【作者】 陈亮；

【导师】 叶章群； 曾晓勇；

【作者基本信息】 华中科技大学 ， 外科学， 2011， 博士

【摘要】 组织工程是一门融合了多种学科知识的综合性学科,是再生医学的重要组成部分。组织工程的研究方法很多,但其基本策略包括种子细胞的选择、支架材料的研制和细胞与支架的相互作用。脂肪组织是一种新的成体干细胞来源。因为取材方便、侵入小、产量大等优点,脂肪干细胞已成为组织工程研究中一线种子细胞。许多研究表明,脂肪组织的生物学特性与解剖部位有关。脂肪干细胞来源于脂肪组织,其取材部位对细胞特性是否也存在这种相关性,目前尚无明确结论。人工合成支架在组织工程研究中的地位越来越重要。高分子聚合物因其稳定而可控的特性应用广泛。聚乳酸质脆,降解度高,而聚己内酯质韧,降解度低,两者不同比例的复合物可提供各种性状的支架。我们利用静电纺丝技术制备了两者的共混物,这种混合物支架由纳米级纤维编织成三维结构,适合模拟细胞外基质。在本研究中,我们比较了不同解剖部位来源的脂肪干细胞特性,结果显示兔腹股沟皮下部位是最佳取材部位。同时研究了聚乳酸/聚己内酯共混支架中两种成分的混合比例对其性能的影响,发现按1/1比例制备的支架表现出更好的性能。在上述研究的基础上,我们以脂肪干细胞为种子细胞,以1/1比例的静电纺丝聚乳酸/聚己内酯共混纤维为支架,通过细胞在支架上的平滑肌诱导,探索其构建组织工程化尿道的可行性。第一部分不同解剖部位来源的脂肪干细胞生物学特性比较目的研究并比较脂肪干细胞取材的解剖部位对脂肪干细胞生物学特性的影响,为脂肪干细胞作为组织工程种子细胞的选择策略提供理论基础。方法5只同龄雌性新西兰大白兔从出生起在同等环境中饲养,4月龄时用于实验研究,平均体质量约2.4kg(2.37～2.42kg).经耳缘静脉麻醉后,每只兔在无菌术下于3个部位手术切取脂肪组织团块,包括腹股沟皮下、颈背部皮下和腹膜后肾周部位。所得脂肪组织经常规分离培养,按来源部位分为三个实验组培养。三组脂肪干细胞分别用以下实验方法比较生物学行为：(1)倒置相差显微镜下形态观察和流式细胞术检测细胞表面标志物CD105和CD166以鉴定细胞；(2)细胞计数法和MTT法检测细胞增殖能力；(3)标准曲线法和活死细胞染色检测细胞活力；(4)组织化学染色(碱性磷酸酶染色和油红染色)、Real-time PCR和Western blot检测成骨、成脂诱导分化能力。结果(1)细胞鉴定：①三个部位的脂肪干细胞均呈典型间充质干细胞形态,肉眼观察未见明显差异；②流式检测三组细胞CD105、CD166标志物,其阳性率未见统计学差异。(2)增殖能力：①4天时皮下组织腹股沟和颈背部细胞数量均高于腹膜后肾周组,差异有显著性(均p<0.05)。两组皮下组织组之间无差异(p>0.05)。7天时三组细胞数量未检出统计学差异(p>0.05)。②单因素方差分析比较三组细胞MTT法测定的增殖曲线,腹股沟皮下和颈背部皮下组细胞都较腹膜后肾周组有统计学差异(均p<0.05),而两者之间无差异(p>0.05)。(3)细胞活力：①腹股沟组、颈背组、腹膜后组活细胞数量逐步递减,组间两两比较均有差异(均p<0.05)。②三组细胞经活死细胞染色计算的活细胞数量各组间比较未见统计学差异(均p>0.05)。(4)分化能力：①定量碱性磷酸酶染色示腹股沟皮下来源脂肪干细胞成骨分化能力优于另两组,差别均有显著性(均p<0.05)。定量油红染色示三组细胞成脂分化能力相似(均p>0.05)。②Real-time PCR检测成骨、成脂分化标志物均有表达,各组间比较显示皮下部位(腹股沟和颈背部)来源的细胞表达量明显高于腹膜后组(p<0.05),而皮下部位中腹股沟组优于颈背部组(p<0.05)。③Western blot检测到各标志物的蛋白表达,与Real-time PCR结果一致。结论(1)脂肪干细胞是一种典型的间充质干细胞,其形态和表型均符合间充质干细胞特征；(2)来源部位对脂肪干细胞的增殖、活力和分化能力有影响,皮下来源的细胞在各方面优于腹膜后来源,同为皮下组织来源的腹股沟部位又优于颈背部位；(3)腹股沟皮下部位是脂肪干细胞现对较理想的取材部位；(4)脂肪干细胞可向骨细胞、脂肪细胞分化,可应用于组织工程研究；(5)脂肪是一种具有高度非均一性的组织,这种非均一性不仅存在于不同个体之间,也存在于同一个体不同部位之间。第二部分静电纺丝聚乳酸/聚己内酯共混纤维支架与脂肪干细胞的生物相容性研究目的研究利用静电纺丝技术制备的聚乳酸/聚己内酯共混纤维的生物相容性,探讨不同的重量混合比对该聚合物支架生物相容性的影响。方法利用静电纺丝技术将聚乳酸(PLLA)和聚己内酯(PCL)的混合物(PLLA/PCL)制备成纳米纤维支架,两种聚合物共混时的重量比为3/1,2/1,1/1(PLLA/PCL)。对这三种不同比例的支架进行形态学、孔隙率、体外降解率的比较；将纳米纤维支架作为异物接种到大鼠背部皮下口袋中,以观察局部炎症反应,评价材料的组织相容性；以脂肪干细胞为种子细胞,接种到三种比例的支架上,检测细胞的增殖、粘附、活力和多向分化(成骨分化、成软骨分化、成脂分化)能力,评价材料的细胞相容性。结果1.形态学观察：(1)纤维直径：3/1支架为1.25±0.27μm,2/1支架为870±45nm,1/1支架为452±24nm,1/1支架较3/1,2/1 NFS差异有非常显著性(均p<0.01)：(2)孔隙率：三种比例支架孔隙率分别为78.3±3.4%,78.7±5.1%和82.2±4.8%,三者间未检出统计学差异；(3)降解率：根据降解曲线显示,PLLA含量越大,支架体外降解率越高。2.组织相容性：三种支架移植均导致炎性细胞浸润,急性炎性反应过程约2周时间,4周后炎症反应消失。3.细胞相容性：脂肪干细胞可在支架上粘附、增殖并多向分化,脂肪干细胞与1/1支架组装的复合体有最好的生物学行为表现。结论(1)PLLA和PCL的共混溶液可经静电纺丝技术制备具有纳米结构的纤维支架；(2)PLLA、PCL混合的重量比率对两者的静纺共混纤维在形态、孔隙率、降解率有影响,1/1比例的材料性能最优；(3)静纺PLLA/PCL支架生物相容性好,可作为组织工程候选支架材料。第三部分尿道组织工程：脂肪干细胞在静电纺丝纳米纤维支架上的平滑肌分化目的研究脂肪干细胞在静电纺丝聚乳酸/聚己内酯共混纳米纤维支架上的平滑肌分化,探索该支架材料作为尿道组织工程的可行性。方法静电纺丝技术制备重量比为1：1的聚乳酸/聚己内酯共混纳米纤维支架。从兔腹股沟皮下分离脂肪干细胞,接种到支架上组装成为复合体。用平滑肌诱导培养基诱导支架上的脂肪干细胞,通过形态学观察、免疫荧光、细胞增殖检测、基因转录、蛋白印记和胶原收缩法比较脂肪干细胞在有无支架条件下诱导的平滑肌细胞有无差别。结果诱导6周后,脂肪干细胞诱导的平滑肌细胞具有平滑肌细胞形态,能够表达α-actin和MHC两种平滑肌标志物,胞体中F-actin含量接近正常平滑肌细胞。细胞增殖显示,诱导3周时,支架上诱导的平滑肌细胞数量较无支架诱导的细胞无差别(p>0.05),而在6周时,两者有非常显著性差别(p<0.01)。基因转录和蛋白表达均显示诱导的脂肪干细胞能够表达平滑肌特异性基因或蛋白,且支架对脂肪干细胞向平滑肌分化无影响。收缩功能检测显示诱导的平滑肌细胞具有很好的收缩能力。结论(1)ASCs可直接在静电纺丝PLLA/PCL纳米纤维支架上诱导分化为具有收缩功能的SMCs;(2)ASCs与NFS构建的复合体可用于尿道组织工程研究；(3)静电纺丝PLLA/PCL纳米纤维可作为平滑肌组织工程候选支架材料。更多还原

【Abstract】 Tissue engineering, an important component of regenerative medicine, is a comprehensive discipline combining many kinds of sciences and technologies. The approaches of tissue engineering may very, but most involve the essential strategies including seed-cells selection, scaffolds design and interaction of cells and scaffolds.Adipose tissue is a new source reservoir for adult stem cells. Because of convenient harvesting, minimal invasion and abundant quantity, adipose-derived stem cells (ASCs) have been regarded as forefront seed-cells in tissue engineering. Many studies have revealed a close relationship between anatomic site and cellular behavior. ASCs originate from adipose tissues. However, the impacts of anatomic site of harvesting on ASCs are obscure till now.The synthetic scaffold plays an increasing role in tissue engineering field. The polymers are preferable for various purposes due to stable and controllable properties. Poly (L-lactic acid) (PLLA) is brittle with high degradation and poly (ε-caprolactone) (PCL) is flexile with low degradation. The composites of PLLA and PCL of different weight ratios offer the scaffolds of different properties. The blends of PLLA and PCL, which we prepared from electrospinning method, are three-dimensional in weaving fibers in nanometer scale, indicating their replacement for extracellular matrix.In this study, we compared the biology of ASCs from three anatomic sites and discovered the subcutaneous inguinal region was the best site for cell harvesting. Moreover, we evaluated the effects of weight ratio of PLLA/PCL blend and confirmed the 1/1 synthesized scaffold had the highest performance. On the basis of the above data, we employed ASCs to differentiate towards smooth muscle cells on the electrospun PLLA/PCL blend nanofibers of 1/1 blending weight ratio to explore the feasibility of such construct for tissue engineered-urinary tract. Objectives To study and compare the impacts of harvesting anatomic site on the biological behaviors of adipose-derived stem cells (ASCs), and to provide theoretical support for the strategy of seed-cell selection in tissue engineering.Materials and Methods Five female New Zealand white rabbits born simultaneously were used in our study. They were raised in the identical environment until to 4 months age old and weighed averagely 2.4kg (ranging from 2.37 to 2.42kg). With the general anesthesia by auricular vein injection, the adipose tissues were resected under the asepsis condition from three different anatomic sites of each rabbit, including subcutaneous inguinal (SI), subcutaneous dorsocervical (SD) and retroperitoneal perinephric (RP) regions. The ASCs obtained from removed tissues were isolated and cultured conventionally and categorized into three experimental groups according to their region of origin. The ASCs of three groups were compared on the biological properties by the following methods:(1) ASCs were characterized by morphological observation under an inverted phase contrast microscope and the expression of surface markers, CD 105 and CD 166, by fluorescence-activated cell sorting (FACS). (2) Cell counting and MTT assay were employed to evaluate proliferation. (3) A known standard curve and Live/Dead cells staining were employed to evaluate viability. (4) Histochemistry staining (ALP staining and Oil red O staining), Real-time PCR and Western blot were employed to evaluate the multilineage differentiation towards to osteogenesis and adipogenesis.Results (1) Characterization of ASCs:①ASCs from three anatomic sites demonstrated typical forms of the mesenchymal stem cells (MSCs). No obvious difference was found by macroscopic observation.②The positive percentages of CD 105 and CD 166 were assessed by FACS and analyzed to nonsignificant difference. (2) Proliferation assay:①On day 4, the average numbers of the ASCs in SI group and SD groups were significantly higher than those in RP group (both p<0.05), with no significant difference between SI and SD group (p>0.05). On day 7, no significant difference was detected among the ASCs from three anatomic sites (p>0.05).②The one way ANOVA was used to compare the growth curves of three groups generated from MTT assay data. The subcutaneous (SI and SD) ASCs had both statistical significance to RP ASCs (both p< 0.05), while nonsignificance was determined between SI and SD ASCs (p>0.05). (3) Viability assay:①The three groups had significant differences in the number of living cells, with a decline from SI to SD to RP ASCs (p<0.05 for these comparisons).②The living cells were counted in five random views of Live/Dead staining. The differences between any two or all of the sites were not statistically significant (all p>0.05). (4) Differentiation capacity:①At the end of osteogenetic culture, the SI ASCs had higher ALP activity than SD or RP cells (both p<0.05). At the end of adipogenic culture, there were no significant differences among the three groups (p>0.05).②All of the gene markers were detected by Real-time PCR during osteogenesis and adipogenesis. In the evaluation for each gene, the expression levels of the subcutaneous (SI and SD) groups were significant higher than that of RP group (both p<0.05). In the comparison between two subcutaneous groups, SI ASCs acted better than SD ASCs (p<0.05).③The results of protein expression levels determined by Western blot were in agreement with Real-time PCR.Conclusions (1) ASCs are a typical type of MSCs, whose morphology and phenotype complied with the MSCs’characteristics; (2) The derived anatomic site has impacts on the proliferation, viability and differentiation capacity of ASCs. The subcutaneous ASCs are preferable than retroperitoneal ASCs, and the SI ASCs are better than SD ASCs. (3) The inguinal region may be a applicable resource reservoir for cell harvesting. (4) The ASCs are capable of multipotential to osteogenesis and adipogeneous, implying their application in tissue engineering. (5) The adipose tissue is highly heterogeneous, both among individuals and also within an individual. PartⅡ. Biocompatibility of electrospun PLLA/PCL blend nanofibrous scaffold and adipose-derived stem cellsObjectives To study the biocompatibility of PLLA/PCL blend fibers prepared from electrospinning method and to discuss the effects of blending weight ration on it.Materials and Methods PLLA and PCL blends with weight ratio of 3/1,2/1 and 1/1 were electrospun into nanofibrous scaffold (NFS). The morphology, porosity and degradation in vitro of such three NFS were compared. The histocompatibility was observed on the inflammatory responses of NFS implanted into subcutaneous pockets of rats. The cytocompatibility was evaluated by proliferation, attachment, viability and multipotential towards to osteogenesis, chondrogensis and adipogenesis.Results 1.Mophology:(1) Average diameter (AD):AD3/1=1.25±0.27μm, AD2/1= 870±45nm, AD1/1=452±24nm. The fibers of 1/1 NFS were significantly thinner than those of 3/1 and 2/1 NFS (both p<0.01); (2) Porosity: The porosity rates of three NFSs were respectively 78.3±3.4%,78.7±5.1% and 82.2±4.8%, with no significant difference among them; (3) Degradation:According to the degradation curve, the rate of degradation was accelerated with the increasing content of PLLA.2. Histocompatibility:The inflammatory responses were all observed during the NFS transplants. The response course was as long as about 2 weeks and vanished at the end of 4 weeks.3. Cytocompatibility:The ASCs were able to attach, grow and differentiate into multilineages on NFSs. The NFS of 1/1 blending ratio was the most suitable scaffold to be fabricated with ASCs.Conclusions (1) The blend composite of PLLA and PCL can be electrospun into nano-scale fibrous scaffold; (2) The weight ratio of PLLA and PCL blending has impacts on morphology, porosity and degradation of electrospun fibrous scaffold; (3) The electrospun PLLA/PCL blend nanofibrous scaffold has good biocompatibility and can be a candidate biomaterial for tissue engineering. PartⅢ. Urethra tissue engineering:leiomyogenic differentiation of adipose-derived stem cells on electrospun nanofibrous scaffoldObjectives To study the leiomyogenic differentiation of adipose-derived stem cells(ASCs) on electrospinning PLLA/PCL blend nanafibrous scaffold and to explore the feasibility of the application of such novel material in urethra tissue engineering.Materials and Methods ASCs obtained from subcutaneous adipose tissue in inguinal region were seeded on the electrospun PLLA/PCL blend nanofibrous scaffold (NFS), which was blended in 1:1 weight ratio, to fabricate ASCs-NFS constructs. The smooth muscle inductive medium was used for ASCs differentiating towards smooth muscle cells (SMCs). The difference between the ASCs-derived smooth muscle cells (AD-SMCs) with and without scaffolds were evaluated by morphological observation, immuno fluorescence, cell proliferation, gene and protein expression assay.Results After 6 weeks, the AD-SMCs demonstrated the typical morphology of SMCs and expressed specific markers, a-actin and myosin heavy chain (MHC). The contents of F-actin of the AD-SMCs were similar with the SMCs. During the proliferation assay, there was no statistical significance between the AD-SMCs with and without the scaffolds (p> 0.05) for 3-week induction. However, great significant difference was detected when it went to the 6th week. Both of the gene transcription and protein expression showed the AD-SMCs were positive to SMCs specific genes or proteins. No effect of the existence of scaffold was found in the course of differentiation from ASCs to SMCs. The AD-SMCs posed good contraction ability by using collagen gel assay.Conclusions (1) ASCs can differentiate into contractile SMCs on PLLA/PCL NFS prepared by electrospinning; (2) The constructs combined ASCs and NFS can be used for the researches of urethra reconstruction; (3) The electrospun PLLA/PCL blend nanofiber can be a candidate material for SMCs tissue engineering.更多还原