發(fā)布時間：2018-05-05 18:21

  本文選題：微流控芯片 + 脂質(zhì)體��； 參考：《浙江工商大學(xué)》2015年碩士論文

【摘要】：脂質(zhì)體是磷脂分子有序排列閉合形成內(nèi)腔為空心的圓球型聚集體。由于磷脂的分子結(jié)構(gòu)包括親水的頭基和疏水的長鏈,故形成的脂質(zhì)體同樣具有兩親性。其內(nèi)部空腔可加載親水性的物質(zhì),疏水鏈之間加載親油性的物質(zhì)。目前脂質(zhì)體的應(yīng)用主要有以下兩個方面：藥物載體和人工細胞模型。傳統(tǒng)制備方法得到的脂質(zhì)體粒徑不均勻,穩(wěn)定性較差,需要通過二次處理才能得到粒徑均一的脂質(zhì)體,但這會降低脂質(zhì)體的利用率；作為人工細胞模型,常用電轉(zhuǎn)染的方法導(dǎo)入外源物質(zhì),用以模擬真實細胞的生命活動。但是常規(guī)的電轉(zhuǎn)染往往需要較高的電壓,且不能動態(tài)監(jiān)測轉(zhuǎn)染過程。微流控芯片的產(chǎn)生為上述兩個問題的解決提供了途徑。 本課題以微流控芯片為主體,搭建了脂質(zhì)體制備平臺和電穿孔動態(tài)監(jiān)測平臺。得到的研究結(jié)果如下： (1)微流控芯片的仿真模擬與制作。應(yīng)用Gambit建立芯片內(nèi)部的結(jié)構(gòu)模型,再用Flunet進行流場模擬。主要考察的因素有微通道的寬度,入口角度以及側(cè)通道的流速。以中央通道流體在混合流體所占的體積比例為參考指標(biāo),得出在微通道的寬度為100μm,入口角度為90°時流體的混合效率最好,側(cè)通道與中央通道的臨界流速比為10。 (2)基于微流控聚焦力學(xué)的方法制備脂質(zhì)體。以1-棕櫚�；�-2-油酰-SN-甘油-3-磷酰膽堿(POPC)、二肉豆蔻酰磷脂酰膽堿(DMPC)為原料,分別考察了不同因素對形成的脂質(zhì)體粒徑及穩(wěn)定性的影響,并用理論公式予以解釋說明。在脂質(zhì)體粒徑方面,考察了側(cè)通道與中央通道體積流速比FRR值、溫度及膽固醇含量的影響：低FRR值時,得到的脂質(zhì)體粒徑較大(900nm-1600nm)但其粒徑分布較寬。而在高FRR值時,得到的粒徑較小,且多分散性較好。脂質(zhì)體粒徑隨著FRR的增加而減��；在20℃～50℃區(qū)間內(nèi),隨著溫度的上升脂質(zhì)體粒徑有下降的趨勢。但DMPC磷脂在230C時粒徑突然增大,這是由于達到了磷脂相變溫度使得膜的彈性模量增大了3-5倍；膽固醇的添加量使得粒徑增大,且在摩爾比為20%的粒徑最大。通過正交實驗得到FRR值對脂質(zhì)體粒徑的影響最大,當(dāng)FRR=4、溫度為20℃、膽固醇添加量為20%時得到的脂質(zhì)體粒徑為276.8±3.2nm,且均一性較好。在脂質(zhì)體穩(wěn)定性方面：高濃度磷脂形成的脂質(zhì)體穩(wěn)定性高于低濃度的；表面活性劑的添加在不影響粒徑的前提下大大提高了脂質(zhì)體的穩(wěn)定性,其中陽離子表面活性劑十二烷基三甲基氯化銨與POPC磷脂按摩爾比10%復(fù)配制得的脂質(zhì)體Zeta電位在13.6±0.54mV和26±1.83mV之間,陰離子表面活性劑十六烷基磷酸鉀與POPC磷脂復(fù)配得到的脂質(zhì)體Zeta電位在-27.38±1.1mV和-32.8±0.39mV之間；膽固醇的添加使脂質(zhì)體穩(wěn)定性增強,在20%的添加量時Zeta電位達到-44.74±0.95mV。此外,還利用CLSM、TEM觀察了不同F(xiàn)RR值下形成脂質(zhì)體的微觀結(jié)構(gòu)。 (3)基于激光共聚焦實時監(jiān)測的電穿孔實驗。以脂質(zhì)體和酵母細胞為實驗對象實現(xiàn)低電壓下的電穿孔。脂質(zhì)體體系：NBD-PE和POPC以0.1%的摩爾比制備形成熒光脂質(zhì)體,在100V、脈沖寬度為200ms、脈沖形式為單脈沖的條件下,在CLSM下選定ROI,觀察熒光強度隨時間變化曲線。由計算得出的理論跨膜電壓是375mV,低于擊穿膜的臨界跨膜電壓,電穿孔沒有成功；酵母細胞體系：用細胞膜紅色熒光探針標(biāo)記酵母細胞,施加相同的電壓以及脈沖寬度,其熒光強度也沒有發(fā)生明顯的變化。為了探究電穿孔成功與否,用8μm的羧基水溶性CdSe/ZnS量子點非特異性標(biāo)記酵母細胞,選定細胞膜為ROI1,在細胞膜極點位置為ROI2,在100V、脈沖寬度為200ms,單脈沖的條件下實現(xiàn)了細胞的電穿孔。
[Abstract]:Liposomes are the spherical spherical aggregates of phospholipid molecules arranged in an orderly arrangement to form a hollow inner cavity. As the molecular structure of phospholipids includes the hydrophilic head and the hydrophobic long chain, the formed liposomes also have two affinity. The internal cavity can load hydrophilic substances, and the hydrophobic chain can load the oil-based substances. There are two main aspects: the drug carrier and the artificial cell model. The traditional preparation method is not uniform in the size of the liposomes, and the stability is poor. It needs two treatments to get the homogeneous liposomes, but this will reduce the utilization rate of the liposomes. As an artificial cell model, the method of electrotransfection is commonly used to import foreign substances. Quality is used to simulate the life activities of real cells. However, conventional electrotransfection often requires high voltage and can not dynamically monitor the transfection process. The production of microfluidic chips provides a way to solve the above two problems.
The microfluidic chip is used as the main body to build a platform for preparing lipid and a dynamic monitoring platform for electroporation.
(1) simulation and fabrication of microfluidic chip. The structure model inside the chip is built with Gambit, and then the flow field is simulated with Flunet. The main factors are the width of the microchannel, the angle of the entrance and the flow velocity of the side channel. When the inlet angle is 90 degrees, the mixing efficiency is the best, and the critical velocity ratio of the side channel to the central channel is 10.. The ratio is 100 m.
(2) the liposomes were prepared based on the microfluidic focusing mechanics. The effects of different factors on the particle size and stability of the liposomes were investigated with 1- palmioyl -2- oil acyl -SN- glycerol -3- phosphachcholine (POPC) and two myrisyl phosphatidylcholine (DMPC), and the theoretical formulas were used to explain the effect of different factors on the particle size of liposomes. The effects of volume velocity ratio (FRR), temperature and cholesterol content on the side channel and central channel were investigated. At low FRR value, the size of the liposomes was larger (900nm-1600nm), but its particle size distribution was wide. At the high FRR value, the particle size was smaller and the polydispersity was better. The particle size of liposomes decreased with the increase of FRR; at 20 C to 50 C In the interval, the particle size of the liposome decreased with the increase of the temperature. But the particle size of DMPC phospholipid increased suddenly at 230C. This was due to the 3-5 times the modulus of the membrane. The addition of cholesterol made the particle size increase and the particle size was the largest in the mole ratio of 20%. The FRR value was obtained by orthogonal experiment. The effect of liposome particle size is the most. When FRR=4, temperature is 20, and the addition of cholesterol is 20%, the size of the liposome is 276.8 + 3.2NM, and the homogenization is better. In the stability of liposomes, the stability of the liposome formed by high concentration phospholipid is higher than that of low concentration; the addition of the surfactant is greatly raised on the premise of not affecting the particle size. The stability of liposomes was higher, in which the cationic surfactant twelve alkyl three methyl ammonium chloride and POPC phospholipid were massaged by 10%. The Zeta potential of the liposomes was between 13.6 + 0.54mV and 26 + 1.83mV. The Zeta potential of the anion surfactant, potassium phosphate potassium phosphate and POPC phospholipid, was -27.38 + 1.1mV and -32 .8 + 0.39mV, the addition of cholesterol enhanced the stability of liposomes, and the Zeta potential reached -44.74 + 0.95mV. at the addition of 20%. CLSM, TEM was used to observe the microstructure of liposomes under different FRR values.
(3) electroporation experiments based on laser confocal real-time monitoring. Liposomes and yeast cells are used to achieve electroporation at low voltage. Liposome system: NBD-PE and POPC are prepared to form fluorescent liposomes at a molar ratio of 0.1%. Under the condition of 100V, pulse width of 200ms and pulse form as single pulse, ROI is selected under CLSM. The calculated fluorescence intensity varies with time. The calculated theoretical cross membrane voltage is 375mV, which is lower than the critical cross membrane voltage of the breakdown membrane. The electroporation is not successful; the yeast cell system: the yeast cells are marked with the red fluorescent probe of the cell membrane, the same voltage and pulse width are applied, and the fluorescence intensity has not changed obviously. To explore the success of electroporation, the non specific yeast cells were labeled with 8 m carboxyl water-soluble CdSe/ZnS quantum dots, the cell membrane was selected as ROI1, the cell membrane pole position was ROI2, and the cell electroporation was realized under the condition of 100V, the pulse width of 200ms, and the single pulse.

【學(xué)位授予單位】：浙江工商大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：TN492

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