纳米给药系统的肿瘤干细胞治疗及机制研究

发布时间：2018-06-22 09:15

本文选题：肿瘤干细胞 + 肿瘤耐药　；参考：《浙江大学》2016年博士论文

【摘要】：近年来的研究发现,肿瘤干细胞的存在可能是肿瘤无法治愈的主要原因。肿瘤干细胞长期处于G0期,对作用于细胞分裂期的多数化疗药物不敏感。肿瘤干细胞具备普通干细胞的性质,受外界环境刺激后可分化形成新的肿瘤细胞,促进肿瘤生长和肿瘤复发。此外肿瘤干细胞较强的成瘤特性,被认为是肿瘤体内转移的主要原因。因此,靶向并清除肿瘤干细胞,成为肿瘤治疗的难点与热点。本研究旨在通过纳米制剂技术,靶向肿瘤干细胞、克服其耐药性,达到杀伤肿瘤干细胞,提高化学药物的抗肿瘤药效的目的。本文的主要内容如下:采用无血清悬浮球培养法,从乳腺癌细胞系MCF-7中富集乳腺癌肿瘤干细胞(Breast cancer stem cells,breast CSCs)。获得的细胞球结构致密、边界清晰,直径约200μm。经检测具备breast CSCs表面分子标记物CD44+/CD24-的细胞比例为36.51%,干性基因Nanog、OCT4及SOX2的转录与蛋白表达均出现上调,耐药蛋白ABCG2表达增强,证实该方法实现了肿瘤干细胞的富集。低分子量壳聚糖硬脂酸嫁接物(Stearic acid-g-chitosan oligosaccharide,CSOSA)形成的糖脂纳米粒在细胞球上具有较强的摄取和渗透能力,可将药物阿霉素(Doxorubicin,DOX)递送到细胞球的内部。酸性磷酸酶试验法(Acid phosphatase assay,APH assay)检测细胞球的存活率,结果表明糖脂载药纳米粒(CSOSA/DOX)比市售盐酸阿霉素制剂(DOX·HCl)有更强的细胞球生长抑制作用,其IC50值分别为1.07和2.64μg/mL。本文分别构建了 MCF-7原位肿瘤动物模型及细胞球原位肿瘤动物模型,研究发现两类肿瘤模型有较大的区别。细胞球肿瘤组织中,细胞成分组成复杂、呈细胞多样性,可致原位乳腺结构重塑,具备一定的耐药性且微环境间质丰富,结构上更接近于原发肿瘤。为使相关研究更接近于临床,本课题采用细胞球肿瘤模型,并选择多周期给药方案。结果显示,第一治疗周期,市售制剂抑瘤效果强于糖脂载药纳米粒。从第二周期开始,市售制剂组肿瘤体积继续增大,糖脂载药纳米粒组肿瘤体积基本不变,两组肿瘤的体积差逐渐缩小。第三周期结束时,糖脂载药纳米组的抗肿瘤效果显著优于市售制剂组(**p0.01)。CSOSA/DOX在杀灭非breast CSCs的同时,可同时杀灭breast CSCs,不会导致肿瘤干细胞的富集;市售制剂对照只能杀灭非breast CSCs,肿瘤干细胞比例由10.95%升高至69.36%。CSOSA/DOX还可通过影响肿瘤相关成纤维细胞活性,减少胶原蛋白的分泌,破坏肿瘤微环境的完整性,导致肿瘤血管数量增多变粗,进一步提高抗肿瘤药效。采用MCF-7肿瘤动物模型,考察多周期给药对肿瘤耐药的影响。研究发现,经过三周期的连续给药治疗,CSOSA/DOX组呈现更强的肿瘤生长抑制作用,其效果优于DOX·HC1。DOX·HC1组肿瘤组织P-gp蛋白表达明显上调,CSOSA/DOX组肿瘤观察不到明显P-gp。采用高剂量脉冲法及低剂量连续刺激法,制备细胞耐药模型,模拟体内产生耐药的过程。DOX·HC1刺激组,细胞对药物的敏感性下降,高剂量与低剂量刺激法获得的细胞其IC50值由0.27±0.04μg/mL升高为0.80±0.03及0.88±0.07μg/mL,耐药能力增强。CSOSA/DOX刺激的细胞,对药物的敏感度变化不大。与体内结果相似,CSOSA/DOX不会诱导细胞P-gp蛋白表达上调。研究发现,CSOSA/DOX不影响mdr1基因的转录水平,而市售制剂可刺激mdr1基因转录P-gp mRNA。低剂量刺激及高剂量刺激上调后的转录水平,分别为敏感MCF-7细胞的6154倍及1191倍,显示CSOSA/DOX通过不增加P-gp蛋白表达水平,减少耐药的发生。脂质纳米载体,以其良好的生物相容性及对难溶性药物较强的包载能力,广泛应用于抗肿瘤治疗。本文选用抗肿瘤药物奥沙利铂(Oxaliplatin,OXA)及肿瘤干细胞特异性药物盐霉素(Salinomycin,SAL)联合治疗肝癌。A54多肽通过PEG链段与脂肪链段十八胺(Octadecylamine,ODA)相连,合成A54-PEG-ODA嫁接物。A54-PEG-ODA嫁接物通过其疏水端插入固体脂质纳米粒(Solid lipid nanoparticle,SLN),构建A54多肽修饰的脂质纳米粒(A54-PEG-SLN)。通过形成奥沙利铂磷脂复合物,实现A54-PEG-SLN对OXA的有效包封。投药量为5%时,载药量和包封率分别为3.46±0.12%和65.3±1.9%。研究表明,,A54多肽修饰的纳米粒,可加快BEL-7402细胞的摄取速度,并具有摄取该细胞的特异选择性。A15适配体可特异性识别肿瘤干细胞标志物CD133分子且亲和力强,本文通过A15的端氨基与NH2-PEG-SLN/SAL脂质纳米粒表面的端氨基反应,制备A15修饰的纳米粒A15-PEG-SLN/SAL。该纳米粒对难溶性药物盐霉素可有效包封,投药量为10%时,载药量及包封率分比为7.77±0.28%和84.42±0.55%。体外细胞药效表明,游离奥沙利铂及A54-PEG-SLN/OXA纳米粒对BEL-7402细胞的IC50值分别为8.11±1.5及16.0±1.2μg/mL;包封后药效下降,可能与药物的释放有关。游离盐霉素对肿瘤干细胞杀伤作用不明显,主要由溶剂二甲亚砜导致;而A15-PEG-SLN/SAL纳米粒肿瘤干细胞药效显著IC50值为0.69±0.015μ/mL。A15-PEG-SLN/SAL纳米粒对BEL-7402细胞杀伤能力较弱,10μg/mL时存活率为80%。考虑到BEL-7402细胞对盐霉素不敏感,在进行体内抗肿瘤药效序贯给药研究时,采用先给予奥沙利铂制剂,杀伤肿瘤敏感细胞,使肿瘤干细胞暴露出治疗位点。当连续给药两个周期后,肿瘤干细胞比例由0.1%升高至3.0%,此时再给予盐霉素制剂。模型动物药效学研究结果显示,A54多肽修饰A54-PEG-SLN/OXA纳米粒的抗肿瘤作用增强,与游离奥沙利铂相当。联合盐霉素给药后,制剂组抗肿瘤药效显著优于游离药物组。本论文研究结果表明,CSOSA/DOX在肿瘤的多周期治疗中,发挥良好的抗肿瘤作用,杀伤肿瘤干细胞,抑制肿瘤复发。A54-PEG-SLN/OXA与A15-PEG-SLN/SAL联合疗法,增强化疗药物OXA的抗肿瘤药效,发挥协同作用,有效治疗肝癌。
[Abstract]:Recent studies have found that the existence of tumor stem cells may be the main reason for the inability of the tumor to be cured. The tumor stem cells are in the G0 phase for a long time and are insensitive to most of the chemotherapeutic drugs that act on the cell division stage. Growth and tumor recurrence. In addition, the strong tumorigenicity of tumor stem cells is considered to be the main cause of metastasis in the tumor. Therefore, targeting and removing tumor stem cells is the difficult and hot spot in the treatment of cancer. The main contents of the antitumor effect of high chemical drugs. The main contents of this article are as follows: using the serum-free suspension ball culture, the mammary cancer cancer stem cells (Breast cancer stem cells, breast CSCs) are enriched from the breast cancer cell line MCF-7. The obtained cell spheres are dense, the boundary is clear, and the diameter of the 200 u M. is detected with the breast CSCs surface points. The cell ratio of the submarker CD44+/CD24- was 36.51%, the transcriptional and protein expression of the dry gene Nanog, OCT4 and SOX2 increased, and the expression of the drug resistant protein ABCG2 was enhanced. It proved that the method realized the enrichment of the cancer stem cells. The glycolipid formed by the low molecular weight chitosan stearic acid receptor (Stearic acid-g-chitosan oligosaccharide, CSOSA) was formed. The nanoparticles have strong uptake and osmotic ability on the cell spheres, which can be delivered to the inside of the cell ball. The acid phosphatase test (Acid phosphatase assay, APH assay) is used to detect the survival rate of the cell ball. The results show that the glycolipid drug loaded nanoparticles (CSOSA/DOX) are compared to the commercial adriamycin hydrochloride (DOX HCl) (DOX. HCl). With a stronger inhibition of cell growth, the IC50 values were 1.07 and 2.64 g/mL. respectively. The MCF-7 in situ tumor animal model and the cell ball in situ tumor animal model were constructed respectively. The study found that the two types of tumor models were different. In the cell spheroid tumor tissue, the cell composition is complex and the cell diversity can cause in situ milk. In order to make the related research more close to the clinic, we adopt the cell ball tumor model and choose the multi cycle drug delivery scheme. The results show that the first treatment period is better than the drug loaded nanoparticles in the first treatment period, from second. At the beginning of the period, the volume of tumor in the market preparation group continued to increase, the tumor volume of the glycolipid drug loaded nanoparticles group was basically unchanged. The volume difference of the two groups was gradually reduced. The anti-tumor effect of the glycolipid drug loaded nanometers was significantly better than the market agent group (**p0.01).CSOSA/ DOX in killing non breast CSCs at the end of the period, and at the same time killing breas at the same time. T CSCs does not lead to the enrichment of tumor stem cells; the market preparation control can only kill non breast CSCs, the proportion of cancer stem cells increased from 10.95% to 69.36%.CSOSA/DOX, which can also affect the activity of tumor related fibroblasts, reduce the secretion of collagen, destroy the integrity of the tumor microenvironment, and lead to the increase of tumor blood vessels. MCF-7 tumor animal model was used to investigate the effect of multi cycle drug delivery on tumor resistance. The study found that after three cycles of continuous administration, the CSOSA/DOX group showed a stronger tumor growth inhibition effect, and the effect was better than that of the DOX HC1.DOX. HC1 group, the expression of P-gp protein was obviously up-regulated, and the CSOSA/DOX group was swollen. The tumor was not observed by P-gp. using high dose pulse method and low dose continuous stimulation method to prepare the cell resistance model, to simulate the drug resistance process in the.DOX. HC1 stimulation group, and to decrease the cell sensitivity to the drug. The IC50 value of the cells obtained by the high dose and low dose stimulation method increased from 0.27 + 0.04 Mu to 0.80 + 0.03 and 0.88 + 0.07 g/. ML, the resistance to.CSOSA/DOX stimulated cells, the sensitivity of the drug changed little. Similar to the results in the body, CSOSA/DOX did not induce the up regulation of P-gp protein expression. The study found that CSOSA/DOX did not affect the transcriptional level of the MDR1 gene, and the market preparation could stimulate the low dose stimulation of the MDR1 gene transcriptional P-gp mRNA. and the up-regulation of high dose stimulation. The post transcriptional level, 6154 times and 1191 times that of sensitive MCF-7 cells, shows that CSOSA/DOX can reduce the occurrence of drug resistance by not increasing the expression of P-gp protein. The lipid nanoscale is widely used in antitumor therapy with its good biocompatibility and its strong encapsulation ability to insoluble drugs. Oxaliplatin (OXA) and tumor stem cell specific drug Salinomycin (SAL) are combined to treat the.A54 polypeptide of liver cancer by connecting to the eighteen amine (Octadecylamine, ODA) of the fat chain segment through the PEG segment, and the A54-PEG-ODA marrying agent is inserted into the solid lipid nanoparticles (Solid lipid) through its water terminal. The A54 polypeptide modified lipid nanoparticles (A54-PEG-SLN) was constructed. By forming the oxaliplatin phospholipid complex, the effective encapsulation of OXA was achieved by A54-PEG-SLN. When the dosage was 5%, the drug loading and encapsulation efficiency were 3.46 + 0.12% and 65.3 + 1.9%. respectively. The A54 polypeptide modified nanoparticles could speed up the uptake of BEL-7402 cells and have the uptake. The specific selective.A15 aptamers of this cell can specifically identify CD133 molecules of tumor stem cell markers and have strong affinity. In this paper, the A15 modified nanoparticles A15-PEG-SLN/SAL., a nano particle of A15 modified nanoparticles, is prepared by the reaction of the amino terminal amino group of the A15 and the NH2-PEG-SLN/SAL lipid nanoparticles on the surface of the lipid nanoparticles. The drug loading and encapsulation ratio Ratio of 7.77 + 0.28% and 84.42 + 0.55%. in vitro showed that the IC50 value of free oxaliplatin and A54-PEG-SLN/OXA nanoparticles to BEL-7402 cells was 8.11 + 1.5 and 16 + 1.2 mu g/mL, respectively, and the drug effect decreased after encapsulation, which may be related to the release of drugs. The effect was not obvious, mainly caused by the solvent two a sulfoxide, and the significant IC50 value of the A15-PEG-SLN/SAL nanoparticle tumor stem cells was 0.69 + 0.015 mu /mL.A15-PEG-SLN/SAL nanoparticles with weak killing ability to BEL-7402 cells. The survival rate of 10 mu g/mL was 80%., considering that the BEL-7402 cells were insensitive to the salt mycophenycin, and the anti-tumor efficacy of the vivo was carried out in the body. In the study of drug administration, oxaliplatin was first given to kill tumor sensitive cells and to expose the tumor stem cells to the treatment site. After two cycles of continuous administration, the proportion of the tumor stem cells increased from 0.1% to 3%, and then the salt mycin preparation was given. The results of the model animal pharmacodynamics study showed that the A54 polypeptide modified the A54-PEG-SLN/OXA nanoparticles. The antitumor effect of the combined oxaliplatin was equal to that of free oxaliplatin. After the combination of salt mycin, the antitumor effect of the preparation group was significantly better than that of the free drug group. The results of this study showed that CSOSA/DOX played a good anti-tumor effect, killed tumor stem cells and inhibited the recurrence of tumor.A54-PEG-SLN/OXA and A15-PEG-SLN in the multi cycle treatment of the tumor. /SAL combined therapy can enhance the antitumor efficacy of OXA and play a synergistic role in the treatment of HCC.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R943

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