| Objectives:To isolate and clone antisense K-ras gene fragments from genomic DNA of pancreatic cancer cell lines BxPC-3 and PC-3,and to construct retroviral recombinants containing antisense K-ras gene fragments( exonl with flanking sequence & exon4B with flanking sequence ).The therapeutic effects and molecular mechanism on pancreatic cancer mediated by the retrovirus are also studied in Vivo and in Vitro. Methods: Two primers containing the digested site of BamHI and EcoRI are designed to amplify antisense K-ras genes'exonl and exon4B with their flanking sequences from genomic DNA of pancreatic cancer cell lines BxPC-3 and PC-3.The product of PCR and pLXSN were digested by BamHI & EcoRI.The gene of interest was inserted into retroviral vector pLXSN with T4 Ligase, the resistant clones were obtained selected by Amp.The recombinant were confirmed by bacterium PCR,agarose electrophoresis after double enzyme( EcoRI & BamHI ) digestion and sequence analysis. Then they were transducted into the retroviral package cell PT-67 by liposome. The resistant cell clones were harvested and enlarged screened with G418 at concentration with 200 ug / ml for 2 weeks.The supernatant were collected which contain high titer retroviral recombinant. Infected by the retrovirus, BxPC-3 and PC-3 cells proliferation and K-ras mRNA were detected by MTT assay and RT-PCR. The expreesion of p21 protein , Fas/FasL, Bax and Bcl-2 were inspection with immunohistochemistry ( SABC method ). Stained by PI and fixed with 70 %ethanol ,BxPC-3 and PC-3 cell cycle and cell apoptosis were tested by flowcytometry ( FCM ).The pLXSN-AS-exonl and pLXSN-AS-exon4B retrovirus 200μL was injected into the location 1 time / day for 3 continious days where the pancreatic cancer cell were inoculated. The weight and volume of the xenograft were analysed when nude mice were treat to death 8 weeks later. Results It is demonstrated by bacterium PCR, double enzyme digestion(EcoRI & BamHI) and seqnece analysis that the exonl,4B with their flanking sequences of antisense K-ras gene were successfully cloned into the vector pLXSN .The recombinant retrovirus concentration were detected by NIH3T3 cell.The concentrtion of pLXSN,pLXSN-AS-exonl and pLXSN-AS-exon4B retrovirus derived from PC-3 cell were 3×109 , 2×108 and 3×108 CFU/L respectively.After infected by pLXSN-AS-exonl and pLXSN-AS-exon4B retrovirus, PC-3 cells proliferation are decreased significantly through MTT assay at 1st ( F=4.716,P<0.05 ) , 2nd ( F=6.914,P<0.05 ) ,3rd ( F=15.118,P<0.05 ) ,4th ( F=16.883,P<0.05 ) ,5th (F=15.134,P<0.05 ) day.Significant deference was not shown between the group of infected by pLXSN and blank controlled group or pLXSN-AS-exonl and pLXSN-AS-exon4B group;There is no significant deference were observed in each group of BxPC-3 cells.The expression of K-ras mRNA was downregulated by RT-PCR assay apparently,but that of BxPC-3 cells is not so obvious as that of PC-3 cells. SABC procedure was used to test the expression of p21 protein of BxPC-3 and PC-3 cells after infected by retrovirus containing antisense K-ras gene fragments.The data show that the expression of p21 protein in PC-3 cells wasdownregulated obviously,but that of BxPC-3 cells has no significant defenrence. PC-3 cells apoptosis were increased significantly after infected by pLXSN-AS-exonl and pLXSN-AS-exon 4B recombinant retrovirus.( pLXSN-AS-exonl group : x2=393.157 P<0.01 ,vs not infected ; x2=448.462,P<0.01, vs pLXSN; pLXSN-AS-exon 4B group: x2=126.737,P<0.01, vs not infected; x2=161.986,P<0.01, vs pLXSN) ,and the expression of apoptosis related gene Bcl-2 was downregulated ,while the expression of Fas was upregulated,thus the ratio of Bcl-2/Bax was descent.On the contrary,FasL expression was the same as before, but no significant deference was discovered in cell line BxPC-3. We also have established the pancreatic cancer xenograft model of nude mice. 200 uL recombinant retrovirus were injected into the location 1 time each day for three continious days where the cancer cells were inoculated. Eight we... |
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