本文選題：肝素前體 + 內(nèi)吞途徑��； 參考：《江南大學(xué)》2017年碩士論文

【摘要】：細(xì)胞對(duì)納米藥物傳遞系統(tǒng)(NDDSs)內(nèi)攝的差異會(huì)影響藥物遞送效率及藥效發(fā)揮。我們?cè)谇捌谟酶嗡厍绑w(heparosan)構(gòu)建NDDSs的研究中發(fā)現(xiàn),其會(huì)選擇性被腫瘤細(xì)胞高效且快速攝取,實(shí)驗(yàn)結(jié)果表明這可能與其內(nèi)攝途徑有關(guān)。為闡明原因,本課題分別對(duì)heparosan及用其構(gòu)建的納米載體在不同類(lèi)型細(xì)胞中的內(nèi)吞途徑、內(nèi)攝效率、胞內(nèi)分布及藥物命運(yùn)進(jìn)行了研究,主要包括如下三方面內(nèi)容:(1)Heparosan的內(nèi)吞途徑及其胞內(nèi)分布研究用熒光探針標(biāo)記heparosan得到HB,并以葡聚糖(Dextran)為對(duì)照。細(xì)胞內(nèi)攝化結(jié)果顯示,較COS7細(xì)胞(經(jīng)SV40病毒轉(zhuǎn)化的非洲綠猴腎成纖維細(xì)胞)而言,MCF-7(人乳腺癌細(xì)胞)、和A549細(xì)胞(人非小肺癌細(xì)胞)和B16(小鼠黑色素瘤細(xì)胞)三種腫瘤細(xì)胞對(duì)HB的內(nèi)攝化具有特異性,且與濃度和孵育時(shí)間正相關(guān)。HB進(jìn)入胞內(nèi)為能量依賴(lài)方式,HB在三種腫瘤細(xì)胞中主要通過(guò)小窩蛋白和巨胞飲途徑被細(xì)胞攝取,HB進(jìn)入細(xì)胞后主要分布于溶酶體中。(2)Heparosan納米粒子的內(nèi)吞途徑及其胞內(nèi)分布研究用疏水性脫氧膽酸修飾heparosan,并通過(guò)熒光標(biāo)記制備得到HD納米粒子,粒徑為170 nm。細(xì)胞內(nèi)攝化實(shí)驗(yàn)結(jié)果表明,正常細(xì)胞和腫瘤細(xì)胞均能高效攝取HD,但其進(jìn)入腫瘤細(xì)胞的能力更強(qiáng)。HD的內(nèi)攝化屬于能量依賴(lài)方式,并且主要通過(guò)網(wǎng)格蛋白介導(dǎo)的內(nèi)吞和巨胞飲途徑被腫瘤細(xì)胞攝取。進(jìn)入細(xì)胞后,HD主要分布于正常細(xì)胞和腫瘤細(xì)胞的溶酶體中。(3)Heparosan納米載體的藥物傳遞研究分別用脫氧膽酸和維生素E修飾heparosan,得到兩親性載體HepD和HepV,其能有效包載阿霉素(DOX)得到HDD和HVD,載藥量分別為4.3%和4.7%。細(xì)胞實(shí)驗(yàn)顯示,HDD和HVD均能快速傳遞DOX進(jìn)入B16細(xì)胞,且隨時(shí)間延長(zhǎng),細(xì)胞中DOX的量增加,能夠有效抑制癌細(xì)胞生長(zhǎng)。綜上所述,heparosan及其構(gòu)建的納米載體在不同種類(lèi)細(xì)胞中具有不同的內(nèi)吞途徑和內(nèi)攝效率,但均分布并聚集在正常細(xì)胞和腫瘤細(xì)胞中。這一結(jié)論對(duì)設(shè)計(jì)溶酶體微環(huán)境敏感的heparosan納米藥物傳遞系統(tǒng)提供了實(shí)驗(yàn)參考。
[Abstract]:The difference in cellular uptake of NDDSs may affect drug delivery efficiency and drug efficacy. In our previous study using heparosan to construct NDDSs, we found that it could be selectively ingested by tumor cells efficiently and quickly, and the results suggested that it might be related to its internal pathway. In order to elucidate the reasons, the endocytosis pathway, uptake efficiency, intracellular distribution and drug fate of heparosan and its nanocarriers in different types of cells were studied. The endocytosis pathway and its intracellular distribution were studied in the following three aspects: heparosan was labeled with fluorescent probe and Dextranan was used as control. The results of cellular internalization showed that, MCF-7 (human breast cancer cell), A549 cell (human non-small lung cancer cell) and B16 (mouse melanoma cell) were more specific to HB than COS7 cells (African green monkey renal fibroblasts transformed by SV40 virus). There is a positive correlation between the concentration and incubation time. HB enters the cell in an energy-dependent manner. HB is mainly absorbed by cells into the cell through the pathway of nest protein and giant cell drink, and mainly distributes in lysosome. Studies on endocytosis pathway and intracellular distribution of particles; modified with hydrophobic deoxycholic acid to prepare HD nanoparticles by fluorescence labeling. The particle size is 170 nm. The results showed that both normal cells and tumor cells could ingest HDin highly, but their ability to enter tumor cells was stronger. HD was an energy-dependent way. And mainly through the griddle protein mediated endocytosis and megalocytosis pathway by tumor cells. Studies on Drug transfer of Heparosan Nano-carriers mainly distributed in lysosomes of normal cells and tumor cells after entering cells; modified with deoxycholic acid and vitamin E to obtain amphiphilic vectors HepD and HepV, which can effectively encapsulate Arabidopsis. HDD and HVD were obtained, the drug loading was 4.3% and 4.7%, respectively. Cell experiments showed that both HDD and HVD could transfer DOX into B16 cells quickly, and the amount of DOX in B16 cells increased with time, which could effectively inhibit the growth of cancer cells. In conclusion, the endocytosis pathway and uptake efficiency were different in different types of cells, but they were distributed and clustered in normal cells and tumor cells. This conclusion provides an experimental reference for the design of heparosan nanopharmaceutical delivery system sensitive to lysosomal microenvironment.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R943

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