人脂肪干细胞在PLGA支架上向肝细胞诱导分化的实验研究

发布时间：2018-06-01 13:09

本文选题：脂肪干细胞 + 聚乳酸羟基乙酸　；参考：《中国人民解放军军事医学科学院》2009年博士论文

【摘要】： 我国是肝病大国,每年因终末期肝病死亡者人数众多。原位肝移植(orthotopic liver transplantation,OLT)是目前根治终末期肝病的唯一方法。但是由于存在供肝严重短缺、免疫排斥和长期服用免疫抑制剂带来多种毒副作用等缺点,其临床应用受到很大限制,因此迫切需要寻找新的治疗方法。肝组织工程的最终目标为通过组织工程的方法再生、再造出有正常代谢功能的肝脏组织,以代偿或替代病变肝组织。因此,肝组织工程的研究和应用将为终末期肝病的治疗带来新的希望。目前肝组织工程的研究仍处于初级阶段,距离临床应用还很遥远,还有很多问题需要解决,如种子细胞的选择,材料的筛选,及组织的血管化等。其中种子细胞和材料问题是首先要解决的基本问题。如何获得大量有功能的肝细胞样细胞是制约肝组织工程发展的瓶颈之一。肝组织工程种子细胞的来源包括成熟肝细胞和干细胞(胚胎干细胞和成体干细胞)。由于成熟肝细胞无法扩增、体外培养易失去活性与功能;而胚胎干细胞存在获取困难、伦理争议和致瘤性风险等诸多限制;相对而言,成体干细胞获取容易、可体外大规模培养扩增,因此,近年来其研究和应用备受关注。成体干细胞中最有代表性的是骨髓来源的间充质干细胞(mesenchymal stem cells,MSCs)。近期研究表明,脂肪组织来源的MSC(即脂肪干细胞,adipose-derived stem cells,ADSCs)与骨髓MSC在生物学性能方面具有极大的相似性,也具有包括肝细胞在内的多向分化潜能,且与其相比更容易获取、取材创伤更小、细胞获得量更大。所以脂肪干细胞有希望成为肝组织工程另一种很有前景的种子细胞来源。理想支架材料的研究和应用是关系到组织工程成功与否的重要因素,因此材料的筛选非常重要。支架材料方面的研究近年来也有较大的进展和突破,而常用于肝组织工程构建的支架材料有聚己内酯(poly varepsilon-caprolactone,PCL)、聚乳酸-羟基乙酸(poly-lactide-co-glycolide,PLGA)、胶原、层粘连蛋白和纤粘连蛋白等。其中通过美国FDA批准的PLGA是目前应用最广泛、最重要的一类人工合成材料,已与多种干细胞结合应用于多种组织工程组织构建的研究。目前,脂肪干细胞在任何三维支架上向肝细胞分化的研究还未见报道,本实验中我们选择人脂肪干细胞作为种子细胞,并首先选定PLGA作为支架,探讨PLGA支架体内、外对人脂肪干细胞向肝细胞分化的影响,为肝组织工程的构建寻找合适的种子细胞和支架材料。首先在二维单层培养体系下用改良的肝细胞诱导液诱导人脂肪干细胞向肝细胞分化。然后将人脂肪干细胞接种在PLGA支架上,体外用相同的肝细胞诱导液培养,观察人脂肪干细胞能否向肝细胞分化。在此基础上进一步探讨PLGA支架物理特性和成纤维细胞共培养对人脂肪干细胞向肝细胞分化的影响。最后观察PLGA支架材料体内对预诱导人脂肪干细胞的影响。研究内容有以下几个主要方面: 一、人脂肪干细胞在二维单层培养体系下向肝细胞的诱导分化分离人脂肪干细胞并从形态、表型和多向分化能力方面进行鉴定。在此基础上,将人脂肪干细胞接种在普通培养板中,用改良的肝细胞诱导液进行诱导。这种改良的诱导液含有肝细胞生长因子(hepatocyte growth factor,HGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)、成纤维细胞生长因子(fibroblast growth factor 4,FGF4)和抑瘤素(oncostatin M,OSM)。诱导14d后,形态、表型和功能鉴定结果显示人脂肪干细胞能够分化为肝细胞样细胞。干细胞诱导过程与非诱导过程细胞的增殖情况不同,而大多数研究关注的是后者,且有文献报道HGF和FGF因子能同时促进细胞的分化和增殖,因此我们观察了此种肝细胞诱导液对细胞增殖的影响。研究结果发现细胞显著增殖,此种肝细胞诱导液在促进人脂肪干细胞向肝细胞分化的同时起到了对细胞扩增的作用。人脂肪干细胞在二维单层培养体系下向肝细胞诱导的成功可为下一步人脂肪干细胞与支架复合后向肝细胞的诱导奠定基础。二、人脂肪干细胞在PLGA三维支架上体外向肝细胞的诱导分化目前,人脂肪干细胞向肝细胞诱导分化的研究仅限于二维环境,在任何三维支架的研究还未见报道。在明确人脂肪干细胞用改良的肝细胞诱导液诱导能够分化成肝细胞样细胞的基础上,本部分研究进一步将人脂肪干细胞与多孔PLGA支架复合,体外用相同的肝细胞诱导液进行诱导,观察多孔PLGA支架体外对脂肪干细胞向肝细胞诱导分化的影响,以寻找适宜人脂肪干细胞向肝细胞分化的三维支架。支架材料和干细胞良好的生物相容性是干细胞能够在支架表面分化的基础,DAPI染色、CCK8检测和扫描电镜观察结果证明PLGA支架和人脂肪干细胞有良好的生物相容性。细胞的生长和增殖在组织工程中非常关键,CCK8检测结果表明接种在PLGA支架上的人脂肪干细胞在前述诱导液作用下同样明显增殖,且显著高于二维环境,不仅再次证实了含有细胞因子HGF、bFGF和FGF4的肝细胞诱导液能够促进细胞增殖,且提示三维环境可能比二维环境更利于细胞增殖。接种于三维支架的细胞其形态受支架类型和细胞种类的影响,我们用扫描电镜观察了接种于多孔PLGA支架的人脂肪干细胞向肝细胞诱导过程细胞形态的变化,发现细胞沿着多孔支架表面伸展,并逐渐融合形成一细胞单层覆盖在支架表面,形态逐渐向多角形改变,很多细胞伸出细胞突触在细胞单层上面互相连接,细胞能够增殖、分化成三维组织样结构。在上述基础上,我们重点探讨了多孔PLGA支架能否支持人脂肪干细胞向肝细胞分化,结果表明细胞能够分化成具有肝细胞特异性形态、表型和功能的肝细胞样细胞。三、成纤维细胞共培养和PLGA支架物理特性对脂肪干细胞向肝细胞分化的影响人脂肪干细胞在多孔PLGA支架上向肝细胞分化过程受很多因素影响。成纤维细胞能够分泌很多利于干细胞向肝细胞分化的细胞外基质和细胞因子,我们采用非接触共培养的方式对成纤维细胞与人脂肪干细胞共培养能否引发其向肝细胞的分化进行了研究。结果发现人真皮成纤维细胞与人脂肪干细胞用普通培养基共培养即可使其表达肝细胞特异性基因,且成纤维细胞共培养可促进肝细胞诱导液诱导的肝细胞特异性基因的表达,表明直接的细胞间接触不是人真皮成纤维细胞介导的人脂肪干细胞向肝细胞分化所必需,成纤维细胞分泌的可溶性细胞因子(如HGF和FGF2)能够通过旁分泌方式为肝细胞分化提供足够的信号,并且能够与肝细胞诱导液发挥相加作用。孔径大小和孔隙率是多孔PLGA支架的重要物理特性,孔径大小主要取决于致孔剂NaCl颗粒的粒径,孔隙率主要取决于NaCl颗粒的比例。前面在研究PLGA支架能否支持人脂肪干细胞向肝细胞分化时制备的多孔PLGA支架其微孔结构特性我们是随机选定的,NaCl粒径为30~50μm,比例为75%(w/w)。为今后更好地设计多孔PLGA支架,我们用不同粒径范围和比例的NaCl颗粒制备了多种微孔特性的PLGA支架,以观察多孔PLGA支架孔径大小和孔径率对人脂肪干细胞向肝细胞分化的影响。细胞形态的扫描电镜观察结果和Alb基因表达的实时定量PCR检测结果表明,NaCl比例固定为75%时,与50μm、50~120μm和200μm相比,NaCl粒径范围为120~200μm的PLGA支架更适合人脂肪干细胞向肝细胞的分化,提示支架孔径过大和过小都不利于细胞的分化;另外,NaCl粒径固定为50~120μm时,与75%和90%相比,NaCl比例为50%的PLGA支架更适合人脂肪干细胞向肝细胞的分化,提示孔隙率的增加也不利于细胞的分化。四、PLGA支架体内对预诱导人脂肪干细胞的影响体内、外环境的差异导致支架材料的体内、外特性不会完全相同,因此在确定PLGA支架体外能够支持人脂肪干细胞向肝细胞分化的基础上有必要进行体内移植实验,以观察PLGA支架体内对预诱导人脂肪干细胞的影响。将人脂肪干细胞与PLGA支架复合,体外用肝细胞诱导液预先诱导14d,然后移植入70%肝切除大鼠体内,14d后观察。部分肝切除的微环境利于干细胞向肝细胞的分化,本实验发现此种微环境下与PLGA支架复合的预诱导人脂肪干细胞仍然存活并维持肝细胞特异性表型的表达,表明PLGA支架体内对植入的细胞并未产生不良影响。但是,体内移植14d后大鼠血清中未检测到人Alb的分泌,这可能与细胞植入的位置和植入细胞数目的不足有关。总之,本实验研究结果表明PLGA支架体内、外均支持人脂肪干细胞向肝细胞的分化,PLGA与人脂肪干细胞复合适合用于肝组织工程的构建。这些研究可为肝组织工程最终应用于临床积累必要的信息。
[Abstract]:Liver transplantation ( OLT ) is the only method for the treatment of end - stage liver disease . However , the ultimate goal of liver tissue engineering is to regenerate the liver tissue with normal metabolic function by tissue engineering . Therefore , the research and application of liver tissue engineering will bring new hope for the treatment of end - stage liver disease .

At present , the research of liver tissue engineering is still in the primary stage , the distance from clinical application is far away , and many problems need to be solved , such as selection of seed cells , screening of materials , and vascularization of tissue . The problem of seed cells and materials is the basic problem to be solved first .

How to obtain a lot of functional hepatocyte - like cells is one of the bottlenecks that restrict the development of liver tissue engineering . The source of liver tissue engineering seed cells includes mature hepatocytes and stem cells ( embryonic stem cells and adult stem cells ) . Since mature hepatocytes cannot be amplified , the culture of embryonic stem cells is easy to lose activity and function . Recent studies have shown that MSCs derived from adipose tissue ( i.e . , adipose - derived stem cells , adipose - derived stem cells , ADSCs ) have great similarities with bone marrow MSCs in their biological properties , and also have multi - directional differentiation potential including hepatocytes , and are easier to acquire than they are .

The research and application of ideal scaffold materials is an important factor affecting the success or failure of tissue engineering , so the research of scaffold materials is very important . The research of scaffold materials has made great progress and breakthrough in recent years , and scaffold materials commonly used in liver tissue engineering are poly - caprolactone ( PCL ) , poly - lactic - glycolic acid ( poly - poly - co - glycolide , PLGA ) , collagen , laminin and fibronectin .

At present , the study of the differentiation of adipose - derived stem cells to hepatocytes in any three - dimensional scaffold has not been reported . In this experiment , we selected human adipose - derived stem cells as seed cells , and first selected PLGA as scaffold to study the effects of PLGA scaffold on the differentiation of human adipose - derived stem cells into hepatocytes .

The main aspects of the study are as follows :

induction and differentiation of human adipose - derived stem cells to hepatocytes in two - dimensional single - layer culture system

The human adipose - derived stem cells were isolated from morphology , phenotype and multi - direction differentiation ability . On this basis , human adipose - derived stem cells were seeded in common culture plates and induced with an improved hepatocyte - inducing solution . After 14 days of induction , morphological , phenotypic and functional identification revealed that human adipose - derived stem cells were able to differentiate into hepatocyte - like cells .

The results showed that HGF and FGF can promote the differentiation and proliferation of the cells at the same time .

Differentiation of human adipose - derived stem cells into liver cells in vitro on PLGA three - dimensional scaffold

At present , the research of human adipose - derived stem cells to liver cell - induced differentiation is limited to two - dimensional environment , and it is not reported in the study of any three - dimensional scaffolds . In order to differentiate into hepatocyte - like cells with the modified hepatocyte - inducing solution induced by human adipose - derived stem cells , the present partial study further studies the influence of human adipose - derived stem cells in vitro on the differentiation of human adipose - derived stem cells into hepatocytes , so as to find a three - dimensional scaffold suitable for differentiation of human adipose - derived stem cells into hepatocytes .

The biocompatibility of scaffold material and stem cells is that the stem cells can be differentiated on the surface of scaffold . The results of DAPI staining , CC8 test and scanning electron microscope show that PLGA scaffold and human adipose - derived stem cells have good biocompatibility . The growth and proliferation of cells are very important in tissue engineering .

Effects of physical properties of co - culture and PLGA scaffold on differentiation of adipose - derived stem cells into hepatocytes

It is found that human dermal fibroblasts and human adipose - derived stem cells are co - cultured with common medium to express the hepatocyte - specific gene .

The pore size and porosity were important physical properties of porous PLGA scaffolds . The pore size was mainly determined by the ratio of pore size and pore size of porous PLGA scaffolds .

Effect of PLGA scaffold on preinduced human adipose - derived stem cells

In vivo , the difference in the external environment causes the body and the outer characteristics of the scaffold material to be not identical , so the in vivo transplantation experiment can be carried out on the basis of determining that the PLGA stent can support the differentiation of human adipose - derived stem cells to liver cells , so as to observe the influence of the PLGA scaffold body on the pre - induced human adipose - stem cells .

Human adipose - derived stem cells were combined with PLGA scaffolds for 14 days in vitro and then transplanted into 70 % hepatectomy rats . After 14 days of transplantation , it was observed that the pre - induced human adipose - derived stem cells combined with PLGA scaffolds were still alive and the expression of hepatocyte - specific phenotype was maintained . However , the secretion of human albumin was not detected in the serum of rats after 14 days in vivo , which could be related to the location of cell implantation and the deficiency of the number of implanted cells .

In conclusion , the results of this study show that the differentiation of human adipose - derived stem cells into liver cells is supported in vitro and in vitro , and the combination of PLGA and human adipose - derived stem cells is suitable for the construction of liver tissue engineering . These studies can be used for the clinical accumulation of necessary information for liver tissue engineering .
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329.2

【参考文献】