發(fā)布時間：2018-03-28 08:43

  本文選題：浙貝黃芩湯　切入點：多藥耐藥　出處：《北京中醫(yī)藥大學》2016年碩士論文

【摘要】：目的1.觀察比較浙貝黃芩湯水提取物、醇提取物、酸水提取物對急性髓系白血病HL60、HL60/ADR細胞增殖及化療藥物敏感性的影響。2.探討野生型p53誘導的磷酸酶1(Wipl)在難治性急性髓系白血病患者外周血中性粒細胞及單個核細胞中的表達情況。3.觀察浙貝黃芩湯醇提取物對急性髓系白血病HL60、HL60/ADR細胞凋亡的影響及其與Wipl表達的相關性。4.探討浙貝黃芩湯醇提取物增加急性髓系白血病HL60、HL60/ADR細胞對阿霉素敏感性與Wipl表達的相關性。方法1.采用MTS法檢測比較浙貝黃芩湯水提取物、醇提取物、酸水提取物對急性髓系白血病敏感細胞HL60、耐藥細胞HL60/ADR的增殖抑制作用,計算各提取物的IC5。值。測定非毒劑量(IC,0)浙貝黃芩湯水提取物、醇提取物、酸水提取物聯(lián)合阿霉素作用后HL60、HL60/ADR細胞阿霉素IC50的變化。2.中性粒細胞分離液分離5例難治性急性髓系白血病患者(難治組)、3例化療敏感的急性髓系白血病患者(緩解組)、3例健康成年人(正常對照組)外周血中性粒細胞及單個核細胞,分別提取總RNA,應用Real-time PCR法檢測Wip1mRNA的表達情況。3.浙貝黃芩湯醇提取物作用于HL60、HL60/ADR細胞,Real-time PCR法檢測Wip1mRNA的表達變化；經(jīng)Annexin/PI雙染后,應用流式細胞儀檢測細胞凋亡率。4.Real-time PCR法檢測HL60、HL60/AD R細胞Wip1mRNA表達的差異。非毒劑量(IC,0)浙貝黃芩湯醇提取物聯(lián)合阿霉素作用HL60、HL60/ADR細胞,Real-time PCR法檢測Wip1mRNA表達的變化。利用Lipofectamine 3000轉(zhuǎn)染試劑將Wipl高表達質(zhì)粒(hWIP1-FLAG-pCMV-Neo-Bam)及對照質(zhì)粒(pCMV-Neo-Bam)轉(zhuǎn)染入HL60細胞,上調(diào)HL60細胞Wip1表達。MTS法檢測Wipl高表達HL60細胞及轉(zhuǎn)染對照質(zhì)粒HL60細胞對阿霉素的敏感性。結(jié)果1.浙貝黃芩湯提取物對HL60細胞的IC50值由低到高依次為醇提取物(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。浙貝黃芩湯水提取物、醇提取物對HL60/ADR細胞的IC50值均大于200ug/ml,浙貝黃芩湯酸水提取物對HL60/ADR細胞的IC50值大于400ug/ml,各提取物對HL60/ADR細胞的工C50值均高于HL60細胞。200ug/m1浙貝黃芩湯水提取物、醇提取物、酸水提取物對HL60/ADR細胞增殖的抑制率分別為(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較差異無統(tǒng)計學意義,P0.05。非毒劑量(IC10)浙貝黃芩湯水提取物、醇提取物聯(lián)合阿霉素作用于HL60細胞,阿霉素的IC50值降低,P0.05,增敏倍數(shù)分別為1.60、2.00倍。非毒劑量(IC10)浙貝黃芩湯醇提取物聯(lián)合阿霉素作用于HL60/ADR細胞,阿霉素的IG50值降低,P0.05,逆轉(zhuǎn)倍數(shù)為1.50倍。2. Real-time PCR檢測結(jié)果顯示難治組外周血中性粒細胞中Wip1mRNA目對內(nèi)參基因β-actin的表達量為0.0969±0.0684,緩解組外周血中性粒細胞中Wip1mRNA相對內(nèi)參基因β-actin的表達量為0.0083±0.0078,正常對照組外周血中性粒細胞中Wip1mRNA相對內(nèi)參基因β-actin的表達量為0.0116(0.0021,0.0118)。Wip1mRNA在難治組外周血中性粒細胞中的表達水平分別較緩解組及正常對照組增高,差異有統(tǒng)計學意義,P0.05；WiplmRNA在緩解組及正常對照組外周血中性粒細胞中的表達水平無統(tǒng)計學差異,P0.05。正常對照組、緩解組、難治組外周血.單個核細胞中Wip1mRNA的表達水平差異無統(tǒng)計學意義,P0.05。3.浙貝黃芩湯醇提取物作用于HL60、HL60/ADR細胞,HL60細胞早期凋亡率及晚期凋亡率增加,P0.05;HL60/ADR細胞早期凋亡率增加,P0.05,晚期凋亡率與對照組相比差異無統(tǒng)計學意義,P0.05。浙貝黃芩湯醇提取物作用后,HL60細胞Wip1mRNA水平下調(diào),HL60/ADR細胞WiplmRNA表達水平無明顯變化。4.WiplmRNA在HL60/ADR細胞中的表達水平低于HL60細胞。與單用阿霉素組相比,非毒劑量(IC10)浙貝黃芩湯醇提取物聯(lián)合阿霉素作用后,HL60細胞WiplmRNA表達上調(diào)、HL60/ADR細胞WiplmRNA表達下調(diào)。Wipl高表達HL60細胞阿霉素的IC50值為0.35±0.09ug/m1,轉(zhuǎn)染對照質(zhì)粒HL60細胞阿霉素的IC50值為0.36±0.17ug/ml,兩組比較差異無統(tǒng)計學意義,P0.05。結(jié)論1.浙貝黃芩湯水提取物、醇提取物、酸水提取物能不同程度的抑制HL60及HL60/ADR細胞增殖,以醇提取物抑制作用最強。浙貝黃芩湯水提取物、醇提取物、酸水提取物對HL60/ADR細胞的抑制作用弱于HL60細胞。非毒劑量(IC,0)浙貝黃芩湯醇提取物能有效增加HL60細胞對阿霉素的敏感性,并部分逆轉(zhuǎn)HL60/ADR細胞對阿霉素的耐藥性。2.Wipl可能在難治性急性髓系白血病患者外周血中性粒細胞中高表達,其在急性髓系白血病中的表達情況仍有待進一步檢測研究。3.浙貝黃芩湯醇提取物能促進HL60、HL60/ADR細胞凋亡,但與Wip1的相關性不明確。4.本實驗未顯示W(wǎng)ipl高表達與HL60細胞化療敏感性的相關性,浙貝黃芩湯醇提取物對HL60/ADR細胞的耐藥逆轉(zhuǎn)作用與Wipl表達未顯示相關性。
[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.

【學位授予單位】：北京中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R733.71

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