本文關(guān)鍵詞： 藍(lán)氏賈第 鞭毛蟲(chóng) 微流控芯片 體外培養(yǎng) 藥物篩選　出處：《大連大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：藍(lán)氏賈第鞭毛蟲(chóng)（Giardia lamblia，簡(jiǎn)稱賈第蟲(chóng)），是一種單細(xì)胞寄生性原蟲(chóng)，可使宿主罹患賈第蟲(chóng)病，該病呈全球性分布。目前的抗賈第蟲(chóng)藥物大都具有嚴(yán)重的副作用，而傳統(tǒng)的體外藥物篩選方法操作復(fù)雜、方法易污染、無(wú)標(biāo)準(zhǔn)化氣體條件，藥物篩選效率低、樣品及試劑消耗大。本研究擬構(gòu)建一個(gè)可以模擬體內(nèi)生長(zhǎng)環(huán)境的體外培養(yǎng)及篩選賈第蟲(chóng)滋養(yǎng)體的集成裝置，為抗賈第蟲(chóng)藥物的研發(fā)提供一個(gè)新的平臺(tái)。 方法：本研究中集成裝置由聚二甲基硅氧烷（polydimethylsiloxane, PDMS）微流控芯片及厭氧培養(yǎng)系統(tǒng)構(gòu)成。其中，PDMS微流控芯片包括濃度梯度生成器（chemicalconcentration gradient generator, CGG）和基于擴(kuò)散的培養(yǎng)室陣列兩部分結(jié)構(gòu)；待測(cè)藥物在CGG的8個(gè)出口可形成濃度梯度，流體通道內(nèi)的培養(yǎng)基通過(guò)擴(kuò)散通道與培養(yǎng)室進(jìn)行交換，維持與體內(nèi)類似的環(huán)境。厭氧培養(yǎng)系統(tǒng)為PDMS微流控芯片提供一個(gè)密閉空間，可按需要提供各種氣體環(huán)境。本研究用熒光染料羅丹明123（Rhodamine123, Rh123）檢測(cè)CGG的功能和基于擴(kuò)散的培養(yǎng)室的擴(kuò)散條件。利用本裝置檢測(cè)體外培養(yǎng)賈第蟲(chóng)滋養(yǎng)體的最佳氣體條件；分別用96孔板和本裝置在最佳氣體條件下，連續(xù)72h培養(yǎng)賈第蟲(chóng)滋養(yǎng)體并進(jìn)行了甲硝唑和替硝唑的藥敏實(shí)驗(yàn)。 結(jié)果：研究結(jié)果表明，CGG可在30s內(nèi)精確迅速地形成8種Rh123濃度梯度；而在25min內(nèi)培養(yǎng)室中Rh123的熒光強(qiáng)度就與流體通道幾乎達(dá)到一致。氣體條件檢測(cè)結(jié)果表明，，3%O2不足以支持賈第蟲(chóng)滋養(yǎng)體的充分生長(zhǎng)，而5%O2時(shí)賈第蟲(chóng)滋養(yǎng)體幾乎完全不能生長(zhǎng)；相比之下，嚴(yán)格的厭氧條件（100%N2）或微氧（1%O2、5%CO2和94%N2）則可使賈第蟲(chóng)滋養(yǎng)體較好的生長(zhǎng)（P0.05），且體外培養(yǎng)賈第蟲(chóng)滋養(yǎng)體的最佳氣體條件為微氧。此外，兩種培養(yǎng)方式都支持賈第蟲(chóng)滋養(yǎng)體48h時(shí)的快速生長(zhǎng)（P0.05）；但72h時(shí)，微流控芯片中的賈第蟲(chóng)滋養(yǎng)體增長(zhǎng)34倍，96孔板中卻僅有23倍增長(zhǎng)（與48h時(shí)相似），即在本裝置中體外培養(yǎng)賈第蟲(chóng)滋養(yǎng)體有更長(zhǎng)的培養(yǎng)周期（P0.05）。兩種培養(yǎng)方式下甲硝唑和替硝唑都對(duì)賈第蟲(chóng)滋養(yǎng)體生長(zhǎng)的抑制具有劑量-效應(yīng)關(guān)系；甲硝唑的IC50值分別為5.13μM和4.52μM，替硝唑的IC50值分別為2.00μM和2.85μM，兩種培養(yǎng)方式的IC50值具有可比性。 結(jié)論：本裝置很好的模擬了賈第蟲(chóng)滋養(yǎng)體體內(nèi)生長(zhǎng)環(huán)境并可用于體外培養(yǎng)和篩選賈第蟲(chóng)滋養(yǎng)體，成功構(gòu)建了高通量篩選抗賈第蟲(chóng)藥物平臺(tái)的第一步，并可廣泛用于其它基于體外培養(yǎng)賈第蟲(chóng)滋養(yǎng)體的實(shí)驗(yàn)。
[Abstract]:Objective: Giardia lamblia (Giardia lamblia) is a single cell parasitic protozoa, which can make the host suffer from Giardia lamblia. The disease has a global distribution. Most of the current anti-Giardia drugs have serious side effects, but the traditional in vitro drug screening methods are complex, the methods are easy to pollute, there are no standardized gas conditions, drug screening efficiency is low. In order to provide a new platform for the development of anti-Giardia drugs, an in vitro culture and screening of Giardia trophozoites was designed to simulate the growth environment in vivo. Methods: the integrated device consisted of polydimethylsiloxane polydimethylsiloxane (PDMS) microfluidic chip and anaerobic culture system. The PDMS microfluidic chip consists of a concentration gradient generator, chemical concentration gradient generator. CGG) and diffusion-based cell array; The concentration gradient could be formed at the 8 exits of CGG, and the medium in the fluid channel could be exchanged with the culture room through the diffusion channel. The anaerobic culture system provides a closed space for PDMS microfluidic chips. The fluorescent dye Rhodamine 123 was used in this study. Rh 123) was used to detect the function of CGG and the diffusion condition of diffusion-based culture room. Giardia trophozoites were cultured with 96 hole plate and this device under the optimum gas condition for 72 hours, and the drug sensitivity tests of metronidazole and tinidazole were carried out. Results: the results showed that eight Rh123 concentration gradients could be formed accurately and rapidly within 30 s. The fluorescence intensity of Rh123 in the culture room was almost the same as that in the fluid channel within 25 minutes. The results of gas condition showed that 3O2 was not enough to support the full growth of Giardia japonica trophozoites. However, Giardia trophozoites almost could not grow at 5O 2. In contrast, strict anaerobic conditions of 100% N _ 2) or 1 O _ 2 / 5 CO _ 2 and 94 N _ 2) could make Giardia trophozoites grow better (P0.05). The best gas condition for cultured Giardia trophozoites in vitro was microoxygen. In addition, both cultures supported the rapid growth of Giardia trophozoites at 48 h. However, at 72 h, the trophozoites in the microfluidic microfluidic chip increased only 23 times (similar to those at 48 h). That is to say, the culture of Giardia trophozoites in vitro has a longer culture period (P0.05). Both metronidazole and tinidazole have a dose-effect relationship to the growth inhibition of Giardia japonica trophozoites. The IC50 values of metronidazole and tinidazole were 5.13 渭 M and 4.52 渭 M, respectively, and the IC50 values of tinidazole were 2.00 渭 M and 2.85 渭 M. The IC50 values of the two cultures were comparable. Conclusion: this device can simulate the growth environment of Giardia japonicus trophozoites in vivo and can be used to culture and screen Giardia trophozoites in vitro. The first step of high throughput screening drug platform against Giardia giardii was successfully constructed. It can be widely used in other experiments based on cultured Giardia japonica trophozoites in vitro.
【學(xué)位授予單位】：大連大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R531.7

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