番荔枝内酯单体Desacetyluvaricin对乙肝相关肝癌细胞TLR4、P53蛋白表达的影响
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摘要
研究目的
通过研究番荔枝内酯单体(Desacetyluvaricin,DES)对乙肝相关肝癌细胞TLR4、P53蛋白表达的影响,从分子生物学水平探讨DES干预乙肝致肝癌的机制,为研制一种新型抗肿瘤药物奠定基础,同时为临床运用番荔枝内酯进行抗癌治疗提供客观依据和一定的理论支持。
研究方法
1、MTT法观察DES对乙肝相关肝癌细胞HepG2.2.15的抑制作用,并考察其量效和时效关系。
2、流式细胞术观察DES对乙肝相关肝癌细胞HepG2.2.15的TLR4、P53蛋白表达影响。
3、激光扫描共聚焦术检测乙肝相关肝癌细胞HepG2.2.15的HBx表达。
研究结果
1、DES作用24h,25μg/ml浓度,即可见药物对乙肝相关肝癌细胞HepG2.2.15出现抑制作用,当作用72h,200μg/ml浓度时,DES对乙肝相关肝癌细胞HepG2.2.15最高抑制率为65%,明显高于50%的有效标准。
2、control组TLR4表达量为37.16%,阴性对照组TLR4表达量为2.60%,两组比较差异有显著性(P＜0.01),DES组TLR4表达量为71.94%,与control组比较差异有显著性(P＜0.01)。control组P53表达量为3.3%,阴性对照组P53表达量为12.3%,两组比较差异有统计学意义(P＜0.05),DES组P53表达量为32.6%,与control组比较差异有显著性(P＜0.01)。
3、control组细胞核大而明显,呈圆形或不规则形,核膜明显,HBx均匀表达于细胞浆里,荧光强度为51.08。DES组细胞体积缩小、变园,胞膜皱缩,核膜不清楚,HBx荧光强度为25.23,与control组比较差异有统计学意义(P＜0.05)。
研究结论
1、DES对乙肝相关肝癌细胞HepG2.2.15增殖呈明显抑制作用,且随药物浓度的提高和作用时间的延长而增强。
2、DES能够诱导乙肝相关肝癌细胞HepG2.2.15凋亡,可能是通过上调P53的表达起作用的。
3、DES抗乙肝相关肝癌细胞的机制可能与上调TLR4的表达,从而激活固有免疫通道,提高机体免疫力有关。
4、HBx有促进肿瘤细胞增殖,抑制肿瘤细胞凋亡的作用。
5、DES抑制HBx的表达。
Object
By researching the effect of Desacetyluvaricin on the expression of TLR4 and P53 protein in the HBV-related human hepatocellular cancer cell.To establish a fundamental base for putting Desacetyluvaricin into a new anticancer drug by disclosing its anticancer mechanism in molecular biologic method.
Methods
1 Observe the inhibitory action of Desacetyluvaricin on the HBV-related human hepato-cellular cancer cell line HepG2.2.15 by MTT assay and find out the correlation of dosage-effect and time-effect.
2 FCM(flow cytometer) was used to analyze the expression of TLR4 and P53 protein of Desacetyluvaricin on the HBV-related human hepatocellular cancer cell line HepG2215.
3 LCSM(laser confocal scanning microscopy) was used to observe the effect of Desacety-luvaricin on the expression of HBx protein in the HBV-related human hepatocellular cancer cell line HepG2.2.15.
Result
1 The HBV-related human hepatocellular cancer cell line HepG2.2.15 has been inhibited after treated by Desacetyluvaricin for 24h and 25μg/ml.The highest inhibitory rate of Desacety-luvaricin on HepG2.2.15 was 65%.It is obviously higher than 50%.
2 The expression of TLR4 in control group and negative control group and DES group were 37.16%and 2.60%and 71.94%respectively,there were distinct difference in three group (P＜0.01).The expression of P53 in DES group and negative control group and control group were 32.6%and 12.3%and 3.3%respectively,there were distinct difference in three group (P＜0.05 or P＜0.01 ).
3 In control group:cell nucleus volume was larger than normal cell.Nuclear membrane was obvious.HBx expressed uniformly in the cytoplasm.The fluorescence intensity of HBx was 51.08.In DES group:cellular volume decreases,membrane crinkles,Nuclear membrane wasn't obvious.The fluorescence intensity of HBx was 25.23.Comparison with the control group was distinct difference(P＜0.05).
Conclusions
1 Desacetyluvaricin had better inhibitory effect on the HBV-related human hepatocellular cancer cell line HepG2215 and the effect was concentration and time-dependent.
2 Desacetyluvaricin induces the apoptosis of the HBV-related hepatocellular cancer cell may be related to upregulating the protein expression of P53.
3 The mechanism of Desacetyluvaricin intervening of the HBV-related hepatocellular cancer cell is related to upregulating the protein expression of TLR4,to activating the inherent immune passageway and to improving the immune ability.
4 HBx can proliferate the HBV-related hepatocellular cancer cell and inhibit the apoptosis of the hepatitis B virus related hepatocellular cancer cell.
5 HBx protein decreases obviously after treated by Desacetyluvaricin on the HBV-related human hepatocellular cancer cell line HepG2215.
通过研究番荔枝内酯单体(Desacetyluvaricin,DES)对乙肝相关肝癌细胞TLR4、P53蛋白表达的影响,从分子生物学水平探讨DES干预乙肝致肝癌的机制,为研制一种新型抗肿瘤药物奠定基础,同时为临床运用番荔枝内酯进行抗癌治疗提供客观依据和一定的理论支持。
研究方法
1、MTT法观察DES对乙肝相关肝癌细胞HepG2.2.15的抑制作用,并考察其量效和时效关系。
2、流式细胞术观察DES对乙肝相关肝癌细胞HepG2.2.15的TLR4、P53蛋白表达影响。
3、激光扫描共聚焦术检测乙肝相关肝癌细胞HepG2.2.15的HBx表达。
研究结果
1、DES作用24h,25μg/ml浓度,即可见药物对乙肝相关肝癌细胞HepG2.2.15出现抑制作用,当作用72h,200μg/ml浓度时,DES对乙肝相关肝癌细胞HepG2.2.15最高抑制率为65%,明显高于50%的有效标准。
2、control组TLR4表达量为37.16%,阴性对照组TLR4表达量为2.60%,两组比较差异有显著性(P＜0.01),DES组TLR4表达量为71.94%,与control组比较差异有显著性(P＜0.01)。control组P53表达量为3.3%,阴性对照组P53表达量为12.3%,两组比较差异有统计学意义(P＜0.05),DES组P53表达量为32.6%,与control组比较差异有显著性(P＜0.01)。
3、control组细胞核大而明显,呈圆形或不规则形,核膜明显,HBx均匀表达于细胞浆里,荧光强度为51.08。DES组细胞体积缩小、变园,胞膜皱缩,核膜不清楚,HBx荧光强度为25.23,与control组比较差异有统计学意义(P＜0.05)。
研究结论
1、DES对乙肝相关肝癌细胞HepG2.2.15增殖呈明显抑制作用,且随药物浓度的提高和作用时间的延长而增强。
2、DES能够诱导乙肝相关肝癌细胞HepG2.2.15凋亡,可能是通过上调P53的表达起作用的。
3、DES抗乙肝相关肝癌细胞的机制可能与上调TLR4的表达,从而激活固有免疫通道,提高机体免疫力有关。
4、HBx有促进肿瘤细胞增殖,抑制肿瘤细胞凋亡的作用。
5、DES抑制HBx的表达。
Object
By researching the effect of Desacetyluvaricin on the expression of TLR4 and P53 protein in the HBV-related human hepatocellular cancer cell.To establish a fundamental base for putting Desacetyluvaricin into a new anticancer drug by disclosing its anticancer mechanism in molecular biologic method.
Methods
1 Observe the inhibitory action of Desacetyluvaricin on the HBV-related human hepato-cellular cancer cell line HepG2.2.15 by MTT assay and find out the correlation of dosage-effect and time-effect.
2 FCM(flow cytometer) was used to analyze the expression of TLR4 and P53 protein of Desacetyluvaricin on the HBV-related human hepatocellular cancer cell line HepG2215.
3 LCSM(laser confocal scanning microscopy) was used to observe the effect of Desacety-luvaricin on the expression of HBx protein in the HBV-related human hepatocellular cancer cell line HepG2.2.15.
Result
1 The HBV-related human hepatocellular cancer cell line HepG2.2.15 has been inhibited after treated by Desacetyluvaricin for 24h and 25μg/ml.The highest inhibitory rate of Desacety-luvaricin on HepG2.2.15 was 65%.It is obviously higher than 50%.
2 The expression of TLR4 in control group and negative control group and DES group were 37.16%and 2.60%and 71.94%respectively,there were distinct difference in three group (P＜0.01).The expression of P53 in DES group and negative control group and control group were 32.6%and 12.3%and 3.3%respectively,there were distinct difference in three group (P＜0.05 or P＜0.01 ).
3 In control group:cell nucleus volume was larger than normal cell.Nuclear membrane was obvious.HBx expressed uniformly in the cytoplasm.The fluorescence intensity of HBx was 51.08.In DES group:cellular volume decreases,membrane crinkles,Nuclear membrane wasn't obvious.The fluorescence intensity of HBx was 25.23.Comparison with the control group was distinct difference(P＜0.05).
Conclusions
1 Desacetyluvaricin had better inhibitory effect on the HBV-related human hepatocellular cancer cell line HepG2215 and the effect was concentration and time-dependent.
2 Desacetyluvaricin induces the apoptosis of the HBV-related hepatocellular cancer cell may be related to upregulating the protein expression of P53.
3 The mechanism of Desacetyluvaricin intervening of the HBV-related hepatocellular cancer cell is related to upregulating the protein expression of TLR4,to activating the inherent immune passageway and to improving the immune ability.
4 HBx can proliferate the HBV-related hepatocellular cancer cell and inhibit the apoptosis of the hepatitis B virus related hepatocellular cancer cell.
5 HBx protein decreases obviously after treated by Desacetyluvaricin on the HBV-related human hepatocellular cancer cell line HepG2215.
引文
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