| 1. Preparation of the oncolytic adenovirus AdhafE1â–³55 selectively killing hepatocarcinoma cells in vitroThe occurrence and progression of malignant tumors is involved in the accumulation of multiple genes, so the effect of gene therapy will be no use if only be relied on the recovery of one or several tumor suppressor genes. One new strategy for treatment of malignant tumors was recently developed by using replication- selective adenovirus (or called selectively oncolytic adenovirus).About 30%-40% of hepatocellular carcinoma (HCC) cells don't secret AFP, but the AFP promoter in these cells still have a trace of activity. And HCC frequently occurs in patients with liver cirrhosis and the prognosis of patients is influenced by the hepatic reserve( the liver regeneration resulted from hepatic stem cells ). Hence the selectivity of antitumor effects for gene therapy is vital for HCC, especially not to hurt the hepatic stem cell.A hybrid promoter, HREAF, in which a 0.4-kb fragment of human vascular endothelial growth factor (VEGF) 5'-flanking sequences containing hypoxia-responsive element (HRE) was fused to the human AFP core promoter AF0.3,was constructed. It can mediate hypoxia-inducible transcriptions of the herpes simplex virus-thymidine kinase (HSV-tk) which was controlled by it in a retroviral vector in high-,low- and non-AFP-producing human HCC cells, but not in non-HCC cells. And HREAF also will prevent the expression of therapeutic genes in hepatic stem cells that also express AFP. So this hybrid promoter HREAF was introduced into the construction of the recombinant oncolytic adenovirus Adhaf El â–³ 55 to direct its selectively replication in HCC cells.One of the characteristic of the selectively oncolytic adenovirus is that if it replicates in this tumor cell and it will only lyse this cell. The promoter HREAF as controller can direct the replication of the oncolytic adenovirus to lyse the HCC cells, and at least to prevent theoretically the damage of the hepatic reserve. Because the regeneration of liver is in order and proper and supplied with sufficient oxygen, it differs from the VEGF expression stimulated not only by hypoxia but also by various cytokines and growth factors in the tissues of hepatitis or cirrhotic liver. And it also differs from the VEGF expression stimulated mainly by hypoxia which is resulted from the excessive growth and unorderly angiogenesis in solid tumor.Basis on the conception above, we constructed HCC selectively oncolytic adenovirus AdhafEl A55 and assess preliminarily its selectively killing ability in HCC cells in vitro. The main results of this part are as follows:1) Cloned the enhancer of VEGF gene, HRE, and the core promoter AFP, AF0.3, from HCC cell genome DNA, then the hybrid promoter HREAF was recombined and verified by sequencing. Transfected the plasmid pShuttle-HREAF-El into HCC cells HepG2, Bel7402, SMMC7721 and lung cancer cells PLA801C,the expression of AdEla gene controlled by HREAF was detectable by RT-PCR in HepG2, Bel7402, SMMC7721 but not in PLA801C after 24h culture under hypoxia-inducible condition, and verified the expression specifity of genes controlled by HREAF in human HCC cells. In this study, plasmids pGEM-HRE, pGEM-AF0.3, pGEM-HREAF and pShuttle-HREAF-El were constructed.2) Cloned adenovirus Adhaf El fragment that has been completely deleted El a 5' transcriptional regulation sequence, constructed the plasmids pGEM-AdEl, pAC-AdE1, pE1-IRES1neo, and identified the El function for starting adenovirus replication. At first, human lung cancer PLA801D cells was transfected with pAC-AdEl mediated by Lipofectamine, then infected with Ad-LacZ, a replication-defective adenovirus. The results suggest that the activity of p-galactosidase in cells transfected with pAC-AdE1 preliminarily proving El function for starting adenovirus replication is significantly higher than the negative contrast cells with pAC-GFP. Following it, subcloned El to thepIRESl.neo to constructed a co-expression vector pAdE1-IRES1.neo. Then pA...
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