發(fā)布時(shí)間：2018-05-29 01:58

  本文選題：心肌梗死 + 脂肪間充質(zhì)干細(xì)胞��； 參考：《北京協(xié)和醫(yī)學(xué)院》2013年博士論文

【摘要】：本課題研究分為兩個(gè)部分。第一部分：研究探索人和小型豬脂肪間充質(zhì)干細(xì)胞獲取、培養(yǎng)、增殖及應(yīng)用CM-Dil標(biāo)記干細(xì)胞的方法和可行性。研究人脂肪間充質(zhì)干細(xì)胞體外分化的能力。研究探討經(jīng)CM-Dil標(biāo)記的人和小型豬脂肪間充質(zhì)干細(xì)胞對(duì)小型豬急性心梗后心肌缺血損傷的治療效果。在第一部分的研究結(jié)果的基礎(chǔ)上,為了進(jìn)一步研究人和小型豬的脂肪間充質(zhì)干細(xì)胞治療小型豬急性心梗后心肌缺血損傷的機(jī)制,進(jìn)行了第二部分的研究。研究探索干細(xì)胞移植后在梗死區(qū)存活、分化及旁分泌作用的情況。 第一部分：脂肪間充質(zhì)干細(xì)胞治療急性心梗后的心室重構(gòu)與心功能評(píng)價(jià)目的： 心肌梗死的主要特點(diǎn)是心臟的血供減少,導(dǎo)致有功能的心臟細(xì)胞壞死。已經(jīng)成為目前社會(huì)主要致病和致死的原因。心臟僅有很有限的自我更新的能力,心肌梗死的不良預(yù)后會(huì)導(dǎo)致心功能衰竭。脂肪間充質(zhì)干細(xì)胞是一種新興的干細(xì)胞,具有分化為多胚層組織細(xì)胞的能力。本研究探討培養(yǎng)、增殖和CM-Dil標(biāo)記脂肪間充質(zhì)干細(xì)胞的可行性,觀察其體外分化的情況。研究探索人和小型豬脂肪間充質(zhì)干細(xì)胞移植后,對(duì)小型豬急性心梗后心室重構(gòu)的治療效果和心臟功能的評(píng)價(jià)。 方法： 收集人和小型豬的脂肪組織,體外培養(yǎng)、增殖脂肪間充質(zhì)干細(xì)胞并且用CM-Dil標(biāo)記。流式細(xì)胞分析儀分析培養(yǎng)出的人脂肪干細(xì)胞表面細(xì)胞標(biāo)志物。應(yīng)用特殊培養(yǎng)基體外誘導(dǎo)人脂肪間充質(zhì)干細(xì)胞分化。 手術(shù)結(jié)扎左冠狀動(dòng)脈前降支制作小型豬急性心梗模型,隨機(jī)分為五組。1.空白組(n=6),直接關(guān)胸,不予任何處理。2.對(duì)照組(n=6),心梗區(qū)及邊緣注射PBS。3.實(shí)驗(yàn)組一(n=6),心梗區(qū)及邊緣注射人脂肪間充質(zhì)干細(xì)胞。4.實(shí)驗(yàn)組二(n=6),心梗區(qū)及邊緣注射同種異體脂肪間充質(zhì)干細(xì)胞。5.實(shí)驗(yàn)組三(n=6),心梗區(qū)及邊緣注射自體脂肪間充質(zhì)干細(xì)胞。 1.手術(shù)前、手術(shù)后2小時(shí)和手術(shù)后6周心臟超聲學(xué)檢查。 2.手術(shù)后2小時(shí)和手術(shù)后6周心肌灌注核素掃描檢查。 3.手術(shù)后6周心梗區(qū)心肌組織Masson三色染色觀察存活心肌量。統(tǒng)計(jì)學(xué)分析比較上述檢測(cè)結(jié)果數(shù)據(jù)。 結(jié)果 人和小型豬的脂肪間充質(zhì)干細(xì)胞均生長(zhǎng)良好,CM-Dil染色明顯。流式細(xì)胞分析儀檢測(cè)發(fā)現(xiàn)培養(yǎng)的人脂肪間充質(zhì)干細(xì)胞符合國(guó)際細(xì)胞治療協(xié)會(huì)(ISCT)對(duì)間充質(zhì)干細(xì)胞的細(xì)胞表型做出的規(guī)定。并且可以體外分化為成熟脂肪細(xì)胞和軟骨細(xì)胞。 干細(xì)胞移植6周后,實(shí)驗(yàn)組一,實(shí)驗(yàn)組二和實(shí)驗(yàn)組三舒張期梗死區(qū)室壁厚度,收縮期梗死區(qū)室壁厚度及心室壁增厚率明顯高于空白組和對(duì)照組(p0.001)。 干細(xì)胞移植6周后,實(shí)驗(yàn)組一、實(shí)驗(yàn)組二和實(shí)驗(yàn)組三心肌灌注面積較前均有改善,且三組之間灌注面積改善無明顯差異(p=0.98)�？瞻捉M和對(duì)照組心肌灌注面積較前具有惡化,兩組之間無明顯差異(p=0.69)。 實(shí)驗(yàn)組一、實(shí)驗(yàn)組二和實(shí)驗(yàn)組三存活心肌量較空白組和對(duì)照組明顯增加,差異具有顯著統(tǒng)計(jì)學(xué)意義(p0.001)。實(shí)驗(yàn)組三存活心肌量較實(shí)驗(yàn)組一、二稍好,差異沒有統(tǒng)計(jì)學(xué)意義(p=0.555)。 結(jié)論 人和小型豬脂肪間充質(zhì)干細(xì)胞獲取,培養(yǎng)及擴(kuò)增簡(jiǎn)單,易于操作。移植脂肪間充質(zhì)干細(xì)胞后不僅可以阻止左心室擴(kuò)張、梗死區(qū)室壁變薄和心室重構(gòu),還可以改善梗死區(qū)缺血心肌的血液灌注。 第二部分：脂肪間充質(zhì)干細(xì)胞修復(fù)急性心肌缺血損傷作用的機(jī)制 目的 在第一部分的基礎(chǔ)上,研究探討脂肪間充質(zhì)干細(xì)胞移植后修復(fù)急性心肌缺血損傷作用的機(jī)制。包括分化為成熟心肌細(xì)胞和血管內(nèi)皮細(xì)胞的能力；旁分泌作用分泌細(xì)胞因子的能力；抑制細(xì)胞凋亡的能力。 方法 結(jié)扎左冠狀動(dòng)脈前降支制作小型豬急性心梗模型,隨機(jī)分為五組。1.空白組(n=6),直接關(guān)胸,不予任何處理。2.對(duì)照組(n=6),心梗區(qū)及邊緣注射PBS。3.實(shí)驗(yàn)組一(n=6),心梗區(qū)及邊緣注射人脂肪間充質(zhì)干細(xì)胞。4.實(shí)驗(yàn)組二(n=6),心梗區(qū)及邊緣注射同種異體脂肪間充質(zhì)干細(xì)胞。5.實(shí)驗(yàn)組三(n=6),心梗區(qū)及邊緣注射自體脂肪間充質(zhì)干細(xì)胞。6周后進(jìn)行以下檢測(cè)： 1) RT-PCR檢測(cè)VEGF、 vWF、 TGF-β3、 HGF、 CXCR4及SDF-1mRNA表達(dá)水平,量化并分析比較結(jié)果。 2)vWF免疫組化染色觀察新生血管密度,TUNEL染色觀察細(xì)胞凋亡情況,量化并分析比較結(jié)果。 3)免疫熒光染色觀察干細(xì)胞移植后存活細(xì)胞量,量化并分析比較結(jié)果；觀察分化成心肌細(xì)胞和血管內(nèi)皮細(xì)胞的情況；觀察新生心肌與原有存活心肌之間形成閏盤連接的情況以及促進(jìn)梗死區(qū)細(xì)胞增殖的情況。 結(jié)果 1)實(shí)驗(yàn)組一、實(shí)驗(yàn)組二和實(shí)驗(yàn)組三VEGF、 vWF、 TGF-β3、 HGF、 CXCR4及SDF-1mRNA表達(dá)水平較空白組和對(duì)照組明顯增加(p0.001),三個(gè)實(shí)驗(yàn)組之間差異沒有統(tǒng)計(jì)學(xué)意義(p=0.97)。 2)實(shí)驗(yàn)組一、實(shí)驗(yàn)組二和實(shí)驗(yàn)組三新生血管密度較空白組和對(duì)照組明顯增加(p0.001)。三個(gè)實(shí)驗(yàn)組之間差異沒有統(tǒng)計(jì)學(xué)意義(p=0.86)。實(shí)驗(yàn)組一、實(shí)驗(yàn)組二和實(shí)驗(yàn)組細(xì)胞凋亡數(shù)明顯低于較空白組和對(duì)照組(p0.001),三個(gè)實(shí)驗(yàn)組之間差異沒有統(tǒng)計(jì)學(xué)意義(p=0.58)。 3)三個(gè)實(shí)驗(yàn)組都有較大量脂肪干細(xì)胞存活,實(shí)驗(yàn)組二和實(shí)驗(yàn)組三存活細(xì)胞量明顯高于實(shí)驗(yàn)組一p0.001)。實(shí)驗(yàn)組二和實(shí)驗(yàn)組三中移植的脂肪間充質(zhì)干細(xì)胞可分化成心肌細(xì)胞和血管內(nèi)皮細(xì)胞,而且新生心肌之間可以形成閏盤連接。未觀察到實(shí)驗(yàn)組一干細(xì)胞分化的情況。實(shí)驗(yàn)組一、實(shí)驗(yàn)組二和實(shí)驗(yàn)組三都觀察到梗死區(qū)有明顯的細(xì)胞增殖,而且發(fā)現(xiàn)CM-Dil陰性細(xì)胞增殖的情況。 結(jié)論 脂肪間充質(zhì)干細(xì)胞移植后可以增加梗死區(qū)新生血管密度,抑制細(xì)胞凋亡；通過分泌細(xì)胞因子促進(jìn)梗死區(qū)新生血管生成,動(dòng)員自身的干細(xì)胞,誘導(dǎo)其分化修復(fù)心肌損傷。同種異體和自體脂肪間充質(zhì)干細(xì)胞移植后可以分化成心肌細(xì)胞和血管內(nèi)皮細(xì)胞,并且形成閏盤連接。
[Abstract]:This study was divided into two parts . The first part : The study explored the method and feasibility of obtaining , culturing , proliferating and applying CM - Dil - labeled stem cells to human and small pig fat mesenchymal stem cells .

The first part : The purpose of the evaluation of ventricular remodeling and cardiac function after treatment of acute myocardial infarction with adipose - derived mesenchymal stem cells :

The main feature of myocardial infarction is that the blood supply of the heart is reduced , which leads to the necrosis of cardiac cells . The heart has limited self - renewal ability , and the poor prognosis of myocardial infarction can lead to heart failure .

Method :

The adipose tissue of human and minipigs was collected , cultured in vitro , expanded to mesenchymal stem cells and labeled with CM - Dil . Flow cytometry was used to analyze the surface cell markers of human adipose - derived stem cells . The differentiation of human adipose - derived mesenchymal stem cells was induced by special culture medium .

The acute myocardial infarction model of small pigs was made by operation ligation of left anterior descending branch of left coronary artery . The model was divided into five groups : 1 . blank group ( n = 6 ) , direct closure and no treatment . 2 . The control group ( n = 6 ) , myocardial infarction area and edge injected PBS . 4 . The experimental group ( n = 6 ) , myocardial infarction area and the edge injected with allogeneic fat mesenchymal stem cells . 5 . The experimental group 3 ( n = 6 ) , myocardial infarction area and edge injected autologous fat mesenchymal stem cells .

1 . Pre - operative , 2 - hour post - surgery , and 6 - week post - operative cardiac ultrasound examination .

2 . After 2 hours after surgery and 6 weeks after surgery , the myocardial perfusion radionuclide scan was examined .

3 . The myocardial tissue of myocardial tissue in the myocardial tissue of 6 weeks after operation was used to observe the survival myocardium . Statistical analysis compared the data of the above - mentioned detection results .

Results

The mesenchymal stem cells of human and minipigs were well - grown , and CM - Dil staining was clear . The flow cytometry showed that cultured human adipose - derived mesenchymal stem cells were in line with the cell phenotype of mesenchymal stem cells by the International Association of Cellular Therapy ( ISCT ) .

After 6 weeks of stem cell transplantation , the thickness of ventricular wall , wall thickness and wall thickness of ventricular wall in experimental group 1 , experimental group 2 and experimental group were significantly higher than those in blank group and control group ( p 0.001 ) .

After 6 weeks of transplantation of the stem cells , the myocardial perfusion area of the experimental group 1 , the experimental group 2 and the experimental group was improved , and there was no significant difference between the three groups ( p = 0.98 ) . The myocardial perfusion area in the blank group and the control group was worse than that in the control group , there was no significant difference between the two groups ( p = 0.69 ) .

There was no significant difference between the experimental group and the experimental group ( p = 0.555 ) .

Conclusion

Human and small pig fat mesenchymal stem cells are obtained , cultured and amplified simply , and are easy to operate . After transplantation of the mesenchymal stem cells , the left ventricle can be prevented from expanding , the wall thinning of the infarct zone and the remodeling of the ventricle can be prevented , and the blood perfusion of the ischemic myocardium in the infarct zone can be improved .

The second part : the mechanism of the repair of acute myocardial ischemia injury by adipose - derived mesenchymal stem cells

Purpose

On the basis of the first part , the mechanism of repairing acute myocardial ischemia injury after transplantation of adipose - derived mesenchymal stem cells was investigated .
The ability to secrete cytokines by parasecretory action ;
Ability to inhibit apoptosis .

method

The acute myocardial infarction model of small pigs was made by ligation of left anterior descending branch of left coronary artery . The model was divided into five groups . 1 . Blank group ( n = 6 ) , myocardial infarction , no treatment . 2 . The control group ( n = 6 ) , myocardial infarction area and the edge injected with PBS . 4 . The experimental group 2 ( n = 6 ) , myocardial infarction area and edge injected autologous fat mesenchymal stem cells . 5 . The experimental group 3 ( n = 6 ) , myocardial infarction area and edge injected autologous fat mesenchymal stem cells . The following tests were performed after 6 weeks :

1 ) RT - PCR was used to detect the expression level of VEGF , VWF , TGF - 尾3 , HGF , CXCR4 and SDF - 1 mRNA , quantify and analyze the results .

2 ) Immunocytochemistry staining was used to observe the density of neovascularization , TUNEL staining was used to observe the apoptosis of the cells , quantify and analyze the results .

3 ) observing the survival cell quantity after the stem cell transplantation by immunofluorescence staining , quantifying and analyzing the comparison result ;
Observe the differentiation into myocardial cells and vascular endothelial cells ;
To observe the relationship between the neonatal myocardium and the original viable myocardium , and to promote the proliferation of the cells in the infarct zone .

Results

1 ) There was no significant difference between the three experimental groups ( p = 0.97 ) . There was no significant difference between the three experimental groups ( p = 0.97 ) in the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the experimental group , the

2 ) There was no significant difference between the three experimental groups ( p = 0.86 ) . There was no significant difference between the three experimental groups ( p = 0.86 ) . There was no significant difference between the experimental groups ( p = 0 . 58 ) .

3 ) There were a lot of adipose - derived stem cells in the three experimental groups , and the three survival cells in the experimental group and the experimental group were significantly higher than that in the experimental group ( P < 0.001 ) . The adipose - derived mesenchymal stem cells transplanted in the experimental group 2 and the experimental group were differentiated into cardiomyocytes and vascular endothelial cells , and the intercalated discs could be formed between the neonatal myocardium . The experimental group 1 , the experimental group 2 and the experimental group 3 observed the obvious cell proliferation in the infarct zone , and found that the CM - Dil - negative cell proliferation was observed .

Conclusion

After transplantation of adipose - derived mesenchymal stem cells , the density of neovascular endothelial cells in the infarct zone can be increased , and the apoptosis of the cells can be inhibited ;
It is possible to differentiate into cardiomyocytes and vascular endothelial cells after transplantation of allogeneic and autologous adipose - derived mesenchymal stem cells , and to form intercalated disk connections .
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R542.22

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 ;Transplantation of autologous adipose-derived stem cells ameliorates cardiac function in rabbits with myocardial infarction[J];Chinese Medical Journal;2007年04期

2 Raquel Taléns-Visconti;Ana Bonora;Ramiro Jover;Vicente Mirabet;Francisco Carbonell;José Vicente Castell;María José Gómez-Lechón;;Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells[J];World Journal of Gastroenterology;2006年36期

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本文編號(hào)：1949058