Liver Stem Cells As Vector For Targeted Gene Therapy Of Hepatocellular Carcinoma.

Objective To investigate the selective tropism of liver stem cells for hepatocellular carcinoma (HCC) cells in vitro co-culture system and in vivo animal model of HCC, and the feasibility of targeted gene therapy for HCC by using hepatic stem cell as vector to deliver therapy gene. MethodsFirstly pAd-CMV-EGFP vector under the control of CMV promoter was constructed by homologous recombination in E.coil BJ 5183, and the recombinant virus was generated in HEK 293 packaging cell line. WB-F344, a kind of rat adult liver stem cells, expanded in vitro were infected with recombinant virus, and infection efficiency, expression of EGFP was assessed by fluorescent microscopy. To establish a cell line which stably, high-level expression EGFP through cloning culture, and the biological characteristics of the cell line were observed and analyzed by fluorescence microscope, immunocytochemistry and experiment of inducing differentiation. Secondly WB-F344 and rat embryonic fibroblasts from primary culture were engineered to express EGFP by recombinant adenoviral-mediated methods, and two kinds of cells marked by EGFP were established, named by WB-EGFP and REF-EGFP. After the HCC cells grew to 40-60% confluence on culture dish with a 10-mm cell-free area, a similar number of WB-EGFP and REF-EGFP were placed in the blank area respectively, without direct contact of two kinds of cells in co-culture system. Then, to observe the movement behavior of WB-EGFP or REF-EGFP in co-culture system with HCC cells constantly.Thirdly Animal model of HCC in rat was produced by replantation of HCC cells, CBRH7919, combining use of immuosuppressive drug. Hepatic stem cells marked by EGFP were injected from caudal veins of rat, and Samples of HCC, pericancerous tissue of liver, normal tissue of liver, kidney, spleen and lung were collected to observe the distribution of the hepatic stem cells in different time by fluorescence microscope, so as to learn whether the hepatic stem cells had the tropism trait for HCC cells. Expression of SDF-1 and c-kit in tissue of liver or tumors were detected by imrnunohistochemistry. Furthermore, hepatic stem cells were transplanted by four approaches, from portal vein, hepatic artery, caudal vein or pericancerous tissue of liver, and then different affections on selective migration, localization and proliferation of the hepatic stem cells in the tumor tissue were observed and analyzed. Results1. Adenovirus vector of pAd-CMV-EGFP was constructed and high titer recombinant virus Ad-EGFP were produced successfully. Adenovirus could deliver EGFP gene to hepatic adult stem cells with high efficiency (about 40-70%). Through cloning culture, a new cell line, named by WB-EGFP, was established, and which could express EGFP after 8-9 passages stably. Furthermore, WB-EGFP expressed biological markers of stem cell, and the proliferation speed, differentiation capability had not been affected.2. WB-EGFP and rat embryonic fibroblasts (REF) were established to express EGFP successfully. The results showed that WB-EGFP cells migrated to the area of HCC cells growth slowly in the co-culture system. The appearance was found not only when WB-EGFP cells were seeded into cell-free area of dish center, but also seeded into blank area at extreme edge of plate. But the trait was not observed in the co-culture system of REF-EGFP and HCC cells, most of REF-EGFP cells still localized in the initial area after 72 hours of incubation. Cell count of WB-EGFP and fibroblasts migrated to the area ofHCC cells growth had significant difference (P<0.01).3. After injected from caudal veins, liver stem cells migrated to HCC lesions.distributing around the margin of the lesion, and diffused to central area of tumors gradually. But there weren't hepatic stem cells with fluorescent marker in pericancerous tissue of liver, normal tissue of liver, kidney, spleen and lung. Cells with c-kit expression increased in HCC (PO.001) and expression of SDF-1 had no difference between HCC and normal liver tissue (P>0.05) ,so signal molecule SDF-1 and receptors on the surface of hepatic stem cell maybe not related about the mechanisms of tropism. Different approaches affected the distribution of liver stem cells, and it was beneficial to selective migration, localization and proliferation of the hepatic stem cells in the tumor tissue to transplant from portal vein (P<0.05). Liver stem cells still showed the trait of selective tropism for HCC cells, and expressed EGFP stably in vivo. ConclusionThe results demonstrate the follows: 1. Target gene could be delivered into hepatic stem cells and expressed stably by recombinant adenoviral-mediated method, and biological characteristics of stem cells could not been affected. Liver stem cells might be used as vector to deliver target gene, and the hepatic stem cells marked by EGFP showed the potential ability as a tracker on the research.2. Adult liver stem cells have the biological behavior of selective tropism for HCC cells in vitro.3. Hepatic stem cells still have the biological behavior of selective migration to HCC cells in animal model, and they could localize and proliferate in the tissue of HCC, expressing target gene stably. These suggest the use of migratory liver stem cells as a delivery vehicle for targeted gene therapy for HCC.