复合转基因EPCs的细胞—细胞外基质复合材料在修复长管状尿道缺损中的实验研究

发布时间：2018-07-10 05:25

本文选题：组织工程 + 慢病毒　；参考：《武汉大学》2014年博士论文

【摘要】：目的：本文研究旨在利用组织工程学原理、细胞分子生物学技术和干细胞技术等,构建一种全新的、复合了包含有广谱抗微生物活性和促血管化作用的抗菌肽LL-37的转基因血管内皮祖细胞和尿路上皮细胞、平滑肌细胞等种子细胞的新型组织工程化尿道组织,从而探索利用该生物材料修复长段管状尿道缺损的可能性。对其中涉及的转基因技术,如慢病毒表达载体的构建、质粒表达的检测、慢病毒的包装及目的细胞的代感染等方法进行探讨,为以后可能的组织工程学技术联合转基因技术奠定基础。同时,鉴于本文涉及的泌尿系组织工程学常用种子细胞,如内皮祖细胞、尿路上皮细胞等,在分离、培养和鉴定等试验方法方面尚存较多争议,遂结合自身经验探讨循外周血途径高效获取内皮祖细胞,通过膀胱黏膜的移行上皮细胞转化培养尿路上皮细胞的方法。方法：(1)慢病毒的包装和病毒滴度的检测。(2)目的基因LL-37过表达慢病毒载体的构建,并进行超纯化的去内毒素抽提,使其具有较高的安全性,同时对构建的载体PGC-FU-LL-37表达情况进行检测。(3)抽取成年新西兰兔股静脉血液,通过密度梯度离心法获取足够量的单个核细胞,并采用碱性成纤维细胞因子、血管内皮细胞生长因子等活性成分诱导单个核细胞向内皮祖细胞转化。(4)制备好的慢病毒转染目的细胞——外周血途径获取的内皮祖细胞,并通过Western Blot等方法对表达情况进行检测。(5)切取成年新西兰兔膀胱组织,剥离黏膜层用于制备尿路上皮细胞,从肌层取活检制备尿路平滑肌细胞,剩余组织用于制备膜片状膀胱脱细胞基质。(6)将制备好的稳定传代的尿路上皮细胞、平滑肌细胞分别种植在膀胱脱细胞基质两侧,待融合至一定程度后将转基因内皮祖细胞种植到上皮细胞侧。(7)将复合了种子细胞的细胞-细胞外基质复合材料包埋于成年新西兰兔腹壁皮下,定期取材行组织学鉴定。(8)将经过包埋处理的细胞-细胞外基质复合材料卷管后行长管状尿道缺损修复替代,并通过静脉尿路造影或逆行尿路造影检测移植物替代效果。结果：(1)RT-PCR等实验室检测方法证实慢病毒表达载体pGC-FU-LL37-GFP构建成功,慢病毒滴度为2.00E+8TU/ml,通过超纯去内毒素抽提方法使得慢病毒载体安全可用。(2)通过外周血途径(股静脉血液)可有效提取单个核细胞,并通过细胞因子将其转化为干细胞性质的内皮祖细胞。(3)通过转基因技术可有效将目的基因抗菌肽LL-37转染进入制备的目的细胞——内皮祖细胞中,免疫荧光法等检测证实转染效率可达90%左右,Western Blot检测证实准基因内皮祖细胞有效表达抗菌肽LL-37。(4)成功分离培养并纯化了兔尿路上皮细胞和平滑肌细胞,且种子细胞增殖迅速、不易老化。(5)自行创立的膀胱脱细胞基质制备技术——高渗盐水-酶消化法-去垢剂洗涤技术可完全脱去组织中的细胞成分,体外复合种子细胞的培养过程中显示出良好的生物相容性,并且可促进种子细胞的增殖分化和在其上的均匀分布。结论：实验室条件可成功构建包含有抗菌肽LL-37的重组慢病毒表达载体pGC-FU-LL37-GFP,通过转基因技术可高效转染目的细胞并使其表达相关目的蛋白。外周血途径亦可成功分离转化得到足够数量的内皮祖细胞,较之普遍采用的骨髓途径获取单个核细胞的方法具有更高的便捷性和可重复性。通过取材膀胱组织,可快速制备尿路上皮细胞、平滑肌细胞,通过纯化扩增可作为合格的组织工程学种子细胞。高渗盐水-酶消化法-去垢剂洗涤法可制备出完全无细胞成分的膀胱脱细胞基质,且保留细胞外基质的生物学活性,在复合制备的种子细胞后,细胞可在基质表面迅速增殖,通过腹壁皮下包埋的方法可成功制备细胞-细胞外基质复合型组织工程学材料,有望应用于长段管状尿道缺损的组织工程学修复应用中,从而达到真正意义上的生物学修复再生。
[Abstract]:Objective: the aim of this study is to construct a new kind of new type of new vascular endothelial progenitor cells, urinary tract epithelial cells, smooth muscle cells, and other seed cells, which contain the broad-spectrum antimicrobial activity and the angiogenic effect of LL-37, using the principle of tissue engineering, cell molecular biology and stem cell technology. In order to explore the possibility of repairing long segment tubular urethral defects with this biomaterial, the possibility of using this biomaterial to repair the tubular urethral defect is explored. The methods involved in the transgenic technology, such as the construction of the Lentivirus Expression Vector, the detection of plasmid expression, the packaging of the lentivirus and the generation of the target cells, are discussed for the future possible tissue engineering. At the same time, there are many controversies in the methods of isolation, culture and identification of common seed cells, such as endothelial progenitor cells, urinary tract epithelial cells, such as endothelial progenitor cells, urinary tract epithelial cells, and so on. Method for culturing and transforming urinary epithelial cells by cystal transitional epithelial cells.
Methods: (1) the packaging of the lentivirus and the detection of the titer of the virus. (2) the construction of the LL-37 overexpression lentivirus vector of the target gene and the ultra purification of the endotoxin extraction to make it highly safe and detect the expression of the constructed carrier PGC-FU-LL-37. (3) the femoral vein blood of adult New Zealand rabbits was extracted by the density ladder. Sufficient amount of mononuclear cells were obtained by degree centrifugation, and the active components such as basic fibroblast and vascular endothelial growth factor were used to induce mononuclear cells to convert to endothelial progenitor cells. (4) a good lentivirus transfected target cells, endothelial progenitor cells obtained from peripheral blood pathway, were prepared by Western Blot and other methods. (5) the adult New Zealand rabbit bladder tissue was cut out, the mucosa was stripped to prepare the urinary tract epithelial cells, the urinary tract smooth muscle cells were prepared from the myometrium, and the remaining tissue was used to prepare the diaphragm like vesical acellular matrix. (6) the stable passages of urinary tract epithelial cells were prepared, and the smooth muscle cells were planted in the bladder. On both sides of the acellular matrix, the transgenic endothelial progenitor cells were planted to the epithelial cell side after a certain degree of fusion. (7) the cell extracellular matrix composite of the seed cells was embedded in the abdominal wall of adult New Zealand rabbits on the abdominal wall. (8) the embedded cell extracellular matrix composites were prepared. Replacement of tube shaped urethral defect after rewinding and replacement of the graft by intravenous urography or retrograde urography.
Results: (1) RT-PCR and other laboratory testing methods confirmed that the Lentivirus Expression Vector pGC-FU-LL37-GFP was successfully constructed, the titer of lentivirus was 2.00E+8TU/ml, and the lentivirus vector was safely used by the ultra pure endotoxin extraction method. (2) the mononuclear cells could be extracted effectively through the peripheral blood pathway (femoral vein blood), and the cell factor could be used by the cytokine. The endothelial progenitor cells transformed into stem cell properties (3) transfection of the target gene antibacterial peptide LL-37 into the intended cell - endothelial progenitor cells by transgene technology, the immunofluorescence assay confirmed that the transfection efficiency was about 90%, and the Western Blot detection confirmed that the para gene endothelial progenitor cells effectively expressed the antibacterial peptide LL-37. (4). The rabbit urinary tract epithelial cells and smooth muscle cells were successfully isolated and purified, and the proliferation of the seed cells was rapid and not easy to age. (5) the preparation technology of the bladder acellular matrix, which was created by ourselves, the technology of hypertonic saline enzyme digestion and detergent washing can completely remove the fine cell components in the tissue and the culture process of the compound seed cells in vitro. It shows good biocompatibility and promotes the proliferation and differentiation of seed cells and the uniform distribution on them.
Conclusion: the recombinant lentivirus expressing vector pGC-FU-LL37-GFP containing antibacterial peptide LL-37 can be successfully constructed in laboratory conditions, and the target cells can be transfected efficiently through transgenic technology and the target protein can be expressed. The peripheral blood pathway can be successfully separated and converted to a sufficient number of inner skin progenitor cells, compared with the commonly used bone marrow pathway. The method of obtaining mononuclear cells is more convenient and repeatable. Urinary tract epithelial cells can be quickly prepared by taking material of the bladder, and smooth muscle cells can be used as qualified tissue engineering seed cells. The completely cell-free bladder removal can be prepared by hypertonic saline enzyme digestion and detergent cleaning. The cell matrix, and the biological activity of the extracellular matrix, can be rapidly proliferated on the surface of the matrix after the compound preparation of the seed cells. The cell extracellular matrix composite tissue engineering materials can be successfully prepared through the subcutaneous embedding of the abdominal wall. It is expected to be applied to the tissue engineering repair of the long segment tubular urethral defect. So as to achieve the true sense of biological regeneration.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R699.6;R318.08

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