Experimental Study Of The Effect Of Rapamycin Liposomes Gutta Inhibiting Corneal Neovascularization

Objective: To prepare rapamycin liposomes gutta,evaluate their effect of inhibiting corneal neovascularization and expression of HIF-1a and VEGF.To invastigate the functional mechanism in inhibiting CNV and offer the theory for inhibiting CNV by decresing HIF-1a and VEGF.Methed: Rapamycin liposomes were prepared by thin film hydration. The major factors on the preparation of Rapamycin liposomes were studied by the method of orthogonal design. 42 Wistar rats were divided into four groups at random,including Rapamycin liposomes treated group(12 rats),blank liposomes treated group(12 rats),blank treated group (12 rats) and normal control group(6 rats). 36 rats were induced by alkali cauterization.After alkali burn, Rapamycin liposomes and blank liposomes were separately droped on the cornea of rats of Rapamycin liposomes treated group and blank liposomes treated group three times per day. 36 alkali cauterized rats were all being dropped with Chloramphenicol Eye Drops four times per day. Then the rats corneal was observed by slitlamp biomicroscope everyday after alkali burn,the length of the corneal neovascularization was measured and the area of corneal neovascularization was caculated.On the 1st,4th,,7th, 14th days after operation, the expression of HIF-1a and VEGF were examined by immunohistochemical method.After that,the optical density value of cells expression HIF-1a and VEGF were measured with the computer image analysis system;meanwhile the HIF-1a mRNA and VEGF mRNA were examined by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR).Results: 1. The body of RAPA liposomes were intact kinds of spheres, the average diameter was 145.2 nm, the envelopment rate was 90.02%. RAPA liposomes could be used for animal's experiment as drip eyes liquid. 2. On the first day after operation, conjunctival congestion,limbus engogement and erosion of corneal epithelium were observed.On the third day, the CNV sprouted as brush from limbus.On the 7th day, the CNV grew to the rim of alkali-buming area.On the 14th day, The CNV covered entirely the entire cornea basically. 3. The CNV in the rapamycin liposomes treated group grew slowly and smaller than that of blank liposomes treated group and blank treated group(P＜0.01).The CNV of the blank liposomes treated group and blank treated group took on almost the same (P＞0.05). 4. VEGF protein and HIF-1a protein were weakly expressed in the cytoplasm of the normal cornea epithelial cells.But after the alkali burn of the cornea,their expression increased dramatically in the epithelial cells,the endothelial cells,the newly formed vascular endothelial cells,and the inflammatory cells. Their expression peaked at the 4th day, decreaced after the 7th day, and significant decreased to near base line after the 14th day.The expression of HIF-1a and VEGF were significantly decreased by Rapamycin. There were significant difference between the Rapamycin liposomes treated group and blank liposomes treated group(P＜0.01),so as Rapamycin liposomes treated group and group(P＜0.01),meanwhile There was not significant difference between blank liposomes treated group and blank treated group (P＞0.05). 5. There were little VEGF mRNA expressed in normal cornea. But after alkali burn, the expression increased,it peaked at the 4th day, decreaced after the 7th day, and significant decreased to near baseline after the 14th day.The expresion of HIF-1a mRNA was detected scrannelly in normal cornea, HIF-1a mRNA expressed increasingly at the 1th day and 4th day after cautery, at the 7th day, it has reached normal.Meanwhile the expression of HIF-1a mRNA were significantly inhibited in the Rapamycin liposomes treated group. There were significantly difference of the expression of VEGF mRNA and HIF-1a mRNA between the rapamycin liposomes treated group and blank liposomes treated group(P＜0.01),Rapamycin liposomes treated group and blank treated group (P＜0.01).Conclusions: 1. liposomes body is an excellent medicine carrier for the RAPA.2. RAPA can obviously suppress the growth of CNV.3. The expression of HIF-1a mRNA, VEGF mRNA,HIF-1a protein,VEGF protein remarkable increase after alkali burn.4. RAPA can significantly suppress the expression of HIF-1a mRNA, VEGFmRNA,HIF-1a protein,VEGF protein.The possibly mechanism of CNV suppression is weakening the VEGFmRNA and the VEGF protein expression by inhibiting the transcription factor- HIF-1a..