發(fā)布時(shí)間：2018-06-24 03:43

  本文選題：顱內(nèi)動(dòng)脈瘤 + 動(dòng)物模型　； 參考：《蘇州大學(xué)》2012年博士論文

【摘要】：第一部分顱內(nèi)動(dòng)脈瘤模型制作的研究 目的：探尋建立穩(wěn)定、快捷、理想的顱內(nèi)動(dòng)脈瘤動(dòng)物模型的方法，同時(shí)進(jìn)一步證明顱內(nèi)動(dòng)脈瘤的發(fā)生、生長(zhǎng)、塑形機(jī)制。 方法：選取48只新西蘭大白兔，隨機(jī)分為A、B、C、D、E五組，A組:新西蘭大白兔8只，結(jié)扎對(duì)側(cè)頸總動(dòng)脈+2%鹽水喂養(yǎng)；B組:兔8只，分叉內(nèi)膜損傷法+A組法；C組:兔10只，胰彈力蛋白酶浸泡法+A組法；D組:兔12只，分叉內(nèi)膜損傷法+胰彈力蛋白酶浸泡法+A組法；E組:兔10只，分叉內(nèi)膜損傷法+胰彈力蛋白酶浸泡法。檢測(cè)A、D、E三組術(shù)前、術(shù)后、術(shù)后4周的血壓；各組于術(shù)后4周行CTA頸部血管造影，然后活體解剖左頸總動(dòng)脈分叉造瘤部位，記錄有無(wú)成瘤情況，并測(cè)量瘤體大��；處死動(dòng)物，切取瘤體標(biāo)本，行病理學(xué)HE染色，光鏡下觀察病理組織學(xué)變化。 結(jié)果：A、D、E三組血壓變化情況：A、D組術(shù)后和術(shù)后4周與術(shù)前相比P0.0l，有顯著性差異；E組術(shù)后和術(shù)后4周與術(shù)前相比P0.0l，無(wú)顯著性差異；A、D組術(shù)后和術(shù)后4周與E組術(shù)后和術(shù)后4周相比P0.0l，有顯著性差異。各組成瘤率比較：C、D、E組間p0.05無(wú)顯著性差異;C、D、E組與A、B組均p0.05有顯著性差異。形成動(dòng)脈瘤體積的比較：D組與E組高度、寬度相比P0.05有顯著性差異,長(zhǎng)度相比P0.05無(wú)顯著性差異;C組與D組長(zhǎng)度、高度、寬度相比均P0.05有顯著性差異。病理組織學(xué)變化:正常動(dòng)脈壁由內(nèi)膜、中膜和外膜構(gòu)成，，各層結(jié)構(gòu)保持完整。動(dòng)脈瘤壁上沒(méi)有內(nèi)膜，結(jié)構(gòu)排列紊亂，瘤壁變薄或厚薄不一，有炎性細(xì)胞浸潤(rùn)。 結(jié)論:頸總動(dòng)脈分叉內(nèi)膜損傷法+胰彈力蛋白酶浸泡+血流動(dòng)力因素誘導(dǎo)法成瘤率相對(duì)偏高，死亡率低，并可獲得體積、病理形態(tài)學(xué)與人類(lèi)顱內(nèi)動(dòng)脈瘤極為相似的動(dòng)脈瘤模型，操作簡(jiǎn)單，損傷小，成瘤周期短，性能穩(wěn)定可靠，通暢率好，制作費(fèi)用低，可作為臨床前期實(shí)驗(yàn)治療腦動(dòng)脈瘤和栓塞材料研究的良好模具。同時(shí)也證實(shí)了顱內(nèi)動(dòng)脈瘤的發(fā)生、生長(zhǎng)塑形及破裂是多種因素綜合作用的結(jié)果。 第二部分PDGF-b、c-Jun、Caspase-3在人腦動(dòng)脈瘤，兔動(dòng)脈瘤模型中的表達(dá)及意義 目的：探討PDGF-b、c-Jun、Caspase-3在人腦動(dòng)脈瘤、兔動(dòng)脈瘤模型中的表達(dá)情況，揭示其在腦動(dòng)脈瘤形成中的潛在作用機(jī)制。 方法：采用免疫組織化學(xué)方法中的Envision法對(duì)22例人顱內(nèi)動(dòng)脈瘤、8例新西蘭大白兔動(dòng)脈瘤模型、6例正常腦動(dòng)脈和6例正常兔頸總動(dòng)脈的石蠟切片標(biāo)本進(jìn)行檢測(cè)PDGF-b、c-Jun、Caspase-3的表達(dá)。用免疫組化laserpix圖像分析系統(tǒng)進(jìn)行圖像分析，獲取PDGF-b、c-Jun、Caspase-3表達(dá)的平均光密度值(mean density)進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果：在22例人顱內(nèi)囊狀動(dòng)脈瘤中，PDGF-b的表達(dá)：強(qiáng)染色6例，明顯染色10例，輕度染色4例，無(wú)染色2例。正常腦動(dòng)脈無(wú)染色。平均光密度值0.272。c-Jun的表達(dá)：強(qiáng)染色12例，明顯染色7例，輕度染色2例，無(wú)染色1例。正常腦動(dòng)脈2例輕度染色。平均光密度值0.437。Caspase-3的表達(dá)：強(qiáng)染色7例，明顯染色9例，輕度染色4例，無(wú)染色2例。正常腦動(dòng)脈無(wú)染色。平均光密度值0.284。統(tǒng)計(jì)學(xué)分析顯示：PDGF-b、c-Jun、Caspase-3與對(duì)照組比較P0.01；PDGF-b與c-Jun的相關(guān)分析:r=0.751，兩者表達(dá)呈正相關(guān)；c-Jun與Caspase-3的相關(guān)分析:r=0.812，兩者表達(dá)呈正相關(guān)。此類(lèi)兔動(dòng)脈瘤模型標(biāo)本PDGF-b、c-Jun、Caspase-3較對(duì)照組均顯示較強(qiáng)表達(dá)，對(duì)照組均陰性表達(dá)。 結(jié)論：PDGF-b、c-Jun、Caspase-3參與腦動(dòng)脈瘤的發(fā)生、生長(zhǎng)、破裂演變過(guò)程，腦動(dòng)脈瘤的形成是多因素綜合作用的結(jié)果，各因素間存在相互影響、相互關(guān)聯(lián)、相互作用。核轉(zhuǎn)錄因子c-Jun可能是顱內(nèi)動(dòng)脈瘤發(fā)生的病理學(xué)因素中血管活性生長(zhǎng)因子、凋亡、酶學(xué)系列變化、炎性反因等損傷機(jī)制中的共同中間環(huán)節(jié)。為臨床開(kāi)發(fā)阻斷顱內(nèi)動(dòng)脈瘤發(fā)生、破裂的新靶點(diǎn)抑制劑提供理論依據(jù)。 第三部分流體切應(yīng)力對(duì)動(dòng)脈內(nèi)皮細(xì)胞PDGF-b、c-Jun、Caspase-3mRNA表達(dá)的影響和意義 目的:探討高流體切應(yīng)力作用下動(dòng)脈內(nèi)皮細(xì)胞PDGF-b、c-Jun、Caspase-3mRNA的表達(dá)水平和規(guī)律,揭示血流動(dòng)力學(xué)因素在顱內(nèi)動(dòng)脈瘤形成演變過(guò)程中的具體分子病理學(xué)機(jī)制. 方法:選擇人胸主動(dòng)脈血管內(nèi)皮細(xì)胞培養(yǎng)、傳代作為內(nèi)皮細(xì)胞功能研究的細(xì)胞源，應(yīng)用體外高切應(yīng)力流場(chǎng)加載機(jī),分別將各組內(nèi)皮細(xì)胞置于切應(yīng)力加載機(jī)的流室腔槽內(nèi)。a.施加高強(qiáng)度流體切應(yīng)力(40dyne/cm2)分別作用0h、1h、4h、6h,然后應(yīng)用RT-PCR技術(shù)檢測(cè)不同作用時(shí)間點(diǎn)內(nèi)皮細(xì)胞PDGF-b、c-Jun、Caspase-3的轉(zhuǎn)錄水平和規(guī)律；b.施加正常強(qiáng)度流體切應(yīng)力(20dyne/cm2)4h仍應(yīng)用RT-PCR技術(shù)檢測(cè)內(nèi)皮細(xì)胞PDGF-b、c-Jun、Caspase-3的轉(zhuǎn)錄水平；c.比較同時(shí)間點(diǎn)（4h）高強(qiáng)度流體切應(yīng)力與正常強(qiáng)度流體切應(yīng)力PDGF-b、c-Jun、Caspase-3的轉(zhuǎn)錄水平的差異性。 結(jié)果：高切應(yīng)力作用下，PDGF-b組：1小時(shí)組內(nèi)皮細(xì)胞PDGF-b轉(zhuǎn)錄水平就顯著升高，4小時(shí)、6小時(shí)較1小時(shí)組有回落，且存在統(tǒng)計(jì)學(xué)差異，但仍較未加載切應(yīng)力(0h)有顯著差異，而6小時(shí)后又有回升趨勢(shì);c-Jun組：未加載切應(yīng)力的內(nèi)皮細(xì)胞組c-Jun mRNA表達(dá)幾乎為零，而在1h、4h、6h時(shí)間點(diǎn)的高切應(yīng)力加載組漸進(jìn)性、與時(shí)間呈正相關(guān)性升高，與未加載切應(yīng)力組比較有顯著差異；隨著切應(yīng)力作用時(shí)間的延長(zhǎng)，6h、4h、1h各組比較均有顯著性差異;Caspase-3組：未加載流場(chǎng)的對(duì)照組(0h組)內(nèi)皮細(xì)胞也檢測(cè)到相對(duì)中等量的Caspase-3mRNA表達(dá)，而在40dyne/cm2的高切應(yīng)力作用下，1h組表達(dá)量明顯升高，較對(duì)照組(0h組)有顯著性差異；但在4h組下降到對(duì)照組水平以下，較對(duì)照組(0h組)亦有顯著性差異；6h組有回升趨勢(shì)，升高到對(duì)照組(0h組)水平以上，仍接近對(duì)照組水平，比較無(wú)統(tǒng)計(jì)學(xué)差異。1h組較4h組、6h組有顯著性差異。 同時(shí)間點(diǎn)（4h）不同強(qiáng)度切應(yīng)力比較：無(wú)流體切應(yīng)力、正常強(qiáng)度流體切應(yīng)力、高強(qiáng)度流體切應(yīng)力PDGF-b、c-Jun的轉(zhuǎn)錄水平的差異有顯著性p0.05;4h時(shí)點(diǎn)Caspase-3組隨著切應(yīng)力增強(qiáng)漸下降且p0.05有顯著性差異。 結(jié)論：高強(qiáng)度的流體切應(yīng)力可誘導(dǎo)人胸主動(dòng)脈內(nèi)皮細(xì)胞內(nèi)PDGF-b mRNA、c-JunmRNA、Caspase-3mRNA的轉(zhuǎn)錄表達(dá)。而且隨著作用時(shí)間延長(zhǎng)1h、4h、6h時(shí)間點(diǎn)各有其特征性表達(dá)規(guī)律。結(jié)合本文第二部分結(jié)果可推測(cè)出高流體切應(yīng)力可能通過(guò)調(diào)節(jié)PDGF-b、c-Jun、Caspase-3的表達(dá)水平，進(jìn)而誘導(dǎo)顱內(nèi)動(dòng)脈瘤的發(fā)生、形成塑形和破裂。在我們的實(shí)驗(yàn)研究中Caspase-3的表達(dá)水平在無(wú)流體切應(yīng)力、正常強(qiáng)度流體切應(yīng)力、高強(qiáng)度流體切應(yīng)力4h時(shí)點(diǎn)隨著切應(yīng)力增強(qiáng)漸下降且有顯著性差異，提示我們需進(jìn)行更多更長(zhǎng)時(shí)點(diǎn)進(jìn)一步深入的研究，來(lái)揭示其作用表達(dá)規(guī)律。
[Abstract]:The study of the first part of the model of intracranial aneurysm
Objective: To explore a stable, quick and ideal animal model of intracranial aneurysms, and further prove the occurrence, growth and shaping mechanism of intracranial aneurysms.
Methods: 48 New Zealand white rabbits were randomly divided into groups of A, B, C, D, E five, group A: 8 New Zealand white rabbits, 8 rabbits were ligated to the lateral common carotid artery, +2% brine, 8 rabbits in group B, and +A group method of bifurcation intima injury, 10 rabbits, 10 rabbits and trypsin immersion method, 12 rabbits, bifurcation intima injury and trypsin immersion method Group E: group E: Rabbit 10, bifurcation intima injury and trypsin soaking. Test the blood pressure of group A, D, E after operation, postoperative, 4 weeks postoperatively; each group performed CTA neck angiography at 4 weeks after operation, then dissected the part of the left common carotid artery bifurcation, recorded the tumor formation, and measured the size of the tumor; the animals were killed and the tumor body marks were executed. The pathological changes were observed by HE staining and histopathological changes were observed under light microscope.
Results: A, D, E three groups of blood pressure changes: A, D group postoperative and 4 weeks after the operation compared with preoperative P0.0l, there were significant differences; E group postoperative and 4 weeks after the operation compared with preoperative P0.0l, no significant difference; A, D group and 4 weeks after the operation and E group, compared with 4 weeks after the 4 weeks, there were significant differences. The significant difference between C, D, E group and A and B group. The comparison of the volume of the aneurysm: the height of the D group and the E group, the width compared with P0.05, there is no significant difference in the length compared with the P0.05; the C group is significantly different from the length, height and width of the D group. The pathological changes: the normal arterial wall is from the intima, middle The membrane and outer membrane are made up. The structure of each layer is intact. There are no intima on the wall of the aneurysm, the structure is arranged in disorder, the wall of the aneurysm is thin or thick, and there is inflammatory cell infiltration.
Conclusion: the common carotid artery branched intima damage method, pancreatic elastase immersion + hemodynamic factor induction method is relatively high, the mortality is low, the volume is low, the pathomorphology and the human intracranial aneurysm are very similar to the aneurysm model. The operation is simple, the injury is small, the cycle of the tumor is short, the performance is stable, the patency rate is good, the cost of patency is good and the production fee is good. Low use can be used as a good mold for the treatment of cerebral aneurysms and embolic materials in preclinical trials. It has also confirmed the occurrence of intracranial aneurysms, and the growth, shaping and rupture are the results of a variety of factors.
The second part is the expression and significance of PDGF-b, c-Jun and Caspase-3 in human cerebral aneurysms and rabbit aneurysm models.
Objective: To investigate the expression of PDGF-b, c-Jun and Caspase-3 in human cerebral aneurysms and rabbit aneurysm models, and to reveal their potential mechanisms in the formation of cerebral aneurysms.
Methods: the expression of PDGF-b, c-Jun and Caspase-3 in 22 human intracranial aneurysms, 8 New Zealand white rabbit aneurysm models, 6 normal cerebral arteries and 6 normal rabbits' common carotid artery were detected by Envision method, and the image analysis was obtained by immunohistochemical laserpix image analysis system. The average optical density (mean density) expressed by PDGF-b, c-Jun and Caspase-3 was analyzed statistically.
Results: in 22 cases of human intracranial saccular aneurysms, the expression of PDGF-b: strong staining in 6 cases, obvious staining in 10 cases, mild staining in 4 cases, without staining in 2 cases, normal cerebral artery without staining. The expression of mean light density value 0.272.c-Jun: strong staining 12 cases, obvious dyeing 7 cases, mild staining 2 cases, no dyeing 1 cases. 2 mild coloring and average light density of normal cerebral artery. The expression of degree value 0.437.Caspase-3: strong staining in 7 cases, obvious staining in 9 cases, mild staining in 4 cases, non staining in 2 cases and normal cerebral artery without staining. The mean light density value 0.284. statistical analysis showed that PDGF-b, c-Jun, Caspase-3 were compared with the control group P0.01; r=0.751, the correlation between PDGF-b and c-Jun was positive correlation; c-Jun and Caspase-3. The correlation analysis: r=0.812, the expression of the two was positive correlation. The rabbit aneurysm model specimens PDGF-b, c-Jun, Caspase-3 were more expressed than the control group, and the control group were all negative.
Conclusion: PDGF-b, c-Jun and Caspase-3 are involved in the occurrence, growth, and rupture process of cerebral aneurysm. The formation of cerebral aneurysms is the result of multiple factors. There are interrelated, interrelated and interrelated factors between the various factors. The nuclear factor c-Jun may be a vasoactive growth factor and apoptosis in the pathological factors of intracranial aneurysm. It provides a theoretical basis for the development of new target inhibitors for the development of intracranial aneurysms and rupture of intracranial aneurysms.
The third part is the effect of fluid shear stress on the expression of PDGF-b, c-Jun and Caspase-3mRNA in arterial endothelial cells.
Objective: To explore the expression level and regularity of PDGF-b, c-Jun and Caspase-3mRNA in arterial endothelial cells under high fluid shear stress, and to reveal the specific molecular pathological mechanism of hemodynamic factors in the process of the formation and evolution of intracranial aneurysms.
Methods: the endothelial cell culture of human thoracic aorta was selected as the source of endothelial cell function study, and the high shear stress field loading machine was applied in vitro. The high intensity fluid shear stress (40dyne/cm2) was applied to.A., 1H, 4h, 6h respectively in the flow chamber slot of the shear stress loader. Then, RT-PCR was applied to RT-PCR, respectively, and then RT-PCR was applied to RT-PCR, respectively. The transcriptional level and regularity of PDGF-b, c-Jun, Caspase-3 of endothelial cells at different time points were measured, and B. applied to normal intensity fluid shear stress (20dyne/cm2) 4H to detect the transcription level of PDGF-b, c-Jun, Caspase-3 of endothelial cells, and C. compare the shear stress of high intensity fluid with normal intensity fluid at the same time point (4h). The differences in transcriptional level of PDGF-b, c-Jun and Caspase-3.
Results: under high shear stress, group PDGF-b: the PDGF-b transcriptional level of endothelial cells in the 1 hour group increased significantly, 4 hours and 6 hours compared with the 1 hours group, and there was a statistical difference, but there was a significant difference compared with the unloaded shear stress (0h), but there was a rebound trend after 6 hours; group c-Jun: c-Jun mRNA of the endothelial cell group that did not load the shear stress. The expression was almost zero, while the progressive stress loading group in 1H, 4H and 6h time points was progressive, with a positive correlation with time, and significant difference compared with the unloaded shear stress group. With the prolongation of the shear stress action time, there were significant differences in 6h, 4H and 1H groups; Caspase-3 group: the control group (0h group) of the unloaded flow field was also endothelial cells. The expression of relatively medium Caspase-3mRNA was detected, and the expression of 1H group increased significantly under the high shear stress of 40dyne/cm2, compared with the control group (0h group), but in 4H group decreased to the control group, compared with the control group (0h group), there was a significant difference between the control group and the control group (Group 0h); the 6h group had a rising trend and increased to the level of the control group (0h group). There was no significant difference between the.1h group and the 4H group. There was significant difference between the 6h group and the control group.
The difference of shear stress at the same time point (4h): no fluid shear stress, normal strength fluid shear stress, high intensity fluid shear stress PDGF-b, c-Jun transcriptional level difference is significant P0.05; 4H time point Caspase-3 group gradually decreases with shear stress and P0.05 has significant difference.
Conclusion: high intensity fluid shear stress can induce the transcriptional expression of PDGF-b mRNA, c-JunmRNA, Caspase-3mRNA in human thoracic aorta endothelial cells. Moreover, with the time of action prolonging 1H, 4h, 6h point, there are characteristic expression rules. Combined with the results of the second part of this article, we can speculate that the high fluid shear stress may be regulated by PDGF-b, c-Jun, Casp. The expression level of ase-3, which leads to the occurrence of intracranial aneurysms, leads to formation and rupture. In our experimental study, the expression level of Caspase-3 is in the absence of fluid shear stress, normal strength fluid shear stress, and high intensity fluid shear stress 4H time points with the shear stress increasing gradually decreasing and significant difference, suggesting that we need to carry on more longer. Further study is carried out to reveal its function and expression pattern.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R743;R-332

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8 王中;金s

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