佛波酯减轻多柔比星心肌毒性及增强抗肿瘤作用的实验研究

发布时间：2018-05-10 23:34

本文选题：TPA + DOX　；参考：《郑州大学》2017年硕士论文

【摘要】：多柔比星(Doxorubicin,DOX)是广谱的抗肿瘤药,广泛用于多种癌症,包括乳腺癌,卵巢癌和前列腺癌。但由于剂量相关的心脏毒性和耐药性的产生使其化疗效果大打折扣,限制了此类药物的长期使用。临床建议使用DOX的累积剂量不宜超过400mg/m~2,然而即使在这些限制的剂量下,也会发生心脏毒性。患恶性肿瘤的儿童在使用DOX治疗后的25年内,患者心脏死亡的相对危险性显著高于正常人群,由DOX诱导的心脏损伤表现为多种形式,但和临床最相关的是扩张型心肌病。因此寻找能够克服DOX心脏毒性同时又能增加DOX抗肿瘤活性的药物有着非常广阔的临床应用前景。12-氧-十四烷酰佛波醇-13-乙酸酯(12-O-teteadecanoylphorbol-13-acetate,TPA)与蛋白激酶C(PKC)具有高度亲和力,通过激活PKC可产生多种生物学效应,如调节基因转录,细胞增殖,分化,存活和程序性凋亡。据文献报道,在心肌缺血及再灌注损伤模型中,TPA激活PKC后可以调节心肌细胞膜Na+-K+-ATP酶活性,增加受损心肌细胞Hsp70的表达,同时也可以作用于线粒体呼吸链,调节能量代谢,从而起到保护受损心肌的作用。因此本研究预考察TPA是否可以拮抗DOX造成的心肌损伤且增加其抗肿瘤活性。1 TPA联合DOX对白血病K562,肺癌A549细胞增殖的影响1.1细胞培养K562细胞用含10%胎牛血清的RM-1640培养基,A549细胞用含10%胎牛血清的DMEM高糖培养基,均在37°C,5%CO2的无菌培养箱中培养,每隔三天传代一次。1.2 MTT法检测细胞增殖MTT法检测药物单用及联合应用情况下,K562及A549细胞抑制率的变化,绘制时效及量效曲线,进行统计学分析,并评价TPA对DOX抗肿瘤作用的影响。2 TPA对DOX诱导的心肌H9C2细胞损伤的影响H9C2细胞培养及抑制率的检测同上,测定细胞培养液中的LDH活性,评价TPA对DOX诱导的心肌H9C2细胞损伤的影响。3 TPA对DOX引起小鼠毒性的影响3.1急性毒性试验中TPA对DOX所致小鼠死亡的影响给予小鼠一次性大剂量尾静脉注射DOX 22mg/kg,五天后给予不同剂量的TPA治疗,观察TPA对DOX所致小鼠死亡率及生存期的影响。3.2 TPA对DOX剂量限制性毒性的影响给予小鼠尾静脉注射DOX 3mg/kg/week,连续12周,建立心肌慢性损伤模型,同时每周给予TPA 25μg/kg三次予以干预。观察小鼠生存状况,测定DOX达到不同累积剂量时心肌酶水平及肝损伤相关指标的变化,并比较各组小鼠心脏病理差异,评价TPA对DOX剂量限制性毒性的影响。4荷瘤小鼠中TPA对DOX的抑瘤率及毒副作用的影响将S180肉瘤细胞细胞移植到BALB/c腋下,瘤体形成后给予TPA及DOX,观察TPA是否可以提高DOX的抑瘤率及减弱其毒性反应。方法5统计方法采用SPSS17.0统计软件对各项数据资料进行统计学分析。动物生存周期采用Graph Pad Prism 5.0进行统计分析。采用单因素方差分析或者独立样本t检验进行各组之间统计学检查。检验水准P为0.05。结果1 TPA对DOX的体外增效作用DOX对K562细胞及A549细胞的抑制均呈现出时间及浓度依赖性,IC50分别为0.88μmol/L及3.42μmol/L。TPA抑制K562细胞生长的IC50为4.316nmol/L,而低浓度的TPA对A549抑制作用不明显,高浓度的TPA对A549有明显的抑制作用。1.62 nmol/L TPA可以显著增强DOX对K562及A549的抑制作用,将DOX抑制K562细胞生长的IC50降低到0.42μmol/L,将抑制A549细胞的IC50降低到1.78μmol/L,与DOX单用组相比,IC50均下降了约50%。2 TPA减弱DOX诱导的心肌H9C2细胞的损伤TPA可以减弱DOX诱导的H9C2细胞的增殖抑制和损伤,1.62 nmol/L TPA可以将DOX抑制H9C2细胞生长的IC50从6.127μmol/L提高到15.099μmol/L,提高了约2.5倍。高浓度的TPA即8.1 nmol/L能显著降低DOX诱导的H9C2细胞LDH的释放,且存在统计学差异(P0.01)。3 TPA对DOX引起小鼠毒性的影响3.1急性毒性实验TPA能够显著降低大剂量DOX引起的小鼠死亡率,延长小鼠的生存时间。TPA 25μg/kg将小鼠死亡率由100%降低到22.2%,并将平均生存天数由9.64±6.72天延长到16.56±3.97天,且存在统计学差异(P0.01)。3.2 TPA对DOX剂量限制性毒性的影响在DOX诱导的慢性心肌损伤模型中,血清肌酸激酶同工酶(Creatine kinase MB,CK-MB)活性随着DOX累积剂量的增加而增加,TPA能明显降低DOX诱导的CK-MB活性的升高。心肌病理结果显示,TPA治疗组明显拮抗了DOX造成的局灶性出血,细胞核溶解及心肌细胞坏死。同时TPA治疗组将DOX引起的动物死亡率由52%降低到16%,将平均生存天数由76.56±22.63天延长到94.16±11.56天,且存在统计学差异(P0.01)。4荷瘤小鼠中TPA对DOX的抑瘤率及毒副作用的影响各组小鼠瘤体重量显示,TPA对肿瘤有一定的抑制作用,且TPA能明显增加DOX的抗肿瘤作用,将DOX的抑瘤率由78.30%增加到89.62%,且存在统计学差异(P0.05)。此外,TPA可以明显改善DOX及瘤体本身造成的小鼠血清CK-MB水平的升高,改善心功能。结论1.TPA可以从体外协同DOX肿瘤细胞的增殖抑制作用,减弱DOX诱导的心肌细胞的损伤。2.TPA可以增加DOX对小鼠肿瘤的抑制作用,并能减轻其心肌毒性。
[Abstract]:Doxorubicin (DOX) is a broad-spectrum antitumor drug that is widely used in a variety of cancers, including breast, ovarian and prostate cancer. But because of the dose related cardiac toxicity and drug resistance, the effect of chemotherapy is discounted and the long-term use of such drugs is limited. The cumulative dose of DOX should not exceed 400mg/. M~2, however, can also be toxic even at these limits. 25 years after the use of DOX, the relative risk of heart death in a child with malignant tumor is significantly higher than that in the normal population. The heart damage induced by DOX is manifested in a variety of forms, but the most related to the bed is dilated cardiomyopathy. Drugs that have been able to overcome DOX's cardiac toxicity and increase DOX antitumor activity have a very broad prospect of clinical application..12- (12-O-teteadecanoylphorbol-13-acetate, TPA) has a high affinity with protein kinase C (PKC) and can produce a variety of biological effects by activating PKC, such as regulating gene transfer. Record, cell proliferation, differentiation, survival and programmed apoptosis. In the model of myocardial ischemia and reperfusion injury, TPA activates the Na+-K+-ATP enzyme activity of the myocardial cell membrane in the myocardial ischemia and reperfusion injury model, and increases the expression of Hsp70 in the damaged myocardial cells, and can also act on the respiratory chain of the cord body, regulate the energy metabolism, and thus protect the damaged heart. Effect of muscle. Therefore, this study examined whether TPA could antagonize myocardial injury caused by DOX and increase its anti tumor activity.1 TPA combined with DOX on leukemic K562 and the proliferation of lung cancer A549 cells. 1.1 cells cultured K562 cells with a RM-1640 medium containing 10% fetal bovine serum, A549 cells using a DMEM high sugar medium containing 10% fetal bovine serum, all in 3 7 C, 5%CO2 culture incubator, every three days, a.1.2 MTT method was used to detect the cell proliferation and MTT method to detect the changes in the inhibition rate of K562 and A549 cells, to draw the aging and dose effect curve, to carry out statistical analysis, and to evaluate the effect of TPA on the anti tumor effect of DOX.2 TPA on DOX induced myocardium The effect of cell injury on H9C2 cell culture and inhibition rate detection, LDH activity in cell culture fluid, the effect of TPA on DOX induced myocardial H9C2 cell damage and the effect of.3 TPA on DOX induced mouse toxicity; 3.1 acute toxicity test in acute toxicity test of TPA on mice caused by DOX in mice gave mice a one-time large dose of tail vein injection. DOX 22mg/kg, five days later, different doses of TPA were given to observe the effect of TPA on the mortality and survival of mice induced by DOX. The effect of.3.2 TPA on the dose restrictive toxicity of DOX was given to the tail vein of the mice, and the mice were injected with DOX 3mg/kg/week for 12 weeks. The model of chronic myocardial injury was established, and TPA 25 mu g/kg was given three times a week at the same time. The changes of myocardial enzyme level and liver injury related indexes when DOX reached different cumulative doses, and the pathological changes of heart were compared in each group, and the effect of TPA on DOX dose restrictive toxicity was evaluated. The effects of TPA on the inhibition rate and side effects of TPA on DOX in.4 bearing mice were transplanted to BALB/c axillary cells in S180 sarcoma cells. After the formation of the tumor, TPA and DOX were given to observe whether TPA could improve the tumor suppressor rate of DOX and weaken its toxic reaction. Method 5 statistical methods were used to analyze the data with SPSS17.0 statistical software. The animal life cycle was statistically analyzed by Graph Pad Prism 5. Single factor analysis of variance or independent sample t test was used. The test level P was 0.05. results 1 TPA to DOX in vitro synergistic effect of DOX on the inhibition of K562 cells and A549 cells showed time and concentration dependence, IC50 respectively 0.88 mol/L and 3.42 micron mol/L.TPA inhibition of K562 cell growth IC50 It is obvious that high concentration of TPA has obvious inhibitory effect on A549,.1.62 nmol/L TPA can significantly enhance the inhibitory effect of DOX on K562 and A549, and reduce the IC50 of DOX inhibition K562 cells to 0.42 mu mol/L. C2 cell damage TPA can weaken the proliferation inhibition and damage of H9C2 cells induced by DOX. 1.62 nmol/L TPA can increase the IC50 from H9C2 cells to 15.099 mu mol/L, which is about 2.5 times higher than that of 15.099 micron. 1) the effect of.3 TPA on the toxicity of DOX in mice 3.1 acute toxicity test TPA can significantly reduce the mortality of mice caused by large dose DOX, prolonging the survival time of the mice by.TPA 25 mu g/kg to reduce the mortality from 100% to 22.2%, and the average survival days from 9.64 + 6.72 days to 16.56 + 3.97 days, and there is a statistical difference (P0.01).3.2 The effect of TPA on the dose limiting toxicity of DOX was increased in the DOX induced chronic myocardial injury model, the activity of serum creatine kinase isoenzyme (Creatine kinase MB, CK-MB) increased with the increase of the cumulative dose of DOX, and TPA significantly reduced the CK-MB activity induced by DOX. The pathological results of the myocardium showed that the TPA treatment group was obviously antagonistic to the result. Focal hemorrhage, nuclear dissolving and cardiac myocyte necrosis. The mortality of DOX induced animals was reduced from 52% to 16% in TPA treatment group, and the average survival time was prolonged from 76.56 + 22.63 days to 94.16 + 11.56 days, and there was statistically significant difference (P0.01) in.4 bearing mice with the effect of TPA on the tumor suppressor rate and side effects of DOX in mice. TPA has a certain inhibitory effect on the tumor, and TPA can significantly increase the anti tumor effect of DOX, increase the inhibitory rate of DOX from 78.30% to 89.62%, and there is a statistical difference (P0.05). In addition, TPA can obviously improve the serum level of CK-MB in the mice caused by DOX and the tumor itself, and improve the cardiac function. Conclusion 1.TPA can be from in vitro association. The inhibition of proliferation of DOX tumor cells and the impairment of.2.TPA induced cardiomyocyte injury by DOX can increase the inhibitory effect of DOX on mice and reduce its myocardial toxicity.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965

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