【摘要】：體內(nèi)大多數(shù)組織由多種細(xì)胞組成,且這些細(xì)胞及其分泌的細(xì)胞外基質(zhì)在組織中有序排列,以發(fā)揮特定的組織功能。隨著組織工程和再生醫(yī)學(xué)的不斷發(fā)展,研究者們一直致力于在體外模擬和重建這些復(fù)雜的組織結(jié)構(gòu),其中圖案化組織工程支架在調(diào)控細(xì)胞形態(tài)、細(xì)胞分布和多細(xì)胞共培養(yǎng)等作用明顯。靜電紡纖維支架具有高的比表面積和類(lèi)似于細(xì)胞外基質(zhì)的結(jié)構(gòu),作為組織工程支架具有很大的優(yōu)勢(shì)。但目前將圖案化技術(shù)與電紡技術(shù)結(jié)合起來(lái)構(gòu)建圖案化纖維支架,并調(diào)控不同種類(lèi)細(xì)胞的空間分布、重建組織結(jié)構(gòu)微環(huán)境的研究仍較少。據(jù)此,本論文設(shè)計(jì)了光刻掩模,運(yùn)用光刻和磁控濺射工藝制得圖案化接收板,采用普通靜電紡絲設(shè)備獲得了圖案化纖維支架。為模擬肝小葉和心肌片層的結(jié)構(gòu),通過(guò)圖案化纖維支架分別實(shí)現(xiàn)了肝細(xì)胞和肝組織中其他相關(guān)細(xì)胞、心肌細(xì)胞和心肌組織中其他相關(guān)細(xì)胞的共培養(yǎng),較好地維持了肝細(xì)胞和心肌細(xì)胞的活力。在此基礎(chǔ)上,將圖案化共培養(yǎng)肝細(xì)胞體系、心肌細(xì)胞體系作為藥物代謝和藥物篩選的體外模型,顯示出較好的體外和體內(nèi)結(jié)果相關(guān)性。利用光刻和磁控濺射工藝制得了圖案化接收板,結(jié)合普通靜電紡絲設(shè)備制備了圖案化纖維膜,方法簡(jiǎn)單,操作性強(qiáng)；圖案的形狀、尺寸和排列方式可控；制得的圖案化纖維膜溝脊結(jié)構(gòu)明顯,圖案區(qū)域的纖維具有較高的定向性。成纖維細(xì)胞在不同尺寸的圖案化纖維支架上培養(yǎng),發(fā)現(xiàn)支架能有效調(diào)節(jié)細(xì)胞的空間分布,使其在預(yù)定的圖案區(qū)域生長(zhǎng),并滲透進(jìn)入纖維支架內(nèi)部；同時(shí)細(xì)胞沿纖維取向生長(zhǎng),分泌的細(xì)胞外基質(zhì)呈現(xiàn)定向分布等特征。合成了乳糖改性聚乳酸,發(fā)現(xiàn)混紡比例為5：5的乳糖改性聚乳酸／聚乳酸-聚乙二醇嵌段共聚物(PELA)復(fù)合支架能有效維持肝細(xì)胞的活力和功能,原代肝細(xì)胞形成了直徑約為60μm的肝細(xì)胞球體。將原代肝細(xì)胞和成纖維細(xì)胞分別接種在上述復(fù)合纖維、PELA纖維的圖案化支架上,通過(guò)圖案化纖維的層層鍥合,實(shí)現(xiàn)了兩種細(xì)胞的共培養(yǎng)。在15天的培養(yǎng)中,與肝細(xì)胞單獨(dú)培養(yǎng)相比,共培養(yǎng)肝細(xì)胞能較好地維持肝細(xì)胞活力、白蛋白分泌、尿素合成和P450酶活力等。在此基礎(chǔ)上,將原代肝細(xì)胞、成纖維細(xì)胞和內(nèi)皮細(xì)胞分別接種于三種不同的圖案化纖維支架中,通過(guò)層層鍥合組裝的方式,成功構(gòu)建了類(lèi)似體內(nèi)肝小葉的基質(zhì)結(jié)構(gòu)。三種細(xì)胞的生長(zhǎng)狀態(tài)較好,均能分泌各自的特征性蛋白,肝細(xì)胞球體內(nèi)生成了膽小管,成纖維細(xì)胞滲透生長(zhǎng)進(jìn)入圖案化纖維支架內(nèi)部,內(nèi)皮細(xì)胞表現(xiàn)出較好的毛細(xì)血管生成能力。以7-芐氧基-4-三氟甲基香豆素、7-甲氧基-4-三氟甲基香豆素和7-羥基香豆素等作為底物,檢測(cè)了圖案化共培養(yǎng)肝細(xì)胞中藥物代謝相關(guān)酶的活性,發(fā)現(xiàn)共培養(yǎng)肝細(xì)胞中CYP3A4. CYP2C9及Ⅱ相代謝酶的活力均能維持在較高水平。采用咪達(dá)唑侖、睪酮、甲苯磺丁脲、華法林和對(duì)乙酰氨基酚藥物等研究了共培養(yǎng)肝細(xì)胞的藥物代謝行為,發(fā)現(xiàn)上述藥物在共培養(yǎng)體系中的代謝清除率接近體內(nèi)數(shù)據(jù),說(shuō)明能較準(zhǔn)確地反映體內(nèi)肝臟的藥物代謝情況。以利福平和谷胱甘肽作為誘導(dǎo)劑,酮康唑和丙磺舒作為抑制劑,證明了共培養(yǎng)肝細(xì)胞能敏感地響應(yīng)藥物的誘導(dǎo)和抑制作用。上述結(jié)果顯示出圖案化共培養(yǎng)肝細(xì)胞體系適合在體外進(jìn)行藥物代謝和毒性的評(píng)價(jià)。制備了PELA/碳納米管的圖案化纖維支架,纖維呈現(xiàn)核殼結(jié)構(gòu),具有一定的導(dǎo)電性。在圖案纖維支架上接種原代心肌細(xì)胞、原代心肌成纖維細(xì)胞,將內(nèi)皮細(xì)胞接種到PELA圖案纖維支架上,通過(guò)圖案化纖維的層層鍥合組裝,構(gòu)建了心肌細(xì)胞平面共培養(yǎng)的微環(huán)境。該體系不僅模擬了心肌組織中不同細(xì)胞的排列分布,更表現(xiàn)出與體內(nèi)心肌相似的生物化學(xué)和電生理特性。針對(duì)目前組織工程化心肌的厚度不夠,為了使所培養(yǎng)的心肌細(xì)胞更接近于體內(nèi)的三維結(jié)構(gòu),設(shè)計(jì)了具有眾多孔道結(jié)構(gòu)的圖案化纖維支架,并模擬心肌細(xì)胞在體內(nèi)生長(zhǎng)所需的力學(xué)微環(huán)境,構(gòu)建了三維心肌組織,三種細(xì)胞均能較好地生長(zhǎng),共培養(yǎng)心肌細(xì)胞能長(zhǎng)時(shí)間維持自發(fā)的跳動(dòng),孔道結(jié)構(gòu)促進(jìn)了細(xì)胞在支架內(nèi)部的滲透生長(zhǎng)和排列,內(nèi)皮細(xì)胞也表現(xiàn)出了血管化能力。采用實(shí)時(shí)拍攝記錄共培養(yǎng)支架上心肌細(xì)胞的搏動(dòng)信號(hào),在體外構(gòu)建的共培養(yǎng)心肌細(xì)胞體系開(kāi)展了藥物篩選的研究。通過(guò)觀察奎尼丁、紅霉素和甲磺胺心定等對(duì)心肌細(xì)胞的作用效果和起效時(shí)間,對(duì)比體內(nèi)和體外測(cè)定的半效應(yīng)濃度(EC50)值,發(fā)現(xiàn)共培養(yǎng)心肌細(xì)胞能準(zhǔn)確地反映藥物在體內(nèi)作用的量效和時(shí)效關(guān)系。進(jìn)一步通過(guò)氟哌啶醇的多次藥物洗脫實(shí)驗(yàn),驗(yàn)證了共培養(yǎng)心肌細(xì)胞體系在藥物篩選中表現(xiàn)出的相關(guān)性、快速性、重復(fù)性、穩(wěn)定性和靈敏性。綜上所述,本論文系統(tǒng)地研究了三維微圖案纖維支架的構(gòu)建、原代肝細(xì)胞共培養(yǎng)體系、原代心肌細(xì)胞共培養(yǎng)體系,在體外獲得了接近于體內(nèi)組織的生理結(jié)構(gòu)和功能,并應(yīng)用于肝藥物代謝及心肌藥物篩選,為藥物研究和應(yīng)用提供了有效的體外篩選手段。
[Abstract]:Most tissues in the body consist of a variety of cells, and these cells and their secreted extracellular matrix are arranged in an orderly manner in the tissue to exert specific tissue functions. With the development of tissue engineering and regenerative medicine, researchers have been committed to simulating and reconstructing these complex tissue structures in vitro, in which the patterning tissue engineering scaffold has obvious effects on regulating cell morphology, cell distribution and multi-cell co-culture. The electrostatic spinning fiber scaffold has a high specific surface area and a structure similar to that of the extracellular matrix, and has great advantages as a tissue engineering scaffold. However, the patterning technology is combined with the electro-spinning technology to build a patterned fiber scaffold, and the spatial distribution of different kinds of cells is regulated, and the research on the reconstruction of the micro-environment of the tissue structure is still relatively small. According to this, the lithography mask is designed, and the patterned receiving plate is fabricated by lithography and magnetron sputtering. The patterned fiber support is obtained by using ordinary electrostatic spinning equipment. In order to simulate the structure of liver lobules and myocardial slices, the co-culture of other relevant cells, cardiac myocytes and other relevant cells in liver tissue and cardiac muscle tissue was achieved by patterning the fiber scaffold, and the viability of hepatocytes and myocardial cells was better maintained. On the basis of this, the co-cultured hepatocyte system and myocardial cell system were used as the in vitro model of drug metabolism and drug screening, which showed better correlation between in vitro and in vivo results. The patterned receiving plate is manufactured by a photoetching and magnetron sputtering process, the patterned fiber film is prepared by combining the common electrostatic spinning equipment, the method is simple, the operability is strong, the shape, the size and the arrangement mode of the pattern are controllable, and the prepared patterned fiber film groove ridge structure is obvious, the fibers of the pattern region have a higher orientation. the fibroblasts are cultured on a patterned fiber support of different sizes, the space distribution of the cells can be effectively adjusted by the support, the cells grow in a predetermined pattern area and penetrate into the interior of the fiber support, and meanwhile, the cells grow along the fiber orientation, The secreted extracellular matrix exhibits characteristics such as directional distribution. Lactose modified poly (lactic acid) was synthesized. It was found that the blend ratio of lactose modified polylactic acid/ polylactic acid-polyethylene glycol block copolymer (PELA) with a blend ratio of 5: 5 could effectively maintain the viability and function of hepatocytes, and primary hepatocytes were formed with hepatocytes with a diameter of about 60. m the primary liver cells and the fibroblasts are respectively inoculated on the patterned support of the composite fiber and the PELA fiber, and the two cells are co-cultured through the layer-by-layer bonding of the patterned fibers. In the 15-day culture, the co-cultured hepatocytes could better maintain the viability of hepatocytes, albumin secretion, urea synthesis and P450 enzyme activity, as compared to the individual culture of hepatocytes. On this basis, the primary liver cells, fibroblasts and endothelial cells were seeded in three different patterned fiber scaffolds. The growth status of the three cells is good, can secrete each characteristic protein, the liver cell ball body generates the bile canaliculus, the fibroblast permeates into the inside of the patterned fiber scaffold, and the endothelial cells show better capillary generating capacity. The activity of drug metabolism-related enzymes in the patterned co-cultured hepatocytes was detected with 7-triethoxy-4-trifluoromethyl coumarin, 7-methoxy-4-trifluoromethyl coumarin and 7-hydroxycoumarin, and CYP3A4 was found in co-cultured hepatocytes. The activity of the metabolizing enzymes of CYP2C9 and 鈪，

本文編號(hào)：2273590