本文選題：造影劑微泡 + 超聲空化效應(yīng)　； 參考：《廣州醫(yī)科大學(xué)》2014年碩士論文

【摘要】：背景 肝臟組織結(jié)構(gòu)脆弱，血流十分豐富，且其體積較大，位置比較固定，因此受到暴力打擊時(shí)容易發(fā)生損傷破裂。 應(yīng)對(duì)肝破裂的急救，臨床上主要采用外科手術(shù)治療，包括紗布?jí)K填塞法、電凝止血法、氬氣止血法、單純縫合修補(bǔ)、肝動(dòng)脈栓塞或結(jié)扎術(shù)、肝部分切除術(shù)等。雖然效果顯著，但是對(duì)患者有較大的手術(shù)損傷，而且，手術(shù)治療常需要一定的設(shè)備及技術(shù)力量的保障，一些危急患者常來(lái)不及進(jìn)行臨床救治就死亡了。通過(guò)超聲造影引導(dǎo)的微創(chuàng)止血方法有止血?jiǎng)┚植孔⑸浞�、�?jīng)皮射頻凝固法、微波凝固法、高強(qiáng)度聚焦超聲法（HIFU）等。這些微創(chuàng)的方法可以獲得較好的止血效果，并且恢復(fù)時(shí)間較短。但是他們都具有一定的局限性。其中，止血?jiǎng)⿲?duì)級(jí)別較大的創(chuàng)傷出血療效并不滿意，且會(huì)引起疼痛。其他三種方法都是通過(guò)局部升溫致組織產(chǎn)生凝固樣壞死來(lái)實(shí)現(xiàn)止血目的。但是當(dāng)局部靶組織溫度迅速升達(dá)70℃以上時(shí)，易引起實(shí)質(zhì)臟器或周圍組織結(jié)構(gòu)變形。而且，因?yàn)榇嬖跓岢列?yīng)使治療效果不佳或治療時(shí)間延長(zhǎng)。另外，上述止血法只能實(shí)現(xiàn)點(diǎn)對(duì)點(diǎn)的止血，這對(duì)較大范圍的出血面治療具有一定的局限性。而HIFU治療過(guò)程中，空化效應(yīng)可以促使凝血塊或均質(zhì)物形成，使溫度升高至80℃以上，從而加強(qiáng)熱效應(yīng)止血作用。 超聲聯(lián)合造影劑微泡不僅能引導(dǎo)上述微創(chuàng)止血過(guò)程，而且激發(fā)的空化效應(yīng)可毀損微小血管而阻斷內(nèi)臟器官的血流（即血管毀損術(shù)），從而實(shí)現(xiàn)對(duì)肝、脾整齊切割傷的止血作用。然而，這些切割傷為I級(jí)損傷，其創(chuàng)面整齊，出血量較少且不穩(wěn)定，無(wú)法排除兩創(chuàng)面相接觸而形成的壓迫性止血及局部血凝塊形成止血作用。以上述研究為基礎(chǔ)，本實(shí)驗(yàn)對(duì)肝組織的另一創(chuàng)傷類型——肝臟斷裂傷（Ⅱ級(jí)外傷）進(jìn)行止血治療，通過(guò)改善非聚焦性脈沖超聲空化治療儀參數(shù)，進(jìn)一步探討超聲聯(lián)合微泡治療的可行性及病理機(jī)制。 目的 超聲聯(lián)合微泡可以阻斷正常肝臟、脾臟的血液循環(huán)，對(duì)肝、脾整齊的切割傷輻照治療后，其創(chuàng)面出血明顯減少。但上述創(chuàng)傷止血實(shí)驗(yàn)的模型為Ⅰ級(jí)外傷，無(wú)法排除兩創(chuàng)面相接觸形成的壓迫性止血及局部血凝塊形成的止血作用�；谝陨涎芯浚緦�(shí)驗(yàn)建立二級(jí)外傷模型——兔肝斷裂傷，通過(guò)觀察新型脈沖式超聲空化治療儀聯(lián)合微泡造影劑脂氟顯對(duì)其創(chuàng)面的止血療效，進(jìn)一步探討超聲聯(lián)合微泡作為一種無(wú)創(chuàng)方法治療肝斷裂傷的可行性以及止血的病理學(xué)機(jī)制。 方法 健康成年雄性新西蘭大白兔14只。14只實(shí)驗(yàn)新西蘭大白兔隨機(jī)分成3組，即超聲微泡組(MEUS)6只、單純治療超聲組(TUS)4只、單純微泡組(MB)組4只。 建立肝斷裂傷模型：麻醉實(shí)驗(yàn)兔并建立靜脈通道，將兔固定于實(shí)驗(yàn)臺(tái)，開(kāi)腹后并將肝臟牽拉出腹腔，剪斷肝臟左葉遠(yuǎn)端制作肝斷裂傷模型。 分組實(shí)驗(yàn)方法：（1）MEUS組：用治療探頭直接垂直輻照傷口創(chuàng)面5min，治療開(kāi)始的4min內(nèi)靜脈勻速推注造影劑微泡，治療時(shí)緩慢勻速移動(dòng)探頭以均勻輻照全整個(gè)傷口；（2）TUS組：經(jīng)耳緣靜脈勻速推注2ml生理鹽水替代造影劑微泡，同時(shí)使用脈沖式超聲治療儀以同樣的方法輻照肝創(chuàng)面；（3）MB組，經(jīng)耳緣靜脈通道4分鐘內(nèi)推注相同劑量的造影劑微泡，同時(shí)超聲治療儀探頭使用同樣的方法置于肝創(chuàng)面假照（但不發(fā)射脈沖）。 創(chuàng)傷出血評(píng)分方法：（1）視覺(jué)評(píng)分法：輻照前后，通過(guò)肉眼觀察，對(duì)創(chuàng)面出血情況進(jìn)行觀察并評(píng)分。（2）稱重法：輻照前后，用吸水濾紙收集創(chuàng)面30s出血量，精密天平進(jìn)行稱重，各組輻照前后出血的差值為創(chuàng)面出血的凈重。 超聲造影灌注情況：輻照治療前后均對(duì)實(shí)驗(yàn)肝進(jìn)行超聲造影，觀察靶區(qū)的造影劑灌注情況進(jìn)行分級(jí)，并進(jìn)行聲學(xué)密度分析（PI,AUC）。 病理學(xué)檢查：治療結(jié)束后，隨機(jī)抽取MEUS組三只實(shí)驗(yàn)兔及所有對(duì)照組進(jìn)行病理學(xué)檢查，余下MEUS組三只實(shí)驗(yàn)兔抗感染飼養(yǎng)48小時(shí)后取輻照靶區(qū)的組織進(jìn)行病理學(xué)檢查。 統(tǒng)計(jì)學(xué)方法：輻照前后出血速度、聲學(xué)密度分析（PI、AUC）的數(shù)據(jù)以平均數(shù)Mean±SD表示，輻照前后的出血視覺(jué)評(píng)分以中位數(shù)Median（Q25，Q75）表示。各組輻照前后的出血速度、峰值強(qiáng)度（PI）、曲線下面積（AUC）的組內(nèi)比較采用配對(duì)t檢驗(yàn)（Paired-Samples T Test），各組之間對(duì)應(yīng)的輻照前后出血速度、PI、AUC的組間比較采用單向方差分析(one-way ANOVA)及多個(gè)均數(shù)間兩兩比較的LSD法進(jìn)行檢驗(yàn)。各組出血視覺(jué)評(píng)價(jià)輻照前后的組內(nèi)比較采用Wilcoxon秩和檢驗(yàn)，各組之間對(duì)應(yīng)的輻照前后出血視覺(jué)評(píng)分組間比較采用Kruskal-Wallis H test及Mann-Whitney U進(jìn)行檢驗(yàn)。輻照前后出血速度與PI、AUC之間，PI與AUC之間采用Pearson分析。輻照前后出血速度與出血視覺(jué)判斷之間采用Spearman分析。P0.05表示為差異有統(tǒng)計(jì)學(xué)意義。所有數(shù)據(jù)均采用SPSS13.0軟件分析。 結(jié)果 一、止血效果 1．出血視覺(jué)評(píng)價(jià)：MEUS組輻照治療后即刻觀察，出血視覺(jué)評(píng)分下降，由4（3,4）分降至1（0.75,1），與治療前比較差異有統(tǒng)計(jì)學(xué)意義（P0.05）；分別與TUS組、MB組治療后比較差異有統(tǒng)計(jì)學(xué)意義（P0.05） 2．出血速度評(píng)價(jià)：將肝創(chuàng)面30s的出血總量除以30s得到平均出血速度。輻照前所有實(shí)驗(yàn)肝出血速度計(jì)量基本一致，三組數(shù)據(jù)兩兩比較差異無(wú)統(tǒng)計(jì)學(xué)意義（P0.05）。輻照后，MEUS組出血速度明顯下降,由0.013±0.004降至0.002±0.001g/s，與治療前比較差異有統(tǒng)計(jì)學(xué)意義（P0.05）；分別與TUS組、MB組治療后比較差異有統(tǒng)計(jì)學(xué)意義（P0.05） 二、超聲造影 1．視覺(jué)判斷：治療前各組肝臟對(duì)應(yīng)區(qū)域、TUS組和MB組的輻照區(qū)域的造影劑灌注良好，評(píng)分為0級(jí)。MEUS組輻照后可見(jiàn)造影劑沿著肝大血管稍緩慢不均勻地進(jìn)入肝組織中，呈明顯大小不等不規(guī)則的充盈缺損區(qū)，，缺損區(qū)的邊緣灌注較低，評(píng)分為Ⅱ級(jí)。 2．聲學(xué)密度分析：輻照前各組峰值強(qiáng)度、曲線下面積比較差異無(wú)顯著意義（P0.05）。輻照后MEUS組的峰值強(qiáng)度（PI）由22.000±6.090%降至9.517±1.380%，曲線下面積(AUC)由2173.983±839.190%s降至872.583±196.409%s，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；分別與TUS組、MB組輻照后的組間比較差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 3．相關(guān)性分析：肝創(chuàng)面輻照后出血速度分別與曲線下面積、峰值強(qiáng)度之間呈正相關(guān)(P0.05)；輻照前后的峰值強(qiáng)度與曲線下面積之間、輻照后視覺(jué)評(píng)分與出血速度之間呈正相關(guān)(P0.05)。 三、病理所見(jiàn) 光鏡下，TUS組、MB組輻照后肝索、肝板結(jié)構(gòu)清楚，肝竇大小均勻，肝竇間見(jiàn)散在紅細(xì)胞分布。MEUS組輻照后病變區(qū)域四種病變混合存在，主要表現(xiàn)為：1肝細(xì)胞渾濁腫脹，壓閉竇狀間隙及肝竇間隙，以臨近創(chuàng)傷面明顯；2廣泛的竇狀間隙、肝竇間及小靜脈內(nèi)出現(xiàn)了大量的紅細(xì)胞淤積，此為稍遠(yuǎn)離創(chuàng)傷面的主要表現(xiàn)；3部分匯管區(qū)結(jié)締組織可見(jiàn)明顯出血；4肝組織出現(xiàn)散在的小灶樣壞死，并見(jiàn)少量炎細(xì)胞浸潤(rùn)。MEUS組飼養(yǎng)48小時(shí)后肉眼見(jiàn)肝臟創(chuàng)傷面灰白，光滑無(wú)出血（即出血速度視覺(jué)評(píng)分0分）；光鏡下所見(jiàn)，在距創(chuàng)傷面約1.5cm范圍內(nèi)的壞死灶較治療后即刻增大，壞死灶外周為變性細(xì)胞帶包繞，變性細(xì)胞區(qū)見(jiàn)少許炎細(xì)胞浸潤(rùn)，病變區(qū)域呈地圖樣。病變區(qū)域與正常肝組織間存在明顯的分界，靠近病變區(qū)域肝組織的肝竇內(nèi)可見(jiàn)少量紅細(xì)胞分布。 結(jié)論 1、超聲聯(lián)合微泡可以有效地阻斷肝臟局部血液循環(huán)。 2、超聲聯(lián)合微泡對(duì)肝斷裂傷輻照治療，使創(chuàng)面出血量減少10倍以上，超聲聯(lián)合微泡有潛力成為臨床治療肝臟斷裂傷（Ⅱ級(jí)損傷）的一種新型、有效、無(wú)創(chuàng)的止血治療方法。 3、超聲聯(lián)合微泡可以實(shí)現(xiàn)創(chuàng)面幾乎停止，出血主要機(jī)制可能是肝細(xì)胞與小血管壁不同程度損傷，肝竇及竇間隙受壓及匯管區(qū)周圍組織出血壓迫等致使靶區(qū)血管的血流停滯或阻斷。 4、本研究使用的超聲治療儀及造影劑微泡輕巧便攜，有潛力成為臨床應(yīng)用于現(xiàn)場(chǎng)急救的一種新方法。
[Abstract]:background
Liver tissue is fragile, blood flow is very rich, and its volume is large and its location is relatively fixed. Therefore, it is easy to break and break when it is attacked by violence.
First aid to cope with liver rupture, surgical treatment is mainly used in clinic, including gauze block filling, electrocoagulation hemostasis, argon gas hemostasis, simple suture repair, hepatic artery embolization or ligation and partial hepatectomy. Although the effect is significant, there are large surgical injuries to the patients, and surgical treatment often requires certain equipment and technique. These minimally invasive methods, such as local injection of hemostatic agents, percutaneous radiofrequency coagulation, microwave coagulation, high intensity focused ultrasound (HIFU), and so on. These minimally invasive methods can obtain better hemostatic effects and recover time. It is short, but they all have some limitations. Among them, the hemostatic agent is not satisfied with the higher level of traumatic bleeding and causes pain. The other three methods are caused by the local warming to produce coagulation necrosis to achieve the aim of hemostasis. But when the temperature of the target tissue of the authorities rapidly rises above 70 centigrade, it is easy to cause the substance. The structure of the viscera or surrounding tissue is deformed. Moreover, because of the existence of heat sink effect, the treatment effect is not good or the time of treatment is prolonged. In addition, the above hemostasis can only achieve point to point hemostasis, which has certain limitations for a large range of bleeding surface treatment. In the course of HIFU treatment, the cavitation effect can cause the formation of clots or homogenate, Raising the temperature to above 80 degrees, thereby enhancing the heat effect of hemostasis.
Ultrasound combined with contrast media can not only guide the above-mentioned minimally invasive hemostasis, but also stimulate the cavitation effect to destroy the tiny blood vessels and block the blood flow of the viscera (vascular damage), thus achieving the hemostasis effect on the liver and spleen neatly cut. However, these cuts are I injury, the wound is neat, the amount of bleeding is less and unstable. The oppressive hemostasis and local blood clot formed by two facial contact can not be excluded. Based on the above study, the experiment has done a hemostatic treatment for another type of liver injury, the liver fracture injury (grade II trauma), and further explores the ultrasonic combination by improving the parameters of the non focused pulse ultrasonic cavitation therapy instrument. The feasibility and pathological mechanism of combined microbubble therapy.
objective
Ultrasound combined with microbubbles can block the normal liver, the blood circulation of the spleen, and the wound bleeding of the liver and spleen neatly cut after irradiation treatment, the bleeding of the wound is obviously reduced. However, the model of the trauma hemostasis experiment is a grade I trauma, which can not exclude the hemostatic effect of the compression hemostasis and the formation of local blood clot formed by two facial contact. Based on the above research, In this experiment, we established a two stage model of traumatic injury of rabbit liver. By observing the hemostatic effect of the new pulse ultrasonic cavitation therapy instrument combined with microbubble contrast agent lipofluorine, the feasibility of ultrasonic combined microbubble as a noninvasive method for the treatment of liver fracture and the pathological mechanism of hemostasis were further explored.
Method
A healthy adult male New Zealand white rabbit 14.14 rabbits were divided into 3 groups randomly, that is, 6 ultrasound microbubbles (MEUS), 4 only treated group (TUS), and 4 group of simple microbubble group (MB).
The model of liver fracture injury was established: the rabbits were anaesthetized and the venous channel was established. The rabbit was fixed on the experimental platform. After opening the abdomen, the liver was pulled out of the abdominal cavity, and the model of liver broken laceration was made by cutting the left lobe of the liver.
Group experiment methods: (1) group MEUS: directly irradiate the wound surface 5min with a therapeutic probe, treatment of the initial 4min intravenous injection of contrast agent microbubbles at the beginning of the treatment, and the slow and uniform moving probe at the time of treatment to irradiate the whole wound evenly; (2) group TUS: 2ml physiological saline was injected into the auricular vein to replace the contrast agent microbubbles, and the pulse was used at the same time. The same method was used to irradiate the liver wound with the same method. (3) in group MB, the same dose of contrast microbubbles were injected within 4 minutes of the auricular vein channel, and the ultrasonic therapeutic probe was used in the same method for the liver wound (but not the pulse).
The scoring method of traumatic bleeding score: (1) visual scoring method: before and after irradiation, the bleeding of the wound was observed and graded by the naked eye. (2) before and after irradiation, the amount of 30s bleeding was collected with the absorbent filter paper, the precision balance was weighed, and the difference between the blood and the blood was the net weight of the wound bleeding before and after irradiation.
Ultrasound contrast perfusion: the experimental liver was contrast-enhanced before and after irradiation, and the contrast agent perfusion in the target area was observed and the acoustic density analysis (PI, AUC) was performed.
Pathological examination: after the treatment, three rabbits were randomly selected from the MEUS group and all the control groups were examined for pathology. The remaining three rabbits in the remaining group of the MEUS rabbits were reared for 48 hours, and the tissues of the irradiated target area were examined for pathological examination.
Statistical methods: the velocity of bleeding before and after irradiation, the data of PI (AUC) was expressed as an average of Mean + SD. The visual score of bleeding before and after irradiation was expressed as Median (Q25, Q75). The rate of bleeding, peak intensity (PI) before and after irradiation, and the area under the curve (AUC) were compared to the paired t test (Paired-Samples T). In each group, the velocity of bleeding before and after irradiation, the comparison of PI, AUC between groups using one-way ANOVA (one-way ANOVA) and the LSD method of 22 comparison among multiple mean numbers, each group of hemorrhagic vision was compared with Wilcoxon rank and test before and after irradiation, and the corresponding inter group ratio of visual scoring before and after irradiation was compared. Kruskal-Wallis H test and Mann-Whitney U were compared. The bleeding speed before and after irradiation, between PI, AUC, PI and AUC were analyzed by Pearson. The difference between the bleeding speed and the visual judgement before and after irradiation was statistically significant. All the data were analyzed by the software.
Result
First, the effect of hemostasis
1. visual evaluation of hemorrhage: immediate observation after irradiation treatment in group MEUS, the visual score of bleeding decreased from 4 (3,4) to 1 (0.75,1), and the difference was statistically significant (P0.05) compared with that before treatment (P0.05), and there was a significant difference between group TUS and group MB (P0.05).
2. evaluation of bleeding speed: the average bleeding rate was obtained by dividing the total amount of hemorrhage of 30s in the liver wound by 30s. All the experimental liver bleeding rates before irradiation were basically the same. There was no statistical difference between the three groups of data 22 (P0.05). After irradiation, the bleeding rate of the MEUS group decreased significantly from 0.013 + 0.004 to 0.002 + 0.001g/s, compared with the pre treatment comparison. The difference was statistically significant (P0.05), and there was significant difference between group TUS and group MB after treatment (P0.05).
Two, contrast-enhanced ultrasound
1. visual judgment: the contrast medium of the liver in each group before treatment, the contrast agent in the irradiated area of group TUS and group MB was well perfused, and the grade 0.MEUS group was irradiated, and the contrast agent entered the liver tissue slightly and slowly along the large blood vessels of the liver. Class II.
2. acoustic density analysis: there was no significant difference between the peak intensity of each group before irradiation and the area under the curve (P0.05). The peak intensity (PI) of group MEUS decreased from 22 + 6.090% to 9.517 + 1.380%, and the area under the curve (AUC) decreased from 2173.983 + 839.190%s to 872.583 + 196.409%s, and the difference was statistically significant (P0.05), respectively, with TUS group and MB group, respectively. The difference between groups after irradiation was statistically significant (P0.05).
3. correlation analysis: there was a positive correlation between the bleeding speed of the liver wound after irradiation and the area under the curve and the peak intensity (P0.05), and between the peak intensity before and after irradiation and the area under the curve, there was a positive correlation between the visual score and the speed of bleeding after irradiation (P0.05).
Three, pathological findings
Under the light microscope, group TUS and group MB irradiated the hepatic cord, the structure of the liver was clear, the size of the hepatic sinusoid was uniform, and the hepatic sinus was scattered in the red blood cell group.MEUS after irradiation, and four kinds of lesions were mixed in the lesion area. The main manifestations were: 1 the turbid swelling of the liver cells, the closed sinus space and the hepatic sinus space, which were adjacent to the traumatic surface; 2 extensive sinus spaces, and the hepatic sinusoids. A large amount of red blood cell deposition was found in the small vein, which was the main manifestation of a little far away from the wound surface; the 3 parts of the connective tissue of the 3 part of the pipe showed obvious bleeding, 4 liver tissue appeared scattered small focal necrosis, and a small amount of inflammatory cells infiltrated the.MEUS group after 48 hours to see the gray white of the wound surface of the liver. In the light microscope, the necrotic foci in the area of about 1.5cm of 1.5cm were increased immediately after the treatment. The necrotic foci were wrapped around the denatured cells, and the degeneration cell area was infiltrated with small inflammatory cells. The lesion area was map like. The lesion area and normal liver tissue were in distinct boundary, close to the hepatic sinusoid of the liver tissue in the lesion area. A small amount of red blood cell distribution was found.
conclusion
1, ultrasound combined with microbubbles can effectively block the local blood circulation in the liver.
2, ultrasound combined with microbubbles can reduce the amount of bleeding of the wound by 10 times. Ultrasound combined with microbubbles has the potential to be a new, effective and noninvasive method of hemostasis for the treatment of liver fracture injury (grade II injury).
3, ultrasonic combined with microbubbles can almost stop the wound. The main mechanism of bleeding may be that the liver cells and the small vessel wall are damaged in different degrees. The pressure of the hepatic sinusoid and the sinus space and the compression of the tissue around the tube area lead to the stagnation or blocking of the blood flow in the target vessels.
4, the ultrasound therapeutic apparatus and contrast agent microbubbles used in this study are portable and portable, and have potential to be a new method for clinical first-aid in the field.

【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R445.1;R657.3

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