The Effect On The Bone Xenograft Tumor Of Prostate Cancer After The Targeted Slience Of NS By Using Interference Plasmid Loaded With Tf-PEI-PEG

Objective:Through the setting of models of the bone xenograft tumor of prostate cancer and observing the effect on xenograft after the targeted slience of NS by using interference plasmid loaded with Tf-PEI-PEG by intravenous, to study the Cellular specificity of shRNA transcription driven by PSMAe/p to silence NS gene expression and the effect of gene silencing on the NS, to further evaluate the gene transfer capability and safety of the non-viral vector Tf-PEG-PEI in vivo by intravenous.Method:(1) Modify the Non-viral vector polyethylenimine (PEI) into the Tf-PEI-PEG; bacterial transformation, amplification, extraction and purification of plasmid were made on the two plasmids pPSMAe/p-UPRT, PSMAe/p-shNS-poly (A), we have the restriction enzyme digestion and sequencing to ensure that the sequence is correct.(2) Culturing the LNCap cell that express PSMA to establish nude mice bone metastasis models. According to different groups, different plasmid and carrier compounds were injected into tail intravenous once every five days, a total of5times. The first group:250μg Tf-PEG-PEI and50μg pPSMAe/p-UPRT; the second group:250μg Tf-PEG-PEI and50μg pPSMAe/p-UPRT; the third group:250μg PEI and50μg PSMAe/p-NS-silencer; the fourth group:250μg Tf-PEG-PEI; the fifth group:50μg PSMAe/p-NS-silencer.(3)To investigate the volume of tumors during therapy. Mice were killed and tumors were obtained, Immunohistochemistry and Western blot were used to detect the expression of NS in each group.(4)The heart, liver, spleen, lung and renal tissues of nude mice were obtained and were detected by HE staining to observe whether appear poisoning or not.Result:(1) The Non-viral vector were synthesized successfully, and identified by1H NMR. Bacterial transformation and amplification, to extract and purify enough two kinds of plasmids. We have the restriction enzyme digestion and sequencing to ensure that the plasmids are correct.(2) the setting of models of he bone xenograft tumor of prostate cancer was successfully. After different plasmid and carrier compounds were injected into tail intravenous, the tumor volume was calculated and the tumor growth curve was drawn. In the course of treatment, the tumor volume of the interference group which used the compound of PSMAe/p-shNS-poly(A) and Tf-PEG-PEI reduced significantly (P<0.05). There is no significant difference in the tumor volume among the other four groups (P>0.05). The tumor tissue underwent NS immunohistochemical staining and NS gene expression was detected by western blot. Comparing the interference group which used the compound of PSMAe/p-shNS-poly(A) and Tf-PEG-PEI with the other four groups, the NS staining positive rate significantly decreased (P<0.05), NS expression was decreased significantly (P<0.05). There was no difference among the rest groups in the NS staining positive rate and in the NS expression (P>0.05).(3) After the heart, liver, spleen, lung and renal tissues of nude mice were detected by HE staining, there was no evidence of poisoningConclusion:In the models of the bone xenograft tumor, PSMAe/p can drive shRNA’ expression with cellular specificity in LNCap cells which express PSMA, targeting NS gene. Tf-PEG-PEI has a high capacity of targeted gene transfer ability by intravenous, it can transfer vitro gene into the prostate cancer tissue from the nude mice bone metastasis tumor model efficiently, and is a safe, efficient RNA interference delivery systems.