结合单抗的载表阿霉素微泡联合超声靶向CSCs治疗多发性骨髓瘤研究

发布时间：2018-04-19 17:00

本文选题：多发性骨髓瘤 + 癌干细胞　；参考：《东南大学》2016年博士论文

【摘要】：多发性骨髓瘤(multiple myeloma,MM)是一种极易复发的致命性造血系统恶性肿瘤,复发后中位生存期仅为5年左右。细胞毒性化学药物治疗、硼替佐米等新型药物治疗、造血干细胞移植等治疗手段虽然延长了患者的中位生存期,但均不能有效解决MM治疗后复发的难题。MM复发和耐药与MM细胞群中存在少量的癌干细胞(cancer stem cells,CSCs)有关。CSCs具有自我更新、多向分化潜能和显著的多药耐药特性,常规治疗药物因未瞄准MM CSCs,无法从根本上控制其增殖与复发。CSCs膜上高表达一种药物外排泵蛋白(ATP-binding cassette transporter G2,ABCG2),其属于ATP结合蛋白转运体家族的第二个成员,广泛存在于正常组织,在机体内承担着一定的生理功能,但在MM等多种恶性肿瘤CSCs中ABCG2表达显著增高,是CSCs耐药的重要标志,可将多种抗癌药物如米托蒽醌、阿霉素等泵出细胞外,ABCG2与CSCs的强耐药特性密切相关。微泡是通过膜壳包裹一定气体形成的包膜微气泡(microbubble,MB)结构,多采用脂质或生物可降解聚合物制成,作为一种新型药物传输载体,具有微泡壳膜安全性高、可通过多种方式携载药物、载药能力强以及超声击破微泡引起的各种生物效应可提高药物传输能力等多种优点。更重要的是,微泡联合超声辐照可实现被动靶向性治疗,将抗肿瘤特异性抗体耦联在载药微泡表面,则可进一步实现主动靶向性治疗。因此,微泡联合超声在恶性肿瘤的治疗方面具有重要应用价值。蒽环类药物表阿霉素(epirubicin,EPI)是临床常用的抗肿瘤化疗药,广泛用于MM、乳腺癌、肉瘤等多种恶性肿瘤。EPI普通剂型不具有肿瘤靶向性,用药后在非肿瘤组织也有较多的分布,导致消化道反应、骨髓抑制、肝肾毒性等各种不良反应,心脏毒性是其剂量限制性毒副反应,显著限制了其临床应用。为解决MM复发、耐药及EPI毒副作用大等问题,本研究以免疫磁珠分选法从人MM细胞系中获得MM CD138-CD34-CSCs,以此为靶标,制备结合ABCG2单抗(mAb)的载EPI脂质微泡,通过mAb靶向高表达ABCG2的MM CSCs,在超声作用下靶向MM释药,以提高肿瘤局部的EPI药物浓度,降低化疗毒副反应;为此,利用结合ABCG2单抗的载EPI脂质微泡体外及荷MM的非肥胖糖尿病/严重的联合型免疫缺陷(nonobese diabetic/severecombined immunodeficient,NOD/SCID)鼠模型研究,探讨该策略对MM的作用效应及其相关机制。目的:探讨结合ABCG2单抗的载EPI微泡联合超声,靶向CSCs治疗MM的效应及其机制,为提高MM患者的疗效、减少复发率及最终根治MM提供新思路、新方法。方法与内容:1.分选与鉴定MM CSCs:采用免疫磁珠分选技术,从人MM细胞系RPMI8226中筛选出表型为CD138-CD34-MM细胞,按课题组先前采用的方法,分别通过体内外实验,重复确证CD138-CD34-MM细胞具有CSCs特性。2.靶向载药微泡的制备与表征:利用生物素-亲和素法将ABCG2mAb与载EPI微泡相结合,制备EPI-MBs+mAb,检测其抗体结合情况和粒径、zeta电位、载药率等理化性质,研究该微泡与MM CSCs的体外靶向结合能力及超声干预下靶向细胞释药能力。3.体外细胞抑制与杀伤实验:实验分PBS对照(Control)组、载EPI微泡(EPI-MBs)组、载EPI微泡靶向MM(EPI-MBs+mAb)组、EPI组、EPI联合超声(EPI+US)组、载EPI微泡联合超声(EPI-MBs+US)组、载EPI微泡靶向MM联合超声(EPI-MBs+mAb+US)共7组。观察不同实验组药物对MM CSCs体外抑制与杀伤效应;并从MM CSCs线粒体膜电位和细胞周期的改变、Caspase-3、Caspase-8和Caspase-9凋亡相关蛋白的表达水平以及透射电镜观察细胞超微结构变化,分析其抗MM CSCs的效应及机制。4.经皮下注射MM CSCs建立MM模型体内治疗实验I:用免疫磁珠分选法从人MM细胞系RPMI8226分离的1 × 106CD138-CD34-细胞,经皮下注射至NOD/SCID鼠。取适量EPI-MBs+mAb和EPI-MBs静脉注入成瘤后鼠体内,病理组织学及超声成像法观察EPI-MBs+mAb对皮下移植瘤的靶向性。将荷瘤鼠随机分为PBS对照(Control)组、EPI组、载EPI靶向微泡(EPI-MBs+mAb)组和载EPI靶向微泡联合超声(EPI-MBs+mAb+US)共4组,每组3只。观察不同实验组尾静脉给药对荷瘤鼠的治疗效应,应用免疫组织化学、HE染色、TUNEL法等病理学方法和Caspase-3、Caspase-9、Bax、Bcl-2、TopoisomeraseⅡα 的 Western blot 检测分析其抗 MM CSCs效应及机制;实验重复1次。5.经尾静脉注射MM CSCs建立MM模型体内治疗实验Ⅱ:用上述方法分离的5 ×106 CD138-CD34-细胞经尾静脉注射至NOD/SCID鼠,约21天后,通过骨密度、肾损伤和尿蛋白动态监测方法,分析鉴定建立荷MMNOD/SCID鼠模型。制备CY7.5标记的EPI-MBs+mAb和EPI-MBs,尾静脉注入荷瘤鼠体内,近红外荧光活体成像法动态观察两种微泡的体内肿瘤靶向性。荷瘤鼠分组情况同体内治疗实验I。观察不同实验组尾静脉给药对荷瘤鼠的治疗效应,并应用细胞学、病理组织学及影像学等技术,探讨其靶向CSCs治疗MM效应及机制。结果:1.RT-PCR、Western Bolt及流式细胞术(FCM)结果显示,免疫磁珠分选获得的CD 138-CD34-MM细胞(MM CSCs),其ABCG2分子表达水平显著增高,与非CD138-CD34-MM细胞(Non-CSCs)比较,差异有统计性意义(P0.05或P0.01);CCK8法、软琼脂克隆形成、Transwell实验结果表明,CD138-CD34-MM CSCs的增殖活性、体外克隆形成能力、细胞迁移和侵袭能力以及在NOD/SCID鼠体内致瘤能力均比Non-CSCs显著增强,差异有统计性意义(P0.05或P0.01)。2.制备的载药微泡靶向MM表征结果显示,EPI-MBs+mAb分散性好、粒径均一、稳定性好、载药率较高,与MM CSCs靶向性结合能力强,超声干预下可靶向RPMI8226细胞释药。3.体外实验结果显示,与对照组比较,EPI-MBs组和EPI-MBs+mAb组无明显抑制MM CSCs增殖效应,其他组则有不同程度的抑制效应,以EPI-MBs+mAb+US组的效果最强。FCM检测发现,抑制效应与凋亡率的实验结果相吻合。Western blot结果表明,EPI-MBs+mAb+US 显著提高 cleaved Caspase-3 和 cleaved Caspase-9 表达,而对诱导凋亡的Caspase-8分子表达无明显影响。4.经皮下注射1×106CD138-CD34-细胞至NOD/SCID鼠,约18天可形成肿瘤;EPI-MBs+mAb在体内能很好的靶向聚集到皮下移植瘤组织并停留较长时间,且具有超声成像功能。与对照组比较,EPI组、EPI-MBs+mAb组和EPI-MBs+mAb+US组都可不同程度的抑制肿瘤体积、延长荷瘤鼠生存期,其中EPI-MBs+mAb+US组,治疗效应最明显。肿瘤免疫组化及Western blot结果表明,EPI-MBs+mAb+US组,能显著促进Caspase-3、Caspase-9、Bax表达,而抑制Bcl-2、增殖细胞核抗原(proliferating cellnucleaantigen,PCNA)、Topoisomerase Ⅱα表达,与其他各组相比,效应最强,差异有统计性意义(P0.05或P0.01)。肿瘤组织HE染色、TUNEL检测结果表明,EPI-MBs+mAb+US组,较其它各治疗组能显著诱导MM CSCs凋亡,差异有统计性意义(P0.05或P0.01)。5.经尾静脉注射注射5× 106CD138-CD34-MM CSCs约21天后,与正常组比较,模型组鼠出现了降低骨密度、溶骨性损害、肾损害和蛋白尿,表明尾静脉注射MM CSCs已成功建立荷MM NOD/SCID小鼠模型。近红外活体荧光成像显示,与EPI-MBs组相比,EPI-MBs+mAb能更好的靶向聚集在脊柱、四肢等肿瘤病灶部位。与模型组比较,各治疗组均能不同程度地减轻荷瘤鼠的骨损害、肾损害以及贫血等症状,差异有统计性意义(P0.05或P0.01),其中,以EPI-MBs+mAb+US组疗效最明显。结论:1.EPI-MBs+mAb+US通过mAb特异的靶向MMCSCs,部分阻断ABCG2药泵活性,在超声干预下释放EPI,发挥其持久抑制MM生长和诱导MM CSCs凋亡;此外,抗体和补体介导的细胞毒效应也可能发挥一定作用。2.EPI-MBs+mAb在荷MM NOD/SCID小鼠体内对MM CSCs具有良好的靶向性,联合超声干预能产生较强的抗MM效应,这为清除MM CSCs、根治MM提供了新的策略。
[Abstract]:Multiple myeloma (multiple myeloma, MM) is a highly recurrent and fatal hematopoietic malignancy. The median survival time is only about 5 years after relapse. Cytotoxic chemical therapy, bortezomizomi, and hematopoietic stem cell transplantation have prolonged the median survival of the patients, but they are not effective. The problem of relapse after MM treatment.MM recurrence and resistance is related to the existence of a small amount of cancer stem cells (cancer stem cells, CSCs) in the MM cell group..CSCs has self renewal, multidirectional differentiation potential and significant multidrug resistance. The conventional therapeutic drug can not fundamentally control the high expression of its proliferation and relapse.CSCs membrane because it does not aim at MM CSCs. A drug ATP-binding cassette transporter G2 (ABCG2), which belongs to the second members of the ATP binding protein transporter family, widely exists in normal tissues and is responsible for certain physiological functions in the body. However, the expression of ABCG2 is significantly increased in a variety of malignant tumor CSCs such as MM and is an important marker of CSCs resistance. Antitumor drugs, such as mitoxantrone, doxorubicin, are pumped out of cells, and ABCG2 is closely related to the strong resistance of CSCs. Microbubbles are coated microbubbles (microbubble, MB), which are formed by a membrane shell, and are made of lipid or biodegradable polymers as a new drug transport carrier, with microbubble membrane safety. More importantly, the combination of microbubbles and ultrasonic irradiation can be used to achieve passive targeting therapy, and the anti tumor specific anti body coupling can be further realized on the surface of drug loaded microbubbles. Active targeting therapy. Therefore, microbubble combined with ultrasound is of great value in the treatment of malignant tumors. Anthracycline epirubicin (EPI) is a commonly used antitumor chemotherapeutic agent. It is widely used in MM, breast cancer, sarcoma and many other malignant tumors,.EPI general dosage forms do not have tumor targeting, and they are used in non tumor group after drug use. The fabric also has more distribution, which leads to various adverse reactions such as digestive tract reaction, bone marrow suppression, hepatorenal toxicity and other adverse reactions. Cardiac toxicity is a dose limiting toxic and side effect, which significantly restricts its clinical application. In order to solve the problems of MM recurrence, drug resistance and large side effects of EPI, this study is to avoid MM CD138-CD34- from the human MM cell line. CSCs was used as a target to prepare EPI lipid microbubbles combined with ABCG2 monoclonal antibody (mAb), target ABCG2 MM CSCs by mAb and target MM under ultrasound to improve the local EPI drug concentration and reduce the toxicity of chemotherapy. A heavy nonobese diabetic/severecombined immunodeficient (NOD/SCID) mouse model was studied to explore the effect of this strategy on MM and its mechanism. Objective: To explore the effect and mechanism of targeting CSCs in combination with ABCG2 mAb, the effect of targeting CSCs in the treatment of MM, to improve the curative effect of MM patients, to reduce the recurrence rate and to reduce the recurrence rate. The final radical cure of MM provides new ideas, methods and contents: 1. sorting and identification of MM CSCs: using immunomagnetic beads sorting technology, the phenotype of CD138-CD34-MM cells was screened from human MM cell line RPMI8226. According to the previous methods used by the group, the CD138-CD34-MM cells were confirmed to have CSCs characteristic.2. targeting load, respectively. Preparation and characterization of drug microbubbles: using biotin avidin method to combine ABCG2mAb with EPI microbubbles to prepare EPI-MBs+mAb, to detect the physical and chemical properties of its antibody binding and particle size, zeta potential, drug loading rate and so on, to study the in vitro binding ability of the microbubbles to MM CSCs and the inhibition of the targeting cell release ability of.3. in vitro under the hyper acoustic intervention. And killing experiment: PBS control (Control) group, EPI microbubble (EPI-MBs) group, EPI microbubble targeted MM (EPI-MBs+mAb) group, EPI group, EPI combined ultrasound (EPI+US) group, carrier EPI micro bubble combined ultrasound (EPI-MBs+US) group. Effect; the expression of mitochondrial membrane potential and cell cycle of MM CSCs, expression level of Caspase-3, Caspase-8 and Caspase-9 apoptosis related proteins and ultrastructural changes of the cells were observed by transmission electron microscopy, and the effect of its anti MM CSCs and mechanism.4. were subcutaneously injected with MM CSCs to establish MM model in vivo treatment experiment I: by immunomagnetic beads sorting method from 1 x 106CD138-CD34- cells isolated from human MM cell line RPMI8226 were injected subcutaneously into NOD/SCID mice. A proper amount of EPI-MBs+mAb and EPI-MBs were injected into the tumor mice. The targeting of EPI-MBs+mAb for subcutaneous transplantation was observed by histopathology and ultrasound imaging. The tumor mice were randomly divided into PBS control (Control) group, EPI group, EPI targeting microtarget. EPI-MBs+mAb group and EPI targeted microbubble combined ultrasound (EPI-MBs+mAb+US) group were 4 groups, each group was 3. Observe the therapeutic effect of the tail vein of different experimental groups on the tumor bearing mice, and use immunohistochemical method, HE staining, TUNEL method and other pathological methods and the Western blot test of Caspase-3, Caspase-9, Bax, Bcl-2, Topoisomerase II A MM CSCs effect and mechanism; experimental repetition of 1 times.5. via tail vein injection of MM CSCs to establish a MM model in vivo treatment II: 5 x 106 CD138-CD34- cells separated by the above method were injected into NOD/SCID mouse through the tail vein, and about 21 days later, through bone density, renal injury and urine protein dynamic monitoring method, analysis and identification of the established MMNOD/SCID rat model. CY7.5 labeled EPI-MBs+mAb and EPI-MBs, the tail vein was injected into the body of the tumor bearing mice. The tumor targeting of the two microbubbles was dynamically observed by the near infrared fluorescence imaging method. The therapeutic effect of the caudal vein on the tumor bearing mice was observed by the intratromaphroditic treatment experiment of the group of tumor mice, and the cytology, the histopathology and the shadow were applied. To explore the MM effect and mechanism of targeting CSCs therapy, the results of 1.RT-PCR, Western Bolt and flow cytometry (FCM) showed that the CD 138-CD34-MM cells (MM CSCs) obtained by the separation of immunomagnetic beads (MM CSCs) had a significant increase in the expression of ABCG2 molecules, and the difference was statistically significant compared with those of non CD138-CD34-MM cells. CCK8, soft agar clones were formed, and the results of Transwell experiment showed that the proliferation activity of CD138-CD34-MM CSCs, the ability to clone and form in vitro, the cell migration and invasion ability and the ability to induce tumor in NOD/SCID mice were significantly higher than that of Non-CSCs, and the difference was statistically significant (P0.05 or P0.01).2. prepared microbubbles targeted to MM. The results showed that EPI-MBs+mAb had good dispersity, uniform particle size, good stability, high drug loading rate and strong binding ability with MM CSCs. In vitro, the experimental results of targeted RPMI8226 cell release by ultrasound intervention showed that, compared with the control group, the EPI-MBs and EPI-MBs+mAb groups did not significantly inhibit the CSCs proliferation effect of MM, and the other groups had different degrees of inhibition effect. It was found that the results of the strongest.FCM test in the EPI-MBs+mAb+US group were found, and the inhibitory effect was consistent with the experimental results of the apoptosis rate. The results of.Western blot showed that EPI-MBs+mAb+US significantly increased the Caspase-9 expression of cleaved Caspase-3 and cleaved, but had no obvious effect on the expression of Caspase-8 molecules induced by apoptosis, and.4. was injected subcutaneous 1 * 106CD138-CD34-. Cells to NOD/SCID mice can form tumors for about 18 days; EPI-MBs+mAb can target a very good target in the body and stay for a long time and have ultrasound imaging function. Compared with the control group, the EPI, EPI-MBs+mAb and EPI-MBs+mAb+US groups can inhibit the tumor volume in varying degrees and prolong the survival period of the tumor bearing mice, of which EPI-M Bs+mAb+US group, the treatment effect is most obvious. The tumor immunization and Western blot results show that the EPI-MBs+mAb+US group can significantly promote Caspase-3, Caspase-9, Bax expression, but inhibit Bcl-2, proliferating cell nuclear antigen (proliferating cellnucleaantigen, PCNA), Topoisomerase II alpha expression, compared with the other groups, the strongest effect, the difference is statistically significant. Significance (P0.05 or P0.01). HE staining of tumor tissue and TUNEL detection results showed that EPI-MBs+mAb+US group, compared with other treatment groups, could significantly induce MM CSCs apoptosis, the difference was statistically significant (P0.05 or P0.01).5. through the tail vein injection of 5 x 106CD138-CD34-MM CSCs for about 21 days, compared with the normal group, the model rats appeared to reduce bone density and dissolve. Bone damage, renal damage and proteinuria showed that the tail vein injection of MM CSCs had successfully established the MM NOD/SCID mouse model. Near infrared living fluorescence imaging showed that compared with the EPI-MBs group, EPI-MBs+mAb could better target the target area of the tumor in the spine and limbs. Compared with the model group, all the treatment groups could reduce the tumor mice to varying degrees. The differences were statistically significant (P0.05 or P0.01) in the symptoms of bone damage, kidney damage and anemia (P0.05 or P0.01). Conclusion: 1.EPI-MBs+mAb+US through mAb specific target MMCSCs, partially blocking the activity of ABCG2 drug pump, releasing EPI under ultrasound intervention, exerting its persistent inhibition of MM growth and inducing MM CSCs apoptosis; furthermore, The cytotoxic effect mediated by antibody and complement may also play a role in the effect of.2.EPI-MBs+mAb on MM CSCs in MM NOD/SCID mice. Combined ultrasound intervention can produce strong anti MM effect, which provides a new strategy for eliminating MM CSCs and eliminating MM.

【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.3

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