順鉑是治療癌症時的常見藥物，但其會有導致耳毒性的副作用。順鉑進入細胞後與水形成複合物，該複合物會阻斷 DNA 複製和轉錄並抑制DNA修復與細胞週期過程。另外順鉑也會產生過量的活性氧且能與抗氧化酶（例如:超氧化物歧化酶和過氧化氫酶）共價結合，減弱內耳抗氧化防禦能力。還原態的菸鹼醯胺腺嘌呤二核苷酸磷酸(Nicotinamide adenine dinucleotide phosphate, NADPH) 氧化酶4(NOX4)是順鉑誘導活性氧((Reactive oxygen species,ROS)產生的重要氧化酶，過去研究證實透過NOX4 siRNA轉染細胞可有效使NOX4基因沉默並且減弱順鉑造成的細胞毒性和ROS產生。本研究建立吸附NOX4 siRNA之溶菌酶微氣泡結合超音波介導技術，施打超音波造成穴蝕效應可開啟細胞膜通透度，使溶菌酶微氣泡作為藥物載體將NOX4 siRNA送入細胞，此技術除了提高轉染效率及避免NOX4 siRNA降解外，也可有效抑制NOX4基因表現與ROS產生。
首先測定順鉑對於HEI-OC1細胞的毒性並檢測順鉑誘發細胞內NOX基因表現，接著製備LyzMBs- NOX4 siRNA其濃度為1.49 ± 0.861 (×108 bubbles/mL)且NOX4 siRNA吸附率為89.25 %；並進行體外細胞實驗 (n=5)，其實驗組別分別為Control (no treatment)組、CDDP(20 µM)組、單純NOX4 siRNA溶液浸泡與超音波組 (NOX4 siRNA+CDDP / NOX4 siRNA+US+CDDP)、NOX4 siRNA溶液混合溶菌酶微氣泡浸泡與超音波組 (LyzMBs+ NOX4 siRNA+CDDP / LyzMBs+ NOX4 siRNA+US+CDDP)、吸附NOX4 siRNA之溶菌酶微氣泡浸泡與超音波組(LyzMBs- NOX4 siRNA+CDDP /LyzMBs- NOX4 siRNA+US+CDDP)。
在體外試驗中，吸附NOX4 siRNA之溶菌酶微氣泡施打超音波後轉染效率提升52.79 %，相較CDDP組其抑制NOX4基因抑制效率提升124.6 %、ROS抑制效率提升75.4 %。在離體組織中，經由超音波微氣泡作用可有效抑制NOX4基因表現，治療因順鉑造成之毛細胞損傷。

Cisplatin is a common drug for treating cancers, but it has the side effect of ototoxicity. After entering into the cell, cisplatin produces a complex with water and the complex blocks DNA replication and transcription and results in the inhibition of DNA repair and cell cycle progression. In addition, cisplatin also produces excess reactive oxygen species(ROS) and can covalently bind to anti-oxidant enzymes (e.g., superoxide dismutase and catalase), weakening the antioxidant defense capacity of the inner ear. NOX4 is an important oxidase for cisplatin-induced ROS production. Past studies have confirmed that NOX4 siRNA transfected cells can effectively silence the NOX4 gene and attenuate the cytotoxicity and ROS production caused by cisplatin. This thesis aims to explore the feasibility of NOX4 siRNA-coated lysozyme microbubble combined with ultrasound for gene therapy. Then, cavitation effect caused by the application of ultrasound can increase the cell membrane permeability and confirm that the lysozyme microbubbles can be used as drugcarriers to deliver NOX4 siRNA into cells. This technology can improve the transfection efficiency and avoid the degradation of NOX4 siRNA. In addition, to improve the transfection efficiency and avoid NOX4 siRNA degradation, this technology was also inhibit used to NOX4 gene expression and ROS production.
Firstly, the toxicity of cisplatin to HEI-OC1 cells was determined and the intracellular NOX gene expression induced by cisplatin was detected. Then, LyzMBs-NOX4 siRNA was prepared at a concentration of 1.49 ± 0.861 (×108 bubbles/mL) and the adsorption rate of NOX4 siRNA was 89.25%; in the in vitro cell experiments (n=5), the experimental groups were divided into Control (no treatment) group、CDDP (cisplatin) (20 µM) group、NOX4 siRNA solution alone soaking and ultrasonic group (NOX4 siRNA+CDDP / NOX4 siRNA+US+ CDDP)、NOX4 siRNA solution mixed with LyzMBs soaking and ultrasonic group (LyzMBs+ NOX4 siRNA+CDDP / LyzMBs+ NOX4 siRNA+US+CDDP)、NOX4 siRNA adsorbed LyzMBs soaking and ultrasonic group (LyzMBs- NOX4 siRNA+CDDP/LyzMBs-NOX4 siRNA+US+CDDP).
In the in vitro test, the transfection efficiency of the LyzMBs-NOX4 siRNA group increased by 52.79 % after ultrasound treatment, compared with the CDDP group, the inhibition efficiency of NOX4 gene inhibition increased by 124.6 % and the ROS inhibition efficiency increased by 75.4 %. In the Organ of Corti explant, microbubbles combined with ultrasound can effectively inhibit the expression of NOX4 gene and reduced cochlear hair cell damage caused by cisplatin.

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