Lentiviral Vector Mediated NK4 Gene-modified Bone-derived Mesenchymal Stem Cells For Gastric Cancer Targeted Therapy

Objective:1. To establish the experimental method for isolating, culturing, and proliferating human bone-derived mesenchymal stem cells (hBMSCs) in vitro;2. To construct NK4 and enhance green fluorescent protein fuse gene of recombinant lentiviral expression vector;3. To investigate the tropism of hBMSCs to gastric cancer in vitro and in vivo;4. To observation the treatment effectiveness of lentiviral vector mediated NK4 gene-modified bone-derived mesenchymal stem cell for gastric cancer and its related mechanism.Methods:1. hBMSCs were isolated and expanded in culture by bone marrow direct plating method and identified by flow cytometry detecting CD34,CD44,CD45,CD 105 and the ability to differentiate into osteocytes; Calcium deposition was detected using alizarin red stain;2. NK4 gene was cloned from HGFcDNA by polymerase chain reaction and identified by gene sequencing. NK4 and enhance green fluorescent protein fuse gene of recombinant lentiviral expression vector was constructed by In-Fusion technique and the expression of GFP was detected by Western blot and observated by fluorescence microscope.The lentiviral titer was detected by real-time quantitative PCR(RT Q-PCR).3. After lentiviral vector expressing NK4 and EGFP fuse gene (Lenti-NK4) and lentiviral vector expressing EGFP (Lenti-GFP) transfected hBMSCs, the optimum(Multiplicity of infection,MOI) was determined by the expression of fluorescence and the transfection efficiency. The expression of NK4 was determined by enzyme linked immunosorbent assay (ELISA) and Western blot.4. To set up tumor xenografts model, MKN45 tumor cells suspension and tumor tissues were inoculated subcutaneously into athymic nude mice; to set up tumor implantation metastasis model, MKN45 tumor cells suspension and tumor tissues cells suspension were injected into the peritoneal cavity of athymic nude mice.5. (1) Cell migration assay was done to determine the tropism of hBMSCs to gastric cancer cells in vitro by Transwells co-culture system. MKN45, GES-1 or culture media were plated on the bottom well of a Transwell plate for 24 h before hBMSCs or hFB were added to the top well. Then the top wells were removed after 24 h and the migrated cells on the bottom side were fixed, stained using crystal violet, and counted in microscope. (2)After subcutaneous tumors had become established, the athymic nude mice were separated into three groups randomly and hBMSCs-GFP, hFB-GFP and Lenti-GFP were injected into tail vein. The athymic nude mice were sacrificed after seven days, the expression of GFP in the tumors and each organ was observed in fluorescence microscope.6. After subcutaneous tumors had become established, thirty two athymic nude mice were separated into four groups randomly, then hBMSCs-NK4, Lenti-NK4, hBMSCs-GFP and PBS were injected into tail vein at 0,7,14. and 21 d. The tumors were measured and volume of tumor was calcutated as n abc/6. The histopathology of tumors was observed by hematoxylin and eosin stain; CD31 and PCNA were detected by immunohistochemistry and apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.RESULTS1. The signs of hBMSCs with positive CD44, CD 105 and negative CD34, CD45 were detected by the flow cytometry, and that is consisted with the characteristics of MSCs. hBMSCs were cultured in medium supplemented with components for osteogenic differentiation. Cells were Alizarin red stain 21 d later. Red-stained calcium deposits were detected in differentiated cultured hBMSCs.2. Recombinant lentiviral expression vector of NK4 and enhance green fluorescent protein fuse gene could express correctly after transfected 293T cells. The titer of Lenti-NK4 is 2x108TU/ml. With time extension and MOI increased, green fluorescence and the secretory volume of NK4 in culture supernatant increased gradually after Lenti-NK4 transfected hBMSCs. The optimum MOI of hBMSCs was 50.The transfection efficiency was 87.8% or 96.5% when Lenti-NK4 or Lenti-GFP transfected hBMSCs with MOI 50. 3. All the mice implanted subcutaneously with 4×106/ml MKN45 cell suspensions or tumor tissues developed subcutaneous tumor nodules and time of tumor formation in the latter method was shorter than in the former method significantly (P<0.05). All the mice injected with 107/ml cells suspension of tissue homogenate into the peritoneal cavity developed metastasis foci.4. (1) Directional migration of hBMSCs through membranes were significantly stimulated by human gastric cancer cells MKN45 in vitro(239.5±54.3 migrated cells compared with GES-1 and medium 43.57±4.6,37.3±4.7) (P<0.01). There was not significantly different between GES-1 and medium (P>0.05). Directional migration of hFB through membranes were not significantly stimulated by human gastric cancer cells MKN45 in vitro(27.7±16.7, migrated cells compared with GES-1 and blank control 16.4±5.1,19.1±6.2) (P>0.05). hBMSCs migrating to MKN45,GES-1 and medium increased significantly compared with hFB (P<0.05). (2) Fluorescent hBMSCs were found within the tumor tissue. No fluorescent signal in tumors was visible in cryosections from hFB control group. Fluorescence in tumor tissue was observed in 1 of 5 mice in Lenti-GFP group. There were significantly differences among three groups (P<0.01). Fluorescence in liver and lung was observed in 1 of 5 mice injected hBMSCs-GFP. There was no fluorescence in heart, kidney and spleen in all mice injected hBMSCs-GFP and the positive rate of fluorescence in tumor was significantly higher than each organ (P<0.05)5,(1) The volume of tumor in hBMSCs-NK4 group was significant small compared with hBMSCs-GFP and PBS groups (P<0.05). The volume of tumor in Lenti-NK4 group was not significant small compared with PBS group (P>0.05) and hBMSCs-GFP group in the first, second and fourth week (P>0.05).The inhibition ratio of tumor was 52.2% and 29.4% in hBMSCs-NK4 and Lenti-NK4 groups. (2) The weigh of tumor in hBMSCs-NK4 was 1.94±0.67g significantly lower than hBMSCs-GFP and PBS groups (P<0.05). There were no significant differences among Lenti-NK4, hBMSCs-GFP and PBS groups (P>0.05). (3) The score of tumor necrosis was 3.63±0.47 in hBMSCs-NK4 group significantly lower than hBMSCs-GFP and PBS groups (P<0.05). There were no significant differences among Lenti-NK4, hBMSCs-GFP and PBS groups (P>0.05). (4) The microvessel density of tumor was 5.6±1.47 in hBMSCs-NK4 group significantly lower than hBMSCs-GFP and PBS groups (P<0.05). There were no significant differences among Lenti-NK4, hBMSCs-GFP and PBS groups (P>0.05).(5) Apoptotic index of tumor cells was 2.69±0.55,1.95±0.91 in hBMSCs-GFP and PBS groups, whereas it significantly increased to 7.31±1.90 in hBMSCs-NK4 groups (P<0.01) and 4.2±1.3 in Lenti-NK4 groups (P<0.05)CONCLUSIONS:1. Highly purified hBMSCs can be obtained by by bone marrow direct plating method.2. hBMSCs can be transfected by lentiviral vector expressing NK4 gene safely and efficiently and the expressin and secretion of NK4 is stable.3. The tropism of hBMSCs to gastric cancer is obvious in vitro and in vivo.4. NK4 gene therapy using hBMSCs as vehicle can inhibit angiopoiesis of tumors and induce tumor cells apoptosis and make tumor necrosis increase, then inhibit gastric cancer growth. MSCs may be suitable delivery vehicles for gastric cancer targeted therapy.