k/AKT pathway was activated by EGF causing an increase of CXCR4-positive cells as well as their chemotaxis.②Affected by EGF and SDF-1,PI-3k/AKT pathway functions in the increase of MMP9, a enzyme closely related to cancer metastasis. Supported by other researches in this field, it is obvious that cancer proliferation, invasion, and metastasis as well as cancer malignant transformation are promoted by the synergistic effect of EGFR and CXCR4. These research results are correspondent with recent clinical reports on CXCR4 and EGFR-positive tumor bearing patients who suffered from high grading tumor, bad prognosis with a disappointing general survival rate. Outcomings indicates that malignant transformation of cancer is a complicated course, which suggests independent inhibition of one certain target is not enough to eliminate cancer, and only if two or more targets are aimed can tumor be thoroughly wiped out.Basing on theory above, we obtain mutant CXCL12 which posses high rivalry activity against CXCR4 by oriented reconstruction on wild CXCL12.Then this new CXCL12 is connected to Decorin mature peptide bridged by antibody heavy chain hinge section, in which way the final Decorin and CXCL12 mutant chimeric protein SDF-1/54-Decorin possessing a dual targeting effect is constructed. This chimeric protein is characterized by the following:It can be concentrated selectively around ovarian cancer, either primartumor or metastasis tissue since it recognizes both EGFR and CXCR4. SDF-1 grad between primartumor and remote specific metastasis organ is interrupted by blocking CXCR4 when it is being binded by rivalry section of mutant SDF-1. Decorin induce specific blockage of EGFR, which further decrease the infiltrative growth and metestate of ovarian cancer.Chimeric gene SDF-1/54-Decorin is inserted into prokaryotic expression vector pET30(+) and successfully expressed as cytorrhyctes in BL21. Active protein with its purity beyond 85% is collected by cytorrhyctes renaturation and purification.In vitro, Molt-4 cell highly expressing CXCR4, A431 cell highly expressing EGFR, SKOV3 highly expressing both are chosen to test the chimeric protein activities in cell which is shown as following:①chimeric protein can induce internalization of EGFR and CXCR4.②chimeric protein can inhibit cancer cell chemotaxis.③chimeric protein can inhibit cancer cell proliferation. Results demonstrate the SDF-1/54-Decorin chimeric protein owning duel effect of both CXCR12 and Decorin could exert relative bioactivities targeting both CXCR4 and EGFR, which provides direct and significant trial support for duel target therapy.更多还原