本文選題：脂肪干細胞 + 聚乳酸羥基乙酸��； 參考：《中國人民解放軍軍事醫(yī)學科學院》2009年博士論文

【摘要】： 我國是肝病大國,每年因終末期肝病死亡者人數(shù)眾多。原位肝移植(orthotopic liver transplantation,OLT)是目前根治終末期肝病的唯一方法。但是由于存在供肝嚴重短缺、免疫排斥和長期服用免疫抑制劑帶來多種毒副作用等缺點,其臨床應用受到很大限制,因此迫切需要尋找新的治療方法。肝組織工程的最終目標為通過組織工程的方法再生、再造出有正常代謝功能的肝臟組織,以代償或替代病變肝組織。因此,肝組織工程的研究和應用將為終末期肝病的治療帶來新的希望。 目前肝組織工程的研究仍處于初級階段,距離臨床應用還很遙遠,還有很多問題需要解決,如種子細胞的選擇,材料的篩選,及組織的血管化等。其中種子細胞和材料問題是首先要解決的基本問題。 如何獲得大量有功能的肝細胞樣細胞是制約肝組織工程發(fā)展的瓶頸之一。肝組織工程種子細胞的來源包括成熟肝細胞和干細胞(胚胎干細胞和成體干細胞)。由于成熟肝細胞無法擴增、體外培養(yǎng)易失去活性與功能;而胚胎干細胞存在獲取困難、倫理爭議和致瘤性風險等諸多限制;相對而言,成體干細胞獲取容易、可體外大規(guī)模培養(yǎng)擴增,因此,近年來其研究和應用備受關注。成體干細胞中最有代表性的是骨髓來源的間充質(zhì)干細胞(mesenchymal stem cells,MSCs)。近期研究表明,脂肪組織來源的MSC(即脂肪干細胞,adipose-derived stem cells,ADSCs)與骨髓MSC在生物學性能方面具有極大的相似性,也具有包括肝細胞在內(nèi)的多向分化潛能,且與其相比更容易獲取、取材創(chuàng)傷更小、細胞獲得量更大。所以脂肪干細胞有希望成為肝組織工程另一種很有前景的種子細胞來源。 理想支架材料的研究和應用是關系到組織工程成功與否的重要因素,因此材料的篩選非常重要。支架材料方面的研究近年來也有較大的進展和突破,而常用于肝組織工程構建的支架材料有聚己內(nèi)酯(poly varepsilon-caprolactone,PCL)、聚乳酸-羥基乙酸(poly-lactide-co-glycolide,PLGA)、膠原、層粘連蛋白和纖粘連蛋白等。其中通過美國FDA批準的PLGA是目前應用最廣泛、最重要的一類人工合成材料,已與多種干細胞結合應用于多種組織工程組織構建的研究。 目前,脂肪干細胞在任何三維支架上向肝細胞分化的研究還未見報道,本實驗中我們選擇人脂肪干細胞作為種子細胞,并首先選定PLGA作為支架,探討PLGA支架體內(nèi)、外對人脂肪干細胞向肝細胞分化的影響,為肝組織工程的構建尋找合適的種子細胞和支架材料。首先在二維單層培養(yǎng)體系下用改良的肝細胞誘導液誘導人脂肪干細胞向肝細胞分化。然后將人脂肪干細胞接種在PLGA支架上,體外用相同的肝細胞誘導液培養(yǎng),觀察人脂肪干細胞能否向肝細胞分化。在此基礎上進一步探討PLGA支架物理特性和成纖維細胞共培養(yǎng)對人脂肪干細胞向肝細胞分化的影響。最后觀察PLGA支架材料體內(nèi)對預誘導人脂肪干細胞的影響。 研究內(nèi)容有以下幾個主要方面: 一、人脂肪干細胞在二維單層培養(yǎng)體系下向肝細胞的誘導分化 分離人脂肪干細胞并從形態(tài)、表型和多向分化能力方面進行鑒定。在此基礎上,將人脂肪干細胞接種在普通培養(yǎng)板中,用改良的肝細胞誘導液進行誘導。這種改良的誘導液含有肝細胞生長因子(hepatocyte growth factor,HGF)、堿性成纖維細胞生長因子(basic fibroblast growth factor,bFGF)、成纖維細胞生長因子(fibroblast growth factor 4,FGF4)和抑瘤素(oncostatin M,OSM)。誘導14d后,形態(tài)、表型和功能鑒定結果顯示人脂肪干細胞能夠分化為肝細胞樣細胞。 干細胞誘導過程與非誘導過程細胞的增殖情況不同,而大多數(shù)研究關注的是后者,且有文獻報道HGF和FGF因子能同時促進細胞的分化和增殖,因此我們觀察了此種肝細胞誘導液對細胞增殖的影響。研究結果發(fā)現(xiàn)細胞顯著增殖,此種肝細胞誘導液在促進人脂肪干細胞向肝細胞分化的同時起到了對細胞擴增的作用。人脂肪干細胞在二維單層培養(yǎng)體系下向肝細胞誘導的成功可為下一步人脂肪干細胞與支架復合后向肝細胞的誘導奠定基礎。 二、人脂肪干細胞在PLGA三維支架上體外向肝細胞的誘導分化 目前,人脂肪干細胞向肝細胞誘導分化的研究僅限于二維環(huán)境,在任何三維支架的研究還未見報道。在明確人脂肪干細胞用改良的肝細胞誘導液誘導能夠分化成肝細胞樣細胞的基礎上,本部分研究進一步將人脂肪干細胞與多孔PLGA支架復合,體外用相同的肝細胞誘導液進行誘導,觀察多孔PLGA支架體外對脂肪干細胞向肝細胞誘導分化的影響,以尋找適宜人脂肪干細胞向肝細胞分化的三維支架。 支架材料和干細胞良好的生物相容性是干細胞能夠在支架表面分化的基礎,DAPI染色、CCK8檢測和掃描電鏡觀察結果證明PLGA支架和人脂肪干細胞有良好的生物相容性。細胞的生長和增殖在組織工程中非常關鍵,CCK8檢測結果表明接種在PLGA支架上的人脂肪干細胞在前述誘導液作用下同樣明顯增殖,且顯著高于二維環(huán)境,不僅再次證實了含有細胞因子HGF、bFGF和FGF4的肝細胞誘導液能夠促進細胞增殖,且提示三維環(huán)境可能比二維環(huán)境更利于細胞增殖。接種于三維支架的細胞其形態(tài)受支架類型和細胞種類的影響,我們用掃描電鏡觀察了接種于多孔PLGA支架的人脂肪干細胞向肝細胞誘導過程細胞形態(tài)的變化,發(fā)現(xiàn)細胞沿著多孔支架表面伸展,并逐漸融合形成一細胞單層覆蓋在支架表面,形態(tài)逐漸向多角形改變,很多細胞伸出細胞突觸在細胞單層上面互相連接,細胞能夠增殖、分化成三維組織樣結構。在上述基礎上,我們重點探討了多孔PLGA支架能否支持人脂肪干細胞向肝細胞分化,結果表明細胞能夠分化成具有肝細胞特異性形態(tài)、表型和功能的肝細胞樣細胞。 三、成纖維細胞共培養(yǎng)和PLGA支架物理特性對脂肪干細胞向肝細胞分化的影響 人脂肪干細胞在多孔PLGA支架上向肝細胞分化過程受很多因素影響。成纖維細胞能夠分泌很多利于干細胞向肝細胞分化的細胞外基質(zhì)和細胞因子,我們采用非接觸共培養(yǎng)的方式對成纖維細胞與人脂肪干細胞共培養(yǎng)能否引發(fā)其向肝細胞的分化進行了研究。結果發(fā)現(xiàn)人真皮成纖維細胞與人脂肪干細胞用普通培養(yǎng)基共培養(yǎng)即可使其表達肝細胞特異性基因,且成纖維細胞共培養(yǎng)可促進肝細胞誘導液誘導的肝細胞特異性基因的表達,表明直接的細胞間接觸不是人真皮成纖維細胞介導的人脂肪干細胞向肝細胞分化所必需,成纖維細胞分泌的可溶性細胞因子(如HGF和FGF2)能夠通過旁分泌方式為肝細胞分化提供足夠的信號,并且能夠與肝細胞誘導液發(fā)揮相加作用。 孔徑大小和孔隙率是多孔PLGA支架的重要物理特性,孔徑大小主要取決于致孔劑NaCl顆粒的粒徑,孔隙率主要取決于NaCl顆粒的比例。前面在研究PLGA支架能否支持人脂肪干細胞向肝細胞分化時制備的多孔PLGA支架其微孔結構特性我們是隨機選定的,NaCl粒徑為30~50μm,比例為75%(w/w)。為今后更好地設計多孔PLGA支架,我們用不同粒徑范圍和比例的NaCl顆粒制備了多種微孔特性的PLGA支架,以觀察多孔PLGA支架孔徑大小和孔徑率對人脂肪干細胞向肝細胞分化的影響。細胞形態(tài)的掃描電鏡觀察結果和Alb基因表達的實時定量PCR檢測結果表明,NaCl比例固定為75%時,與50μm、50~120μm和200μm相比,NaCl粒徑范圍為120~200μm的PLGA支架更適合人脂肪干細胞向肝細胞的分化,提示支架孔徑過大和過小都不利于細胞的分化;另外,NaCl粒徑固定為50~120μm時,與75%和90%相比,NaCl比例為50%的PLGA支架更適合人脂肪干細胞向肝細胞的分化,提示孔隙率的增加也不利于細胞的分化。 四、PLGA支架體內(nèi)對預誘導人脂肪干細胞的影響 體內(nèi)、外環(huán)境的差異導致支架材料的體內(nèi)、外特性不會完全相同,因此在確定PLGA支架體外能夠支持人脂肪干細胞向肝細胞分化的基礎上有必要進行體內(nèi)移植實驗,以觀察PLGA支架體內(nèi)對預誘導人脂肪干細胞的影響。 將人脂肪干細胞與PLGA支架復合,體外用肝細胞誘導液預先誘導14d,然后移植入70%肝切除大鼠體內(nèi),14d后觀察。部分肝切除的微環(huán)境利于干細胞向肝細胞的分化,本實驗發(fā)現(xiàn)此種微環(huán)境下與PLGA支架復合的預誘導人脂肪干細胞仍然存活并維持肝細胞特異性表型的表達,表明PLGA支架體內(nèi)對植入的細胞并未產(chǎn)生不良影響。但是,體內(nèi)移植14d后大鼠血清中未檢測到人Alb的分泌,這可能與細胞植入的位置和植入細胞數(shù)目的不足有關。 總之,本實驗研究結果表明PLGA支架體內(nèi)、外均支持人脂肪干細胞向肝細胞的分化,PLGA與人脂肪干細胞復合適合用于肝組織工程的構建。這些研究可為肝組織工程最終應用于臨床積累必要的信息。
[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 .
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R329.2

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相關期刊論文 前1條

1 岳慧敏;張磊;王韞芳;梁峰;管利東;李紹青;閆舫;南雪;白慈賢;林峰;顏永年;裴雪濤;;人骨髓來源的間充質(zhì)干細胞在聚乳酸羥基乙酸材料上增殖及向內(nèi)皮細胞分化的研究[J];科學通報;2006年10期

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本文編號：1964291