Gene Therapy Of Human Hepatocellular Carcinoma By Human Interferon-α2b Gene Modified Bone Marrow Mesenchymal Stem Cells

Part OneConstruction of recombinant over-expression plasmid containing human interferon-α2b gene and its expression in human bone marrow mesenchymal stem cells[Objective] To construct the recombinant over-expression plasmid pcDNA3.1 containing human IFN-α2b gene. To investigate the IFN-α2b gene expression in pcDNA3.1-IFN-α2b transfected human bone marrow mesenchymal stem cells (BMSC), and to investigate the property of the IFN-α2b gene modified BMSC in vitro.[Methods]According to gene bank, primer premier 5.0 was introduced to design the specific primer of huan IFN-α2b gene. Total RNA was extracted from BMSC by Trizol-A+methods from BMSC. Full-length cDNA fragments of IFN-α2b were expanded with reverse transcription PCR (RT-PCR) from the total RNA of BMSC. The acquired cDNA of IFN-α2b was identified by agarose gel electrophoresis and recycled. The amplified cDNA fragment of IFN-α2b and plasmid pcDNA3.1 were digested with endonuclease Hind Ⅲ and Xho Ⅰ, and connected to pcDNA3.1 to achieve pcDNA3.1-IFN-α2b. The recombinant plasmid pcDNA3.1-IFN-α2b was identified by RT-PCR and gene sequencing. Recombinant plasmid pcDNA3.1-IFN-α2b was transfected into human BMSC by liposome Lipofectamine 2000. The BMSC transfected with empty plasmid was used as control. The transfection efficiency was determined by mRNA expression of IFN-α2b by qPCR and the IFN-α2b levels in the culture supernatants by ELISA. Morphology of transfencted and untransfected BMSC were observed under nverted phase contrast microscope. The proliferative ability of IFN-α2b gene modified BMSC was tested by MTT assay. The expression of CD44, CD90, CD35 and CD45 on the cell surface of transfected BMSC was tested by flow cytometry. The transfected BMSC were induced to differentiate into adipocytes and osteoblasts.[Results] The recombinant plasmid of pcDNA3.1-IFN-α2b were identified by RT-PCR identification, the length of PCR product from recombinant plasmid pcDNA3.1-IFN-α2b is consistent with inserted gene fragment of 567bp. According to gene sequencing analyses, the sequence identified was in accordance with the sequence published in the gene bank.DFN-α2b gene was successfully transfected into human BMSC by qPCR assay, with almost four fold mRNA levels of IFN-α2b in BMSC-IFN-α2b than in control BMSC and BMSC transfected with empty plasmid. The secretion of IFN-α2b was significantly increased in the culture media of BMSC-IFN-α2b compared with control BMSC, with a secretion peak in 3d after transfection. The BMSC-IFN-α2b cell shared the similar spindle-shaped fusiform morphology and surface markers, proliferative ability compared with control cells. Finally, the IFN-α2b transfected BMSC still can be induced to differentiate into adipocytes and osteoblasts.[Conclusions] The recombinant plasmid of pcDNA3.1-IFN-α2b was successfully constructed by RT-PCR. The IFN-α2b gene can be effectively transfected into human BMSC. The human IFN-α2b gene modified BMSC still maintain their characters as mesenchymal stem cells.Part TwoGene therapy of human hepatocellular carcinoma by human Interferon-α2b gene modified bone marrow mesenchymal stem cells[Objectives] To investigate the inhibitory effects of BMSC-IFN-α2b on human HepG2 and Huh7 hepatocellular carcinoma cell lines in vitro. To investigate the growth inhibitory effects of BMSC-IFN-α2b on human HCC in vivo and to discuss the possible mechanism.[Methods]1. In vitro study1.1 BMSC-IFN-α2b and its conditioned media inhibit HCC cell in vitro.Recombinant plasmid containing human IFN-α2b gene was constructed and transfected into human bone marrow mesenchymal stem cells (BMSC) using LipofectAmine2000 reagent. The conditioned media (CM) were collected from culturing BMSC transfected with IFN-α2b or empty plasmid. Human HepG2 and Huh7 HCC cells were cocultured with BMSC-vector and BMSC-IFN-α2b, respectively, and conditioned media from BMSC-vector and BMSC-IFN-α2b were applied to HepG2 and Huh7 HCC cells, respectively. HepG2 and Huh7 HCC cells cultured in H-DMEM were used as control.48h later, MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay were used to determine the vitality of HepG2 and Huh7 HCC cells.1.2 BMSC-IFN-α2b inhibits HCC cell growth in vitro.HepG2 and Huh7 HCC cells were divided into four groups:(1) Control group: H-DMEM was applied; (2) BMSC-CM group:Conditioned media from BMSC was applied; (3) IFN-α2b treated group:IFN-α2b was added to H-DMEM at a concentration of 500IU/ml; (4) BMSC-IFN-α2b-CM group:Conditioned media from BMSC-IFN-α2b was used to culture HCC cells. HepG2 and Huh7 cells were treated with different treatments, and subdivided into 12h,24h and 48h subgroup. MTT assay were used to determine the vitality. Bromodeoxyuridine labeling was used. Cell cycle was analyzed by flow cytometry. Cell apoptosis was analyzed by flow cytometry with Annexin V/PI dual-staining. Quantitative reverse transcription polymerase chain reaction was used to determine the mRNA expression of Notch1, Hes1 and Hes7. Western blot was used to evaluate the protein expression of Notch1, Hes1 and Hes7.2. In vivo studyXenografted tumor model were established by subcutaneously injecting HepG2 HCC cells to the left flank of NOD/SCID nude mice. The tumor bearing mice were randomly divided into four groups:BMSC-IFN-α2b treated group (n=3), BMSC treated group (n=3), IFN-α2b treated group (n=3), and PBS treated group (n=3). 1×1007 DAPI labeled BMSC or BMSC-IFN-∝2b or 100μl of PBS were injected into the tail vein of mice 2 weeks after HCC inoculation, and 5×106IU/kg of PEG-IFN-α2b was used intravenousely in IFN-α2b treatment group.After inoculation, the tumor growth was examined every three days. On day 21, the animals were sacrificed and tumors were collected and weighed. Fluorescent microscopy was used to trace DAPI labeled BMSC or BMSC-IFN-α2b in tumor tissues. Immunohistochemistry was used to determine the expression of Ki67 and IFN-α2b in tumor tissue and Notch1 by immunofluorescent staining. Cell apoptosis were analyzed by flow cytometry with Annexin V/PI dual-staining. Quantitative reverse transcription polymerase chain reaction was used to determine the mRNA expression of Notchl, Hes1 and Hes7. Western blot analysis was used to evaluate the protein expression of Notchl, Hes1 and Hes7.[Results]1. In vitro study1.1 BMSC-IFN-α2b and its conditioned media inhibit HCC cell in vitro.Significant lower cell vitality in each coculture group were observed than that of control group for the two HCC cell lines (P=0.000). BMSC-IFN-α2b group had lower vitality than that of BMSC coculture group(P<0.05). Significant lower cell vitality in each conditioned media treated group were also observed than that of control group for the two HCC cell lines (P=0.000), and BMSC-IFN-α2b-CM group had lower vitality than BMSC-CM group (P<0.05). Meanwhile, there were no significant vitality difference between BMSC for the two cell lines (P>0.05). The same results also observed for BMSC-IFN-α2b coculture group and its conditioned media treated group.1.2 BMSC-IFN-α2b inhibits HCC cell growth in vitro.According to MTT assay, the control group of HepG2 and Huh7 HCC cells showed significant higher vitality than the other three experimental groups in each indicated time (12h,24h.48h, P=0.000). Lower cell vitality were observed in BMSC-IFN-α2b-CM group than that of BMSC-CM group and IFN-α2b group at each time point(P<0.01), and significant difference among each time(12h,24h,48h) were observed in BMSC-IFN-α2b-CM group(P=0.000).According to cell cycle analysis by flow cytometry, there were no difference in the distribution of G0/G1 phase, S phase and G2/M phase cell in 12h for both HepG2 and Huh7 HCC cells(P>0.05). IFN-α2b group in 24h had significant higher proportion of G0/G1 phase cell than control group, BMSC group and BMSC-IFN-α2b-CM group for the two cell lines (P<0.01), but had significant lower G2/M phase cell than the other three experimental groups(P<0.05). When treated for 24h, Huh7 HCC cell in IFN-α2b treated group had lower S phase cell than that of control group, BMSC-CM group and BMSC-IFN-α2b-CM group(P<0.05). And Huh7 HCC cell in BMSC-IFN-α2b-CM group had significant higher proportion of G2/M phase cell than in IFN-α2b group and BMSC-CM group(P<0.05). When treated for 48h, both HepG2 and Huh7 cells in control group had significant higher proportion of G2/M phase cell than the other three experimental groups(P<0.05).AnnexinV/PI dual-staining by flow cytometry showed that, both HepG2 and Huh7 HCC cells in control group had significant lower apoptosis rate not only in early-stage but also in later-stage than that of BMSC-CM group, IFN-α2b group and BMSC-IFN-α2b-CM group(P<0.05). Significant higher apoptosis rate in later-stage were observed in BMSC-IFN-a2b-CM group than that of IFN-α2b group and BMSC-CM group(P=0.000).Brdu immunofluorescent staining revealed HepG2 HCC cell in BMSC-IFN- α2b-CM group had significant lower Brdu positive cell than control group, BMSC-CM group and IFN-α2b group in each indicated time point(12h,24h,48h, P< 0.01), and lower positive cell were observed in 24h than in 12h, and lower in 48h than in 24h(P<0.05). Huh7 HCC cell in control group had significant higher Brdu positive cell at each time than the other three groups(P<0.05). Huh7 HCC cell in BMSC-IFN-α2b-CM group had significant lower positive cell in 24h and 48h than that of BMSC-CM group and IFN-α2b group(P<0.05), with significant difference among each time were observed in BMSC-IFN-α2b-CM group(P<0.01).qPCR analysis showed that HepG2 cell in control group had significant higher Notch 1, Hes1 and Hes7-mRNA expression at each indicated time than that of BMSC-CM group,IFN-α2b group and BMSC-IFN-α2b-CM group(P<0.05). HepG2 HCC cell in BMSC-IFN-α2b-CM group showed significant lower Notchl, Hes1 and Hes7-mRNA expression in each time than that of BMSC-CM group and IFN-α2b group (P=0.000), with significant difference among 12h、24h and 48h within group(P=0.000). Huh7 HCC cell in BMSC-IFN-α2b-CM group also showed significant lower Notch1, Hes1 and Hes7-mRNA expression in each indicated time than that of BMSC-CM group and IFN-α2b group (P=0.000), with significant lower in 24h and 48h than in 12h(P<0.05).According to western blot analysis, both HepG2 and Huh7 cells in control group had significant higher Notch1,Hesl and Hes7 proteins expression in each indicated time than that of BMSC-CM group, IFN-α2b group and BMSC-IFN-α2b-CM group(P <0.05). And BMSC-IFN-α2b-CM group had significant lower Notchl, Hes1 and Hes7 protein expression in each indicated time than that of BMSC-CM group, IFN-α2b group for the two types of cancer cell lines(P=0.000).2. In vivo studyControl group had significant larger tumor weight than BMSC group, IFN-α2b group and BMSC-IFN-α2b group(P<0.05). BMSC-IFN-α2b group had smaller tumor weight than that of BMSC group and IFN-α2b group(P<0.05). A small number of DAPI labeled BMSC and BMSC-IFN-α2b in tumor tissue from BMSC or BMSC-IFN-α2b transplant group were observed by luminescence microscope, but only scattered IFN-α2b positive cells in BMSC-IFN-α2b group with immunohisto-chemistry. Higher Ki67 expression in control group than in BMSC group, IFN-α2b group and BMSC-IFN-α2b group(P<0.05). Ki67 expression in BMSC-IFN-α2b group was significant lower than BMSC group and IFN-α2b group(P<0.05).Cell apoptosis analysis revealed significant lower early-stage and later-stage apoptosis cell in control group than in BMSC group, IFN-α2b group and BMSC-IFN-α2b group(P<0.05), but no difference among BMSC group, IFN-α2b group and BMSC-IFN-α2b group. BMSC-IFN-α2b group had highest apoptosis cell of later-stage, which was significant higher than BMSC group and IFN-α2b group(P <0.05). According to Notch1 immunofluorescent staining in tumor tissue, control group had significant higher Notch1 expression than in BMSC group, IFN-α2b group and BMSC-IFN-α2b group(P<0.05). Notch1 expression in BMSC-IFN-α2b group was significant lower than in BMSC group and IFN-α2b group(P<0.05).According to qPCR analysis in tumor tissue, control group showed significant higher Notch1, Hes1 and Hes7-mRNA expression than in BMSC group, IFN-α2b group and BMSC-IFN-α2b group(P<0.05). BMSC-IFN-α2b group showed significant lower Notch1, Hes1 and Hes7-mRNA expression than BMSC group and IFN-α2b group(P<0.05). Western blot analysis revealed significant higher Notchl, Hes1 and Hes7 protein expression in control group than in BMSC group, IFN-α2b group and BMSC-IFN-α2b groups(P<0.05). BMSC-IFN-α2b group showed significant lower expression of Notch1, Hes1 and Hes7 protein than BMSC group and IFN-α2b group(P<0.05).IFN-α2b gene modified BMSCs[Conclusions] Both Human BMSC and IFN-α2b can inhibit the proliferation and induce apoptosis of HCC cells in vitro and in vivo, and the effects was strengthened by IFN-α2b gene modified BMSC through negatively regulating the Notch1-Hes1 signal pathway. Those results suggested that human IFN-α2b gene modified BMSC may be used as an effective therapeutic strategy for HCC.