An Experimental Study On Inhibitory Effect Of ChM-â…  On Angiogenesis Of Osteosarcoma

Objective (1)To obtain the gene of human Chondromodulin-I gene and express and purify it in procaryotic expression system. (2)To study expression of ChM-I in cartilage matrix and discuss the mechanism of angiogenesis in cartilage .(3)To construct the nude mice model of osteosarcoma and study the inhibitory effect of chondromodulin-I on angiogenesis on nude mice in vivo . (4)To observe in vitro changes of human umbilical vein endothelial cells after confrontation with chondromodulin-I.Methods (1)A fragment of human Chondromodulin-I gene was amplified from tissue by RT-PCR, and cloned into procaryotic expression vector pET28a(+). The recombinant plasmid pET (ChM-I) was transformed into E.coli B121(DE3) and expressed under the induction of IPTG. The expression product fused with 6 ×His at N-terminal was analyzed by Western blotting, and purified by using Ni2 +2NTA ion exchange resin. The purity of ChM-I protein was analyzed by SDS-PAGE. (2)The ChM-I protein was detected using immunohistochemistry ,and ChM-I mRNA in situ hybridization .(3)The nude mice were hypodermic injected with the MG-63 cells of osteosarcoma for constructing the nude mice mode of osteosarcoma. The changes of growth and morphous was observed that the nude mice were propagated after 4th passage. The identification of cell cycle of the transplanted osteosarcoma cells, the stable passage model, was taken by flow cytometry. And then, we injected ChM- I around the transplanted tumor to block it. The nude mice were executed in 5 weeks in order to accounting area density of capillary under light microscope. The results were compared with control group by T-Test with SPSS. (4)The endotheliocytes from human umbilical vein were gotten in childbearing and enlymatic perfusion digestion twice using trypsinase (0.12 %) and cultured with chondromodulin-1 in vitro. We observed the morphologic characteristics of endothelial cells under the inverted microscope , and antihemophilic the factor Ⅷ antigen by immunohistochemical test for HUVEC identification.Results (1) Human ChM-I cDNA was obtained , and the expression plasmid pET (ChM-I) was constructed successfully. Western blot analysis showed that human ChM-I with 25kDa molecular weight was expressed in E. coli at 37 ℃. The purity of the recombined CHM-I was more than 90 % after purification using Ni2 +2NTA ion exchange resin. (2)Low power view indicating the cartilage-specific immunolocalization of ChM-I by the anti-ChM-I antibody. High power views showing strongly stained ChM-I in the inter-territorial matrix in the proliferating cartilage zone; as well as the resting cartilage zone and the upper hypertrophic cartilage zone.The absence of ChM-I staining became evident in the lower hypertrophic and calcified cartilage zones. Preimmune rabbit IgG exhibited no positive staining as shown by the high power view of the proliferating cartilage zone .Low power view indicating the cartilage-specific expression of ChM-I mRNA from the resting cartilage zone to the upper hypertrophic cartilage zone using 35S-labeled cDNA probe. High resolution autoradiographs using the ~3H-labeled cDNA probe showing intense signals of ChM-I mRNA in the proliferating cartilage zone , but very few signals in calcified chondrocytes , The specificity of the hybridization signals was confirmed by RNase treatment of the sections before hybridization with the DNA probe, only a background level of signals was found over the cells in the proliferating cartilage zone.(3)We got human umbilical vein endotheliocytes in vitro , and confirmed them by immunity fluorescence. Well-purified HUVEC with few toxic effects were obtained by two staged digestion of trypsinase. HUVEC in confluent primary culture reproduce the epithelioid organization of the vascular lining in situ, endothelial cells may look like cobble stones or long spindle shaped. The marked structure of junctional integrity and substratum attached to one side of cells may be a supplement to identify HVEC .This marked structure disappear when HUVEC cultured with chondromodulin- I .(4)We obtained the nude mice model of osteosarcoma which propagating stably and found distinguished difference in the area density of capillary between the injected ChM-I group and control group (P < 0.01).Conclusion (1) The study laid a foundation of further study on the biological activity and application of human Chondromodulin-I protein. (2)ChM-I protein was accumulated in the inter-territorial space of cartilage matrix .the distribution of ChM-I in cartilage was clearly distinct from that of bFGF, suggesting that the FGF-containing pericellular space of chondrocytes was wrapped within a barrier of ChM-I. This unique localization pattern may explain how ChM-I confers an anti-angiogenic property on cartilage. The accumulation of ChM-I protein was reduced or absent in the lower hypertrophic and calcified zone of cartilage in parallel with the loss of its gene expression. (3)HUVEC cell line can be got from infant umbilical cord by primary cultivation, and chondromodulin-I can directly prohibit morphological and functional changes in endothelial cells.(4)ChM-I is an inhibitor on angiogenesis on the nude mice model in vivo. It is significance actively that ChM-I can prohibit invasion and metastasis of osteosarcoma through reducing the amounts of area density of capillary, and raise survival rate of patients who get sick of osteosarcoma.