本文選題：動(dòng)態(tài)灌注培養(yǎng) + 晚期內(nèi)皮祖細(xì)胞　； 參考：《昆明醫(yī)科大學(xué)》2016年博士論文

【摘要】：[目的]探討在體外通過動(dòng)態(tài)灌注系統(tǒng)培養(yǎng)晚期內(nèi)皮祖細(xì)胞-脫鈣骨基質(zhì)復(fù)合體,對(duì)晚期內(nèi)皮祖細(xì)胞在脫鈣骨基質(zhì)上的鋪展粘附、增殖、分化和基質(zhì)孔隙中成管能力的影響及其可能的機(jī)制。探討動(dòng)態(tài)灌注預(yù)血管化的晚期內(nèi)皮祖細(xì)胞-脫鈣骨基質(zhì)復(fù)合體的血管在體內(nèi)與受區(qū)血管可能的融合機(jī)制。[方法]1.密度梯度離心法分離培養(yǎng)人外周血晚期內(nèi)皮祖細(xì)胞,并通過細(xì)胞形態(tài)觀察,流式細(xì)胞檢測(cè),結(jié)合凝集素及攝取乙�；兔芏戎鞍讓�(shí)驗(yàn)和Matrigel基質(zhì)膠體外成管實(shí)驗(yàn)對(duì)細(xì)胞進(jìn)行綜合鑒定。分別用10/30/50/70/100 MOI值的慢病毒介導(dǎo)eGFP試轉(zhuǎn)染OECs,計(jì)算細(xì)胞轉(zhuǎn)染效率,MTT比色法繪制不同MOI值下晚期內(nèi)皮祖細(xì)胞的生長(zhǎng)曲線,并與對(duì)照組細(xì)胞進(jìn)行比較,優(yōu)選出最優(yōu)的MOI值,挑選耐嘌呤霉素單細(xì)胞克隆并擴(kuò)增培養(yǎng)建立穩(wěn)定細(xì)胞株,并判斷其對(duì)晚期內(nèi)皮祖細(xì)胞的增殖、細(xì)胞表面標(biāo)記及體外成管特性有無影響。2.構(gòu)建體外動(dòng)態(tài)灌注培養(yǎng)系統(tǒng),將eGFP慢病毒穩(wěn)定轉(zhuǎn)染晚期內(nèi)皮祖細(xì)胞株接種于脫鈣骨基質(zhì)上,分別進(jìn)行靜態(tài)接種培養(yǎng)、動(dòng)態(tài)灌注培養(yǎng)和抑制劑實(shí)驗(yàn),通過熒光顯微鏡實(shí)時(shí)動(dòng)態(tài)觀察eGFP標(biāo)記的晚期內(nèi)皮祖細(xì)胞在脫鈣骨基質(zhì)上的鋪展粘附和增殖情況,于培養(yǎng)第6天掃描電子顯微鏡下觀察細(xì)胞在脫鈣骨基質(zhì)孔隙中的體外成管情況,RT-PCR及Western blot檢測(cè)分析不同培養(yǎng)條件下的細(xì)胞CD34、VEGFR-1、VEGFR-2、VE-cadherin、Tie2和VEGF的mRNA及蛋白表達(dá)水平。3.建立裸鼠背部血管化動(dòng)態(tài)觀察窗模型,結(jié)合尾靜脈注射德克薩斯紅標(biāo)記的右旋糖酐標(biāo)記血漿,光鏡及熒光顯微鏡下觀察微血管,比較單純觀察窗模型及其結(jié)合德克薩斯紅標(biāo)記的右旋糖酐在微血管觀察中的優(yōu)劣勢(shì),并記錄微血管開始顯影時(shí)間,最佳觀測(cè)時(shí)間及顯影持續(xù)時(shí)間。往裸鼠背部血管化動(dòng)態(tài)觀察窗中植入靜態(tài)培養(yǎng)和動(dòng)態(tài)灌注培養(yǎng)的OECs-DBM支架,分別在植入后第1、3、5、7、9、12天熒光顯微鏡下動(dòng)態(tài)觀察并計(jì)數(shù)支架邊緣長(zhǎng)入血管數(shù)量,計(jì)算支架邊緣及中心區(qū)熒光累積光密度值來反映支架獲得的血液灌注量。在植入后第12天切取支架及其周圍組織進(jìn)行硬組織切片HE染色,在光鏡及熒光顯微鏡下觀察支架上熒光細(xì)胞的分布及其與受體血管融合情況。[結(jié)果]1.晚期血管內(nèi)皮祖細(xì)胞表現(xiàn)為典型的鵝卵石樣形態(tài),能較為特異的結(jié)合凝集素及攝取乙酰化低密度脂蛋白,在Matrigel基質(zhì)膠上可形成血管管腔樣結(jié)構(gòu),流式細(xì)胞儀檢測(cè)顯示其表達(dá)細(xì)胞表面標(biāo)記CD34和VEGFR-2,基本不表達(dá)CD133。當(dāng)MOI=50時(shí),慢病毒介導(dǎo)的eGFP轉(zhuǎn)染晚期內(nèi)皮祖細(xì)胞轉(zhuǎn)染效率較高且對(duì)細(xì)胞生長(zhǎng)增殖無影響,篩選出的eGFP-OECs穩(wěn)定轉(zhuǎn)染細(xì)胞株在熒光顯微鏡下發(fā)出明亮穩(wěn)定的綠色熒光,細(xì)胞的生物學(xué)形狀及功能保持穩(wěn)定。2.與靜態(tài)培養(yǎng)組相比較,動(dòng)態(tài)灌注組脫鈣骨基質(zhì)上的細(xì)胞密度和細(xì)胞投射面積更大,掃描電鏡觀察顯示動(dòng)態(tài)灌注培養(yǎng)組在脫鈣骨基質(zhì)孔隙中形成三維血管腔樣結(jié)構(gòu)的數(shù)量多于靜態(tài)培養(yǎng)組。RT-PCR及Western blot檢測(cè)顯示,與靜態(tài)培養(yǎng)組相比,動(dòng)態(tài)灌注培養(yǎng)組細(xì)胞CD34 mRNA表達(dá)及蛋白合成下調(diào),內(nèi)皮細(xì)胞標(biāo)記VEGFR-1、VEGFR-2、VE-cadherin和Tie2mRNA表達(dá)及蛋白合成上調(diào)。抑制劑實(shí)驗(yàn)顯示PI3K和mTOR抑制劑能夠抑制流體剪切應(yīng)力促進(jìn)細(xì)胞鋪展粘附、增殖、分化和體外成管的作用。3.成功構(gòu)建裸鼠背部血管化觀察窗模型。與單純觀察窗模型相比,其結(jié)合德克薩斯紅標(biāo)記的右旋糖酐在微血管觀察中更為精確。OECs-DBM支架邊緣新長(zhǎng)入血管計(jì)數(shù)結(jié)果顯示兩組支架植入后周圍都有微血管長(zhǎng)入,隨著培養(yǎng)時(shí)間的延長(zhǎng),長(zhǎng)入的微血管數(shù)量增多。在支架植入后第3、5、7天,動(dòng)態(tài)灌注組的新長(zhǎng)入血管數(shù)目較靜態(tài)培養(yǎng)組多。動(dòng)態(tài)灌注組的長(zhǎng)入血管數(shù)量上升速率較靜態(tài)灌注組快。動(dòng)態(tài)灌注組支架邊緣區(qū)域的熒光累積光密度值在所有觀察時(shí)間點(diǎn)均高于靜態(tài)培養(yǎng)組支架,而兩組的支架中心區(qū)熒光累積光密度值差異更為顯著。硬組織切片HE染色顯示兩組支架外均未見綠色熒光細(xì)胞。[結(jié)論]通過動(dòng)態(tài)灌注系統(tǒng)給予脫鈣骨基質(zhì)上晚期內(nèi)皮祖細(xì)胞持續(xù)不斷的流體剪切應(yīng)力,能夠經(jīng)由PI3K/Akt/mTOR信號(hào)傳導(dǎo)通路促進(jìn)晚期內(nèi)皮祖細(xì)胞在脫鈣骨基質(zhì)上的鋪展粘附、增殖、向成熟內(nèi)皮細(xì)胞分化和基質(zhì)孔隙中三維成管的能力,促進(jìn)脫鈣骨基質(zhì)體外預(yù)血管化。與靜態(tài)培養(yǎng)組相比,體外動(dòng)態(tài)灌注培養(yǎng)預(yù)血管化的OECs-DBM支架能以長(zhǎng)入式融合的方式與受區(qū)血管融合,更快更充分的獲得受區(qū)的血液灌注,研究成果可能用于促進(jìn)組織工程產(chǎn)品的臨床應(yīng)用。
[Abstract]:[Objective] to explore the effect and possible mechanism of advanced endothelial progenitor cells (endothelium progenitor cells, decalcified bone matrix complex) in vitro by dynamic perfusion system on the ability of spreading and adhesion, proliferation, differentiation and matrix pore formation on decalcified bone matrix. A possible fusion mechanism between the blood vessels of the matrix complex in the body and the recipient vessels. [method]1. density gradient centrifugation was used to isolate and culture the advanced endothelial progenitor cells of human peripheral blood, and through cell morphology observation, flow cytometry, binding of lectin and uptake of acetylated LDL and Matrigel matrix colloidal outer tube test. The 10/30/50/70/100 MOI value of eGFP was used to transfect OECs, and the transfection efficiency was calculated. MTT colorimetric method was used to plot the growth curve of late endothelial progenitor cells under different MOI values. Compared with the control group, the optimal MOI value was optimized, and the single cell clone and amplification of purinomycin were selected and amplified and established. To stabilize the cell line and determine its proliferation to advanced endothelial progenitor cells, the cell surface markers and the characteristics of the in vitro culture have no effect on the.2. construction in vitro, and the eGFP lentivirus stable transfection of late endothelial progenitor cells is inoculated on the decalcified bone matrix for static inoculation, dynamic perfusion culture and inhibitor. The spread and adhesion of eGFP labeled advanced endothelial progenitor cells on decalcified bone matrix were observed by fluorescence microscopy in real time. The cell formation in vitro of decalcified bone matrix was observed under sixth days of scanning electron microscope, and RT-PCR and Western blot were used to detect CD34, V of cells under different culture conditions. EGFR-1, VEGFR-2, VE-cadherin, Tie2 and VEGF mRNA and protein expression level.3. established the dynamic observation window model of the nude mouse back vascularization, combined with the tail vein injection of Texas red labeled dextran plasma, light microscope and fluorescence microscope to observe the microvascular, compared the simple observation window model and the right of Texas red label right The advantages and disadvantages of maleic anhydride in microvascular observation, and the recording time of the microvasculature, the best observation time and the duration of development were recorded. The static and dynamic perfusion OECs-DBM scaffolds were implanted into the dynamic observation window of the back vascularization of nude mice, and the dynamic observation and count branches were observed on the 1,3,5,7,9,12 day fluorescence microscope after the implantation. The number of blood vessels on the edge of the frame and the value of fluorescence accumulated light density of the stent were calculated to reflect the amount of blood perfusion obtained by the scaffold. The stent and its surrounding tissue were cut by HE staining at twelfth days after implantation. The distribution of fluorescein on the scaffold and the fusion relationship with the receptor vessel were observed under the light and fluorescence microscope. [results] [results]1. advanced vascular endothelial progenitor cells showed a typical cobblestone like morphology, which could be more specific binding lectin and acetylated LDL, and could form vascular lumen like structure on Matrigel matrix gel. Flow cytometry showed that the expression of cell surface markers CD34 and VEGFR-2, basically not CD133. when MOI= was expressed. At 50, the transfection efficiency of lentivirus mediated eGFP transfected advanced endothelial progenitor cells was higher and no effect on cell growth and proliferation. The selected eGFP-OECs stable transfected cell lines emitted bright and stable green fluorescence under the fluorescence microscope, and the biological shape and function of the cells remained stable and.2. was compared with the static culture group, and the dynamic perfusion group was removed. The cell density and the cell projection area on the calcium bone matrix were larger. The scanning electron microscope showed that the number of three-dimensional vascular cavities formed in the pore of decalcified bone matrix was more than that in the static culture group.RT-PCR and Western blot. Compared with the static culture group, the expression of CD34 mRNA and the egg in the dynamic perfusion culture group were compared with that of the static culture group. White synthesis down-regulation, endothelial cell markers VEGFR-1, VEGFR-2, VE-cadherin and Tie2mRNA expression and up regulation of protein synthesis. Inhibitor experiments showed that PI3K and mTOR inhibitors could inhibit fluid shear stress and promote cell spreading and adhesion, proliferation, differentiation and in vitro formation of tubes..3. successfully constructed the nude mouse back vascularization observation window model. Compared with the window model, it combined with the Texas red labeled dextran in the microvascular observation more accurate.OECs-DBM stent edge new long entry blood vessel count results showed that two groups of stent implantation around the microvascular length, with the extension of culture time, the increase of the number of microvasculature increased. After the stent implantation at the 3,5,7 day, dynamic irrigation. The number of new long entry vessels in the injection group was more than that in the static culture group. The increase rate of the amount of blood vessels in the dynamic perfusion group was faster than that in the static perfusion group. The fluorescence cumulative light density value of the stent edge area in the dynamic perfusion group was higher than that in the static culture group at all observation time points, but the difference of the fluorescence cumulative light density value of the two groups was more different. HE staining of hard tissue sections showed that no green fluorescent cells were found outside the two groups. [Conclusion] the continuous fluid shear stress was given to late endothelial progenitor cells on the decalcified bone matrix through the dynamic perfusion system, and could promote the spread and adhesion of late inner progenitor cells to the decalcified bone matrix via the PI3K/Akt/mTOR signal transduction pathway. Proliferation, the ability to differentiate into mature endothelial cells and the ability of three-dimensional tube formation in the matrix pores to promote the prevascularization of the decalcified bone matrix in vitro. Compared with the static culture group, the prevascularized OECs-DBM scaffold in vitro can fuse with the recipient blood vessels in a long entry mode, and study the blood perfusion faster and more fully. The results may be used to promote the clinical application of tissue engineering products.

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R68;R318.08

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相關(guān)期刊論文 前1條

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本文編號(hào)：1896066