猪骨髓间充质干细胞成骨诱导分化后免疫原性的实验研究
发布时间：2019-07-04 10:54
【摘要】： 研究背景及意义:严重创伤、感染、肿瘤等原因所致的骨缺损在临床治疗中非常常见。目前常用的治疗方法是植骨术,而严重的骨缺损用现有的方法难以获得满意的疗效,骨组织工程的兴起为解决大段骨缺损的难题提供了新的途径。骨髓间充质干细胞(mesenchymal stem cell,MSCs)被认为是骨组织工程最佳的种子细胞。因为这种细胞取材方便,可通过穿刺获得,创伤小,取材后并发症少,细胞培养增殖快,在体内外成骨诱导环境下可分化为骨组织,而且具有多向分化潜能;此外,MSCs也容易分离和培养。尽管自体MSCs或成骨细胞用于动物及临床个体化治疗已经取得了良好的结果,但MSCs在骨髓中含量很少,并随着年龄的增长减少;现有方法体外迅速扩增困难。而且一些自身免疫性疾病患者,其MSCs增殖能力明显减弱。随着培养代数增加可能出现“反分化”现象和致瘤性,从而失去了细胞应有的功能和安全性。因此,自体骨髓间充质干细胞的应用会受来源和数量限制,难以满足临床“随取随用”的要求。建立同种异体MSCs种子细胞库是解决这些问题的捷径,也是组织工程从实验室走向产业化的重要前提。近年来,国内外研究发现MSCs具有免疫调节作用,已往低等级动物模型也证明同种异体种子细胞及其构建的组织工程肌腱、软骨、骨组织植入体内免疫反应轻微,不足以影响工程化组织植入体内后的修复功能。因此,同种异体骨组织工程研究前景充满潜力和希望。 目的:(1)猪骨髓分离培养MSCs并在体外诱导成骨细胞,并对其细胞生物学特性进行观察,探讨MSCs最佳的培养方法和条件;(2)研究MSCs成骨诱导分化后在不同条件下对外周血单个核细胞增殖的影响及细胞因子分泌情况;研究MSCs免疫原性及IFN-γ对其免疫原性的影响,探讨MSCs免疫调节机制,为体内实验提供依据;(3)成骨诱导分化后MSCs与脱钙骨基质材料复合构建组织工程骨,植入猪皮下并和单纯脱钙骨基质材料植入进行比较,以了解同种异体组织工程骨在体内的免疫排斥反应程度及异位成骨能力。从而了解以MSCs为种子细胞的同种异体组织工程骨移植的可行性。 方法: 1.猪骨髓间充质干细胞生物学特性(1)无菌条件下在小香猪髂嵴处穿刺抽取2-5m1骨髓,经密度梯度离心分离提纯MSCs,分别培养在含5%胎牛血清的A-DMEM培养液和10%胎牛血清F12-DMEM培养液里。观察第3d、第5d的CFU-F数量、最大传代次数、细胞长满时间、FACS检测第3代细胞的CD14、CD29、CD44、CD45、SLA-I、SLA-II表达。(2)比较MSCs及DOC细胞贴壁率、生长曲线、生长周期。(3)用碱性磷酸酶染色、Von-Kossa染色、茜素红法、骨钙素免疫组化染色鉴定成骨诱导分化后的MSCs。2.猪骨髓间充质干细胞成骨诱导分化后免疫原性实验研究(1)以未分化的骨髓间充质干细胞为对照,采用流式细胞技术分别检测未诱导、成骨诱导的MSCs、MSCs+IFN-γ、DOC+IFN-γSLA分子的表达;(2)采用RT-PCR技术分别检测DOC、未分化的MSCs、MSCs+IFN-γ组、DOC+IFN-γ的SLA基因表达情况;(3)采用混合淋巴反应观察①不同数量级的DOC对PBMC增殖的影响;②DOC对经丝分裂原刺激的PBMC增殖的影响;③DOC经IFN-γ预处理后体外对单向混合淋巴反应体系影响;④DOC经IFN-γ预处理后体外对双向混合淋巴反应体系的影响。(4)检测MSCs和DOC未经过和经过IFN-γ处理后其培养上清TGF-β1和IL-10的分泌情况3.同种异体组织工程骨猪皮下植入免疫排斥及异位成骨实验研究(1)对新鲜猪胫骨采用脱脂、脱钙、脱蛋白方法制备猪DBM材料并在光镜下和电镜下观察形态学和组织学结构;(2)体外组织工程骨构建并在光镜下和电镜下细胞附着,生长,基质分泌情况。(3)以DBM材料为对照,将同种异体组织工程骨植入15头免疫功能健全的异体小香猪背侧脊柱旁左侧皮下作为实验组,对侧植入单纯的脱钙骨基质猪皮下,采用HE和Masson染色观测1w、2w、4w、8w、12w异位成骨情况,并用ELISA法检测术后局部组织和外周血1w、2w、4w、8w、12w的IL-2及其TNF-α表达水平。 结果:1.猪骨髓间充质干细胞及成骨诱导分化后生物学特性(1)在A-DMEM与F12-DMEM培养液培养的MSCs具有相同特征:细胞呈纺锤形或三角形,类似纤维细胞,旋涡样排列;第3代细胞FACS检测结果显示:分离培养的细胞CD29、CD44、SLA-I表达强阳性,而CD14、CD34、SLA-II阴性。与F12-DMEM培养液培养的MSCs相比,A-DMEM培养的MSCs、原代培养3d、5d贴壁生长的细胞克隆数较多、原代细胞生长至80-100%所需时间较短,最大传代次数多。(2) MSCs及DOC细胞贴壁率无明显差别,生长曲线MSCs倍增时间为36.8h,DOC为38.9h。MSCs和DOC细胞周期比较,MSCs细胞周期中S+G2比例较多,G1比例较少。表明:MSCs生长增殖速度较DOC快。(3)成骨诱导14d,Von-Kossa染色见细胞间质有大量的钙盐沉积;ALP染色显示细胞呈85%阳性;茜素红法见到团块状细胞中有钙盐沉积;免疫细胞化学检测见到骨钙素阳性表达细胞。2.猪骨髓间充质干细胞成骨诱导分化后免疫原性实验研究(1) FACS检测结果显示:MSCs+IFN-γ组、DOC+IFN-γ组SLA-I表达上调(P0.05),SLA-II表达明显上调(P0.01)。(2) RT-PCR结果显示:DOC组、MSCs+IFN-γ组、DOC+IFN-γ组SLA-I(P1,P14)表达上调(P0.05),SLA-II(DRA,DRB,DQA,DQB)表达明显上调(P0.01)。(3)①大于1×104数量级以上DOC不能刺激PBMC增殖,低于1×104数量级对PBMC有增殖作用。抑制作用与细胞数量成正相关。②DOC能抑制经PHA刺激的PBMC增殖。③DOC经IFN-γ预处理后仍然能抑制PHA和Con-A刺激的PBMC增殖。④DOC经IFN-γ预处理后体外对双向混合淋巴反应体系(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),但经IFN-γ刺激后,MSCs分泌的TGF-β1水平明显高于未经IFN-γ刺激的MSCs,而经IFN-γ刺激后DOC分泌的TGF-β1明显低于未经IFN-γ刺激的DOC。3.同种异体组织工程骨猪皮下植入免疫排斥及异位成骨实验研究(1)制备猪DBM材料保持天然的网状结构;(2)成骨诱导分化后MSCs与脱钙骨基质材料复合7天显示细胞附着于材料表面和孔隙内壁,并分裂增殖数目倍增。SEM观察,细胞排列规则,周围有细胞外基质分泌。(3)所有小香猪术后无发热、畏寒等全身反应。取材所见术后1、2、4周双侧植入物周围均见轻微组织反应,但于8、12周逐渐消失。组织学观察,异位成骨以软骨内化骨为主。 结论:(1)猪MSCs成骨能力强,在成骨诱导条件下可向成骨细胞分化,表达碱性磷酸酶和骨钙素,具有作为种子细胞的潜力。(2)体外实验表明猪MSCs的诱导成骨后仍保持低免疫原性,在炎前细胞因子刺激下其免疫原性可能增强,但其可能通过分泌一些具有免疫调节作用的细胞因子来调控免疫反应。(3)体内实验表明同种异体组织工程骨植入具有较好的异位成骨效果。但早期可引发宿主轻微的免疫反应,但随时间延长,免疫反应逐渐消失。总之,同种异体MSCs作为种子细胞与DBM材料复合培养可能是体外组织工程骨构建的一种较好选择。
[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.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
本文编号：2509864
[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.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
【参考文献】
相关期刊论文 前10条
1 任杰,杨爽;生物医用材料——聚乳酸的表面改性研究进展[J];北京生物医学工程;2004年04期
2 史培良,胡平,顾晓明,高峰,杨维东,毛天球;聚羟基丁酸酯与骨髓基质细胞构建组织工程骨的实验研究[J];解放军医学杂志;2001年04期
3 陈瀛,李子荣,林朋,张黎;骨组织工程支架材料表面修饰的试验研究[J];生物医学工程研究;2003年03期
4 吴群,熊平,陈实,冯书堂,龚非力;近交系海南五指山猪SLA经典I类和II类分子序列分析[J];现代免疫学;2004年01期
5 柴岗,张艳,王敏,胡晓洁,吴娟娟,刘伟,崔磊,曹谊林;人骨髓基质细胞诱导成骨细胞和脱钙骨粘附率的研究[J];实用美容整形外科杂志;2003年02期
6 李彦林,杨志明,解慧琪,秦廷武,黄富国;生物衍生组织工程骨支架材料的制备及理化特性[J];生物医学工程学杂志;2002年01期
7 王小勇,席庆,毛天球,曹罡,付振虹;同种异体成骨细胞移植的免疫学研究[J];中国临床康复;2003年02期
8 王身国,贝建中;组织工程细胞支架及其相关技术的研究[J];中国创伤骨科杂志;2000年04期
9 陈剑飞,林坚平,吴多能,姚伦龙,蔡海莉,王清华,姚捷;异种骨基质明胶(BMG)的抗原性研究[J];中华创伤骨科杂志;2004年07期
10 杨志明,余希杰,解慧琪,陈旭,屈艺;不同来源成骨细胞生物学特性的比较研究[J];中华创伤杂志;2001年01期
，本文编号：2509864
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2509864.html
最近更新
教材专著
