Primary Study Of Vasculogenic Mimicry In Retinalblastoma

Objective To explore the existence of Vasculogenic mimicry in human Retinalblastoma; to explore the effect of hypoxia on inducing retinalblastoma cell to transform to endothelioid cell and form vasculogenic mimicry through three-dimension culture in vitro; and to build shRNA virus vector targeting at hypoxia inducing factor-la using RNAi technology; to observe the effect of hypoxia inducing retinalblastoma cell to transform to endothelioid cell.Methods 1. To collect human Retinalblastoma sample; to observe whether there are Vasculogenic mimicry in human Retinalblastoma, the relationship among VMD, tumor staging and differentiation through paraffin section, HE staining and PAS/CD34 double staining; to observe expression of ABCG2, AC133, HIF-1α, EphA2 and MMP-2.2. Using slow virus vector-Lentivirus to construct shRNA-virus vector targeting at HI-la, transfect Y79 cells and screen out stable-tranfected by anti-neo. To detect the expression of HIF-la by RT-PCR and Western Blot.3. Y79 cells were cultured under the hypoxia circumstance in vitro, morphological changes of Y79 cells were observed with inverted microscope. Analyzed specific cell markers, banding Dil-acLDL of tumor's endothelial cells-like using inununofluorescenc, observed the ability of VM formation at the three-dimension medium, detected the expression of HIF-la, EphA2 and MMP-2 by Real-time PCR and Western Blot.Results 1. VM which is PAS positive and CD34 negative, were observed in human Retinalblastoma paraffin section, statistical analyze results display that VM positive rate correlates with tumor differentiation and have nothing to do with tumor staging. ABCG2 and AC133 positive cells can be observed in the RB tissue with VM, and their positive cell percentage are significantly higher than that in the RB tissue without VM. The expression of HIF-la, EphA2 and MMP-2 are higher in the RB tissue with VM.2. Sequence testing results display that the construction of shRNA-virus vector targeting at HI-la was succeed. The expression of HI-la almost be completely silenced in the stable-transfected Y79 cells. 3. In vitro, after the hypoxia culture, some Y79 cells became stick-wall growth like endothelium, express AC133 and obtain the ability of banding Dil-acLDL. At the three-dimension medium, hypoxia can induce Y79 cells to format VM. The expression of HIF-la, EphA2 and MMP-2 were significantly increased after the hypoxia culture. However, all above mentioned changes were not observed in cells stably transfected shRNA-virus vector targeting at HIF-la.Conclusion 1.VM exists in the low differentiated RB tissue, VM density correlates with tumor differentiation and have nothing to do with tumor staging.2. Constructed shRNA-virus vector targeting at HIF-la can efficiently degradate the endogenous HI-la mRNA.3. Hypoxia is one of the most inducing factors of VM in RB tissue, shRNA targeted at HIF-1αhas the effect of inhibit the formation of VM induced by hypoxia in Y79 cells.4.The endothelial-like cell may be come from the stem cells of RB.