| Part I Parameter Optimization on Ultrasonically Activated Hematoporphyrin Inhibit Neovascularization of Chicken Chorioallantoic Membrane (CAM)Objective : To observe the effect of ultrasonically activated hematoporphyrin on neovascularization of CAM and optimize the experiment parameters.Methods: Use selected chicken embryos to do the experiments on ultrasonically activated hematoporphyrin inhibiting neovascularization of CAM. After taking pictures on the CAM specimens by digital camera, the effects on the CAM new blood vessel in blank group, ultrasound group, hematoporphyrin group and hematoporphyrin SDT group were detected by counting of neovascularization, then the ultrasonic parameters and the dose of hematoporphyrin were determined. Observe the optimal parameter of ultrasonically activated hematoporphyrin inhibiting neovascularization of CAM. Keeping away from the light when adding hematoporphyrin.Results: Blood vessel counting method showed: when fixed frequency was 1MHz, sound intensity was 1.5W, expose time was 0s, 30s, 60, 90s, the number of new blood vessels of CAM in the experimental groups was 60.08±18.48, 62.27±26.02, 43.66±9.38, 30.19±8.75. Ultrasound itself can inhibit neovascularization of CAM and the inhibiting effect increased according to the expose time. When fixed frequency was 1MHZ, expose time was 90s, sound intensity was 0W/cm2, 0.5W/cm2, 1.0W/cm2, 1.5W/cm2, the number of new blood vessels of CAM in the experimental groups was 60.19±20.30, 55.61±21.79, 56.20±22.46, 37.98±6.88. 1.5W group had significantly statistical difference by contrasting with the blank group.(P<0.01)Ultrasound itself can inhibit neovascularization of CAM when the sound intensity was 1.5W. When the concentration of hematoporphyrin was 0μg/ml, 25μg/ml, 50μg/ml, 75μg/ml, the number of new blood vessels of CAM in the experimental groups had no statistical difference by contrasting with the blank group.(P>0.01)Hematoporphyrin had no effect on neovascularization of CAM. When the fixed frequency was 1MHZ, expose time was 90s, sound intensity was 1.5W/cm2, the concentration of hematoporphyrin was 0μg/ml, 25μg/ml, 50μg/ml, 75μg/ml, the number of new blood vessels of CAM in the experimental groups was 35.69±9.45, 25.44±8.93, 17.07±6.31, 17.62±5.11. The experimental groups had statistical difference by contrasting with the blank group(.P<0.01)Hematoporphyrin concentration 50μg/ml group had no statistical difference by contrasting with 75μg/ml group. ( P>0.01 ) Hematoporphyrin SDT had significant effect on neovascularization of CAM. When the concentration of hematoporphyrin was 50μg/ml, the inhibiting effect was greatest.Conclusion: The optimal parameters of ultrasonically activated hematoporphyrin inhibiting neovascularization of CAM are sound intensity: 1.5W/cm2, expose time: 90s, concentration of hematoporphyrin: 50μg/ml. This experiment selects the optimal parameters. Part II Experiment on the Effect of Ultrasonically Activated Hematoporphyrin to Inhibit Neovascularization of Chicken Chorioallantoic Membrane (CAM)Objective: To observe the inhibiting effect of Hematoporphyrin SDT treatment on neovascularizationMethods: By rating method, software DFY, staining method HE, observe the effect of ultrasonically activated hematoporphyrin to inhibit neovascularization of CAM.Results: Rating result showed: the degree of vascular growth in chick embryo of every group was different. It was tested that hematoporphyrin group had no statistical difference by contrasting with the blank group.(P>0.01)It indicated that hematoporphyrin itself had no effect on neovascularization on the whole. Ultrasound group, hematoporphyrin SDT group had significantly statistical difference by contrasting with the hematoporphyrin group.(P<0.01)Ultrasound group and hematoporphyrin SDT group had significantly inhibiting effect on neovascularization of CAM, among which 3+, 4+ of hematoporphyrin SDT group increased more than that of ultrasound group and had statistical difference.(P<0.01)It indicated that hematoporphyrin SDT group had more inhibiting effect than ultrasound group. Data in DFY showed: percentage of blood vessel area in in blank group, hematoporphyrin group, ultrasound group and hematoporphyrin SDT group were: 88.88±7.43,85.67±9.42,74.82±16.92,60.83±11.63 Ultrasound group and hematoporphyrin SDT group had significantly statistical difference by contrasting with the blank group.(P<0.01)Ultrasound group had significantly statistical difference by contrasting with the hematoporphyrin SDT group. ( P<0.01 ) The hematoporphyrin group had no statistical difference by contrasting with the blank group. Data of staining method HE showed: the density of blood vessels in blank group, hematoporphyrin group, ultrasound group and hematoporphyrin SDT group were respectively: 8.50±2.57,7.21±2.05,4.82±1.43,1.59±0.26.Ultrasound group and hematoporphyrin SDT group had significantly statistical difference by contrasting with the blank group. (P<0.01)The hematoporphyrin group had no statistical difference by contrasting with the blank group(P>0.01).Conclusion: Sonodynamic effect of ultrasonically activated hematoporphyrin can inhibit neovascularization of CAM significantly. PART III the mechanism study on Ultrasonically Activated Hematoporphyrin Inhibit Neovascularization of Chicken Chorioallantoic Membrane (CAM)Objective: To detect the possible mechanism of sonodynamic effect of ultrasonically activated hematoporphyrin to inhibit neovascularization of CAM.Methods: Preliminarily observe the mechanism of of sonodynamic effect of ultrasonically activated hematoporphyrin to inhibit neovascularization of CAM through immunohistochemical methods of VEGF.Results: Data of immunohistochemical methods showed: the average optical of VEGF in blank group, hematoporphyrin group, ultrasound group and hematoporphyrin SDT group were 0.46±0.07,0.40±0.10,0.26±0.05,0.14±0.07. The hematoporphyrin group had no statistical difference by contrasting with the blank group(P>0.01). Ultrasound group and hematoporphyrin SDT group had significantly statistical difference by contrasting with the blank group(P<0.01). Ultrasound group had significantly statistical difference by contrasting with the hematoporphyrin SDT group(P<0.01).Conclusion: VEGF expression of CAM was inhibited significantly by sonodynamic effect of ultrasonically activated hematoporphyrin,which is One of the possible mechanisms of neovascularization of CAM being inhibited.
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