【作者】 刘洁；

【导师】 谢民强；

【作者基本信息】 南方医科大学 ， 耳鼻咽喉头颈外科学， 2011， 硕士

【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(P>0.05),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(P>0.05)。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(P>0.05),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。更多还原

【Abstract】 Laryngeal carcinoma is one of the common malignant tumors that attack human head and neck, accounting for approximately 1% of overall malignant tumors. In China, the incidence rate of laryngeal carcinoma is about 1.5-3 per 10 million people. In the sites of ear, nose and throat, its incidence rate is just lower than that of nasopharyngeal carcinoma and or nasal cavity and sinus carcinoma with second in norther China and third in souther. As the early symptoms of laryngeal cancer is not obvious, its diagnosis is often delayed to advanced stage, especially in supraglottic cancer and hypopharyngeal cancer. Besides, in advanced laryngeal cancer, the rate of lymph node metastasis is high, while the surgical total resection rate is low, and the 5-year survival rate is less than 30%. Moreover, the patients who underwent total laryngectomy will lose speech function in all their life. The leading causes of death of patients with laryngeal carcinoma are local recurrence and cervical lymph node metastasis. The present treatments like surgery, radiotherapy or chemotherapy have little effctiveness on those patients with advanced laryngeal carcinoma. As a result, it has long been a problem for clinicians to improve the survival rate and life quality of patients after advanced laryngeal cancer treatments. Molecular targeted therapy is a new means of cancer treatment, specifically aiming at the target molecules of cancer cell which play a key role in tumorigenesis and development, or taking advantage of overexpression receptor of cancer cell surface, delivered targeting drug to the tumor cells through the coupling of special ligands to treat cancer and reduce systemic side-effects. The folate receptor as a molecular target for cancer therapy has already had its clinical application. Its ligand is a small molecular weight of vitamin folic acid, which is highly expressed in the tumor cell surface, whereas it has almost no expression in most normal tissues. Because of the high affinity between folic acid and the folate receptor, folic acid can be conjugated with drug and targeted to the tumor. Preliminary studies of our group have found the high expression of folate receptor in laryngeal cancer and successfully prepared folate modified magnetic nanoparticles loaded cisplatin and loaded targeting gene, which show satisfying results in the treatment of nasopharyngeal carcinoma and matrix metalloproteinase-2 gene silencing in laryngeal cancer cells. However, the stability of drug and capacity of drug loading are still not desirable. This study focuses on optimizing the drug preparation technology. With laryngeal cancer cells Hep-2 as the research object, it evaluates targeting property and therapeutic effect of this molecular targeting medicine optimized through drug intake and in vitro inhibit experiments. The study is divided into three parts as follows.The first part stresses on the optimization and characterization of the preparation process of folate modified magnetic nanoparticles loaded cisplatin.In order to achieve preparation process optimization, the ratio of glyco toluenesulfonyl chloride and PEG was increased from 4～5:1 to 6:1 during the preparation of amine-terminated poly-ethylene; as a result, the reaction was sped up significantly. Besides, during the folate carboxyl activation process, the proportion of each component was optimized, and the coupling of amino PEG was increased. Characterization results showed that the folate modified magnetic nanoparticles loaded cisplatin optimized had a good stability and magnetic magnetic responsiveness. Its average hydrodynamic diameter was 110.9+1.7 nm, zeta potential was-26.45±1.26 mV, cisplatin content was 1.3 mg/ml, iron content was about 1.39 mg/ml, and maximum saturation magnetization was 22.2 emu/g.The second part focuses on the study of the targeting property of folate modified magnetic nanoparticles loaded cisplatin for laryngeal cancer cells.In this study, the folic acid receptor-positive laryngeal cancer Hep-2 cells was used as the research subject, and folate receptor-positive nasopharyngeal carcinoma cells HNE-1 and folate receptor-negative nasopharyngeal carcinoma cell CNE-2 was used as control. The study investigated the targeting property of folate modified magnetic nanoparticles loaded cisplatin (FA-CDDP-MNPs) through the nanomedicine uptake of Hep-2 cells, Prussian iron staining and transmission electron microscopy. The results showed that folate modifide magnetic nanoparticles(FA-MNPs) and FA-CDDP-MNPs were susceptible uptaked by folate receptor-positive carcinoma cells, Hpe-2 and HNE-1, not susceptible uptaked by folate receptor expression negative CNE-2, and the magnetic nanoparticles were present in the cytoplasm after cellular uptake, which showed that FA-CDDP-MNPs has a good molecular targeting.The third part stresses on folate modified magnetic nanoparticles loaded cisplatin for laryngeal cancer cells in vitro inhibition effects:The study used MTT, flow cytometry and transmission electron microscopy to detect the in vitro inhibition effects and cytotoxicity of FA-CDDP-MNPs, CDDP, and FA-MNPs on Hep-2 laryngeal carcinoma cells. MTT Results showed that both the FA-CDDP-MNPs and CDDP were obvious dose dependent and time dependent for the inhibition rate to Hep-2. When the concentration of cisplatin is 8μg/ml and cultured for 48 h, the inhibition rate of FA-CDDP-MNPs is 82.2% and that of CDDP reached as high as 93.3%. There was no significant difference between them (P> 0.05), and the FA-MNPs had no effect on the growth of Hep-2.The flow cytometry results was consistent with that of MTT. Hep-2 cells were cultured for 48 hours with FA-CDDP-MNPs and CDDP respectively. Cells were obvious apoptosis, and apoptosis rate ware in positive proportion to the increase of drug concentration. However, there was no significant difference between the two kinds of drug (P>0.05). Low concentrations all resulted in cells G0/G1 phase blocking, and high concentration did not affect cell cycle, while the same concentration of FA-MNPs did not affect apoptosis of Hep-2, but made the cell cycle shift to S phase.TEM results showed that the morphology of the cells which uptaked FA-CDDP-MNPs were changed significantly, while the ones which uptaked FA-MNPs displayed no change in cell morphology. The above results suggested that in vitro FA-CDDP-MNPs had the same effects on inhibiting the growth of laryngeal cancer Hep-2 cells just as cisplatin did alone, while the carrier itself had no cytotoxicity, and could be uptaked by Hep-2, but high concentration might affect the cell cycle.更多还原