CAJ | 학술논문

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WestemBlot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 μm0l·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.
목적:암세포막상P-gp당단백적과량표체시종류다약항성적주요궤제.인체내편마P-gp당단백적기인중부유mdr1섭급다약항성.본연구중설계료침대mdr1적반의핵산여아매소적우련물,병차대기세포독성진행료고찰.동시대우련물대인표피암세포주KB-A-1내적P-gp단백적표체야주료분자수평상적연구.방법:사용MTT법고찰우련물대KB-A-1세포적독성.용HPLC고찰우련물대세포내아매소적적루량적영향.대우P-gp단백표체적변화,주요시통과RT-PCR급WestemBlot방법진행료연구.결과:우련물적세포독성비과핵감산고.재저제량적우련물(0.5 μm0l·L-1)적작용하,세포대아매소적민감성제고.우련물능유효적제고세포내아매소적적루량.병차종RT-PCR급WesternBlot간,우련물처리후적세포내P-gp표체최소.결론:선용합괄적기단,대반의핵산진행결구수식능구교호적증강반의핵산적성능.채용아매소작위우련기단진관증강료세포독성,단시재경대정도상증강료기억제P-gp단백적효력,제고료종류내약성적역전배수,구유일정적잠재응용개치.
Over-expression of P-glycoprotein(P-gp) on the surface of tumor cells that works as a membrane pump enhancing the drug efflux is regarded as a main candidate mechanism of multidrug resistance(MDR). And mdr1 is one of the two main genes that encode P-gp in human cells. In this study, a novel conjugate made of mdr 1-anti-sense oligodeoxynucleotide(ODN) and a potent anticancer drug doxorubicin was constructed. The cytotoxicity of the conjugate as well as the effect of the conjugate on the modulation of P-gp-mediated MDR in KB-A-1 cell lines was studied. The molecular mechanism of its regulation effect was also investigated. METHODS: Multidrug resistant KB-A-1 cell lines were used. MTF was applied to measure the cytotoxicity of the conjugate and doxorubicin to KB-A-1 cells.Effect of the conjugate on intracellular doxorubicin accumulation was determined by HPLC. RT-PCR and Western blot were used to determine the expression of mdr 1 gene and P-glycoprotein in KB-A-1 cells. RESULRTS: The results showed that the conjugate exhibited more cytotoxicity than ODN, and also it could promote the cytotoxicity of doxorubicin to KB-A-1 cells. The IC50 of doxorubicin dropped from 24 to 18 μg·ml-1. The results of RT-PCR and Western blot analysis illuminated that conjugate might down-regulate the P-gp expression. CONCLUSION: All the findings suggest that the conjugate is more potential for efficient reversal of MDR phenomenon than free ODN.The study indicated that such a conjugate should be a new feasible and efficient anti-sense reagent both in lab research and further in clinical therapy.