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目的 :癌细胞膜上P gp糖蛋白的过量表达是肿瘤多药抗性的主要机制。人体内编码P gp糖蛋白的基因中仅有mdr1涉及多药抗性。本研究中设计了针对mdr1的反义核酸与阿霉素的偶联物 ,并且对其细胞毒性进行了考察。同时对偶联物对人表皮癌细胞株KB A 1内的P gp蛋白的表达也做了分子水平上的研究。方法 :使用MTT法考察偶联物对KB A 1细胞的毒性。用HPLC考察偶联物对细胞内阿霉素的积累量的影响。对于P gp蛋白表达的变化 ,主要是通过RT PCR及WesternBlot方法进行了研究。结果 :偶联物的细胞毒性比寡核苷酸高。在低剂量的偶联物 (0 .5 μmol·L-1)的作用下 ,细胞对阿霉素的敏感性提高。偶联物能有效的提高细胞内阿霉素的积累量。并且从RT PCR及WesternBlot看 ,偶联物处理后的细胞内P gp表达最少。结论 :选用合适的基团 ,对反义核酸进行结构修饰能够较好的增强反义核酸的性能。采用阿霉素作为偶联基团尽管增强了细胞毒性 ,但是在更大程度上增强了其抑制P gp蛋白的效力 ,提高了肿瘤耐药性的逆转倍数 ,具有一定的潜在应用价值。
OBJECTIVE: Overexpression of P gp glycoprotein on cancer cell membrane is the main mechanism of tumor multidrug resistance. Of the genes in humans that code for the gp glycoprotein, only mdrl is involved in multidrug resistance. In this study, conjugates of mdr1 antisense and doxorubicin were designed and their cytotoxicity was investigated. At the same time, the conjugate was also studied molecularly on the expression of P gp protein in human epidermal carcinoma cell line KB A 1. Methods: The toxicity of the conjugate to KB A 1 cells was investigated using MTT assay. The effect of the conjugate on the accumulation of intracellular doxorubicin was investigated by HPLC. The changes of P gp protein expression were mainly studied by RT PCR and Western Blot method. Results: The conjugates are more cytotoxic than oligonucleotides. The sensitivity of cells to doxorubicin increased with the low dose of conjugate (0. 5 μmol·L-1). Conjugates can effectively increase the accumulation of intracellular doxorubicin. And from RT PCR and WesternBlot see, the conjugates treated cells P gp expression is the least. Conclusion: The structure of antisense oligonucleotides can be used to enhance the performance of antisense oligonucleotides. The use of doxorubicin as a coupling group enhances the cytotoxicity, but enhances its potency of inhibiting P gp protein to a greater extent and increases the reversal multiple of tumor resistance, which has a certain potential value.