慢病毒介导的TACO基因特异性siRNA对巨噬细胞抗结核分枝杆菌的影响及其相关机制分析-临床检验诊断学专业毕业论文.docxVIP

IV IV 摘要 3. 研究证实 PSRT1 可显著增强巨噬细胞杀伤胞内 MTB 的能力。 4. 研究发现抑制 TACO 的表达可有效促进巨噬细胞内含 MTB 的吞噬体与 溶酶体的融合，提示 TACO 的存在可能是阻碍巨噬细胞内 MTB 吞噬体成熟的主 要机制之一。 5. 研究显示抑制 TACO 的表达对巨噬细胞的自噬水平无明显影响，提示 PSRT1 促进 MTB 吞噬体与溶酶体融合的功能并不是通过自噬-溶酶体途径实现 的，具体机制有待进一步的研究。 关键词：富含色氨酸天冬氨酸的膜蛋白； 小干扰 RNA；慢病毒；抑菌作用； 自噬；吞噬溶酶体 Abst Abstract PAGE PAGE VI ABSTRACT Objective: To construct and screen for lentiviral interference vector ,which can be specifically expressed TACO genes siRNA . To reseach and explore the effect and mechanism in the inhibition of macrophage intracellular bacteria. To provide basis and direction for subsequent reseach of targeted against Mtb infection in vivo. Method: Choosing three target sequence, According to TACO gene mRNA sequence, designing corresponding shRNA in accordance with siRNA design principle and a negative contrast shRNA sequences by random disturb. For each shRNA each to design a pair of single oligodeoxynucleotide,the express double-stranded DNA is formed after low temperature annealing.Digesting the PSICOR lentiviral vector plasmid by restriction enzymes.Inserting annealed double-stranded DNA into the incision,getting the plasmid of PSICOR-TACO-siRNA. Confirmed correctly by enzyme digestion and sequencing identgification,determine its naming PSRT 1-4,the pSRT4 lentivirus expression plasmid is negative contrast shRNA. Packaging virus particles by mixing the restructured lentiviral interference vector(PSRT1~4), the envelope protein plasmids pMD2.G and the lentiviral packaging plasmids psPAX2,according to a certain percentage.The supernatant was collected containing lentiviral particles, concentrated virus particles after centrifugal ultrafiltration （Named LV - pSRT1-4, respectively），Then,infected 293T cells by hole dilution method, via measuring and counting the number of the fluorescence expression cell, calculated virus drops degree.The packed virus particles infected RAW264.7 cell in suitable titer for 48 hours and 72 hours,Cracking cells extracted total RNA and