【作者】 陈宏远；

【导师】 蔡绍皙； 蔡绍晖； 杜军；

【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),属于七次跨膜的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)。高水平表达于肿瘤细胞表面的CXCR4作为乳腺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乳腺癌等肿瘤疾病的转移。因而,通过抑制肿瘤细胞表面CXCR4的功能性表达将为有效防治乳腺癌转移提供新的途径。本课题首次采取细胞内趋化因子(intrakine)突变体策略,构建含有SDF-1的突变体SDF-1α/54与内质网定位片段KDEL的嵌合基因的重组真核表达载体和腺病毒表达载体,其表达的细胞内趋化因子突变体SDF-1α/54/KDEL蛋白可在细胞内与同时与新合成的靶受体CXCR4产生特异性结合,并滞留于内质网形成复合物,随后被泛素蛋白等途径分解。抑制CXCR4在细胞表面的表达,从而阻止了CXCR4/ SDF-1与乳腺癌转移有关的通路,研究结果将为探究以CXCR4为靶点的肿瘤基因治疗提供直接的实验依据。目的:①采用RT-PCR等方法从健康成人骨髓组织中克隆人基质细胞衍生因子(hSDF-1)野生型成熟肽编码序列(SDF-1wt) ,定向插入原核克隆表达质粒pET-30a(+),构建其原核表达系统。以此为基础进行删除C末端结构域的缺失突变,为实施细胞内趋化因子突变体提供物质基础;②构建人SDF-1α细胞内趋化因子突变体SDF-1α/54/KDEL的重组真核表达质粒,并考察其对T细胞性白血病细胞株Molt-4细胞表面受体CXCR4表达的影响,及对细胞趋化和生长的影响,为表型敲除肿瘤细胞表面受体CXCR4以抑制肿瘤的转移提供初步实验依据;③构建携带含细胞内趋化因子突变体SDF-1α/54/KDEL基因片段的重组腺病毒Ad-SDF-1α/54/KDEL,考察其对乳腺癌细胞株MCF-7的CXCR4表型敲除效应,及其对乳腺癌细胞的生长、趋化和侵袭作用的影响;④建立在裸鼠体内肿瘤细胞高表达CXCR4的乳腺癌动物模型,为后续考察重组腺病毒Ad-SDF-1α/54/KDEL对CXCR4表型敲除作用和荷瘤小鼠肿瘤的影响奠定基础。方法:①采用RT-PCR从人骨髓基质细胞克隆野生型hSDF-1wt基因并进行缺少突变产生C端α-螺旋结构域缺失的hSDF-1α/54基因,将二者直接插入原核表达载体pET-30a(+),构建hSDF-1wt和hSDF-1α/54的原核表达系统后,转化大肠杆菌表达BL21(DE3),IPTG进行诱导表达。②以SDF-WT- PET30a(+)重组质粒为模板,PCR扩增SDF-1α/54基因,并在其C末端引入对应于内质网定位信号序列4肽KDEL的编码基因序列,形成细胞内趋化因子突变体SDF-1α/54/KDEL。随后将其亚克隆至真核克隆/表达质粒pcDNA3.1/Myc-His(-)A和pEGFP-C3,构建出两组重组表达质粒:pcDNA3.1/SDF-1α/54/KDEL和pEGFP/SDF-1α/54 / KDEL。测序正确的重组子经提取质粒和纯化后用脂质体转染Cos-7细胞,用Western blot鉴定突变体SDF-1α/54/KDEL的功能性表达,并用激光共聚焦显微镜观察SDF-1/54α/KDEL的融合蛋白在质粒转染后细胞内的定位情况。然后用电穿孔法将经质粒大量提取并纯化的重组体pcDNA3.1/SDF-1α/54/KDEL和pEGFP/SDF-1α/54/KDEL,瞬时转染Molt-4后,用流式细胞仪(FCM)检测细胞膜表面CXCR4含量的变化。FCM检测转染后Molt-4生长情况,趋化性实验检测SDF-1α/54/KDEL转染后对Molt-4表面受体CXCR4的影响。③应用PCR的方法制备含SDF-1α/54/KDEL基因的DNA片段,分别双酶切SDF-1α/54/KDEL和空的穿梭质粒pAdTrack-CMV,再将二者连接,将SDF-1α/54 KDEL亚克隆至腺病毒穿梭质粒载体,构建成pAdTrack-CMV-SDF-1α/54/KDEL,再利用宿主细菌内同源重组的方法将其与E1缺失的腺病毒骨架质粒pAdEasy-1进行同源重组,构建成重组腺病毒质粒Ad-SDF-1α/54/KDEL。鉴定正确后,利用Lipofectamine 2000介导将腺病毒Ad-SDF-1α/54/KDEL感染细胞转染293细胞,大约7天出现病毒空斑。提取重组感染腺病毒的293细胞的mRNA进行SDF-1α/54/KDEL基因转录的RT-PCR鉴定,并配合GFP表达鉴定观察SDF-1α/54/KDEL蛋白的表达。通过细胞传代收获大量扩增的重组腺病毒,测定重组腺病毒的感染复数(MOI)值。体外细胞实验鉴定Ad-SDF-1α/54/KDEL的表型敲除活性:流式细胞仪观察细胞膜CXCR4变化情况; MTT法考察Ad-SDF-1α/54/KDEL对乳腺癌细胞MCF-7增殖作用的影响;趋化性实验检测Ad-SDF-1α/54/KDEL对MCF-7细胞的趋化活性的影响;明胶酶谱和侵袭实验探索重组腺病毒的Ad-SDF-1α/54/KDEL对MCF-7细胞侵袭的影响。④通过裸鼠皮下脂肪垫移植高转移性乳腺癌细胞株MDA-MB-231建立乳腺癌转移动物模型,检测荷瘤小鼠原发瘤和转移瘤各组织的SDF-1α和CXCR4的表达情况,为考察Ad-SDF-1α/54/KDEL表型敲除CXCR4对乳腺癌的转移的影响作好准备。结果:①利用RT-PCR方法从骨髓基质细胞中克隆出人野生型SDF-1(SDF-1wt)基因,对SDF-1进行了删除C端α螺旋结构域的缺失突变形成其突变体SDF-1α/54,测序结果表明SDF-1wt克隆及突变均达到既定目的。②双酶切和测序结果表明SDF-1α/54/KDEL基因重组真核载体pcDNA3.1/ Myc-His(-)A和pEGFP-C3构建成功,Western blot证实转染后的Cos-7细胞有突变体SDF-1α/54/KDEL嵌合基因的表达,进一步的融合蛋白亚细胞定位进一步显示, SDF-1α/54/KDEL和EGFP的融合蛋白主要定位在内质网。两种重组真核表达载体通过电穿孔转染Molt-4细胞后,均可明显降低胞膜表面CXCR4表达量,细胞阳性率降低了90%以上, SDF-1α/54/KDEL和SDF-1α/KDEL对CXCR4的表型敲除效果无显著差异。流式细胞术(FCM)检测电穿孔瞬时转染后Molt-4增殖并未受到影响;趋化性实验提示,瞬时转染pcDNA3.1/SDF-1α/54/KDEL 48h后,Molt-4细胞的趋化作用受到抑制。③构建了腺病毒穿梭质粒pAdTrack-CMV-SDF-1α/54/KDEL,与腺病毒骨架质粒pAdEasy-1共转染293细胞获得了重组腺病毒pAd-SDF-1α/54/KDEL。RT-PCR和和GFP表达鉴定表明包装后的病毒有细胞内趋化因子突变体SDF-1α/54/KDEL基因的转录和表达。重组腺病毒经扩增后,测定其感染复数MOI为100时,对MCF-7感染效率为100%。感染Ad-SDF-1α/54/KDEL 5d和10d后的流式细胞仪检测结果表明Ad-SDF-1α/54/KDEL能够显著抑制MCF-7细胞CXCR4表达,MOI在200以内时,对细胞的生长无抑制作用。MOI为100时,Ad -SDF-1α/54/KDEL可显著抑制MCF-7的趋化性和侵袭性。体外实验表明构建重组腺病毒Ad-SDF-1α/54/KDEL具有对肿瘤细胞表型敲除的效应。④MDA-MB-231乳腺癌细胞接种裸鼠BALB/C后第7天在接种部位可见结节,通过体内驯化传代3代以后肿瘤移植成功率(成瘤率)为100%。病理学检测结果,肝、肺等组织等组织CXCR4阳性,RT-PCR检测鼠SDF-1α在脾、肝、肺组织有表达,初步建立了进行Ad-SDF-1α/54/KDEL活性的乳腺癌体内实验动物模型。结论:①克隆了出人基质细胞衍生因子-1(hSDF-1)基因,并通过基因缺失突变删除了C末端α螺旋结构域,获得了突变体SDF-1α/54,并构建了其大肠杆菌表达系统。②以真核克隆/表达载体pcDNA3.1/Myc-His(-)A和pEGFP-C3为基础构建的两种重组真核表达载体在Cos-7细胞表达SDF-1α/54/KDEL,具有在基因水平实施CXCR4表型敲除的功能,并能抑制Molt-4细胞的趋化性且不影响该细胞的生长。表型敲除效率不受SDF-1αC末端α螺旋结构域缺失的影响。其可能机制是:转染后合成的SDF-1α/54/KDEL新蛋白,运至高尔基复合体时由于KDEL的内质网定位作用,新蛋白不会向细胞膜运转而分泌到细胞外,而是滞留于内质网。由于SDF-1α/54/KDEL保留了与CXCR4结合的主要结构,仍具备识别和结合CXCR4受体的能力。SDF-1α/54/KDEL蛋白便可以在细胞内结合新合成的或内在化的CXCR4形成复合物,随后被泛素-蛋白酶体等途径降解,从而抑制了受体CXCR4在细胞膜上的表达,达到了表型敲除的效果。③重组腺病毒Ad-SDF-1α/54/KDEL能够在乳腺癌细胞株MCF-7中感染和表达、滴度高、增殖快、作用高效、毒副作用小,具有对MCF-7受体CXCR4表型敲除的作用。通过感染乳腺癌MCF-7细胞,表型敲除的效应较重组真核表达载体持续时间长,并能在体外抑制乳腺癌细胞的趋化和侵袭性。其表型敲除机制可能与前述真核表达质粒相同,推测其具有抑制乳腺癌细胞转移的作用。④通过裸鼠皮下脂肪垫移植建立的乳腺癌转移动物模型,成功率高,肿瘤保持了人乳腺癌表达CXCR4/SDF-1的活性,可以用于Ad-SDF-1α/54/KDEL体内活性研究。更多还原

【Abstract】 Chemokine receptor 4 (CXCR4), as one of the seven-transmembrane G-protein-coupled receptor family, is a kind of cell membrane protein, whose chemokine that can bind and activate to which was only known to be Stromal cell-Derived Factor-1 (SDF-1). Due to the interaction between CXCR4 expressed highly on tumor cells and SDF-1 directly involving in the metastasis of tumor disease such as breast cancer, it maybe a new approach to effectively prevent the metastasis of breast cancer by blocking the functional expression of CXCR4 on the surface of tumor cell. In this study, on the basis relationship of CXCR4/SDF-1αstructure-activity relationship, vie an intrakine mutant strategy which contain SDF-1αmutant SDF-1α/54 gene and an endoplasmic reticulum (ER) retention signal (KDEL) to block the expression of CXCR4, the SDF-1α/54/KDEL chimeric gene were amplified and then subcloned into the eukaryotic expression vectors and the adenovirus vector respectively. The newly fusion protein SDF-1α/54/KDEL could be intracellularly expressed in the ER, and simultaneously could specifically bind to the newly-synthetized target receptor CXCR4 and consequently remain in ER, the compouds followed by the ubiquitin proteasome pathway or lysosomal pathway decomposition and so on, by which knockout the expression of CXCR4, thus inhibits cancer metastasis function phenotype. The results will also provide direct experimental evidence to explore CXCR4 as a target for the cancer gene therapy.Objectives:①The mature wild-type peptide-colding sequence (SDF-1wt) of hSDF was cloned from health adult myeloid tissue by RT-PCR, then its prokaryotic expression was constructed by orientally inserting into the prokaryotic expression plasmid pET-30a(+) . Furthermore, deletion mutation was applied by losing the structural domain of C extermial to form SDF-1α/54, which provided the substance for the practice of the intrakine mutant.②To construct the eukaryotic expression plasmid of SDF-1αmutant recombinant SDF-1α/54/KDEL, and investigate its effect on CXCR4 in T-cell leukemia cell line Molt-4 cells as well as the activity of SDF-1α/54/KDEL protein, all of which could provide feasibility for the knockout phenotype of tumor CXCR4 to inhibit tumor metastasis. ③To construct recombinant adenovirus expression vector Ad-SDF-1α/54/KDEL and inspecting their phenotype knockout activity such as the cell growth, chemokines and invasion for breast cancer cell line MCF-7.④To establish a proper model of human breast carcinoma with highly expressing CXCR4 in nude mice for preparation of the effects with purified Ad-SDF-1α/54/KDEL Methods:①Human SDF-1α/54 gene with the depletion ofα-helix at the C-terminal domain acquired by site-directed mutagenesis was obtained from wild-type hSDF-1wt gene with RT-PCR, and then hSDF-1wt and hSDF-1α/54 were inserted into the prokaryotic expression vector pET-30a(+) respectively and sequencely transform the E coli to express BL21(DE3) with the induction of IPTG..②SDF-1α/54 gene was amplified by PCR from SDF-WT- PET30a(+) plasmid, and the coding gene of KDEL corresponding to the rention signal sequence in ER was introduced into its C-terminal. SDF-1α/54/KDEL gene was subcloned into the eukaryotic expression plasmid of pcDNA3.1/Myc-His(-)A and pEGFP-C3 to construct two of expression plasmids: pcDNA3.1/SDF-1α/54/KDEL and pEGFP /SDF-1/54α/ KDEL. Exactly sequencing recons was used to transfect Cos-7 cell with liposome after plasmid isolation and purification, and the functional expression of SDF-1α/54/KDEL mutant protein was assayed by Western blot, as well as the location of fusion protein containing SDF-1/54α/KDEL in the plasmid transfected cell was observed by laser confocal microscopy. The recombinant vector of pcDNA3.1/SDF-1α/ 54/KDEL and pEGFP/SDF-1/54α/KDEL with large plasmids isolation abstraction and purification by electroporation were to transiently transfect CXCR4-positive Molt-4 human T-cell leukemic cell line, and the level of CXCR4 on cell surface was checked by FCM. The growth state of Molt-4 after transfection was examined by FCM, and the phaenotypic knockout activity of Molt-4 after transfected with SDF-1α/54/KDEL was proved by chemotaxis experiment.③SDF-1α/54/KDEL gene was amplifyed by PCR from pcDNA3.1/SDF-1α/54/ KDEL vector, and vacuous shuttle plasmid pAdTrack-CMV were cut by XbaI and XhoI respectively, following the connection of them. SDF-1α/54/KDEL was subcloned into the shuttle plasmid vector to construct pAdTrack-CMV-SDF-1α/54/KDEL, and then it was homologously recombinated with pAdEasy-1 by the means of insider homologous recombination of host bacterium to construct Ad-SDF-1α/54/KDEL. After exact assessment, Ad-SDF-1α/54/KDEL was intransfected in 293-cell mediated by Lipofectamine 2000, virus plaque appeared in 7 days after the transfection. The mRNA was extractedand then assayed by RT-PCR and SDF-1α/54/KDEL expression cluld be observed by GFP. Flourescence Recombinant AD virus was bulkly harvested by passages and the MOI value was assayed. The phenotypic knockout activity of Ad-SDF-1α/54/KDEL was exerted with in vitro. Observation Ad-SDF-1 /54/KDEL on breast cancer cell proliferation of MCF-7 by MTT methods; Ad-SDF-1α/54/KDEL on MCF-7 cell chemotactic activity; Gelatin zymography and invasion experimental verification the inhibitory effect of recombinant adenovirus Ad-SDF-1α/54/KDEL on MCF-7 cell.④The animal model of breast cancer metastasis was established by transplanting MDA-MB-231 cell into the subcutaneous fat pad of nude mice to detect the expression of SDF-1 and CXCR4 in the primary tumor tissue and metastatic tumor tissue in tumor-bearing mice, with an aim to get ready to investigate the metastasis role of Ad-SDF-1α/54/KDEL for the transfer of breast cancer.Results:①Human SDF-1 wild-type (SDF-1wt) gene was amplified by RT-PCR from adult bone marrow stromal cells; Using PCR to delete the SDF-1 C-terminalα-helix domain. The sequencing results showed SDF-1wt cloning and mutation is correct.②Enzymes digestion and sequencing results demonstrate successful construction of SDF-1α/54/KDEL gene recombinate eukaryotic vector: pcDNA3.1/Myc-His (-) A and pEGFP-C3. Western blot analysis confirmed transfected Cos-7 cells with the mutant SDF-1 /54/KDEL chimeric gene expression and further fusion protein intracellular localization. Furthermore, SDF-1α/54/KDEL and EGFP fusion protein was localized ER. Two recombinate eukaryotic expression vectors were transfected by electroporation to Molt-4 cell, this could significantly downregulation the expression of CXCR4. The positive rate of cells and fluorescence intensity are more than 90%, SDF-1α/KDEL and SDF-1α/54/KDEL effect have no significant difference. Molt-4 proliferation has not been affected after transient transfection detected by Flow cytometry (FCM); Chemotaxis experiments suggest that Molt-4 cells chemotactic role is inhibited.③The adenovirus shuttle vector pAdTrack-CMV-SDF-1α/54/KEL was constructed, and the recombinant adenovirus Ad-SDF-1α/54/KDEL was obtained by co-intransfecting pAdEasy-1 into 293 cells. RT-PCR and GFP expression identification showed there is the transcription and expression of SDF-1α/54/KDEL in the packaged virus. The plural MOI infection of amplified Adenoviruses was 0~200, and its infection efficiency for MCF-7 was 100% when MOI was 100. FCM analysis revealed that Ad-SDF-1α/54/KDEL infected within 10 days could still significantly down-regulate the expression of CXCR4 in MCF-7 cells. Cell growth and proliferation were not influenced to a certain extent of MOI 200. When MOI was 100, MCF-7 cells infected by Ad-SDF-1α/54/KDEL for 3 days can significantly inhibit its chemotaxis and invasion.④Nodules can be seen at the transplanting sites after implantting MDA-MB-231 breast cancer cells into nude mice BALB/C for seven days. The successful rate of tumor transplantation was 100% after the cancer cells were vivo domesticated and passaged for three generation. Pathology test results showed the positive expression of CXCR4 in the tissue of liver and lungs. RT-PCR of SDF-1αdisplayed it expressed in the tissue of spleen, liver, lung of nude mice, all of which contributed for the primary establishment of in vivo animal model for the Ad-SDF-1α/54/KDEL activity of breast cancer.Conclusions:①Human stromal cell derived factor-1 (hSDF-1) gene was successfully cloned, and the acquirement of SDF-1α/54 mutant could be done through deleting the C-terminalα-helix domain with the technique of gene deletion mutant, and so dose E. coli expression system..②SDF-1α/54/KDEL expressed in two different Eukaryotic expression vectors has the ability of CXCR4 phenotype knockout at gene-level after the transfection of Molt-4 cells by electroporation, and also has the ability to inhibit chemotaxis of Molt-4 cells and has no effect on the growth of Molt-4 cells. The activity of knockout phenotype were not affected by the loss of C-terminalα-helix domain of SDF-1α.The possible mechanism maybe that the post-transfected newly-synthesized SDF-1αprotein, due to the ER location function of KDEL, when it was transferred to Goergi, will not be transferred toward cell membrane and secreted out but remain in ER. Because the main structure of SDF-1α/54/KDEL with the ability to bind to CXCR4 has been reserved, it still has the ability to recognize and bind to CXCR4. Thus SDF-1α/54/KDEL protein could combine the newly synthesized or internalization CXCR4 to form a compound, then the compound may be breakdown by the pathway of ubiquitin proteolysis or other intracellular mechanism, resulting in the expression inhibition of CXCR4 on the cell membrane and the achievement of phenotypic knockout. ③Recombinate adenovirus Ad-SDF-1α/54/KDEL has a good activity of phenotypic knockout for breast cancer cells MCF-7, which was characteristics of time-prolonged action, high-titer, quick proliferation, high efficiency for action and low toxicity, thus make it to be an effective tool carrier for the breast cancer metastasis research. The inhibition function of CXCR4 knockout phenotype in vitro for breast cancer cell chemotaxis and invasion is based on the CXCR4 inhibition at the target.④A human breast carcinoma model in nude mice was established, which reserved certain biological features of CXCR4/SDF-1,therefore it may be an available model for the further research of the activity of Ad-SDF-1α/54/KDEL in vivo.更多还原