Morphological Studies On The Anti-angiogenesis In Human Osteosarcoma-bearing Nude Mice

Objective: Tumor growth is dependent on the angiogenic factors which induces angiogenesis.Vascular endothelial growth factor (VEGF) is an endothelial cell-specefic mitogen and an angiogenesis inducer in vivo. VEGF plays an important role in angiogenesis of osteosarcoma. The aim of present study is to establish the model of osteosarcoma subcutaneous xenograft in nude mice and use recombinant-adenovirus-VEGF-RNA interfering to block the production of VEGF in vivo, and to discuss the impact on the development of osteosarcoma by the blockage of endogenous production of VEGF. Thus, it can provide information for the anti-angiogenesis gene therapy by the target of VEGF.Methods: The osteosarcom axenografts were established by subcutaneous injection of human MG63 osteosarcoma cells into 45 nude mice (Balb/c) which weighed 15-20 grams, and were 4-8 weeks old. Every mouse was injected to three million cells. These mice were divided randomly into 3 groups, A: AD-VEGF-siRNA group, 15 mice, B: AD-EGFP group, 15 mice, C: PBS group, 15 mice. The mice were treated with AD-VEGF-siRNA, AD-EGFP and PBS respectively intra-tumor at 8^th, 10^th, 12^th,14^th, 16^th day after the implantation of MG63 cells.The drug was injected at 200ul each time and the total dose of virus recombinant was 2×10⁹pfu. The next indexes were detected.The volume of tumor was measured every two days after the first injection and the growth curve was drawn.Two mice chosen from each group randomly at 18^th day after subcutaneous injection of human MG63 osteosarcoma cells were checked with MRI to observe the filling of blood stream in osteosarcoma. All remaining mice were sacrificed at 19^th day after injection. The tumor samples were collected and weighted. These indexs were dectected such as sample observation and weight, HE staining, VEGF and CD31 immunocytochemical analysis. Ultrastructure of vasculogenic mimicry by electronic microscope was observed in one sample chosen from each group randomly.Results: 1. Observation of tumor sample and HE staining: tumors could be seen at 5^th day after the implantation of MG63 ells. Osteosarcoma Xenografts in AD-VEGF-siRNA group were small, hemispherical, partly bleeding and ulcer. HE staining showed few number of tumor cells, big cell interspace, few blood capillaries, whereas, tumors in the two control groups were big. HE staining showed large amount of cells, small cell interspace, little stroma, plenty of blood capillaries.2.The development of tumor growth: the tumors of AD-VEGF-siRNA group grew slowly till to the end of drug injection. At 18^th day the average volume of AD-VEGF-siRNA group was (0.3132±0.1793) cm³, whereas the volumes of other two control groups were (1.2130±0.2904) cm³ and (1.4303±0.9475) cm³. At 19^th day the tumor weight of AD-VEGF-siRNA group were (0.7473±0.2071) g, whereas, the tumor weight of other two control groups were (1.1899±0.2741)g and (1.1861±0.3496)g respectively. Comparison among groups showed significant differences (P＜0.05). 3. The numers of microvessel density (MVD) labeled by CD31 immunocytochemical analysis among AD-VEGF-siRNA group and other two control groups of were 5.79±0.34, 10.59±1.06 and 10.98±0.45. Comparison among groups showed significant differences (P＜0.05). 4. Expression of VEGF analysis: positive values of VEGF protein in each group were 235228.09±123244.41, 964192.40±90479.62 and 981298.00±213590.50. Comparison among groups showed significant differences (P＜0.05).5.Correlation analysis of MVD and VEGF: the Pesrson correlation coefficient was 0.9989, P=0.0302. They were positively correlated. 6. Filling of blood stream in osteosarcoma by MRI: time intensity curve showed the descenting range and maximum slope in AD-VEGF-siRNA group had decreased more than those in PBS group. 7. Observation of vessel mimicry: numbers of VM in each group were 1.4000±0.5477, 2.6000±0.5477, 2.6000±0.8944. Comparison among groups showed significant differences (P＜0.05). 8. Transmission electronic microscope proved that vasculogenic mimicy existed in osteosarcoma.Conclusions: 1. AD-VEGF-siRNA may effectively block the growth of osteosarcoma from macroscopic views and microscopic views. 2. Immunocytochemical analysis shows AD-VEGF-siRNA may inhabit the expression of VEGF, and interfere with new vessels' angiogenesis. 3. VEGF has a close relationship with MVD in osteosarcoma. 4. Filling of blood stream in osteosarcoma by MRI also shows AD-VEGF-siRNA may interfere with new vessels' angiogenesis. This shows the value of MRI in the morphological studies on anti-angiogenesis. 5. The animal experiments also testify vasculogenic mimicy existed in osteosarcoma. AD-VEGF-siRNA may well be interfere with vasculogenic mimicy through affecting the blood supply of osteosarcoma indirectly.