本文選題：寡聚脫氧核苷酸 + HMGB1�。� 參考：《吉林大學(xué)》2014年碩士論文

【摘要】：目的： 本研究旨在研究MS1（9一種AG型單鏈寡聚脫氧核苷酸）對CpG ODN刺激RAW264.7細(xì)胞引起的HMGB1蛋白表達(dá)水平及分泌的影響，進(jìn)而闡明MS19減輕H1N1流感病毒誘導(dǎo)的小鼠急性肺損傷模型病理損傷的治療機制。 方法： 1、實驗分為對照組、CpG ODN刺激組、MS19刺激組、CpG ODN+MS19刺激組，顯微鏡下觀察各組在刺激不同時間后對小鼠RAW264.7細(xì)胞生長狀態(tài)的影響。 2、用實時熒光定量PCR方法檢測MS19對CpG ODN刺激RAW264.7細(xì)胞HMGB1mRNA水平的影響。 3、用免疫熒光法檢測MS19對CpG ODN刺激RAW264.7細(xì)胞HMGB1蛋白定位的影響。 4、用Western blot方法檢測MS19對CpG ODN刺激RAW264.7細(xì)胞胞內(nèi)HMGB1蛋白量的影響。 結(jié)果： 1、顯微鏡下觀察各組刺激RAW264.7細(xì)胞不同時間后細(xì)胞的生長狀態(tài)：各組細(xì)胞用CpG ODN、MS19、CpG ODN+MS19刺激RAW264.7細(xì)胞6h/12h/18h后細(xì)胞生長狀態(tài)與對照組相比無明顯異常，細(xì)胞均貼壁生長，密度均勻，折光度強，數(shù)量明顯增多，細(xì)胞狀態(tài)良好，形態(tài)大體一致，輪廓清楚。 2、實時熒光定量PCR結(jié)果顯示CpG ODN刺激組、MS19刺激組及MS19聯(lián)合CpD ODN刺激組刺激RAW264.7細(xì)胞不同時間段后，HMGB1mRNA水平與對照組對比無明顯差異。 3、免疫熒光法觀察MS19作用18h時對CpG ODN刺激的RAW264.7細(xì)胞HMGB1蛋白定位影響，可見CpG ODN刺激組與對照組對比，兩組細(xì)胞內(nèi)、外均可見綠色熒光，，CpG ODN刺激組細(xì)胞外熒光強度較對照組強。MS19刺激組與對照組對比，對照組細(xì)胞內(nèi)、外均可見綠色熒光，而MS19刺激組綠色熒光主要定位在細(xì)胞內(nèi)及細(xì)胞膜處。CpG ODN刺激組與CpG ODN聯(lián)合MS19刺激組對比，兩組細(xì)胞內(nèi)、外均見綠色熒光，而CpG ODN聯(lián)合MS19刺激組熒光定位以細(xì)胞內(nèi)為主。 4、用western blot法檢測胞內(nèi)HMGB1蛋白量，結(jié)果顯示：用相應(yīng)ODN刺激48h后，CpG ODN刺激組、MS19刺激組及CpG ODN聯(lián)合MS19刺激組細(xì)胞內(nèi)HMGB1蛋白量與對照組比較無明顯差異；刺激72h后，CpG ODN刺激組與對照組比較細(xì)胞內(nèi)HMGB1蛋白減少，MS19刺激組及CpG ODN聯(lián)合MS19刺激組與對照組比較細(xì)胞內(nèi)HMGB1蛋白增多，CpG ODN聯(lián)合MS19刺激組細(xì)胞內(nèi)HMGB1蛋白量處于CpGODN刺激組與MS19刺激組細(xì)胞內(nèi)HMGB1蛋白量之間。 結(jié)論： 1、MS19對RAW264.7細(xì)胞的細(xì)胞生長狀態(tài)無明顯影響。 2、MS19對RAW264.7細(xì)胞HMGB1mRNA水平無明顯影響。 3、MS19能夠抑制CpG ODN刺激RAW264.7細(xì)胞分泌的HMGB1蛋白自胞內(nèi)向胞外轉(zhuǎn)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R511.7

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