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  本文關鍵詞：血小板粘附的SPR傳感檢測方法研究　出處：《重慶大學》2016年碩士論文　論文類型：學位論文

  更多相關文章： 血小板功能 表面等離體激元共振傳感器 剪切率 基質蛋白 齒狀通道

【摘要】：隨著我國經濟發(fā)展和生活水平的提高,心血管系統(tǒng)疾病越來越成為嚴重威脅人民健康的一個重要因素。其中,血栓性疾病發(fā)病率的上升趨勢尤為明顯。這類疾病的發(fā)病機制是與病理性血栓形成有關。目前預防血栓形成是這類疾病的一種重要防治手段。抗血小板藥物的藥理機制就是抑制血小板的粘附和聚集。在抗血小板新藥研發(fā)中,藥物對不同患者的生物效應并不一致,使得監(jiān)測個體患者的抗血小板治療藥物最佳治療劑量,最大限度地降低血栓和減少出血變得尤為重要。因此,各類體外檢測血小板功能的方法和儀器被廣泛應用于抗血小板治療的監(jiān)測中。但是,傳統(tǒng)的血小板功能檢測裝置及方法普遍存在操作復雜、用時長、需標記、精度不高、價格昂貴等缺點,限制了其推廣運用。為解決這一問題,本文選用了表面等離體激元共振傳感技術用于血小板功能的研究。該分析技術具有高靈敏度、無需標記、操作簡單、高通量等優(yōu)勢,通過測定血小板的粘附值來評估血小板功能的改變,從而實現抗血小板治療中藥物的監(jiān)測。本文首先利用COMSOL仿真軟件分析了幾組具有不同寬度及高度的微通道。經過仿真研究通道底部剪切率的分布情況,確定出了一組剪切率分布最為均勻的通道并完成了芯片的加工制作。在氣相及液相中分別測試了血小板在膠原上的粘附情況。實驗結果表明,血液流經通道后,金膜表面折射率有所增加,SPR角增大,通過計算SPR角的增量就可算得血小板粘附的具體數值。這證明了SPR傳感器用于研究以粘附值為參考標準的血小板功能檢測的可行性。同時,該技術能實現通道任意位置的精確檢測,定量分析血小板在通道中的分布情況,而且實驗重復性高,分析區(qū)域被劃分成的多個小分析點差異性小。采用多點分析的方法可以在一次實驗中就實現傳統(tǒng)分析需要多次檢測的效果,大大提高了測試分析的效率。通過比較分析不同測試條件下的血小板粘附檢測方法,發(fā)現液相中的血小板粘附檢測效果更好。在液相條件下,檢測了不同因素對血小板粘附的影響,分別從剪切率和不同基質蛋白方面展開研究。結果表明,在一定范圍內,血小板粘附值隨剪切率的增加而增加,在500s-1時,粘附值達到2.958ng/mm2;此后,繼續(xù)增加剪切率,粘附值逐漸下降。在不同濃度的多種基質蛋白實驗中發(fā)現,血小板的粘附依賴于膠原蛋白溶液濃度,濃度越大,粘附就越多;而纖維蛋白原分子的結構決定了血小板的粘附值;血清蛋白的惰性使得血小板幾乎不與其發(fā)生結合反應。隨后,設計了一種齒狀通道作為實驗模型,用于分析體內血管狹窄時血小板粘附的分布狀況。實驗中發(fā)現,在低剪切率下,在齒狀通道的任何位置血小板的粘附值都要高于中高剪切率,且隨著剪切率增加,通道中游區(qū)血小板數量逐漸低于上下游。最后,將不同濃度的阿司匹林作用于正常人的血液,結果發(fā)現阿司匹林用量越多,血小板粘附量就越低,說明藥物的用量是與抑制程度成正比的。用二磷酸腺苷(ADP)試劑激活血小板來模擬血栓性疾病患者血小板的活化狀態(tài)。施加不同濃度的阿司匹林抑制劑后,低劑量的阿司匹林并不能完全抑制血小板的粘附,粘附值仍高于正常水平,藥物藥效不明顯;加大用量后,粘附值逐漸降低,趨于正常。但在高劑量下,粘附明顯較低,說明血小板的凝血功能在減弱,在體內會發(fā)生出血事件。
[Abstract]:With the development of our country's economy and the improvement of the living standard, the disease of cardiovascular system has become an important factor that seriously threatens the people's health. Among them, the trend of the incidence of thrombotic diseases is particularly obvious. The pathogenesis of these diseases is related to the formation of pathological thrombus. At present, the prevention of thrombosis is an important means of prevention and treatment of these diseases. The pharmacological mechanism of antiplatelet drugs is to inhibit the adhesion and aggregation of platelets. In the development of antiplatelet drugs, the biological effects of drugs on different patients are not consistent, which makes monitoring individual patients' optimal treatment dose of antiplatelet drugs, minimizing thrombosis and reducing bleeding. Therefore, various methods and instruments for the detection of platelet function in vitro are widely used in the monitoring of antiplatelet therapy. However, the traditional platelet function testing devices and methods generally have many shortcomings, such as complex operation, long time use, marking, low accuracy and high price, which limits their popularization and application. In order to solve this problem, this paper uses a surface plasmon resonance sensing technique to study the function of platelets. The analysis technology has the advantages of high sensitivity, no labeling, simple operation, high throughput and so on. By measuring the platelet adhesion value, we can evaluate the change of platelet function, so as to achieve the monitoring of drugs in antiplatelet therapy. In this paper, the COMSOL simulation software is used to analyze several groups of microchannels with different width and height. Through the simulation of the distribution of the shear rate at the bottom of the channel, a group of channels with the most uniform distribution of shear rate are determined and the fabrication of the chip is completed. The adhesion of platelets on collagen was tested in the gas phase and the liquid phase. The experimental results showed that after the passage of blood, the refractive index of the gold film increased and the SPR angle increased. By calculating the increment of SPR angle, the specific value of platelet adhesion could be calculated. This proves that the SPR sensor is used to study the feasibility of platelet function detection with the reference standard of adhesion. At the same time, the technology can detect accurately any position of the channel, quantitatively analyze the distribution of platelets in the channel, and the experimental repeatability is high. The analysis area is divided into multiple small analysis points with little difference. The method of multipoint analysis can be used in an experiment to realize the effect of multiple detection in the traditional analysis, which greatly improves the efficiency of the test analysis. By comparing and analyzing the method of platelet adhesion detection under different test conditions, it is found that the effect of platelet adhesion detection in the liquid phase is better. In liquid phase, the effect of different factors on platelet adhesion was detected, and the shear rate and different matrix protein were studied. The results showed that within a certain range, the platelet adherence value increased with the increase of shear rate. When 500s-1, the adhesion value reached 2.958ng/mm2. Thereafter, the shear rate continued to increase, and the adhesion value gradually decreased. Found in a variety of experiments with different concentrations of matrix protein, platelet adhesion on collagen solution concentration, the greater the concentration, the more structure and adhesion; fibrinogen molecules determines the platelet adhesion value; serum protein and its inertness makes almost no platelet binding reaction. Subsequently, a dentate channel was designed as an experimental model to analyze the distribution of platelet adhesion during vascular stenosis in the body. In experiment, it was found that at low shear rate, the adhesion value of platelets at any location of dentate channel was higher than that of mid high shear rate, and with the increase of shear rate, the number of platelets in the middle passage of the channel was gradually lower than that of the upstream and downstream. Finally, different concentrations of aspirin were applied to the blood of normal people. It was found that the more the aspirin dosage, the lower the platelet adherence, which indicated that the dosage of drugs was directly proportional to the degree of inhibition. Platelet activation by adenosine two (ADP) reagents is used to simulate the activation of platelet in patients with thrombotic disease. After applying different concentration of aspirin inhibitor, low dose aspirin did not completely inhibit platelet adhesion. The adhesion value was still higher than the normal level. The drug efficacy was not obvious. After increasing the dosage, the adhesion value gradually decreased and tended to be normal. But at high dose, the adhesion is obviously lower, indicating that the blood coagulation function of the platelets is weakened, and the bleeding event will occur in the body.
【學位授予單位】：重慶大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R54

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