基于微流控芯片平台的基因诊断和细胞—药物相互作用研究
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摘要
本论文以微流控芯片为基本平台,结合临床医学研究对象进行了基因诊断和细胞-药物相互作用研究,为微流控芯片的生物医学应用进行了有益的尝试。
用自行设计研制的微流控芯片系统进行了基因诊断研究,包括:⑴利用生物信息学方法分析了SARS病毒基因组并选择特异性序列设计引物,以反转录多重PCR与微流控芯片电泳结合完成了18例SARS临床样品的基因诊断;⑵设计和制作了一种集成有等速电泳预浓缩和筛分电泳分离的微流控芯片,其检测限较传统电泳芯片降低了300倍。将该芯片与基因亚型序列特异性PCR方法联合使用,完成了200例乙型肝炎病毒基因分型研究。研究结果表明采用这种芯片可以缩短PCR扩增时间,提高检测灵敏度。上述基因诊断研究证实微流控芯片具有高灵敏度、高分离效率、快速分离和低样品消耗等特点,可作为基因诊断的一种潜在平台。
用自行设计研制的微流控芯片系统进行了细胞-药物相互作用研究。该芯片具有5组微流控梯度形成通路和30个细胞培养池,集成了细胞种植、培养、不同种药物梯度浓度刺激、荧光标记和荧光信号检测等功能单元。利用这种微流控芯片研究了乳腺癌MCF-7细胞内谷胱甘肽含量与化学治疗敏感性的关系,结果显示三氧化二砷和N-乙酰丝氨酸分别具有降低和升高细胞内谷胱甘肽水平的作用,同时还显示细胞内谷胱甘肽的消耗可以改善肿瘤细胞的化疗敏感性。
This thesis focuses on developing microfluidic chips for biomedical applications on genetic diagnosis and cell-drug interaction. The in-house built microfluidic system was applied in genetic diagnosis.
(1) We analyzed SARS coronavirus genome with bioinformatics tools and selected sequence specific region for primer design, and applied microfluidic chip electrophoresis, in combination with reverse transcription-multiplex PCR, in genetic diagnosis of 18 SARS cases; (2) We designed and fabricated a microfluidic chip integrated isotachophoresis preconcentration and sieving electrophoresis separation, which can attain 300 times lower detection limit than that of conventional electrophoresis microchip. The microfluidic chip-based analysis, in combination with subtype sequence specific PCR, was used in genotyping of 200 Hepatitis B virus cases. This method provided genotyping information in reduced PCR amplification time with higher detection rate when compared with conventional methods. The above work showed that microfluidic chip for genetic diagnosis has advantages of high sensitivity, high resolution, fast separation and low consumption. It has the potential to be used in clinical genetic diagnosis.
Microfluidic chip was also used in study on cell-drug interaction. We designed and fabricated a microfluidic chip featuring parallel-gradient generating networksfor studying cell-drug interaction. The microfluidic chip contains 5 gradient generators and 30 cell culture chambers where the resulted chemical concentration gradients are delivered to stimulate on-chip cultured cells. This microfluidic chip exploits the advantage of lab-on-a-chip technology by integrating cell seeding, culture, stimulation and staining into a single chip. The microfluidic chip was applied in studying the role of reduced glutathione (GSH) in MCF-7 cells’chemotherapy sensitivity. Suppression of intracellular GSH by treatment with arsenic trioxside has been shown to increase chemotherapy sensitivity; conversely, elevation of GSH production by treatment with N-acetyl cysteine leads to increased drug resistance. The results indicated that high GSH level has negative effect on chemotherapeutic sensitivity, and depletion of cellular GSH may serve as an effective way to improve chemotherapy sensitivity. The microfluidic chip is able to perform multiple chemical stimulating and multiparametric cell analysis with easy operation, thus holds great potential for extrapolation to the high-throughput drug screening.
用自行设计研制的微流控芯片系统进行了基因诊断研究,包括:⑴利用生物信息学方法分析了SARS病毒基因组并选择特异性序列设计引物,以反转录多重PCR与微流控芯片电泳结合完成了18例SARS临床样品的基因诊断;⑵设计和制作了一种集成有等速电泳预浓缩和筛分电泳分离的微流控芯片,其检测限较传统电泳芯片降低了300倍。将该芯片与基因亚型序列特异性PCR方法联合使用,完成了200例乙型肝炎病毒基因分型研究。研究结果表明采用这种芯片可以缩短PCR扩增时间,提高检测灵敏度。上述基因诊断研究证实微流控芯片具有高灵敏度、高分离效率、快速分离和低样品消耗等特点,可作为基因诊断的一种潜在平台。
用自行设计研制的微流控芯片系统进行了细胞-药物相互作用研究。该芯片具有5组微流控梯度形成通路和30个细胞培养池,集成了细胞种植、培养、不同种药物梯度浓度刺激、荧光标记和荧光信号检测等功能单元。利用这种微流控芯片研究了乳腺癌MCF-7细胞内谷胱甘肽含量与化学治疗敏感性的关系,结果显示三氧化二砷和N-乙酰丝氨酸分别具有降低和升高细胞内谷胱甘肽水平的作用,同时还显示细胞内谷胱甘肽的消耗可以改善肿瘤细胞的化疗敏感性。
This thesis focuses on developing microfluidic chips for biomedical applications on genetic diagnosis and cell-drug interaction. The in-house built microfluidic system was applied in genetic diagnosis.
(1) We analyzed SARS coronavirus genome with bioinformatics tools and selected sequence specific region for primer design, and applied microfluidic chip electrophoresis, in combination with reverse transcription-multiplex PCR, in genetic diagnosis of 18 SARS cases; (2) We designed and fabricated a microfluidic chip integrated isotachophoresis preconcentration and sieving electrophoresis separation, which can attain 300 times lower detection limit than that of conventional electrophoresis microchip. The microfluidic chip-based analysis, in combination with subtype sequence specific PCR, was used in genotyping of 200 Hepatitis B virus cases. This method provided genotyping information in reduced PCR amplification time with higher detection rate when compared with conventional methods. The above work showed that microfluidic chip for genetic diagnosis has advantages of high sensitivity, high resolution, fast separation and low consumption. It has the potential to be used in clinical genetic diagnosis.
Microfluidic chip was also used in study on cell-drug interaction. We designed and fabricated a microfluidic chip featuring parallel-gradient generating networksfor studying cell-drug interaction. The microfluidic chip contains 5 gradient generators and 30 cell culture chambers where the resulted chemical concentration gradients are delivered to stimulate on-chip cultured cells. This microfluidic chip exploits the advantage of lab-on-a-chip technology by integrating cell seeding, culture, stimulation and staining into a single chip. The microfluidic chip was applied in studying the role of reduced glutathione (GSH) in MCF-7 cells’chemotherapy sensitivity. Suppression of intracellular GSH by treatment with arsenic trioxside has been shown to increase chemotherapy sensitivity; conversely, elevation of GSH production by treatment with N-acetyl cysteine leads to increased drug resistance. The results indicated that high GSH level has negative effect on chemotherapeutic sensitivity, and depletion of cellular GSH may serve as an effective way to improve chemotherapy sensitivity. The microfluidic chip is able to perform multiple chemical stimulating and multiparametric cell analysis with easy operation, thus holds great potential for extrapolation to the high-throughput drug screening.
引文
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