Construction Of Engineered Cells With TNF-α Releasable Micro-vesicle And Its Anti-tumor Growth Effects

Objective: Cancer has become a common disease which seriously threaten people's lives and health all over the world. Recently, some cytokines have been proved definite anti-tumor effect, such as tumor necrosis factor. However, there are several demerits that hamper the clinical application widely. The first is short biological half-time in vivo, at the same time, even with large dose administration there is low concentration in tumor area, which results in unsatisfied anti-tumor effect, as well as serious adverse side effects. Based on genetically engineered cells microencapsulated preparation platform, using pre-established methods, this study was aimed to construct engineered cells with APA microencapsulated TNF-α/293 cells. Then, in vitro, we observed the characteristics of APA microencapsulated TNF-α/293 cells and its inhibition on tumor proliferation. On the one hand, exerting the micro biological pump effects of a capsule cells continuously secreting tumor necrosis factor-α, maintaining the high concentration of cytokines in the tumor tissue, and producing the continuously tumor killing and growth inhibiting effects. On the other hand, exerting the immune rejection preventing and proliferation limiting effects of capsule cells with high growth of APA microcapsules, building foundation for further use of the microencapsulated cell transplantation with this gene.Materials and Methods: (1) In this study, we used gene clone technology to build human tumor necrosis factor-secretingαplasmid PSNAV2.0 in the eukaryotic expression vector. TNF-αpackaged efficiently expressed RAAV2-TNF-αadeno-associated virus. Using Lipofectin2000 cationic liposome method, sequencing right PSNAV2.0-hTNF-αvector, and transfected into human embryonic kidney cells, identified TNF-αprotein which was secretoried by transfected cells, using G418 resistance screening method, constructed a TNF-α/293 cell line which can stablly secreting human tumor necrosis factorαs,by RT-PCR, Western blot, ELISA and flow Cytometry techniques, detected the expression and secretion of tumor necrosis factor;(2)Observed the cultured cells and its anti-tumor growth effects in vitro, in the 0 / 293 cells group, Hek293 cells Group, TNF-α/293 cell culture medium was concentrated group of treatment, adding LOVO cells and HepG2 cell culture plates at different time point test the optical density in 490nm by MTT assay; (3) in vitro Culture, the comparison group of APA microencapsulated TNF-α/293 5 high-dose group, APA microencapsulated TNF-α/293 middle dose group, APA microencapsulated TNF-α/293 low dose group and negative group APA micro-Sacs 0 / 293 cells, positive drug added TNF-αfactor (200ng/ml), adding LOVO cells and HepG2 cells culture plate, different time point test optical density in 490nm by MTT assay.Results: (1) the use of PCR amplification method, we get the pcDNA-hTNF-αin the hTNF-αgene. The target gene was inserted into the vector PSNA2.0 (7115bp) EcoRI and SalI restriction sites between the two enzymes to construct PSNAV2.0-hTNF-αplasmid. Monoclonal hTNF-α/293 cell lines secreted human tumor necrosis factorαsustainablly after 48 hours; (2) in vitro, Hek-293 cells and 0 / 293 cells have no inhibit effect on the HepG2 cells and LOVO cells; group absorbance value of TNF-α/293 cells and TNF-αcontrol were lower than 0 / 293 cell groups and Hek-293 cell group at 24h, 48h, 72h, the difference was significant ; that TNF-α/293 LOVO cells and cell groups on the proliferation of HepG2 cells, significant inhibition; (3) APA microcapsules in vitro of 0 / 293 cells and cell groups on the HepG2 cell proliferation LOVO no inhibitory effect, P > 0.05 for the difference was not statistically significant; APA microencapsulated TNF-α/293 cells, high-dose group, and TNF-αpositive group in 24h, 48h, 72h APA microencapsulated absorbance value lower than 0 / 293 cell group , P <0.05 as significant difference, indicating TNF-α/293 cells, high-dose group and the positive control group LOVO cells and HepG2 cells was significantly inhibited.Conclusion: we constructed genetically engineered cells hTNF-α/293 successfully which secreted tumor necrosis factorαsustainablly in human embryonic kidney cell lines ; preliminary proved that when APA microencapsulated cells cocultured with and liver cancer and colon cancer tumor cells, it can produce the effect of inhibiting tumor cell proliferation, and its effect is in a dose-dependent; hTNF-α/293 cells produce the protein which diffuses out of APA microcapsules ,the protein are free to play outside of the membrane anti-tumor activity, APA microencapsulated hTNF-α/293 adjuvant treatment of tumor cells is expected to become the new technology and worth further study.