Targeted Therapy Of Endothelial Progenitor Cells With PTEN Gene Transfection On Liver Cancer In Nude Mice

Objective:To analyse the segregation and cultivation methods of EPCs which derive from the marrow of mice; to contrast the biological activities of early EPCs with later ones so that a solid experimental foundation can be laid for future research.Methods:Mononuclear cells were segregated from the marrow of mice through density gradient centrifugation and then cultivated on human fibronectin coated plates. After that, these cells growed under the stimulus of vacular endothelial growth factors and recombinant human epithelial growth factors were purified the early and late EPCs. Cell structures of the early and late EPCs as well as the uptake rates of FITC-UEA-I,DiI-ac-LDL were compared respectively. Later the expression of common cell surface markers was detected by means of flow cytometry; the differences of the cell proliferation between two groups were measured through MTT; the differences of cell invasiveness between two groups were detected using Transwell and the differences in the ability to form new vessels between two groups were also compared through the experiment of tube formation assay in vitro.Results:EPCs, both the early and late ones, were successfully separated from the marrow of mice. The growth of EPCs during the early stage beared the characteristic of colony formation while during the terminal stage EPCs growed like paving stones. EPCs, both in the early and late ones could intake FITC-UEA-I and Dil-ac-LDL and there was no remarkable difference in the double positive chromosomal cells. During the advanced stage, a higher expression of VEGF-2in EPCs was displayed (p<0.05) while the expression of CD133in early EPCs was higher (p<0.05). Late EPCs exhibited a greater ability to proliferate and to form new vessels (p<0.05) while early EPCs show a greater ability to transfer (p<0.05).Conclusion:It is a workable method with obvious advantages to separate from mice marrow EPCs, which take on different biological features during the early and late stages. EPCs of different stages can be selected in accordance with the research objectives, thus providing a theoretical basis for further research and future experiments. Objective:To find out the distribution of fluorescence-labeled EPCs within the transplanted tumour and other vital internal organs of nude mice.Methods:First we established the implanted subcutaneous hepatic carcinoma tumors in nude mice by means of HepG2before injecting EPCs labeled with4'6-diamidino-2-phenylindole, DAPI through their tail veins. Then the distributions of EPCs labeled with different fluorescence within the transplanted tumors and other important organs were observed. Meanwhile, the exhibitions of CD133, HIF-la, SDF-1and VEGF within transplanted tumors and other important organs during various periods were detected using RT-PCR.Results:The distribution of EPCs labeled with DAPI within the subcutaneous hepatic carcinoma tumors of nude mice was much greater than that in other important organs (p<0.05) and was next to little in the liver. The exhibitions of CD133, HIF-la, SDF-1and VEGF were obviously higher than that in other important internal organs. Conclusion:It has been proved in our experiment that EPCs do possess targeted chemotaxis towards liver cancer, thus having great potential to be the the carrier for the targeted therapy of tumors. Objective:To discover the apoptosis and the inhibiting function of EPCs containing common PTEN towards the growth of subcutaneous transplanted tumors of nude mice; to explore the feasibility of exploiting EPCs as the carrier for the targeted therapy of liver cancerMethods:Liposome assay was used to transfer plasmids containing wild PTEN into EPCs and HUVEC, which were injected into the nude mice via tail veins. Meanwhile, the nude mice were injected with EPCs as the control group. The volumes and weight changes were observed, tumor-inhibiting rates calculated respectively. Later, the expressions of PTEN, Bad and Bcl-2were measured in the tumors of either group respectively by means of Western. Besides, Tunel was exploited to detect the apoptosis rate of cells in tumour tissue.Results:The tumor inhibition rate of EPCs injected with gWT-PTEN/pGZ21dxZ was56.85%, which was apparently higher than the other groups (p<0.05). The expression of PTEN and Bad in the PTEN-EPCs group was much higher than the other groups while the expression of Bcl-2was remarkably lower than the other groups (p<0.05). Detected by means of Tunel, the apoptosis rate of cells in the PTEN-EPCs group was19.42±4.12%, evidently higher than the other groups (p<0.05). Conclusion:EPCs can be used as the carrier for the targeted therapy of liver cancer and display desirable tumor-inhibiting effects through the exploitation of PTEN, thus providing prospects for the treatment of liver cancer.