| Part I The relationship between cell ERCC1expression and the resistance.Objective:Nearly twenty years lung cancer rates are on the rise, already up to70/10.In China, the mortality of lung cancer is the biggest rise in malignant tumors.In the treatment of lung cancer, cisplatin is the most widely usedchemotherapy drugs, but because of the primary resistance and secondaryresistance being and appear, make the platinum drugs in clinical use in thecourse of treatment effect is abate, the serious influence the effect ofchemotherapy.Cisplatin is widely used in non-small cell lung cancer, testicular cancer,ovarian cancer treatment, etc. Cisplatin on tumor cells poison effect is mainlythrough the formation of platinum-DNA adduct, influence cell DNAreplication and transcription, leading to DNA damage. And in the process ofclinical chemotherapy tumor cells on the drug resistance of clinicalchemotherapy failure is the direct or indirect cause. Multidrug resistancemechanism mainly includes: membrane glycoprotein mediated; Enzymesmediated; Regulation of gene mediated apoptosis and abnormal DNA repair avariety of causes of the drug resistance. The nucleotide excise repair (NER) isthe maintain genome function integration, repair the damage caused by thecarcinogenic factors in the process of cancer and an important link. NER wayis a complicated process, involving more than30proteins and peptides,proteins can be divided into two groups: a group of action for DNA damagerecognition and removal, such as ERCC1and XPA, etc.another group forDNA helicase, such as ERCC2, ERCC3, etc. In NER, DNA damageidentification and resection for limiting step, so the way of NER ERCC1is animportant factor. In recent years the ERCC1research can be divided into two classes: on theone hand in cancer tissues ERCC1is a cut of the gene, the low expression maybe related to the occurrence of cancer related. On the other hand, ERCC1geneis considered influence lung cancer platinum chemotherapy curative effect andprognosis of related gene. The expression and cancer chemotherapy,especially and use to platinum based chemotherapy was negatively correlated.This part experiment discusses the small doses cisplatin act on human lungadenocarcinoma A549and A549/DDP cells strains, observer the ERCC1geneexpression, and reveals ERCC1playd the important role in lungadenocarcinoma platinum resistance, to provide a new thinking of the reasonabout explain the platinum drugs to lung cancer drug resistance.Methods:1. The cell sources: This study used in the A549and A549/DDP cell lineswere kinds of human lung adenocarcinoma tissues.Cell lines have certaininvasive and resistance.2. The A549and A549/DDP cell lines cultured with RPMI-1640nutrient solution, the latter in the training process need to use lowconcentration DDP (2μ g/ml) maintain its resistance.3. The experiment group and intervention: According to the requirementof experiment is divided into A549cell blank control group, A549cellcultured with20mg/L cisplatin action group, and drug resistance cell lineA549/DDP group.4. MTT assay were used to detect A549/DDP cell line’s relativeresistance index (RI).5. Application of reverse transcription PCR (RT-PCR) and Westernblotting test (Western Blot) method detected ERCC1mRNA and proteinexpression.Results:1. Using MTT absorption method to detect the cell line in490nmmeasurement absorbance value (OD), According to the formula to calculatethe two strains of the cell survival rate, using the linear weighted regression equation for cell line of median effective inhibition concentration (IC50).Results data show: A549/DDP cell line for cisplatin drug IC50was203.85±9.745μ mol/L, A549cell line for cisplatin drug IC50was20.98±3.785μ mol/L, the former relative resistance index was9.72times.2. In the gene and protein level were testing two plant cell differentERCC1expression strength, According to the preliminary experimentalresults, we choose20mg/L concentration of cisplatin as A549cellconditioning concentration, After24hours conditioned medium incubation,A549cell line ERCC1mRNA and protein level have been increased, ERCC1mRNA expression level rose from33.12%to80.57%, ERCC1protein levelrose from37.86%to71.47%, but with resistance cell line A549/DDP ERCC1of mRNA and protein expression level is still low compared.differences aresignificant (P<0.05).Conclusions:A549and A549/DDP cell treated by small dose of cisplatin canincrease cell ERCC1gene expression, and at the same time,ERCC1gene maybe involved in the formation of platinum resistance.Part II Java Brucea joint siRNA-ERCC1gene reversal resistanceObjective:In recent years, RNAi technology were applicated in cells, the animalsand the level, anti-tumor, anti-virus and so on various experimental research,showed wide prospect of clinical application. RNA interference technology isabout gene function and gene therapy of new research hot spot. RNAi issimilar to the effect of gene knockout, Compared with the traditional antisenseRNA technology, RNAi design is simple, low cost, the cycle is short. RNAispecificity, targeted, inhibition effect, in the reaction process only causedsRNA homologous mRNA degradation, and different source of other genesis not affected. RNAi also have biological efficiency, a small amount ofsiRNA fragment transfection into target cells can effectively closed targetedgene that RNA interference in the process of catalytic amplification way, its inhibition ratio used alone antisense RNA to several times higher.This part in the study were used to Java brucea fruit oil emulsion, RNAitechnology silence ERCC1and combining both observed after drug resistancein the cell line ERCC1expression change, At a cellular level ERCC1proof innon-small cell lung cancer platinum resistance process, and to search forreversal lung cancer platinum resistance effective clinical approach.Methods:1. Cell source and culture: the application of the platinum resistanceA549/DDP cell source and cultivate in this part is same as the part I of thistext.2. The experiment group and intervention: According to the differentdrugs and transfection condition, the cell is divided into0.16mg/L Javabrucea fruit oil action group, siRNA-ERCC1action group and Java bruceafruit oil joint RNA interference action group, at the same time will not do anyprocessing A549/DDP cells as blank control group.3. Application of RT-PCR and Western Blot method detecting variousintervention group A549/DDP cells ERCC1mRNA and protein expression.4. Using MTT method to calculate the multiple drug resistance. of thetreated cells.Results:1.Detect the three experimental cell groups different ERCC1expressionstrength from the gene and protein two levels.We choose the cells for furtherexperiment after all the cells cultured by Java brucea or siRNA fragmenttransfected for24hours. The results showed that blank control group, Javabrucea fruit oil action group, siRNA-ERCC1action group and Java bruceafruit oil joint RNA interference action group of ERCC1mRNA expressionstrength for:100.00%,75.56±2.34%,68.18±2.88%,17.02±3.70%. Eachgroups’ expression intensity of ERCC1protein:100.00%,80.67±1.48%,70.66±2.84%,15.18±1.53%. differences are significant (P<0.05).2. Using MTT method to detect the experimental group cell line in490nm place measurement absorbance value (OD), according to the formula of the cell line half effective inhibition concentration (IC50). Results showed thatfour groups cells’ IC50are:203.85u mol/L,186.10u mol/L,159.15u mol/L,143.33u mol/L. The resistance ratio formula calculation, we concluded thatthe cell resistance ratio of9.72times from fell to8.87times,7.59times,6.83times. differences are significant (P<0.05).Conclusions:1. Java brucea fruit oil emulsion and RNA interference technology caneffectively and significantly reduce the ERCC1gene expression, especially inthe joint application can obviously reversal A549/DDP cells to cisplatinresistance.2. ERCC1can serve as effective targets for platinum resistance reversalfor the clinical treatment of lung cancer in the future to provide more effectivesolutions. |
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