【摘要】： 研究背景及意義:嚴(yán)重創(chuàng)傷、感染、腫瘤等原因所致的骨缺損在臨床治療中非常常見。目前常用的治療方法是植骨術(shù),而嚴(yán)重的骨缺損用現(xiàn)有的方法難以獲得滿意的療效,骨組織工程的興起為解決大段骨缺損的難題提供了新的途徑。骨髓間充質(zhì)干細(xì)胞(mesenchymal stem cell,MSCs)被認(rèn)為是骨組織工程最佳的種子細(xì)胞。因?yàn)檫@種細(xì)胞取材方便,可通過穿刺獲得,創(chuàng)傷小,取材后并發(fā)癥少,細(xì)胞培養(yǎng)增殖快,在體內(nèi)外成骨誘導(dǎo)環(huán)境下可分化為骨組織,而且具有多向分化潛能;此外,MSCs也容易分離和培養(yǎng)。盡管自體MSCs或成骨細(xì)胞用于動(dòng)物及臨床個(gè)體化治療已經(jīng)取得了良好的結(jié)果,但MSCs在骨髓中含量很少,并隨著年齡的增長減少;現(xiàn)有方法體外迅速擴(kuò)增困難。而且一些自身免疫性疾病患者,其MSCs增殖能力明顯減弱。隨著培養(yǎng)代數(shù)增加可能出現(xiàn)“反分化”現(xiàn)象和致瘤性,從而失去了細(xì)胞應(yīng)有的功能和安全性。因此,自體骨髓間充質(zhì)干細(xì)胞的應(yīng)用會(huì)受來源和數(shù)量限制,難以滿足臨床“隨取隨用”的要求。建立同種異體MSCs種子細(xì)胞庫是解決這些問題的捷徑,也是組織工程從實(shí)驗(yàn)室走向產(chǎn)業(yè)化的重要前提。近年來,國內(nèi)外研究發(fā)現(xiàn)MSCs具有免疫調(diào)節(jié)作用,已往低等級(jí)動(dòng)物模型也證明同種異體種子細(xì)胞及其構(gòu)建的組織工程肌腱、軟骨、骨組織植入體內(nèi)免疫反應(yīng)輕微,不足以影響工程化組織植入體內(nèi)后的修復(fù)功能。因此,同種異體骨組織工程研究前景充滿潛力和希望。 目的:(1)豬骨髓分離培養(yǎng)MSCs并在體外誘導(dǎo)成骨細(xì)胞,并對(duì)其細(xì)胞生物學(xué)特性進(jìn)行觀察,探討MSCs最佳的培養(yǎng)方法和條件;(2)研究MSCs成骨誘導(dǎo)分化后在不同條件下對(duì)外周血單個(gè)核細(xì)胞增殖的影響及細(xì)胞因子分泌情況;研究MSCs免疫原性及IFN-γ對(duì)其免疫原性的影響,探討MSCs免疫調(diào)節(jié)機(jī)制,為體內(nèi)實(shí)驗(yàn)提供依據(jù);(3)成骨誘導(dǎo)分化后MSCs與脫鈣骨基質(zhì)材料復(fù)合構(gòu)建組織工程骨,植入豬皮下并和單純脫鈣骨基質(zhì)材料植入進(jìn)行比較,以了解同種異體組織工程骨在體內(nèi)的免疫排斥反應(yīng)程度及異位成骨能力。從而了解以MSCs為種子細(xì)胞的同種異體組織工程骨移植的可行性。 方法: 1.豬骨髓間充質(zhì)干細(xì)胞生物學(xué)特性(1)無菌條件下在小香豬髂嵴處穿刺抽取2-5m1骨髓,經(jīng)密度梯度離心分離提純MSCs,分別培養(yǎng)在含5%胎牛血清的A-DMEM培養(yǎng)液和10%胎牛血清F12-DMEM培養(yǎng)液里。觀察第3d、第5d的CFU-F數(shù)量、最大傳代次數(shù)、細(xì)胞長滿時(shí)間、FACS檢測(cè)第3代細(xì)胞的CD14、CD29、CD44、CD45、SLA-I、SLA-II表達(dá)。(2)比較MSCs及DOC細(xì)胞貼壁率、生長曲線、生長周期。(3)用堿性磷酸酶染色、Von-Kossa染色、茜素紅法、骨鈣素免疫組化染色鑒定成骨誘導(dǎo)分化后的MSCs。2.豬骨髓間充質(zhì)干細(xì)胞成骨誘導(dǎo)分化后免疫原性實(shí)驗(yàn)研究(1)以未分化的骨髓間充質(zhì)干細(xì)胞為對(duì)照,采用流式細(xì)胞技術(shù)分別檢測(cè)未誘導(dǎo)、成骨誘導(dǎo)的MSCs、MSCs+IFN-γ、DOC+IFN-γSLA分子的表達(dá);(2)采用RT-PCR技術(shù)分別檢測(cè)DOC、未分化的MSCs、MSCs+IFN-γ組、DOC+IFN-γ的SLA基因表達(dá)情況;(3)采用混合淋巴反應(yīng)觀察①不同數(shù)量級(jí)的DOC對(duì)PBMC增殖的影響;②DOC對(duì)經(jīng)絲分裂原刺激的PBMC增殖的影響;③DOC經(jīng)IFN-γ預(yù)處理后體外對(duì)單向混合淋巴反應(yīng)體系影響;④DOC經(jīng)IFN-γ預(yù)處理后體外對(duì)雙向混合淋巴反應(yīng)體系的影響。(4)檢測(cè)MSCs和DOC未經(jīng)過和經(jīng)過IFN-γ處理后其培養(yǎng)上清TGF-β1和IL-10的分泌情況3.同種異體組織工程骨豬皮下植入免疫排斥及異位成骨實(shí)驗(yàn)研究(1)對(duì)新鮮豬脛骨采用脫脂、脫鈣、脫蛋白方法制備豬DBM材料并在光鏡下和電鏡下觀察形態(tài)學(xué)和組織學(xué)結(jié)構(gòu);(2)體外組織工程骨構(gòu)建并在光鏡下和電鏡下細(xì)胞附著,生長,基質(zhì)分泌情況。(3)以DBM材料為對(duì)照,將同種異體組織工程骨植入15頭免疫功能健全的異體小香豬背側(cè)脊柱旁左側(cè)皮下作為實(shí)驗(yàn)組,對(duì)側(cè)植入單純的脫鈣骨基質(zhì)豬皮下,采用HE和Masson染色觀測(cè)1w、2w、4w、8w、12w異位成骨情況,并用ELISA法檢測(cè)術(shù)后局部組織和外周血1w、2w、4w、8w、12w的IL-2及其TNF-α表達(dá)水平。 結(jié)果:1.豬骨髓間充質(zhì)干細(xì)胞及成骨誘導(dǎo)分化后生物學(xué)特性(1)在A-DMEM與F12-DMEM培養(yǎng)液培養(yǎng)的MSCs具有相同特征:細(xì)胞呈紡錘形或三角形,類似纖維細(xì)胞,旋渦樣排列;第3代細(xì)胞FACS檢測(cè)結(jié)果顯示:分離培養(yǎng)的細(xì)胞CD29、CD44、SLA-I表達(dá)強(qiáng)陽性,而CD14、CD34、SLA-II陰性。與F12-DMEM培養(yǎng)液培養(yǎng)的MSCs相比,A-DMEM培養(yǎng)的MSCs、原代培養(yǎng)3d、5d貼壁生長的細(xì)胞克隆數(shù)較多、原代細(xì)胞生長至80-100%所需時(shí)間較短,最大傳代次數(shù)多。(2) MSCs及DOC細(xì)胞貼壁率無明顯差別,生長曲線MSCs倍增時(shí)間為36.8h,DOC為38.9h。MSCs和DOC細(xì)胞周期比較,MSCs細(xì)胞周期中S+G2比例較多,G1比例較少。表明:MSCs生長增殖速度較DOC快。(3)成骨誘導(dǎo)14d,Von-Kossa染色見細(xì)胞間質(zhì)有大量的鈣鹽沉積;ALP染色顯示細(xì)胞呈85%陽性;茜素紅法見到團(tuán)塊狀細(xì)胞中有鈣鹽沉積;免疫細(xì)胞化學(xué)檢測(cè)見到骨鈣素陽性表達(dá)細(xì)胞。2.豬骨髓間充質(zhì)干細(xì)胞成骨誘導(dǎo)分化后免疫原性實(shí)驗(yàn)研究(1) FACS檢測(cè)結(jié)果顯示:MSCs+IFN-γ組、DOC+IFN-γ組SLA-I表達(dá)上調(diào)(P0.05),SLA-II表達(dá)明顯上調(diào)(P0.01)。(2) RT-PCR結(jié)果顯示:DOC組、MSCs+IFN-γ組、DOC+IFN-γ組SLA-I(P1,P14)表達(dá)上調(diào)(P0.05),SLA-II(DRA,DRB,DQA,DQB)表達(dá)明顯上調(diào)(P0.01)。(3)①大于1×104數(shù)量級(jí)以上DOC不能刺激PBMC增殖,低于1×104數(shù)量級(jí)對(duì)PBMC有增殖作用。抑制作用與細(xì)胞數(shù)量成正相關(guān)。②DOC能抑制經(jīng)PHA刺激的PBMC增殖。③DOC經(jīng)IFN-γ預(yù)處理后仍然能抑制PHA和Con-A刺激的PBMC增殖。④DOC經(jīng)IFN-γ預(yù)處理后體外對(duì)雙向混合淋巴反應(yīng)體系(hPBMC+pPBMC)PBMC增殖有抑制作用。(4) MSCs和DOC均能分泌TGF-β1和IL-10,DOC分泌的IL-10水平高于MSCs(P0.01),但經(jīng)IFN-γ刺激后,MSCs分泌的TGF-β1水平明顯高于未經(jīng)IFN-γ刺激的MSCs,而經(jīng)IFN-γ刺激后DOC分泌的TGF-β1明顯低于未經(jīng)IFN-γ刺激的DOC。3.同種異體組織工程骨豬皮下植入免疫排斥及異位成骨實(shí)驗(yàn)研究(1)制備豬DBM材料保持天然的網(wǎng)狀結(jié)構(gòu);(2)成骨誘導(dǎo)分化后MSCs與脫鈣骨基質(zhì)材料復(fù)合7天顯示細(xì)胞附著于材料表面和孔隙內(nèi)壁,并分裂增殖數(shù)目倍增。SEM觀察,細(xì)胞排列規(guī)則,周圍有細(xì)胞外基質(zhì)分泌。(3)所有小香豬術(shù)后無發(fā)熱、畏寒等全身反應(yīng)。取材所見術(shù)后1、2、4周雙側(cè)植入物周圍均見輕微組織反應(yīng),但于8、12周逐漸消失。組織學(xué)觀察,異位成骨以軟骨內(nèi)化骨為主。 結(jié)論:(1)豬MSCs成骨能力強(qiáng),在成骨誘導(dǎo)條件下可向成骨細(xì)胞分化,表達(dá)堿性磷酸酶和骨鈣素,具有作為種子細(xì)胞的潛力。(2)體外實(shí)驗(yàn)表明豬MSCs的誘導(dǎo)成骨后仍保持低免疫原性,在炎前細(xì)胞因子刺激下其免疫原性可能增強(qiáng),但其可能通過分泌一些具有免疫調(diào)節(jié)作用的細(xì)胞因子來調(diào)控免疫反應(yīng)。(3)體內(nèi)實(shí)驗(yàn)表明同種異體組織工程骨植入具有較好的異位成骨效果。但早期可引發(fā)宿主輕微的免疫反應(yīng),但隨時(shí)間延長,免疫反應(yīng)逐漸消失�？傊�,同種異體MSCs作為種子細(xì)胞與DBM材料復(fù)合培養(yǎng)可能是體外組織工程骨構(gòu)建的一種較好選擇。
[Abstract]:Background and significance: bone defects due to severe trauma, infection, and tumor are very common in clinical treatment. At present, the common treatment method is bone grafting, and the serious bone defect is difficult to obtain satisfactory curative effect by the existing method, and the rise of the bone tissue engineering provides a new way for solving the problem of the large-segment bone defect. Mesenchymal stem cells (MSCs) are considered as the best seed cells in bone tissue engineering. Because the cell is convenient to obtain, the cell can be obtained by puncture, the wound is small, the complication is less after the material is obtained, the cell culture is rapid in proliferation, the bone tissue can be differentiated into bone tissue under the external bone-induced environment in the body, and the cell has the multi-directional differentiation potential; in addition, the MSCs can be easily separated and cultured. Although autologous MSCs or osteoblasts have obtained good results for the individual treatment of animals and clinical, the content of MSCs in the bone marrow is very small, and with the increase of age, the existing method is difficult to rapidly expand in vitro. In addition, the proliferation ability of MSCs was significantly decreased in some patients with autoimmune diseases. With the increase of the culture algebra, "anti-differentiation" and tumorigenicity may occur, thus losing the function and safety of the cells. Therefore, the application of autologous bone marrow mesenchymal stem cells can be limited by source and quantity, and it is difficult to meet the requirements of clinical "on-demand". The establishment of a seed cell library of the allogeneic MSCs is a shortcut to solve these problems, and it is also an important prerequisite for the construction of the tissue engineering from the laboratory to the industrialization. In recent years, it has been found that MSCs have the immunomodulating effect, and the allogenic seed cells and the tissue engineering tendon, cartilage and bone tissue in the tissue engineered by the same allogenic seed cells and the constructed tissue engineering tendon, cartilage and bone tissue have been found to be slightly in vivo. Not to affect the repair function after the engineered tissue is implanted in the body. Therefore, the research prospect of allogenic bone tissue engineering is full of potential and hope. Objective: (1) MSCs were isolated and cultured in vitro and cultured in vitro, and the biological characteristics of the cells were observed, and the best tissue culture of MSCs was discussed. (2) To study the effect of MSCs on the proliferation of peripheral blood mononuclear cells and the secretion of cytokines under different conditions. The results provide a basis for the experiment. (3) After the osteogenic induction and differentiation, the MSCs are combined with the demineralized bone matrix material to construct the tissue engineering bone, and the bone is implanted into the pigskin and compared with the pure decalcified bone matrix material, so as to understand the degree of the immune rejection in the bone of the allogenic tissue engineering bone in the body, and Ectopic osteogenic ability. It is known that MSCs are the bone of the same allogenic tissue of the seed cells. the feasibility of transplantation Methods:1. The biological characteristics (1) of bone marrow-derived mesenchymal stem cells (MSCs) in pigs were used to puncture the bone marrow of 2-5 ml at the tip of the small-sized pig. The MSCs were purified by density gradient centrifugation, and cultured in A-DMEM medium containing 5% fetal bovine serum and 10% fetal bovine blood, respectively. CD14, CD29, CD44, CD45, S of the third generation of cells were detected by FACS in the F12-DMEM medium. Observe the number of CFU-F of the 3rd day, the 5th day, the maximum number of passages, the time of the cells, and the FACS to detect the CD14, CD29, CD44, CD45, S of the third generation of cells. LA-I, SLA-II expression. (2) MSCs and DOC cells were compared (3) staining with alkaline phosphatase, Von-Kossa staining, red-red method, and immunohistochemical staining of osteocalcin. Bone-induced differentiation of bone marrow-derived mesenchymal stem cells (MSCs) induced the differentiation of bone marrow-derived mesenchymal stem cells. (1) The non-induced, osteogenic-induced MSCs, MSCs, IFN-1, DOC + were detected by flow cytometry. (3) The effect of DOC on proliferation of PBMC was observed by using mixed lymph reaction to observe the effect of DOC on proliferation of PBMC. The effect of the original stimulation on the proliferation of PBMCs; the effect of the ONDOC on the one-way mixed lymphatic reaction system in vitro after the treatment with IFN-I; and the in vitro response of the EDOC via the IFN-preprocess The effect of the two-way mixed lymphatic reaction system. (4) The detection of MSCs and DOC did not pass through and the culture supernatant TGF-1 was cultured after the treatment with IFN-1. Secretion of 1 and IL-10 3. Implanted immune rejection and ectopic osteogenesis in bone pigskin of allogenic tissue engineering (1) Degreasing, decalcification and deproteinization were used to prepare the porcine DBM material and under the light microscope and under the light microscope. The morphology and the histological structure were observed under the microscope. (2) The in vitro tissue engineering bone was constructed and fine under the light microscope and under the electron microscope. Cell adhesion, growth, and matrix secretion. (3) In contrast with DBM material, the left side of the dorsal spinal column of the allogenic pig with 15-head immune function was implanted subcutaneously as the experimental group, and the other side was implanted with pure decalcium bone matrix pigskin and stained with HE and Masson. The ectopic bone formation of 1w, 2w, 4w, 8w, and 12w was observed, and the IL-2, 2w, 4w, 8w and 12w of the local tissue and peripheral blood were detected by ELISA. -2 and its T Results:1. The biological characteristics (1) of the bone marrow-derived mesenchymal stem cells and the osteogenesis-induced differentiation of the pig have the same characteristics in the culture of A-DMEM and F12-DMEM medium: the cells are fusiform or triangular, similar to the fibroblast and the vortex-like arrangement, and the third generation of cells FA The results of CS test showed that the expression of CD29, CD44, SLA-I was positive in the isolated cultured cells. In contrast to the MSCs cultured with F12-DMEM medium, the number of cells cultured in A-DMEM was much higher than that of MSCs cultured in F12-DMEM medium, and the primary cells were grown to 8. The time required for 0-100% was shorter and the maximum number of passages was more. (2) There was no significant difference in the adherence of MSCs and DOC cells, the doubling time of the growth curve was 36.8 h, and the DOC was 38.9 h. The cell cycle of MSCs and DOC was compared with that of the MSCs. The proportion of S + G2 in the cell cycle is more, and the proportion of G1 is higher. The results showed that the growth rate of MSCs was higher than that of the DOC. (3) The proliferation of MSCs was faster than that of the DOC. (3) There was a large amount of calcium salt deposition in the cells of the cells. The staining of ALP showed that the cells were 85% positive. The results of FACS analysis showed that the expression of SLA-I was up-regulated in the MSCs + IFN-1 group, DOC + IFN-1 group (P0.05). The results showed that the expression of SLA-II was up-regulated (P0.01). (2) The results of RT-PCR showed that the expression of SLA-I (P1, P14) was up-regulated (P0.05), SLA-II (DRA, DRB, D). The expression of QA and DQB was up-regulated (P0.01). (3) The DOC could not stimulate the proliferation of PBMC. Lower than 1-104 order of magnitude for PBM C has a proliferative effect. The inhibitory effect is positively related to the number of cells. The DOC can inhibit the proliferation of PBMCs stimulated by PHA. Inhibition of the proliferation of PBMCs stimulated by PHA and Con-A. (4) MSCs and DOC were able to secrete TGF-1 and IL-10, and the level of IL-10 secreted by DOC was higher than that of MSCs (P0.01). The level was significantly higher than that of the non-IFN-stimulated MSCs, and the TGF-1 secreted by the DOC after IFN-mediated stimulation 1. It is significantly lower than that of the non-IFN-1-stimulated DOC.3. The study of the implantation of immune rejection and ectopic osteogenesis in the bone pigskin of the allogenic tissue engineering (1) The preparation of the porcine DBM material maintains a natural network structure; (2) the osteogenic induction and differentiation of the MSCs and the decalcified bone matrix material Compound 7-day display of cell attachment On the surface of the material and the inner wall of the pores and the multiplication of the division of the proliferation. Observation, cell arrangement rules, extracellular matrix secretion around. ( 3) No fever, aversion to cold and other whole body reaction after operation of all the small fragrant pigs.1,2 and 4 weeks after operation. A slight tissue reaction was observed, but was gradually eliminated at 8 and 12 weeks. The results showed that (1) The osteogenic ability of the pig MSCs was strong, and the bone formation was fine under the condition of osteogenesis induction. Cell differentiation, expression of alkaline phosphatase and osteocalcin, with the potential as a seed cell. (2) In vitro experiments show that the induction of porcine MSCs remains low, and its immunogenicity may be enhanced in the presence of pro-inflammatory cytokines, but may the immune response can be regulated by the secretion of a number of cytokines that have an immunomodulatory effect. (3) the body The results of the internal experiment show that the allogenic tissue engineering bone implantation has better ectopic osteogenic effect, but the early stage can In general, the allogenic MSCs were used as seed cells and D.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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