發(fā)布時間：2018-06-25 17:14

  本文選題：丹皮酚 + 雷公藤內(nèi)酯醇�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2012年博士論文

【摘要】：皮膚惡性黑素瘤(CMM)因其易轉(zhuǎn)移、對細(xì)胞毒藥物耐受等特點(diǎn)而被認(rèn)為是最具侵襲性和致死率最高的腫瘤之一。近年來該病的發(fā)病率和死亡率持續(xù)增高,但仍沒有理想的治療藥物。因此,尋求治療黑素瘤的新型藥物十分必要。雷公藤內(nèi)酯醇及丹皮酚作為具有多種藥理活性的中藥單體,已在多項(xiàng)研究中被證實(shí)具有抗腫瘤作用,但抗黑素瘤的作用尚未見報道。本研究旨在探討此兩種中藥單體在抗黑素瘤方面發(fā)揮的作用及其機(jī)制。 (1)丹皮酚和雷公藤內(nèi)酯醇對黑素瘤細(xì)胞增殖的影響 利用CCK8方法檢測了藥物作用后對黑素瘤A375和M14細(xì)胞增殖抑制率的影響,并用PI染色-流式細(xì)胞術(shù)檢測了藥物對兩種黑素瘤細(xì)胞細(xì)胞周期的影響。結(jié)果顯示,0.5mM,1mM,2mM,4mM,8mM丹皮酚作用24,48,72h均對A375細(xì)胞有抑制作用,各時間點(diǎn)的半數(shù)抑制濃度(IC50)分別約為5.19mmM,1.50mM和1.09mM；丹皮酚對M14細(xì)胞24,48和72h的半數(shù)抑制濃度(IC50)分別約為4.11mM,1.60mM和1.17mM；且可以將細(xì)胞阻滯在G2/M期。12.5nM,25nM,50nM,100nM雷公藤內(nèi)酯醇作用24,48,72h對A375細(xì)胞也均有抑制作用,其IC50分別約為84.46nM,33.00nM和8.53nM；雷公藤內(nèi)酯醇對M14細(xì)胞24、48和72h的半數(shù)抑制濃度(IC50)分別約為130.90nM,35.90nM和6.50nM；而雷公藤內(nèi)酯醇是將細(xì)胞阻滯在S期。 (2)丹皮酚和雷公藤內(nèi)酯醇對黑素瘤細(xì)胞凋亡的影響 利用Hoechst33258染色,熒光顯微鏡下觀察細(xì)胞的形態(tài),以流式細(xì)胞術(shù)檢測藥物作用下細(xì)胞的凋亡率變化。結(jié)果表明,分別以5mM丹皮酚作用24h,或30nM雷公藤內(nèi)酯醇作用48h后,黑素瘤A375和M14細(xì)胞均出現(xiàn)凋亡的形態(tài)學(xué)變化。以0,1.25,2.5,5mM丹皮酚作用24h后,A375細(xì)胞的早期凋亡率分別為3.11%±0.53%,13.74%士1.73%,25.95%±0.57%和46.44%±0.81%；M14細(xì)胞的早期凋亡率則分別為1.00%±0.08%,2.00%±0.01%,2.99%±0.29%和14.73%±0.94%,各作用組與對照組相比均有統(tǒng)計(jì)學(xué)意義(P0.05)。0nM,10nM,20nM,30nM雷公藤內(nèi)酯醇作用48h后,A375細(xì)胞的凋亡率分別為4.20%±0.09%,19.50%±0.32%,36.57%±3.85%和58.68%±2.03%；M14細(xì)胞的早期凋亡率則分別為2.92%±0.17%,20.99%±0.40%,34.28%±2.04%和63.38%-±0.71%,各作用組與對照組相比均有統(tǒng)計(jì)學(xué)意義(P0.05)。兩種化合物誘導(dǎo)凋亡作用均呈劑量依賴關(guān)系。 (3)丹皮酚和雷公藤內(nèi)酯醇對黑素瘤細(xì)胞凋亡機(jī)制的研究 在上述(1)和(2)中,觀察到A375細(xì)胞對丹皮酚和雷公藤內(nèi)酯醇的誘導(dǎo)凋亡作用較M14細(xì)胞敏感,為此在本部分實(shí)驗(yàn)中,選擇A375細(xì)胞進(jìn)行藥物誘導(dǎo)黑素瘤細(xì)胞凋亡機(jī)制的相關(guān)研究。本部分實(shí)驗(yàn)中檢測了caspase3, caspase8和caspase9活性的變化,并利用Western Blot測定p53和NF-κB及其相關(guān)蛋白水平的變化。實(shí)驗(yàn)結(jié)果顯示,1.25mM,2.5mM,5mM丹皮酚作用于黑素瘤細(xì)胞株A375細(xì)胞24h后,caspase3、caspase8及caspase9的活性均發(fā)生具有統(tǒng)計(jì)學(xué)意義的升高；p53及Bax的蛋白水平隨藥物濃度的增加而升高,而NF-κB、Bcl-2、Bcl-XL的蛋白水平隨藥物濃度增加而降低,表明丹皮酚對黑素瘤A375細(xì)胞的促凋亡作用不僅通過caspase和NF-κB通路,還通過激活p53通路誘導(dǎo)凋亡。以10nM,20nM,30nM雷公藤內(nèi)酯醇作用于黑素瘤A375細(xì)胞株48h后,caspase3和caspase9的活性隨濃度的升高而增加,而caspase8的活性則隨濃度的升高而略有下降；NF-κB、Bcl-2、 Bcl-XL的蛋白水平隨藥物濃度增加而降低,表明雷公藤內(nèi)酯醇僅通過caspase和NF-κB通路誘導(dǎo)黑素瘤A375細(xì)胞凋亡。 (4)丹皮酚和雷公藤內(nèi)酯醇對黑素瘤細(xì)胞侵襲的影響及其機(jī)制的研究 侵襲、轉(zhuǎn)移是惡性腫瘤最本質(zhì)的特性。本研究采用細(xì)胞劃痕實(shí)驗(yàn)及transwell實(shí)驗(yàn),觀察藥物對A375細(xì)胞侵襲性和遷移能力的影響,結(jié)果表明,兩種中藥單體均對黑素瘤A375和M14細(xì)胞具有抑制侵襲的作用。以1.25mM丹皮酚作用24h后,A375細(xì)胞的遷移距離由對照組的197.00±11.14縮短至46.67±5.69,M14細(xì)胞的遷移距離則由對照組的172.67±9.29縮短至33.00±6.24,藥物作用組與對照組結(jié)果比較差異均具顯著統(tǒng)計(jì)學(xué)意義(P0.01)；分別以1.25mM,2.5mM,5mmM丹皮酚作用24h后,穿過人工基底膜的A375細(xì)胞數(shù)由對照組的180.67±13.65分別減少至131.67±10.41,74.33±3.21和33.33±4.16；穿膜的M14細(xì)胞數(shù)也由對照組的306.00±7.55依次減少至253.67±14.01,150.33±9.6和55.33±5.69,各組與相應(yīng)對照組比均有明顯統(tǒng)計(jì)學(xué)差異(P0.01)。10nM雷公藤內(nèi)酯醇作用48h后,A375胞的遷移距離由對照組的286.67±17.62縮短至185.67±7.09,M14細(xì)胞的遷移距離則由對照組的415.67±19.76縮短至213.33±10.02,藥物作用組與對照組結(jié)果比較差異均具顯著統(tǒng)計(jì)學(xué)意義(P0.01)；分別以10nM,20nM,30nM雷公藤內(nèi)酯醇作用48h后,穿過人工基底膜的A375細(xì)胞數(shù)由對照組的183.00±10.82分別減少為143.67±7.77,60.67±4.04和27.33±2.52；穿膜的M14細(xì)胞數(shù)也由對照組的129.67±7.23減少至80.33±2.51,65.00±5.00和20.33±1.53,各組與相應(yīng)對照組比均有明顯統(tǒng)計(jì)學(xué)差異(P0.01)。 本研究以Western Blot、Real-time PCR檢測uPA和VEGF的基因表達(dá)和蛋白水平的變化,以探討這兩種藥物影響人黑素瘤腫瘤細(xì)胞侵襲性和遷移能力的可能的機(jī)制,結(jié)果表明,丹皮酚對uPA和VEGF的基因表達(dá)量隨濃度增加逐漸升高：0mM(對照組),1.25mM,2.5mM和5mM的結(jié)果分別為uPA：0.20±0.02,0.28±0.01,0.38±0.01和1.97±0.21；VEGF:0.59±0.06,0.79±0.04,2.27±0.02和3.61±0.21,而uPA和VEGF的蛋白水平明顯降低,0mM(對照組),1.25mM,2.5mM和5mM組的uPA表達(dá)與β-actin的灰度比值分別為1.01、0.89、0.68、和0.46,其VEGF表達(dá)與β-actin的灰度比值分別為1.13、1.00、0.92和0.52。雷公藤內(nèi)酯醇對uPA和VEGF的基因表達(dá)量隨濃度增加逐漸升高：0nM(對照組),10nM,20nM和30nM的結(jié)果分別為uPA：0.20±0.02,0.41±0.00,0.95±0.05和5.84±0.16；VEGF:0.59±0.06,0.45±0.11,0.89±0.08和1.00±0.03,而uPA和VEGF的蛋白水平明顯降低,0nM(對照組),10nM,20nM和30nM組的uPA表達(dá)與β-actin的灰度比值分別為1.01、0.81、0.44和0.15,其VEGF表達(dá)與β-actin的灰度比值分別為1.13、1.46、1.34和0.96。表明丹皮酚和雷公藤內(nèi)酯醇可能通過降低VEGF和uPA的蛋白水平而抑制黑素瘤細(xì)胞的侵襲。 結(jié)論 丹皮酚和雷公藤內(nèi)酯醇均可以抑制人黑素瘤細(xì)胞A375和M14增殖,其中丹皮酚的抑制機(jī)制可能與抑制黑素瘤細(xì)胞在G2/M周期阻止細(xì)胞有絲分裂有關(guān),而雷公藤內(nèi)酯醇的作用機(jī)制可能與抑制細(xì)胞在S期抑制細(xì)胞DNA復(fù)制有關(guān)。 丹皮酚和雷公藤內(nèi)酯醇能劑量依賴性地誘導(dǎo)人黑素瘤A375和M14細(xì)胞凋亡。其中雷公藤內(nèi)酯醇通過caspase和NF-κB通路誘導(dǎo)人黑素瘤細(xì)胞凋亡；而丹皮酚不僅通過caspase和NF-κB通路,還通過激活p53通路誘導(dǎo)黑素瘤A375細(xì)胞凋亡。 丹皮酚和雷公藤內(nèi)酯醇均可通過抑制VEGF和uPA的蛋白表達(dá)抑制黑素瘤細(xì)胞的侵襲和遷移。 丹皮酚和雷公藤內(nèi)酯醇均具有抗黑素瘤的作用,具有皮膚黑素瘤治療新藥的研發(fā)前景。
[Abstract]:Cutaneous malignant melanoma (CMM) is considered to be one of the most invasive and fatal tumors because of its easy metastasis and tolerance to cytotoxic drugs. In recent years, the incidence and mortality of the disease have continued to increase, but there is still no ideal treatment. Therefore, it is necessary to seek new drugs for the treatment of melanoma. And paeonol, a Chinese medicine monomer with a variety of pharmacological activities, has been proved to have antitumor effect in a number of studies, but the role of anti melanoma has not yet been reported. The purpose of this study is to explore the role and mechanism of these two kinds of monomers in the anti melanoma.
(1) effects of Paeonol and triptolide on the proliferation of melanoma cells
The effect of drug effect on the proliferation inhibition of melanoma A375 and M14 cells was detected by CCK8, and the effect of drugs on the cell cycle of two melanoma cells was detected by PI staining flow cytometry. The results showed that 0.5mM, 1mM, 2mM, 4mM, and 8mM paeonol had inhibitory effect on A375 cells, and half of the time points were inhibited. The concentration (IC50) was about 5.19mmM, 1.50mM and 1.09mM, and the inhibitory concentration (IC50) of Paeonol on M14 cells 24,48 and 72h (IC50) was about 4.11mM, 1.60mM and 1.17mM, respectively. M, 33.00nM and 8.53nM; the median inhibitory concentration (IC50) of triptolide to M14 cells 24,48 and 72h (IC50) was approximately 130.90nM, 35.90nM and 6.50nM, while triptolide was blocked in the S phase.
(2) effects of Paeonol and triptolide on the apoptosis of melanoma cells
Using Hoechst33258 staining, the morphology of cells was observed under the fluorescence microscope, and the apoptosis rate of cells under the action of drug was detected by flow cytometry. The results showed that the apoptosis of melanoma A375 and M14 cells was changed after the action of 5mM paeonol acting 24h or 30nM triptolide acting as 48h. The early apoptosis rates of A375 cells were 3.11% + 0.53%, 13.74% 1.73%, 25.95% + 0.57% and 46.44% + 0.81%, respectively, and the early apoptosis rates of M14 cells were 1% + 0.08%, 2% + 0.01%, 2.99% + 13.74%, respectively, and all the groups were statistically significant (P0.05).0nM, 10nM, 20nM, and 30nM After 48h, the apoptosis rates of A375 cells were 4.20% + 0.09%, 19.50% + 0.32%, 36.57% + 3.85% and 58.68% + 2.03% respectively. The early apoptosis rate of M14 cells was 2.92% + 0.17%, 20.99% + 0.40%, 34.28% + 2.04% and 63.38%- +, respectively. All the groups were statistically significant compared with the control group (P0.05). Quantity dependence.
(3) the mechanism of Paeonol and triptolide on the apoptosis of melanoma cells
In the above (1) and (2), A375 cells were observed to be sensitive to the apoptosis of Paeonol and triptolide than M14 cells. Therefore, in this part of the experiment, the mechanism of apoptosis induced by A375 cells was selected to induce the apoptosis of melanoma cells. In this part, the changes in the activity of Caspase3, caspase8 and caspase9 were detected, and the benefits were detected in this part of the experiment. The changes in the levels of p53 and NF- kappa B and their related proteins were measured by Western Blot. The experimental results showed that 1.25mM, 2.5mM, and 5mM paeonol acted on the A375 cell 24h of the melanoma cell line, Caspase3, caspase8 and its activity increased statistically, and the level of protein and protein increased with the increase of drug concentration. The protein levels of kappa B, Bcl-2, and Bcl-XL decrease with the increase of drug concentration, indicating that the apoptosis promoting effect of Paeonol on melanoma A375 cells not only passes through the caspase and NF- kappa B pathway, but also induces apoptosis by activating p53 pathway. The activity of caspase8 decreased slightly with the increase of concentration, and the protein level of NF- kappa B, Bcl-2, Bcl-XL decreased with the increase of drug concentration, indicating that triptolide induced the apoptosis of melanoma A375 cells only by caspase and NF- kappa B pathway.
(4) effects of Paeonol and triptolide on the invasion of melanoma cells and its mechanism
Invasion and metastasis are the most essential characteristics of malignant tumor. In this study, the effects of drugs on the invasiveness and migration of A375 cells were observed by cell scratch test and Transwell experiment. The results showed that all two kinds of Chinese medicine monomers had inhibitory effects on melanoma A375 and M14 cells. A375 cell migration after 1.25mM paeonol acted as 24h. The distance from 197 + 11.14 of the control group was shortened to 46.67 + 5.69, and the migration distance of M14 cells was shortened from 172.67 + 9.29 to 33 + 6.24 in the control group. The difference between the drug action group and the control group was statistically significant (P0.01). The number of A375 cells passing through the artificial basement membrane after the action of 1.25mM, 2.5mM, 5mmM with 24h was the number of A375 cells. The 180.67 + 13.65 of the control group decreased to 131.67 + 10.41,74.33 + 3.21 and 33.33 + 4.16, and the number of M14 cells in the membrane also decreased to 253.67 + 14.01150.33 + 9.6 and 55.33 + 5.69 in the control group, and the ratio of each group to the corresponding control group was significantly different (P0.01).10nM after 48h of triptolide, A375 cell The migration distance was shortened from 286.67 + 17.62 to 185.67 + 7.09 in the control group. The migration distance of M14 cells was shortened from 415.67 + 19.76 to 213.33 + 10.02 in the control group. The difference between the drug group and the control group was statistically significant (P0.01). 10nM, 20nM, 30nM triptolide acted after 48h and passed through the artificial basement membrane, respectively. The number of A375 cells decreased from 183 + 10.82 of the control group to 143.67 + 7.77,60.67 + 4.04 and 27.33 + 2.52, and the number of M14 cells in the membrane also decreased from 129.67 + 7.23 to 80.33 + 2.51,65.00 + 5 and 20.33 + 1.53 in the control group, and there were significant differences in the ratio of each group to the corresponding control group (P0.01).
In this study, Western Blot and Real-time PCR were used to detect the gene expression and protein levels of uPA and VEGF, to explore the possible mechanisms of these two drugs to affect the invasiveness and migration of human melanoma tumor cells. The results showed that the gene expression of paeonol to uPA and VEGF increased gradually with the concentration of uPA and VEGF: 0mM (control group), 1.25mM, 2.5. The results of mM and 5mM were uPA:0.20 + 0.02,0.28 + 0.01,0.38 + 0.01 and 1.97 + 0.21 respectively, VEGF:0.59 + 0.06,0.79 + 0.04,2.27 + 0.02 and 3.61 + 0.21, while the protein levels of uPA and VEGF decreased obviously, 0mM (control group). The ratio of tin to 1.13,1.00,0.92 and 0.52. increased with the concentration of uPA and VEGF, respectively: 0nM (control group), the results of 10nM, 20nM and 30nM were uPA:0.20 + 0.02,0.41 + 0.00,0.95 + 0.05 and 5.84 + 0.16, respectively. The protein levels of 0nM (control group), 0nM (control group), 10nM, 20nM and 30nM were 1.01,0.81,0.44 and 0.15, respectively. The ratio of VEGF expression to beta -actin was 1.13,1.46,1.34 and 0.96., indicating that paeonol and triptolide may pass down the level of VEGF and protein and inhibit melanoma. The invasion of the cell.
conclusion
Both paeonol and triptolide can inhibit the proliferation of A375 and M14 in human melanoma cells. The inhibition mechanism of paeonol may be related to inhibiting the mitosis of melanoma cells in the G2/M cycle, and the mechanism of triptolide may be related to the inhibition of cell DNA replication in S cells.
Paeonol and triptolide can induce apoptosis of human melanoma A375 and M14 cells in a dose-dependent manner. In which triptolide induces apoptosis of human melanoma cells through caspase and NF- kappa B pathway; and paeonol not only induces the apoptosis of melanoma A375 cells by activating the caspase and NF- kappa B pathway.
Paeonol and triptolide can inhibit the invasion and migration of melanoma cells by inhibiting the expression of VEGF and uPA protein.
Paeonol and triptolide both have the effect of anti melanoma, and have the research and development prospect of new drugs for the treatment of cutaneous melanoma.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R285.5;R739.5

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