聚L-谷氨酸/壳聚糖材料在脂肪组织工程以及胆管癌三维培养模型中应用的实验研究
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摘要
第一部分聚L-谷氨酸/壳聚糖材料在脂肪组织工程中应用的实验研究
目的
每年全世界有数百万的患者因创伤、肿瘤切除手术和先天性发育缺陷等原因会导致的大面积软组织缺损,大范围的软组织缺损是无法自愈的,需要通过手术进行等方法修复重建。组织修复重建的主要目的不仅仅是恢复正常的组织结构,更重要的是通过对缺损的修复,恢复正常的组织功能,减轻患者焦虑、自卑等不良心理。
传统的软组织修复重建方法主要包括游离脂肪移植,复合组织瓣移植或人工合成替代物的植入等方式,它们都存在着各自的缺点,如游离脂肪移植成活率低、容易吸收(体积降低40%-60%)；复合组织瓣移植,对供区组织损伤大,操作复杂,不易塑形；人工合成替代物,如硅胶等可能会引起机体不同程度的排异反应,长期效果可能不理想。
随着细胞生物学与生物材料科学的发展,以种子细胞、支架材料构建组织为基本特征的组织工程学应运而生,为解决组织乃至器官缺损提供了新的思路。脂肪组织工程的基本策略就是利用种子细胞与具有生物相容性和生物降解性的生物材料结合经过生长因子诱导构建脂肪组织。成功构建组织工程化脂肪的关键在于：①种子细胞的选择和获得；②具有良好生物降解性和组织相容性的三维支架材料；③种子细胞增殖和分化的微环境。
本实验以人脂肪干细胞(adipose tissue-derived adult stem cells, ADSC)作为种子细胞,以水溶性聚L-谷氨酸/壳聚糖(poly[L-glutamic acid]/chitosan, PLGA/CS)静电络合而成的新型多孔材料作为支架材料,构建组织工程化脂肪。探讨新型支架材料聚L-谷氨酸/壳聚糖的细胞相容性及与ADSC体外及体内成脂的状况,证实人ADSC能够在水溶性聚L-谷氨酸/壳聚糖支架材料上构建组织工程化脂肪,为脂肪组织工程增添新的内容。
方法
一、人脂肪干细胞体外成脂分化潜能的研究
1.应用酶消化法从吸脂手术的患者中获取新鲜脂肪组织,提取ADSCs,体外培养扩增,取第3代ADSCs用于实验研究。
2.流式细胞术检测ADSCs分化群表面抗原CD13、CD14、CD34、CD44、 CD45、CD49d、CD90、CD105、CD106和CD166的表达。
3.利用不同的生长因子体外诱导,对ADSCs成骨、成脂、成软骨三向分化性能进行鉴定。
4.体外诱导ADSCs成脂分化21天,观察细胞形态学变化,Oil-red O染色及其定量检测细胞内脂滴的形成情况。
二、ADSCs于PLGA/CS支架材料体外成脂的研究
1.以聚乳酸PLA微球(直径在200-300-tm)作为致孔剂,利用冷冻干燥技术和静电络合技术制成球形孔结构的PLGA/CS支架材料。
2.用扫描电镜(scanning electron microscope, SEM)和荧光染料DiL标记的方法定性检测ADSCs在PLGA/CS支架材料上粘附和增殖情况。
3.通过Hoechst33258染色的DNA定量方法,绘制成脂分化诱导组和未诱导组的ADSCs在PLGA/CS支架材料上的增殖曲线。
4.通过定量检测细胞/材料复合物乳酸脱氢酶的释放,评价PLGA/CS材料对ADSCs的细胞毒性。
5.用Oil-red O染色法,定量检测ADSCs接种到PLGA/CS材料经成脂诱导后细胞内脂滴形成情况,并与末诱导组进行对比。
6.用RT-PCR法检测诱导组和未诱导组的ADSCs接种PLGA/CS支架材料后的脂特异性基因的表达情况,包括过氧化物酶体增生物激活物受体(peroxisome proliferator-activated receptor gamma2, PPARγ2)、脂肪酸结合蛋白(fatty acid-binding protein, aP2)、脂蛋白脂肪酶(lipoprotein lipase, LPL)、脂连接蛋白(adiponectin)。
三、ADSCs在PLGA/CS支架材料体内成脂研究
以ADSC接种在PLGA/CS支架材上后体外成脂诱导2周为实验组,以空白PLGA/CS支架材料为对照组。12只SCID裸鼠随机分为两组,每组6只,将实验组和对照组材料分别移植于裸鼠皮下。移植6周后取出植入物,测量其体积变化来观察PLGA/CS支架材料降解情况；OCT包埋进行冰冻切片,Oil-red O染色检测其脂肪形成情况。
结果
1.成功的从脂肪抽吸术获取的新鲜脂肪组织中,原代培养出ADSCs,细胞活性良好,增殖旺盛。流式细胞术的检测结果显示,实验所用P3ADSCs的CD13、CD34、CD44、CD90、CD105、CD166以及CD49d阳性表达,CD14、CD45与CD31阴性表达；鉴定结果显示其具有成骨、成软骨和成脂的三向分化潜能。成脂诱导过程中,细胞形态由“长梭形”向圆形脂肪细胞转化,Oil-red O染色及其定量测定显示随时间增加,细胞内脂滴形成逐渐增多。
2. PLGA/CS材料外观呈多孔海绵状,孔径均一,孔间连通性良好,孔隙率>90%,对ADSCs显示出良好的细胞相容性；SEM与DiL染色的共聚焦显微镜观察显示ADSCs于PLGA/CS支架材料上粘附、增殖良好；PLGA/CS支架材料的细胞毒性对接种的ADSCs影响不大。Oil-red O染色及其定量检测显示实验组脂滴累积明显高于对照组；RT-PCR结果显示,实验组成脂特异性基因表达均增加,且明显高于对照组。
3.裸鼠体内移植物6周后取出,可见移植物体积完整,有明显的血管长入；体积测量结果显示,实验组和对照组体积均有不同程度降低,分别为初始体积的90.5%和82.3%,两组间差别无统计学意义(p>0.05)。冰冻切片Oil-red O染色显示实验组有新生脂肪形成,而对照组没有。
结论
1. ADSCs具有成脂分化潜能,是脂肪组织工程理想的种子细胞。
2. ADSCs在PLGA/CS支架材料上能较好粘附、增殖以及体外和体内的成脂分化,说明PLGA/CS支架材料对ADSCs具有良好的生物相容性和促成脂分化的潜能,表明PLGA/CS可能是一种理想的脂肪组织工程细胞支架材料。
3. PLGA/CS支架材料在裸鼠体内可以降解,表明其具有生物可降解性,但材料的降解率与体内脂肪形成的动力学关系仍需要进一步研究。
创新性及研究的意义
本研究首次将聚L-谷氨酸/壳聚糖静电络合而成的支架材料应用于脂肪组织工程中研究,成功的完成了ADSCs在PLGA/CS支架材料上体外和体内脂肪组织的构建,为组织工程支架以及整个脂肪组织工程的发展增加了新的实验资料。
第二部分聚L-谷氨酸／壳聚糖材料在胆管癌三维培养模型中应用的实验研究
目的
胆管癌(Cholangiocarcinoma, CC)泛指起源于胆管上皮的恶性肿瘤,发病率仅低于肝细胞肝癌,排在肝胆系统肿瘤中的第二位。根据解剖部位不同,胆管癌可分为肝外胆管癌和肝内胆管癌。由于胆管癌发病隐匿,早期症状不明显,发病时已近中晚期,手术切除治愈率低,预后不良。
流行病学调查显示,近年来胆管癌的发病率明显有升高趋势,逐渐成为重要的恶性肿瘤致死的病因,日益受到了国内外学者越来越广泛的关注。尽管诊断和治疗方法不断的进步,胆管癌的预后依然不容乐观。建立适宜的研究模型,充分了解胆管癌发生和发展的分子机制,对胆管癌的治疗和预后改善具有重要意义。
上皮间质转化(epithelial to mesenchymal transition, EMT)是指细胞从上皮形态到间质成纤维形态的转化过程,主要表现为上皮特点的失去和移动能力的获得。其重要的改变包括E-钙粘素(E-cadherin)介导的细胞间粘连、其他上皮标记和细胞极性的丧失,同时伴随着移动能力的获得和细胞骨架重组。EMT不仅发生在胚胎形成早期和器官组织慢性纤维化过程中,也发生在肿瘤局部侵袭,远处转移和药物抵抗的过程中。肿瘤的浸润过程,肿瘤细胞发生EMT,彼此分离,移动入基底膜,侵袭到临近的结缔组织中。目前已证实很多肿瘤的扩散和转移都是通过EMT实现的,如乳腺癌、卵巢癌、肺癌,胆管癌等。EMT的过程受到很多环境因素和信号通路的调控。在这些信号通路中,转化生长因子-β1(transforming growth factor, TGF-β1)是EMT发生及肿瘤和其他病理现象发展中最重要的诱导因子。
本研究中,我们首先在二维(two dimensional,2D)模型中探讨了TGF-β1对肝内胆管癌细胞HCCC9810EMT过程的影响,然后利用PLGA/CS体外建立肿瘤细胞三维(three dimensional,3D)培养模型,观察了3D模型对HCCC9810细胞生长、增殖及侵袭能力的影响,不仅为胆管癌的基因治疗提供了靶点,同时将组织工程理念引领到肿瘤研究中,为肿瘤的生物学特性和治疗方法的研究提供了新的思路。
方法
1.以TGF-β110ng/ml浓度体外2D诱导HCCC9810细胞,设为实验组,正常生长培养液培养的HCCC9810细胞为对照组,采用光学显微镜观察细胞形态的变化；用体外划痕试验(wound healing assay)检测细胞移动能力的改变；免疫荧光定性检测上皮特性指标,包括E-cadherin、β-连环蛋白(β-catenin)和角蛋白(Pan-cytokeratins, Pan-CK),以及间质特性指标,包括波形蛋白(Vimentin)和纤维粘连蛋白(Fibronectin)的变化以及骨架蛋白F-actin的重组情况；WB和real-time PCR法检测上皮和间质特性指标的蛋白和mRNA水平的表达情况。
2.将HCCC9810细胞接种于PLGA/CS材料上,建立PLGA/CS胆管癌三维培养模型,用SEM和荧光染料DiL标记的方法定性检测HCCC9810细胞在PLGA/CS材料上粘附和生长情况；用Hoechst33528染色DNA定量检测HCCC9810细胞在PLGA/CS材料上的增殖情况,并与2D结果进行对比；用real-time PCR法检测上皮特性指标(包括E-cadherin和β-catenin)和间质特性指标(Fibronectin、Vimentin和N-钙粘素[N-cadherin])以及TGF-β1的mRNA表达情况。
结果
1.体外2D培养下,TGF-β1诱导HCCC9810细胞逐渐失去上皮“铺路石’形状,转化成成纤维样“长梭形”形状；划痕试验显示实验组的细胞获得移动能力；免疫荧光结果显示胆管癌细胞不表达E-cadherin,而β-catenin由诱导前细胞膜表达变成诱导后细胞质中也有表达,Pan-CK诱导后表达降低；诱导后间质细胞标志Vimentin和Fibronectin明显增强,同时细胞骨架蛋白F-actin发生重组；WB结果显示,上皮细胞特性标志中,E-cadherin无表达,β-catenin和Pan-CK诱导后表达不同程度降低,而间质细胞特性标志中,Vimentin和Fibronectin表达明显增加,N-cadherin表达未有明显变化；real-time PCR结果显示mRNA水平上皮细胞特性标志中,E-cadherin和β-catenin诱导后表达显著性降低,间质细胞特性标志Vimentin, Fibronectin和N-cadherin均不同程度增加,均具有统计学意义(p<0.05)。
2. HCCC9810细胞接种在PLGA/CS材料上表现为粘附、增殖良好；DNA定量检测结果显示细胞于材料上的增殖速率较2D培养速度降低,这更加符合肿瘤体内生长的真实情况；real-time PCR结果显示,细胞接种材料后,E-cadherin和β-catenin的mRNA表达随时间逐渐降低,第21天表达显著降低(p<0.05),Fibronectin和vimentin于细胞接种材料后14天和21天mRNA表达明显增高(p<0.05); N-cadherin的mRNA表达未见明显变化；TGF-β1的mRNA水平于细胞接种材料14天和21天后表达明显增加(p<0.05)。
结论
1. TGF-β1能够诱导胆管癌发生EMT,证实TGF-β1及其信号通路在胆管癌的侵袭和转移中具有挥重要作用,可作为胆管癌基因治疗的靶点。
2.组织工程支架材料PLGA/CS可用于体外3D肿瘤细胞培养模型的建立,较传统2D培养模式更真实的反应肿瘤体内生长情况,且长时间培养,肿瘤细胞白分泌TGF-β1,使细胞移动和侵袭能力增加,符合胆管癌体内自分泌或旁分泌TGF-β1实现局部浸润和远处转移的特点。
创新性及研究意义
1.证实了TGF-β1能够诱导胆管癌发生EMT,提出TGF-β1可作为胆管癌基因治疗的靶点；
2.组织工程支架材料PLGA/CS可用于体外3D肿瘤细胞培养模型的建立,为肿瘤侵袭的的研究提供更加新的研究方法。
Part1Application of porous scaffold poly (L-glutamic acid)/chitosan on adipose tissue engineering
Objective
Large soft-tissue defects from traumatic injury, oncologic resection and congenital deformities do not repair spontaneously. The defects and resulting contour deformities lead to abnormal cosmesis, affect the emotional well-being of patients, and may impair function (e.g., limb range of motion). Current standard treatments primarily involve tissue transplantation, including composite tissue flaps and synthetic substitutes. However, limitations are donor-site morbidity, unpredictable outcomes due to graft resorption over time (40%-60%volume loss), allergic reactions, and fibrous capsule contraction, as well as complications and costly surgery.
The development of adipose tissue engineering has made it an attractive approach with great potential for repairing large soft-tissue defects. The basic strategy of adipose tissue engineering involves the utilization of artificial of natural extracellular matrix as scaffolds in combination with specific types of cells under the stimulation of growth factors to restore defects structurally and functionally. The key points in adipose tissue engineering were composed with the selection of cell, biomaterials with biocompatibility and biodegradation as well as the microenvironment for growth and differentiation of cell.
In this study, we constructed tissue-engineered adipose with adipose tissue-derived adult stem cells and a novel porous scaffold poly (L-glutamic acid)/chitosan (PLGA/CS) which was improved with electrostatic interaction between carboxyl groups of water soluble PLGA and amido groups of CS using the phase separation method. Besides, we investigated the biological characteristics of PLGA/CS and the attractive potential of PLGA/CS as scaffold for tissue engineering.
Methods
1. The adipogenic differentiation potential of ADSCs
(1) We harvested ADSCs from fresh human lipoaspirates obtained from patients who had undergone abdominal liposuction with enzyme digestion method. After the standard culture in vitro, passage2cells were used in the study.
(2) The expression of cluster differentiation (CD) markers on ADSCs was revealed by flow cytometry, including CD13、CD14、CD34、CD44、CD45、CD49d、CD90、 CD105, CD106and CD166.
(3) Multi-lineage differentiation of ADSCs was reveal by ALP staining, the expression of collagen type11and Oil-red O staining.
(4) The changes of morphologic features and intracellular lipid vacuoles were observed by light microscope and Oil-red O staining respectively.
2. The adipogenic differentiation of ADSCs on PLGA/CS in vitro
(1) PLGA/CS was prepared with PLA microspheres (diameter200-300μm) as porogens using freeze drying and static electricity method.
(2) Scanning electron microscope (SEM) observation and fluorescent DiL labeling were carried out to reveal the attachment and growth of ADSCs on PLGA/CS.
(3) The quantitative assay of cell proliferation with time was detected by DNA assay.
(4) The cytotoxicity of PLGA/CS was revealed by the quantity assay for the expression of lactate dehydrogenase (LDH).
(5) Oil-red O staining was carried out to assess the intracellular lipid accumulation of ADSCs on PLGA/CS with or without adipogenic induction.
(6) RT-PCR was carried out to reveal the expression of adipocyte-specific and insulin-regulated genes, including peroxisome proliferatior-activated receptor y2(PPARy2), fatty acid-binding protein (aP2), lipoprotein lipase (LPL) and adiponectin of ADSCs on PLGA/CS with or without adipogenic induction.
3. In vivo adipose tissue formation
After2weeks of exposure to adipogenic medium in vitro, ADSC-seeded scaffolds were implanted into the dorsum of severe combined immunodeficient (SCID) mice (6weeks old); acellular scaffolds were a control (n=6each group). After6weeks, animals were sacrificed and implants were harvested. To examine whether volume-stable adipose tissue could be harvested, tissue volumes were measured by immersing the tissue in PBS in a graded pipette. The volume change of implants was the ratio of the volume of implants retrieved at6weeks to the initial volume. Furthermore, to demonstrate newly formed fat tissue, the constructs retrieved in both groups were flash-frozen in Tissue-Tek OCT freezing medium for Oil-red O staining.
Results
1. In2D culture, ADSCs isolated from fresh human lipoaspirates showed uniform fibroblast-like morphologic features. Flow cytometry revealed the positive expression of CD13, CD34, CD44, CD90, CD105, CD166, CD49d and negative expression of CD14, CD45and CD31; Multi-lineage differentiation of ADSCs was confirmed by ALP staining, the expression of collagen type Ⅱ and Oil-red O staining. Under non-differentiation culture, ADSCs retained their fibroblast-like spindle shapes, with negative staining for Oil-red O at7and14days. On incubation with adipogenic medium, cells resembled adipocytes， with a rounded appearance and a time-dependent increase in intracellular lipid vacuoles, with positive staining for Oil-red O at7and14days. Increased staining for Oil-red O demonstrated increased adipogenic differentiation of ADSCs with increased exposure time. After1and2weeks, Oil-red O staining was higher for differentiated ADSCs than controls (p<0.05).
2. The PLGA/CS scaffold appeared as a soft and porous spongy-like mass. The porosity of this scaffold was greater than90%. SEM demonstrated the even distribution and abundance of ECM deposition surrounding PLGA/CS scaffolds at7days after seeding. Quantitative Hoechst33258assay revealed greatly increased number of cell-seeded constructs in both groups. Oil-red O staining was greater for cell-seeded scaffolds exposed to adipogenic medium for21days than non-induced constructs (p<0.05). RT-PCR revealed increased expression of adipocyte-specific and insulin-regulated genes.
3. Six weeks after implantation, gross inspection revealed intact and well-vascularized cell-seeded scaffolds. Moreover, cell-scaffold constructs appeared to shrink, which had decreased to90.5%and82.3%of the original volume in the cell-seeded and acellular groups, respectively (p>0.05). Oil-red O staining demonstrated adipose tissue formation within adipogenic-differentiated cell-seeded scaffolds after6weeks' implantation, with little adipose tissue formed within acellular implants.
Conclusion
1. ADSCs are an ideal autologous cell source for adipose tissue engineering.
2. The PLGA/CS scaffold had good biocompatibility and biodegradation for cell adhesion and proliferation for tissue engineering, and it could be a promising novel scaffold material for tissue engineering.
3. Even though the adipose-like tissue was confirmed in vivo study, a further study is needed to fabricate porous scaffolds with optimal ration of PLGA and CS which can match the kinetics of new adipose formation in vivo.
Originality
We introduced a novel porous scaffold poly (L-glutamic acid)/chitosan (PLGA/CS) which was improved with electrostatic interaction between carboxyl groups of water soluble PLGA and amido groups of CS using the phase separation method and finished the first use of such scaffold for adipose tissue engineering.
Part2Application of porous scaffold poly (L-glutamic acid)/chitosan for tumor3D-cultured model
Objective
Cholangiocarcinoma (CC) is the second most common from of primary liver cancer after hepatocellular carcinoma. CC is classified anatomically into extrahepatic cholangiocarcinoma (ECC) and intrahepatic cholangiocarcinoma (ICC). Despite advances in diagnosis and treatment, the prognosis of CC has not yet been resolved, because it is difficult to make an early diagnosis and standard therapy is not very effective. CC is generally characterized by strong proliferation, invasion, and early metastasis. To improve the prognosis, we require a fuller understanding of the molecular mechanisms behind its proliferation and progression.
Epithelial-mesenchymal transition (EMT) is an intricate process by which epithelial cells lose their epithelial characteristics and acquire a mesenchymal-like phenotype. This dramatic phenotypic changes involve the loss of E-cadherin-mediated cell-cell adhesion and additional prototypic epithelial markers, as well as the loss of apical-basal polarity, concomitantly with the acquisition of a motile behavior and a profound reorganization of the cytoskeleton. EMT was related to early embryogenesis, organ fibrosis as well as the process of tumor progression including invasion, metastasis and therapeutic resistance. EMT provides tumor cells with the ability to dissociate from each other and degrade and to actively migrate into the basal membrane and invade the adjacent connective tissues. ECM has been proved to relate to the tumor progression such as breast, prostate, ovarian, lung cancer. A great number of environment factors and signals have been described that induce EMT. Among the signaling pathways, transforming growth factor-(3(TGF-β) is probably the best characterized inducer of EMT in development, cancer and other pathologies.
In this study, we first investigated the effect of TGF-β1on EMT of HCCC9810cell in2D culture model to provide the target for gene therapy. Besides, we translated tissue engineered scaffold PLGA/CS into3D cancer-cultured model to reveal the effect of such3D model on the growth and invasion of cancer.
Methods
1. In standard monolayer cultures, HCCC9810was cultured within induced medium composed with10ng/ml TGF-β1,1%FBS and RPMI1640, the cells cultured in growth medium was served as control group; the expected morphological changes were detected by direct microscopic observation. The simple wound-healing migration assay was carried out to assess the motility behavior of cells. The organization at cellular levels of the individual EMT markers (i.e. Vimentin, Fibronectin for mesenchymal marker; E-cadherin, β-catenin and Pan-CK for epithelial marker) was revealed by immunofluorescence assay, while the protein and mRNA levels were detected by WB and real-time PCR respectively.
2. HCCC9810cells were seeded onto porous scaffold PLGA/CS. Scanning electron microscope (SEM) observation and fluorescent DiL labeling were carried out to reveal the attachment and growth of cells on PLGA/CS; the quantitative assay of cell proliferation with time was detected by DNA assay. The mRNA levels of EMT-related markers and TGF-β1was detected by real-time PCR.
Results
1. Under microscopic observation, the cells in induced group exhibited the loss of cell-cell contacts and cobblestone morphology, appearance of elongated mesenchymal features along with growth as individual cells; the wound healing assay showed the motility behaviror of cells cultured in induced medium. Immunophenotypical characterization in2D cultures showed the expression of β-catenin transformed from the membrane into cytoplasm; no expression of E-cadherin; down-regulated expression of Pan-CK; the higher expression of mesenchymal markers fibronectin and vimentin after induction; F-actin cytoskeleton reorganization was observed. Real-time PCR showed down-regulated expression of E-cadherin and β-catenin; the up-regulated expression of fibronectin, vimentin and N-cadherin. WB showed no expression of E-cadherin; the less expression of β-catenin; the higher expression of fibronectin, vimentin and N-cadherin which were similar to the results of IF and real-time PCR.
2. SEM demonstrated the even distribution and abundance of ECM deposition surrounding PLGA/CS scaffolds. Quantitative Hoechst33258assay revealed greatly increased number of cell-seeded constructs, whereas the proliferation rate is less than that of2D cultured cell. The results from3D model reflected the real growth state of tumor in vivo. Real-time PCR showed the expression of E-cadherin and β-catenin was significantly decreased after21days post seeding (p<0.05); the expression of vimentin and fibronectin was significantly increased after14and21days post seeding (p<0.05); interestingly, the expression of TGF-β1was increased after14and21days post seeding (p<0.05).
Conclusion
1. We confirmed that HCCC9810cells underwent EMT process induced by TGF-β1. The expected morphological changes and the expected changes in EMT-related markers do occur at the cellular, protein and mRNA level.
2. We translated tissue engineering technology platforms into cancer research and provided more physiological models for cancer research. We found such scaffold-based3D culture model could promote the autocrine of TGF-β1to improve the EMT and tumor invasion.
Originality
1. TGF-β1may become the target for gene therapy of cholangiocarcinoma.
2. Tissue-engineered scaffold PLGA/CS was first utilized for cancer research, and the
3D culture model could be promising tool for tumor invasion and metastasis as well as the development of therapeutic drugs.
目的
每年全世界有数百万的患者因创伤、肿瘤切除手术和先天性发育缺陷等原因会导致的大面积软组织缺损,大范围的软组织缺损是无法自愈的,需要通过手术进行等方法修复重建。组织修复重建的主要目的不仅仅是恢复正常的组织结构,更重要的是通过对缺损的修复,恢复正常的组织功能,减轻患者焦虑、自卑等不良心理。
传统的软组织修复重建方法主要包括游离脂肪移植,复合组织瓣移植或人工合成替代物的植入等方式,它们都存在着各自的缺点,如游离脂肪移植成活率低、容易吸收(体积降低40%-60%)；复合组织瓣移植,对供区组织损伤大,操作复杂,不易塑形；人工合成替代物,如硅胶等可能会引起机体不同程度的排异反应,长期效果可能不理想。
随着细胞生物学与生物材料科学的发展,以种子细胞、支架材料构建组织为基本特征的组织工程学应运而生,为解决组织乃至器官缺损提供了新的思路。脂肪组织工程的基本策略就是利用种子细胞与具有生物相容性和生物降解性的生物材料结合经过生长因子诱导构建脂肪组织。成功构建组织工程化脂肪的关键在于：①种子细胞的选择和获得；②具有良好生物降解性和组织相容性的三维支架材料；③种子细胞增殖和分化的微环境。
本实验以人脂肪干细胞(adipose tissue-derived adult stem cells, ADSC)作为种子细胞,以水溶性聚L-谷氨酸/壳聚糖(poly[L-glutamic acid]/chitosan, PLGA/CS)静电络合而成的新型多孔材料作为支架材料,构建组织工程化脂肪。探讨新型支架材料聚L-谷氨酸/壳聚糖的细胞相容性及与ADSC体外及体内成脂的状况,证实人ADSC能够在水溶性聚L-谷氨酸/壳聚糖支架材料上构建组织工程化脂肪,为脂肪组织工程增添新的内容。
方法
一、人脂肪干细胞体外成脂分化潜能的研究
1.应用酶消化法从吸脂手术的患者中获取新鲜脂肪组织,提取ADSCs,体外培养扩增,取第3代ADSCs用于实验研究。
2.流式细胞术检测ADSCs分化群表面抗原CD13、CD14、CD34、CD44、 CD45、CD49d、CD90、CD105、CD106和CD166的表达。
3.利用不同的生长因子体外诱导,对ADSCs成骨、成脂、成软骨三向分化性能进行鉴定。
4.体外诱导ADSCs成脂分化21天,观察细胞形态学变化,Oil-red O染色及其定量检测细胞内脂滴的形成情况。
二、ADSCs于PLGA/CS支架材料体外成脂的研究
1.以聚乳酸PLA微球(直径在200-300-tm)作为致孔剂,利用冷冻干燥技术和静电络合技术制成球形孔结构的PLGA/CS支架材料。
2.用扫描电镜(scanning electron microscope, SEM)和荧光染料DiL标记的方法定性检测ADSCs在PLGA/CS支架材料上粘附和增殖情况。
3.通过Hoechst33258染色的DNA定量方法,绘制成脂分化诱导组和未诱导组的ADSCs在PLGA/CS支架材料上的增殖曲线。
4.通过定量检测细胞/材料复合物乳酸脱氢酶的释放,评价PLGA/CS材料对ADSCs的细胞毒性。
5.用Oil-red O染色法,定量检测ADSCs接种到PLGA/CS材料经成脂诱导后细胞内脂滴形成情况,并与末诱导组进行对比。
6.用RT-PCR法检测诱导组和未诱导组的ADSCs接种PLGA/CS支架材料后的脂特异性基因的表达情况,包括过氧化物酶体增生物激活物受体(peroxisome proliferator-activated receptor gamma2, PPARγ2)、脂肪酸结合蛋白(fatty acid-binding protein, aP2)、脂蛋白脂肪酶(lipoprotein lipase, LPL)、脂连接蛋白(adiponectin)。
三、ADSCs在PLGA/CS支架材料体内成脂研究
以ADSC接种在PLGA/CS支架材上后体外成脂诱导2周为实验组,以空白PLGA/CS支架材料为对照组。12只SCID裸鼠随机分为两组,每组6只,将实验组和对照组材料分别移植于裸鼠皮下。移植6周后取出植入物,测量其体积变化来观察PLGA/CS支架材料降解情况；OCT包埋进行冰冻切片,Oil-red O染色检测其脂肪形成情况。
结果
1.成功的从脂肪抽吸术获取的新鲜脂肪组织中,原代培养出ADSCs,细胞活性良好,增殖旺盛。流式细胞术的检测结果显示,实验所用P3ADSCs的CD13、CD34、CD44、CD90、CD105、CD166以及CD49d阳性表达,CD14、CD45与CD31阴性表达；鉴定结果显示其具有成骨、成软骨和成脂的三向分化潜能。成脂诱导过程中,细胞形态由“长梭形”向圆形脂肪细胞转化,Oil-red O染色及其定量测定显示随时间增加,细胞内脂滴形成逐渐增多。
2. PLGA/CS材料外观呈多孔海绵状,孔径均一,孔间连通性良好,孔隙率>90%,对ADSCs显示出良好的细胞相容性；SEM与DiL染色的共聚焦显微镜观察显示ADSCs于PLGA/CS支架材料上粘附、增殖良好；PLGA/CS支架材料的细胞毒性对接种的ADSCs影响不大。Oil-red O染色及其定量检测显示实验组脂滴累积明显高于对照组；RT-PCR结果显示,实验组成脂特异性基因表达均增加,且明显高于对照组。
3.裸鼠体内移植物6周后取出,可见移植物体积完整,有明显的血管长入；体积测量结果显示,实验组和对照组体积均有不同程度降低,分别为初始体积的90.5%和82.3%,两组间差别无统计学意义(p>0.05)。冰冻切片Oil-red O染色显示实验组有新生脂肪形成,而对照组没有。
结论
1. ADSCs具有成脂分化潜能,是脂肪组织工程理想的种子细胞。
2. ADSCs在PLGA/CS支架材料上能较好粘附、增殖以及体外和体内的成脂分化,说明PLGA/CS支架材料对ADSCs具有良好的生物相容性和促成脂分化的潜能,表明PLGA/CS可能是一种理想的脂肪组织工程细胞支架材料。
3. PLGA/CS支架材料在裸鼠体内可以降解,表明其具有生物可降解性,但材料的降解率与体内脂肪形成的动力学关系仍需要进一步研究。
创新性及研究的意义
本研究首次将聚L-谷氨酸/壳聚糖静电络合而成的支架材料应用于脂肪组织工程中研究,成功的完成了ADSCs在PLGA/CS支架材料上体外和体内脂肪组织的构建,为组织工程支架以及整个脂肪组织工程的发展增加了新的实验资料。
第二部分聚L-谷氨酸／壳聚糖材料在胆管癌三维培养模型中应用的实验研究
目的
胆管癌(Cholangiocarcinoma, CC)泛指起源于胆管上皮的恶性肿瘤,发病率仅低于肝细胞肝癌,排在肝胆系统肿瘤中的第二位。根据解剖部位不同,胆管癌可分为肝外胆管癌和肝内胆管癌。由于胆管癌发病隐匿,早期症状不明显,发病时已近中晚期,手术切除治愈率低,预后不良。
流行病学调查显示,近年来胆管癌的发病率明显有升高趋势,逐渐成为重要的恶性肿瘤致死的病因,日益受到了国内外学者越来越广泛的关注。尽管诊断和治疗方法不断的进步,胆管癌的预后依然不容乐观。建立适宜的研究模型,充分了解胆管癌发生和发展的分子机制,对胆管癌的治疗和预后改善具有重要意义。
上皮间质转化(epithelial to mesenchymal transition, EMT)是指细胞从上皮形态到间质成纤维形态的转化过程,主要表现为上皮特点的失去和移动能力的获得。其重要的改变包括E-钙粘素(E-cadherin)介导的细胞间粘连、其他上皮标记和细胞极性的丧失,同时伴随着移动能力的获得和细胞骨架重组。EMT不仅发生在胚胎形成早期和器官组织慢性纤维化过程中,也发生在肿瘤局部侵袭,远处转移和药物抵抗的过程中。肿瘤的浸润过程,肿瘤细胞发生EMT,彼此分离,移动入基底膜,侵袭到临近的结缔组织中。目前已证实很多肿瘤的扩散和转移都是通过EMT实现的,如乳腺癌、卵巢癌、肺癌,胆管癌等。EMT的过程受到很多环境因素和信号通路的调控。在这些信号通路中,转化生长因子-β1(transforming growth factor, TGF-β1)是EMT发生及肿瘤和其他病理现象发展中最重要的诱导因子。
本研究中,我们首先在二维(two dimensional,2D)模型中探讨了TGF-β1对肝内胆管癌细胞HCCC9810EMT过程的影响,然后利用PLGA/CS体外建立肿瘤细胞三维(three dimensional,3D)培养模型,观察了3D模型对HCCC9810细胞生长、增殖及侵袭能力的影响,不仅为胆管癌的基因治疗提供了靶点,同时将组织工程理念引领到肿瘤研究中,为肿瘤的生物学特性和治疗方法的研究提供了新的思路。
方法
1.以TGF-β110ng/ml浓度体外2D诱导HCCC9810细胞,设为实验组,正常生长培养液培养的HCCC9810细胞为对照组,采用光学显微镜观察细胞形态的变化；用体外划痕试验(wound healing assay)检测细胞移动能力的改变；免疫荧光定性检测上皮特性指标,包括E-cadherin、β-连环蛋白(β-catenin)和角蛋白(Pan-cytokeratins, Pan-CK),以及间质特性指标,包括波形蛋白(Vimentin)和纤维粘连蛋白(Fibronectin)的变化以及骨架蛋白F-actin的重组情况；WB和real-time PCR法检测上皮和间质特性指标的蛋白和mRNA水平的表达情况。
2.将HCCC9810细胞接种于PLGA/CS材料上,建立PLGA/CS胆管癌三维培养模型,用SEM和荧光染料DiL标记的方法定性检测HCCC9810细胞在PLGA/CS材料上粘附和生长情况；用Hoechst33528染色DNA定量检测HCCC9810细胞在PLGA/CS材料上的增殖情况,并与2D结果进行对比；用real-time PCR法检测上皮特性指标(包括E-cadherin和β-catenin)和间质特性指标(Fibronectin、Vimentin和N-钙粘素[N-cadherin])以及TGF-β1的mRNA表达情况。
结果
1.体外2D培养下,TGF-β1诱导HCCC9810细胞逐渐失去上皮“铺路石’形状,转化成成纤维样“长梭形”形状；划痕试验显示实验组的细胞获得移动能力；免疫荧光结果显示胆管癌细胞不表达E-cadherin,而β-catenin由诱导前细胞膜表达变成诱导后细胞质中也有表达,Pan-CK诱导后表达降低；诱导后间质细胞标志Vimentin和Fibronectin明显增强,同时细胞骨架蛋白F-actin发生重组；WB结果显示,上皮细胞特性标志中,E-cadherin无表达,β-catenin和Pan-CK诱导后表达不同程度降低,而间质细胞特性标志中,Vimentin和Fibronectin表达明显增加,N-cadherin表达未有明显变化；real-time PCR结果显示mRNA水平上皮细胞特性标志中,E-cadherin和β-catenin诱导后表达显著性降低,间质细胞特性标志Vimentin, Fibronectin和N-cadherin均不同程度增加,均具有统计学意义(p<0.05)。
2. HCCC9810细胞接种在PLGA/CS材料上表现为粘附、增殖良好；DNA定量检测结果显示细胞于材料上的增殖速率较2D培养速度降低,这更加符合肿瘤体内生长的真实情况；real-time PCR结果显示,细胞接种材料后,E-cadherin和β-catenin的mRNA表达随时间逐渐降低,第21天表达显著降低(p<0.05),Fibronectin和vimentin于细胞接种材料后14天和21天mRNA表达明显增高(p<0.05); N-cadherin的mRNA表达未见明显变化；TGF-β1的mRNA水平于细胞接种材料14天和21天后表达明显增加(p<0.05)。
结论
1. TGF-β1能够诱导胆管癌发生EMT,证实TGF-β1及其信号通路在胆管癌的侵袭和转移中具有挥重要作用,可作为胆管癌基因治疗的靶点。
2.组织工程支架材料PLGA/CS可用于体外3D肿瘤细胞培养模型的建立,较传统2D培养模式更真实的反应肿瘤体内生长情况,且长时间培养,肿瘤细胞白分泌TGF-β1,使细胞移动和侵袭能力增加,符合胆管癌体内自分泌或旁分泌TGF-β1实现局部浸润和远处转移的特点。
创新性及研究意义
1.证实了TGF-β1能够诱导胆管癌发生EMT,提出TGF-β1可作为胆管癌基因治疗的靶点；
2.组织工程支架材料PLGA/CS可用于体外3D肿瘤细胞培养模型的建立,为肿瘤侵袭的的研究提供更加新的研究方法。
Part1Application of porous scaffold poly (L-glutamic acid)/chitosan on adipose tissue engineering
Objective
Large soft-tissue defects from traumatic injury, oncologic resection and congenital deformities do not repair spontaneously. The defects and resulting contour deformities lead to abnormal cosmesis, affect the emotional well-being of patients, and may impair function (e.g., limb range of motion). Current standard treatments primarily involve tissue transplantation, including composite tissue flaps and synthetic substitutes. However, limitations are donor-site morbidity, unpredictable outcomes due to graft resorption over time (40%-60%volume loss), allergic reactions, and fibrous capsule contraction, as well as complications and costly surgery.
The development of adipose tissue engineering has made it an attractive approach with great potential for repairing large soft-tissue defects. The basic strategy of adipose tissue engineering involves the utilization of artificial of natural extracellular matrix as scaffolds in combination with specific types of cells under the stimulation of growth factors to restore defects structurally and functionally. The key points in adipose tissue engineering were composed with the selection of cell, biomaterials with biocompatibility and biodegradation as well as the microenvironment for growth and differentiation of cell.
In this study, we constructed tissue-engineered adipose with adipose tissue-derived adult stem cells and a novel porous scaffold poly (L-glutamic acid)/chitosan (PLGA/CS) which was improved with electrostatic interaction between carboxyl groups of water soluble PLGA and amido groups of CS using the phase separation method. Besides, we investigated the biological characteristics of PLGA/CS and the attractive potential of PLGA/CS as scaffold for tissue engineering.
Methods
1. The adipogenic differentiation potential of ADSCs
(1) We harvested ADSCs from fresh human lipoaspirates obtained from patients who had undergone abdominal liposuction with enzyme digestion method. After the standard culture in vitro, passage2cells were used in the study.
(2) The expression of cluster differentiation (CD) markers on ADSCs was revealed by flow cytometry, including CD13、CD14、CD34、CD44、CD45、CD49d、CD90、 CD105, CD106and CD166.
(3) Multi-lineage differentiation of ADSCs was reveal by ALP staining, the expression of collagen type11and Oil-red O staining.
(4) The changes of morphologic features and intracellular lipid vacuoles were observed by light microscope and Oil-red O staining respectively.
2. The adipogenic differentiation of ADSCs on PLGA/CS in vitro
(1) PLGA/CS was prepared with PLA microspheres (diameter200-300μm) as porogens using freeze drying and static electricity method.
(2) Scanning electron microscope (SEM) observation and fluorescent DiL labeling were carried out to reveal the attachment and growth of ADSCs on PLGA/CS.
(3) The quantitative assay of cell proliferation with time was detected by DNA assay.
(4) The cytotoxicity of PLGA/CS was revealed by the quantity assay for the expression of lactate dehydrogenase (LDH).
(5) Oil-red O staining was carried out to assess the intracellular lipid accumulation of ADSCs on PLGA/CS with or without adipogenic induction.
(6) RT-PCR was carried out to reveal the expression of adipocyte-specific and insulin-regulated genes, including peroxisome proliferatior-activated receptor y2(PPARy2), fatty acid-binding protein (aP2), lipoprotein lipase (LPL) and adiponectin of ADSCs on PLGA/CS with or without adipogenic induction.
3. In vivo adipose tissue formation
After2weeks of exposure to adipogenic medium in vitro, ADSC-seeded scaffolds were implanted into the dorsum of severe combined immunodeficient (SCID) mice (6weeks old); acellular scaffolds were a control (n=6each group). After6weeks, animals were sacrificed and implants were harvested. To examine whether volume-stable adipose tissue could be harvested, tissue volumes were measured by immersing the tissue in PBS in a graded pipette. The volume change of implants was the ratio of the volume of implants retrieved at6weeks to the initial volume. Furthermore, to demonstrate newly formed fat tissue, the constructs retrieved in both groups were flash-frozen in Tissue-Tek OCT freezing medium for Oil-red O staining.
Results
1. In2D culture, ADSCs isolated from fresh human lipoaspirates showed uniform fibroblast-like morphologic features. Flow cytometry revealed the positive expression of CD13, CD34, CD44, CD90, CD105, CD166, CD49d and negative expression of CD14, CD45and CD31; Multi-lineage differentiation of ADSCs was confirmed by ALP staining, the expression of collagen type Ⅱ and Oil-red O staining. Under non-differentiation culture, ADSCs retained their fibroblast-like spindle shapes, with negative staining for Oil-red O at7and14days. On incubation with adipogenic medium, cells resembled adipocytes， with a rounded appearance and a time-dependent increase in intracellular lipid vacuoles, with positive staining for Oil-red O at7and14days. Increased staining for Oil-red O demonstrated increased adipogenic differentiation of ADSCs with increased exposure time. After1and2weeks, Oil-red O staining was higher for differentiated ADSCs than controls (p<0.05).
2. The PLGA/CS scaffold appeared as a soft and porous spongy-like mass. The porosity of this scaffold was greater than90%. SEM demonstrated the even distribution and abundance of ECM deposition surrounding PLGA/CS scaffolds at7days after seeding. Quantitative Hoechst33258assay revealed greatly increased number of cell-seeded constructs in both groups. Oil-red O staining was greater for cell-seeded scaffolds exposed to adipogenic medium for21days than non-induced constructs (p<0.05). RT-PCR revealed increased expression of adipocyte-specific and insulin-regulated genes.
3. Six weeks after implantation, gross inspection revealed intact and well-vascularized cell-seeded scaffolds. Moreover, cell-scaffold constructs appeared to shrink, which had decreased to90.5%and82.3%of the original volume in the cell-seeded and acellular groups, respectively (p>0.05). Oil-red O staining demonstrated adipose tissue formation within adipogenic-differentiated cell-seeded scaffolds after6weeks' implantation, with little adipose tissue formed within acellular implants.
Conclusion
1. ADSCs are an ideal autologous cell source for adipose tissue engineering.
2. The PLGA/CS scaffold had good biocompatibility and biodegradation for cell adhesion and proliferation for tissue engineering, and it could be a promising novel scaffold material for tissue engineering.
3. Even though the adipose-like tissue was confirmed in vivo study, a further study is needed to fabricate porous scaffolds with optimal ration of PLGA and CS which can match the kinetics of new adipose formation in vivo.
Originality
We introduced a novel porous scaffold poly (L-glutamic acid)/chitosan (PLGA/CS) which was improved with electrostatic interaction between carboxyl groups of water soluble PLGA and amido groups of CS using the phase separation method and finished the first use of such scaffold for adipose tissue engineering.
Part2Application of porous scaffold poly (L-glutamic acid)/chitosan for tumor3D-cultured model
Objective
Cholangiocarcinoma (CC) is the second most common from of primary liver cancer after hepatocellular carcinoma. CC is classified anatomically into extrahepatic cholangiocarcinoma (ECC) and intrahepatic cholangiocarcinoma (ICC). Despite advances in diagnosis and treatment, the prognosis of CC has not yet been resolved, because it is difficult to make an early diagnosis and standard therapy is not very effective. CC is generally characterized by strong proliferation, invasion, and early metastasis. To improve the prognosis, we require a fuller understanding of the molecular mechanisms behind its proliferation and progression.
Epithelial-mesenchymal transition (EMT) is an intricate process by which epithelial cells lose their epithelial characteristics and acquire a mesenchymal-like phenotype. This dramatic phenotypic changes involve the loss of E-cadherin-mediated cell-cell adhesion and additional prototypic epithelial markers, as well as the loss of apical-basal polarity, concomitantly with the acquisition of a motile behavior and a profound reorganization of the cytoskeleton. EMT was related to early embryogenesis, organ fibrosis as well as the process of tumor progression including invasion, metastasis and therapeutic resistance. EMT provides tumor cells with the ability to dissociate from each other and degrade and to actively migrate into the basal membrane and invade the adjacent connective tissues. ECM has been proved to relate to the tumor progression such as breast, prostate, ovarian, lung cancer. A great number of environment factors and signals have been described that induce EMT. Among the signaling pathways, transforming growth factor-(3(TGF-β) is probably the best characterized inducer of EMT in development, cancer and other pathologies.
In this study, we first investigated the effect of TGF-β1on EMT of HCCC9810cell in2D culture model to provide the target for gene therapy. Besides, we translated tissue engineered scaffold PLGA/CS into3D cancer-cultured model to reveal the effect of such3D model on the growth and invasion of cancer.
Methods
1. In standard monolayer cultures, HCCC9810was cultured within induced medium composed with10ng/ml TGF-β1,1%FBS and RPMI1640, the cells cultured in growth medium was served as control group; the expected morphological changes were detected by direct microscopic observation. The simple wound-healing migration assay was carried out to assess the motility behavior of cells. The organization at cellular levels of the individual EMT markers (i.e. Vimentin, Fibronectin for mesenchymal marker; E-cadherin, β-catenin and Pan-CK for epithelial marker) was revealed by immunofluorescence assay, while the protein and mRNA levels were detected by WB and real-time PCR respectively.
2. HCCC9810cells were seeded onto porous scaffold PLGA/CS. Scanning electron microscope (SEM) observation and fluorescent DiL labeling were carried out to reveal the attachment and growth of cells on PLGA/CS; the quantitative assay of cell proliferation with time was detected by DNA assay. The mRNA levels of EMT-related markers and TGF-β1was detected by real-time PCR.
Results
1. Under microscopic observation, the cells in induced group exhibited the loss of cell-cell contacts and cobblestone morphology, appearance of elongated mesenchymal features along with growth as individual cells; the wound healing assay showed the motility behaviror of cells cultured in induced medium. Immunophenotypical characterization in2D cultures showed the expression of β-catenin transformed from the membrane into cytoplasm; no expression of E-cadherin; down-regulated expression of Pan-CK; the higher expression of mesenchymal markers fibronectin and vimentin after induction; F-actin cytoskeleton reorganization was observed. Real-time PCR showed down-regulated expression of E-cadherin and β-catenin; the up-regulated expression of fibronectin, vimentin and N-cadherin. WB showed no expression of E-cadherin; the less expression of β-catenin; the higher expression of fibronectin, vimentin and N-cadherin which were similar to the results of IF and real-time PCR.
2. SEM demonstrated the even distribution and abundance of ECM deposition surrounding PLGA/CS scaffolds. Quantitative Hoechst33258assay revealed greatly increased number of cell-seeded constructs, whereas the proliferation rate is less than that of2D cultured cell. The results from3D model reflected the real growth state of tumor in vivo. Real-time PCR showed the expression of E-cadherin and β-catenin was significantly decreased after21days post seeding (p<0.05); the expression of vimentin and fibronectin was significantly increased after14and21days post seeding (p<0.05); interestingly, the expression of TGF-β1was increased after14and21days post seeding (p<0.05).
Conclusion
1. We confirmed that HCCC9810cells underwent EMT process induced by TGF-β1. The expected morphological changes and the expected changes in EMT-related markers do occur at the cellular, protein and mRNA level.
2. We translated tissue engineering technology platforms into cancer research and provided more physiological models for cancer research. We found such scaffold-based3D culture model could promote the autocrine of TGF-β1to improve the EMT and tumor invasion.
Originality
1. TGF-β1may become the target for gene therapy of cholangiocarcinoma.
2. Tissue-engineered scaffold PLGA/CS was first utilized for cancer research, and the
3D culture model could be promising tool for tumor invasion and metastasis as well as the development of therapeutic drugs.
引文
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