本文選題：心肌梗死 + 干細(xì)胞�。� 參考：《南方醫(yī)科大學(xué)》2016年博士論文

【摘要】：隨著人們生活水平的提升,近年來(lái)心血管疾病,特別是心肌梗死的發(fā)病率呈逐年上升趨勢(shì),心肌梗死仍然是我國(guó)致死率和致殘率最高的疾病之一。盡管近年來(lái)人們對(duì)心肌梗死的認(rèn)識(shí)和診療水平都取得了長(zhǎng)足進(jìn)展,血管重建術(shù)(包括經(jīng)皮冠脈成形術(shù)和冠脈旁路移植術(shù))和相關(guān)藥物的使用很大程度上降低心肌梗死患者的死亡率,但這些措施無(wú)法解決心肌梗死后心肌細(xì)胞喪失這一關(guān)鍵問(wèn)題。干細(xì)胞移植治療心肌梗死是最有希望使心肌再生的方法,是近十余年來(lái)該研究領(lǐng)域的熱點(diǎn),未來(lái)發(fā)展的主要方向之一。骨髓間充質(zhì)干細(xì)胞(Mesenchymal stem cells, MSCs)由于其獲取和擴(kuò)增方便,有多向分化能力,可以自體移植及異體移植有免疫豁免功能等優(yōu)點(diǎn)而倍受青睞,是近年來(lái)用于臨床研究最常見(jiàn)的干細(xì)胞之一。但是,臨床試驗(yàn)結(jié)果表明,干細(xì)胞移植治療后遠(yuǎn)期獲益不明顯,全因死亡率等主要臨床終點(diǎn)改善不佳。究其原因主要是干細(xì)胞移植后面臨心肌局部血流的沖擊和細(xì)胞壞死后產(chǎn)生的過(guò)氧化物損傷,導(dǎo)致干細(xì)胞的滯留率和存活率都很低。如能為移植的干細(xì)胞提供一個(gè)暫時(shí)的“安身的場(chǎng)所”,并對(duì)干細(xì)胞的功能進(jìn)行調(diào)控,增強(qiáng)其抵抗心梗局部過(guò)氧化微環(huán)境損傷,則有希望提高干細(xì)胞的存活和滯留,從而發(fā)揮其應(yīng)有的作用�？勺⑸湫孕募〗M織工程(Injectable cardiac tissue engineering)是將可注射性支架材料通過(guò)心內(nèi)膜或心外膜局部注射到心肌病變位置進(jìn)行治療。一般是指具有三維網(wǎng)絡(luò)樣結(jié)構(gòu)的水凝膠,具有一定的韌性和粘稠度,富含水分達(dá)95%以上,可以攜帶藥物或者細(xì)胞進(jìn)行治療。研究表明,水凝膠可以作為細(xì)胞移植的載體,能夠粘附包裹移植細(xì)胞,在一定程度上減少因?yàn)闈B漏或者血流沖擊造成的細(xì)胞流失,增加細(xì)胞在目標(biāo)位點(diǎn)的滯留時(shí)間和數(shù)量而發(fā)揮其應(yīng)有的作用；另外,用可注射性水凝膠進(jìn)行心肌組織工程治療可以增加室壁厚度,為心室壁提供一個(gè)機(jī)械支撐力,從而減少心室張力,改善心室重構(gòu),改善心功能,當(dāng)水凝膠的機(jī)械力學(xué)性質(zhì)類(lèi)似于健康心肌,這個(gè)效果更顯著。有生物學(xué)功能的生物材料能夠根據(jù)心梗后的病理生理變化及干細(xì)胞功能需求進(jìn)一步優(yōu)化,近年來(lái)受到研究者的青睞,如促進(jìn)血管新生的功能性多肽水凝膠、具有靶向作用的生物納米材料、具有抗氧化作用的水凝膠等。冠脈梗塞后由于局部心肌缺血缺氧,或者由于血管重建再通導(dǎo)致的再灌注損傷,心肌細(xì)胞及成纖維細(xì)胞等在代謝過(guò)程中可以產(chǎn)生一系列活性氧簇(Reactive oxygen species, ROS),主要包括：O2-、H2O2、HO2·和·OH等,對(duì)心肌細(xì)胞和外源性移植的干細(xì)胞都可以造成嚴(yán)重?fù)p傷。如能開(kāi)發(fā)出具有抗氧化作用的水凝膠,不僅能發(fā)揮水凝膠的機(jī)械支撐作用,而且可以有效降低梗死區(qū)局部ROS水平,保護(hù)細(xì)胞免受損傷,提高細(xì)胞移植后的存活率。葛根素(化學(xué)名為8-p-D-葡萄糖吡喃糖-7,4'-二羥基異黃酮,puerarin)是從野葛的干燥根中提取分離得到的主要活性成分,是一種異黃酮類(lèi)化合物。研究顯示：葛根素能夠清除超氧化物陰離子,抑制由過(guò)氧化氫引起的紅細(xì)胞溶血損傷和脂質(zhì)過(guò)氧化物的生成；能保護(hù)過(guò)氧化誘導(dǎo)的內(nèi)皮細(xì)胞和PC12細(xì)胞損傷,有很好的保護(hù)細(xì)胞,抗過(guò)氧化的作用；基礎(chǔ)實(shí)驗(yàn)研究表明葛根素這種保護(hù)細(xì)胞與抗凋亡的作用的可能機(jī)制為：(1)葛根素分子可以提供質(zhì)子耦合機(jī)體過(guò)量的ROS,直接破壞H2O2分子,降低細(xì)胞內(nèi)的過(guò)氧化水平,從而減少其對(duì)細(xì)胞的損傷；(2)提高M(jìn)SCs的內(nèi)源性抗氧化酶,比如SOD酶、GSH-PX等酶的活性,提高細(xì)胞自身抗氧化防御系統(tǒng)對(duì)過(guò)氧化陰離子的清除能力；(3)葛根素能直接參與損傷細(xì)胞DNA的修復(fù)。因此,面對(duì)細(xì)胞移植后心梗局部的過(guò)氧化微環(huán)境損傷,葛根素有望能夠調(diào)控干細(xì)胞移植后的存活。如能開(kāi)發(fā)出葛根素類(lèi)水凝膠進(jìn)行心肌組織工程治療,不僅能夠?yàn)楣Ｈ笮氖姨峁C(jī)械支撐力,包載移植細(xì)胞減少細(xì)胞的流失,而且可以發(fā)揮葛根素抗氧化保護(hù)細(xì)胞的作用,進(jìn)一步增加移植細(xì)胞的滯留和存活,使其發(fā)揮最大功效。載藥水凝膠的構(gòu)建一般包括化學(xué)鍵鍵合和物理包裹的形式,在前者中藥物既成為藥物釋放的對(duì)象又是載體的一部分,能夠明顯提高藥物包載率,但是較后者在材料的選擇和制作工藝上有更高的要求。我們課題組在嘗試采用葛根素鍵合多肽構(gòu)建水凝膠的過(guò)程中發(fā)現(xiàn),葛根素本身不進(jìn)行任何的化學(xué)修飾就能夠通過(guò)簡(jiǎn)易的工藝和促發(fā)方式自組裝形成水凝膠。結(jié)合葛根素的化學(xué)結(jié)構(gòu)及其他成膠體系的成膠原理,我們推測(cè)葛根素水凝膠(Puerarin hydrogel, PG)的成膠原理可能為：葛根素分子結(jié)構(gòu)中既含有親水的葡萄吡喃糖結(jié)構(gòu)也有相對(duì)疏水的異黃酮結(jié)構(gòu),在常溫下處于微溶物質(zhì),在加熱下可以使其完全溶解,冷卻時(shí)可以通過(guò)分子間苯環(huán)堆疊力、疏水作用力和氫鍵作用自組裝形成具有三維纖維網(wǎng)絡(luò)的膠體結(jié)構(gòu)。葛根素水凝膠是迄今為止發(fā)現(xiàn)的第一種能自己形成小分子水凝膠的天然藥物。葛根素水凝膠的發(fā)現(xiàn)具有明顯的優(yōu)勢(shì)：(1)由于成份只有葛根素本身,因此其藥物包載率是100%,是藥物包載率最高的藥物載體,其藥物釋放是通過(guò)膠體的緩慢降解實(shí)現(xiàn)；(2)制作工藝和成膠方式簡(jiǎn)便；(3)通過(guò)加熱冷卻后約數(shù)分鐘成膠,為其可注射性創(chuàng)造條件。接著,課題組對(duì)葛根素水凝膠進(jìn)行研究,主要包括水凝膠的物理化學(xué)性能表征和生物學(xué)相容性檢測(cè),體外觀察葛根素水凝膠在過(guò)氧化環(huán)境下保護(hù)MSCs的能力,葛根素水凝膠攜帶MSCs體內(nèi)移植大鼠心肌梗死模型的研究。研究分以下三部分進(jìn)行：第一部分：葛根素水凝膠的物理化學(xué)性能表征和生物學(xué)相容性檢測(cè)我們對(duì)葛根素水凝膠表征時(shí)觀察到,其最低成膠濃度為1%,并且為均勻的透明頭膠體,隨著膠體濃度增高,其透明性有所減弱,6%時(shí)形成乳白色的不透明的水凝膠；通過(guò)透射電鏡表征水凝膠的自組裝形貌：葛根素水凝膠為三維網(wǎng)絡(luò)狀結(jié)構(gòu),各個(gè)濃度的水凝膠均形成較長(zhǎng)的納米纖維,纖維直徑大約為10-50nm,1%、2%和4%形成的纖維長(zhǎng)度及直徑均相似；力學(xué)性能表征發(fā)現(xiàn),葛根素水凝膠是非頻率依賴(lài)性,而是應(yīng)力依賴(lài)型膠體,2%和4%的葛根素水凝膠處于穩(wěn)定狀態(tài)時(shí)的力學(xué)強(qiáng)度約為10K.Pa,這與心肌組織的力學(xué)強(qiáng)度相似,適合進(jìn)行心肌組織工程研究；體外藥物釋放實(shí)驗(yàn)發(fā)現(xiàn)：在生理環(huán)境下,2%和4%濃度的水凝膠在前12h以較快的速率釋放葛根素藥物,藥物可以持續(xù)釋放12h以上；細(xì)胞相容性實(shí)驗(yàn)表明：CCK-8檢測(cè)結(jié)果顯示葛根素水凝膠與心肌細(xì)胞和心肌成纖維細(xì)胞均有良好的細(xì)胞相容性,無(wú)明顯細(xì)胞毒害作用；第二部分：葛根素水凝膠在過(guò)氧化環(huán)境下保護(hù)MSCs的能力研究及機(jī)制探討本實(shí)驗(yàn)采用100μmol/L H2O2干預(yù)1h作為外源性ROS模擬體內(nèi)心肌梗死后的過(guò)氧化環(huán)境。實(shí)驗(yàn)分5組進(jìn)行：正常MSCs組、H2O2干預(yù)組、H2O2+2%PG組、H2O2+4%PG組和H2O2+6%PG組。通過(guò)檢測(cè)細(xì)胞內(nèi)的SOD活性、MDA含量和ROS熒光探針DHE的水平可以反映細(xì)胞內(nèi)ROS的含量,與正常MSCs組比較,H202干預(yù)組細(xì)胞內(nèi)MDA含量和SOD活性增加,DHE紅色熒光數(shù)量明顯增加,而2%、4%和6%濃度的葛根素水凝膠可以顯著減少SOD活性和MDA含量,DHE紅色熒光明顯減少；用CCK-8檢測(cè)細(xì)胞存活和增殖,培養(yǎng)基中LDH的釋放反映細(xì)胞毒性的大小,AO/EB染色從細(xì)胞形態(tài)學(xué)上觀察細(xì)胞的存活和凋亡,Annexin V-FITC/PI雙染凋亡檢測(cè)法檢測(cè)細(xì)胞的凋亡率,這些檢測(cè)結(jié)果表明H202誘導(dǎo)后細(xì)胞存活減少、死亡增加,2%、4%和6%的葛根素水凝膠均可明顯降低氧化應(yīng)激后MSCs的凋亡率,增加細(xì)胞存活；Western blot分析顯示2%、4%和6%的葛根素水凝膠可使抑制細(xì)胞凋亡蛋白Bcl-2表達(dá)增加和促凋亡蛋白Bax和Caspase-3表達(dá)減少,從蛋白水平印證了細(xì)胞形態(tài)學(xué)的變化。這些結(jié)果均表明H202誘導(dǎo)細(xì)胞ROS的產(chǎn)生及細(xì)胞存活減少、凋亡增多,而通過(guò)2%、4%和6%的葛根素水凝膠培養(yǎng)細(xì)胞均可以抑制ROS的產(chǎn)生,保護(hù)細(xì)胞減少細(xì)胞凋亡,其機(jī)制可能是通過(guò)減少促凋亡蛋白Bax和Caspase-3的表達(dá)和增加抑制細(xì)胞凋亡蛋白Bcl-2的表達(dá)產(chǎn)生作用。第三部分：葛根素水凝膠在MSCs移植治療大鼠心肌梗死中的作用及機(jī)制研究在本部分研究中,我們將可注射性葛根素水凝膠攜帶MSCs移植治療大鼠心肌梗死模型,以觀察其對(duì)移植細(xì)胞的滯留存活、大鼠心功能和心臟結(jié)構(gòu)的改善作用。實(shí)驗(yàn)采用心臟超聲對(duì)移植后心功能的變化進(jìn)行評(píng)價(jià)；HE染色、Masson染色檢測(cè)移植后心臟組織的形態(tài)學(xué)變化,包括室壁厚度,心梗面積和心肌纖維化程度；用免疫熒光評(píng)價(jià)心梗部位心肌和血管新生情況,Western blot檢測(cè)抗凋亡蛋白和促凋亡蛋白的變化。結(jié)果發(fā)現(xiàn)：細(xì)胞移植后4周,4%PG+MSCs組較單純MSCs組的細(xì)胞滯留及存活明顯增加；移植治療術(shù)后4周,心彩超心功能檢測(cè)提示4%PG+MSCs組、MSCs組和4%PG水凝膠聯(lián)合MSCs組均能改善左室射血分?jǐn)?shù)(LVEF),增加心室短軸縮短率(FS),但4%PG+MSCs組改善更加明顯；組織形態(tài)學(xué)檢測(cè)顯示4%PG、MSCs組和4%PG+MSCs組均能改善心梗后室壁厚度,減少心梗面積,減少心肌纖維化程度,減少心肌損傷,增加血管新生,增加心肌組織數(shù)量,且葛根素水凝膠聯(lián)合MSCs治療組的效果更加明顯。綜述所述,本研究成功制備葛根素水凝膠,其成份只有葛根素,是現(xiàn)今發(fā)現(xiàn)的第一種能自組裝形成水凝膠的天然藥物,其具有與心肌組織相近的力學(xué)強(qiáng)度,能夠平穩(wěn)釋放葛根素藥物達(dá)12小時(shí)以上,具有良好的心肌細(xì)胞和心肌成纖維細(xì)胞相容性；葛根素是一種強(qiáng)抗氧化劑,可以減少H2O2誘導(dǎo)的細(xì)胞ROS水平,增加細(xì)胞存活,減少細(xì)胞凋亡率及細(xì)胞凋亡相關(guān)蛋白的產(chǎn)生,其機(jī)制可能與其抑制凋亡有關(guān)；葛根素水凝膠能夠提高移植細(xì)胞在心梗局部的滯留和存活,促進(jìn)MSCs改善心功能和心室重構(gòu)的治療效果,其機(jī)制可能是通過(guò)抗氧化,減少細(xì)胞凋亡,促進(jìn)血管新生,改善心梗微環(huán)境的作用修復(fù)心梗組織。本研究從干細(xì)胞移植治療的不足這一關(guān)鍵臨床問(wèn)題出發(fā),緊密結(jié)合生物材料學(xué)與心肌組織工程學(xué),研制了葛根素水凝膠并對(duì)其抗過(guò)氧化、保護(hù)細(xì)胞的生物學(xué)效用進(jìn)行研究,并包裹干細(xì)胞移植治療動(dòng)物心梗模型,闡明其改善心梗微環(huán)境與促進(jìn)心梗修復(fù)的作用及可能機(jī)制,對(duì)于心肌組織工程研究中支架材料的選擇和設(shè)計(jì)以及未來(lái)心肌梗死臨床治療具有重要指導(dǎo)意義。
[Abstract]:With the improvement of people's living standards, the incidence of cardiovascular disease, especially myocardial infarction, is increasing year by year. Myocardial infarction is still one of the most fatal diseases in our country. Although people have made great progress in the level of understanding and diagnosis of myocardial infarction in recent years, vascular reconstruction (including percutaneous coronary intervention) The use of coronary angioplasty and coronary bypass graft) and the use of related drugs greatly reduce the mortality of patients with myocardial infarction, but these measures do not solve the key problem of myocardial cell loss after myocardial infarction. Stem cell transplantation for myocardial infarction is the most promising method for cardiac muscle regeneration. It has been the study for more than ten years. Mesenchymal stem cells (MSCs), which is one of the most common stem cells used in recent years, is one of the most common stem cells for clinical research. The results of clinical trials showed that the long-term benefit of stem cell transplantation was not obvious, and the main clinical endpoints such as the death rate were not well improved. The main reason is that the stem cell transplantation faces the impact of local blood flow and the peroxide damage after cell necrosis, which leads to the low retention and survival rate of stem cells. The stem cells provide a temporary "safe place" and regulate the function of the stem cells to enhance their resistance to the injury of the partial peroxidation microenvironment of the myocardial infarction, so as to improve the survival and retention of the stem cells, and thus play its due role. Injectable cardiac tissue engineering is an injectable myocardium. Injectable scaffolds are treated by local injection of the endocardium or epicardium to the location of the myocardial lesion. Generally, it refers to a hydrogel with a three-dimensional network structure, with a certain toughness and consistency, more than 95% moisture content, and can be used to carry drugs or cells for treatment. Research shows that hydrogel can be used as a cell transplant. The carrier is able to adhere to the transplanted cells, to some extent, to reduce the loss of cells caused by leakage or blood flow impact, and to increase the cell's retention time and quantity at the target site. In addition, the use of injectable hydrogel for myocardial tissue engineering can increase the thickness of the ventricular wall and provide the ventricular wall. It provides a mechanical support to reduce ventricular tension, improve ventricular remodeling, and improve cardiac function. When the mechanical properties of hydrogel are similar to that of healthy myocardium, this effect is more significant. Biological materials with biological functions can be further optimized according to the pathophysiological changes after myocardial infarction and the demand for stem cell function. People have the favor, such as functional polypeptide hydrogels that promote angiogenesis, biological nanomaterials with targeted effects, hydrogels with antioxidative action, etc. after coronary infarction, cardiac myocytes and fibroblasts can be metabolized in the metabolic process due to ischemia and anoxia of local myocardium, or reperfusion injury caused by revascularization and revascularization. A series of Reactive oxygen species (ROS), including O2-, H2O2, HO2 and OH, can cause serious damage to both cardiac and exogenous stem cells. For example, the ability to develop hydrogel with antioxidant activity can not only play the mechanical support of hydrogel, but also effectively reduce the infarct area. Local ROS level protects cells from damage and improves survival after cell transplantation. Ge Gensu (chemical named 8-p-D- glucosamine -7,4'- two hydroxyl isoflavone, puerarin) is a major active component extracted from the dried root of Pueraria lobata, a isoflavone compound. Anions, which inhibit the hemolytic injury of erythrocytes and the formation of lipid peroxide caused by hydrogen peroxide, can protect endothelial cells and PC12 cells induced by peroxide, protect cells and resist peroxidation; basic experimental study shows that the possible mechanism of Puerarin protective cells and anti apoptosis is: ( 1) the Ge Gensu molecule can provide a proton coupled body overdose of ROS, which directly destroys H2O2 molecules, reduces the level of peroxidation in cells and reduces its damage to cells. (2) increase the activity of endogenous antioxidant enzymes, such as SOD enzymes, GSH-PX and other enzymes in MSCs, and improve the scavenging energy of the cell's own antioxidant defense system to the peroxidation anions. (3) (3) puerarin can be directly involved in the repair of damaged cells. Therefore, in the face of partial peroxidation microenvironment injury after transplantation, puerarin may be able to regulate the survival of stem cells after transplantation. For example, the development of Puerarin hydrogel for myocardial tissue engineering can not only provide mechanical support for the infarcted ventricles. The inclusion of transplanted cells reduces the loss of cells and can play the role of the antioxidant protective cells of puerarin to further increase the retention and survival of the transplanted cells, making it the most effective. The construction of the hydrogel usually includes the form of chemical bond bonding and physical encapsulation. In the former, the drug is the object of drug release. As a part of the carrier, it can obviously improve the drug loading rate, but the latter has a higher requirement on the selection and fabrication of the material. In the process of trying to construct the hydrogel with Puerarin bond polypeptide, we found that the puerarin itself can pass the simple process and the promoter without any chemical modification. In combination with the chemical structure of puerarin and the glue forming principle of other gelatine systems, we speculate that the gelation principle of Puerarin hydrogel (PG) may be: the structure of the puerarin molecular structure contains both the hydrophilic grape Piran structure and the hydrophobic isoflavone structure, which is in the micro solution at normal temperature. Substances, which can be completely dissolved in heating, can form a colloid structure with a three-dimensional fiber network through the intermolecular piled force, hydrophobic interaction and hydrogen bonding. The puerarin hydrogel is the first natural drug to form a small sub hydrogel. Now it has obvious advantages: (1) because the ingredient is only puerarin itself, the drug loading rate is 100%, the drug loading rate is the highest, its drug release is achieved through the slow degradation of colloid; (2) the preparation process and the gelling method is simple and simple; (3) through the heating and cooling for a few minutes after the gelation, for its injectable creation bar Then, the team studied the puerarin hydrogel, including the physical and chemical characterization and biological compatibility detection of the hydrogel, in vitro observation of the ability of the puerarin hydrogel to protect the MSCs under the peroxidation environment. The Ge Gensu hydrogel carried the rat model of myocardial infarction with MSCs in vivo. The study was divided into three parts. The first part: physical and chemical characterization and biological compatibility test of Puerarin hydrogel, we observed that the minimum gel concentration was 1% when the Ge Gensu hydrogel was characterized, and it was a uniform transparent head colloid, with the increase of colloid concentration, its transparency decreased and the opaque hydrogel formed in milk white at 6%. The self-assembled morphology of the hydrogel was characterized by transmission electron microscopy: the gelatin hydrogel was a three-dimensional network structure, and the hydrogels of each concentration formed longer nanofibers. The fiber diameter was about 10-50nm, the length and diameter of the fibers formed in 1%, 2% and 4% were all similar. The mechanical properties showed that the puerarin hydrogel was a non frequency dependence. It is a stress dependent colloid. The mechanical strength of the 2% and 4% puerarin hydrogels at a stable state is about 10K.Pa, which is similar to the mechanical strength of the myocardium. It is suitable for the study of myocardial tissue engineering. In vitro drug release experiments found that in the physiological environment, the 2% and 4% concentration hydrogels are released at a faster rate in the front 12h. Puerarin drugs could continuously release more than 12h, and the cytocompatibility test showed that the results of CCK-8 detection showed that the puerarin hydrogel had good cytocompatibility with myocardial cells and myocardial fibroblasts, and there was no obvious cytotoxic effect. The second part: the ability to protect MSCs under the peroxidation environment of Puerarin hydrogel. The study and mechanism discussion used 100 mol/L H2O2 intervention 1H as a exogenous ROS to simulate the peroxidation environment after myocardial infarction in vivo. The experiment was divided into 5 groups: normal MSCs group, H2O2 intervention group, H2O2+2%PG group, H2O2+4%PG group and H2O2+6%PG group. The activity of SOD, MDA content and ROS fluorescence probe could be reflected by the detection of the cell SOD activity. The intracellular ROS content was compared with the normal MSCs group. The intracellular MDA content and SOD activity increased in the H202 intervention group, and the number of DHE red fluorescence increased significantly. The 2%, 4% and 6% puerarin hydrogels could significantly reduce the SOD activity and MDA content, and the DHE red fluorescence decreased obviously; CCK-8 was used to detect the cell survival and proliferation, and the LDH release in the medium was reversed. The size of enantiomer toxicity, AO/EB staining was used to observe cell survival and apoptosis from cell morphology, and Annexin V-FITC/PI double staining apoptosis detection method to detect cell apoptosis rate. These results showed that H202 induced cell survival decreased and death increased. The 2%, 4% and 6% puerarin hydrogels could significantly reduce the apoptosis of MSCs after oxidative stress. Western blot analysis showed that 2%, 4% and 6% puerarin hydrogels could inhibit the increased expression of apoptotic protein Bcl-2 and decrease the expression of apoptotic protein Bax and Caspase-3. The changes of cell morphology were confirmed from protein level. These results showed that H202 induced the production of ROS in cell and the decrease of cell survival and apoptosis. Increase, and the 2%, 4% and 6% puerarin hydrogel cultured cells can inhibit the production of ROS and protect cells to reduce apoptosis. The mechanism may be by reducing the expression of apoptotic protein Bax and Caspase-3 and increasing the expression of apoptosis protein Bcl-2. The third part: the puerarin hydrogel is treated with MSCs transplantation In this part of this study, we used injectable puerarin hydrogel to carry MSCs transplantation in the treatment of rat myocardial infarction model in order to observe the survival of the transplanted cells, the function of heart and the structure of the heart in rats. The morphological changes of cardiac tissue after transplantation were detected by HE staining and Masson staining, including the thickness of the wall, the area of myocardial infarction and the degree of myocardial fibrosis. The changes of myocardial and vascular neovascularization were evaluated by immunofluorescence, and the changes of anti apoptotic protein and apoptotic egg white were detected by Western blot. The results were found to be 4 weeks after the transplantation, 4%PG+ The cell retention and survival in group MSCs was significantly higher than that in group MSCs, and 4 weeks after transplantation, cardiac function test suggested that group 4%PG+MSCs, MSCs group and 4%PG hydrogel combined MSCs group could improve left ventricular ejection fraction (LVEF) and shorten the short axis shortening rate (FS), but the improvement of the 4% PG+MSCs group was more obvious; histomorphological detection showed 4%P. G, group MSCs and group 4%PG+MSCs can improve the thickness of the ventricular wall, reduce the area of myocardial infarction, reduce the degree of myocardial fibrosis, reduce the myocardial damage, increase the angiogenesis and increase the number of myocardial tissue, and the effect of the puerarin hydrogel combined with the MSCs treatment group is more obvious. Puerarin, the first natural drug that can be self assembled to form a hydrogel, has a mechanical strength similar to that of the myocardium. It can smoothly release Puerarin for more than 12 hours and has a good compatibility between myocardial cells and myocardial fibroblasts. Puerarin is a strong antioxidant and can reduce H2O2 induction. Cell ROS level can increase cell survival, reduce cell apoptosis rate and apoptosis related protein production, and its mechanism may be related to its inhibition.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R542.22

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