叶酸靶向载顺铂磁性纳米药物治疗喉癌的体外实验研究

发布时间：2018-03-06 21:15

本文选题：喉癌　切入点：分子靶向　出处：《南方医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：喉癌是头颈部较常见恶性肿瘤,我国的发病率约为1.5-3/10万人口,约占全身恶性肿瘤的1%,在耳鼻咽喉科仅次于鼻咽癌和鼻腔、鼻窦癌,北方居第二位,南方居第三位。喉癌早期症状不明显,常常延误至晚期才‘被明确诊断,特别是声门上型癌和喉咽癌。晚期癌淋巴结转移率高,手术全切率低,5年生存率不到30%,而且患者终身失去言语功能。喉癌局部复发和颈淋巴结转移是死亡的主要原因。晚期癌无论手术、放疗还是化疗,效果均不满意,如何提高晚期喉癌治疗后的生存率和生活质量一直是困扰临床医生的难题。分子靶向治疗是一种新的肿瘤治疗手段,它能够特异性地作用于肿瘤发生发展中起关键作用的靶分子,或者利用肿瘤高表达的受体,通过配体将偶联的药物靶向输送到肿瘤细胞,从而达到治疗肿瘤、减少全身毒副作用的目的。叶酸受体作为肿瘤治疗的一个分子靶点已进入临床应用研究,其配体是一种小分子量的维生素叶酸,后者在肿瘤细胞膜表面高度表达；而在绝大多数正常组织中几乎不表达。利用叶酸和叶酸受体的高度亲和性,可将与叶酸偶联的药物靶向至肿瘤。我们课题组前期研究发现喉癌高表达叶酸受体,并已成功制备了叶酸靶向载顺铂和载基因磁性纳米药物,在喉癌基质金属蛋白酶2基因沉默和鼻咽癌的治疗研究中取得良好效果。但发现药物的稳定性和载药量尚不满意,本研究在此基础上对药物制备工艺进行优化,并以喉癌细胞Hep-2作为研究对象,通过药物摄取和体外抑制实验评价这种优化后的叶酸分子靶向载顺铂磁性纳米药物的靶向性和治疗效果。共分三个部分：第一部分,叶酸分子靶向载顺铂磁性纳米药物制备工艺的优化及表征制备工艺优化主要是在端氨基聚乙二醇制备过程中将对甲苯磺酰氯与聚乙二醇的比例从4-5：1提高至6：1,使反应速度明显加快。叶酸的羧基活化过程中优化了各组分的比例,与端氨基聚乙二醇的耦合量增加。表征结果显示：改进后的叶酸分子靶向载顺铂磁性纳米药物平均水动力学直径为110.9±1.7nm, zeta电位为-26.45±1.26 mV,顺铂含量为1.3mg/ml,铁含量约为.1.39mg/ml,最大饱和磁化强度为22.2emu/g,具有良好的稳定性和磁响应性。第二部分,叶酸分子靶向载顺铂磁性纳米药物对喉癌细胞的靶向性研究以叶酸受体阳性喉癌Hep-2细胞作研究对象,叶酸受体阳性鼻咽癌细胞HNE-1和叶酸受体阴性鼻咽癌细胞CNE-2作对照,通过细胞摄取铁染色、透射电镜考察叶酸分子靶向载顺铂磁性纳米药物(FA-CDDP-ASA-MNPs)的靶向性。结果显示：叶酸分子靶向磁性纳米载体及其载顺铂磁性纳米药物均易被叶酸受体表达阳性的喉癌细胞Hpe-2和鼻咽癌细胞HNE-1摄取,而不易被叶酸受体表达阴性的CHE-2摄取,纳米药物被细胞摄取后存在于细胞浆中,结果表明其具有良好的分子靶向性。第三部分,叶酸分子靶向载顺铂磁性纳米药物对喉癌细胞的体外抑制效应采用MTT、流式细胞分析和透射电镜方法检测FA-CDDP-ASA-MNPs、CDDP和FA-ASA-MNPs对喉癌细胞Hep-2的体外抑制效应和细胞毒性。MTT结果显示：FA-CDDP-ASA-MNPs和CDDP二者对Hep-2的抑制率均存在明显的剂量依赖性和时间依赖性,到顺铂含量为8μg/ml作用48 h时,FA-CDDP-ASA-MNPs的抑制率达到82.2%,CDDP高达93.3%,二者之间差异无显著性(P0.05),而FA-ASA-MNPs对Hep-2的生长无影响。流式细胞仪检测结果与MTT一致,FA-CDDP-ASA-MNPs、CDDP分别与Hep-2细胞共培养48小时后,细胞均发生明显凋亡,且凋亡率随药物浓度增加而升高,但二者之间差异无显著性(P0.05)。低浓度时均导致细胞G0/G1期阻滞,高浓度时不影响细胞周期(P0.05),而同样浓度的FA-ASA-MNPs不影响Hep-2凋亡,但可使细胞周期向S期转移。透射电镜结果表明摄取FA-CDDP-ASA-MNPs的细胞出现明显凋亡形态改变,而摄取FA-ASA-MNPs的细胞形态无改变。以上结果提示,顺铂与我们制备的叶酸分子靶向磁性纳米载体连接后仍然具有与单纯顺铂同样的体外抑制喉癌Hep-2细胞生长的效应,而载体本身无细胞毒性,且能被Hep-2摄取,高浓度时可能影响细胞周期。
[Abstract]:Laryngeal cancer is common in head and neck malignant tumor incidence in our country is about 1.5-3/10 million people, accounting for about 1% of all malignant tumors, in otolaryngology after nasopharyngeal and nasal sinus cancer, ranking second in the north, South China ranks third. Early symptoms of laryngeal cancer is not obvious, is often delayed to advanced stage the diagnosis, especially in supraglottic cancer and hypopharyngeal cancer. Cancer lymph node metastasis rate, total resection rate is low, the 5 year survival rate of less than 30%, and the patients lost lifelong speech function. The local recurrence of laryngeal carcinoma and cervical lymph node metastasis is the main cause of death. Both advanced cancer surgery, radiotherapy or chemotherapy the results are not satisfactory, and how to improve the survival rate and quality of life after treatment of advanced laryngeal cancer plagued clinicians. Molecular targeted therapy is a new therapeutic method in the treatment of cancer, it can be specifically applied to the tumor occurrence and development Target molecules play a key role, or high expression by tumor receptor, drug target coupling to delivery to tumor cells by ligand, so as to achieve the treatment of tumor, reduce systemic side effects. A molecular target of folate receptor as a tumor has entered into clinical application, its ligand is a small molecular weight of vitamin folic acid, which is highly expressed in the tumor cell membrane surface; and almost no expression in most normal tissues. High affinity folate and folate receptor can be used, drug target and folate conjugated to tumor. Our previous study found that high expression of folate receptor in laryngeal carcinoma, and has the successful preparation of folate targeted drug cisplatin and gene loaded magnetic nanoparticles, achieved good results in laryngeal carcinoma matrix metalloproteinase 2 gene silencing and treatment of nasopharyngeal carcinoma. But it was found that the stability of drugs And the drug is still not satisfied, on the basis of drug preparation process was optimized, and the laryngeal carcinoma cell Hep-2 as the research object, through drug intake and in vitro experimental evaluation of folic acid molecular target of the optimized to cisplatin loaded magnetic nanoparticles targeting and therapeutic effect. Is divided into three parts:
The first part, optimization and characterization of the preparation of cisplatin magnetic nanoparticles by molecular targeting of folic acid molecules
Optimized preparation process is mainly in the end preparation process, the proportion of amino polyethylene glycol p-toluene sulfonyl chloride and polyethylene glycol increased from 4-5:1 to 6:1, the reaction rate was significantly accelerated. Folic acid carboxyl activation process to optimize the proportion of each component, and increase the amount of coupling of amino terminated polyethylene glycol. The characterization results showed that folic acid molecular target after the cisplatin loaded magnetic nanoparticles the average hydrodynamic diameter was 110.9 + 1.7nm, the zeta potential is -26.45 + 1.26 mV, cisplatin content is 1.3mg/ml, the iron content is about.1.39mg/ml, the maximum saturation magnetization is 22.2emu/g, with stability and good magnetic response.
The second part, the study of the targeting of folic acid molecular targeting cisplatin magnetic nanomaterials on laryngeal cancer cells
The folate receptor positive Hep-2 cancer cells as the research object, folate receptor positive nasopharyngeal carcinoma cell HNE-1 and folate receptor negative nasopharyngeal carcinoma CNE-2 cells as control, the cellular uptake of iron staining, transmission electron microscopy study of folic acid molecular targeted cisplatin loaded magnetic nanoparticles (FA-CDDP-ASA-MNPs) targeting. The results showed that folic acid targeted nanocarriers and cisplatin loaded magnetic nanoparticles are susceptible to folate receptor positive expression in laryngeal carcinoma cells Hpe-2 and nasopharyngeal carcinoma cell HNE-1 uptake, and not susceptible to folate receptor negative CHE-2 uptake, nano drug taken up by the cells in the cytoplasm. The results showed that it has excellent molecular targeting.
The third part, the inhibitory effect of folic acid molecular targeting cisplatin magnetic nanomaterials on laryngeal cancer cells in vitro
By MTT, flow cytometry and transmission electron microscopy method for detection of FA-CDDP-ASA-MNPs, CDDP and FA-ASA-MNPs of Hep-2 laryngeal carcinoma cells in vitro and the effect of cytotoxicity of.MTT showed that FA-CDDP-ASA-MNPs and CDDP two inhibitory rate of Hep-2 were obvious dose and time dependence to cisplatin was 8 g/ml 48 h when FA-CDDP-ASA-MNPs, the inhibition rate reached 82.2%, up to 93.3% CDDP, no significant differences between the two (P0.05), while FA-ASA-MNPs has no effect on the growth of Hep-2. The results of flow cytometry and MTT, FA-CDDP-ASA-MNPs, CDDP were co cultured with Hep-2 cells after 48 hours, cells underwent apoptosis, and the apoptosis rate increased with the concentration increased, but no significant differences between the two (P0.05). Low concentrations led to cell arrest in G0/G1 phase and high concentration does not affect the cell cycle (P0.05), and the same concentration The degree of FA-ASA-MNPs does not influence the apoptosis of Hep-2, but can be transferred to the S phase of cell cycle. The results of TEM showed that the change of apoptosis morphology uptake in FA-CDDP-ASA-MNPs cells, whereas the uptake of FA-ASA-MNPs cell morphology did not change. The above results indicate that cisplatin and we prepared folic acid molecular target is connected to the magnetic nano carrier still has effect in vitro the same with cisplatin only inhibit the growth of human laryngeal carcinoma Hep-2 cells, while the carrier itself has no toxicity, and can be Hep-2 uptake at high concentration may affect the cell cycle.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.65

【引证文献】