| ObjectivePart 1 : to study the therapeutic effect of the triamcinolone acetonide ( TA) on the different stages of proliferative retinopathy and it' s interaction with vascular endothelial growth factor( VEGF).Part 2 : to observe the inhibit effect of Triamcinolone Acetonide to the proliferation of monkey choroid - retinal endothelial cells ( RF/6 A ) in hypoxia and in normal conditions.Part 3 : the influence of TA on the expression of VEGF of retinal and choroid endothiels of Rhesus monkey under hypoxic condition. Discussion of the therapeutic mechanism of TA on retinal and choroid neovascular response.MethodsPart 1 : to study the therapeutic effect of the triamcinolone acetonide (TA) on the different stages of proliferative retinopathy and it' s interaction with vascular endothelial growth factor ( VEGF). Grouping;choose 80 healthy infant C57BL6 mice [PN7 (postnatal d 7) 160 eyes in total] , and randomly divide them into 4 groups: pre -therapeutic group(PN7) , treated before placed in the incubator ) ( n = 20 ) , late - therapeutic group (treated after incubator nursing, PN12)(n=20) ,normal control group(n =20) , control group with oxygen supplement (n = 20). models of vascular proliferation retinopathy were established.Animal model: for normal control group, infant mice and the mother mouseare bred in room air condition, while for those mice in the other 3 groups , are placed in an certain infant incubator where the oxygen flow was adjusted ( about 1. 50 ~ 2. 50L/min) to keep the concentration in the incubator at 75 ±2%. Oxygen concentration was measured using a CY -100 digital Oxygen Analyzer ( Li-da instrument factory, jiande, zhejiang ) and was checked at least four times daily during the period of exposure. On PN12( after 5 days in this high concentration condition) animals were removed from the oxygen and placed in room air. triamcinolone acetonicleljxl(0. O4mg) was administered by intravitreal injection on day 7 in the pretreatment group ( received treatment before oxygen exposure). The late -treatment group (after oxygen exposure) received TA at the same dose on day 12. and the same volume of BSS was injected into the other eye of the mice both in these two groups as a control . Retinal neovascularization was examined by fluorescein dextran angiography. Both eyeballs of newborn mice were enucleated for performing pathological sections and were studied by immunohistochemical method in order to count the nuclei of proliferative retinal vessels and to investigate the expression of VEGF in the retina.Part 2 : to observe the inhibit effect of Triamcinolone Acetonide to the proliferation of monkey choroid - retinal endothelial cells ( RF/6 A) in hypoxia and in normal conditions, drug concentration and groups: (1)normal control group(2) normal low concentration group ?normal median concentration group <3)normal high concentration group ?hypoxia control group ?hypoxia low concentration group (7) hypoxia median concentration group ? hypoxia high concentration group.Define the hypoxia model According to the method introduced by ku-wabara, we also made some improvement. Get a dry pot , remove the glass stopper by using a rubber stopper . make two holes in the rubber stopper, one for gas inlet, the other for gas outlet. The 6 - well tissue culture plates is in the pot, ensure the obturation of the pot by Vaseline. The pot was perfused with 95% N2;5% CO2 thought the inlet at the velocity of 2 -3L/minutes at the temperature of 37^. Make sure the concentration of oxygen is below 1% by using the oxygen analyzer ( made by Lida equipment factory, Jiande , Zhejiang, China) . Close the inlet and outlet by using hemostat, Then cultured in the 37 X, in-cubator for 4 hours.In normal control group, the RF/6 A cells are cultured under normal condition, without drugs. In hypoxia control group ,the RF/6 A cells are cultured under hypoxic condition and without drugs. In normal or hypoxia low \median \high concentration group, the concentration of TA are 0. 05mg/ml,0. lmg/ml, lmg/ ml. add Triamcinolone Acetonide into the cultured choroid - retinal endothelial cells of rhesus monkey (RF/6 A) , measure the effect of Triamcinolone Acetonide on the cellular activity by MTT, observe its effect on cellular proliferation and apoptosis by flow cytometry ( FCM).Part 3 : the influence of TA on the expression of VEGF of retinal and choroid endothiels of Rhesus monkey under hypoxic condition. Discussion of the therapeutic mechanism of TA on retinal and choroid neovascular response. Dosage and grouping: (T)normal control group (5)normal therapeutic group ?hypoxic control group (4)hypoxic therapeutic group. Normal control group means RF/6 A cells without any drugs. Hypoxic control group : RF/6 A cells in hypoxic condition without any drugs, concentration of normal and hypoxic therapeutic groups are both lmg/ml. We observe the influence of TA to the expression of VEGF of RF/6A cells by immunohistochemistry image analysis and half fix quantified RT -PCR.ResultsPart 1: the number of nuclei of retinal neovascular endothiels and the total OD value of VEGF: normal control group (nuclei number 0. 04 i 0. 19;total OD value36.15 ± 3. 11) , pre - therapeutic group (nuclei number 7. 82 ± 1.15;total OD value86. 36 ±2. 89) , control group with oxygen supplement ( nuclei number 23.57 ± 1.95;total OD value212. 35 ± 5. 93 ) , late - therapeutic group ( nuclei number 17.55 ± 1.75;total OD valuel32. 29 ±6. 38 ). There are obvious significances between pre - therapeutic group and its control group, late -therapeutic group and its control group, as well as compared with control group with oxygen supplement. P <0.01 oPart 2: hypoxia will relatively reduce the number of cells in S - phase of acell cycle and meanwhile increase the proportion of cells in G2 - M phase. Tri-amcinolone Acetonide has a great effect on the cell cycle of choroid - retinal en-dothelial cells of rhesus monkey and it can induce apoptosis of endothelial cells. It relatively increases S - phase cells and reduces G2 - M phase cells in both normal and hypoxia condi tions, which indicates its role in blocking cell cycle from S - phase to G2 - M phase and reducing mitosis.Part 3 : the staining intensity of VEGF protein in cellular plasma is obviously enhanced under hypoxic condition when detected by immunohistochemistry. But, definitely, the intensity decreased when TA are added. The OD value of VEGF protein under normal room air is 276. 82 ±8. 63;normal room air with TA : 137.08 ±10. 18;hypoxia: 406. 09 ±8. 82;hypoxia with TA: 244. 15 ± 12.77o Half fix quantified RT - PCR indicates the relative expression of VEGF mRNA in RF/6A cells of normal control group is 1. 38 ±0. 05;that of hypoxic control group is 5.41 ± 0. 06;that of normal therapeutic group is 0. 70 ± 0. 06;that of hypoxic therapeutic group is 4. 23 ±0. 05. By these two means, we found obvious significances between hypoxic control group and normal control group, hypoxic control group and hypoxic therapeutic group, normal control group and normal therapeutic group(P= 0. 000).Conclusions *TA prevents the retinal neovascular response in the early stage;hypoxia promotes the proliferation of retinal endothiels and the expression of VEGF in RF/6A cells. TA inhibits the retinal neovascular response and the expression of VEGF. TA effects on the cell cycle ,thus , inhibits the cellular energy metabolism , induces apoptosis, decreases the expression of VEGF in RF/6A and consequently , inhibits the proliferation of RF/6A. |
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