本文選題：雙氫青蒿素　切入點(diǎn)：耐藥結(jié)核　出處：《西南醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:通過選取納米殼聚糖作為藥物載體,將雙氫青蒿素制備成納米殼聚糖雙氫青蒿素,作用于被耐藥結(jié)核桿菌感染的巨噬細(xì)胞,探究其對(duì)感染耐藥結(jié)核桿菌巨噬細(xì)胞磷脂酰肌醇信號(hào)通路的影響。方法:(1)采用恒溫磁力攪拌器,制備納米殼聚糖雙氫青蒿素。(2)選取通過結(jié)核基因芯片法與酸羅氏藥敏培養(yǎng)法共同鑒別出的耐藥株為實(shí)驗(yàn)對(duì)象。(3)選取耐RFP、INH的雙耐株及單耐低濃度INH株各一株,通過結(jié)核分枝桿菌藥敏試劑盒檢測(cè)納米殼聚糖雙氫青蒿素聯(lián)合RFP或INH是否對(duì)耐藥結(jié)核分枝桿菌有抑制作用。(4)選取人單核-巨噬細(xì)胞U937用含10%FBS的DMEM高糖培養(yǎng)基置于37℃、5%CO2、飽和濕度的培養(yǎng)箱中培養(yǎng)。(5)選取對(duì)數(shù)生長(zhǎng)期菌株,調(diào)整菌液濃度后按照細(xì)胞比細(xì)菌10:1的比例將細(xì)胞與細(xì)菌共培養(yǎng)24h制備結(jié)核感染細(xì)胞模型。(6)利用免疫熒光鑒定復(fù)制模型。(7)實(shí)驗(yàn)分組:空白對(duì)照組、MTB感染組、MTB+納米殼聚糖雙氫青蒿素組(藥物終濃度為10ug/ml、50ug/ml、100ug/ml)。(8)通過Fura2-AM檢測(cè)各組U937細(xì)胞內(nèi)的游離鈣離子濃度。(9)使用超微量Ca~(2+)-ATP酶試劑盒檢測(cè)各組U937細(xì)胞的Ca~(2+)-ATP酶活性。(10)采用人PLC、CaM酶聯(lián)免疫分析試劑盒檢測(cè)各組U937細(xì)胞的PLC、CaM活性。結(jié)果:(1)RFP或INH聯(lián)合納米殼聚糖雙氫青蒿素對(duì)耐RFP、INH雙耐株及單耐低濃度INH株均有抑菌作用。(2)在顯微鏡下觀察結(jié)核感染細(xì)胞模型U937細(xì)胞內(nèi)有標(biāo)記的綠色熒光,證明構(gòu)建結(jié)核分枝桿菌感染U937模型成功。(3)細(xì)胞內(nèi)鈣離子濃度測(cè)定顯示,與MTB感染組相比,當(dāng)加入50ug/ml、100ug/ml的納米殼聚糖雙氫青蒿素時(shí),MTB+納米殼聚糖雙氫青蒿素組細(xì)胞內(nèi)的游離鈣離子濃度升高(P0.05)。(4)Ca~(2+)-ATP酶活性檢測(cè)顯示,與MTB感染組相比,當(dāng)加入50ug/ml、100ug/ml的納米殼聚糖雙氫青蒿素時(shí),MTB+納米殼聚糖雙氫青蒿素組細(xì)胞的Ca~(2+)-ATP酶活力下降(P0.01)。(5)PLC活性檢測(cè)顯示,與MTB感染組相比,MTB+納米殼聚糖雙氫青蒿素組細(xì)胞的PLC活力上升(P0.01)。(6)Ca M活性檢測(cè)顯示,與MTB感染組相比,當(dāng)加入50ug/ml、100ug/ml的納米殼聚糖雙氫青蒿素時(shí),MTB+納米殼聚糖雙氫青蒿素組細(xì)胞的Ca M活力上升(P0.01)。結(jié)論:(1)納米殼聚糖雙氫青蒿素有較強(qiáng)的協(xié)同相應(yīng)抗結(jié)核藥或逆轉(zhuǎn)結(jié)核菌耐藥性的作用。(2)在一定濃度范圍內(nèi),納米殼聚糖雙氫青蒿素可能通過磷脂酰肌醇信號(hào)通路升高感染MTB巨噬細(xì)胞的Ca~(2+)濃度。(3)在一定濃度范圍內(nèi),納米殼聚糖雙氫青蒿素殺傷耐藥結(jié)核的機(jī)制可能是加強(qiáng)吞噬體-溶酶體的融合。(4)在治療耐藥結(jié)核研究領(lǐng)域中,納米殼聚糖雙氫青蒿素有望成為其新藥。
[Abstract]:Aim: to prepare dihydroartemisinin from dihydroartemisinin by selecting nano-chitosan as drug carrier and to act on macrophages infected by drug-resistant Mycobacterium tuberculosis. To explore its effect on phosphatidylinositol signaling pathway in macrophages infected with drug-resistant Mycobacterium tuberculosis. Preparation of nano-chitosan dihydroartemisinin. 2) the drug resistant strains identified by the method of nodule gene chip and Roche drug sensitivity culture were selected as the experimental object. Using Mycobacterium tuberculosis drug sensitivity kit to detect whether chitosan dihydroartemisinin combined with RFP or INH has inhibitory effect on drug-resistant Mycobacterium tuberculosis.) Human monocyte-macrophage U937 was selected for DMEM high glucose medium containing 10s. Incubate in a incubator with saturated humidity at 37 鈩，

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