转染IL-18基因联合二萜生物碱对舌鳞癌细胞Tscca的增殖抑制与促凋亡作用

发布时间：2018-02-26 22:48

本文关键词： IL-18 二萜生物碱联合用药舌鳞状细胞癌增殖凋亡　出处：《锦州医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的构建白介素-18(IL-18)真核表达载体,通过转染技术将IL-18基因转染入舌鳞癌细胞Tscca,同时分别加入不同浓度的二萜生物碱(DA),分别采用流式细胞术、MTT实验检测细胞凋亡与增殖抑制情况,最后通过检测Akt/p-Akt通路探究IL-18联合二萜生物碱对舌鳞细胞Tscca可能凋亡机制。方法1、根据Gen Bank中IL-18 DNA序列,设计并合成IL-18基因特异性引物;2、提取人新鲜血液中单个核淋巴细胞(PBMC),提PBMC的总RNA,RT-PCR方法获得其c DNA,PCR扩增目的片段,构建PEGFPN3-IL-18真核表达质粒,经酶切和测序均鉴定正确后瞬时转染Tscca细胞,荧光显微镜下观察质粒的转染效率;3、DMSO溶解二萜生物碱,调整其浓度为0.2 mg/ml、0.4 mg/ml、0.6 mg/ml;4、MTT法及流式细胞术检测空白对照组(未处理Tscca细胞)、IL-18组(仅转染IL-18)、单独二萜生物碱组(分为0.2 mg/ml、0.4 mg/ml、0.6 mg/ml 3组)及联合组(分为IL-18+0.2 mg/ml、IL-18+0.4 mg/ml、IL-18+0.6 mg/ml 3组)对舌鳞癌细胞Tscca生长的影响与细胞凋亡情况;5、Western blot实验检测各组细胞信号调节激酶Akt/p-Akt的蛋白水平,分析IL-18联合二萜生物碱是否在此条通路发挥一定作用从而抑制舌癌细胞Tscca的增殖。结果1、成功构建PEGFPN3-IL-18真核表达质粒;2、成功转染舌鳞癌细胞,且荧光镜下观察转染PEGFPN3-IL-18真核表达质粒的Tscca细胞较未转染细胞凋亡增加;3、二萜生物碱浓度为0.2、0.4、0.6 mg/ml时,随浓度增大Tscca细胞凋亡增多,且均有p0.05,差异有统计学意义;4、IL-18与二萜生物碱联合对Tscca细胞作用48 h后,较单独使用二萜生物碱抑制作用明显,且呈浓度依赖性,均有p0.05,差异有统计学意义;5、IL-18联合二萜生物碱还可剂量依赖性地降低p-Akt的蛋白水平,各组均有p0.05,差异有统计学意义,而总Akt水平几乎不变。结论1、二萜生物碱对Tscca细胞有增殖抑制及促凋亡作用,且呈浓度依赖性;2、IL-18联合二萜生物碱对舌鳞癌细胞Tscca有协同抑制作用,且抑制作用较单独使用IL-18或二萜生物碱均较强;3、IL-18联合二萜生物碱在抑制p-Akt的表达及活化这条信号通路上发挥一定作用从而抑制舌癌细胞Tscca的增殖并促进其凋亡。
[Abstract]:Objective to construct the eukaryotic expression vector of interleukin-18 (IL-18) and transfect the IL-18 gene into tongue squamous carcinoma cells (TSCCA), and add different concentrations of diterpenoid alkaloid (diterpene alkaloid) to Tscca. flow cytometry (FCM) was used to detect the apoptosis and proliferation inhibition of TSCA cells. Finally, the Akt/p-Akt pathway was detected to explore the possible apoptotic mechanism of IL-18 combined with diterpene alkaloids on Tscca of tongue squamous cells. Method 1. According to the IL-18 DNA sequence of Gen Bank, A IL-18 gene specific primer was designed and synthesized to extract mononuclear lymphocytes from human fresh blood. The total RNA-RT-PCR method was used to amplify the target fragment of PBMC, and the eukaryotic expression plasmid of PEGFPN3-IL-18 was constructed. Tscca cells were transiently transfected by enzyme digestion and sequencing. The transfection efficiency of the plasmid was observed by fluorescence microscope and the diterpenoid alkaloids were dissolved by DMSO. Adjusting its concentration to 0.2 mg / ml / ml 0.4 mg / ml / ml ~ (0.6 mg / ml) and flow cytometry to detect blank control group (untreated Tscca cell / IL-18 group (only transfected with IL-18), single diterpenoid alkaloid group (0.2 mg / ml / ml ~ 0.4 mg / ml ~ (0.6 mg/ml) group) and combined group (IL-18 0.2 mg / ml ~ (-1) IL-18 ~ 0.4 mg / ml ~ (0.6) mg/ml). The effect on the growth of Tscca and apoptosis of tongue squamous carcinoma cells were analyzed by Western blot assay. The protein level of signal regulated kinase (Akt/p-Akt) was detected by Western blot assay. To analyze whether IL-18 combined with diterpene alkaloid plays a certain role in this pathway to inhibit the proliferation of Tscca in tongue cancer cells. Results 1. The eukaryotic expression plasmid of PEGFPN3-IL-18 was successfully constructed and transfected into tongue squamous cell carcinoma cells successfully. The apoptosis of Tscca cells transfected with PEGFPN3-IL-18 eukaryotic expression plasmid was higher than that of untransfected cells under fluorescence microscope. When the concentration of diterpene alkaloid was 0.2 ~ 0.40.60 mg/ml, the apoptosis of Tscca cells increased with the increase of the concentration of diterpene alkaloid. There was a significant difference in p0.05. the inhibitory effect of IL-18 and diterpene alkaloids on Tscca cells was more obvious than that of diterpenoid alkaloids alone for 48 h, and in a dose-dependent manner. The difference was statistically significant (P 0.05). IL-18 combined with diterpenoid alkaloid could also decrease the protein level of p-Akt in a dose-dependent manner. Conclusion 1. Diterpene alkaloids can inhibit the proliferation and promote apoptosis of Tscca cells in a dose-dependent manner. In a concentration-dependent manner, diterpene alkaloids have synergistic inhibitory effects on Tscca in tongue squamous carcinoma cells. The inhibitory effect was stronger than that of IL-18 or diterpenoid alkaloids alone. The combination of IL-18 and diterpene alkaloids could inhibit the expression and activation of p-Akt, thus inhibit the proliferation of Tscca and promote the apoptosis of tongue cancer cells.
【学位授予单位】：锦州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.86

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