本文關(guān)鍵詞： IL-18 二萜生物堿 聯(lián)合用藥 舌鱗狀細胞癌 增殖 凋亡　出處：《錦州醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的構(gòu)建白介素-18(IL-18)真核表達載體,通過轉(zhuǎn)染技術(shù)將IL-18基因轉(zhuǎn)染入舌鱗癌細胞Tscca,同時分別加入不同濃度的二萜生物堿(DA),分別采用流式細胞術(shù)、MTT實驗檢測細胞凋亡與增殖抑制情況,最后通過檢測Akt/p-Akt通路探究IL-18聯(lián)合二萜生物堿對舌鱗細胞Tscca可能凋亡機制。方法1、根據(jù)Gen Bank中IL-18 DNA序列,設(shè)計并合成IL-18基因特異性引物;2、提取人新鮮血液中單個核淋巴細胞(PBMC),提PBMC的總RNA,RT-PCR方法獲得其c DNA,PCR擴增目的片段,構(gòu)建PEGFPN3-IL-18真核表達質(zhì)粒,經(jīng)酶切和測序均鑒定正確后瞬時轉(zhuǎn)染Tscca細胞,熒光顯微鏡下觀察質(zhì)粒的轉(zhuǎn)染效率;3、DMSO溶解二萜生物堿,調(diào)整其濃度為0.2 mg/ml、0.4 mg/ml、0.6 mg/ml;4、MTT法及流式細胞術(shù)檢測空白對照組(未處理Tscca細胞)、IL-18組(僅轉(zhuǎn)染IL-18)、單獨二萜生物堿組(分為0.2 mg/ml、0.4 mg/ml、0.6 mg/ml 3組)及聯(lián)合組(分為IL-18+0.2 mg/ml、IL-18+0.4 mg/ml、IL-18+0.6 mg/ml 3組)對舌鱗癌細胞Tscca生長的影響與細胞凋亡情況;5、Western blot實驗檢測各組細胞信號調(diào)節(jié)激酶Akt/p-Akt的蛋白水平,分析IL-18聯(lián)合二萜生物堿是否在此條通路發(fā)揮一定作用從而抑制舌癌細胞Tscca的增殖。結(jié)果1、成功構(gòu)建PEGFPN3-IL-18真核表達質(zhì)粒;2、成功轉(zhuǎn)染舌鱗癌細胞,且熒光鏡下觀察轉(zhuǎn)染PEGFPN3-IL-18真核表達質(zhì)粒的Tscca細胞較未轉(zhuǎn)染細胞凋亡增加;3、二萜生物堿濃度為0.2、0.4、0.6 mg/ml時,隨濃度增大Tscca細胞凋亡增多,且均有p0.05,差異有統(tǒng)計學意義;4、IL-18與二萜生物堿聯(lián)合對Tscca細胞作用48 h后,較單獨使用二萜生物堿抑制作用明顯,且呈濃度依賴性,均有p0.05,差異有統(tǒng)計學意義;5、IL-18聯(lián)合二萜生物堿還可劑量依賴性地降低p-Akt的蛋白水平,各組均有p0.05,差異有統(tǒng)計學意義,而總Akt水平幾乎不變。結(jié)論1、二萜生物堿對Tscca細胞有增殖抑制及促凋亡作用,且呈濃度依賴性;2、IL-18聯(lián)合二萜生物堿對舌鱗癌細胞Tscca有協(xié)同抑制作用,且抑制作用較單獨使用IL-18或二萜生物堿均較強;3、IL-18聯(lián)合二萜生物堿在抑制p-Akt的表達及活化這條信號通路上發(fā)揮一定作用從而抑制舌癌細胞Tscca的增殖并促進其凋亡。
[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.
【學位授予單位】：錦州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.86

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