本文關(guān)鍵詞： 熊果酸 隱形納米脂質(zhì)體 葉酸受體靶向 組織分布 促凋亡 人口腔表皮樣癌 抑瘤作用　出處：《華中科技大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：研究目的： 熊果酸(Urosolic acid, UA)具有廣泛的生物學(xué)效應(yīng)和良好的抗癌效果,但因其水溶性差且生物利用度低,使其廣泛應(yīng)用受到限制。本研究首先制備熊果酸長(zhǎng)循環(huán)脂質(zhì)體,再利用葉酸與葉酸受體之間的特異性結(jié)合能力和某些腫瘤細(xì)胞表面葉酸受體過度表達(dá)的特點(diǎn),用葉酸修飾的脂質(zhì)材料制備葉酸靶向熊果酸脂質(zhì)體,極大地提高了熊果酸脂質(zhì)體在腫瘤部位的富集,在解決熊果酸溶解性和生物利用度問題的同時(shí),增加腫瘤靶區(qū)組織里的藥物濃度,延長(zhǎng)其作用時(shí)間,提高抗癌效果,降低藥物的毒副作用,為臨床腫瘤的有效治療提供一種新的思路和方法。 研究方法： 在本課題的研究中,首先采用經(jīng)典的脂質(zhì)體制備方法薄膜分散法分別制備非靶向熊果酸長(zhǎng)循環(huán)脂質(zhì)體(non-targeted PEGylated liposomal UA, PL-UA)和葉酸受體靶向熊果酸長(zhǎng)循環(huán)脂質(zhì)體(folate-receptor targeted liposomal UA, FTL-UA),同時(shí)考察熊果酸脂質(zhì)體的理化性質(zhì)如粒徑、載藥量、包封率、電位和穩(wěn)定性等各項(xiàng)因素。 選擇合適的腫瘤靶向細(xì)胞,探討熊果酸脂質(zhì)體的細(xì)胞靶向性及體外抑瘤作用,分析PL-UA和FTL-UA對(duì)人口腔表皮樣癌(KB)細(xì)胞抑制作用上的差異性：利用細(xì)胞增殖抑制實(shí)驗(yàn)(MTT)分析熊果酸脂質(zhì)體對(duì)KB腫瘤細(xì)胞生長(zhǎng)抑制作用；Hoechst33258熒光染色分析細(xì)胞凋亡的形態(tài)學(xué)變化；流式細(xì)胞術(shù)分析熊果酸脂質(zhì)體對(duì)細(xì)胞凋亡率的影響和細(xì)胞周期分布的變化；通過Annexin V-FITC/PI雙染的細(xì)胞凋亡試驗(yàn),定量分析藥物誘導(dǎo)細(xì)胞凋亡的具體情況。 考察UA脂質(zhì)體在小鼠體內(nèi)藥物代謝動(dòng)力學(xué)及組織分布的特征：通過對(duì)小鼠血漿、心、肝、脾、胃、腎等組織的藥物含量進(jìn)行分析,考察熊果酸隱形納米脂質(zhì)體、游離藥物在體內(nèi)長(zhǎng)循環(huán)作用、組織分布上的差異性,為進(jìn)一步研究葉酸受體靶向脂質(zhì)體在腫瘤治療中的應(yīng)用提供依據(jù)。 考察熊果酸脂質(zhì)體葉酸靶向治療荷瘤鼠人口腔表皮樣癌的作用：在前述實(shí)驗(yàn)研究的基礎(chǔ)上進(jìn)一步證實(shí)FTL-UA是否具有良好的抗腫瘤效果,利用KB細(xì)胞表面大量表達(dá)葉酸受體的特征,以balb/c裸鼠為研究對(duì)象,建立荷瘤鼠人口腔表皮樣癌KB腫瘤模型,通過對(duì)該模型尾靜脈注射FTL-UA,觀察其靶向治療荷瘤鼠人口腔表皮樣癌的效果及對(duì)荷瘤鼠生存周期的影響,為FTL-UA進(jìn)一步應(yīng)用于臨床腫瘤靶向治療提供依據(jù)。 實(shí)驗(yàn)結(jié)果： 采用薄膜分散法制備的FTL-UA及PL-UA的平均粒徑分別為(160.1±12.5) nm,(151.4士16.1)m;分散系數(shù)為0.196±0.052,0.168±0.066,粒度分布窄,大小均勻；其電位分別為(-21.24±4.2)mV,(-23.15±6.7)mV;包封率為(88.9±7.9)%,(86.7土12.1)%。在4℃放置3個(gè)月后,能維持較好的穩(wěn)定性。 在體外,MTT結(jié)果顯示FTL-UA對(duì)KB細(xì)胞有明顯的殺傷作用,其抑制率呈明顯時(shí)間-劑量依賴性,FTL-UA組的IC50值低于PL-UA組(作用72h后的IC50值分別為22.05μM和146.3μM)；細(xì)胞周期分布的結(jié)果顯示FTL-UA主要使KB細(xì)胞阻滯于S期,即腫瘤細(xì)胞的DNA合成期；另外,熒光染色和流式細(xì)胞術(shù)分析也證實(shí)了FTL-UA能誘導(dǎo)KB細(xì)胞凋亡。 給6周齡左右小鼠尾靜脈注射20mg/Kg藥物,通過藥物代謝動(dòng)力學(xué)各參數(shù)和組織分布的分析,熊果酸脂質(zhì)體的半衰期、藥-時(shí)曲線下面積、表觀分布容積、平均滯留時(shí)間和清除率較單純給予游離熊果酸有顯著性差異,表現(xiàn)出明顯的體內(nèi)緩釋和長(zhǎng)循環(huán)效應(yīng)。同時(shí),熊果酸脂質(zhì)體在肝、腎中含量高于其他組織,在其他組織中分布很少,僅有少量組織殘留。 建立balb/c裸鼠KB腫瘤模型,尾靜脈分別注射4.5mg/Kg的游離UA.PL-UA及FTL-UA,每隔一天注射一次,連續(xù)給藥5次。其結(jié)果顯示FTL-UA組中每只荷瘤鼠的腫瘤表面有明顯的壞死空洞形成,瘤體逐漸萎縮,明顯抑制腫瘤生長(zhǎng),生存周期也高于其他組。 實(shí)驗(yàn)結(jié)論： 本實(shí)驗(yàn)研究結(jié)果提示,FTL-UA有望作為新型的化療藥物在人口腔癌化療方面發(fā)揮積極的作用。除了改善熊果酸的溶解性外,還可增加藥物療效,延長(zhǎng)體內(nèi)循環(huán)時(shí)間,顯著抑制腫瘤生長(zhǎng),發(fā)揮其抗腫瘤效應(yīng)。本研究中所獲得的實(shí)驗(yàn)結(jié)果也為FTL-UA作為化療藥物進(jìn)一步應(yīng)用于臨床腫瘤的治療奠定了基礎(chǔ)。通過對(duì)該新劑型的研究,化療藥物的葉酸受體靶向治療有可能成為腫瘤綜合治療的新方法之一。
[Abstract]:The purpose of the study is:
Ursolic acid (Urosolic acid UA) has a wide range of biological effects and good anticancer effect, but because of its poor water solubility and low bioavailability, its application was limited. Ursolic acid was firstly prepared long circulating liposomes in this study, using specific characteristics between folate and folate receptor binding ability and some tumor cell surface receptor overexpression with folic acid, folic acid modified lipid material preparation of folate targeted liposomes of ursolic acid, greatly improve the ursolic acid liposome in the enrichment of the site of the tumor, in the solution of ursolic acid solubility and bioavailability of the problem at the same time, increase the drug concentration in the tumor tissues in the target area and the role of time, improve the anti-cancer effect, reduce the side effects of drugs, to provide a new idea and method for effective clinical treatment of tumors.
Research methods:
In this study, firstly, the classic preparation methods of liposome were prepared by film dispersion method and non targeting of ursolic acid long circulating liposomes (non-targeted PEGylated liposomal UA, PL-UA) and folate receptor targeting of ursolic acid long circulating liposomes (folate-receptor targeted liposomal UA, FTL-UA), and the effects of ursolic acid liposome the physicochemical properties such as particle size, drug loading, encapsulation efficiency, various factors of potential and stability.
Choose appropriate to the tumor target cells, target cells of ursolic acid liposomes to the antitumor effect and in vitro analysis of PL-UA and FTL-UA on human oral epidermoid carcinoma (KB) cell difference inhibition: inhibition of cell proliferation (MTT) analysis of the inhibitory effects of ursolic acid liposome on KB tumor cells growth; morphological changes of cell apoptosis analysis by Hoechst33258 staining; analysis of ursolic acid liposome on apoptosis and changes of cell cycle distribution by flow cytometry; cell apoptosis test Annexin V-FITC/PI double staining, quantitative analysis of the specific situation of drug induced apoptosis.
The effects of UA liposomes in vivo drug metabolism and pharmacokinetics characteristics of mice and tissue distribution in mice by plasma, heart, liver, spleen, stomach, kidney and other tissues of the drug content of ursolic acid, stealth nano liposomes and free drug in the body long cycle, differences in tissue distribution, provide the basis for to the application of liposome in the treatment of tumor and further study on folate receptor targeting.
To the treatment of nude mice bearing human oral epidermoid carcinoma of the effects of ursolic acid liposome target: folic acid based on the experimental study on further confirm whether FTL-UA has a good anti-tumor effect by KB cell surface expression characteristics of folate receptor, balb/c in nude mice as the research object, the establishment of human oral epidermoid tumor bearing mice kind of KB cancer model, based on the model of tail vein injection of FTL-UA to observe the targeted treatment of nude mice bearing human oral epidermoid carcinoma and its effect on the survival period of tumor bearing mice, FTL-UA for the further clinical application of tumor targeted therapy provides the basis.
Experimental results:
The average particle by film dispersion of FTL-UA and PL-UA prepared by the diameters of (160.1 + 12.5) nm, (151.4 + 16.1) m; dispersion coefficient is 0.196 + 0.052,0.168 + 0.066, narrow size distribution and uniform size; the potential respectively (-21.24 + 4.2) mV, (-23.15 + 6.7) mV package; was (88.9 + 7.9)% and (86.7 + 12.1)%. For 3 months at 4 DEG C, can maintain good stability.
In vitro, MTT results showed that FTL-UA has obvious killing effect on KB cells, the inhibition rate was time dose dependent, FTL-UA group's IC50 value is lower than that of group PL-UA (72h after the IC50 values were 22.05 M and 146.3 M); cell cycle distribution showed that FTL-UA KB cells. The arrest in S phase, DNA synthesis of tumor cells; in addition, fluorescence staining and flow cytometry analysis also confirmed that FTL-UA could induce apoptosis of KB cells.
For about 6 week old mice tail vein injection of 20mg/Kg drugs, through the analysis of pharmacokinetic parameters and tissue distribution, liposome half-life, area under concentration time curve, the apparent volume of distribution, the average residence time and clearance were treated free of ursolic acid has significant differences, showing sustained release in vivo the obvious and long circulating effect. At the same time, liposome in the liver, kidney was higher than that in other tissues, rarely found in other tissues, only a small amount of tissue residues.
The establishment of balb/c KB nude mice tumor model, free UA.PL-UA and FTL-UA 4.5mg/Kg were injected into the tail vein injection, every day once, continuous administration of 5 times. The results showed that the surface of the tumor in the FTL-UA group each mice had obvious necrosis of tumor cavity, gradually atrophic, inhibit tumor growth, survival period is also higher than other groups.
Experimental conclusions:
The results of this study suggest that FTL-UA may be a novel chemotherapeutic drugs play a positive role in human oral cancer chemotherapy. In addition to improve the solubility of ursolic acid, but also can increase the curative effect, prolong the circulation time in vivo, inhibit tumor growth, exert its anti-tumor effect. The experimental results obtained in this study also laid the foundation for the treatment of FTL-UA as a drug for clinical application of tumor. Through the study of the new formulation, target chemotherapy to folate receptor therapy may become a new method of tumor therapy.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96;R94

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本文編號(hào)：1449251