本文選題：TPA + DOX　； 參考：《鄭州大學》2017年碩士論文

【摘要】：多柔比星(Doxorubicin,DOX)是廣譜的抗腫瘤藥,廣泛用于多種癌癥,包括乳腺癌,卵巢癌和前列腺癌。但由于劑量相關的心臟毒性和耐藥性的產(chǎn)生使其化療效果大打折扣,限制了此類藥物的長期使用。臨床建議使用DOX的累積劑量不宜超過400mg/m~2,然而即使在這些限制的劑量下,也會發(fā)生心臟毒性�；紣盒阅[瘤的兒童在使用DOX治療后的25年內(nèi),患者心臟死亡的相對危險性顯著高于正常人群,由DOX誘導的心臟損傷表現(xiàn)為多種形式,但和臨床最相關的是擴張型心肌病。因此尋找能夠克服DOX心臟毒性同時又能增加DOX抗腫瘤活性的藥物有著非常廣闊的臨床應用前景。12-氧-十四烷酰佛波醇-13-乙酸酯(12-O-teteadecanoylphorbol-13-acetate,TPA)與蛋白激酶C(PKC)具有高度親和力,通過激活PKC可產(chǎn)生多種生物學效應,如調(diào)節(jié)基因轉(zhuǎn)錄,細胞增殖,分化,存活和程序性凋亡。據(jù)文獻報道,在心肌缺血及再灌注損傷模型中,TPA激活PKC后可以調(diào)節(jié)心肌細胞膜Na+-K+-ATP酶活性,增加受損心肌細胞Hsp70的表達,同時也可以作用于線粒體呼吸鏈,調(diào)節(jié)能量代謝,從而起到保護受損心肌的作用。因此本研究預考察TPA是否可以拮抗DOX造成的心肌損傷且增加其抗腫瘤活性。1 TPA聯(lián)合DOX對白血病K562,肺癌A549細胞增殖的影響1.1細胞培養(yǎng)K562細胞用含10%胎牛血清的RM-1640培養(yǎng)基,A549細胞用含10%胎牛血清的DMEM高糖培養(yǎng)基,均在37°C,5%CO2的無菌培養(yǎng)箱中培養(yǎng),每隔三天傳代一次。1.2 MTT法檢測細胞增殖MTT法檢測藥物單用及聯(lián)合應用情況下,K562及A549細胞抑制率的變化,繪制時效及量效曲線,進行統(tǒng)計學分析,并評價TPA對DOX抗腫瘤作用的影響。2 TPA對DOX誘導的心肌H9C2細胞損傷的影響H9C2細胞培養(yǎng)及抑制率的檢測同上,測定細胞培養(yǎng)液中的LDH活性,評價TPA對DOX誘導的心肌H9C2細胞損傷的影響。3 TPA對DOX引起小鼠毒性的影響3.1急性毒性試驗中TPA對DOX所致小鼠死亡的影響給予小鼠一次性大劑量尾靜脈注射DOX 22mg/kg,五天后給予不同劑量的TPA治療,觀察TPA對DOX所致小鼠死亡率及生存期的影響。3.2 TPA對DOX劑量限制性毒性的影響給予小鼠尾靜脈注射DOX 3mg/kg/week,連續(xù)12周,建立心肌慢性損傷模型,同時每周給予TPA 25μg/kg三次予以干預。觀察小鼠生存狀況,測定DOX達到不同累積劑量時心肌酶水平及肝損傷相關指標的變化,并比較各組小鼠心臟病理差異,評價TPA對DOX劑量限制性毒性的影響。4荷瘤小鼠中TPA對DOX的抑瘤率及毒副作用的影響將S180肉瘤細胞細胞移植到BALB/c腋下,瘤體形成后給予TPA及DOX,觀察TPA是否可以提高DOX的抑瘤率及減弱其毒性反應。方法5統(tǒng)計方法采用SPSS17.0統(tǒng)計軟件對各項數(shù)據(jù)資料進行統(tǒng)計學分析。動物生存周期采用Graph Pad Prism 5.0進行統(tǒng)計分析。采用單因素方差分析或者獨立樣本t檢驗進行各組之間統(tǒng)計學檢查。檢驗水準P為0.05。結(jié)果1 TPA對DOX的體外增效作用DOX對K562細胞及A549細胞的抑制均呈現(xiàn)出時間及濃度依賴性,IC50分別為0.88μmol/L及3.42μmol/L。TPA抑制K562細胞生長的IC50為4.316nmol/L,而低濃度的TPA對A549抑制作用不明顯,高濃度的TPA對A549有明顯的抑制作用。1.62 nmol/L TPA可以顯著增強DOX對K562及A549的抑制作用,將DOX抑制K562細胞生長的IC50降低到0.42μmol/L,將抑制A549細胞的IC50降低到1.78μmol/L,與DOX單用組相比,IC50均下降了約50%。2 TPA減弱DOX誘導的心肌H9C2細胞的損傷TPA可以減弱DOX誘導的H9C2細胞的增殖抑制和損傷,1.62 nmol/L TPA可以將DOX抑制H9C2細胞生長的IC50從6.127μmol/L提高到15.099μmol/L,提高了約2.5倍。高濃度的TPA即8.1 nmol/L能顯著降低DOX誘導的H9C2細胞LDH的釋放,且存在統(tǒng)計學差異(P0.01)。3 TPA對DOX引起小鼠毒性的影響3.1急性毒性實驗TPA能夠顯著降低大劑量DOX引起的小鼠死亡率,延長小鼠的生存時間。TPA 25μg/kg將小鼠死亡率由100%降低到22.2%,并將平均生存天數(shù)由9.64±6.72天延長到16.56±3.97天,且存在統(tǒng)計學差異(P0.01)。3.2 TPA對DOX劑量限制性毒性的影響在DOX誘導的慢性心肌損傷模型中,血清肌酸激酶同工酶(Creatine kinase MB,CK-MB)活性隨著DOX累積劑量的增加而增加,TPA能明顯降低DOX誘導的CK-MB活性的升高。心肌病理結(jié)果顯示,TPA治療組明顯拮抗了DOX造成的局灶性出血,細胞核溶解及心肌細胞壞死。同時TPA治療組將DOX引起的動物死亡率由52%降低到16%,將平均生存天數(shù)由76.56±22.63天延長到94.16±11.56天,且存在統(tǒng)計學差異(P0.01)。4荷瘤小鼠中TPA對DOX的抑瘤率及毒副作用的影響各組小鼠瘤體重量顯示,TPA對腫瘤有一定的抑制作用,且TPA能明顯增加DOX的抗腫瘤作用,將DOX的抑瘤率由78.30%增加到89.62%,且存在統(tǒng)計學差異(P0.05)。此外,TPA可以明顯改善DOX及瘤體本身造成的小鼠血清CK-MB水平的升高,改善心功能。結(jié)論1.TPA可以從體外協(xié)同DOX腫瘤細胞的增殖抑制作用,減弱DOX誘導的心肌細胞的損傷。2.TPA可以增加DOX對小鼠腫瘤的抑制作用,并能減輕其心肌毒性。
[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.

【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R965

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