超声激活血卟啉声动力作用抑制鸡胚绒毛尿囊膜新生血管形成的实验研究

发布时间：2018-06-14 20:24

本文选题：鸡胚绒毛尿囊膜 + 超声　；参考：《重庆医科大学》2008年硕士论文

【摘要】： 第一部分超声激活血卟啉声动力作用抑制绒毛尿囊膜新生血管形成的参数优化目的:观察超声激活血卟啉声动力作用对鸡胚绒毛尿囊膜新生血管形成的影响并优化其实验参数。方法:采用所选发育天数的鸡胚进行超声激活血卟啉声动力作用抑制绒毛尿囊膜新生血管形成的实验,将CAM标本用数码相机拍照后,通过血管生成计数法检测空白对照组、单纯超声组、单纯血卟啉组及血卟啉SDT组对鸡胚绒毛尿囊膜(CAM)新生血管的影响,并以此确定超声参数和血卟啉剂量,观察超声激活血卟啉声动力作用抑制鸡胚CAM新生血管生成的最佳参数。加入血卟啉时应严格暗室避光操作。结果:血管计数法显示:当固定频率为1MHz,声强为1.5W,辐照时间为0s、30s、60s、90s时,各实验组CAM血管数量分别为60.08±18.48、62.27±26.02、43.66±9.38、30.19±8.75,可见单纯超声对CAM血管生成有抑制作用,且抑制作用随辐照时间增加而增强;当固定频率为1MHZ,辐照时间为90S ,声强各组分别为0w/cm2、0.5w/cm2、1.0w/cm2、1.5w/cm2,各实验组CAM血管数量分别为60.19±20.30、55.61±21.79、56.20±22.46、37.98±6.88,1.5w组与空白对照组比较差异有显著统计学意义(P0.01),可见当单纯超声辐照声强为1.5w/cm2时,对CAM血管生成有抑制作用;当单纯血卟啉浓度为0μg/ml、25μg/ml、50μg/ml、75μg/ml时,各实验组CAM血管数量与空白对照组比较差异无统计学意义(P0.01)。说明单纯血卟啉对CAM新生血管的生成无影响;当固定频率为1MHZ,辐照时间为90s ,声强为1.5w/cm2,各组血卟啉浓度为0μg/ml、25μg/ml、50μg/ml、75μg/ml时,各实验组CAM血管数量分别为35.69±9.45、25.44±8.93、17.07±6.31、17.62±5.11。各实验组与对照组相比较差异有统计学意义,(P0.01),其中50μg/ml与75μg/ml组相比较差异无统计学意义,(P0.01)。说明血卟啉SDT对CAM新生血管的生成有明显影响,当血卟啉浓度达50μg/ml时其抑制效应最大。结论:超声激活血卟啉抑声动力作用制鸡胚CAM新生血管的最佳参数为超声辐照声强为1.5 w/cm2 ,辐照时间为90S,血卟啉浓度为50μg/ml。本实验选择此最佳参数。第二部分超声激活血卟啉声动力作用抑制绒毛尿囊膜新生血管形成实验目的:观察和检测超声激活血卟啉声动力作用对新生血管形成的抑制作用。方法:采用等级评定方法、DFY软件、HE染色法观察超声激活血卟啉声动力作用对CAM新生血管形成的抑制作用。结果:等级评定结果显示:各组中鸡胚的血管生长状态等级不同,经检验发现,空白组和血卟琳组之间差异无显著统计学意义(P0.01),说明单纯血卟啉基本不影响血管新生;单纯超声组和血卟啉SDT组与之相比较差异均有显著统计学意义(P0.01),说明单纯超声组和血卟啉SDT组有明显抑制鸡胚CAM上新生血管形成的作用,其中血卟啉SDT组的3+ ,4+明显比单纯超声组增多,其差异有显著统计学意义(P〈0.01),说明血卟啉SDT组抑制血管生成效应强于单纯超声组;DFY数据显示:空白对照组、单纯血卟啉组、单纯超声组、血卟啉SDT组的血管面积百分比分别为:88.88±7.43、85.67±9.42、74.82±16.92、60.83±11.63。与空白对照组相比,单纯超声组、血卟啉SDT组与之差异有显著统计学意义(P0.01);单纯超声组与血卟啉SDT组之间比较,差异有显著统计学意义(P0.01);单纯血卟啉组与空白对照组比较差异无显著统计学意义; HE染色数据显示:空白组、单纯血卟啉组、单纯超声组、血卟啉SDT组的血管密度分别为8.50±2.57、7.21±2.05、4.82±1.43、1.59±0.26。与空白对照组相比,单纯超声组、血卟啉SDT组与之差异有显著统计学意义(P0.01),单纯超声组与血卟啉SDT组之间比较,差异有显著统计学意义(P0.01),单纯血卟啉组与空白组比较差异无显著统计学意义(P0.01)。结论:超声激活血卟啉声动力作用显著抑制鸡胚绒毛尿囊膜新生血管形成。第三部分超声激活血卟啉声动力作用抑制绒毛尿囊膜新生血管形成的机理研究目的:观察和检测超声激活血卟啉声动力作用抑制鸡胚绒毛尿囊膜新生血管形成的可能机理。方法:应用VEGF免疫组化的方法初步观察超声激活血卟啉声动力作用抑制鸡胚绒毛尿囊膜新生血管的作用机理。结果:免疫组化数据显示:空白组、血卟啉组、单纯超声组、血卟啉SDT组的VEGF平均光密度分别为0.46±0.07、0.40±0.10、0.26±0.05、0.14±0.07。与空白对照组相比,血卟啉组差异无显著统计学意义(P0.01);单纯超声组、血卟啉SDT组与之差异有显著统计学意义(P0.01);单纯超声组与血卟啉SDT组之间比较,差异有显著统计学意义(P0.01)。结论:超声激活血卟啉声动力作用对VEGF表达具有显著的抑制作用,这可能是其抑制鸡胚绒毛尿囊膜新生血管形成的机理之一。
[Abstract]:Part one: ultrasound activated hematoporphyrin sonodynamic effect to optimize the parameters of chorioallantoic membrane neovascularization.
Objective: To observe the effect of sonodynamic activation of hematoporphyrin on angiogenesis of chick embryo chorioallantoic membrane and optimize its experimental parameters.
Methods: the embryo of the selected development days was used to suppress the formation of the neovascularization of the chorionic ananus membrane by ultrasonic activation of hematoporphyrin. After taking photos of the CAM specimens with a digital camera, the blank control group was detected by the angiogenic count method, the simple ultrasound group, the simple hematoporphyrin group and the blood porphyrin SDT group were used to the chick chorioallantoic membrane (CAM). The effect of the neovascularization, and in order to determine the ultrasonic parameters and the dose of the blood porphyrin, observe the best parameters of ultrasonic activation of the sonodynamic effect of hematoporphyrin on the formation of CAM neovascularization in chicken embryo.
Results: the blood vessel counting method showed that when the fixed frequency was 1MHz, the sound intensity was 1.5W, the irradiation time was 0s, 30s, 60s, 90s, the number of CAM vessels in the experimental groups was 60.08 + 18.48,62.27 + 26.02,43.66 + 9.38,30.19 + 8.75 respectively, and the simple ultrasound could inhibit the CAM angiogenesis, and the inhibition effect was enhanced with the increase of irradiation time; when the fixed frequency was fixed. For 1MHZ, the irradiation time was 90S and the intensity of sound intensity was 0w/cm2,0.5w/cm2,1.0w/cm2,1.5w/cm2 respectively. The number of CAM vessels in each group was 60.19 + 20.30,55.61 + 21.79,56.20 + 6.88,1.5w group, and the difference was significant (P0.01) compared with that of the blank control group (P0.01). When the concentration of hematoporphyrin was 0 g/ml, 25 g/ml, 50 u g/ml, 75 g/ml, there was no significant difference between the number of CAM vessels in the experimental group and the blank control group (P0.01). It showed that the pure blood porphyrin had no effect on the formation of the neovascularization of CAM; when the fixation frequency was 1MHZ, the irradiation time was 90s, the sound intensity was 1.5w/cm2, each group of blood was 1.5w/cm2. When the concentration of porphyrin was 0 g/ml, 25 g/ml, 50 u g/ml, 75 g/ml, the difference of the number of CAM vessels in the experimental groups was 35.69 + 9.45,25.44 + 6.31,17.62 + 5.11., respectively, compared with the control group. (P0.01), the 50 mu g/ml was not statistically significant compared with the 75 micron g/ml group. The formation of neovascularization has a significant effect, and its inhibitory effect is the largest when the concentration of hematoporphyrin reaches 50 mu g/ml.
Conclusion: the best parameters of ultrasonic activation of hematoporphyrin in the inhibition of CAM neovascularization in chicken embryo are ultrasonic intensity of 1.5 w/cm2, irradiation time 90S, and blood porphyrin concentration of 50 g/ml..
The second part is to activate the hematoporphyrin by ultrasound to inhibit the neovascularization of chorioallantoic membrane.
Objective: To observe and detect the inhibitory effect of sonodynamic effect of ultrasound activated hematoporphyrin on neovascularization.
Methods: the inhibitory effect of ultrasound activated hematoporphyrin sonodynamic action on the angiogenesis of CAM was observed by the grading method, DFY software and HE staining.
Results: the grade evaluation showed that the blood vessel growth status of the chicken embryos in each group was different, and the difference between the blank group and the blood porphyrin group had no significant statistical significance (P0.01), indicating that the simple hematoporphyrin had no influence on the angiogenesis, and there was significant difference in the difference between the simple ultrasound group and the blood porphyrin SDT group (P0. 01) the effect of the simple ultrasound group and the hematoporphyrin SDT group on the formation of new blood vessels on the chicken embryo CAM was obviously inhibited, and the 3+ and 4+ in the hematoporphyrin SDT group were significantly higher than those in the simple ultrasound group, and the difference was statistically significant (P < 0.01), indicating that the inhibitory effect of the blood porphyrin SDT group on the angiogenesis effect was stronger than that in the simple ultrasound group; DFY data showed that the blank control was a blank control. Group, the percentage of blood vessel area in the simple hematoporphyrin group, the simple ultrasound group and the blood porphyrin SDT group were 88.88 + 7.43,85.67 + 9.42,74.82 + 16.92,60.83 + 11.63. compared with the blank control group. The difference of the blood porphyrin SDT group was statistically significant (P0.01), and the difference between the simple ultrasound group and the blood porphyrin SDT group was significant. Statistical significance (P0.01); there was no significant difference between the simple hematoporphyrin group and the blank control group; the HE staining data showed that the blood vessel density in the blank group, the simple hematoporphyrin group, the simple ultrasound group and the blood porphyrin SDT group were 8.50 + 2.57,7.21 + 2.05,4.82 + 1.43,1.59 + 0.26., respectively, compared with the blank control group, and the simple ultrasound group and the hematoporphyrin SDT There was significant difference in the difference between the group and the group (P0.01). The difference between the simple ultrasound group and the blood porphyrin SDT group was statistically significant (P0.01). There was no significant difference in the difference between the simple hematoporphyrin group and the blank group (P0.01).
Conclusion: sonodynamic activation of hematoporphyrin significantly inhibits neovascularization of chick chorioallantoic membrane.
The third part is to study the mechanism of sonodynamic effect of ultrasound activated porphyrin on chorioallantoic membrane angiogenesis.
Objective: To observe and detect the mechanism of sonodynamic effect of ultrasound activated hematoporphyrin on the formation of chick chorioallantoic membrane neovascularization.
Methods: VEGF immunohistochemical method was used to observe the mechanism of ultrasound activated hematoporphyrin sonodynamic inhibition on chick embryo chorioallantoic membrane neovascularization.
Results: the immunohistochemical data showed that the average optical density of VEGF in the blank group, the hematoporphyrin group, the simple ultrasound group and the hematoporphyrin SDT group was 0.46 + 0.07,0.40 + 0.10,0.26 + 0.05,0.14 0.07., respectively, compared with the blank control group, and there was no significant difference between the blood porphyrin group and the blank control group (P0.01), and the difference between the hematoporphyrin SDT group and the group of pure ultrasound group was statistically significant. Significance (P0.01); the difference between the simple ultrasound group and the blood porphyrin SDT group was statistically significant (P0.01). Conclusion: the sonographic activation of the hematoporphyrin by ultrasound has a significant inhibition on the expression of VEGF.
This may be one of the mechanisms that inhibit the formation of neovascularization in chick chorioallantoic membrane.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R312;R73-36

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