iPS细胞诱导分化及组织工程心肌构建的研究

发布时间：2018-05-28 06:27

本文选题：胚胎干细胞 + iPS细胞　；参考：《第四军医大学》2012年博士论文

【摘要】：胚胎干细胞（ESC）与诱导性多能干细胞（iPSC）由于具有自我更新和强大分化潜能，在心肌再生方面有着极大的应用前景。与ESC不同，iPSC可以由病人的皮肤组织重编程获取，经过一系列诱导分化，有可能建立大批量同源心肌细胞而无伦理争议和免疫排斥。因此，以iPSC高效诱导分化的心肌细胞替代活体心肌细胞作为种子细胞来源，采用组织工程技术体外构建心肌组织并联合组织移植进行心肌修复与再生，逐渐成为干细胞心肌再生领域研究的重要发展趋势。 ESC和iPSC可在体内外（在体内体外均可以）分化为功能性的心肌细胞，但它们自发诱导分化为心肌细胞的效率很低，这种低下的诱导效率难以满足细胞替代治疗以及体外构建组织工程心肌的需求。近年来，，尽管ESC的心肌分化取得了令人振奋的研究成果，但长期分化中关于这种ESC源性心肌细胞的成熟和功能维持仍然缺乏进一步的研究。从干细胞到新生的中胚层再到心脏的原始细胞最终到终末分化的心肌细胞，涉及一系列细胞生长、生物发育的复杂调控过程。细胞-细胞（cell-cell）、细胞-基质（cell-matrix）的相互作用可能是细胞高效诱导分化的关键。共培养细胞通过细胞-细胞的相互作用可以形成有利于干细胞定向分化的微环境。基于这一推测，本课题拟通过联合维生素C（Vc）诱导和细胞共培养建立ESC的心肌分化模型。利用建立的ESC心肌分化模型观察其对iPSC心肌分化的影响，探索共培养促进iPSC心肌分化的机制和高效诱导分化的方法；基于组织工程技术探讨有效的心脏组织脱细胞的方法和iPSC在组织工程心肌构建中的应用价值。 1）共培养心肌分化模型的建立目的：观察Vc诱导下小鼠胚胎成纤维细胞（MEF）或新生心肌细胞（NCM）共培养对ESC心肌分化的影响，建立共培养心肌分化的模型。方法：小鼠ESC通过悬滴培养形成EB，培养液添加0.1mmol/L的Vc来诱导其往心肌细胞分化。从实验动物提取MEF和NCM作为共培养细胞，利用插入式培养皿建立EB与共培养细胞的非接触共培养系统。观察培养过程中EB的形态学变化，检测心脏特异基因的表达，评估不同培养环境的心肌分化率差异。结果：在ESC分化第8d镜下可观察到跳动的EB。跳动的EB可表达心肌细胞特异性标记物GATA4、MLC-2V、CX43，且cTnI、CX43细胞免疫荧光染色阳性。在ESC的分化过程中MEF或NCM共培养可以增加ESC分化中后期的跳动的EB所占百分比，促进心肌细胞的形成，其中NCM共培养的作用更为显著。共培养促进GATA4、MLC-2V、CX43表达上调，GATA6表达下调。cTnI和CX43的免疫荧光检测提示共培养环境下心肌细胞分化的成熟度有所提高，同样以NCM共培养的作用更为显著。结论：MEF或NCM与EB共培养促进了ESC的心肌分化并有助于长期分化状态的维持，与MEF共培养相比，NCM共培养的作用更为明显。 2）共培养诱导iPSC向心肌细胞分化及其机制目的：观察共培养心肌细胞分化模型对iPSC心肌细胞分化的影响，建立iPSC心肌细胞分化的高效分化模型，并探索其分化机制。方法：小鼠iPSC通过悬滴培养形成EB，培养液添加0.1mmol/L的Vc以诱导其往心肌细胞分化。EB转入非接触共培养系统与MEF或NCM共培养，观察培养过程中EB的形态学变化，半定量PCR检测Oct-4、GATA4、Nkx2.5、ANF、CX43的表达，定量PCR追踪GATA4、ANF在分化4d、8d、12d、16d、20d、24d、28d、32d的表达。行β-肾上腺素能受体刺激观察分化细胞的功能状况，FCM和BrdU分析检测共培养条件下iPSC分化中期及后期的iPSCM增殖情况。行半定量PCR检测integrin α1、integrin α2受体的表达。在细胞培养过程中，添加integrinα1受体阻断剂，观察其对心脏特异因子（aMHC）及心脏转录因子（NKX2.5、Mef2C、GATA4）表达的影响。结果：与ESC相同，在iPSC分化的第8d，显微镜下可观察到EB出现跳动的区域。Oct-4是未分化iPSC的标记物，在分化过程，Oct-4的表达逐渐下降直至消失。EB可表达心肌特异性标记物GATA4、Nkx2.5、ANF、CX43，MEF及NCM共培条件促进GATA4、Nkx2.5、ANF、CX43的表达上调。进一步采用定量PCR方法追踪GATA4和ANF的表达，发现NCM共培养能够更好地维持其表达。行β-肾上腺素能受体刺激实验提示，相对于MEF共培养，NCM共培养更能保持分化后期细胞的生理活性。FCM和BrdU分析提示MEF和NCM共培养可以促进分化后期的细胞增殖。α1β1integrin抑制剂可影响iPSCM后期的基因表达，导致心脏特异因子（aMHC）及心脏转录因子（NKX2.5、Mef2C、GATA4）表达下降。结论：MEF或NCM共培养促进iPSC心肌分化作用主要体现在分化的后期，其机制与共培养维持了integrin受体信号通路的激活从而促进iPSC分化的心肌细胞增殖有关。 3）脱细胞基质制备及组织工程心肌初步构建目的：基于组织工程技术探讨有效的心脏脱细胞的方法和在组织工程心肌构建中的价值。方法：摘取成年兔子的心脏，PBS冲洗干净，通过SDS洗涤及酶消法去除组织内细胞，获得脱细胞基质，HE染色评价脱细胞情况，材料冻干后辐照消毒备用。利用共培养分化模型由ESC/iPSC制备大批量心肌细胞群，以差速贴壁法获得相对纯度的心肌细胞；以其为种子细胞，约106~7/ml的密度种植和注射到支架材料，DMEM培养液（含10%胎牛血清）培养。分别在培养1周、2周检测材料支架种植心肌细胞后的变化，并与种植前比较。免疫组化检测辅肌动蛋白(α-actinin)的表达。结果：SDS洗涤和酶消法可以去除组织表面的细胞，然而行HE染色却发现，SDS洗涤法消化后细胞基质稍有破坏，组织内仍残留有细胞核成分；而酶消法基本将细胞外基质保存完整，细胞成分已经去除干净。将ESC/iPSC分化的心肌细胞作为种子细胞，以106~107/ml的细胞密度种植和注射到基质材料。在细胞种植一周，基质材料表面已经覆盖有团状的细胞，内部的一些区域已经有细胞长入；在细胞种植两周后，行HE染色发现基质材料表面已经完全覆盖细胞，基质材料内部液长满了细胞；行a-actinin的免疫组织化学染色，发现这些细胞是a-actinin阳性，提示均为心肌细胞。结论：酶消化之后添加NaOH可以完全去除心脏组织中的细胞成分，而保留基本完整的胞外基质，可供细胞重新长入；iPSC分化的心肌细胞可作为种子细胞，应用于组织工程心肌的构建。
[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 伪1 and integrin 伪2 was detected by semi - quantitative polymerase chain reaction ( PCR ) . The expression of integrin 伪1 , integrin 伪2 receptor was detected by semi - quantitative PCR . The effects of integrin 伪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 ( 伪 - 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 .
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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