本文選題：胚胎干細(xì)胞 + iPS細(xì)胞��； 參考：《第四軍醫(yī)大學(xué)》2012年博士論文

【摘要】：胚胎干細(xì)胞（ESC）與誘導(dǎo)性多能干細(xì)胞（iPSC）由于具有自我更新和強(qiáng)大分化潛能，在心肌再生方面有著極大的應(yīng)用前景。與ESC不同，iPSC可以由病人的皮膚組織重編程獲取，經(jīng)過一系列誘導(dǎo)分化，有可能建立大批量同源心肌細(xì)胞而無倫理爭(zhēng)議和免疫排斥。因此，以iPSC高效誘導(dǎo)分化的心肌細(xì)胞替代活體心肌細(xì)胞作為種子細(xì)胞來源，采用組織工程技術(shù)體外構(gòu)建心肌組織并聯(lián)合組織移植進(jìn)行心肌修復(fù)與再生，逐漸成為干細(xì)胞心肌再生領(lǐng)域研究的重要發(fā)展趨勢(shì)。 ESC和iPSC可在體內(nèi)外（在體內(nèi)體外均可以）分化為功能性的心肌細(xì)胞，但它們自發(fā)誘導(dǎo)分化為心肌細(xì)胞的效率很低，這種低下的誘導(dǎo)效率難以滿足細(xì)胞替代治療以及體外構(gòu)建組織工程心肌的需求。近年來，，盡管ESC的心肌分化取得了令人振奮的研究成果，但長期分化中關(guān)于這種ESC源性心肌細(xì)胞的成熟和功能維持仍然缺乏進(jìn)一步的研究。從干細(xì)胞到新生的中胚層再到心臟的原始細(xì)胞最終到終末分化的心肌細(xì)胞，涉及一系列細(xì)胞生長、生物發(fā)育的復(fù)雜調(diào)控過程。細(xì)胞-細(xì)胞（cell-cell）、細(xì)胞-基質(zhì)（cell-matrix）的相互作用可能是細(xì)胞高效誘導(dǎo)分化的關(guān)鍵。共培養(yǎng)細(xì)胞通過細(xì)胞-細(xì)胞的相互作用可以形成有利于干細(xì)胞定向分化的微環(huán)境�；谶@一推測(cè)，本課題擬通過聯(lián)合維生素C（Vc）誘導(dǎo)和細(xì)胞共培養(yǎng)建立ESC的心肌分化模型。利用建立的ESC心肌分化模型觀察其對(duì)iPSC心肌分化的影響，探索共培養(yǎng)促進(jìn)iPSC心肌分化的機(jī)制和高效誘導(dǎo)分化的方法；基于組織工程技術(shù)探討有效的心臟組織脫細(xì)胞的方法和iPSC在組織工程心肌構(gòu)建中的應(yīng)用價(jià)值。 1）共培養(yǎng)心肌分化模型的建立 目的：觀察Vc誘導(dǎo)下小鼠胚胎成纖維細(xì)胞（MEF）或新生心肌細(xì)胞（NCM）共培養(yǎng)對(duì)ESC心肌分化的影響，建立共培養(yǎng)心肌分化的模型。 方法：小鼠ESC通過懸滴培養(yǎng)形成EB，培養(yǎng)液添加0.1mmol/L的Vc來誘導(dǎo)其往心肌細(xì)胞分化。從實(shí)驗(yàn)動(dòng)物提取MEF和NCM作為共培養(yǎng)細(xì)胞，利用插入式培養(yǎng)皿建立EB與共培養(yǎng)細(xì)胞的非接觸共培養(yǎng)系統(tǒng)。觀察培養(yǎng)過程中EB的形態(tài)學(xué)變化，檢測(cè)心臟特異基因的表達(dá)，評(píng)估不同培養(yǎng)環(huán)境的心肌分化率差異。 結(jié)果：在ESC分化第8d鏡下可觀察到跳動(dòng)的EB。跳動(dòng)的EB可表達(dá)心肌細(xì)胞特異性標(biāo)記物GATA4、MLC-2V、CX43，且cTnI、CX43細(xì)胞免疫熒光染色陽性。在ESC的分化過程中MEF或NCM共培養(yǎng)可以增加ESC分化中后期的跳動(dòng)的EB所占百分比，促進(jìn)心肌細(xì)胞的形成，其中NCM共培養(yǎng)的作用更為顯著。共培養(yǎng)促進(jìn)GATA4、MLC-2V、CX43表達(dá)上調(diào)，GATA6表達(dá)下調(diào)。cTnI和CX43的免疫熒光檢測(cè)提示共培養(yǎng)環(huán)境下心肌細(xì)胞分化的成熟度有所提高，同樣以NCM共培養(yǎng)的作用更為顯著。 結(jié)論：MEF或NCM與EB共培養(yǎng)促進(jìn)了ESC的心肌分化并有助于長期分化狀態(tài)的維持，與MEF共培養(yǎng)相比，NCM共培養(yǎng)的作用更為明顯。 2）共培養(yǎng)誘導(dǎo)iPSC向心肌細(xì)胞分化及其機(jī)制 目的：觀察共培養(yǎng)心肌細(xì)胞分化模型對(duì)iPSC心肌細(xì)胞分化的影響，建立iPSC心肌細(xì)胞分化的高效分化模型，并探索其分化機(jī)制。 方法：小鼠iPSC通過懸滴培養(yǎng)形成EB，培養(yǎng)液添加0.1mmol/L的Vc以誘導(dǎo)其往心肌細(xì)胞分化。EB轉(zhuǎn)入非接觸共培養(yǎng)系統(tǒng)與MEF或NCM共培養(yǎng)，觀察培養(yǎng)過程中EB的形態(tài)學(xué)變化，半定量PCR檢測(cè)Oct-4、GATA4、Nkx2.5、ANF、CX43的表達(dá)，定量PCR追蹤GATA4、ANF在分化4d、8d、12d、16d、20d、24d、28d、32d的表達(dá)。行β-腎上腺素能受體刺激觀察分化細(xì)胞的功能狀況，F(xiàn)CM和BrdU分析檢測(cè)共培養(yǎng)條件下iPSC分化中期及后期的iPSCM增殖情況。行半定量PCR檢測(cè)integrin α1、integrin α2受體的表達(dá)。在細(xì)胞培養(yǎng)過程中，添加integrinα1受體阻斷劑，觀察其對(duì)心臟特異因子（aMHC）及心臟轉(zhuǎn)錄因子（NKX2.5、Mef2C、GATA4）表達(dá)的影響。 結(jié)果：與ESC相同，在iPSC分化的第8d，顯微鏡下可觀察到EB出現(xiàn)跳動(dòng)的區(qū)域。Oct-4是未分化iPSC的標(biāo)記物，在分化過程，Oct-4的表達(dá)逐漸下降直至消失。EB可表達(dá)心肌特異性標(biāo)記物GATA4、Nkx2.5、ANF、CX43，MEF及NCM共培條件促進(jìn)GATA4、Nkx2.5、ANF、CX43的表達(dá)上調(diào)。進(jìn)一步采用定量PCR方法追蹤GATA4和ANF的表達(dá)，發(fā)現(xiàn)NCM共培養(yǎng)能夠更好地維持其表達(dá)。行β-腎上腺素能受體刺激實(shí)驗(yàn)提示，相對(duì)于MEF共培養(yǎng)，NCM共培養(yǎng)更能保持分化后期細(xì)胞的生理活性。FCM和BrdU分析提示MEF和NCM共培養(yǎng)可以促進(jìn)分化后期的細(xì)胞增殖。α1β1integrin抑制劑可影響iPSCM后期的基因表達(dá)，導(dǎo)致心臟特異因子（aMHC）及心臟轉(zhuǎn)錄因子（NKX2.5、Mef2C、GATA4）表達(dá)下降。 結(jié)論：MEF或NCM共培養(yǎng)促進(jìn)iPSC心肌分化作用主要體現(xiàn)在分化的后期，其機(jī)制與共培養(yǎng)維持了integrin受體信號(hào)通路的激活從而促進(jìn)iPSC分化的心肌細(xì)胞增殖有關(guān)。 3）脫細(xì)胞基質(zhì)制備及組織工程心肌初步構(gòu)建 目的：基于組織工程技術(shù)探討有效的心臟脫細(xì)胞的方法和在組織工程心肌構(gòu)建中的價(jià)值。 方法：摘取成年兔子的心臟，PBS沖洗干凈，通過SDS洗滌及酶消法去除組織內(nèi)細(xì)胞，獲得脫細(xì)胞基質(zhì)，HE染色評(píng)價(jià)脫細(xì)胞情況，材料凍干后輻照消毒備用。利用共培養(yǎng)分化模型由ESC/iPSC制備大批量心肌細(xì)胞群，以差速貼壁法獲得相對(duì)純度的心肌細(xì)胞；以其為種子細(xì)胞，約106~7/ml的密度種植和注射到支架材料，DMEM培養(yǎng)液（含10%胎牛血清）培養(yǎng)。分別在培養(yǎng)1周、2周檢測(cè)材料支架種植心肌細(xì)胞后的變化，并與種植前比較。免疫組化檢測(cè)輔肌動(dòng)蛋白(α-actinin)的表達(dá)。 結(jié)果：SDS洗滌和酶消法可以去除組織表面的細(xì)胞，然而行HE染色卻發(fā)現(xiàn)，SDS洗滌法消化后細(xì)胞基質(zhì)稍有破壞，組織內(nèi)仍殘留有細(xì)胞核成分；而酶消法基本將細(xì)胞外基質(zhì)保存完整，細(xì)胞成分已經(jīng)去除干凈。將ESC/iPSC分化的心肌細(xì)胞作為種子細(xì)胞，以106~107/ml的細(xì)胞密度種植和注射到基質(zhì)材料。在細(xì)胞種植一周，基質(zhì)材料表面已經(jīng)覆蓋有團(tuán)狀的細(xì)胞，內(nèi)部的一些區(qū)域已經(jīng)有細(xì)胞長入；在細(xì)胞種植兩周后，行HE染色發(fā)現(xiàn)基質(zhì)材料表面已經(jīng)完全覆蓋細(xì)胞，基質(zhì)材料內(nèi)部液長滿了細(xì)胞；行a-actinin的免疫組織化學(xué)染色，發(fā)現(xiàn)這些細(xì)胞是a-actinin陽性，提示均為心肌細(xì)胞。 結(jié)論：酶消化之后添加NaOH可以完全去除心臟組織中的細(xì)胞成分，而保留基本完整的胞外基質(zhì)，可供細(xì)胞重新長入；iPSC分化的心肌細(xì)胞可作為種子細(xì)胞，應(yīng)用于組織工程心肌的構(gòu)建。
[Abstract]:Embryonic stem cells ( ESC ) and induced pluripotent stem cells ( iPSC ) have great potential for application in myocardial regeneration due to their self - renewal and strong differentiation potential . Unlike ESC , iPSC can be obtained by reprogramming the skin tissue of the patient .

Conclusion : ESC and iPSC can be differentiated into functional cardiomyocytes in vitro ( in vivo and in vitro ) , but they spontaneously induce differentiation into cardiomyocytes , which is difficult to meet the need of cell replacement therapy and in vitro construction of tissue engineering myocardium .
Based on the tissue engineering technique , the effective method of cardiac tissue removal and the application value of iPSC in the construction of tissue engineering myocardium were discussed .

1 ) Establishment of co - cultured myocardial differentiation model

Objective : To observe the effect of Vc - induced co - culture of mouse embryonic fibroblasts ( MEFs ) or neonatal cardiomyocytes ( NCM ) on the myocardial differentiation of ESC .

Methods : The mouse ESCs were cultured by suspension culture to form EB , and the culture solution was added with 0.1 mmol / L Vc to induce the differentiation of myocardial cells . The cells were extracted from the experimental animals , and the non - contact co - culture system of EB and co - cultured cells was established by using the inserted culture dish . The morphological changes of EB in the culture were observed , the expression of specific genes in the heart was detected , and the difference of myocardial differentiation rate was assessed in different culture environments .

Results : EB can be observed under the microscope of ESC differentiation . EB may express myocardial cell specific markers GATA4 , MLC - 2V , CX43 . In the course of differentiation of ESC , the percentage of EB in the middle and late stages of ESC differentiation can be increased , and the formation of myocardial cells is promoted . In the co - culture , the expression of GATA4 , MLC - 2V , CX43 is up - regulated , and the expression of GATA6 is downregulated .

Conclusion : The co - culture of MEFs or NCM with EB promotes the myocardial differentiation of ESCs and contributes to the maintenance of long - term differentiation state , which is more obvious than that of the co - culture of NCM .

2 ) Co - culture induced the differentiation of iPSC into cardiomyocytes and its mechanism

Objective : To observe the effect of co - cultured myocardial cell differentiation model on myocardial cell differentiation of iPSC , to establish an efficient differentiation model of myocardial cell differentiation in iPSC , and to explore its differentiation mechanism .

Methods : The expression of integrin 偽1 and integrin 偽2 was detected by semi - quantitative polymerase chain reaction ( PCR ) . The expression of integrin 偽1 , integrin 偽2 receptor was detected by semi - quantitative PCR . The effects of integrin 偽1 receptor blocking agent on cardiac specific factor ( aMHC ) and cardiac transcription factor ( NKX2.5 , Mef2C , GATA4 ) were observed .

Results : In the same manner as ESC , it was observed that the presence of EB was observed under microscope on the 8th day of the differentiation of iPSC . Oct - 4 was a marker of undifferentiated iPSC . The expression of GATA4 , X2.5 , ANE , CX43 was increased by using quantitative PCR method .

Conclusion : The co - culture of MEFs or NCM promotes the differentiation of iPSC , and its mechanism and co - culture maintain the activation of integrin receptor signaling pathway to promote the proliferation of myocardial cells in the differentiation of iPSC .

3 ) Preparation of acellular matrix and preliminary construction of tissue engineering myocardium

Objective : To explore the methods of effective cardiac degranulation and its value in the construction of tissue engineering myocardium based on tissue engineering technology .

Methods : The hearts of adult rabbits were washed with PBS and washed with PBS . The cells in tissues were removed by SDS - washing and enzymatic digestion .
The expression of secondary actin ( 偽 - actinin ) was detected by immunohistochemistry .

Results : SDS - washing and enzymatic digestion could remove the cells on the surface of the tissue , however , HE staining showed that the cell matrix was damaged slightly after the SDS - washing method , and the cell nuclear components remained in the tissue .
The cells of ESC / iPSC were cultured and injected into matrix material at the density of 106 - 107 / ml .
After two weeks of cell implantation , HE staining showed that the surface of the matrix material had completely covered the cells , and the interior of the matrix material was full of cells ;
Immunohistochemical staining of a - actinin found that these cells were a - actinin positive , suggesting that both were cardiomyocytes .

Conclusion : NaOH can completely remove cellular components in cardiac tissue after enzymatic digestion , while preserving the basic intact extracellular matrix , which can be used for cell re - entry ;
iPSC differentiated cardiomyocytes can be used as seed cells for the construction of tissue engineering myocardium .
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 歐東波;陳瑞;鄭強(qiáng)蓀;郭菁菁;郭萬剛;劉雄濤;;胚胎干細(xì)胞在體外模擬心肌生長環(huán)境中向心肌分化[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2009年04期

本文編號(hào)：1945665