Experiment On Relationship Between Drug-resistance Of Human Liver Carcinoma Cell And Aldose Reductase Like-1

Objective: After transfected by aldose reductase like-1 gene(ARL-l), We established human heptocellular carcinoma cell Hne(HepG2/ARL-l) which expressed ARL-1 highly and stably. The effect of ARL-1 on cell growth and apoptosis induced by anticancer drug consisting of active aldehyde group were observed. To get the different gene expression profiles between the transfected cell line and the control cell line. To study the relationship between ARL-1 and drug resistance of human heptocellular carcinoma cell in molecular level, which supplied the theoretical basis for us to chose drug and entact treatment plan in clinical.Method: Eukaryotic expression vectors containing ARL-1 gene cDNA were transfected into human hetptocellular carcinoma cell Hne(HepG2) mediated by lipofectAMINE, the transfected cell was screened by G418. The study group(HepG2/ARL-l) Which expressed ARL-1 highly and stably, the control group was non-transfected cell(HepG2). The level of mRNA and protein in study group were estimated by reverse transcription polymerase chain reaction(RT-PCR), flow cytometric analysis(FCS) and immunohistochemistry individually. The effect of ARL-1 on cell growth was measured by growth curve. E-ADM and MMC were experimental drug consisting of active aldehyde group. 5-Fu which does not consist of active aldehyde group was positive control. The level of L-lactate dehydrogenase(LDH) was detected to examine cytotoxicity induced by anticancer drug, MTT assay was used to detect cell viability. The rate of apoptostic cell was estimated by flow cytometric analysis(FCS). DNA fragment of apoptosis cell were identified with agarose gel elecctrophoretogram. The cDNA chip of human heptocellular carcinoma consisting of 1629 genes was used to detected the different expression profiles between two groups. The mRNA of multidrug resistance and ubiqutin were analysed by RT-PCR to verify the result of cDNA chips.Result The mRNA level of ARL-1 and the protein level of ARL-1 in study group were higher than that in control group, it verified that ARL-1 was transfected into HepG2 successfully. Growth curve showed that ARL-1 had no effect on cell growth. Cytotoxicity induced by E-ADM and MMC in study group was much lowerthan that in control group, Cytotoxicity induced by 5-Fu in both groups were not different sinificantly. Cell viability affected by E-ADM and MMC in study group was much higher than that in control group, Cell viability affected by 5-Fu in both groups were not different significantly. The rate of apoptostic cell induced by E-ADM and MMC in study group were lower significantly than that in control group (P<0.05) , The rate of apoptostic cell induced by 5-Fu in both groups were not different significantly (P>0.05) . The DNA electrophoretogram in control group represented DNA ladder, the characteristic patten which was tested for DNA-cleavaged in nucleiod. Compared with control group, 8 kinds of gene expression were elevated in study group and 13 kinds of gene expression were decreased in study group, which were correlated with protein metabolism, signal pathway and drug resistant. The mRN A level of MDR1 and Ubiquitin were elevated in study group , it was consistent with the result of cDNA chips. Conclusion:1 After the HepG2 cell line was transfected by ARL-1 Successfully, the HepG2/ARL-l cell line which expressed ARL-1 highly and stably was established.2 ARL-1 gene can reduce the cytotoxicity of anti-cancer drug consisting of active aldehyde group and suppress apoptosis induced by anti-cancer drug consisting of active aldehyde group. That might be the main reason that human heptocellular carcinoma cell acquired the drug-resistance to anticancer drug consisting of active aldehyde group.3 After transfected by ARL-1, gene expression profile of HepG2 was changed.