蓝氏贾第虫体外培养和筛选的微流控平台构建

发布时间：2018-01-24 22:37

本文关键词： 蓝氏贾第鞭毛虫微流控芯片体外培养药物筛选　出处：《大连大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：蓝氏贾第鞭毛虫（Giardia lamblia，简称贾第虫），是一种单细胞寄生性原虫，可使宿主罹患贾第虫病，该病呈全球性分布。目前的抗贾第虫药物大都具有严重的副作用，而传统的体外药物筛选方法操作复杂、方法易污染、无标准化气体条件，药物筛选效率低、样品及试剂消耗大。本研究拟构建一个可以模拟体内生长环境的体外培养及筛选贾第虫滋养体的集成装置，为抗贾第虫药物的研发提供一个新的平台。方法：本研究中集成装置由聚二甲基硅氧烷（polydimethylsiloxane, PDMS）微流控芯片及厌氧培养系统构成。其中，PDMS微流控芯片包括浓度梯度生成器（chemicalconcentration gradient generator, CGG）和基于扩散的培养室阵列两部分结构；待测药物在CGG的8个出口可形成浓度梯度，流体通道内的培养基通过扩散通道与培养室进行交换，维持与体内类似的环境。厌氧培养系统为PDMS微流控芯片提供一个密闭空间，可按需要提供各种气体环境。本研究用荧光染料罗丹明123（Rhodamine123, Rh123）检测CGG的功能和基于扩散的培养室的扩散条件。利用本装置检测体外培养贾第虫滋养体的最佳气体条件；分别用96孔板和本装置在最佳气体条件下，连续72h培养贾第虫滋养体并进行了甲硝唑和替硝唑的药敏实验。结果：研究结果表明，CGG可在30s内精确迅速地形成8种Rh123浓度梯度；而在25min内培养室中Rh123的荧光强度就与流体通道几乎达到一致。气体条件检测结果表明，，3%O2不足以支持贾第虫滋养体的充分生长，而5%O2时贾第虫滋养体几乎完全不能生长；相比之下，严格的厌氧条件（100%N2）或微氧（1%O2、5%CO2和94%N2）则可使贾第虫滋养体较好的生长（P0.05），且体外培养贾第虫滋养体的最佳气体条件为微氧。此外，两种培养方式都支持贾第虫滋养体48h时的快速生长（P0.05）；但72h时，微流控芯片中的贾第虫滋养体增长34倍，96孔板中却仅有23倍增长（与48h时相似），即在本装置中体外培养贾第虫滋养体有更长的培养周期（P0.05）。两种培养方式下甲硝唑和替硝唑都对贾第虫滋养体生长的抑制具有剂量-效应关系；甲硝唑的IC50值分别为5.13μM和4.52μM，替硝唑的IC50值分别为2.00μM和2.85μM，两种培养方式的IC50值具有可比性。结论：本装置很好的模拟了贾第虫滋养体体内生长环境并可用于体外培养和筛选贾第虫滋养体，成功构建了高通量筛选抗贾第虫药物平台的第一步，并可广泛用于其它基于体外培养贾第虫滋养体的实验。
[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.
【学位授予单位】：大连大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R531.7

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