雙氫青蒿素調(diào)節(jié)核糖體蛋白L23抑制胃癌MKN45細胞增殖的機制研究
發(fā)布時間：2019-04-03 12:02
【摘要】：目的:研究雙氫青蒿素(dihydroartemisinin,DHA)對胃癌MKN45細胞增殖、凋亡的影響,并探索其可能的機制,明確DHA在胃癌治療中的可行性及其作用的發(fā)揮與核糖體蛋白L23(Ribosome protein L23,RPL23)活性狀態(tài)是否存在關(guān)系。方法:(1)根據(jù)Pubmed人RPL23基因序列,設(shè)計3條引物,通過脂質(zhì)體轉(zhuǎn)染化學合成的RPL23-si RNA干擾片段,使用實時熒光定量PCT(Real-time PCR,RT-PCR)、蛋白印記實驗(Western blot,WB)進行干擾效率鑒定;(2)使用不同濃度梯度的DHA作用MKN45細胞,MTT實驗檢測細胞增殖情況,確定DHA的半數(shù)抑制濃度(Half maximal inhibitory concentration,IC50);(3)使用DHA及si RNA轉(zhuǎn)染共處理MKN45細胞,將實驗分為4組:對照組(NC組)、RPL23-si RNA組、DHA組、DHA+RPL23-si RNA組,用甲基噻唑基四唑(Methylcyclopentadienyl Manganese Tricarbonyl,MTT)實驗測定細胞增殖、流式細胞術(shù)(Flow cytometry,FCM)測定細胞凋亡及細胞周期,激光共聚焦掃描顯微鏡(Confocal laser scanning microscopy,CLSM)觀察細胞中RPL23、鼠雙微體2(Murine double minute 2,MDM2)蛋白定位,WB實驗檢測RPL23、MDM2、B細胞易位基因2(B cell translation gene 2,BTG2)、細胞周期蛋白D1(Cyclin D1)表達;(4)實驗數(shù)據(jù)采取均數(shù)±標準差(?),SPSS 19.0進行統(tǒng)計分析。結(jié)果:(1)構(gòu)建并篩選出最佳RPL23-si RNA干擾片段:RT-PCR鑒定結(jié)果顯示,正常細胞組、對照組、si RNA1組、si RNA2組、si RNA3組5組中RPL23m RNA表達水平具有具有顯著統(tǒng)計學差異(P0.01),多重比較發(fā)現(xiàn),si RNA1組干擾效率明顯高于RPL23-si RNA2組(P0.01)及RPL23-si RNA3組(P0.01),WB鑒定與上述結(jié)果一致,可以認為,si RNA1是最佳干擾序列,可用于后續(xù)實驗。(2)DHA IC50值的確定:MTT實驗檢測了不同濃度梯度的DHA對于MKN45增殖的影響,MKN45的抑制率與藥物濃度之間呈現(xiàn)出劑量依賴性,隨著藥物濃度的增加,MKN45細胞的抑制率隨之增加,當DHA濃度為160um/L時,細胞增殖抑制率為50%左右,說明,160um/L可作為DHA的IC50值。(3)MTT實驗及FCM實驗顯示,DHA可明顯抑制胃癌MKN45增殖,誘導(dǎo)細胞凋亡,NC組、RPL23-si RNA組、DHA組、DHA+RPL23-si RNA組細胞的平均凋亡率之間存在統(tǒng)計學差異(P0.01)。細胞周期實驗顯示,4組細胞數(shù)在G1期、S期均表現(xiàn)出統(tǒng)計學差異(P0.01),RPL23抑制表達后,MKN45細胞的G1期細胞數(shù)目減少,S期細胞數(shù)目增多,說明RPL23-si RNA促進了細胞周期G1/S期轉(zhuǎn)化,而經(jīng)過DHA再次處理后,則出現(xiàn)了不同程度的G1/S期轉(zhuǎn)化抑制。用激光共聚焦顯微鏡觀察到,正常的MKN45細胞中,RPL23主要表達在細胞漿,MDM2則主要表達于細胞核漿,使用RPL23-si RNA轉(zhuǎn)染后,細胞漿中的RPL23明顯降低,而MDM2則出現(xiàn)了細胞核中的濃聚,而DHA處理后,兩組細胞中的RPL23再次出現(xiàn)了胞漿中的不同程度濃聚,而MDM2則在細胞核漿中出現(xiàn)了熒光減弱。WB實驗觀察到4組細胞中RPL23(P0.01)、MDM2(P0.01)、Cyclin D1(P0.01)、BTG2(P0.01)均存在統(tǒng)計學差異。結(jié)論:DHA可通過RPL23-MDM2信號通路上調(diào)RPL23、BTG2表達、抑制MDM2、Cyclin D1表達發(fā)揮抗胃癌MKN45細胞增殖、誘導(dǎo)細胞凋亡的作用。
[Abstract]:Objective: to study the effect of dihydroartemisinin (dihydroartemisinin,DHA) on proliferation and apoptosis of gastric cancer MKN45 cells and explore its possible mechanism, and to clarify the feasibility of DHA in the treatment of gastric cancer and its function and the expression of ribosomal protein L23 (Ribosome protein L23, Whether there is a relationship between the active state of RPL23 or not. Methods: (1) according to the sequence of human RPL23 gene of Pubmed, three primers were designed and transfected into chemically synthesized RPL23-si RNA interference fragments by liposome. Real-time fluorescence quantitative PCT (Real-time PCR,RT-PCR) and protein imprinting test (Western blot, were used. WB) for the identification of interference efficiency; (2) MKN45 cells were treated with DHA with different concentration gradient. The proliferation of MKN45 cells was detected by MTT assay, and the 50% inhibitory concentration of DHA (Half maximal inhibitory concentration,IC50) was determined. (3) MKN45 cells were transfected with DHA and si RNA, and were divided into 4 groups: control group (NC group), RPL23-si RNA group, DHA RPL23-si RNA group, and the proliferation of cells was measured by methylthiazolyl tetrazolium (Methylcyclopentadienyl Manganese Tricarbonyl,MTT assay. Flow cytometry (Flow cytometry,FCM) was used to detect apoptosis and cell cycle, laser confocal scanning microscope (Confocal laser scanning microscopy,CLSM) was used to observe the localization of RPL23, mouse double microsomes 2 (Murine double minute 2, MDM 2) protein, and WB assay was used to detect RPL23,MDM2,. B cell translocation gene 2 (B cell translation gene 2, BTG2) and cyclin D1 (Cyclin D1) expression; (4) the experimental data were statistically analyzed by mean 鹵standard deviation (?), SPSS 19.0). Results: (1) the optimal RPL23-si RNA interference fragment was constructed and screened. The results of RT-PCR identification showed that the normal cell group, control group, si RNA1 group, si RNA2 group, and normal cell group, control group, si RNA1 group, si RNA2 group. The expression level of RPL23m RNA in si RNA3 group was significantly higher than that in RPL23-si RNA2 group (P0.01) and RPL23-si RNA3 group (P0.01). Multiple comparisons showed that the interference efficiency in si RNA1 group was significantly higher than that in RPL23-si RNA2 group (P0.01) and RPL23-si RNA3 group (P0.01). WB identification is consistent with the above results, it can be considered that si RNA1 is the best interference sequence and can be used in subsequent experiments. (2) determination of DHA IC50 value: MTT test detected the effect of DHA of different concentration gradient on MKN45 proliferation. There was a dose-dependent relationship between the inhibition rate of MKN45 and the drug concentration. With the increase of the drug concentration, the inhibition rate of MKN45 cells increased. When the concentration of DHA was 160um/L, the inhibition rate of cell proliferation was about 50%, which indicated that the inhibitory rate of cell proliferation was about 50%. 160um/L can be used as the IC _ (50) value of DHA. (3) MTT and FCM test showed that DHA could significantly inhibit MKN45 proliferation and induce apoptosis of gastric cancer, NC group, RPL23-si RNA group, DHA group, NC group, RPL23-si RNA group, DHA group. There was statistical difference between the average apoptosis rate of DHA RPL23-si RNA group (P0.01). Cell cycle test showed that the number of MKN45 cells in G1 phase and S phase showed statistical difference (P0.01). After the expression of RPL23 was inhibited, the number of G1 phase cells in MKN45 cells decreased, and the number of S phase cells increased. The results showed that RPL23-si RNA promoted G1 / S phase transformation in cell cycle, but after retreatment with DHA, there were different degrees of G1 / S phase transformation inhibition. It was observed by confocal laser microscopy that RPL23 was mainly expressed in cytoplasm and MDM2 was mainly expressed in nuclear cytoplasm in normal MKN45 cells. After transfected with RPL23-si RNA, the RPL23 in cytoplasm decreased significantly. On the other hand, MDM2 showed the accumulation of nucleus, and after DHA treatment, the RPL23 in the two groups again showed different degrees of accumulation in the cytoplasm, and the concentration of RPL23 in the cytoplasm of the two groups of cells was similar to that of the control group. RPL23 (P0.01), MDM2 (P0.01) and BTG2 (P0.01) were observed in the four groups of cells by WB test. The fluorescence of MDM2 decreased in the nuclear plasma of the cells, and there were significant differences between the four groups (P0.01). Conclusion: DHA can up-regulate the expression of RPL23,BTG2 through RPL23-MDM2 signaling pathway, inhibit the expression of MDM2,Cyclin D1 and play an anti-proliferation and apoptosis-inducing role in gastric cancer MKN45 cells.
【學位授予單位】：湖北中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.2
本文編號：2453194
[Abstract]:Objective: to study the effect of dihydroartemisinin (dihydroartemisinin,DHA) on proliferation and apoptosis of gastric cancer MKN45 cells and explore its possible mechanism, and to clarify the feasibility of DHA in the treatment of gastric cancer and its function and the expression of ribosomal protein L23 (Ribosome protein L23, Whether there is a relationship between the active state of RPL23 or not. Methods: (1) according to the sequence of human RPL23 gene of Pubmed, three primers were designed and transfected into chemically synthesized RPL23-si RNA interference fragments by liposome. Real-time fluorescence quantitative PCT (Real-time PCR,RT-PCR) and protein imprinting test (Western blot, were used. WB) for the identification of interference efficiency; (2) MKN45 cells were treated with DHA with different concentration gradient. The proliferation of MKN45 cells was detected by MTT assay, and the 50% inhibitory concentration of DHA (Half maximal inhibitory concentration,IC50) was determined. (3) MKN45 cells were transfected with DHA and si RNA, and were divided into 4 groups: control group (NC group), RPL23-si RNA group, DHA RPL23-si RNA group, and the proliferation of cells was measured by methylthiazolyl tetrazolium (Methylcyclopentadienyl Manganese Tricarbonyl,MTT assay. Flow cytometry (Flow cytometry,FCM) was used to detect apoptosis and cell cycle, laser confocal scanning microscope (Confocal laser scanning microscopy,CLSM) was used to observe the localization of RPL23, mouse double microsomes 2 (Murine double minute 2, MDM 2) protein, and WB assay was used to detect RPL23,MDM2,. B cell translocation gene 2 (B cell translation gene 2, BTG2) and cyclin D1 (Cyclin D1) expression; (4) the experimental data were statistically analyzed by mean 鹵standard deviation (?), SPSS 19.0). Results: (1) the optimal RPL23-si RNA interference fragment was constructed and screened. The results of RT-PCR identification showed that the normal cell group, control group, si RNA1 group, si RNA2 group, and normal cell group, control group, si RNA1 group, si RNA2 group. The expression level of RPL23m RNA in si RNA3 group was significantly higher than that in RPL23-si RNA2 group (P0.01) and RPL23-si RNA3 group (P0.01). Multiple comparisons showed that the interference efficiency in si RNA1 group was significantly higher than that in RPL23-si RNA2 group (P0.01) and RPL23-si RNA3 group (P0.01). WB identification is consistent with the above results, it can be considered that si RNA1 is the best interference sequence and can be used in subsequent experiments. (2) determination of DHA IC50 value: MTT test detected the effect of DHA of different concentration gradient on MKN45 proliferation. There was a dose-dependent relationship between the inhibition rate of MKN45 and the drug concentration. With the increase of the drug concentration, the inhibition rate of MKN45 cells increased. When the concentration of DHA was 160um/L, the inhibition rate of cell proliferation was about 50%, which indicated that the inhibitory rate of cell proliferation was about 50%. 160um/L can be used as the IC _ (50) value of DHA. (3) MTT and FCM test showed that DHA could significantly inhibit MKN45 proliferation and induce apoptosis of gastric cancer, NC group, RPL23-si RNA group, DHA group, NC group, RPL23-si RNA group, DHA group. There was statistical difference between the average apoptosis rate of DHA RPL23-si RNA group (P0.01). Cell cycle test showed that the number of MKN45 cells in G1 phase and S phase showed statistical difference (P0.01). After the expression of RPL23 was inhibited, the number of G1 phase cells in MKN45 cells decreased, and the number of S phase cells increased. The results showed that RPL23-si RNA promoted G1 / S phase transformation in cell cycle, but after retreatment with DHA, there were different degrees of G1 / S phase transformation inhibition. It was observed by confocal laser microscopy that RPL23 was mainly expressed in cytoplasm and MDM2 was mainly expressed in nuclear cytoplasm in normal MKN45 cells. After transfected with RPL23-si RNA, the RPL23 in cytoplasm decreased significantly. On the other hand, MDM2 showed the accumulation of nucleus, and after DHA treatment, the RPL23 in the two groups again showed different degrees of accumulation in the cytoplasm, and the concentration of RPL23 in the cytoplasm of the two groups of cells was similar to that of the control group. RPL23 (P0.01), MDM2 (P0.01) and BTG2 (P0.01) were observed in the four groups of cells by WB test. The fluorescence of MDM2 decreased in the nuclear plasma of the cells, and there were significant differences between the four groups (P0.01). Conclusion: DHA can up-regulate the expression of RPL23,BTG2 through RPL23-MDM2 signaling pathway, inhibit the expression of MDM2,Cyclin D1 and play an anti-proliferation and apoptosis-inducing role in gastric cancer MKN45 cells.
【學位授予單位】：湖北中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.2
【參考文獻】
相關(guān)期刊論文 前10條
1 彭文苗;張志敏;胡萌;余麗芳;章必成;饒智國;秦傳蓉;;RPL23-siRNA干擾片段的構(gòu)建及篩選[J];實用腫瘤學雜志;2016年06期
2 馬敏;劉德山;梁爾順;李曉東;成偉;隗�；�;;扶正消瘕方聯(lián)合化療治療晚期胃癌的臨床療效[J];中國老年學雜志;2016年22期
3 彭文苗;秦傳蓉;張志敏;胡萌;饒智國;;核糖體蛋白L23參與腫瘤進程的研究[J];國際腫瘤學雜志;2016年11期
4 許文彬;陳遠能;張濤;陳玉;李華燕;平倩;;清熱化濕方調(diào)節(jié)PI3K-AKT信號誘導(dǎo)自噬維持胃黏膜穩(wěn)態(tài)防治幽門螺桿菌感染相關(guān)胃癌的機制[J];中華中醫(yī)藥學刊;2016年10期
5 尤小蘭;錢海鑫;秦磊;王元杰;李文琦;連彥軍;趙小軍;徐寧;黃傳江;程之逸;劉貴遠;;進展期胃癌根治術(shù)中腹腔動脈干灌注化療的應(yīng)用研究[J];中華胃腸外科雜志;2016年09期
6 賈叢偉;孫洋;張婷婷;盧朝輝;陳杰;;miR-125a-5p對胰腺癌細胞增殖、凋亡和細胞周期的影響[J];中國醫(yī)學科學院學報;2016年04期
7 張向前;張敏;張靜;王艷梅;虎小毅;孫宏飛;童東東;熊曉蕃;趙正豪;;過表達miR-26b抑制MKN-45人胃癌細胞的增殖[J];細胞與分子免疫學雜志;2016年08期
8 張敬偉;段冬梅;任中海;;復(fù)方苦參注射液與順鉑腹腔灌注化療聯(lián)合治療胃癌惡性腹水[J];中國實驗方劑學雜志;2016年11期
9 宋卓;蘇春雨;徐競男;李杰;;扶正解毒法防治胃癌的理論基礎(chǔ)及循證依據(jù)[J];中醫(yī)雜志;2016年10期
10 樊夢嬌;千年松;戴廣海;;胃癌靶向治療與免疫治療的新進展[J];現(xiàn)代腫瘤醫(yī)學;2016年11期
相關(guān)碩士學位論文 前1條
1 李劍;內(nèi)質(zhì)網(wǎng)應(yīng)激在雙氫青蒿素誘導(dǎo)前列腺癌PC-3細胞凋亡中的作用研究[D];重慶醫(yī)科大學;2011年
，本文編號：2453194
本文鏈接：http://sikaile.net/yixuelunwen/zlx/2453194.html
最近更新
教材專著
