Experimental Studies On Pigment Epithelium-derived Factor

Pigment epithelium derived-factor, was identified in the conditioned medium of cultured fetal human retinal pigment epithelial cells as an extracellular neurotrophic agent capable of inducing neurite outgrowth in cultured human retinoblastoma cells in 1989. It was isolated from human retinal pigment epithelial cells as a 50kDa protein in 1991. Pigment epithelium-derived factor (PEDF), a neurotrophic protein, is a member of the serine protease inhibitor supergene family, but does not inhibit proteinases. PEDF is a protein having neurotrophic, neuronotrophic and gliastatic characteristics. It was found to be a most potent inhibitor of angiogenesis in 1999. PEDF acts in the opposite way on the endothelial cells that are forming new vessels, inducing their apoptosis .Although PEDF is particularly highly expressed in the vitreous and cornea, its mRNA is, however also found in most normaltissues and tumors. PEDF has been shown to be a highly effective endogenous inhibitor of angiogenesis in animal and cell culture models. Currently, there are no antiangiogenic treatments available for patients with neovascularization disease, such as cancer, age-related macular degeneration (AMD), corneal neovascularization (CNV)and arthritis, and so on. PEDF is a natural angiogenesis inhibitor that (1) targets only new vessel growth, (2) can be administered therapeutically as a soluble protein or by viral-mediated gene transfer, (3) is stable and nontoxic when used in injection, and (4) is more potent than other well-characterized angiogenesis inhibitors. PEDF can inhibit new vessels which making it a candidate for use in angiogenesis diseases. The research on anti-angiogenesis activity of PEDF is not more. The purpose of our study is (l)To obtain the recombinant plasmid pET28a/PEDF and to purify recombinant PEDF. (2)To Investigate the influence of recombinant PEDF on endothelial cells proliferation, migration activity and the effect of recombinant PEDF on the angiogenesis of chick chorioallantic membrane(CAM) and rabbit cornea injured by alkali. (3)To prepare the injective microcapsules of recombinant PEDF and to observe the inhibition effect on chick chorioallantic membrane angiogenesis and tumor and angiogenesis in C57BL/6J mice loaded with melanoma. Main results: 1. Total RNA was extracted from a human embryo. The full-length PEDF cDNA was obtained by RT-PCR. The cDNA was cloned into vector pET28a and transformed into E. CoJiBL2l and obtain the recombinant plasmid pET28a/PEDF and then to express recombinant PEDF protein . In this study, we found a possible new sub-type of human PEDF DNA different from the human PEDFDNA that issued by GeneBank. 2. Soluble recombinant PEDF was well expressed in M9 medium about 2 hrs after inducing with lmmol/L IPTG at 25°C. 3. Novagen' s pET System has become recognized one of the most powerful approaches available for producing recombinant protein .The pET28a vectors carry an N-terrainal His-Tag configuration plus an optional C-terminal His-Tag sequence .We made our pET28a vectors carry an N-terminal His-Tag and express fusion protein .The His'Tag sequence is very useful as a fusion partner for purification of target proteins and increasing the probability of target proteins biological activity in general. We pi/rified soluble recombinant PEDF by IMAC(Ni2+-NTA) in one step and peptides were purified to homogeneity. 4. In vitro angiogenesis assay, we observe the recombinant PEDF' s ability to inhibit the migration and proliferation of human capillary endothelial cells toward angiogenic bFGF(lng/ml). The results is that recombinant PEDF suppressed bFGF-induced human capillary endothelial cell migration and proliferation in a dose-dependent manner. 1u g/ml recombinant PEDF suppressed bFGF-induced proliferation by 56. 2% (KO. 05) and migration by 75. 5% {K0. 05) respectively at 48h in culture. The effect of recombinant PEDF protein on neovascularization invivo was evaluated using a CAM model and rabbit corneal model. In CAM assay, the neovascularization numbers (22.40 + 6.36) of 1 U g/CAM recombinant PEDF is more different from negativecontrol (54.70+12.67) (J°<0. 05) . In rabbits corneal model, the neovascularization areas(37. 26+11. 58 mm2) of 5u g recombinant PEDF on 21th day is much reduced than negative control (65.40 +9.94 mm2) (P <0. 05). The mechanism of recombinant PEDF is to inhibit endothelial cells and makes them apoptosis which targets only new vessel growth and not the normal capillaries. 5. To investigate the effects of recombinant PEDF on rabbit corneal alkali burns by drops of recombinant PEDF(2. 5ug each drop, 4 times per day) and injection of recombinant PEDF(20 li g, each other day). The results revealed that there was no significant difference(P =0. 361)between the two ways. The reason may be that the biochemical characteristics, pH and electrons. 6. For the first time , the slow-release microcapsules of recombinant PEDF for injection with various concentration(100 ii g/ml , 200y g/ml, 400u g/ml) was prepared. 7. In the CAM assay, the slow-release microcapsules of recombinant PEDF(100u g/ml ) for injection resulted in marked inhibition of the CAM neovascularization. 8. In animals tests, the growth of tumor was potently suppressed by the slow-release microcapsule of recombinant PEDF for injection at various concentration at dose-dependent. Melanoma was inhibited by 66.08% with the 400 n g/ml slow-release microcapsule of recombinant PEDF for injection on 22th day. 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