| Choriocarcinoma is a malignant trophoblastic tumor, a long-term clinical treatment with chemotherapy, the cure rate of up to 80% -90%, but the occurrence of multidrug resistance and tumor recurrence is still the leading cause of death in patients with choriocarcinoma. Therefore, to effectively control the occurrence of multi-drug resistance and relapse has become the key to the treatment of choriocarcinoma. At present, the development of drug resistance reversal agents toxicity limit its clinical application, because the tumor cells and the complexity of resistance mechanisms, making only inhibition, blocking a single mechanism is not sufficient to eliminate drug-resistant MDR. Chinese medicine is rich in resources, the role of targets, and, therefore, to find a new high efficiency, low toxicity of anticancer drugs and reversal agents of Chinese medicine has become the focus of the study of tumor drug resistance. In this study, multidrug resistance based on the molecular mechanisms have, through the MTT assay Shikonin of JAR / MTX reversed the role of the resistance, Western blot method BCRP protein and P-glycoprotein expression, RT-PCR method in the BCRP mRNA level of mdr1 gene expression, flow cytometry Hochest33342 and Rh123 fluorescence value in the cell changes, and further studies in vitro reversal of Shikonin choriocarcinoma JAR / MTX resistance mechanism of cells. Objective : Discussion Shikonin resistant cells of human choriocarcinoma JAR / MTX resistance reversal effect, JAR / MTX resistance mechanism of cells, choriocarcinoma cell Shikonin reversal mechanism of drug resistance.Methods:Determined through the MTT Shikonin of JAR / MTX cells, the survival rate of≥80% of the concentration of drug resistance reversal of the concentration of C, MTT assay MTX, Shikonin of JAR, JAR / MTX cells, half lethal dose of IC50, MTT assay MTX to JAR / MTX + Shikonin (concentration C) half lethal dose of IC50, come to JAR / MTX on JAR resistance index, JAR / MTX + shikonin (C) of the JAR and the resistance index for the resistance Shikonin multiples. Experimental Packet:①JAR;②JAR + Shikonin;③JAR / MTX;④JAR / MTX + MTX;⑤JAR / MTX + Shikonin;⑥JAR / MTX + MTX + Shikonin. Shikonin MTT law concentration drawn from the concentration of resistance reversal Shikonin C, MTX for 2ug/ml. Western blot and RT-PCR method was used to observe BCRP, P-gp in the six-cell protein levels and mRNA expression levels. Experimental Packet:①JAR;②JAR + Shikonin;③JAR / MTX;④JAR / MTX + shikonin, shikonin concentration MTT method derived from the concentration of resistance reversal Shikonin C, MTX for 2ug/ml. 4 groups were detected by flow cytometry, respectively, by adding cells in Hochest33342 and PI double staining with Rh123 intracellular fluorescence value after the change in intensity. Results:1. Shikonin of JAR / MTX reversed the role of drugsMTT obtained JAR cells MTX, half lethal dose Shikonin IC50, respectively MTX IC50JAR≈1.3ug/ml, Shikonin IC50 JAR≈2ug/ml; MTT obtained JAR / MTX cells MTX, Shikonin Su-half lethal dose IC50, respectively MTXIC50JAR/MTX≈9.5ug/ml, Shikonin IC50 JAR / MTX≈2ug/ml; come through MTT method 0.2ug/ml Shikonin for cell survival when≥80%, that the low dose of Chinese medicine with or without cytotoxic cytotoxicity, defined as the concentration of drug resistance reversal; MTT obtained JAR / MTX + Shikonin 0.2ug/ml half lethal dose MTX cells IC50, for MTXIC50JAR/MTX + Purple luteolin≈1.8ug/ml; JAR / MTX resistance of JAR index for the 7.3, JAR / MTX + Shikonin (0.2ug/ml) of JAR resistance index for the 1.4, the resistance Shikonin multiples about 5.3.2. Western blot method of protein levels observed in shikonin, MTX and combined the two drugs when BCPR and breast cancer resistance protein P-glycoprotein expression level of Western blot detection of six groups of cells in the protein expression level of BCRP, the groups can be seen in the Department 230KD-specific protein bands and IP3RI Office 42KD internalβ-actin protein band. Carried out on the gray strip analysis and calculation of protein bands IP3RI gray value /β-actin protein band gray value found④>③,⑥>⑤ >①>②,③⑥compared with no significant difference; Western blot detection of six groups of cells in the protein levels of P-glycoprotein expression, the six was no significant difference between groups.3.RT-PCR in the mRNA level of observation Shikonin, MTX and combined the two drugs when BCPR and breast cancer resistance protein P-glycoprotein expression level of RT-PCR detection of six groups of cells in the mRNA expression level of BCRP,④>③,⑥>⑤>①>②,③⑥compared with no significant difference; RT-PCR detection of six groups of cells in the mRNA level of P-glycoprotein expression between the six groups was no significant difference.4. 2 cells detected by flow cytometry and not in Canada Shikonin substrate when the cells change in fluorescence intensityThe use of flow cytometry to Hochest33342 and PI double staining observed with the two cells are not in Canada at the time Shikonin intracellular fluorescence intensity, whether or JAR cell JAR / MTX cells, can be so Shikonin of intracellular Hochest33342 increase in fluorescence intensity values,②>④,①>③,④and no significant difference between①; the use of flow cytometry with Rh123 and PI double staining observed with the two cells, not in Canada at the time Shikonin intracellular fluorescence intensity values, whether or JAR cell JAR / MTX cells, can be so Shikonin intracellular fluorescence intensity of RH123 increases,②>④,①>③,④and no significant difference between①.Conclusion:1.Shikonin reversible in vitro drug-resistant choriocarcinoma cells JAR / MTX resistance to MTX, Shikonin resistance of choriocarcinoma cells JAR / MTX has reversed the role of resistance.2. Drug-resistant choriocarcinoma cells JAR / MTX resistance to MTX and the BCRP protein and mRNA expression related to, MTX-resistant cells can activate the choriocarcinoma JAR / MTX activity of the BCRP protein.3. Shikonin resistance of choriocarcinoma cells JAR / MTX resistance reversal of one of the mechanisms for the reduction of BCRP protein and mRNA expression can be resumed Shikonin resistant choriocarcinoma cells JAR / MTX on MTX sensitivity.4. Drug-resistant choriocarcinoma cells JAR / MTX to MTX resistance P-glycoprotein and mdr1 eggs and changes in gene expression has nothing to do, but with the P-glycoprotein related activity increased egg.5. Shikonin resistance of choriocarcinoma cells JAR / MTX resistance reversal of one of the mechanisms for the reduced egg cell P-glycoprotein function.In short, further evidence of this experiment Shikonin resistance of choriocarcinoma cells JAR / MTX resistance reversal of role, and further reveals the reversal of its molecular mechanisms of drug resistance through inhibition of BCRP protein and mRNA expression, reduced BCRP protein and P-glycoprotein functions of eggs. By detecting clinical BCRP protein and P-glycoprotein expression in eggs to detect whether the choriocarcinoma cell resistance to MTX, in view of the beneficial effect of a variety of the human body as a novel agent to reverse the traditional Chinese medicine, may become known as the treatment of Shikonin A new drug-resistant choriocarcinoma means. Therefore, the experimental study of the treatment of patients with choriocarcinoma resistance has provided new clues.
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