本文選題：多發(fā)性骨髓瘤 + 癌干細胞��； 參考：《東南大學(xué)》2016年博士論文

【摘要】：多發(fā)性骨髓瘤(multiple myeloma,MM)是一種極易復(fù)發(fā)的致命性造血系統(tǒng)惡性腫瘤,復(fù)發(fā)后中位生存期僅為5年左右。細胞毒性化學(xué)藥物治療、硼替佐米等新型藥物治療、造血干細胞移植等治療手段雖然延長了患者的中位生存期,但均不能有效解決MM治療后復(fù)發(fā)的難題。MM復(fù)發(fā)和耐藥與MM細胞群中存在少量的癌干細胞(cancer stem cells,CSCs)有關(guān)。CSCs具有自我更新、多向分化潛能和顯著的多藥耐藥特性,常規(guī)治療藥物因未瞄準MM CSCs,無法從根本上控制其增殖與復(fù)發(fā)。CSCs膜上高表達一種藥物外排泵蛋白(ATP-binding cassette transporter G2,ABCG2),其屬于ATP結(jié)合蛋白轉(zhuǎn)運體家族的第二個成員,廣泛存在于正常組織,在機體內(nèi)承擔著一定的生理功能,但在MM等多種惡性腫瘤CSCs中ABCG2表達顯著增高,是CSCs耐藥的重要標志,可將多種抗癌藥物如米托蒽醌、阿霉素等泵出細胞外,ABCG2與CSCs的強耐藥特性密切相關(guān)。微泡是通過膜殼包裹一定氣體形成的包膜微氣泡(microbubble,MB)結(jié)構(gòu),多采用脂質(zhì)或生物可降解聚合物制成,作為一種新型藥物傳輸載體,具有微泡殼膜安全性高、可通過多種方式攜載藥物、載藥能力強以及超聲擊破微泡引起的各種生物效應(yīng)可提高藥物傳輸能力等多種優(yōu)點。更重要的是,微泡聯(lián)合超聲輻照可實現(xiàn)被動靶向性治療,將抗腫瘤特異性抗體耦聯(lián)在載藥微泡表面,則可進一步實現(xiàn)主動靶向性治療。因此,微泡聯(lián)合超聲在惡性腫瘤的治療方面具有重要應(yīng)用價值。蒽環(huán)類藥物表阿霉素(epirubicin,EPI)是臨床常用的抗腫瘤化療藥,廣泛用于MM、乳腺癌、肉瘤等多種惡性腫瘤。EPI普通劑型不具有腫瘤靶向性,用藥后在非腫瘤組織也有較多的分布,導(dǎo)致消化道反應(yīng)、骨髓抑制、肝腎毒性等各種不良反應(yīng),心臟毒性是其劑量限制性毒副反應(yīng),顯著限制了其臨床應(yīng)用。為解決MM復(fù)發(fā)、耐藥及EPI毒副作用大等問題,本研究以免疫磁珠分選法從人MM細胞系中獲得MM CD138-CD34-CSCs,以此為靶標,制備結(jié)合ABCG2單抗(mAb)的載EPI脂質(zhì)微泡,通過mAb靶向高表達ABCG2的MM CSCs,在超聲作用下靶向MM釋藥,以提高腫瘤局部的EPI藥物濃度,降低化療毒副反應(yīng);為此,利用結(jié)合ABCG2單抗的載EPI脂質(zhì)微泡體外及荷MM的非肥胖糖尿病/嚴重的聯(lián)合型免疫缺陷(nonobese diabetic/severecombined immunodeficient,NOD/SCID)鼠模型研究,探討該策略對MM的作用效應(yīng)及其相關(guān)機制。目的:探討結(jié)合ABCG2單抗的載EPI微泡聯(lián)合超聲,靶向CSCs治療MM的效應(yīng)及其機制,為提高MM患者的療效、減少復(fù)發(fā)率及最終根治MM提供新思路、新方法。方法與內(nèi)容:1.分選與鑒定MM CSCs:采用免疫磁珠分選技術(shù),從人MM細胞系RPMI8226中篩選出表型為CD138-CD34-MM細胞,按課題組先前采用的方法,分別通過體內(nèi)外實驗,重復(fù)確證CD138-CD34-MM細胞具有CSCs特性。2.靶向載藥微泡的制備與表征:利用生物素-親和素法將ABCG2mAb與載EPI微泡相結(jié)合,制備EPI-MBs+mAb,檢測其抗體結(jié)合情況和粒徑、zeta電位、載藥率等理化性質(zhì),研究該微泡與MM CSCs的體外靶向結(jié)合能力及超聲干預(yù)下靶向細胞釋藥能力。3.體外細胞抑制與殺傷實驗:實驗分PBS對照(Control)組、載EPI微泡(EPI-MBs)組、載EPI微泡靶向MM(EPI-MBs+mAb)組、EPI組、EPI聯(lián)合超聲(EPI+US)組、載EPI微泡聯(lián)合超聲(EPI-MBs+US)組、載EPI微泡靶向MM聯(lián)合超聲(EPI-MBs+mAb+US)共7組。觀察不同實驗組藥物對MM CSCs體外抑制與殺傷效應(yīng);并從MM CSCs線粒體膜電位和細胞周期的改變、Caspase-3、Caspase-8和Caspase-9凋亡相關(guān)蛋白的表達水平以及透射電鏡觀察細胞超微結(jié)構(gòu)變化,分析其抗MM CSCs的效應(yīng)及機制。4.經(jīng)皮下注射MM CSCs建立MM模型體內(nèi)治療實驗I:用免疫磁珠分選法從人MM細胞系RPMI8226分離的1 × 106CD138-CD34-細胞,經(jīng)皮下注射至NOD/SCID鼠。取適量EPI-MBs+mAb和EPI-MBs靜脈注入成瘤后鼠體內(nèi),病理組織學(xué)及超聲成像法觀察EPI-MBs+mAb對皮下移植瘤的靶向性。將荷瘤鼠隨機分為PBS對照(Control)組、EPI組、載EPI靶向微泡(EPI-MBs+mAb)組和載EPI靶向微泡聯(lián)合超聲(EPI-MBs+mAb+US)共4組,每組3只。觀察不同實驗組尾靜脈給藥對荷瘤鼠的治療效應(yīng),應(yīng)用免疫組織化學(xué)、HE染色、TUNEL法等病理學(xué)方法和Caspase-3、Caspase-9、Bax、Bcl-2、TopoisomeraseⅡα 的 Western blot 檢測分析其抗 MM CSCs效應(yīng)及機制;實驗重復(fù)1次。5.經(jīng)尾靜脈注射MM CSCs建立MM模型體內(nèi)治療實驗Ⅱ:用上述方法分離的5 ×106 CD138-CD34-細胞經(jīng)尾靜脈注射至NOD/SCID鼠,約21天后,通過骨密度、腎損傷和尿蛋白動態(tài)監(jiān)測方法,分析鑒定建立荷MMNOD/SCID鼠模型。制備CY7.5標記的EPI-MBs+mAb和EPI-MBs,尾靜脈注入荷瘤鼠體內(nèi),近紅外熒光活體成像法動態(tài)觀察兩種微泡的體內(nèi)腫瘤靶向性。荷瘤鼠分組情況同體內(nèi)治療實驗I。觀察不同實驗組尾靜脈給藥對荷瘤鼠的治療效應(yīng),并應(yīng)用細胞學(xué)、病理組織學(xué)及影像學(xué)等技術(shù),探討其靶向CSCs治療MM效應(yīng)及機制。結(jié)果:1.RT-PCR、Western Bolt及流式細胞術(shù)(FCM)結(jié)果顯示,免疫磁珠分選獲得的CD 138-CD34-MM細胞(MM CSCs),其ABCG2分子表達水平顯著增高,與非CD138-CD34-MM細胞(Non-CSCs)比較,差異有統(tǒng)計性意義(P0.05或P0.01);CCK8法、軟瓊脂克隆形成、Transwell實驗結(jié)果表明,CD138-CD34-MM CSCs的增殖活性、體外克隆形成能力、細胞遷移和侵襲能力以及在NOD/SCID鼠體內(nèi)致瘤能力均比Non-CSCs顯著增強,差異有統(tǒng)計性意義(P0.05或P0.01)。2.制備的載藥微泡靶向MM表征結(jié)果顯示,EPI-MBs+mAb分散性好、粒徑均一、穩(wěn)定性好、載藥率較高,與MM CSCs靶向性結(jié)合能力強,超聲干預(yù)下可靶向RPMI8226細胞釋藥。3.體外實驗結(jié)果顯示,與對照組比較,EPI-MBs組和EPI-MBs+mAb組無明顯抑制MM CSCs增殖效應(yīng),其他組則有不同程度的抑制效應(yīng),以EPI-MBs+mAb+US組的效果最強。FCM檢測發(fā)現(xiàn),抑制效應(yīng)與凋亡率的實驗結(jié)果相吻合。Western blot結(jié)果表明,EPI-MBs+mAb+US 顯著提高 cleaved Caspase-3 和 cleaved Caspase-9 表達,而對誘導(dǎo)凋亡的Caspase-8分子表達無明顯影響。4.經(jīng)皮下注射1×106CD138-CD34-細胞至NOD/SCID鼠,約18天可形成腫瘤;EPI-MBs+mAb在體內(nèi)能很好的靶向聚集到皮下移植瘤組織并停留較長時間,且具有超聲成像功能。與對照組比較,EPI組、EPI-MBs+mAb組和EPI-MBs+mAb+US組都可不同程度的抑制腫瘤體積、延長荷瘤鼠生存期,其中EPI-MBs+mAb+US組,治療效應(yīng)最明顯。腫瘤免疫組化及Western blot結(jié)果表明,EPI-MBs+mAb+US組,能顯著促進Caspase-3、Caspase-9、Bax表達,而抑制Bcl-2、增殖細胞核抗原(proliferating cellnucleaantigen,PCNA)、Topoisomerase Ⅱα表達,與其他各組相比,效應(yīng)最強,差異有統(tǒng)計性意義(P0.05或P0.01)。腫瘤組織HE染色、TUNEL檢測結(jié)果表明,EPI-MBs+mAb+US組,較其它各治療組能顯著誘導(dǎo)MM CSCs凋亡,差異有統(tǒng)計性意義(P0.05或P0.01)。5.經(jīng)尾靜脈注射注射5× 106CD138-CD34-MM CSCs約21天后,與正常組比較,模型組鼠出現(xiàn)了降低骨密度、溶骨性損害、腎損害和蛋白尿,表明尾靜脈注射MM CSCs已成功建立荷MM NOD/SCID小鼠模型。近紅外活體熒光成像顯示,與EPI-MBs組相比,EPI-MBs+mAb能更好的靶向聚集在脊柱、四肢等腫瘤病灶部位。與模型組比較,各治療組均能不同程度地減輕荷瘤鼠的骨損害、腎損害以及貧血等癥狀,差異有統(tǒng)計性意義(P0.05或P0.01),其中,以EPI-MBs+mAb+US組療效最明顯。結(jié)論:1.EPI-MBs+mAb+US通過mAb特異的靶向MMCSCs,部分阻斷ABCG2藥泵活性,在超聲干預(yù)下釋放EPI,發(fā)揮其持久抑制MM生長和誘導(dǎo)MM CSCs凋亡;此外,抗體和補體介導(dǎo)的細胞毒效應(yīng)也可能發(fā)揮一定作用。2.EPI-MBs+mAb在荷MM NOD/SCID小鼠體內(nèi)對MM CSCs具有良好的靶向性,聯(lián)合超聲干預(yù)能產(chǎn)生較強的抗MM效應(yīng),這為清除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.

【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R733.3

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