本文選題：雞胚絨毛尿囊膜 + 超聲��； 參考：《重慶醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 第一部分超聲激活血卟啉聲動(dòng)力作用抑制絨毛尿囊膜新生血管形成的參數(shù)優(yōu)化 目的:觀察超聲激活血卟啉聲動(dòng)力作用對(duì)雞胚絨毛尿囊膜新生血管形成的影響并優(yōu)化其實(shí)驗(yàn)參數(shù)。 方法:采用所選發(fā)育天數(shù)的雞胚進(jìn)行超聲激活血卟啉聲動(dòng)力作用抑制絨毛尿囊膜新生血管形成的實(shí)驗(yàn),將CAM標(biāo)本用數(shù)碼相機(jī)拍照后,通過(guò)血管生成計(jì)數(shù)法檢測(cè)空白對(duì)照組、單純超聲組、單純血卟啉組及血卟啉SDT組對(duì)雞胚絨毛尿囊膜(CAM)新生血管的影響,并以此確定超聲參數(shù)和血卟啉劑量,觀察超聲激活血卟啉聲動(dòng)力作用抑制雞胚CAM新生血管生成的最佳參數(shù)。加入血卟啉時(shí)應(yīng)嚴(yán)格暗室避光操作。 結(jié)果:血管計(jì)數(shù)法顯示:當(dāng)固定頻率為1MHz,聲強(qiáng)為1.5W,輻照時(shí)間為0s、30s、60s、90s時(shí),各實(shí)驗(yàn)組CAM血管數(shù)量分別為60.08±18.48、62.27±26.02、43.66±9.38、30.19±8.75,可見(jiàn)單純超聲對(duì)CAM血管生成有抑制作用,且抑制作用隨輻照時(shí)間增加而增強(qiáng);當(dāng)固定頻率為1MHZ,輻照時(shí)間為90S ,聲強(qiáng)各組分別為0w/cm2、0.5w/cm2、1.0w/cm2、1.5w/cm2,各實(shí)驗(yàn)組CAM血管數(shù)量分別為60.19±20.30、55.61±21.79、56.20±22.46、37.98±6.88,1.5w組與空白對(duì)照組比較差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01),可見(jiàn)當(dāng)單純超聲輻照聲強(qiáng)為1.5w/cm2時(shí),對(duì)CAM血管生成有抑制作用;當(dāng)單純血卟啉濃度為0μg/ml、25μg/ml、50μg/ml、75μg/ml時(shí),各實(shí)驗(yàn)組CAM血管數(shù)量與空白對(duì)照組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.01)。說(shuō)明單純血卟啉對(duì)CAM新生血管的生成無(wú)影響;當(dāng)固定頻率為1MHZ,輻照時(shí)間為90s ,聲強(qiáng)為1.5w/cm2,各組血卟啉濃度為0μg/ml、25μg/ml、50μg/ml、75μg/ml時(shí),各實(shí)驗(yàn)組CAM血管數(shù)量分別為35.69±9.45、25.44±8.93、17.07±6.31、17.62±5.11。各實(shí)驗(yàn)組與對(duì)照組相比較差異有統(tǒng)計(jì)學(xué)意義,(P0.01),其中50μg/ml與75μg/ml組相比較差異無(wú)統(tǒng)計(jì)學(xué)意義,(P0.01)。說(shuō)明血卟啉SDT對(duì)CAM新生血管的生成有明顯影響,當(dāng)血卟啉濃度達(dá)50μg/ml時(shí)其抑制效應(yīng)最大。 結(jié)論:超聲激活血卟啉抑聲動(dòng)力作用制雞胚CAM新生血管的最佳參數(shù)為超聲輻照聲強(qiáng)為1.5 w/cm2 ,輻照時(shí)間為90S,血卟啉濃度為50μg/ml。本實(shí)驗(yàn)選擇此最佳參數(shù)。 第二部分超聲激活血卟啉聲動(dòng)力作用抑制絨毛尿囊膜新生血管形成實(shí)驗(yàn) 目的:觀察和檢測(cè)超聲激活血卟啉聲動(dòng)力作用對(duì)新生血管形成的抑制作用。 方法:采用等級(jí)評(píng)定方法、DFY軟件、HE染色法觀察超聲激活血卟啉聲動(dòng)力作用對(duì)CAM新生血管形成的抑制作用。 結(jié)果:等級(jí)評(píng)定結(jié)果顯示:各組中雞胚的血管生長(zhǎng)狀態(tài)等級(jí)不同,經(jīng)檢驗(yàn)發(fā)現(xiàn),空白組和血卟琳組之間差異無(wú)顯著統(tǒng)計(jì)學(xué)意義(P0.01),說(shuō)明單純血卟啉基本不影響血管新生;單純超聲組和血卟啉SDT組與之相比較差異均有顯著統(tǒng)計(jì)學(xué)意義(P0.01),說(shuō)明單純超聲組和血卟啉SDT組有明顯抑制雞胚CAM上新生血管形成的作用,其中血卟啉SDT組的3+ ,4+明顯比單純超聲組增多,其差異有顯著統(tǒng)計(jì)學(xué)意義(P〈0.01),說(shuō)明血卟啉SDT組抑制血管生成效應(yīng)強(qiáng)于單純超聲組;DFY數(shù)據(jù)顯示:空白對(duì)照組、單純血卟啉組、單純超聲組、血卟啉SDT組的血管面積百分比分別為:88.88±7.43、85.67±9.42、74.82±16.92、60.83±11.63。與空白對(duì)照組相比,單純超聲組、血卟啉SDT組與之差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01);單純超聲組與血卟啉SDT組之間比較,差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01);單純血卟啉組與空白對(duì)照組比較差異無(wú)顯著統(tǒng)計(jì)學(xué)意義; HE染色數(shù)據(jù)顯示:空白組、單純血卟啉組、單純超聲組、血卟啉SDT組的血管密度分別為8.50±2.57、7.21±2.05、4.82±1.43、1.59±0.26。與空白對(duì)照組相比,單純超聲組、血卟啉SDT組與之差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01),單純超聲組與血卟啉SDT組之間比較,差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01),單純血卟啉組與空白組比較差異無(wú)顯著統(tǒng)計(jì)學(xué)意義(P0.01)。 結(jié)論:超聲激活血卟啉聲動(dòng)力作用顯著抑制雞胚絨毛尿囊膜新生血管形成。 第三部分超聲激活血卟啉聲動(dòng)力作用抑制絨毛尿囊膜新生血管形成的機(jī)理研究 目的:觀察和檢測(cè)超聲激活血卟啉聲動(dòng)力作用抑制雞胚絨毛尿囊膜新生血管形成的可能機(jī)理。 方法:應(yīng)用VEGF免疫組化的方法初步觀察超聲激活血卟啉聲動(dòng)力作用抑制雞胚絨毛尿囊膜新生血管的作用機(jī)理。 結(jié)果:免疫組化數(shù)據(jù)顯示:空白組、血卟啉組、單純超聲組、血卟啉SDT組的VEGF平均光密度分別為0.46±0.07、0.40±0.10、0.26±0.05、0.14±0.07。與空白對(duì)照組相比,血卟啉組差異無(wú)顯著統(tǒng)計(jì)學(xué)意義(P0.01);單純超聲組、血卟啉SDT組與之差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01);單純超聲組與血卟啉SDT組之間比較,差異有顯著統(tǒng)計(jì)學(xué)意義(P0.01)。結(jié)論:超聲激活血卟啉聲動(dòng)力作用對(duì)VEGF表達(dá)具有顯著的抑制作 用,這可能是其抑制雞胚絨毛尿囊膜新生血管形成的機(jī)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R312;R73-36

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