抗MRP3单链抗体与sTRAIL融合蛋白的克隆表达及其对多形性胶质母细胞瘤的靶向凋亡诱导作用研究

发布时间：2018-06-04 21:37

本文选题：多形性胶质母细胞瘤 + 多药耐药相关蛋白3　；参考：《大连医科大学》2012年博士论文

【摘要】：多形性胶质母细胞瘤（GBM）是成人最常见、恶性度最高的原发脑肿瘤，WHO将其归类为Ⅳ级星形细胞瘤。尽管手术技术不断进步，放疗结合新一代化疗药物的应用，GBM患者的预后仍然极差，中位生存期仅为15个月左右，迫切需要新的治疗方法。近年提出针对肿瘤抗原的“抗体导向”治疗新理念，以抗体为载体，将药物导向肿瘤病灶，提高药物疗效，减少药物毒副作用，代表肿瘤免疫治疗的新方向。多药耐药相关蛋白3（MRP3）在人类GBM实体瘤和细胞系中均呈高水平表达，且表达于肿瘤细胞膜表面。相反，MRP3在正常脑组织中不表达。因此，MRP3可作为抗体导向治疗GBM的理想分子靶点。2010年，Kuan等利用噬菌体展示技术，分离得到抗MRP3的人源重组单链抗体（scFv），能够与人类MRP3胞外区N端的氨基酸残基特异性结合，是抗体导向治疗GBM的理想抗体分子。肿瘤坏死因子相关凋亡诱导配体（TRAIL），又称凋亡素-2配体（APO-2L），属TNF超家族成员，通过与死亡受体TRAIL-R1（DR4）和TRAIL-R2（DR5）结合，激活外源性凋亡途径，诱导caspase依赖的细胞凋亡。TRAIL具有显著的凋亡肿瘤细胞凋亡活性而对正常细胞没有毒性，被视为最具有临床应用前景的抗癌药物之一。天然TRAIL为膜结合型，其胞外区含有蛋白酶作用位点，可以从细胞膜上被剪切下来，或通过mRNA剪接作用，形成可溶性TRAIL（sTRAIL），仍然保留天然TRAIL的选择性凋亡诱导活性。本研究利用基因重组技术，首次将抗MRP3的单链抗体与仅对肿瘤细胞有很强杀伤作用的sTRAIL通过柔性氨基酸连接臂(Gly4-Ser)3相连，构建抗MRP3的单链抗体与sTRAIL的融合蛋白，命名为antiMRP3(scFv)-sTRAIL，以期通过antiMRP3(scFv)与MRP3的特异结合，加强sTRAIL在GBM细胞膜表面的富集，达到靶向诱导GBM凋亡的目的。本论文将从以下三部分阐述antiMRP3(scFv)-sTRAIL融合蛋白对GBM的靶向凋亡诱导作用及其机制。第一部分antiMRP3(scFv)-sTRAIL融合蛋白的诱导表达、纯化及其生物学活性鉴定利用pMAL原核表达系统诱导表达与纯化antiMRP3(scFv)-sTRAIL融合蛋白，并鉴定其生物学活性。通过重组PCR技术分别扩增antiMRP3(scFv)和sTRAIL基因片段，插入原核表达质粒pMAL-c2，构建重组表达质粒pMAL-antiMRP3(scFv)-sTRAIL，经菌落PCR、酶切鉴定及DNA测序证明构建的重组质粒与预期完全一致。将重组质粒转化感受态E.coli BL21，IPTG诱导表达，得到带有麦芽糖结合蛋白（MBP）标签肽的antiMRP3(scFv)-sTRAIL融合蛋白，经Amylose Resin亲和层析柱纯化，SDS-PAGE鉴定纯化的融合蛋白。结果显示，在92kD处观察到纯化的融合蛋白条带。融合蛋白在E.coli BL21中的表达率约30%，纯化的融合蛋白纯度达95%。以MRP3阳性U251多形性胶质母细胞瘤细胞为研究对象，用MTT法分别测定不同浓度antiMRP3(scFv)-sTRAIL融合蛋白和MBP蛋白作用24h后对U251细胞的增殖抑制率，结果表明antiMRP3(scFv)-sTRAIL融合蛋白可显著抑制U251细胞的增殖并呈浓度依赖性，而MBP蛋白无明显作用。应用IC50剂量（62.5nmol/L）的antiMRP3(scFv)-sTRAIL融合蛋白作用U251细胞24h后，倒置显微镜下观察U251细胞形态的改变。antiMRP3(scFv)-sTRAIL融合蛋白处理组U251细胞在形态学上呈现明显细胞凋亡改变，而MBP蛋白组和未处理组细胞未发生异常形态改变。上述结果表明成功构建了pMAL-antiMRP3(scFv)-sTRAIL重组质粒，并实现了原核表达与纯化antiMRP3(scFv)-sTRAIL融合蛋白。初步的生物学功能鉴定表明该融合蛋白具有显著诱导U251多形性胶质母细胞瘤细胞凋亡的活性，为进一步研究其对U251细胞的靶向凋亡诱导作用和机制奠定了基础。第二部分antiMRP3(scFv)-sTRAIL融合蛋白对U251多形性胶质母细胞瘤的靶向凋亡诱导作用通过荧光激活细胞分选技术（FACS）分析antiMRP3(scFv)-sTRAIL融合蛋白与MRP3阳性U251细胞和MRP3阴性Jurkat细胞的抗原特异性结合。结果显示antiMRP3(scFv)-sTRAIL融合蛋白可特异性结合至MRP3阳性U251细胞膜表面，其结合可被亲代antiMRP3(scFv)竞争性阻断，而antiMRP3(scFv)-sTRAIL融合蛋白与MRP3阴性Jurkat细胞未见明显细胞膜表面结合，表明antiMRP3(scFv)-sTRAIL融合蛋白与U251细胞的结合为MRP3抗原特异性。观察应用亲代antiMRP3(scFv)竞争性阻断antiMRP3(scFv)-sTRAIL与MRP3的靶向结合，或TRAIL活性中和抗体mAb2E5中和sTRAIL活性，对antiMRP3(scFv)-sTRAIL融合蛋白凋亡诱导活性的影响。用MTT法检测应用不同浓度antiMRP3(scFv)-sTRAIL作用于U251细胞24h后的细胞存活率，结果显示antiMRP3(scFv)-sTRAIL呈现较强杀伤U251细胞作用，在24h内随融合蛋白浓度增加，U251细胞存活率逐渐降低。应用antiMRP3(scFv)或mAb2E5预孵育后，antiMRP3(scFv)-sTRAIL对U251细胞的杀伤作用被显著抑制，各浓度梯度下均无明显的杀伤活性，MBP蛋白对U251细胞的存活率没有影响。用流式细胞仪检测IC50剂量（62.5nmol/L）的antiMRP3(scFv)-sTRAIL作用8h、12h、24h后U251细胞的凋亡情况，与亲代antiMRP3(scFv)和未处理组对比得出，antiMRP3(scFv)-sTRAIL作用8h时U251细胞即发生明显凋亡，24h内细胞凋亡率随诱导时间延长而上升。应用antiMRP3(scFv)或mAb2E5预孵育，antiMRP3(scFv)-sTRAIL对U251细胞的凋亡诱导作用被完全阻断，结果与MTT检测相符。上述结果表明，作为抗体导向的免疫毒素，antiMRP3(scFv)-sTRAIL融合蛋白仅与MRP3阳性U251细胞靶向结合并诱导其凋亡，其结合为MRP3抗原特异性。antiMRP3(scFv)-sTRAIL融合蛋白的凋亡诱导活性可被亲代antiMRP3(scFv)竞争性抑制或被TRAIL活性中和抗体阻断，由此证明antiMRP3(scFv)-sTRAIL通过antiMRP3(scFv)单链抗体导向部分特异性结合MRP3抗原，实现靶向诱导GBM细胞凋亡，其凋亡诱导活性是由sTRAIL部分介导的，而非靶向结合的antiMRP3(scFv)-sTRAIL不具备明显凋亡诱导活性。第三部分antiMRP3(scFv)-sTRAIL融合蛋白对TRAIL-R1和TRAIL-R2的激活作用 TRAIL-R1（DR4）和TRAIL-R2（DR5）是介导外源性细胞凋亡途径的两种死亡受体，，其中DR5在多种肿瘤中的表达水平高于DR4，因而在诱导肿瘤凋亡中处于主导地位。研究表明，天然膜结合型TRAIL可激活TRAIL-R1和TRAIL-R2两种死亡受体，而可溶性sTRAIL只保留了对TRAIL-R1的激活作用。为分析antiMRP3(scFv)-sTRAIL对U251细胞两种死亡受体的激活情况，用real-time PCR法首先分析U251细胞中TRAIL-R1和TRAIL-R2的mRNA表达水平，结果表明TRAIL-R1和TRAIL-R2在U251细胞中均呈阳性表达，其中TRAIL-R2的表达水平高于TRAIL-R1。用抗-TRAIL-R1和抗-TRAIL-R2阻断抗体孵育U251细胞1h，分别单独或同时阻断TRAIL-R1和TRAIL-R2，然后应用IC50剂量的antiMRP3(scFv)-sTRAIL处理U251细胞24h，流式细胞仪检测细胞凋亡率，分析antiMRP3(scFv)-sTRAIL对两种死亡受体的激活作用。结果显示，阻断TRAIL-R1凋亡率下降28%，阻断TRAIL-R2凋亡率下降35%，同时阻断TRAIL-R1和TRAIL-R2凋亡率下降46%。最后，用Western blot方法验证了antiMRP3(scFv)-sTRAIL融合蛋白激活外源性凋亡途径，诱导caspase依赖的细胞凋亡。上述结果表明，antiMRP3(scFv)-sTRAIL融合蛋白与靶抗原特异性结合将sTRAIL由可溶性形式转变为天然TRAIL的膜结合型形式，不仅能够激活TRAIL-R1，而且恢复了其对TRAIL-R2的激活能力，增强sTRAIL的凋亡诱导活性，从而获得增强的抗肿瘤作用。antiMRP3(scFv)-sTRAIL融合蛋白通过结合TRAIL-R1和/或TRAIL-R2两种死亡受体，激活外源性细胞凋亡途径，诱导caspase依赖的细胞凋亡。
[Abstract]:Glioblastoma multiforme (GBM) is the most common and most malignant primary brain tumor in adults. WHO is classified as grade IV astrocytoma. Although the surgical technique is progressing, radiotherapy combined with the application of a new generation of chemotherapeutic drugs, the prognosis of GBM patients is still very poor, the median survival time is only about 15 months, and a new treatment is urgently needed. In recent years, a new concept of "antibody oriented" therapy for tumor antigen is proposed. Using antibody as the carrier, it will guide the tumor to improve the efficacy of the drug, reduce the side effects of the drug, and represent the new direction of the tumor immunotherapy.
Multidrug resistance related protein 3 (MRP3) expressed high level in human GBM solid tumor and cell line, and expressed on the surface of tumor cell membrane. On the contrary, MRP3 was not expressed in normal brain tissue. Therefore, MRP3 can be used as an ideal molecular target for antibody directed treatment of GBM for.2010 years. Kuan and other uses phage display technology to isolate the human source of anti MRP3. Recombinant single chain antibody (scFv) can bind specifically to the amino acid residues at the N end of the extracellular domain of human MRP3, and is an ideal antibody molecule for antibody directed therapy of GBM.
The tumor necrosis factor related apoptosis inducing ligand (TRAIL), also known as apoptin -2 ligand (APO-2L), is a member of the TNF superfamily. By binding to the death receptor TRAIL-R1 (DR4) and TRAIL-R2 (DR5), the apoptosis pathway is activated, and the apoptosis.TRAIL of the caspase dependent apoptosis.TRAIL has significant apoptotic cell apoptosis activity, but not to normal cells. Toxicity is considered as one of the most promising anticancer drugs. Natural TRAIL is a membrane binding type, and its extracellular domain contains protease action sites, which can be cut off from the cell membrane or through mRNA splicing to form soluble TRAIL (sTRAIL), and still retain the selective apoptosis inducing activity of Titian TRAIL.
In this study, we used gene recombination technology to combine the anti MRP3 single chain antibody with the sTRAIL only with the tumor cells which had a strong killing effect through the flexible amino acid connection arm (Gly4-Ser) 3, to construct a fusion protein of the single chain antibody against MRP3 and sTRAIL, named antiMRP3 (scFv) -sTRAIL, in order to combine antiMRP3 (scFv) with MRP3. To enhance the concentration of sTRAIL on the surface of GBM cell membrane to achieve the goal of targeting GBM apoptosis, this paper will explain the target apoptosis inducing effect and mechanism of antiMRP3 (scFv) -sTRAIL fusion protein on GBM from the following three parts.
Part one: induction, purification and biological activity identification of antiMRP3 (scFv) -sTRAIL fusion protein
AntiMRP3 (scFv) -sTRAIL fusion protein was expressed and purified by pMAL prokaryotic expression system, and its biological activity was identified. AntiMRP3 (scFv) and sTRAIL gene fragments were amplified by recombinant PCR technology, and the plasmid pMAL-c2 was inserted into the prokaryotic expression plasmid, and the recombinant expression plasmid pMAL-antiMRP3 (scFv) -sTRAIL was constructed. The recombinant plasmid was fully consistent with the expectation. The recombinant plasmid was transformed into the receptive E.coli BL21, IPTG was induced, and the antiMRP3 (scFv) -sTRAIL fusion protein with maltose binding protein (MBP) tagged peptide was purified by Amylose Resin affinity chromatography column, and the purified fusion protein was identified by SDS-PAGE. The results showed that the fusion protein was observed in 92kD. The purified fusion protein band was expressed. The expression rate of fusion protein in E.coli BL21 was about 30%, and the purity of purified fusion protein was 95%.
Using MRP3 positive U251 polymorphous glioblastoma cells as the research object, the proliferation inhibition rate of antiMRP3 (scFv) -sTRAIL fusion protein and MBP protein at different concentrations was measured by MTT method. The results showed that antiMRP3 (scFv) -sTRAIL fusion protein could significantly inhibit the proliferation and concentration dependence of the cells. The antiMRP3 (scFv) -sTRAIL fusion protein of IC50 dose (62.5nmol/L) acted on U251 cell 24h, and the morphology of U251 cells was observed under the inverted microscope. The apoptosis of U251 cells in the.AntiMRP3 (scFv) -sTRAIL fusion protein treated group was obviously changed in morphology, but the cells and the untreated groups did not occur. Abnormal morphologic changes.
The results showed that the recombinant plasmid of pMAL-antiMRP3 (scFv) -sTRAIL was successfully constructed and the prokaryotic expression and purification of antiMRP3 (scFv) -sTRAIL fusion protein was realized. The preliminary biological function identification showed that the fusion protein could induce the apoptosis of U251 multiform glioblastoma cells and further study its U251 cells. It lays the foundation for the inducing effect and mechanism of apoptosis.
The second part of antiMRP3 (scFv) -sTRAIL fusion protein induces apoptosis in U251 glioblastoma multiforme.
The antigen specific binding of antiMRP3 (scFv) -sTRAIL fusion protein with MRP3 positive U251 cells and MRP3 negative Jurkat cells was analyzed by fluorescence activated cell sorting (FACS). The results showed that the antiMRP3 (scFv) -sTRAIL fusion protein could be combined specifically to the surface of the MRP3 positive cell membrane. The combination of antiMRP3 (scFv) -sTRAIL fusion protein and MRP3 negative Jurkat cells showed no apparent adhesion to the cell membrane surface, indicating that the binding of antiMRP3 (scFv) -sTRAIL fusion protein with U251 cells was specific for MRP3 antigen.
The effects of antiMRP3 (scFv) on the targeting of antiMRP3 (scFv) -sTRAIL and MRP3, or the activity of TRAIL activity and the neutralization of the antibody mAb2E5 neutralization sTRAIL, on the apoptosis induced activity of antiMRP3 (scFv) -sTRAIL fusion protein were observed. The results showed that antiMRP3 (scFv) -sTRAIL showed a strong killing effect on U251 cells, and the survival rate of U251 cells decreased with the increase of fusion protein concentration in 24h. The killing effect of antiMRP3 (scFv) -sTRAIL on the cells was significantly inhibited after the incubation of antiMRP3 (scFv) or mAb2E5, and there was no obvious killing activity under the concentration gradient. P protein had no effect on the survival rate of U251 cells. The apoptosis of 8h, 12h and 24h U251 cells after antiMRP3 (scFv) -sTRAIL in IC50 dose (scFv) -sTRAIL was detected by flow cytometry. The induction time was prolonged and increased. The apoptosis induction of antiMRP3 (scFv) -sTRAIL on U251 cells was completely blocked by antiMRP3 (scFv) or mAb2E5 preincubation, and the results were consistent with the MTT detection.
These results suggest that as an antibody directed immuno toxin, antiMRP3 (scFv) -sTRAIL fusion protein only combines with MRP3 positive U251 cells and induces apoptosis. The apoptosis inducible activity of the MRP3 antigen specific.AntiMRP3 (scFv) -sTRAIL fusion protein can be suppressed competitively by antiMRP3 (scFv) or be neutralized by TRAIL activity and antibody. Blocking, it is proved that antiMRP3 (scFv) -sTRAIL can induce apoptosis of GBM cells by targeting specific binding of MRP3 antigen by antiMRP3 (scFv) single chain antibody, and its apoptosis induction activity is mediated by sTRAIL part, but the non targeting antiMRP3 (scFv) -sTRAIL does not possess obvious apoptosis induced activity.
The third part is the activation effect of antiMRP3 (scFv) -sTRAIL fusion protein on TRAIL-R1 and TRAIL-R2.
TRAIL-R1 (DR4) and TRAIL-R2 (DR5) are two kinds of death receptors that mediate exogenous apoptotic pathways, in which the expression level of DR5 is higher than that of DR4, so it is dominant in inducing tumor apoptosis. The study shows that the natural membrane binding TRAIL can activate the two death receptors of TRAIL-R1 and TRAIL-R2, and the soluble sTRAIL only remains. The activation of TRAIL-R1 was activated. In order to analyze the activation of antiMRP3 (scFv) -sTRAIL to two kinds of death receptors in U251 cells, the mRNA expression level of TRAIL-R1 and TRAIL-R2 in U251 cells was analyzed by real-time PCR method. U251 cell 1H was incubated with anti -TRAIL-R1 and anti -TRAIL-R2 antibodies, and TRAIL-R1 and TRAIL-R2 were blocked separately or simultaneously. Then U251 cell 24h was treated with IC50 dose antiMRP3 (scFv) -sTRAIL, and the apoptotic rate was detected by flow cytometry. The activation of two death receptors was analyzed. The apoptosis rate of L-R1 decreased by 28%, the apoptosis rate of blocking TRAIL-R2 decreased by 35%, and the apoptosis rate of TRAIL-R1 and TRAIL-R2 decreased 46%. at the same time. The Western blot method was used to verify that antiMRP3 (scFv) -sTRAIL fusion protein activates exogenous apoptosis pathway and induces apoptosis of caspase dependent cells.
The above results show that the specific binding of antiMRP3 (scFv) -sTRAIL fusion protein and target antigen transforms sTRAIL from soluble form into a membrane binding form of natural TRAIL. It can not only activate TRAIL-R1 but also restore its activation ability to TRAIL-R2, enhance the apoptosis inducing activity of sTRAIL, and thus obtain enhanced anti-tumor effect.AntiMRP3. (scFv) -sTRAIL fusion protein activates the exogenous apoptotic pathway by inducing the death of caspase dependent cells by binding to two death receptors, TRAIL-R1 and / or TRAIL-R2.
【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392;R739.41

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