| More than 100,000 people died from hepatocarcinoma. one of the most common malignancies, every year in our country and the number across the world is 2.5 times of it. A lot of them accept surgery, the best way to cure it. and undergo combined therapy include of chemotherapy. But the effect of chemotherapy is far away from satisfition because hepatocarcinoma is more resistant than other solid tumors. How to make hepatocarcinoma sensitive to chemotherapy is a hot spot in the area of hepatocarcinoma therapy. Telomerase is a specialized reverse transcriptase composed of an RNA subunit (hTR) that provides the template for the telomere synthesis reaction and a rateilimiting catalytic protein subunit (hTERT). The hTR is conserved expressing in all the cells so the telomerase activity is relating to the level of hTERT expression. And many study showed that telomerase is essential to most tumor cells. And the data of our lab revealed more than 85% hepatocarcinoma with much stronger telomerase activity than cirrhosis. Based on the other surprising observation that normal hepatocelluar tissue without telomerase activity, many researchers propose that telomerase plays an important role in the initiation and development of hepatocarcinoma. More and more scientists are spurred to find a new way, using telomerase as a target, to attack hepatocarcinoma by some reasons mentioned above. A part of them find inhibition of telomerase activity via antisense nuclei. But the tumor cells go into crisis over a long period of time depending on telomere length. Clinical applicant of this method is in the air for the low efficient of antisense nuclei and delay of effect. RNAi, the new mechanic found recently, maybe the new way to achieve high efficiency and safety. Gene silencing mediated by double-stranded RNA (dsRNA) is a sequence-specific, highly conserved mechanism in eukaryotes. Only a few molecules double-stranded RNA can trigger gene with homologous sequence silence through a series of enzymes. In our study, we investigate the possibility of inhibition of hTERT by adenovirus-mediated siRNA enhancing hepatocarcinoma chemosensitivity. 1. The preparation and construction of Ad-si/hTERTAd-si/hTERT can be constructed by the means of recombination technique frompSUPER-EGFP and Ad-Easy system. A synthetic oligonucleotide coding for interfering hTERT was cloned into pSUPER-EGFP. After sequence confirmation, a fragement with shRNA expression box was released with EcoRI+Hind III and ligated into pCDNA3.1+. The fragement releseaed from pCDNA3.1+-si/hTERT with Hind III+XbaI was cloned into TRACK, named as TRACK-si/hTERT. Digesting TRACK-si/hTERT with endonuclease Pme I. the linear plasmid was transfected into Ad-BJ5183 to produce adenovirus vector. After getting the righ clone, we digest it overnight with Pac I and transfect into 293 cells to pakeage and amplify virals. We purified viral by cesium chloride density after amplified. The titer of Ad-si/hTERT was 1010 PUF/ml by UV and store at -80C.2. Ad-si/hTERT enhance hepatocarcinoma cell line SMMC-7721 chemosensitivity in vitroWe want to test the effect of Ad-si/hTERT plus cisplatin on 7721 in vitro in this part. We find the Ad-si/hTERT can highly infect 7721 cells by means of fluorescence microscopy. And we sure this adenovirus can suppress the expression of hTERT and the activity of telomerase after experiments such as RT-PCR. Western-blot and TRAP. But the cell growth curve and apoptosis rate are no significant different from the control groups. Other results from MTT and FACS show that the survival and cell cycle of 7721 were seriously effected by cisplatin by without cell apoptosis. To our surprising, when we treat 7721 with Ad-si/hTERT plus cisplatin. most of the cells apoptosis in 48 hours. Comparing of the low apoptosis rate of the control groups, we sure that Ad-si/hTERT makes 7721 cells more sensitive to cisplatin.3. Ad-si/hTERT enhance hepatocarcinoma chemosensitivity in vivoOur data from Ad-si/hTERT plus cisplatin reveal that telomerase disable makes 7721 more se... |
PDF Full Text Request