纳米壳聚糖双氢青蒿素对感染耐药结核杆菌巨噬细胞磷脂酰肌醇信号通路的影响

发布时间：2018-03-06 16:23

  本文选题：双氢青蒿素　切入点：耐药结核　出处：《西南医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:通过选取纳米壳聚糖作为药物载体,将双氢青蒿素制备成纳米壳聚糖双氢青蒿素,作用于被耐药结核杆菌感染的巨噬细胞,探究其对感染耐药结核杆菌巨噬细胞磷脂酰肌醇信号通路的影响。方法:(1)采用恒温磁力搅拌器,制备纳米壳聚糖双氢青蒿素。(2)选取通过结核基因芯片法与酸罗氏药敏培养法共同鉴别出的耐药株为实验对象。(3)选取耐RFP、INH的双耐株及单耐低浓度INH株各一株,通过结核分枝杆菌药敏试剂盒检测纳米壳聚糖双氢青蒿素联合RFP或INH是否对耐药结核分枝杆菌有抑制作用。(4)选取人单核-巨噬细胞U937用含10%FBS的DMEM高糖培养基置于37℃、5%CO2、饱和湿度的培养箱中培养。(5)选取对数生长期菌株,调整菌液浓度后按照细胞比细菌10:1的比例将细胞与细菌共培养24h制备结核感染细胞模型。(6)利用免疫荧光鉴定复制模型。(7)实验分组:空白对照组、MTB感染组、MTB+纳米壳聚糖双氢青蒿素组(药物终浓度为10ug/ml、50ug/ml、100ug/ml)。(8)通过Fura2-AM检测各组U937细胞内的游离钙离子浓度。(9)使用超微量Ca~(2+)-ATP酶试剂盒检测各组U937细胞的Ca~(2+)-ATP酶活性。(10)采用人PLC、CaM酶联免疫分析试剂盒检测各组U937细胞的PLC、CaM活性。结果:(1)RFP或INH联合纳米壳聚糖双氢青蒿素对耐RFP、INH双耐株及单耐低浓度INH株均有抑菌作用。(2)在显微镜下观察结核感染细胞模型U937细胞内有标记的绿色荧光,证明构建结核分枝杆菌感染U937模型成功。(3)细胞内钙离子浓度测定显示,与MTB感染组相比,当加入50ug/ml、100ug/ml的纳米壳聚糖双氢青蒿素时,MTB+纳米壳聚糖双氢青蒿素组细胞内的游离钙离子浓度升高(P0.05)。(4)Ca~(2+)-ATP酶活性检测显示,与MTB感染组相比,当加入50ug/ml、100ug/ml的纳米壳聚糖双氢青蒿素时,MTB+纳米壳聚糖双氢青蒿素组细胞的Ca~(2+)-ATP酶活力下降(P0.01)。(5)PLC活性检测显示,与MTB感染组相比,MTB+纳米壳聚糖双氢青蒿素组细胞的PLC活力上升(P0.01)。(6)Ca M活性检测显示,与MTB感染组相比,当加入50ug/ml、100ug/ml的纳米壳聚糖双氢青蒿素时,MTB+纳米壳聚糖双氢青蒿素组细胞的Ca M活力上升(P0.01)。结论:(1)纳米壳聚糖双氢青蒿素有较强的协同相应抗结核药或逆转结核菌耐药性的作用。(2)在一定浓度范围内,纳米壳聚糖双氢青蒿素可能通过磷脂酰肌醇信号通路升高感染MTB巨噬细胞的Ca~(2+)浓度。(3)在一定浓度范围内,纳米壳聚糖双氢青蒿素杀伤耐药结核的机制可能是加强吞噬体-溶酶体的融合。(4)在治疗耐药结核研究领域中,纳米壳聚糖双氢青蒿素有望成为其新药。
[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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