本文選題：組織工程 + 慢病毒　； 參考：《武漢大學(xué)》2014年博士論文

【摘要】：目的：本文研究旨在利用組織工程學(xué)原理、細(xì)胞分子生物學(xué)技術(shù)和干細(xì)胞技術(shù)等,構(gòu)建一種全新的、復(fù)合了包含有廣譜抗微生物活性和促血管化作用的抗菌肽LL-37的轉(zhuǎn)基因血管內(nèi)皮祖細(xì)胞和尿路上皮細(xì)胞、平滑肌細(xì)胞等種子細(xì)胞的新型組織工程化尿道組織,從而探索利用該生物材料修復(fù)長段管狀尿道缺損的可能性。對其中涉及的轉(zhuǎn)基因技術(shù),如慢病毒表達(dá)載體的構(gòu)建、質(zhì)粒表達(dá)的檢測、慢病毒的包裝及目的細(xì)胞的代感染等方法進(jìn)行探討,為以后可能的組織工程學(xué)技術(shù)聯(lián)合轉(zhuǎn)基因技術(shù)奠定基礎(chǔ)。同時,鑒于本文涉及的泌尿系組織工程學(xué)常用種子細(xì)胞,如內(nèi)皮祖細(xì)胞、尿路上皮細(xì)胞等,在分離、培養(yǎng)和鑒定等試驗方法方面尚存較多爭議,遂結(jié)合自身經(jīng)驗探討循外周血途徑高效獲取內(nèi)皮祖細(xì)胞,通過膀胱黏膜的移行上皮細(xì)胞轉(zhuǎn)化培養(yǎng)尿路上皮細(xì)胞的方法。 方法：(1)慢病毒的包裝和病毒滴度的檢測。(2)目的基因LL-37過表達(dá)慢病毒載體的構(gòu)建,并進(jìn)行超純化的去內(nèi)毒素抽提,使其具有較高的安全性,同時對構(gòu)建的載體PGC-FU-LL-37表達(dá)情況進(jìn)行檢測。(3)抽取成年新西蘭兔股靜脈血液,通過密度梯度離心法獲取足夠量的單個核細(xì)胞,并采用堿性成纖維細(xì)胞因子、血管內(nèi)皮細(xì)胞生長因子等活性成分誘導(dǎo)單個核細(xì)胞向內(nèi)皮祖細(xì)胞轉(zhuǎn)化。(4)制備好的慢病毒轉(zhuǎn)染目的細(xì)胞——外周血途徑獲取的內(nèi)皮祖細(xì)胞,并通過Western Blot等方法對表達(dá)情況進(jìn)行檢測。(5)切取成年新西蘭兔膀胱組織,剝離黏膜層用于制備尿路上皮細(xì)胞,從肌層取活檢制備尿路平滑肌細(xì)胞,剩余組織用于制備膜片狀膀胱脫細(xì)胞基質(zhì)。(6)將制備好的穩(wěn)定傳代的尿路上皮細(xì)胞、平滑肌細(xì)胞分別種植在膀胱脫細(xì)胞基質(zhì)兩側(cè),待融合至一定程度后將轉(zhuǎn)基因內(nèi)皮祖細(xì)胞種植到上皮細(xì)胞側(cè)。(7)將復(fù)合了種子細(xì)胞的細(xì)胞-細(xì)胞外基質(zhì)復(fù)合材料包埋于成年新西蘭兔腹壁皮下,定期取材行組織學(xué)鑒定。(8)將經(jīng)過包埋處理的細(xì)胞-細(xì)胞外基質(zhì)復(fù)合材料卷管后行長管狀尿道缺損修復(fù)替代,并通過靜脈尿路造影或逆行尿路造影檢測移植物替代效果。 結(jié)果：(1)RT-PCR等實(shí)驗室檢測方法證實(shí)慢病毒表達(dá)載體pGC-FU-LL37-GFP構(gòu)建成功,慢病毒滴度為2.00E+8TU/ml,通過超純?nèi)?nèi)毒素抽提方法使得慢病毒載體安全可用。(2)通過外周血途徑(股靜脈血液)可有效提取單個核細(xì)胞,并通過細(xì)胞因子將其轉(zhuǎn)化為干細(xì)胞性質(zhì)的內(nèi)皮祖細(xì)胞。(3)通過轉(zhuǎn)基因技術(shù)可有效將目的基因抗菌肽LL-37轉(zhuǎn)染進(jìn)入制備的目的細(xì)胞——內(nèi)皮祖細(xì)胞中,免疫熒光法等檢測證實(shí)轉(zhuǎn)染效率可達(dá)90%左右,Western Blot檢測證實(shí)準(zhǔn)基因內(nèi)皮祖細(xì)胞有效表達(dá)抗菌肽LL-37。(4)成功分離培養(yǎng)并純化了兔尿路上皮細(xì)胞和平滑肌細(xì)胞,且種子細(xì)胞增殖迅速、不易老化。(5)自行創(chuàng)立的膀胱脫細(xì)胞基質(zhì)制備技術(shù)——高滲鹽水-酶消化法-去垢劑洗滌技術(shù)可完全脫去組織中的細(xì)胞成分,體外復(fù)合種子細(xì)胞的培養(yǎng)過程中顯示出良好的生物相容性,并且可促進(jìn)種子細(xì)胞的增殖分化和在其上的均勻分布。 結(jié)論：實(shí)驗室條件可成功構(gòu)建包含有抗菌肽LL-37的重組慢病毒表達(dá)載體pGC-FU-LL37-GFP,通過轉(zhuǎn)基因技術(shù)可高效轉(zhuǎn)染目的細(xì)胞并使其表達(dá)相關(guān)目的蛋白。外周血途徑亦可成功分離轉(zhuǎn)化得到足夠數(shù)量的內(nèi)皮祖細(xì)胞,較之普遍采用的骨髓途徑獲取單個核細(xì)胞的方法具有更高的便捷性和可重復(fù)性。通過取材膀胱組織,可快速制備尿路上皮細(xì)胞、平滑肌細(xì)胞,通過純化擴(kuò)增可作為合格的組織工程學(xué)種子細(xì)胞。高滲鹽水-酶消化法-去垢劑洗滌法可制備出完全無細(xì)胞成分的膀胱脫細(xì)胞基質(zhì),且保留細(xì)胞外基質(zhì)的生物學(xué)活性,在復(fù)合制備的種子細(xì)胞后,細(xì)胞可在基質(zhì)表面迅速增殖,通過腹壁皮下包埋的方法可成功制備細(xì)胞-細(xì)胞外基質(zhì)復(fù)合型組織工程學(xué)材料,有望應(yīng)用于長段管狀尿道缺損的組織工程學(xué)修復(fù)應(yīng)用中,從而達(dá)到真正意義上的生物學(xué)修復(fù)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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R699.6;R318.08

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