發(fā)布時間：2018-08-04 20:15

【摘要】：腦膠質(zhì)瘤是中樞神經(jīng)系統(tǒng)最常見的顱腦內(nèi)腫瘤,發(fā)生于神經(jīng)外胚層,世界衛(wèi)生組織數(shù)據(jù)顯示,腦膠質(zhì)瘤是35歲以下中青年第2號腫瘤殺手,每年全球約有18~60萬中青年被惡性腦膠質(zhì)瘤奪去生命。膠質(zhì)瘤體呈強侵襲性生長特點,外科手術(shù)不易分開腫瘤組織和正常組織,導致手術(shù)治療效果不佳。術(shù)后的放化療策略對于腦膠質(zhì)瘤的治療具有非常重要的意義。但是由于血腦屏障(blood brain barrier,BBB)的生理屏障作用,使得化療藥物難以透過BBB,進而到達腫瘤組織起到殺傷腫瘤細胞的作用。經(jīng)特異性配體修飾的靶向納米遞藥系統(tǒng)可降低化療藥物的副作用,增加藥物在病變部位的蓄積,因此在腫瘤治療領(lǐng)域具有重要意義。目前大多數(shù)納米載體均為人工合成,作為外源性物質(zhì)具有免疫原性。仿生型納米遞藥系統(tǒng)是一種天然微粒系統(tǒng),可以模擬體內(nèi)物質(zhì)或其他病原體,具有較低的免疫原性和良好的靶向性。仿生納米遞藥系統(tǒng)可以分為三大類,分別是基于細胞、內(nèi)源性蛋白和病原體的遞藥系統(tǒng)。以脂蛋白為基礎(chǔ)構(gòu)建的仿生型納米給藥系統(tǒng)是其中重要的一類,具有良好的生物相容性,同時其結(jié)構(gòu)中的靶向配體,可與受體結(jié)合,具有天然的靶向能力,從而可獲得良好的靶向作用。膠質(zhì)瘤干細胞(glioma stem cells,GSCs)是腦膠質(zhì)瘤中具有自我更新和多向分化潛能的細胞,該細胞與腫瘤的侵襲性生長、復(fù)發(fā)、耐藥性有關(guān)。GSCs的存在使膠質(zhì)瘤對傳統(tǒng)的放療和化療產(chǎn)生耐受,并且保持著自我更新與增殖,誘導膠質(zhì)瘤的復(fù)發(fā)。因此針對GSCs進行腫瘤的靶向治療具有重要意義。鹽霉素(salinomycin,SAL)被報道可以選擇性殺傷多種腫瘤干細胞,同時有研究表明鹽霉素對腫瘤細胞也有很好的殺傷作用。本課題構(gòu)建一種靶向性的仿生納米載體包載鹽霉素,生物相容性良好,通過腦靶向功能肽Angiopep-2和LDL共同介導透過BBB進入腦部,靶向于膠質(zhì)瘤表面的LDL和LRP-1受體,實現(xiàn)雙極靶向遞藥,殺傷膠質(zhì)瘤細胞和膠質(zhì)瘤干細胞,同時抑制膠質(zhì)瘤干細胞來源的血管內(nèi)皮細胞,阻斷腫瘤新生組織的血液供應(yīng),實現(xiàn)多靶點抗膠質(zhì)瘤作用。課題第一部分,采用兩種膠質(zhì)瘤細胞系SHG-44和U87細胞,采用無血清懸浮培養(yǎng)基從膠質(zhì)瘤細胞中分離培養(yǎng)出懸浮的細胞球,稱為腫瘤微球體(tumorsphere,TS),并通過免疫熒光法驗證兩種腫瘤微球體均表達膠質(zhì)瘤干細胞標記物CD133和巢蛋白(Nestin);流式細胞法檢測CD133陽性率,SHG-44 TS中陽性細胞百分比41.48%,u87ts中陽性細胞百分比17.3%,和文獻報道膠質(zhì)瘤干細胞生長特性和特異性標記物的表達情況一致,表明無血清培養(yǎng)基方法得到的腫瘤微球體即為膠質(zhì)瘤干細胞。而后分別考察sal對兩種膠質(zhì)瘤細胞系和gscs的毒性作用,結(jié)果表明sal對膠質(zhì)瘤細胞和gscs均具有殺傷作用,但shg-44ts和u87ts對sal更為敏感。課題第二部分,從人血液中提取分離得到低密度脂蛋白(lowdensitylipoprotein,ldl),通過傅里葉紅外光譜對其進行鑒定,bca法測得ldl的濃度為1.67mg/ml。將angiopep-2肽修飾于ldl上得到ldl-angiopep-2(ldl-ang),介導其跨越bbb,通過核心重組法制備載sal的ldl納米粒,即ldl-sal-ang。對處方進行優(yōu)化,包封率為35.63±2.65%,載藥量為5.83±0.7%,粒徑和電位分別為22.95±1.58nm和-5.98±0.20mv。課題第三部分,考察載鹽霉素低密度脂蛋白納米粒體外抗腫瘤細胞和腫瘤干細胞活性,激光共聚焦顯微鏡觀察膠質(zhì)瘤細胞和gscs對載熒光探針dii納米粒(ldl-sal-dii-ang和ldl-sal-dii)的攝取情況。膠質(zhì)瘤細胞和gscs對ldl-sal-dii-ang的攝取強度均高于ldl-sal-dii,并存在受體的競爭性抑制,證明ldl的核心包載sal和表面angiopep-2的連接不影響納米粒與ldlr的結(jié)合。細胞毒性試驗證明ldl和ldl-ang對細胞毒性較低�？疾靗dl-sal-ang和ldl-sal對膠質(zhì)瘤細胞和gscs的毒性和誘導細胞凋亡情況。兩種納米粒對膠質(zhì)瘤干細胞的毒性均強于sal,其中l(wèi)dl-sal-ang具有最強細胞毒性作用。在shg-44細胞、u87細胞、shg-44ts和u87ts中,ldl-sal-ang的ic50值分別為0.17μm,3.66μm,0.16μm和0.04μm,同時誘導膠質(zhì)瘤細胞及膠質(zhì)瘤干細胞凋亡能力最強。考察納米粒對膠質(zhì)瘤細胞及gscs微球體形成的影響試驗中,ldl-sal-ang組形成的微球數(shù)量最少,且球體疏松,說明納米粒在較低藥物濃度下就可抑制膠質(zhì)瘤干細胞的自我更新。培養(yǎng)膠質(zhì)瘤干細胞來源的血管內(nèi)皮細胞,免疫熒光法進行鑒定,觀察納米粒對細胞小管形成能力和遷移能力的影響,結(jié)果表明ldl-sal-ang對細胞的小管形成和遷移抑制作用最為顯著,說明ldl納米粒通過作用于膠質(zhì)瘤干細胞對其新生血管生成產(chǎn)生抑制作用。課題第四部分,考察了載鹽霉素低密度脂蛋白納米粒的腦轉(zhuǎn)運特性。考察人腦微血管內(nèi)皮細胞(humanbrainmicrovesselendothelialcell,hbmec)對ldl-dii-ang和ldl-dii的攝取情況,hbmec對納米粒的攝取強度隨時間延長而增強。采用transwell小室共培養(yǎng)模型,考察納米粒在hbmec單層細胞的滲透情況,模擬其透bbb作用,結(jié)果表明ldl-dii-ang滲透率優(yōu)于ldl-dii,達到80%。同時考察了納米粒被transwell小室下室膠質(zhì)瘤細胞及gscs所攝取情況,結(jié)果表明ldl納米粒經(jīng)Angiopep-2修飾后,增強跨BBB能力,提高細胞攝取,增強了抗腫瘤活性。制備載熒光探針DiR的納米粒(LDL-DiR-Ang和LDL-Di R)。在裸鼠腦尾狀核注射膠質(zhì)瘤細胞懸液,制備裸鼠荷原位腦膠質(zhì)瘤模型。動物活體成像觀察載DiR納米粒在小鼠體內(nèi)的分布。與LDL-DiR相比,LDL-Di R-Ang在腦部聚集更多,熒光更強,具有良好的腦靶向性。同時荷原位瘤裸鼠尾靜脈注射LDL-DiI-Ang和LDL-DiI,取荷瘤腦組織冰凍切片,激光共聚焦顯微鏡下觀察,結(jié)果與活體成像一致。取ICR小鼠,空白LDL和LDL-Ang連續(xù)給藥七天,取各組織進行CD68免疫組化染色,驗證納米粒無明顯組織毒性。課題第五部分,進行載鹽霉素低密度脂蛋白納米粒的體內(nèi)藥代動力學和組織分布研究。將ICR小鼠分為三組,SAL、LDL-SAL和LDL-SAL-Ang組,觀察不同組的SAL血藥濃度隨時間變化的情況和組織分布的情況。結(jié)果表明LDL-SAL和LDL-SAL-Ang均具有一定緩釋作用,半衰期顯著延長,藥時曲線下面積顯著增加。LDL-SAL-Ang在腦組織中的蓄積高于其他兩組,腦組織對其攝取也顯著高于其他兩組,證明LDL-SAL-Ang具有較強的腦靶向能力。課題第六部分,考察載鹽霉素低密度脂蛋白納米粒的體內(nèi)抗腫瘤活性。首先制備裸鼠荷原位腦膠質(zhì)瘤模型,將納米粒與一線抗膠質(zhì)瘤藥物替莫唑胺(TMZ)聯(lián)合給藥,給藥組分為8組:對照組、SAL組、TMZ組、LDL-SAL組、LDL-SAL-Ang組、SAL+TMZ組、LDL-SAL+TMZ組和LDL-SAL-Ang+TMZ組。給藥結(jié)束后腦組織切片行免疫組化染色,觀察腫瘤部位膠質(zhì)瘤細胞凋亡情況和血管新生情況,觀察每組剩余小鼠生存狀態(tài)和體重變化情況。結(jié)果表明LDL-SAL-Ang可促進原位腦膠質(zhì)瘤中膠質(zhì)瘤細胞凋亡并抑制血管新生,改善小鼠的生存狀態(tài),延長生存時間。給藥組小鼠生存時間和狀態(tài)均有延長和改善,其中LDL-SAL-Ang+TMZ組小鼠中位生存期長達47天,相對于對照組延長67.8%,體重下降最為緩慢,同時腫瘤細胞凋亡顯著,新生血管數(shù)量最少,在給藥組中顯示最優(yōu)的治療效果,表明LDL-SAL-Ang具有體內(nèi)抗腫瘤活性,與TMZ聯(lián)用效果更佳,可有效治療腦膠質(zhì)瘤。以上研究結(jié)果表明,本課題構(gòu)建的腦靶向給藥系統(tǒng)LDL-SAL-Ang在體內(nèi)外具有較強的抗膠質(zhì)瘤干細胞作用,同時具有雙極腦靶向特性,透過BBB后靶向于腫瘤組織,實現(xiàn)多靶點抗腦膠質(zhì)瘤作用,為腦膠質(zhì)瘤的化療提供新思路和實驗基礎(chǔ)。
[Abstract]:Glioma is the most common Craniocerebral Tumor in the central nervous system and occurs in the neuroectoderm. The WHO data show that glioma is the No. second tumor killer of young and middle-aged people under 35 years of age. Every year, about 18~60 million of young people worldwide are killed by malignant glioma. The glioma is characterized by strong invasive growth and surgery is not easy. Apart from tumor tissue and normal tissue, the effect of surgical treatment is not good. The postoperative radiotherapy and chemotherapy strategy is of great significance for the treatment of glioma. But because of the physiological barrier of blood brain barrier (BBB), it makes the chemotherapeutic drugs difficult to penetrate into the tumor tissue to kill the tumor cells by BBB. The targeted nano drug delivery system modified by specific ligands can reduce the side effects of chemotherapeutic drugs and increase the accumulation of drugs in the lesion site. Therefore, it is of great significance in the field of tumor treatment. Most of the nanoscale carriers are synthesized by artificial synthesis and have immunogenicity as a exogenous substance. The bionic nano delivery system is a one. The natural particle system, which can simulate substance or other pathogens in the body, has a lower immunogenicity and good targeting. The bionic nano drug delivery system can be divided into three major categories, which are based on cell, endogenous protein and pathogen delivery system. The biomimetic nano drug delivery system based on lipoprotein is important. A class of glioma stem cells (GSCs) is a cell with self renewal and multidirectional differentiation potential in glioma, and the cell and tumor invasiveness. Growth, recurrence, and drug resistance related to the presence of.GSCs make glioma tolerant to traditional radiotherapy and chemotherapy, and maintain self renewal and proliferation and induce recurrence of glioma. Therefore, targeted therapy for GSCs is of great significance. Salinomycin (SAL) has been reported to be able to selectively kill a variety of tumor stem cells. At the same time, some studies have shown that salt mycin also has a good killing effect on tumor cells. This topic constructs a targeted biomimetic nanoscale carrier containing salt mycin, which has good biocompatibility, through the brain targeting functional peptide Angiopep-2 and LDL into the brain through BBB, targeting the LDL and LRP-1 receptors on the surface of the gelatin tumor to achieve bipolar targeting. Drug delivery, killing glioma cells and glioma stem cells, simultaneously inhibiting the vascular endothelial cells derived from glioma stem cells, blocking the blood supply of neoplastic tissue, and achieving the anti glioma effect of multiple targets. In the first part, two glioma cell lines, SHG-44 and U87 cells, were used in the serum-free suspension culture medium from glioma cells. The cell spheres were isolated and cultured in the medium, called tumorsphere (TS). By immunofluorescence, two kinds of tumor microspheres were demonstrated to express the markers CD133 and nestin (Nestin) of glioma stem cells. The positive rate of CD133 was detected by flow cytometry, the percentage of positive cells in SHG-44 TS and the percentage of positive cells in u87ts were 17.3%. The growth characteristics of glioma stem cells and the expression of specific markers were consistent with the literature. It showed that the tumor microspheres obtained by the serum-free medium were glioma stem cells. Then the toxic effects of SAL on two glioma cell lines and GSCs were examined respectively. The results showed that Sal had a killing effect on glioma cells and GSCs. But shg-44ts and u87ts are more sensitive to sal. In the second part, we extract and separate low density lipoprotein (lowdensitylipoprotein, LDL) from human blood and identify them by Fourier transform infrared spectroscopy. The concentration of LDL is 1.67mg/ml. by BCA method, and angiopep-2 peptide is trimed on LDL to get ldl-angiopep-2 (ldl-ang), which is mediated by 1.67mg/ml.. The preparation of SAL loaded LDL nanoparticles, namely ldl-sal-ang., was optimized by the core recombination method, the encapsulation efficiency was 35.63 + 2.65%, the drug loading was 5.83 + 0.7%, the particle size and potential were 22.95 + 1.58nm and -5.98 + 0.20mv., respectively, and the activity of anti tumor cells and tumor stem cells in vitro of low density lipoprotein nanoparticles was investigated. The uptake of DiI nanoparticles (ldl-sal-dii-ang and ldl-sal-dii) by glioma cells and GSCs was observed by laser confocal microscopy. The uptake of ldl-sal-dii-ang in glioma cells and GSCs was higher than that of ldl-sal-dii, and there was a competitive inhibition of the receptor, which proved that the connection between Sal and surface angiopep-2 of LDL was not connected to LDL. The combination of nanoparticles and LDLR. Cytotoxicity tests show that LDL and ldl-ang have low cytotoxicity. The toxicity of ldl-sal-ang and ldl-sal to glioma cells and GSCs and the induction of cell apoptosis. The toxicity of two nanoparticles to glioma stem cells is stronger than that of Sal, and ldl-sal-ang has the strongest cytotoxic effect. In the cells, U87 cells, shg-44ts and u87ts, the IC50 values of ldl-sal-ang were 0.17 mu m, 3.66 m, 0.16 mu m and 0.04 micron respectively. At the same time, the apoptosis ability of glioma cells and glioma stem cells was the strongest. It is indicated that nanoparticles can inhibit the self renewal of glioma stem cells at low drug concentration. The culture of vascular endothelial cells derived from glioma stem cells, identification of immunofluorescence, and the effect of nanoparticles on the formation and migration of cell tubules are observed. The results show that ldl-sal-ang has an inhibitory effect on the formation and migration of tubules. In the fourth part, the brain transport properties of low density lipoprotein nanoparticles (humanbrainmicrovesselendothelialcell, HBMEC) of human brain microvascular endothelial cells (humanbrainmicrovesselendothelialcell, HBMEC) were investigated in the fourth part of the study. The uptake of HBMEC nanoparticles increased with time. The Transwell cell co culture model was used to investigate the permeability of the nanoparticles in HBMEC monolayer cells, and the BBB effect was simulated. The results showed that the ldl-dii-ang permeability was better than that of ldl-dii, and the nanoparticle was detected by 80%. in the Transwell small chamber glioma at the same time. The results of cell and GSCs uptake showed that the LDL nanoparticles were modified by Angiopep-2 to enhance the ability to cross the BBB, improve the cell uptake and enhance the anti-tumor activity. The nanoparticles (LDL-DiR-Ang and LDL-Di R) loaded with the fluorescent probe DiR (LDL-DiR-Ang and LDL-Di R) were prepared in the nude mouse caudate nucleus by injection of glioma cell suspension to prepare the nude mouse in situ glioma model. The distribution of DiR nanoparticles in mice was observed by imaging. Compared with LDL-DiR, LDL-Di R-Ang had more aggregation in the brain, stronger fluorescence and better brain targeting. At the same time, LDL-DiI-Ang and LDL-DiI were injected into the tail vein of the nude mice. The frozen section of the tumor bearing brain tissue was taken under the laser confocal microscope, and the results were in accordance with the living body imaging. ICR mice, blank LDL and LDL-Ang were continuously given for seven days, and each tissue was stained by CD68 immunohistochemical staining to verify that the nanoparticles had no obvious histotoxicity. The fifth part of the subject was to study the pharmacokinetics and tissue distribution of the low density lipoprotein nanoparticles in the body, and the ICR mice were divided into three groups, SAL, LDL-SAL and LDL-SAL-Ang. The blood concentration of SAL in different groups was changed with time and tissue distribution. The results showed that both LDL-SAL and LDL-SAL-Ang had a sustained release effect, the half-life was prolonged significantly, the area under the curve of the drug time increased significantly and the accumulation of.LDL-SAL-Ang in the brain tissue was higher than that of the other two groups, and the uptake of the brain tissue was significantly higher than that of the other two groups. LDL-SAL-Ang has strong brain targeting ability. In the sixth part, the antitumor activity of low density lipoprotein nanoparticles was investigated in the body. First, the nude mice bearing in situ glioma model was prepared, and the nanoparticles were combined with the first line antiglioma drug temozolomide (TMZ), and the drug group was divided into 8 groups: the control group, the SAL group, the TMZ group, and the LDL-SA. Group L, group LDL-SAL-Ang, group SAL+TMZ, group LDL-SAL+TMZ, and LDL-SAL-Ang+TMZ group. After the end of the administration, the brain tissue sections were immunohistochemical staining, the apoptosis and angiogenesis of glioma cells at the tumor site were observed, and the survival and weight changes of the remaining mice in each group were observed. The results showed that LDL-SAL-Ang could promote the in situ glioma in situ. The apoptosis of glioma cells and inhibition of angiogenesis, improve the survival state of mice and prolong the survival time. The survival time and state of the mice were prolonged and improved. The median survival period of the LDL-SAL-Ang+TMZ group was 47 days longer than that of the control group, which was 67.8% and the decrease was the slowness, and the apoptosis of the tumor cells was significant. The number of blood vessels is the least, which shows the best therapeutic effect in the drug delivery group. It shows that LDL-SAL-Ang has antitumor activity in the body and better combined with TMZ, which can effectively treat glioma. The above results show that the brain targeting drug delivery system, which is constructed by the project, has a strong anti glioma stem cell effect in vivo and in vivo and in vitro, and has a good effect on LDL-SAL-Ang. It has the characteristics of bipolar brain targeting, which targets the tumor tissue through BBB, and achieves multiple targets against glioma. It provides a new idea and experimental basis for the chemotherapy of brain glioma.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R943

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