本文選題：脂肪干細(xì)胞 + 聚乳酸羥基乙酸�。� 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)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(yán)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(diǎn),其臨床應(yīng)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(biāo)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(yīng)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(jí)階段,距離臨床應(yīng)用還很遙遠(yuǎn),還有很多問題需要解決,如種子細(xì)胞的選擇,材料的篩選,及組織的血管化等。其中種子細(xì)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(xì)胞樣細(xì)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(xì)胞的來源包括成熟肝細(xì)胞和干細(xì)胞(胚胎干細(xì)胞和成體干細(xì)胞)。由于成熟肝細(xì)胞無法擴(kuò)增、體外培養(yǎng)易失去活性與功能;而胚胎干細(xì)胞存在獲取困難、倫理爭(zhēng)議和致瘤性風(fēng)險(xiǎn)等諸多限制;相對(duì)而言,成體干細(xì)胞獲取容易、可體外大規(guī)模培養(yǎng)擴(kuò)增,因此,近年來其研究和應(yīng)用備受關(guān)注。成體干細(xì)胞中最有代表性的是骨髓來源的間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)。近期研究表明,脂肪組織來源的MSC(即脂肪干細(xì)胞,adipose-derived stem cells,ADSCs)與骨髓MSC在生物學(xué)性能方面具有極大的相似性,也具有包括肝細(xì)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(xì)胞獲得量更大。所以脂肪干細(xì)胞有希望成為肝組織工程另一種很有前景的種子細(xì)胞來源。 理想支架材料的研究和應(yīng)用是關(guān)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(jìn)展和突破,而常用于肝組織工程構(gòu)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(zhǔn)的PLGA是目前應(yīng)用最廣泛、最重要的一類人工合成材料,已與多種干細(xì)胞結(jié)合應(yīng)用于多種組織工程組織構(gòu)建的研究。 目前,脂肪干細(xì)胞在任何三維支架上向肝細(xì)胞分化的研究還未見報(bào)道,本實(shí)驗(yàn)中我們選擇人脂肪干細(xì)胞作為種子細(xì)胞,并首先選定PLGA作為支架,探討PLGA支架體內(nèi)、外對(duì)人脂肪干細(xì)胞向肝細(xì)胞分化的影響,為肝組織工程的構(gòu)建尋找合適的種子細(xì)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(xì)胞誘導(dǎo)液誘導(dǎo)人脂肪干細(xì)胞向肝細(xì)胞分化。然后將人脂肪干細(xì)胞接種在PLGA支架上,體外用相同的肝細(xì)胞誘導(dǎo)液培養(yǎng),觀察人脂肪干細(xì)胞能否向肝細(xì)胞分化。在此基礎(chǔ)上進(jìn)一步探討PLGA支架物理特性和成纖維細(xì)胞共培養(yǎng)對(duì)人脂肪干細(xì)胞向肝細(xì)胞分化的影響。最后觀察PLGA支架材料體內(nèi)對(duì)預(yù)誘導(dǎo)人脂肪干細(xì)胞的影響。 研究內(nèi)容有以下幾個(gè)主要方面: 一、人脂肪干細(xì)胞在二維單層培養(yǎng)體系下向肝細(xì)胞的誘導(dǎo)分化 分離人脂肪干細(xì)胞并從形態(tài)、表型和多向分化能力方面進(jìn)行鑒定。在此基礎(chǔ)上,將人脂肪干細(xì)胞接種在普通培養(yǎng)板中,用改良的肝細(xì)胞誘導(dǎo)液進(jìn)行誘導(dǎo)。這種改良的誘導(dǎo)液含有肝細(xì)胞生長因子(hepatocyte growth factor,HGF)、堿性成纖維細(xì)胞生長因子(basic fibroblast growth factor,bFGF)、成纖維細(xì)胞生長因子(fibroblast growth factor 4,FGF4)和抑瘤素(oncostatin M,OSM)。誘導(dǎo)14d后,形態(tài)、表型和功能鑒定結(jié)果顯示人脂肪干細(xì)胞能夠分化為肝細(xì)胞樣細(xì)胞。 干細(xì)胞誘導(dǎo)過程與非誘導(dǎo)過程細(xì)胞的增殖情況不同,而大多數(shù)研究關(guān)注的是后者,且有文獻(xiàn)報(bào)道HGF和FGF因子能同時(shí)促進(jìn)細(xì)胞的分化和增殖,因此我們觀察了此種肝細(xì)胞誘導(dǎo)液對(duì)細(xì)胞增殖的影響。研究結(jié)果發(fā)現(xiàn)細(xì)胞顯著增殖,此種肝細(xì)胞誘導(dǎo)液在促進(jìn)人脂肪干細(xì)胞向肝細(xì)胞分化的同時(shí)起到了對(duì)細(xì)胞擴(kuò)增的作用。人脂肪干細(xì)胞在二維單層培養(yǎng)體系下向肝細(xì)胞誘導(dǎo)的成功可為下一步人脂肪干細(xì)胞與支架復(fù)合后向肝細(xì)胞的誘導(dǎo)奠定基礎(chǔ)。 二、人脂肪干細(xì)胞在PLGA三維支架上體外向肝細(xì)胞的誘導(dǎo)分化 目前,人脂肪干細(xì)胞向肝細(xì)胞誘導(dǎo)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(bào)道。在明確人脂肪干細(xì)胞用改良的肝細(xì)胞誘導(dǎo)液誘導(dǎo)能夠分化成肝細(xì)胞樣細(xì)胞的基礎(chǔ)上,本部分研究進(jìn)一步將人脂肪干細(xì)胞與多孔PLGA支架復(fù)合,體外用相同的肝細(xì)胞誘導(dǎo)液進(jìn)行誘導(dǎo),觀察多孔PLGA支架體外對(duì)脂肪干細(xì)胞向肝細(xì)胞誘導(dǎo)分化的影響,以尋找適宜人脂肪干細(xì)胞向肝細(xì)胞分化的三維支架。 支架材料和干細(xì)胞良好的生物相容性是干細(xì)胞能夠在支架表面分化的基礎(chǔ),DAPI染色、CCK8檢測(cè)和掃描電鏡觀察結(jié)果證明PLGA支架和人脂肪干細(xì)胞有良好的生物相容性。細(xì)胞的生長和增殖在組織工程中非常關(guān)鍵,CCK8檢測(cè)結(jié)果表明接種在PLGA支架上的人脂肪干細(xì)胞在前述誘導(dǎo)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(shí)了含有細(xì)胞因子HGF、bFGF和FGF4的肝細(xì)胞誘導(dǎo)液能夠促進(jìn)細(xì)胞增殖,且提示三維環(huán)境可能比二維環(huán)境更利于細(xì)胞增殖。接種于三維支架的細(xì)胞其形態(tài)受支架類型和細(xì)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(xì)胞向肝細(xì)胞誘導(dǎo)過程細(xì)胞形態(tài)的變化,發(fā)現(xiàn)細(xì)胞沿著多孔支架表面伸展,并逐漸融合形成一細(xì)胞單層覆蓋在支架表面,形態(tài)逐漸向多角形改變,很多細(xì)胞伸出細(xì)胞突觸在細(xì)胞單層上面互相連接,細(xì)胞能夠增殖、分化成三維組織樣結(jié)構(gòu)。在上述基礎(chǔ)上,我們重點(diǎn)探討了多孔PLGA支架能否支持人脂肪干細(xì)胞向肝細(xì)胞分化,結(jié)果表明細(xì)胞能夠分化成具有肝細(xì)胞特異性形態(tài)、表型和功能的肝細(xì)胞樣細(xì)胞。 三、成纖維細(xì)胞共培養(yǎng)和PLGA支架物理特性對(duì)脂肪干細(xì)胞向肝細(xì)胞分化的影響 人脂肪干細(xì)胞在多孔PLGA支架上向肝細(xì)胞分化過程受很多因素影響。成纖維細(xì)胞能夠分泌很多利于干細(xì)胞向肝細(xì)胞分化的細(xì)胞外基質(zhì)和細(xì)胞因子,我們采用非接觸共培養(yǎng)的方式對(duì)成纖維細(xì)胞與人脂肪干細(xì)胞共培養(yǎng)能否引發(fā)其向肝細(xì)胞的分化進(jìn)行了研究。結(jié)果發(fā)現(xiàn)人真皮成纖維細(xì)胞與人脂肪干細(xì)胞用普通培養(yǎng)基共培養(yǎng)即可使其表達(dá)肝細(xì)胞特異性基因,且成纖維細(xì)胞共培養(yǎng)可促進(jìn)肝細(xì)胞誘導(dǎo)液誘導(dǎo)的肝細(xì)胞特異性基因的表達(dá),表明直接的細(xì)胞間接觸不是人真皮成纖維細(xì)胞介導(dǎo)的人脂肪干細(xì)胞向肝細(xì)胞分化所必需,成纖維細(xì)胞分泌的可溶性細(xì)胞因子(如HGF和FGF2)能夠通過旁分泌方式為肝細(xì)胞分化提供足夠的信號(hào),并且能夠與肝細(xì)胞誘導(dǎo)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(xì)胞向肝細(xì)胞分化時(shí)制備的多孔PLGA支架其微孔結(jié)構(gòu)特性我們是隨機(jī)選定的,NaCl粒徑為30~50μm,比例為75%(w/w)。為今后更好地設(shè)計(jì)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(duì)人脂肪干細(xì)胞向肝細(xì)胞分化的影響。細(xì)胞形態(tài)的掃描電鏡觀察結(jié)果和Alb基因表達(dá)的實(shí)時(shí)定量PCR檢測(cè)結(jié)果表明,NaCl比例固定為75%時(shí),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(xì)胞向肝細(xì)胞的分化,提示支架孔徑過大和過小都不利于細(xì)胞的分化;另外,NaCl粒徑固定為50~120μm時(shí),與75%和90%相比,NaCl比例為50%的PLGA支架更適合人脂肪干細(xì)胞向肝細(xì)胞的分化,提示孔隙率的增加也不利于細(xì)胞的分化。 四、PLGA支架體內(nèi)對(duì)預(yù)誘導(dǎo)人脂肪干細(xì)胞的影響 體內(nèi)、外環(huán)境的差異導(dǎo)致支架材料的體內(nèi)、外特性不會(huì)完全相同,因此在確定PLGA支架體外能夠支持人脂肪干細(xì)胞向肝細(xì)胞分化的基礎(chǔ)上有必要進(jìn)行體內(nèi)移植實(shí)驗(yàn),以觀察PLGA支架體內(nèi)對(duì)預(yù)誘導(dǎo)人脂肪干細(xì)胞的影響。 將人脂肪干細(xì)胞與PLGA支架復(fù)合,體外用肝細(xì)胞誘導(dǎo)液預(yù)先誘導(dǎo)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(xì)胞向肝細(xì)胞的分化,本實(shí)驗(yàn)發(fā)現(xiàn)此種微環(huán)境下與PLGA支架復(fù)合的預(yù)誘導(dǎo)人脂肪干細(xì)胞仍然存活并維持肝細(xì)胞特異性表型的表達(dá),表明PLGA支架體內(nèi)對(duì)植入的細(xì)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(cè)到人Alb的分泌,這可能與細(xì)胞植入的位置和植入細(xì)胞數(shù)目的不足有關(guān)。 總之,本實(shí)驗(yàn)研究結(jié)果表明PLGA支架體內(nèi)、外均支持人脂肪干細(xì)胞向肝細(xì)胞的分化,PLGA與人脂肪干細(xì)胞復(fù)合適合用于肝組織工程的構(gòu)建。這些研究可為肝組織工程最終應(yīng)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(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329.2

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相關(guān)期刊論文 前1條

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