| Objectives1. Recombinant plasmid vector pET (pCL-neo-Egr-1-tk) was constructed byfusing of Egr-1 (early growth response-1) promoter to the upstream of tk gene2. To transfect human hepatocellular carcinoma cell lines (SMMC-7721) with recombinant plasmid vector pET by the way of lipofectamine as a delivery system and named as SMMC/ET, after irradiated by Co60- γ rays, the tkmRNA expression in the transfected cell line and the killing effect in vitro of tk gene on the cell line (SMMC/ET) were observed when the prodrug GCV (ganciclovir) administered3. The animal model of balb/c nude mice was established by injected subcutaneously with exponentially growing transfected cell lines for further research of the killing effect on hepatocellular carcinoma in vivo by radio-inducible suicide gene after exposure to a Co60- γ raysMethods1. The plasmid pHS V-106 which containing tk gene was cut with Eco r I andPvuII, the 1.9 kb full-length tkcDNA was obtained at the same time.Plasmid pEO which containing Egr-1 promoter was cut with Xba I , complementing with klenow and cutting again with Eco r I , then the pE part was obtained, ligated pE part with tk gene by T4 DNA ligase and acquired pCL-neo-Egr-1-tk recombinants, named as pET. The control plasmid vector pCT (pCL-neo-CMV-tk) with CMVpromoter was also constructed by subcloned tk gene into pCL-neo plasmid vector which under the downstream of CMV promoter.2. Plasmid vector pET, pCT, pCL-neo were transfected respectively into SMMC-7721 cell lines with lipofectamine as a delivery system . The cloned cells had been selected with G418 and named as SMMC/ET, SMMC/CT and SMMC/CL cell lines respectively. The SMMC/ET cell was accepted irradiation by Co60- Y rays in 0, 5, 7.5, 10, 15, 20Gy dosagerespectively which 0Gy dosage act as control group.The polymerase chain reaction (PCR) and RT-PCR analysis were used to determine the tkmRNA expression in SMMC/ET cell line.3. Cells of SMMC-7721, SMMC/CL, SMMC/CT and SMMC/ET were accepted with l0Gy dosage C06? Y rays irradiation and SMMC-7721 cellline acted as control group, twelve hours after irradiation, prodrug GCV was used in the four groups and the viabilityof cells were determined by the method of MTT, for the purpose, we can research the killing effect of radio- inducible suicide gene (pET) on the hepatocellular carcinoma celllines in vitro.4. All the 24 balb/c nude mice were injected subcutaneously with exponentially growing cell lines SMMC-7721, SMMC/CL, SMMC/CT and SMMC/ET and divided into four groups. SMMC-7721 group act as control group. Each animal was injected continuously for fourteen days with GCV intraperitoneally when tumor diameter reach 0.6cm, At the same time, all the animal were accepted with l0Gy dosage Co60- Y raysirridation twice. After the therapy, killing all the animals and observing the volume and weight of tumor, furthermore, the mean tumor inhibition rate was analyzed and the pathological results of tumor was observed.Results1. The successful construction of recombinant plasmid vector pET and pCT was proved after it was cut by Eco r I and Not I .2. After irradiation in different γ-ray dosage, the tkmRNA expression of transfected cell lines (SMMC/ET) was increased markedly as compared to control group (55.2 ?7.2)%, especially in 15Gy dosage (117.2?11.1)% CP=0.000), suggesting that the Egr-1 promoter linked upstream to tkcDNAcan be activated by Co60- Y rays and enhance tk gene expression in vitro.3. After irradiation (l0Gy), the SMMC/ET and SMMC/CT cell lines can be killed by prodrug GCV significantly compare to other two groups CP=0.000). The viability of SMMC/ET group also is higher than that of SMMC/CT group (p=0.008). It indicatede that tk gene itself can kill thetransfected cell lines when prodrug GCV exist, furthermore, tk gene therapy under the control of radio-inducible promoter Egr-1 is more efficient.4. All the 24 nude mice appeared inoculating tumor successfully, there was no dif... |
