PKCα对肝癌药物敏感性作用的研究
摘要
【目的】
肝细胞癌(hepatocellular carcinoma,HCC)是我国常见恶性肿瘤。诱发肝癌的因素很多,其中病毒性肝炎、肝硬化是重要病因。化疗是肝癌主要治疗手段,肝癌细胞对抗癌药物产生耐药现象是导致化疗失败的主要原因,是肝癌临床治疗中的一大难题。肿瘤的多药耐药性(multidrug resistance,MDR)由多种机制调控,研究认为, MDR表型的出现与P-糖蛋白(P-glycoprotein,P-gp)的过表达相关,P-gp发挥着药物泵功能,将药物泵出细胞膜外。蛋白激酶C ( protein kinaseC,PKC)的同工酶,特别是PKCα,对肿瘤细胞MDR表型的出现或维持可能起着决定性作用。因此研究PKCα对肝癌细胞的抗癌药物敏感性的影响,有助于我们了解肝癌耐药的发生机制,为克服肝癌临床治疗中的MDR提供实验依据。
【方法】
1.将体外合成的载有目的片段的DNA链整合进反转录病毒载体。
2.将反转录病毒载体热转化入大肠杆菌中培养,药物筛选阳性克隆后,提取无内毒素质粒。
3.按一定比例将无内毒素质粒与病毒包装细胞混合后,在电转仪下,将反转录病毒载体电转入病毒包装细胞。
4.药物筛选2星期后,将阳性克隆扩大培养。
5.用病毒上清液感染人HepG2细胞。
6.药物筛选后,挑选阳性克隆细胞扩大培养,并将其与PMA处理的HepG2细胞及未受干预的HepG2细胞做药物敏感性及生长速度比较。
【结果】
1.Westernblot显示:与对照组(未受干预的HepG2细胞)相比,感染siRNA片段者PKCα的表达显著减少;而感染空载体者无显著变化; PMA可以刺激细胞内PKCα的表达。
2.siRNA和PMA对HepG2细胞生长速度的影响实验表明,与对照组相比,转染siRNA的HepG2细胞生长速度明显减慢;PMA处理组速度略快;空载体组无显著变化。
3.药物杀伤结果:经过6次反复MTT后,计算各组对表阿霉素的IC50值。对照组为10.2200±0.7805;转染siRNA组6.4450±0.8549;转染空载体组10.1500±0.9343,PMA处理组11.5400±1.3647。LSD-t检验结果显示,与对照组相比,siRNA处理组、PMA处理组对表阿霉素的药物敏感性变化均有统计学意义上的差异(P<0.05),而空载体组与HepG2组比较无统计学意义上的差异。
【结论】
1.实验中所采用的siRNA片段对细胞内PKCα的表达有明显的干扰作用,极大地降低了HepG2细胞中PKCα的表达。
2.细胞内PKCα表达升高时,肝癌细胞对药物的敏感性减弱,耐药性增强;当细胞内PKCα降低时,肝癌细胞对药物的敏感性增强,耐药性减弱。提示抑制肝癌细胞中PKCα表达,可以降低肝癌细胞对药物的耐受性,增强化疗效果。
【objective】
HCC(hepatocellular carcinoma)is one of the important malignant tumor. There are many factors inducing hepatoma. The most common reason is virus hepatitis and liver cirrhosis. Generalized chems is one of the important therapy of the hepotoma. The generalized chems often failed in clinic because the hepatocellular carcinoma resisted to the multi-antitumor-drug. So the treat of hepatoma is still a hang-up. Many factors cause MDR. Many researchers thought that MDR appearance related to P-gp hyper-expression. People think that the P-gp educing the medicine pumping function is the most important reason. The P-gp can pump the medicine out of cells. PKC isozymes, especially PKCα, can influence the occurrence of the tumoer cell MDR. So it is helpful to find out the mechanisms of hepatoma multidrug resistant that the hepatoma’drug sensitivity changs when we chang the PKCαexpression. The research gave the clinical therapy of hepatoma-drug-resistant experiment basement.
【Methods】
1. Extraorgan synthetic DNA chain which including the aim frag integrated into retrovirus vector.
2. The retrovirus vector which including the aim DNA integrated into colibacillus. After the drug screening ,we got out the non-endotoxin plasmid.
3. Mingled the non-endotoxin plasmid and virus-incasing cells together, using the electricity transfer instrument made the retrovirus vector integrating into the virus-incasing cells.
4. After two weeks drug screening, we macroculltured the masccline cells.
5. Using the virus supernate infected the HepG2.
6. After two weeks drug screening, we macroculltures the live cells. Compared the drug sensitivity and growth velocity of the PMA treated HepG2 and siRNA treated HepG2 to the no-interfereing HepG2.
【Results】
1. Westernlot: The PKCαexpression was very little in siRNA interference’s. It was no difference in idling interference’s. The PMA could stimulate the expression of PKCα.
2. The growth velocity of the siRNA interference’s HepG2 was obviously stepping down ,whereas the gowth velocity of the PMA interference’s HepG2 was faster than the no-interference HepG2. The growth velocity of the idling interference’s HepG2 was no change.
3. MTT result: Doing the experiment six times, we caculated the IC50 of every group to the EPI. Conteol group 10.2200±0.7805; siRNA interference group 6.4450±0.8549; idling interference group 10.1500±0.9343, PMA interference group 11.5400±1.3647. LSD-t analysis: compared to the control group, the siRNA interference group’s and the PMA interference group’s drug sensitivity to the EPI were all changing and had the satistical significance(p<0.05), while the idling interference had no difference.【Conclusion】
1. The siRNA frag used in this experiment was obviously cutting down the expression of PKCαof the HepG2.
2. When the expression of PKCαstepped up, the drug sensitivity of the HCC was weakened; when the expression of PKCαcut down, the drug sensitivity of the HCC was strengthened and the tolerance to the drug was down regulated. To hint, suppressing the expression of the PKCαmaybe depress the toleration of HCC to antitumor drug and strengthen the Chems effect.
肝细胞癌(hepatocellular carcinoma,HCC)是我国常见恶性肿瘤。诱发肝癌的因素很多,其中病毒性肝炎、肝硬化是重要病因。化疗是肝癌主要治疗手段,肝癌细胞对抗癌药物产生耐药现象是导致化疗失败的主要原因,是肝癌临床治疗中的一大难题。肿瘤的多药耐药性(multidrug resistance,MDR)由多种机制调控,研究认为, MDR表型的出现与P-糖蛋白(P-glycoprotein,P-gp)的过表达相关,P-gp发挥着药物泵功能,将药物泵出细胞膜外。蛋白激酶C ( protein kinaseC,PKC)的同工酶,特别是PKCα,对肿瘤细胞MDR表型的出现或维持可能起着决定性作用。因此研究PKCα对肝癌细胞的抗癌药物敏感性的影响,有助于我们了解肝癌耐药的发生机制,为克服肝癌临床治疗中的MDR提供实验依据。
【方法】
1.将体外合成的载有目的片段的DNA链整合进反转录病毒载体。
2.将反转录病毒载体热转化入大肠杆菌中培养,药物筛选阳性克隆后,提取无内毒素质粒。
3.按一定比例将无内毒素质粒与病毒包装细胞混合后,在电转仪下,将反转录病毒载体电转入病毒包装细胞。
4.药物筛选2星期后,将阳性克隆扩大培养。
5.用病毒上清液感染人HepG2细胞。
6.药物筛选后,挑选阳性克隆细胞扩大培养,并将其与PMA处理的HepG2细胞及未受干预的HepG2细胞做药物敏感性及生长速度比较。
【结果】
1.Westernblot显示:与对照组(未受干预的HepG2细胞)相比,感染siRNA片段者PKCα的表达显著减少;而感染空载体者无显著变化; PMA可以刺激细胞内PKCα的表达。
2.siRNA和PMA对HepG2细胞生长速度的影响实验表明,与对照组相比,转染siRNA的HepG2细胞生长速度明显减慢;PMA处理组速度略快;空载体组无显著变化。
3.药物杀伤结果:经过6次反复MTT后,计算各组对表阿霉素的IC50值。对照组为10.2200±0.7805;转染siRNA组6.4450±0.8549;转染空载体组10.1500±0.9343,PMA处理组11.5400±1.3647。LSD-t检验结果显示,与对照组相比,siRNA处理组、PMA处理组对表阿霉素的药物敏感性变化均有统计学意义上的差异(P<0.05),而空载体组与HepG2组比较无统计学意义上的差异。
【结论】
1.实验中所采用的siRNA片段对细胞内PKCα的表达有明显的干扰作用,极大地降低了HepG2细胞中PKCα的表达。
2.细胞内PKCα表达升高时,肝癌细胞对药物的敏感性减弱,耐药性增强;当细胞内PKCα降低时,肝癌细胞对药物的敏感性增强,耐药性减弱。提示抑制肝癌细胞中PKCα表达,可以降低肝癌细胞对药物的耐受性,增强化疗效果。
【objective】
HCC(hepatocellular carcinoma)is one of the important malignant tumor. There are many factors inducing hepatoma. The most common reason is virus hepatitis and liver cirrhosis. Generalized chems is one of the important therapy of the hepotoma. The generalized chems often failed in clinic because the hepatocellular carcinoma resisted to the multi-antitumor-drug. So the treat of hepatoma is still a hang-up. Many factors cause MDR. Many researchers thought that MDR appearance related to P-gp hyper-expression. People think that the P-gp educing the medicine pumping function is the most important reason. The P-gp can pump the medicine out of cells. PKC isozymes, especially PKCα, can influence the occurrence of the tumoer cell MDR. So it is helpful to find out the mechanisms of hepatoma multidrug resistant that the hepatoma’drug sensitivity changs when we chang the PKCαexpression. The research gave the clinical therapy of hepatoma-drug-resistant experiment basement.
【Methods】
1. Extraorgan synthetic DNA chain which including the aim frag integrated into retrovirus vector.
2. The retrovirus vector which including the aim DNA integrated into colibacillus. After the drug screening ,we got out the non-endotoxin plasmid.
3. Mingled the non-endotoxin plasmid and virus-incasing cells together, using the electricity transfer instrument made the retrovirus vector integrating into the virus-incasing cells.
4. After two weeks drug screening, we macroculltured the masccline cells.
5. Using the virus supernate infected the HepG2.
6. After two weeks drug screening, we macroculltures the live cells. Compared the drug sensitivity and growth velocity of the PMA treated HepG2 and siRNA treated HepG2 to the no-interfereing HepG2.
【Results】
1. Westernlot: The PKCαexpression was very little in siRNA interference’s. It was no difference in idling interference’s. The PMA could stimulate the expression of PKCα.
2. The growth velocity of the siRNA interference’s HepG2 was obviously stepping down ,whereas the gowth velocity of the PMA interference’s HepG2 was faster than the no-interference HepG2. The growth velocity of the idling interference’s HepG2 was no change.
3. MTT result: Doing the experiment six times, we caculated the IC50 of every group to the EPI. Conteol group 10.2200±0.7805; siRNA interference group 6.4450±0.8549; idling interference group 10.1500±0.9343, PMA interference group 11.5400±1.3647. LSD-t analysis: compared to the control group, the siRNA interference group’s and the PMA interference group’s drug sensitivity to the EPI were all changing and had the satistical significance(p<0.05), while the idling interference had no difference.【Conclusion】
1. The siRNA frag used in this experiment was obviously cutting down the expression of PKCαof the HepG2.
2. When the expression of PKCαstepped up, the drug sensitivity of the HCC was weakened; when the expression of PKCαcut down, the drug sensitivity of the HCC was strengthened and the tolerance to the drug was down regulated. To hint, suppressing the expression of the PKCαmaybe depress the toleration of HCC to antitumor drug and strengthen the Chems effect.
引文
1.叶任高、陆再英等,内科学第六版人民卫生出版社2004年5月,450-457
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