Objective To construct the recombinant of hepatocellular carcinoma-targeting adenovirus containing r-Caspase-3 gene and provide the gene therapic strategy for hepatocellular carcinoma. Methods The pAdTrack-EAFP-PALB was constructed and the r-Caspase-3 gene was subcloned into the vector. The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183 cells. The candidate clone was analyzed by restriction endonuclease digestion and sequencing, and then pAdEasy-EAFP-PALB/r-Caspase-3 vector was digested with PacⅠand transfected into AD293 cells for packaging and amplifying, recombinant virus was constructed successfully. Infection titer and efficiency of recombinant virus were monitored by green fluorescent protein (GFP) expression. The expression of r-Caspase-3 in infected HepG2 cells was detected by RT-PCR and Western blot. The apoptosis of HepG2 cells was detected by SRB dyeing method. Results Shuttle vector pAdTrack-EAFP-PALB/r-Caspase-3 was correct after identification by restriction endonuclease analysis and sequencing. By PCR and PacⅠ restriction endonuclease analysis， the homologous recombinant of pAdEasy-EAFP-PALB/r-Caspase-3 was successful. The expression of GFP was observed when linearized pAdEasy-EAFP-PALB/r-Caspase-3 was transfected into AD293 cells. AD293 cells could be infected repeatedly by recombinant adenovirus. The expression of r-Caspase-3 gene on HepG2 cells was detected by RT-PCR and Western-blot methods respectively, which confirmed that the Ad-EAFP-PALB/r-Caspase-3 was constructed successfully. The specificity of Ad-EAFP-PALB/r-caspase-3 which targeting induced hepatocellular carcinoma cells was founded by SRB dyeing test. Conclusion The Recombinant of hepatocellular carcinoma-targeting adenovirus containing r-Caspase-3 gene was constructed successfully and which established the foundation of r-Caspase-3 gene therapy in future research to hepatocellular carcinoma.
【摘要】 目的 篩選人源喉癌Hep-2細胞株特異結合的短肽，作為喉癌靶向治療的載體。 方法 體外培養Hep-2細胞株作為靶細胞，人正常喉黏膜上皮細胞為吸附細胞；用噬菌體展示十二肽庫進行3輪差減篩選，隨機挑取10個噬菌體克隆進行測序；采用酶聯免疫吸附（enzyme linked immunosorbent assay，ELISA）法鑒定噬菌體與Hep-2細胞的結合活性；通過免疫熒光鑒定喉癌細胞特異性結合肽（F2）噬菌體陽性克隆與喉癌細胞結合的特異性。 結果 經過3輪篩選后，噬菌體在靶細胞Hep-2上出現明顯富集；ELISA分析鑒定顯示5個陽性克隆能與Hep-2細胞特異結合，其中F2噬菌體克隆對喉癌細胞的結合靶向性明顯高于對照細胞（Plt;0.05）； 免疫熒光顯色顯示，F2能特異性地與喉癌細胞結合。 結論 利用噬菌體展示肽庫技術，可以成功篩選到F2，其可能成為喉癌靶向治療的載體。【Abstract】 Objective To obtain the polypeptides specifically bound to laryngeal squamous cell carcinoma line (Hep-2) and use it as a potential therapeutic vector targeting laryngeal squamous cell carcinoma patients. Methods With the Hep-2 cells as the target cells and human normal laryngeal squamous epithelial cells (HNLE cells) as the absorber cells, 3 rounds of panning from a Ph.D.-12TM phage-display peptide library were carried out. Ten randomly selected phage clones were sent for sequence detection. The affinity of phage clones was detected by enzyme-linked immunosorbent assay (ELISA). The positive phage clones (F2) specifically bound to Hep-2 were identified by immunofluorescence detection. Results After 3 rounds of screening, 5 positive phage clones showed specific binding to Hep-2 cells and the affinity of positive phage clones (F2) was significantly higher than that of the control groups (Plt;0.05). The results of immunofluorescence detection indicated that F2 could be specifically bound to Hep-2. Conclusions Phage display peptide libraries technique can successfully screen the peptide specifically bound to Hep-2 cell line. Thus, it provides a potential vector for targeting therapy of laryngeal squamous cell carcinoma patients.
ObjectiveTo investigate the lymphatic targeting of 5-fluorouracil (5-FU) carbon nanoparticles in rats. Methods5-FU concentration in lymphoid tissue of rats was determined by reversed phase high performance liquid chromatography after intraperitoneal injection of 5-FU carbon nanoparticle and 5-FU ordinary form (20 mg/kg body weight). Results5-FU concentration of lymphoid tissue in the 5-FU carbon nanoparticle group was higher than that in the 5-FU ordinary form group, and could sustain a longer time. Conclusion5-FU carbon nanoparticles injection can improve the drug concentration of target lymphatic organs, also has a good lymphatic targeting
Objective To study the effects of pcDNA3/AFP/TK/Angio fusion gene targeting therapy for human primary liver cancer in nude mice implanted with SMMC-7721. Methods Human liver cancer cell line SMMC-7721 was implanted subcutaneously in nude mice to establish experiment model. Animals bearing liver cancer were randomly divided into five groups: control group， vector group， GCV (ganciclovir) group， pcDNA3/TK/Angio group; pcDNA3/AFP/TK/Angio group. Different plasmids were directly injected into tumors and GCV was intraperitoneally administrated simultaneously according to different groups. The growth of tumors was observed and the pathology was examined as well. Serum AFP level was measured by radioimmunology, the ultrastructural change of tumor cells was studied by using electron microscopy, the expressions of MVD and VEGF were respectively detected with immunohistochemistry and the cell apoptosis in situ was detected by TUNEL. Results The success rate to establish subcutaneous implanted liver cancer model in nude mice was 100%. The tumor volume, serum AFP level, VEGF and MVD expressions of pcDNA3/TK/Angio group and pcDNA3/AFP/TK/Angio group were lower than those in control group, vector group and GCV group (P＜0.05) and more apoptosis cells could be observed. While the tumor volume, serum AFP level, VEGF and MVD expressions of pcDNA3/AFP/TK/Angio group was lower than those in pcDNA3/TK/Angio group (P＜0.05); and apoptosis index was higher than that of the latter (P＜0.05).Conclusion pcDNA3/AFP/TK/Angio fusion gene inhibits the growth of tumor remarkably and becomes a promising new biological agent to treat human primary liver cancer.
ObjectiveTo construct tumor specific tubercle bacillus antigen Ag85A gene lentiviral vector driven by murine telomerase catalytic subunit promoter (PmTERT), paving the way for further research in tumor targeting immuno-gene therapy. MethodsPmTERT was amplified by PCR method, with murine genomic DNA as template. Then, transcriptional activities of PmTERT in various murine and human cell strains were studied by luciferase assay. Ag85A expression lentiviral vectors driven by cytomegalo virus (CMV) promoter and PmTERT respectively (pLVX-Ag85ACMV and pLVX-Ag85A-PmTERT) were constructed with nucleic acid cloning approach. And above recombinants were verified with DNA sequencing and Western blot. ResultsLucifease assay revealed that 331 bp PmTERT cloned in present research had transcriptional activity in murine Lewis lung cancer cells, human lung adenocarcinoma cells A549, and human esophageal cancer cells EC-109, while no transcriptional activity in murine fibroblasts NIH3T3 and human embryo fibroblasts MRC-5. Western blot revealed expression of Ag85A in pLVX-Ag85A-CMV transfected Lewis and NIH3T3 cells, pLVX-Ag85A-PmTERT transfected Lewis cells, no expression in pLVX-Ag85A-PmTERT transfected NIH3T3 cells. ConclusionPmTERT has tumor specific transcriptional activity. Ag85A gene can express selectively in tumor cells, driven by PmTERT.