脐带间充质干细胞体外扩增研究及其分泌细胞因子的高通量筛查
发布时间：2018-08-26 12:31
【摘要】：研究背景：间充质干细胞（Mesenchymalstemcells,MSCs）也称为多潜能间充质基质细胞，为一种非造血成体干细胞，在组织中分布广泛，具有自我更新和多向分化能力。由于具有免疫调节、多向分化潜能、易于获取、体外增殖快、冻存后活性损失小、免疫原性低并且无伦理问题及毒副作用等特点已经在临床上被广泛用于多种疾病的治疗性研究。成体骨髓来源的MSCs（Bonemarrow-derivedMSCs,BM-MSCs）是研究最早且较为深入的MSCs，但由于抽取骨髓对供者损伤较大、易受病毒感染、细胞数量和增殖/分化能力随年龄显著下降等原因限制了BM-MSCs的临床应用。大量研究已表明，多种组织均含有MSCs，如脐带（Umbilicalcord,UC）、脂肪等。而脐带作为胎儿娩出后的医疗废弃物，具有易于获取、伦理限制小、体外易于扩增及培养等优势。因此，本实验拟对脐带来源的MSCs（UC-derivedMSCs,UC-MSCs）与成人BM-MSCs的生物学特性进行比较，以期望获得更佳的MSCs种子细胞来源。同时，许多研究均表明，MSCs分泌的细胞因子在MSCs治疗效应中发挥至关重要的作用，但目前尚无UC-MSCs细胞因子表达谱的研究报道。因此，本实验拟采用细胞因子抗体芯片对不同代次UC-MSCs分泌的细胞因子进行高通量筛选，为UC-MSCs传代次数的确定并为后续治疗机制的研究奠定理论基础。 目的：在MSCs临床应用中，为种子细胞来源提供实验依据；我们比较了体外分离培养UC-和BM-MSCs在形态特征、增殖能力、表面标志和诱导分化能力等方面的差异；为给体外长期培养UC-MSCs的稳定性提供实验证据，我们进一步研究了体外长期培养UC-MSCs的生物特性及活性变化等特点；为了确定体外培养UC-MSCs的较佳传代次数并为治疗机制提供一定的理论依据，我们对不同代次的UC-MSCs体外分泌的细胞因子进行细胞因子抗体芯片筛查和表达谱的差异比较。 方法：我们分别应用酶消化法和密度梯度离心法分离UC-MSCs和BM-MSCs，并用贴壁培养方法进一步纯化两种来源MSCs。采用显微镜技术观察比较了两者的形态特点；应用细胞计数法检测了两者的增殖能力；通过体外连续传代试验研究两者的传代能力；采用流式细胞术分析了两者免疫表型；通过染色法鉴定比较体外诱导UC-和BM-MSCs向脂肪、软骨和成骨细胞分化能力。我们进一步对UC-MSCs进行体外连续培养，检测了细胞生物特性和功能，并采用细胞因子抗体芯片对不同代次UC-MSCs的上清液进行了高通量筛查（目标蛋白为174种细胞因子，RayBio），根据荧光强度信号情况，筛选出MSCs上清液中阳性表达的细胞因子，并采用统计学方法分析不同代次UC-MSCs间的分泌阳性细胞因子的信号差异。 结果：细胞形态结果显示，UC-和BM-MSCs细胞形态相似，均表现为长梭型、贴壁生长；体外长期传代试验表明，BM-MSCs体外增殖能力较差，传代至9~10代基本失去增殖能力，而UC-MSCs增殖及更新能力明显强于BM-MSCs，传至15代时仍保持原有的特性；细胞表型结果表明，，UC-和BM-MSCs具有均一的免疫表型：不表达造血和内皮标志CD34、CD45、CD11b、CD31以及免疫共刺激分子CD80、CD86和MHCⅡ类分子（HLA-DR），表达间质细胞标记CD44、CD105和CD166和CD73。但BM-MSCs的CD80显著高于UC-MSCs的（P=0.0137）；诱导分化结果显示，UC-和BM-MSCs均可向脂肪、成骨和软骨三系分化；细胞因子抗体芯片检测结果表明，UC-MSCs上清液中阳性分泌的细胞因子有102种（强阳性34种，一般阳性68种），主要涉及促细胞迁移类（24.5%）、受体类（20.6%）、促细胞生长和分化类（20.6%）和免疫调节类（12.7%）；统计分析结果表明，第3、5和9代UC-MSCs分泌的细胞因子有显著统计学差异的只有11种：TNF-beta（P=0.0147）、IL-12p70（P=0.0265）、MSP-alpha（P=0.0289）、HCC-4/CCL16（P=0.027）、IP-10/CXCL10（P=0.0202）、GITR（P=0.024）、PDGFRalpha（P=0.0277）、VEGFR2（P=0.038）、IL-9（P=0.0291）、MIP-1beta/CCL4（P=0.0497）和CNTF（P=0.0073）。 结论：UC-MSCs能替代BM-MSCs作为临床上MSCs的种子细胞；不同代次UC-MSCs分泌的绝大多数细胞因子水平无差异；蛋白芯片能以极小的样本量检测高通量的细胞因子，是筛查MSCs细胞因子表达谱比较理想的技术方法。
[Abstract]:BACKGROUND: Mesenchymal stem cells (MSCs), also known as pluripotent mesenchymal stromal cells, are non-hematopoietic adult stem cells. They are widely distributed in tissues and have the ability of self-renewal and multi-differentiation. Adult bone marrow-derived MSCs (BM-MSCs) are the earliest and more in-depth MSCs. However, bone marrow extraction is more harmful to the donor, susceptible to viral infection, and increases the number of cells. The clinical application of BM-MSCs is limited by the decrease of reproductive/differentiation ability with age. Many studies have shown that many kinds of tissues contain MSCs, such as umbilical cord (UC), fat and so on. The biological characteristics of umbilical cord derived MSCs (UC-derived MSCs) and adult BM-MSCs were compared in order to obtain a better source of MSCs seed cells. In this study, cytokines secreted by different generations of UC-MSCs were screened by cytokine antibody chips in order to determine the number of passages of UC-MSCs and lay a theoretical foundation for the follow-up study of treatment mechanism.
OBJECTIVE: To provide experimental evidence for the origin of seed cells in the clinical application of MSCs; We compared the morphological characteristics, proliferation, surface markers and differentiation induction ability of UC-MSCs and BM-MSCs isolated and cultured in vitro; To provide experimental evidence for the stability of long-term cultured UC-MSCs in vitro, we further studied the in vitro growth of UC-MSCs. In order to determine the best passage times of UC-MSCs cultured in vitro and provide a theoretical basis for the treatment mechanism, we screened cytokines secreted by different generations of UC-MSCs in vitro and compared the differences of cytokine antibody chip and expression profiles.
METHODS: UC-MSCs and BM-MSCs were separated by enzyme digestion and density gradient centrifugation, and further purified by adherent culture. The morphological characteristics of UC-MSCs and BM-MSCs were observed and compared by microscopy, their proliferation ability was detected by cell counting, and the proliferation of UC-MSCs and BM-MSCs was studied by continuous passage test in vitro. The ability of UC-and BM-MSCs to differentiate into adipose, cartilage and osteoblasts in vitro was compared by staining. We further cultured UC-MSCs in vitro, detected their biological characteristics and functions, and used cytokine antibody chips for different generations. The supernatant of secondary UC-MSCs was screened by high-throughput screening (the target protein was 174 cytokines, RayBio). According to the fluorescence intensity signal, the positive cytokines expressed in the supernatant of secondary UC-MSCs were screened. The signal difference of secreting positive cytokines between different generations of UC-MSCs was analyzed by statistical method.
Results: Cell morphology showed that UC-and BM-MSCs were similar in morphology, both of which were spindle-shaped and adherent to the wall. Long-term passage test in vitro showed that BM-MSCs had poor proliferative ability and basically lost proliferative ability from passage 9 to 10, while UC-MSCs had stronger proliferative and renewable ability than BM-MSCs, and still maintained their original characteristics at passage 15. The results of cell phenotype showed that UC-and BM-MSCs had homogeneous immunophenotype: CD34, CD45, CD11b, CD31, CD80, CD86 and HLA-DR were not expressed in UC-and BM-MSCs, but CD80 of BM-MSCs was significantly higher than that of UC-MSCs (P=0.0137). The results showed that both UC-and BM-MSCs could differentiate into adipose, osteogenic and cartilage trilineage; cytokine antibody chip assay showed that there were 102 cytokines (34 strongly positive, 68 generally positive) secreted in the supernatant of UC-MSCs, mainly involved in promoting cell migration (24.5%), receptors (20.6%), promoting cell growth and differentiation (20.6%) and immunity. Statistical analysis showed that only 11 cytokines secreted by UC-MSCs in the 3rd, 5and 9generations were significantly different: TNF-beta (P = 0.0147), IL-12p70 (P = 0.0265), MSP-alpha (P = 0.0289), HCC-4/CCL16 (P = 0.027); IP-10/CXCL10 (P = 0.0202), GITR (P = 0.024), GITR (P = 0.0.024), PDGFRalpha (P = 0.77 (P = 0.77), IL-12p70 (P = 0.77), VEGF R2 (P = 0.0265), MSP = 0.0265, MSP-alpha (P = 0.02890.0289), HCC-4/CCL16 (P 0.0291), MI P-1beta/CCL4 (P=0.0497) and CNTF (P=0.0073).
CONCLUSION: UC-MSCs can replace BM-MSCs as the seed cells of MSCs in clinic; the levels of most cytokines secreted by different generations of UC-MSCs are not different; protein chip can detect high-throughput cytokines with very small sample size, which is an ideal technique for screening the cytokine expression profiles of MSCs.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
本文编号：2204877
[Abstract]:BACKGROUND: Mesenchymal stem cells (MSCs), also known as pluripotent mesenchymal stromal cells, are non-hematopoietic adult stem cells. They are widely distributed in tissues and have the ability of self-renewal and multi-differentiation. Adult bone marrow-derived MSCs (BM-MSCs) are the earliest and more in-depth MSCs. However, bone marrow extraction is more harmful to the donor, susceptible to viral infection, and increases the number of cells. The clinical application of BM-MSCs is limited by the decrease of reproductive/differentiation ability with age. Many studies have shown that many kinds of tissues contain MSCs, such as umbilical cord (UC), fat and so on. The biological characteristics of umbilical cord derived MSCs (UC-derived MSCs) and adult BM-MSCs were compared in order to obtain a better source of MSCs seed cells. In this study, cytokines secreted by different generations of UC-MSCs were screened by cytokine antibody chips in order to determine the number of passages of UC-MSCs and lay a theoretical foundation for the follow-up study of treatment mechanism.
OBJECTIVE: To provide experimental evidence for the origin of seed cells in the clinical application of MSCs; We compared the morphological characteristics, proliferation, surface markers and differentiation induction ability of UC-MSCs and BM-MSCs isolated and cultured in vitro; To provide experimental evidence for the stability of long-term cultured UC-MSCs in vitro, we further studied the in vitro growth of UC-MSCs. In order to determine the best passage times of UC-MSCs cultured in vitro and provide a theoretical basis for the treatment mechanism, we screened cytokines secreted by different generations of UC-MSCs in vitro and compared the differences of cytokine antibody chip and expression profiles.
METHODS: UC-MSCs and BM-MSCs were separated by enzyme digestion and density gradient centrifugation, and further purified by adherent culture. The morphological characteristics of UC-MSCs and BM-MSCs were observed and compared by microscopy, their proliferation ability was detected by cell counting, and the proliferation of UC-MSCs and BM-MSCs was studied by continuous passage test in vitro. The ability of UC-and BM-MSCs to differentiate into adipose, cartilage and osteoblasts in vitro was compared by staining. We further cultured UC-MSCs in vitro, detected their biological characteristics and functions, and used cytokine antibody chips for different generations. The supernatant of secondary UC-MSCs was screened by high-throughput screening (the target protein was 174 cytokines, RayBio). According to the fluorescence intensity signal, the positive cytokines expressed in the supernatant of secondary UC-MSCs were screened. The signal difference of secreting positive cytokines between different generations of UC-MSCs was analyzed by statistical method.
Results: Cell morphology showed that UC-and BM-MSCs were similar in morphology, both of which were spindle-shaped and adherent to the wall. Long-term passage test in vitro showed that BM-MSCs had poor proliferative ability and basically lost proliferative ability from passage 9 to 10, while UC-MSCs had stronger proliferative and renewable ability than BM-MSCs, and still maintained their original characteristics at passage 15. The results of cell phenotype showed that UC-and BM-MSCs had homogeneous immunophenotype: CD34, CD45, CD11b, CD31, CD80, CD86 and HLA-DR were not expressed in UC-and BM-MSCs, but CD80 of BM-MSCs was significantly higher than that of UC-MSCs (P=0.0137). The results showed that both UC-and BM-MSCs could differentiate into adipose, osteogenic and cartilage trilineage; cytokine antibody chip assay showed that there were 102 cytokines (34 strongly positive, 68 generally positive) secreted in the supernatant of UC-MSCs, mainly involved in promoting cell migration (24.5%), receptors (20.6%), promoting cell growth and differentiation (20.6%) and immunity. Statistical analysis showed that only 11 cytokines secreted by UC-MSCs in the 3rd, 5and 9generations were significantly different: TNF-beta (P = 0.0147), IL-12p70 (P = 0.0265), MSP-alpha (P = 0.0289), HCC-4/CCL16 (P = 0.027); IP-10/CXCL10 (P = 0.0202), GITR (P = 0.024), GITR (P = 0.0.024), PDGFRalpha (P = 0.77 (P = 0.77), IL-12p70 (P = 0.77), VEGF R2 (P = 0.0265), MSP = 0.0265, MSP-alpha (P = 0.02890.0289), HCC-4/CCL16 (P 0.0291), MI P-1beta/CCL4 (P=0.0497) and CNTF (P=0.0073).
CONCLUSION: UC-MSCs can replace BM-MSCs as the seed cells of MSCs in clinic; the levels of most cytokines secreted by different generations of UC-MSCs are not different; protein chip can detect high-throughput cytokines with very small sample size, which is an ideal technique for screening the cytokine expression profiles of MSCs.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
【参考文献】
相关期刊论文 前1条
1 张卫光;间充质干细胞与生物人工肝的研究进展[J];解剖科学进展;2004年03期
本文编号：2204877
本文链接：https://www.wllwen.com/xiyixuelunwen/2204877.html
最近更新
教材专著
