| Background and objectiveThe inception rate of pancreatic cancer with a obvious tendency of increasing worldwide in recent years , for which the traditional therapy is mainly by operation exairesis combined with the radiotherapy and chemotherapy, accounts 1% to 5% of malignant tumor. However ,improving of operation methods, changing of strategy for radiotherapy and chemotherapy, employing of combined therapy still make satisfied results ,for death rate of patients with pancreas cancer is still with no tend to decrease and the five year survival rate is lower than 5%.With improvement of molecular biology and genetic immunology, new ideas of nosogenesis for pancreas cancer from the -aspect of gene and immune provide new ways and methods for treatment of pancreas cancer. Gene therapy for pancreas cancer is thought the new mode for tumor treatment followed by traditional therapies like operation ,radiotherapy and chemotherapy . Experts and scholars in every country are proceeding the research in the biological behaviors, finding genetic changes in pancreas tumors play a main role in the infiltration and diversion of pancreas cancers which is also a process of gradually accumulative genetic changes. Telomere is a structure at the terminal end of linear chromosome in eucaryotic cells which is composed of telomere DNA and telomere binding protein .Telomerase which is a kind of RNA-dependent DNA polymerase, is plasmonucleic acid protease made up of RNA and protein. The active telomerase is composed of a hTR and some protein ingredients. Human telomerase RNA has replication template for telomere DNA. The protein ingredients contain the hTERT and hTEP1.hTR and hTEP1 express in tissues of many health adults while hTERT which play a vital role in the process of cell canceration expresses in cells with telomerase activity in the process of immortalization . The fact that activity can be detected in most of the tumors while it express negatively in the normal body tissues, services as a cue that telomerase is a kind of tumor marker generally existing. The research in diagnosis for pancreas cancers by detecting telomerase in the pancreatic fluid advanced a lot with a finding that telomerase activity is still higher than it in chronic pancreatitis and benign tumors .Telomerase gene could be thought as one significant member of oncogenes. hTERT has 1132 amino-acid residue with a molecular weight of 127kDa.The telomerase activity which is greatly related with the expression level of hTERT gene can be regulated by many transcription factors , oncogenes and anti-oncogenes. Recent researches suggest that high expression of hTERT could inhibit cell apoptosis and cause cell immortalization by multi-differential stimulus .Blocking or down regulation of hTERT expression can cause decurtation of telomerase and damage of chromosome stability ,which inhibits cell growth ,encourages cell apoptosis and reverse the malignant phenotype of tumor cells.RNA interference is a kind of self-defense phenomenon in the eukaryotic cells which mainly takes place after genetic transcription or in the process of mRNA modifying and translation in other words with a character of specific targeting ,high performance and duration. Early RNAi experiments mostly apply siRNA of chemical synthesis. The fact that siRNA can cause degradation of corresponding RNA with specificity by RISC and continuously play a marked effect on newly produced RNA makes siRNA characterized by the merit of less requirement for medicine, high efficiency, long persistence and great specificity.This research ,by applying the gene engineering technique , insert human hTERT-siRNA sequence into psilencer4.1CMV neo so as to construct recombinant plasmid psilencer4.1CMVneo-hTERT-siRNA and study the recombinant plasmid function on growth inhibiting of human pancreatic cancer endosomaticly and exosomaticly to establish foundation for research in the clinical application of siRNA targeting hTERT. So far,there is still no report about research in the function of psilencer4.1CMVneo-hTERT-siRNA recombinant plasmid making psilencer4.1CMVneo as its carrier on anti-human pancreatic cancers. Methods1. design the hTERT-siRNA ,apply the designed siRNA targeting hTERT to BLASTTM analysis in GenBank and select the sequence; synthesize the expression template of siRNA ;cut the pSilencer4.1TM-CMV neo carrier free of loading with enzyme HindIII and BamHI in turn ;reclaim the large fragments after enzyme cutting by quick purification and reclamation kit of DNA fragments; conduct the carrier dephosphorylation after enzyme cutting; construct the expression carrier of hTERT-siRNA -siRNA; conduct the enzyme cutting and sequencing evaluating of recombinant plasmid.2. transfer the pSilencer4.1 -CMV neo-hTERT-siRNA to BxPC-3 cells and select the positive cells; detect the hTER gene expression 48h after grouping by RT-PCR; detect its effect on the cell vigor 48h afer recombinant plasmid transfection to BxPC-3 cells by MTT ; detect the cell cycle and cell apoptosis in each group after 48h by flow cytometry( group T1, group T2, group Lipofectamine, group mismatching and group cell control);detect the expression of telomerase protein in each group by western-blot after 48h.3. tumor transferring in nude mice and grouping for experiment: set the model of subcutaneously transplantation tumor from human primary pancreatic cancer in nude mice by subcutaneously transferring 0.2ml in the back of nude mice and divide the nude mice bearing cancer randomly into four groups when the tumor diameter grow to 5mm (1)tumor control group (2) no-load carrier group (group of no-load pSilencer4.1-CMVneo plasmid ):directly inject un-load pSilencer4.1-CMVneo plasmid into the tumor on the day of grouping (20ug/0.1ml each mice) and then inject once every ten days for three times.(3)group T1 (group of psilencer4.1-CMVneo-hTERT1-siRNA recombinant plasmid): directly inject psilencer4.1-CMVneo-hTERT2-siRNA recombinant plasmid in the tumor on the day of grouping (20ug/0.1ml each mice ) and then inject once every ten days for three times. After group handling of nude mice bearing cancer for 30 days ,measure the size of tumor .evaluate the gross tumor volume and then weigh the tumor weight with electronic balance after executation by neck dislocation . Observe the subcutaneous tumor histology character in each nude mice group by light microscope with HE stain and the pathomorphism changes in the heart ,liver, lien, lung kidney and pancreatic tissue or whether these organs have metastatic lesion .Observe the fine structure changes in the tumor of each group by electronic microscope. Detect the expression of hTERT ,Bcl-2 and BaxmRNA in the tumor of each group by RT-PCR. Detect the expression of Bcl-2,Bax, p53, VEGF, telomerase protein in tumor of each group by immunohistochemistry. Detect the expression of telomerase protein in tumors of each group by Western-Blot.Results1. The synthetic T1,T2 which are two pairs of oligonucleotide single strands form double strands by renaturation .Conduct electrophoresis in 2% agarose gel. The result shows the two fragments both have a length of 55bps and display the clear straps in the agarose gel electropherogram .Identify the recombinant plasmid initially by agarose gel electrophoresis: In accord with the experiment prediction, recombinant plasmid takes on the positive strap at the expected site in the agarose gel electropherogram, which proves the initial identification is true. The result of sequencing shows that the expression template of siRNA has been successfully constructed on the pSilence4.1CMV carrier and the sequence is completely correct.2. The result by agarose gel electrophoresis shows : the hTERT-mRNA expression of BxPC-3 in group of recombinant pSilencer4.1-CMV neo-hTERT- siRNA T1 and T2 has significantly declined compared with the expression in group Lp, Nc and c .No obvious strap displays in the group GAPDH. The result by MMT shows T1 and T2 have an obvious effect on growth inhibiting of BxPC-3 while group Lipofectamine, negative control group and the cell control group not on the whole. Compared with the group Lipofectamine negative control group and the cell control group ,the difference in group T1 and T2 is obvious and has statistical significance(P<0.05).The difference is not obvious between group T1 and T2(P>0.05).The difference by group comparison among the three groups is not significant (P >0.05).The result by flow cytometry shows :the fact of the BxPC-3 cell strain in group T1 and T2 is that the cell ratio at the stage of G0/G1 increases, the cell ratio at stage of S and G2/M decreases and the cell proliferation vigor has obviously declined. The cell proliferation vigor has no obvious changes in group Lipofectamine ,cell control group and mismatch group. The apoptosis ratio of BxPC-3 in group Lipofectamine ,cell control group and mismatch group is comparatively low while the ratio in group T1 and T2 has obviously increased. The fact of cells in each group by Western-Blot is that reference GAPDH is a strap uniformly developed while telomerase protein was a developed strap with different densities. The telomerase protein expression of BxPC-3 in group Lipofectamine, cell control group and mismatch group is comparatively high while the expression in group T1 and T2 has obviously decreased.3. After group handling of nude mice bearing cancer in each group for 30 days, the survival rate of nude mice is 100%.Observe by naked eyes after executing all the nude mice : the body surfac and celiac lymph nodes have on diversion ,neither have the abdominal membrane ,heart, liver ,lien Jung ,kidney and pancreatic tissue .There is still no abdominal dropsy. The character of tumor control group and no-load group by light microscope with HE stain that the tumors grow in state of sublobe nodosity with comparatively tenacious texture and integrated envelope is basically same to character of initial cell line .The fact of the transplantation tumors in group T1 and T2 by HE stain is that tumor cell density decreases, the cells shrinks, connective tissues in interstitial substance increases causing slight changes of fibrosis and parts of the cancer nest have lamellar cell necrosis with different degree. Some sporadic necrosis cancer nests have no cell structures. The pathomorphism changes of the organs above in the four nude mice groups by HE stain are basically normal Morphologic changes. 30 days after the group handling , the difference in the volume and weight of the transplantation in each nude mice group is comparatively obvious and difference in group T1 and T2 is obvious compared with tumor control group and no-load carrier group (P <0.01).The difference is not obvious between group T1 and T2 , (P >0.05) neither is the difference between tumor control group and no-load group(P>0.05).The tumor ultramicrostructure in each nude mice group: the fact of BxPC-3 cell in tumor control group and no-load carrier group is that microvillus-like evection is many on the cell surface, the cell nucleus is not regular but large , pseudo-inclusion body locates intranuclear ,The nucleoli have enlarged obviously with irregular shape , nucleoli sticks to the caryotheca tightly and euchromatin is in great abundance, cell organs is comparatively less , microfilament-like substance exists in some cells, chondriosome are not many, crista mitochondriales are not in large number and regular arrangement, rough endoplasmic reticulum can be seen with a low number, microfilament-like substances exist in some cells ,phenomena of caryocinesia is common and cell nuclear-cytoplasmic ratio is large which are themorphologic characteristics of typical malignant tumors. Fine structure of BxPC-3 in group T1 and T2 have obvious changes that microvillus on the cell surface have reduced or disappeared ,some of the chondrosom have swelled , cytoplasm densities have increased at the same time , nuclear chromatin concentrates to two or some large boluses gathering at the edge of nuclear envelope followed by nucleus breaking into fragments and apoptotic body can visible.30 days after grouping handling , the difference in the mRNA expression of hTERT, Bcl-2 ,Bax in group T1 and T2 is obvious compared with group tumor control group and un-load carrier group(P <0.01). The difference between group T1 and T2 is not obvious (P >0.05),neither is the difference between tumor control group and no-load carrier group (P >0.05). The difference in protein expression of Bcl-2, Bax, Telomerase , p53,VEGF in group T1 and T2 is obvious compared with tumor control group and no-load carrier group(P <0.01).The difference between T1 and T2 is not obvious(P>0.05) , neither is the difference between tumor control group and no-load carrier group (P >0.05). The result by Western-Blot shows after the function of pSilencer4.1-CMVneo-hTERT-siRNA recombinant plasmid the density of Telomerase protein expression strap in group T1 and T2 has obviously declined compared with the tumor control group and no-load carrier group and the difference in protein expression is comparatively significant while the difference neither between group T1 and group T2 nor between tumor control group and no-load carrier group is obvious.Conclusions1. The sequence design of siRNA targeting hTERT gene is reasonable and successfully produces two fragments hTERT1-siRNA and hTERT2-siRNA both of which have a length of 55bps.2. Result from enzyme cutting and sequence detecting proves expression templates of hTERT1-siRNA and hTERT2-siRNA have been successfully constructed in pSilence4.1CMV neo carrier ,which produces two recombinant plasmids Silencer4.1-CMV neo-hTERT1-siRNA and Silencer4.1-CMV neo -hTERT2-siRNA with correct sequence.3. Recombinant plasmid Silencer4.1 -CMV neo -hTERT-siRNA can cause degradation targeting BxPC-3 pancreatic cancer cells and down regulation of hTERT mRNA and telomerase protein expression and have favorable RNAi silence effect.4. Recombinant plasmid Silencer4.1-CMV neo-hTERT-siRNA has certain anti-pancreatic cancer effect that it can obviously inhibit growth of BxPC-3 pancreatic cancer cells, block cell cycle and cause cell apoptosis.5. Recombinant plasmid Silencer4.1-CMV neo-hTERT-siRNA has internal anti-pancreatic cancer effect that it can effectively inhibit growth of subcutaneously transplantation tumor from BxPC-3 pancreatic cancer cells in nude mice and cause apoptosis of pancreatic tumor cells.6. The fact that recombinant plasmid Silencer4.1 -CMV neo -hTERT-siRNA can cause obviou down regulation of anti-apoptosis gene hTERT mRNA, Bcl-2 mRNA and protein, protein of mtp53 and telomerase expression an up regulation of mRNA and protein expression of Bax serves as a cue that its anti-pancreatic cancer effect may be played by causing apoptosis of pancreatic cancer cells.7. Recombinant plasmid Silencer4.1-CMV neo-hTERT-siRNA can obviously cause down regulation of VEGF protein expression in transplantation tumor in nude mice and then it may have inhibiting effect on tumor vascularizaiotn.8. Certain dependability between hTERT gene expression and expression of some oncogenes and anti-oncogenes requires further approvement.
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