AG型单链寡聚脱氧核苷酸对CpG ODN刺激RAW264.7细胞分泌HMGB1的影响

发布时间：2018-06-15 02:35

本文选题：寡聚脱氧核苷酸 + HMGB1　；参考：《吉林大学》2014年硕士论文

【摘要】：目的：本研究旨在研究MS1（9一种AG型单链寡聚脱氧核苷酸）对CpG ODN刺激RAW264.7细胞引起的HMGB1蛋白表达水平及分泌的影响，进而阐明MS19减轻H1N1流感病毒诱导的小鼠急性肺损伤模型病理损伤的治疗机制。方法： 1、实验分为对照组、CpG ODN刺激组、MS19刺激组、CpG ODN+MS19刺激组，显微镜下观察各组在刺激不同时间后对小鼠RAW264.7细胞生长状态的影响。 2、用实时荧光定量PCR方法检测MS19对CpG ODN刺激RAW264.7细胞HMGB1mRNA水平的影响。 3、用免疫荧光法检测MS19对CpG ODN刺激RAW264.7细胞HMGB1蛋白定位的影响。 4、用Western blot方法检测MS19对CpG ODN刺激RAW264.7细胞胞内HMGB1蛋白量的影响。结果： 1、显微镜下观察各组刺激RAW264.7细胞不同时间后细胞的生长状态：各组细胞用CpG ODN、MS19、CpG ODN+MS19刺激RAW264.7细胞6h/12h/18h后细胞生长状态与对照组相比无明显异常，细胞均贴壁生长，密度均匀，折光度强，数量明显增多，细胞状态良好，形态大体一致，轮廓清楚。 2、实时荧光定量PCR结果显示CpG ODN刺激组、MS19刺激组及MS19联合CpD ODN刺激组刺激RAW264.7细胞不同时间段后，HMGB1mRNA水平与对照组对比无明显差异。 3、免疫荧光法观察MS19作用18h时对CpG ODN刺激的RAW264.7细胞HMGB1蛋白定位影响，可见CpG ODN刺激组与对照组对比，两组细胞内、外均可见绿色荧光，，CpG ODN刺激组细胞外荧光强度较对照组强。MS19刺激组与对照组对比，对照组细胞内、外均可见绿色荧光，而MS19刺激组绿色荧光主要定位在细胞内及细胞膜处。CpG ODN刺激组与CpG ODN联合MS19刺激组对比，两组细胞内、外均见绿色荧光，而CpG ODN联合MS19刺激组荧光定位以细胞内为主。 4、用western blot法检测胞内HMGB1蛋白量，结果显示：用相应ODN刺激48h后，CpG ODN刺激组、MS19刺激组及CpG ODN联合MS19刺激组细胞内HMGB1蛋白量与对照组比较无明显差异；刺激72h后，CpG ODN刺激组与对照组比较细胞内HMGB1蛋白减少，MS19刺激组及CpG ODN联合MS19刺激组与对照组比较细胞内HMGB1蛋白增多，CpG ODN联合MS19刺激组细胞内HMGB1蛋白量处于CpGODN刺激组与MS19刺激组细胞内HMGB1蛋白量之间。结论： 1、MS19对RAW264.7细胞的细胞生长状态无明显影响。 2、MS19对RAW264.7细胞HMGB1mRNA水平无明显影响。 3、MS19能够抑制CpG ODN刺激RAW264.7细胞分泌的HMGB1蛋白自胞内向胞外转移。
[Abstract]:Aim: to investigate the effects of MS1O9, an AG type oligodeoxynucleotide, on the expression and secretion of HMGB1 protein in RAW264.7 cells stimulated by CpG ODN. Furthermore, the therapeutic mechanism of MS19 to alleviate the pathological injury induced by H1N1 influenza virus in mice was elucidated. Methods: 1. The experiment was divided into control group (CpG ODN stimulation group) and MS19 stimulation group (CpG ODN MS19 stimulation group). The growth state of RAW264.7 cells was observed under microscope. 2The effect of MS19 on the HMGB1 mRNA level of RAW264.7 cells stimulated by CpG ODN was detected by real-time fluorescent quantitative PCR. 3 the HMGB1 mRNA level of RAW264.7 cells stimulated by CpG ODN was detected by real-time fluorescent quantitative PCR. The effect of MS19 on the localization of HMGB1 protein in RAW264.7 cells stimulated by CpG ODN was detected by fluorescence staining. 4 the effect of MS19 on the HMGB1 protein content in RAW264.7 cells stimulated by CpG ODN was detected by Western blot assay. Results: 1. The growth state of RAW264.7 cells stimulated by CpG ODN MS19 and CpG ODN MS19 at different time was observed under microscope. The growth state of RAW264.7 cells stimulated by CpG ODN19 CpG ODN MS19 was not significantly abnormal compared with that of the control group. The density is uniform, the diopter is strong, the number is obviously increasing, the cell is in good condition, and the shape is roughly the same. 2The results of real-time fluorescence quantitative PCR showed that there was no significant difference in mRNA level of HMGB1 between MS19 stimulated group and MS19 combined with CpD ODN stimulation group after RAW264.7 cells were stimulated by CpG ODN at different time points compared with control group. The effect of MS19 on the localization of HMGB1 protein in RAW264.7 cells stimulated by CpG ODN for 18 h was observed by immunofluorescence assay. The intensity of extracellular fluorescence in the CpG ODN stimulated group was stronger than that in the control group and the control group. Green fluorescence was observed in both the control group and the control group. However, the green fluorescence in MS19 stimulated group was mainly located in the cell and cell membrane. Compared with CpG ODN plus MS19 stimulation group, green fluorescence was found in both cells and outside of the two groups. The fluorescence localization of CpG ODN combined with MS19 was mainly intracellular. 4. The intracellular HMGB1 protein was detected by western blot assay. The results showed that there was no significant difference in HMGB1 protein content between CpG ODN group and CpG ODN plus MS19 group after 48h stimulation with corresponding ODN. HMGB1 protein decreased in CpG ODN group and CpG ODN combined with MS19 stimulation group increased HMGB1 protein in CpG ODN combined with MS19 stimulation group compared with control group after 72 h. The intracellular HMGB1 protein content of CpG ODN combined with MS19 stimulation group was at CpG ODN stimulation group, and the intracellular HMGB1 protein content of CpG ODN combined with MS19 stimulation group was at CpG ODN stimulation group. The amount of HMGB1 protein in the cells of group M S 19 and group M S 19 was increased. Conclusion: (1) MS19 has no effect on the growth state of RAW264.7 cells, but MMS19 has no effect on HMGB1 mRNA level in RAW264.7 cells. 3. CpG ODN-stimulated HMGB1 protein from RAW264.7 cells can be inhibited by CpG ODN.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R511.7

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