【摘要】：目的:探討苦參素(Oxymatrine,OMT)對結(jié)腸癌細(xì)胞HCT-8及其對長春新堿耐藥的HCT-8/VCR細(xì)胞的增殖抑制作用及對化療藥的多藥耐藥逆轉(zhuǎn)作用及可能分子機(jī)制。方法:(1)采用四甲基偶氮唑藍(lán)法(Methyl Thiazolyl Tetrazolium,MTT)檢測HCT-8/VCR細(xì)胞對化療藥長春新堿(Vincristine,VCR)、順鉑(Cisplatin,CDDP)及5-氟尿嘧啶(5-Fluorouracil,5-Fu)的多藥耐藥性。(2)采用MTT法檢測苦參素對結(jié)腸癌細(xì)胞HCT-8及耐藥細(xì)胞HCT-8/VCR的增殖抑制作用及1mg/ml苦參素作用后對各化療藥耐藥性的變化。(3)實(shí)驗(yàn)分為HCT-8組、HCT-8/VCR組及HCT-8/VCR+苦參素組,苦參素組經(jīng)1mg/ml苦參素作用48小時(shí)后,于倒置顯微鏡下觀察各組細(xì)胞的形態(tài),流式細(xì)胞術(shù)檢測各組細(xì)胞內(nèi)Rho123的熒光強(qiáng)度,qPCR檢測ABCB1基因表達(dá),Western blot法檢測ABCB1基因編碼的P-gp蛋白表達(dá)。結(jié)果:(1)MTT結(jié)果顯示,HCT-8細(xì)胞對化療藥VCR,CDDP及5-Fu的IC50分別為25.63?3.13,12.33?2.01,2.89?0.62,耐藥細(xì)胞HCT-8/VCR為320.95?6.44,45.95?4.76,18.32?3.91,兩組相比差異均有統(tǒng)計(jì)學(xué)意義(P0.001),HCT-8/VCR細(xì)胞對VCR,CDDP及5-Fu的耐藥倍數(shù)分別為12.52,3.73,6.34,提示該細(xì)胞為多藥耐藥細(xì)胞,可用于耐藥相關(guān)實(shí)驗(yàn)。(2)苦參素可抑制耐藥細(xì)胞HCT-8/VCR及其親本細(xì)胞HCT-8的增殖,呈濃度依賴性(P0.05),選擇抑制率低于10%的1mg/ml苦參素濃度作用48h作為后續(xù)逆轉(zhuǎn)耐藥相關(guān)實(shí)驗(yàn)。(3)經(jīng)1mg/ml苦參素作用后,HCT-8/VCR細(xì)胞對化療藥VCR,CDDP及5-Fu的敏感性增加,各化療藥IC50均降低(P0.01),各化療藥耐藥性逆轉(zhuǎn)倍數(shù)分別為3.44,2.15,2.04。倒置顯微鏡下顯示苦參素作用組細(xì)胞生長速度變慢,細(xì)胞漂浮死亡增加,可見空泡,貼壁性差。qPCR結(jié)果顯示,HCT-8組及苦參素作用組ABCB1 mRNA表達(dá)均低于HCT-8-VCR組(P0.01);Western blot結(jié)果顯示,HCT-8組及苦參素作用組中P-gp蛋白表達(dá)也明顯低于HCT-8-VCR組(P0.05)。流式細(xì)胞術(shù)顯示,苦參素作用組平均熒光強(qiáng)度為(23.45±0.64)%,高于HCT-8/VCR組(8.3±2.12)%,差異有統(tǒng)計(jì)學(xué)意義(P0.05).結(jié)論:苦參素可抑制結(jié)腸癌細(xì)胞增殖并逆轉(zhuǎn)結(jié)腸癌細(xì)胞的多藥耐藥性,其機(jī)制可能與抑制ABCB1基因及其編碼的P-gp的表達(dá),提高細(xì)胞內(nèi)藥物濃度有關(guān)。
[Abstract]:Aim: to investigate the inhibitory effect of matrine (Oxymatrine,OMT) on the proliferation of colon cancer cell line HCT-8 and vincristine resistant HCT-8/VCR cell line and the reversal of multidrug resistance to chemotherapeutic agents and its possible molecular mechanism. Methods: (1) (Methyl Thiazolyl Tetrazolium,MTT was used to detect the chemotherapeutic agents of HCT-8/VCR cells, including vincristine (Vincristine,VCR), cisplatin (Cisplatin,CDDP) and 5-Fluorouracil (5-Fluorouracil). (2) MTT assay was used to detect the inhibitory effect of matrine on the proliferation of colon cancer cell line HCT-8 and drug resistant cell line HCT-8/VCR and the change of drug resistance of 1mg/ml matrine to various chemotherapeutic agents. (3) the experiment was divided into HCT-8 group, HCT-8/VCR group and HCT-8/VCR matrine group. After treated with 1mg/ml matrine for 48 hours, the morphology of cells in each group was observed under inverted microscope, and the fluorescence intensity of Rho123 in each group was detected by flow cytometry. QPCR was used to detect the expression of ABCB1 gene., Western blot method was used to detect the expression of P-gp protein encoded by ABCB1 gene. Results: (1) MTT results showed that the IC50 of HCT-8 cells to the chemotherapeutic drugs VCR,CDDP and 5-Fu were 25.63 ~ 3.1312.332.01and 2.890.62, respectively. The HCT-8/VCR of drug-resistant cells was 320.95 / 6.44 (45.95 / 4.76) and 18.32 / 3.91, respectively. There was significant difference between the two groups (P0.001), and there was a significant difference between the two groups (P0.001), and the difference between HCT-8/VCR cells and VCR, was significant (P0.001). The multiples of drug resistance of CDDP and 5-Fu were 12.52 ~ 3.73 ~ 6.34, respectively, which suggested that the cells were multidrug resistant and could be used in drug-resistance related experiments. (2) Matrine could inhibit the proliferation of HCT-8/VCR and its parent cells HCT-8. In a concentration-dependent manner (P0.05), the concentration of 1mg/ml, whose inhibitory rate was less than 10%, was selected for 48h as a subsequent reversal of drug-resistance related experiments. (3) after treated with 1mg/ml matrine, HCT-8/VCR cells were treated with the chemotherapeutic drug VCR,. The sensitivity of CDDP and 5-Fu was increased, the IC50 of each chemotherapeutic drug was decreased (P0.01), and the reversal times of drug resistance of each chemotherapeutic drug was 3.44 ~ 2.15 ~ 2.04respectively. The inverted microscope showed that the cell growth rate was slower, the cell floating death increased, the vacuole was visible, and the adhesiveness was poor in the matrine group. QPCR results showed that the growth rate of the cells in the matrine group was lower than that in the control group. The expression of ABCB1 mRNA in HCT-8 group and matrine treated group was lower than that in HCT-8-VCR group (P0.01). Western blot results showed that the expression of P-gp protein in HCT-8 group and matrine treated group was significantly lower than that in HCT-8-VCR group (P0.05). Flow cytometry showed that the average fluorescence intensity of the matrine treated group was (23.45 鹵0.64)%, higher than that of the HCT-8/VCR group (8.3 鹵2.12)%, the difference was statistically significant (P0.05). Conclusion: matrine can inhibit the proliferation of colon cancer cells and reverse the multidrug resistance of colon cancer cells. The mechanism may be related to the inhibition of the expression of ABCB1 gene and its encoded P-gp and the increase of intracellular drug concentration.
【學(xué)位授予單位】：右江民族醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.3

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