浙贝黄芩汤对急性髓系白血病化疗敏感性的影响

发布时间：2018-03-28 08:43

本文选题：浙贝黄芩汤　切入点：多药耐药　出处：《北京中医药大学》2016年硕士论文

【摘要】：目的1.观察比较浙贝黄芩汤水提取物、醇提取物、酸水提取物对急性髓系白血病HL60、HL60/ADR细胞增殖及化疗药物敏感性的影响。2.探讨野生型p53诱导的磷酸酶1(Wipl)在难治性急性髓系白血病患者外周血中性粒细胞及单个核细胞中的表达情况。3.观察浙贝黄芩汤醇提取物对急性髓系白血病HL60、HL60/ADR细胞凋亡的影响及其与Wipl表达的相关性。4.探讨浙贝黄芩汤醇提取物增加急性髓系白血病HL60、HL60/ADR细胞对阿霉素敏感性与Wipl表达的相关性。方法1.采用MTS法检测比较浙贝黄芩汤水提取物、醇提取物、酸水提取物对急性髓系白血病敏感细胞HL60、耐药细胞HL60/ADR的增殖抑制作用,计算各提取物的IC5。值。测定非毒剂量(IC,0)浙贝黄芩汤水提取物、醇提取物、酸水提取物联合阿霉素作用后HL60、HL60/ADR细胞阿霉素IC50的变化。2.中性粒细胞分离液分离5例难治性急性髓系白血病患者(难治组)、3例化疗敏感的急性髓系白血病患者(缓解组)、3例健康成年人(正常对照组)外周血中性粒细胞及单个核细胞,分别提取总RNA,应用Real-time PCR法检测Wip1mRNA的表达情况。3.浙贝黄芩汤醇提取物作用于HL60、HL60/ADR细胞,Real-time PCR法检测Wip1mRNA的表达变化；经Annexin/PI双染后,应用流式细胞仪检测细胞凋亡率。4.Real-time PCR法检测HL60、HL60/AD R细胞Wip1mRNA表达的差异。非毒剂量(IC,0)浙贝黄芩汤醇提取物联合阿霉素作用HL60、HL60/ADR细胞,Real-time PCR法检测Wip1mRNA表达的变化。利用Lipofectamine 3000转染试剂将Wipl高表达质粒(hWIP1-FLAG-pCMV-Neo-Bam)及对照质粒(pCMV-Neo-Bam)转染入HL60细胞,上调HL60细胞Wip1表达。MTS法检测Wipl高表达HL60细胞及转染对照质粒HL60细胞对阿霉素的敏感性。结果1.浙贝黄芩汤提取物对HL60细胞的IC50值由低到高依次为醇提取物(141.06±11.29ug/ml)、水提取物(177.28±4. OOug/ml)、酸水提取物(217.66±16.78 ug/m1),P0.05。浙贝黄芩汤水提取物、醇提取物对HL60/ADR细胞的IC50值均大于200ug/ml,浙贝黄芩汤酸水提取物对HL60/ADR细胞的IC50值大于400ug/ml,各提取物对HL60/ADR细胞的工C50值均高于HL60细胞。200ug/m1浙贝黄芩汤水提取物、醇提取物、酸水提取物对HL60/ADR细胞增殖的抑制率分别为(27.28±4.69)%、(37.67±3.43)%、(25.39±4.98)%,其中醇提取物抑制率高于水提取物及酸水提取物,P0.05；水提取物及酸提取物抑制率比较差异无统计学意义,P0.05。非毒剂量(IC10)浙贝黄芩汤水提取物、醇提取物联合阿霉素作用于HL60细胞,阿霉素的IC50值降低,P0.05,增敏倍数分别为1.60、2.00倍。非毒剂量(IC10)浙贝黄芩汤醇提取物联合阿霉素作用于HL60/ADR细胞,阿霉素的IG50值降低,P0.05,逆转倍数为1.50倍。2. Real-time PCR检测结果显示难治组外周血中性粒细胞中Wip1mRNA目对内参基因β-actin的表达量为0.0969±0.0684,缓解组外周血中性粒细胞中Wip1mRNA相对内参基因β-actin的表达量为0.0083±0.0078,正常对照组外周血中性粒细胞中Wip1mRNA相对内参基因β-actin的表达量为0.0116(0.0021,0.0118)。Wip1mRNA在难治组外周血中性粒细胞中的表达水平分别较缓解组及正常对照组增高,差异有统计学意义,P0.05；WiplmRNA在缓解组及正常对照组外周血中性粒细胞中的表达水平无统计学差异,P0.05。正常对照组、缓解组、难治组外周血.单个核细胞中Wip1mRNA的表达水平差异无统计学意义,P0.05。3.浙贝黄芩汤醇提取物作用于HL60、HL60/ADR细胞,HL60细胞早期凋亡率及晚期凋亡率增加,P0.05;HL60/ADR细胞早期凋亡率增加,P0.05,晚期凋亡率与对照组相比差异无统计学意义,P0.05。浙贝黄芩汤醇提取物作用后,HL60细胞Wip1mRNA水平下调,HL60/ADR细胞WiplmRNA表达水平无明显变化。4.WiplmRNA在HL60/ADR细胞中的表达水平低于HL60细胞。与单用阿霉素组相比,非毒剂量(IC10)浙贝黄芩汤醇提取物联合阿霉素作用后,HL60细胞WiplmRNA表达上调、HL60/ADR细胞WiplmRNA表达下调。Wipl高表达HL60细胞阿霉素的IC50值为0.35±0.09ug/m1,转染对照质粒HL60细胞阿霉素的IC50值为0.36±0.17ug/ml,两组比较差异无统计学意义,P0.05。结论1.浙贝黄芩汤水提取物、醇提取物、酸水提取物能不同程度的抑制HL60及HL60/ADR细胞增殖,以醇提取物抑制作用最强。浙贝黄芩汤水提取物、醇提取物、酸水提取物对HL60/ADR细胞的抑制作用弱于HL60细胞。非毒剂量(IC,0)浙贝黄芩汤醇提取物能有效增加HL60细胞对阿霉素的敏感性,并部分逆转HL60/ADR细胞对阿霉素的耐药性。2.Wipl可能在难治性急性髓系白血病患者外周血中性粒细胞中高表达,其在急性髓系白血病中的表达情况仍有待进一步检测研究。3.浙贝黄芩汤醇提取物能促进HL60、HL60/ADR细胞凋亡,但与Wip1的相关性不明确。4.本实验未显示Wipl高表达与HL60细胞化疗敏感性的相关性,浙贝黄芩汤醇提取物对HL60/ADR细胞的耐药逆转作用与Wipl表达未显示相关性。
[Abstract]:Objective To observe the treatment of 1. ethanol extract of Scutellaria baicalensis extract, water, acid water extract on acute myeloid leukemia HL60,.2. cell proliferation and chemosensitivity of HL60/ADR of wild type p53 induced phosphatase 1 (Wipl).3. to observe the treatment of Scutellaria Decoction extract on acute myeloid leukemia HL60 expression in refractory acute myeloid the leukemia patient peripheral blood neutrophils and mononuclear cells,.4. influence HL60/ADR cell apoptosis and the expression of Wipl on the treatment of alcohol extract of Scutellaria Decoction increased in acute myeloid leukemia HL60 cells, the correlation between HL60/ADR and Wipl expression on adriamycin sensitivity. Methods 1. using MTS assay to compare the treatment of Scutellaria Decoction extract. Acid alcohol extract, water extract on acute myeloid leukemia HL60 sensitive cells, inhibition of HL60/ADR cells proliferation, the calculation of each extract Determination of IC5. value. Non toxic dose (IC, 0) of ethanol extract of Scutellaria baicalensis extract, water treatment, acid water extract combined with adriamycin after HL60,.2. changes in HL60/ADR cells of adriamycin IC50 neutral granulocyte were isolated in 5 cases of refractory acute myeloid leukemia patients (refractory group), 3 cases of acute spinal cord chemotherapy the Department of leukemia patients (remission group) and 3 healthy adults (normal control group) peripheral blood neutrophils and mononuclear cells were extracted from the total RNA, HL60, HL60/ADR cells detected by Wip1mRNA Real-time PCR method.3. expression studies of alcohol extract of Scutellaria decoction, the Wip1mRNA expression of Real-time by PCR Annexin/PI; by double staining,.4.Real-time PCR assay HL60 application rate of flow cytometry to detect cell apoptosis, expression of HL60/AD R in Wip1mRNA cells. Non toxic dose (IC, 0) treatment for alcohol extract of Scutellaria decoction combined with adriamycin With HL60, HL60/ADR cells, Real-time PCR were detected by Wip1mRNA expression. Using Lipofectamine 3000 transfection reagent Wipl high expression plasmid (hWIP1-FLAG-pCMV-Neo-Bam) and the control plasmid (pCMV-Neo-Bam) was transfected into HL60 cells, upregulation of HL60 expression of Wip1 high expression of HL60 cells and.MTS method to detect Wipl plasmid transfection control sensitivity of HL60 cells to adriamycin. IC50 1. quality of Scutellaria Decoction extract on HL60 cell values ranged from low to high as the alcohol extract (141.06 + 11.29ug/ml), water extract (177.28 + 4. OOug/ml), acid water extract (217.66 + 16.78 ug/m1), P0.05. treatment of Scutellaria Decoction water extract, alcohol extract of IC50 HL60/ADR cells were higher than 200ug/ml, IC50 treatment the water extract of Scutellaria Decoction acid on HL60/ADR cells was greater than 400ug/ml, C50 of the extracts on HL60/ADR cells was higher than that of HL60 cells.200ug/m1 treatment of Scutellaria Decoction Alcohol extract, extract, inhibition on HL60/ADR cell proliferation rate of acid water extract respectively (27.28 + 4.69)% and (37.67 + 3.43)% and (25.39 + 4.98)%, the ethanol extract inhibition rate higher than that of water extract and acid water extract, water extract and acid extract P0.05; the inhibition rate was not statistically significant the difference of P0.05., non toxic dose (IC10) treatment of Scutellaria Decoction water extract, alcohol extract combined with adriamycin on HL60 cells, adriamycin IC50 value decreased, P0.05 sensitivity were 1.60,2.00 times. Non toxic dose (IC10) treatment of alcohol extract of Scutellaria decoction combined with adriamycin on HL60/ADR cells and adriamycin the decrease of IG50 value. P0.05,.2. Real-time reversal multiples of 1.50 PCR showed that the refractory group the expression of peripheral blood neutrophil Wip1mRNA of reference gene beta -actin was 0.0969 + 0.0684, remission group Wip1mRNA peripheral blood neutrophils The relative expression of reference gene beta -actin was 0.0083 + 0.0078, normal control group the expression of peripheral blood neutrophil Wip1mRNA in relative reference gene beta -actin was 0.0116 (0.0021,0.0118) expression levels of.Wip1mRNA in refractory group peripheral blood neutrophils respectively compared with remission group and normal control group were statistically increased the significance of differences, P0.05; WiplmRNA in the remission group and normal control group the expression level of peripheral blood neutrophils in P0.05. was no significant difference between the normal control group, the remission group and refractory group in peripheral blood. No statistically significant difference in Wip1mRNA expression in mononuclear cells, HL60 HL60/ADR cells, P0.05.3. treatment of Scutellaria Decoction effect of alcohol extract of HL60 cells, the early and late apoptosis increased P0.05; early apoptosis rate of HL60/ADR cells increased, P0.05, late apoptosis rate compared with the control group had no significant difference, P0.05. The treatment effect of alcohol extract of Scutellaria decoction, HL60 cells decreased levels of Wip1mRNA expression in HL60/ADR cells, no significant change of WiplmRNA.4.WiplmRNA expression level in HL60/ADR cells than HL60 cells. Compared with adriamycin group, non toxicity dose (IC10) treatment effect of alcohol extract of Scutellaria decoction combined with adriamycin, HL60 expression was up-regulated in WiplmRNA cells, HL60/ADR the WiplmRNA expression of.Wipl cells with high expression of HL60 cells of adriamycin IC50 was 0.35 + 0.09ug/m1, HL60 cell transfection plasmid IC50 value of doxorubicin was 0.36 + 0.17ug/ml, two groups had no statistically significant difference, P0.05. extract conclusion 1. Fritillaria Scutellaria Decoction water extract, acid, water extract can inhibit the proliferation of HL60/ADR cells and HL60 in different degree with the strongest inhibitory effect of alcohol extract, alcohol extract of Fritillaria thunbergii extract of Scutellaria baicalensis. Soup, inhibition, acid water extract on HL60/ADR cells Weak in HL60 cells. Non toxic dose (IC, 0) the treatment of alcohol extract of Scutellaria decoction can effectively increase the sensitivity of HL60 cells to adriamycin, and partially reversed HL60/ADR resistance of.2.Wipl cells to adriamycin could be highly expressed in the treatment of refractory acute myeloid leukemia patients with peripheral blood neutrophils, its expression in acute myeloid leukemia remains to be further research on detection of.3. treatment of alcohol extract of Scutellaria decoction can promote HL60 cell apoptosis, HL60/ADR and Wip1, but the correlation is not clear.4. in this experiment did not show the correlation of high expression of Wipl and HL60 cell sensitivity to chemotherapy, the treatment of alcohol extract of Scutellaria Decoction reversal effect and Wipl expression on HL60/ADR cells no relationship.

【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.71

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