本文選題：鹽酸阿霉素 + 整合素ανβ3��； 參考：《東南大學(xué)》2016年博士論文

【摘要】：研究目的：整合素ανβ3在血管生成相關(guān)疾病中發(fā)揮著重要作用,可被視為血管生成的一個(gè)特征性標(biāo)記物。本研究制備了整合素ανβ3親和肽P1c(本實(shí)驗(yàn)室發(fā)現(xiàn)并制備的結(jié)締組織生長(zhǎng)因子多肽,命名為P1c)修飾、PEG化的鹽酸阿霉素長(zhǎng)循環(huán)脂質(zhì)體,以期通過(guò)靶向腫瘤細(xì)胞和腫瘤新生血管中過(guò)度表達(dá)的整合素ανβ3受體,增加藥物向腫瘤部位的轉(zhuǎn)運(yùn),達(dá)到特異性地治療腫瘤的目的,從而為血管生成相關(guān)疾病的診療提供新方案。研究方法：1、采用薄膜分散法和硫酸銨梯度法制備P1c修飾的鹽酸阿霉素長(zhǎng)循環(huán)脂質(zhì)體,并對(duì)其粒徑、外觀形態(tài)、包封率、偶聯(lián)率及體外釋放度等理化表征進(jìn)行考察。2、選用高表達(dá)整合素ανβ3的人腦膠質(zhì)瘤U87MG細(xì)胞及低表達(dá)整合素ανβ3的人乳腺癌MCF-7細(xì)胞作為體外考察的細(xì)胞模型,研究該載體的體外靶向性及抗腫瘤活性。通過(guò)流式細(xì)胞實(shí)驗(yàn)和激光共聚焦實(shí)驗(yàn)研究U87MG細(xì)胞及MCF-7細(xì)胞細(xì)胞對(duì)P1c靶向及非靶向鹽酸阿霉素脂質(zhì)體的攝取,通過(guò)CCK-8法檢測(cè)脂質(zhì)體的細(xì)胞毒性。3、建立裸鼠U87MG腦膠質(zhì)瘤模型,考察P1c靶向鹽酸阿霉素長(zhǎng)循環(huán)脂質(zhì)體的體內(nèi)抗腫瘤效果,鹽酸阿霉素、非靶向鹽酸阿霉素脂質(zhì)體和生理鹽水作為實(shí)驗(yàn)對(duì)照,以腫瘤體積作為主要考察指標(biāo),并對(duì)腫瘤組織進(jìn)行免疫組化及免疫熒光等病理學(xué)檢查,比較各組制劑的抗腫瘤效果。研究結(jié)果：1、P1c修飾的鹽酸阿霉素脂質(zhì)體和非靶向脂質(zhì)體平均粒徑分別為131.2 m和128.4nm,Zeta電位分別為-19.7±2.8 mV和-33.1±1.6 mV。透射電鏡觀察脂質(zhì)體形態(tài)圓整且分散均勻,包封率均在95%以上,P1c偶聯(lián)率為66.8±1.6%,體外釋放結(jié)果表明所制備的脂質(zhì)體在37℃磷酸鹽緩沖液(pH7.4)中有明顯的緩釋效果。2、流式細(xì)胞實(shí)驗(yàn)發(fā)現(xiàn),將脂質(zhì)體與細(xì)胞共同孵育1小時(shí)后,在U87MG細(xì)胞中,P1c修飾的鹽酸阿霉素長(zhǎng)循環(huán)脂質(zhì)體組的平均熒光強(qiáng)度高于非靶向脂質(zhì)體組及ανβ3單克隆抗體阻斷組(P0.05),在MCF-7細(xì)胞各脂質(zhì)體組的熒光強(qiáng)度無(wú)明顯差異(P0.05),激光共聚焦實(shí)驗(yàn)與流式細(xì)胞分析結(jié)果一致。體外細(xì)胞毒性實(shí)驗(yàn)結(jié)果顯示,在U87MG細(xì)胞,Plc修飾的鹽酸阿霉素長(zhǎng)循環(huán)脂質(zhì)體組的細(xì)胞毒性高于非靶向脂質(zhì)體組,在MCF-7細(xì)胞各脂質(zhì)體組細(xì)胞毒性無(wú)明顯差異。在兩種腫瘤細(xì)胞,游離鹽酸阿霉素的細(xì)胞結(jié)合能力和細(xì)胞毒性均明顯高于各脂質(zhì)體組。3、U87MG腫瘤模型鼠體內(nèi)藥效學(xué)結(jié)果顯示,Plc修飾的鹽酸阿霉素脂質(zhì)體組、非靶向鹽酸阿霉素脂質(zhì)體組和鹽酸阿霉素溶液組的抑瘤率分別為48.08%、31.03%和22.9%,腫瘤組織的免疫組化及免疫熒光結(jié)果顯示Plc修飾的鹽酸阿霉素脂質(zhì)體組整合素ανβ3和CD31表達(dá)量最低,表明Plc修飾的鹽酸阿霉素脂質(zhì)體具有較好的抗腫瘤活性。研究結(jié)論：以P1c多肽修飾的長(zhǎng)循環(huán)脂質(zhì)體作為藥物載體,能夠通過(guò)靶向腫瘤細(xì)胞和腫瘤新生血管中過(guò)度表達(dá)的整合素ανβ3受體,提高藥物的體內(nèi)抗腫瘤作用,是一種具有應(yīng)用前景的主動(dòng)靶向藥物傳遞系統(tǒng)。
[Abstract]:Objective: integrin V beta 3 plays an important role in angiogenesis related diseases and can be considered as a characteristic marker of angiogenesis. This study prepared the integrin alpha v beta 3 affinity peptide P1c (the connective tissue growth factor polypeptide, named P1c), which was discovered and prepared in our laboratory, and PEG hydrochloride adriamycin long circulation fat Plastids, in order to increase the transport of integrin alpha v beta 3 by targeting tumor cells and tumor neovascularization, increase the transport of drugs to the tumor site, and achieve the purpose of specific treatment of tumors, thus providing a new scheme for the diagnosis and treatment of vascular related diseases. Research methods: 1, preparation of membrane dispersion and ammonium sulfate gradient method. P1c modified adriamycin long circulating liposome was investigated, and its particle size, appearance, encapsulation rate, coupling rate, and in vitro release were investigated for.2. The human glioma U87MG cells with high expression of integrin V beta 3 and human breast cancer MCF-7 cells with low expression of integrin V beta 3 were selected as the cell models for the study. The targeting and antitumor activity of the carrier in vitro was studied by flow cytometry and laser confocal experiment to study the uptake of P1c targeting and non targeting adriamycin liposomes by U87MG cells and MCF-7 cells. The cytotoxic.3 of liposomes was detected by CCK-8, and U87MG glioma model was established in nude mice, and P1c target doxorubicin was examined by P1c. The antitumor effect of long circulating liposomes in vivo, adriamycin hydrochloride, non targeting doxorubicin liposomes and physiological saline as experimental control, tumor volume as the main investigation index, and immunohistochemical and immunofluorescence examination of tumor tissue, compare the antitumor effect of each group. The results of the study: 1, P1c modification The average particle size of doxorubicin and non targeted liposomes was 131.2 m and 128.4nm respectively. The Zeta potential was -19.7 + 2.8 mV and -33.1 + 1.6 mV. respectively. The liposomes were round and distributed evenly, the encapsulation efficiency was above 95% and the P1c coupling rate was 66.8 + 1.6%. The release results showed that the prepared liposomes were at 37 C. In the acid salt buffer solution (pH7.4), there was a significant release effect of.2. Flow cytometry found that after incubating the liposomes with the cells for 1 hours, the average fluorescence intensity of the P1c modified adriamycin long circulating liposome group was higher than that of the non targeted liposome group and the alpha Nu beta 3 monoclonal antibody block group (P0.05) in the U87MG cells, and in the MCF-7 cell lipids. The fluorescence intensity of the plastid group was not significantly different (P0.05), and the laser confocal experiment was consistent with the flow cytometry. In vitro cytotoxicity test showed that in U87MG cells, the cytotoxicity of Plc modified adriamycin long circulating liposome group was higher than that of non targeted liposome group, and there was no significant difference in cytotoxicity between the liposomes of MCF-7 cells. The cell binding capacity and cytotoxicity of doxorubicin in the two tumor cells were significantly higher than those of the liposome group.3. The pharmacodynamic results of the U87MG tumor model mice showed that the Plc modified adriamycin liposome group, the non targeting doxorubicin liposome group and the salt acid adriamycin group were 48.08%, 31 respectively. 3% and 22.9%, the immunohistochemical and immunofluorescence results of the tumor tissue showed that the expression of integrin alpha v beta 3 and CD31 in the adriamycin liposome group of Plc modified hydrochloric acid was the lowest, indicating that the adriamycin liposome with Plc modified adriamycin had better anti-tumor activity. Overexpression of the integrin - V beta 3 receptor in tumor cells and neovascularization is an active targeting drug delivery system, which improves the antitumor effect of the drug in vivo.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R943

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