本文選題：喉癌　切入點(diǎn)：分子靶向　出處：《南方醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：喉癌是頭頸部較常見惡性腫瘤,我國的發(fā)病率約為1.5-3/10萬人口,約占全身惡性腫瘤的1%,在耳鼻咽喉科僅次于鼻咽癌和鼻腔、鼻竇癌,北方居第二位,南方居第三位。喉癌早期癥狀不明顯,常常延誤至晚期才‘被明確診斷,特別是聲門上型癌和喉咽癌。晚期癌淋巴結(jié)轉(zhuǎn)移率高,手術(shù)全切率低,5年生存率不到30%,而且患者終身失去言語功能。喉癌局部復(fù)發(fā)和頸淋巴結(jié)轉(zhuǎn)移是死亡的主要原因。晚期癌無論手術(shù)、放療還是化療,效果均不滿意,如何提高晚期喉癌治療后的生存率和生活質(zhì)量一直是困擾臨床醫(yī)生的難題。分子靶向治療是一種新的腫瘤治療手段,它能夠特異性地作用于腫瘤發(fā)生發(fā)展中起關(guān)鍵作用的靶分子,或者利用腫瘤高表達(dá)的受體,通過配體將偶聯(lián)的藥物靶向輸送到腫瘤細(xì)胞,從而達(dá)到治療腫瘤、減少全身毒副作用的目的。葉酸受體作為腫瘤治療的一個分子靶點(diǎn)已進(jìn)入臨床應(yīng)用研究,其配體是一種小分子量的維生素葉酸,后者在腫瘤細(xì)胞膜表面高度表達(dá)；而在絕大多數(shù)正常組織中幾乎不表達(dá)。利用葉酸和葉酸受體的高度親和性,可將與葉酸偶聯(lián)的藥物靶向至腫瘤。我們課題組前期研究發(fā)現(xiàn)喉癌高表達(dá)葉酸受體,并已成功制備了葉酸靶向載順鉑和載基因磁性納米藥物,在喉癌基質(zhì)金屬蛋白酶2基因沉默和鼻咽癌的治療研究中取得良好效果。但發(fā)現(xiàn)藥物的穩(wěn)定性和載藥量尚不滿意,本研究在此基礎(chǔ)上對藥物制備工藝進(jìn)行優(yōu)化,并以喉癌細(xì)胞Hep-2作為研究對象,通過藥物攝取和體外抑制實驗評價這種優(yōu)化后的葉酸分子靶向載順鉑磁性納米藥物的靶向性和治療效果。共分三個部分： 第一部分,葉酸分子靶向載順鉑磁性納米藥物制備工藝的優(yōu)化及表征 制備工藝優(yōu)化主要是在端氨基聚乙二醇制備過程中將對甲苯磺酰氯與聚乙二醇的比例從4-5：1提高至6：1,使反應(yīng)速度明顯加快。葉酸的羧基活化過程中優(yōu)化了各組分的比例,與端氨基聚乙二醇的耦合量增加。表征結(jié)果顯示：改進(jìn)后的葉酸分子靶向載順鉑磁性納米藥物平均水動力學(xué)直徑為110.9±1.7nm, zeta電位為-26.45±1.26 mV,順鉑含量為1.3mg/ml,鐵含量約為.1.39mg/ml,最大飽和磁化強(qiáng)度為22.2emu/g,具有良好的穩(wěn)定性和磁響應(yīng)性。 第二部分,葉酸分子靶向載順鉑磁性納米藥物對喉癌細(xì)胞的靶向性研究 以葉酸受體陽性喉癌Hep-2細(xì)胞作研究對象,葉酸受體陽性鼻咽癌細(xì)胞HNE-1和葉酸受體陰性鼻咽癌細(xì)胞CNE-2作對照,通過細(xì)胞攝取鐵染色、透射電鏡考察葉酸分子靶向載順鉑磁性納米藥物(FA-CDDP-ASA-MNPs)的靶向性。結(jié)果顯示：葉酸分子靶向磁性納米載體及其載順鉑磁性納米藥物均易被葉酸受體表達(dá)陽性的喉癌細(xì)胞Hpe-2和鼻咽癌細(xì)胞HNE-1攝取,而不易被葉酸受體表達(dá)陰性的CHE-2攝取,納米藥物被細(xì)胞攝取后存在于細(xì)胞漿中,結(jié)果表明其具有良好的分子靶向性。 第三部分,葉酸分子靶向載順鉑磁性納米藥物對喉癌細(xì)胞的體外抑制效應(yīng) 采用MTT、流式細(xì)胞分析和透射電鏡方法檢測FA-CDDP-ASA-MNPs、CDDP和FA-ASA-MNPs對喉癌細(xì)胞Hep-2的體外抑制效應(yīng)和細(xì)胞毒性。MTT結(jié)果顯示：FA-CDDP-ASA-MNPs和CDDP二者對Hep-2的抑制率均存在明顯的劑量依賴性和時間依賴性,到順鉑含量為8μg/ml作用48 h時,FA-CDDP-ASA-MNPs的抑制率達(dá)到82.2%,CDDP高達(dá)93.3%,二者之間差異無顯著性(P0.05),而FA-ASA-MNPs對Hep-2的生長無影響。流式細(xì)胞儀檢測結(jié)果與MTT一致,FA-CDDP-ASA-MNPs、CDDP分別與Hep-2細(xì)胞共培養(yǎng)48小時后,細(xì)胞均發(fā)生明顯凋亡,且凋亡率隨藥物濃度增加而升高,但二者之間差異無顯著性(P0.05)。低濃度時均導(dǎo)致細(xì)胞G0/G1期阻滯,高濃度時不影響細(xì)胞周期(P0.05),而同樣濃度的FA-ASA-MNPs不影響Hep-2凋亡,但可使細(xì)胞周期向S期轉(zhuǎn)移。透射電鏡結(jié)果表明攝取FA-CDDP-ASA-MNPs的細(xì)胞出現(xiàn)明顯凋亡形態(tài)改變,而攝取FA-ASA-MNPs的細(xì)胞形態(tài)無改變。以上結(jié)果提示,順鉑與我們制備的葉酸分子靶向磁性納米載體連接后仍然具有與單純順鉑同樣的體外抑制喉癌Hep-2細(xì)胞生長的效應(yīng),而載體本身無細(xì)胞毒性,且能被Hep-2攝取,高濃度時可能影響細(xì)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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R739.65

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 李維;靶向FOLR1的脫氧核酶增加鼻咽癌對紫杉醇敏感性研究[D];中南大學(xué);2012年

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本文編號：1576534