人脐带间充质干细胞促进严重烧伤大鼠创面愈合及其机制的研究

发布时间：2018-01-29 13:47

本文关键词： 人脐带间充质干细胞烧伤创面烧伤血清 Notch信号　出处：《中国人民解放军医学院》2014年博士论文　论文类型：学位论文

【摘要】：目的烧伤是平时、战时的常见外伤，突发性成批烧伤的救治效果与维护社会稳定密切相关。挽救严重烧伤患者生命的关键环节是尽早封闭创面。近些年的研究发现间充质干细胞（Mesenchymal Stem Cells，MSCs）能够有效修复损伤组织器官的结构和功能。基于MSCs有效修复重建损伤组织器官的功能，本研究拟探讨MSCs对严重烧伤创面的修复潜能。因此，将分离培养的人脐带间充质干细胞（humanUmbilical Cord Mesenchymal Stem Cells，hUCMSCs）移植入严重烧伤大鼠体内，探讨hUCMSCs对严重烧伤大鼠创面愈合的调控作用及机制，从而为临床严重烧伤创面的治疗提供新的思路和实验依据。方法(1)采用3种酶消化法（单纯胶原酶Ⅱ消化法、胶原酶Ⅱ+胰蛋白酶消化法、胶原酶Ⅱ+透明质酸酶消化法）及组织块贴壁法分离培养hUCMSCs。免疫荧光染色法/流式鉴定细胞的表面标志物CD105、CD90、CD44、CD73、HLA-I、CD45、HLA-DR、CD31和vWF；油红O染色、阿利新蓝染色和Von Kossa钙沉积法鉴定细胞成脂、成软骨和成骨的分化潜能；倒置相差显微镜和透射电镜观察细胞的形态及超微结构；MTT法检测不同代细胞的增殖情况以及hUCMSCs对T细胞的调节作用；碘化丙啶（PI）染色和流式检测不同代hUCMSCs的细胞周期。(2)收集特重度烧伤患者血清和正常对照血清。使用10%胎牛血清（10%FBS）、10%正常对照血清（10%HCS）和10%烧伤3d患者血清（10%BPS3d）培养体系培养hUCMSCs。AO-EB染色法/流式检测三组细胞的凋亡情况；倒置相差显微镜观察三组细胞的生长密度；MTT法检测三组细胞的增殖情况；碘化丙啶（PI）染色和流式检测三组的细胞周期；β-gal染色检测三组细胞的衰老情况。(3)取成年雄性Wistar大鼠126只，随机分成假伤组，烧伤组和烧伤+hUCMSCs移植组。将GFP标记的hUCMSCs或PBS通过尾静脉注入对应组大鼠体内。Image Pro Plus软件评估大鼠创面的愈合率；小动物活体荧光成像系统（BLI）检测GFP-hUCMSCs在大鼠体内的迁移情况；PCR技术检测创面是否表达人类特异性DNA；免疫组化染色检测创面炎症细胞浸润程度、新生微血管数量以及I、III型胶原表达；激光多普勒血流仪评估创面的血流；ELISA法检测创面促炎因子、抗炎因子、VEGF、I型胶原、III型胶原的含量。(4)实验分为3组：10%正常对照血清(10%HCS)、10%烧伤3d患者血清(10%BPS3d)和10%烧伤3d患者血清+Notch信号特异性抑制剂DAPT/GSI(DAPT/GSI+BPS)或者分为5组：10%HCS、10%BPS3d、10%HCS+20ng/mL VEGF、10%HCS+20ng/mL bFGF和10%HCS+20ng/mLVEGF+20ng/mL bFGF培养hUCMSCs。MTT法和台盼蓝染色计数检测hUCMSCs的存活和增殖情况； PI染色和流式检测细胞的增殖周期；Western Blot检测周期蛋白D(Cyclin D)的表达情况；ELISA法检测血清中VEGF、bFGF等细胞因子的含量；免疫荧光染色法检测VEGFR1和bFGFR2的表达情况；Western Blot和qRT-PCR检测Notch信号关键分子Notch-1和Hes-1的表达情况。结果(1)四种方法均可获得hUCMSCs，但胶原酶Ⅱ+透明质酸酶消化法获得细胞数量较多，且获得细胞的增殖速率较快。因此，胶原酶Ⅱ+透明质酸酶消化法是一种高效提取hUCMSCs的方法。获得的细胞阳性表达间质系表面标志物，但不表达内皮系和造血系表面标志物。传代到3代时，细胞的形态为长梭形，呈旋涡状生长。透射电镜示该细胞的核大且不规则，核仁明显，胞浆较少。使用特定诱导液诱导，其可向脂肪细胞、成软骨和成骨细胞分化。细胞周期的结果显示，80%以上的细胞处于静止期(G0～G1期)，符合较原始干细胞的特征。综合以上结果确定分离得到的细胞是hUCMSCs。MTT的结果示，，各代的hUCMSCs经1d潜伏期，2d-6d是对数增殖期，于第7d时开始出现不同程度的接触抑制而进入平台期。此外，hUCMSCs能显著抑制由植物血凝素(PHA)诱导同种异体T细胞的增殖。(2)10%FBS、10%HCS和10%BPS3d培养体系培养的hUCMSCs形态和结构均正常，未见细胞核着橘红色荧光的凋亡细胞或死亡细胞；流式的结果也验证了此结果。MTT的结果示，从第2d至第6d，10%BPS3d组细胞的增殖速度和增殖细胞量显著快于和多于其它两组；倒置相差显微镜观察细胞增殖生长的融合结果同MTT。10%BPS3d组细胞增殖期（S）的比例显著高于其它两组，而衰老细胞的百分率则显著低于其他两组。综上所述，严重烧伤血清中含有一些保护细胞且促进细胞增殖的细胞因子，因此将hUCMSCs移植治疗严重烧伤动物模型是可行的。(3)将GFP-hUCMSCs静脉移植入严重烧伤大鼠体内，其可随着血液循环迁移到达烧伤创面，显著降低创面炎症细胞浸润程度、显著降低创面促炎因子IL-1，IL-6，TNF-α水平和显著增加抗炎因子IL-10和TNF-α的刺激基因-6（TSG-6）水平。此外，hUCMSCs还可显著增加创面新生微血管的数量和VEGF水平以及显著上调创面中I型胶原、III型胶原比例，进而加速了严重烧伤创面的愈合过程。(4)与10%HCS相比，10%BPS3d可显著促进hUCMSCs快速增殖和显著促进hUCMSCs的细胞周期从静止期进入了增殖期，也可显著升高Cyclin D的表达水平以及可显著升高Notch信号关键分子Notch-1和Hes-1mRNA和蛋白的表达水平，而给予Notch信号特异性抑制剂DAPT/GSI后，则可显著降低10%BPS3d诱导的hUCMSCs增殖和显著降低Notch-1和Hes-1的表达水平。BPS中VEGF和bFGF的含量显著高于其它细胞因子，且hUCMSCs表达了VEGF和bFGF的受体。分别应用以及联合应用VEGF和bFGF的抗体，发现联合应用二者的抗体可以显著降低由10%BPS3d诱导hUCMSCs的增殖，相反添加外源性联bFGF和VEGF可显著诱导hUCMSCs的增殖和显著上调Notch-1和Hes-1mRNA和蛋白的表达水平。说明严重烧伤血清中的bFGF和VEGF是促进hUCMSCs的关键因子。结论hUCMSCs能显著加速严重烧伤大鼠创面的愈合。其次是严重烧伤患者血清中bFGF和VEGF激活了Notch信号通路进而促进hUCMSCs的存活和增殖，这也可能是hUCMSCs在严重烧伤大鼠创面微环境中存活、增殖和发挥促愈合功能的机制之一。
[Abstract]:To burn is common in peacetime, wartime trauma, the treatment effect of sudden mass burn is closely related to the maintenance of social stability. The key is to save lives in patients with severe burn wound closure as soon as possible. In recent years, the study found that mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) can effectively repair the structure and function of tissues and organs. MSCs effective repair and reconstruction of tissue and organ function damage based on, this study intends to explore the potential of MSCs for severe burn wound repair. Therefore, the isolation and culture of human umbilical cord mesenchymal stem cells (humanUmbilical Cord Mesenchymal Stem Cells, hUCMSCs) transplanted in severely burned rats, to investigate the regulatory effect and mechanism of hUCMSCs on wound healing in severe burn in rats, so as to provide new ideas and experimental basis for the clinical treatment of severe burn wounds.
Methods (1) using 3 enzyme digestion method (simple collagenase digestion method, collagenase and trypsin, collagenase and hyaluronidase digestion) were isolated and cultured hUCMSCs. immunofluorescence staining by flow cytometry / surface markers of CD105 cells, and tissue explant method, CD90, CD44, CD73 HLA-I, CD45, HLA-DR, CD31, and vWF; oil red O staining, alcian blue staining and Von Kossa calcium deposition identification of adipogenic, chondrogenic and osteogenic differentiation potential; inverted phase contrast microscope and transmission electron microscope to observe the morphology and ultrastructure of cells; MTT method to detect cell proliferation and hUCMSCs generation regulation of T cells; propidium iodide (PI) staining and flow cytometry to detect the cell cycle of different generations of hUCMSCs. (2) collected in severe burn patients serum and normal serum. 10% fetal bovine serum (10%FBS) and 10% normal control serum (10%HCS) and 10% burn The serum of patients with 3D injury (10%BPS3d) in vitro culture system hUCMSCs.AO-EB staining flow cytometry / apoptosis cells in three groups; inverted growth density was observed in the three groups cells microscope; the proliferation of MTT was detected in three groups of cells; propidium iodide (PI) staining and flow cytometry cell cycle in three groups; aging detection of three groups of beta cells -gal staining. (3) adult male Wistar 126 rats were randomly divided into sham injury group, burns group and +hUCMSCs group. GFP labeled hUCMSCs or PBS by intravenous injection of corresponding group rats.Image Pro Plus software to evaluate the healing rate of rats was small; animal in vivo fluorescence imaging system (BLI) for detection of GFP-hUCMSCs in the migration of rats; detection of wound whether PCR expression of human specific DNA; immunohistochemical staining was used to detect the wound inflammation cells infiltration, angiogenesis The number and I expression of type III collagen; laser Doppler flowmetry evaluation of wound blood flow; proinflammatory cytokines, detection of wound ELISA anti-inflammatory factor, VEGF, collagen type I, collagen type III. (4) were divided into 3 groups: 10% normal control serum (10%HCS), 3D (10% in serum of patients with burn 10%BPS3d) and 10% 3D patients with burn serum +Notch signal specific inhibitor DAPT/GSI (DAPT/GSI+BPS) or divided into 5 groups: 10%HCS, 10%BPS3d, 10%HCS+20ng/mL VEGF, 10%HCS+20ng/mL bFGF and 10%HCS+20ng/mLVEGF+20ng/mL bFGF in cultured hUCMSCs.MTT assay and trypan blue staining counting detection hUCMSCs survival and proliferation; PI staining and flow cytometry to detect the cell cycle; Western Blot detection of cyclin D (Cyclin D) expression; VEGF in serum were measured by ELISA, the content of bFGF and other cytokines; immunofluorescence staining was used to detect VEGFR1 expression of bFGFR2 and W; The expression of the key molecules of Notch signals, Notch-1 and Hes-1, was detected by estern Blot and qRT-PCR.
Results (1) the four methods can get hUCMSCs, but collagenase and hyaluronic acid from enzyme digestion and cell number, cell proliferation rate. Therefore, collagenase II + hyaluronic acid enzyme digestion method is an efficient method for hUCMSCs extraction. The positive expression of cell surface markers of mesenchymal lineage get, but not the expression of endothelial and hematopoietic markers. The passage to the 3 generation, the morphology of the cells were fusiform, showing the vortex like growth. The transmission electron microscope showed that the cells with large nuclei and irregular nucleoli, less cytoplasm. Use specific induction medium induced, the fat to cells, chondrogenic and osteogenic differentiation. The cell cycle showed that more than 80% of the cells in the stationary phase (G0 ~ G1), with the more primitive stem cell characteristics. Based on the above results to determine the isolated cells is the result of hUCMSCs.MTT shows, the generation of hUCMSCs by 1D potential Ummer, 2d-6d is the logarithmic growth phase, began to appear different degree of contact inhibition and into the platform on the 7d. In addition, hUCMSCs significantly inhibited by phytohemagglutinin (PHA) induced by allogeneic T cell proliferation. (2) 10%FBS, 10%HCS and 10%BPS3d culture hUCMSCs the morphology and structure of the culture system were normal. Neclei with orange red fluorescence in apoptotic cells or dead cells; flow cytometry results also verified the results of.MTT showed that from the 2D to the 6D, the proliferation rate and cell proliferation capacity of 10%BPS3d cells were significantly higher than the group and more than the other two groups; observation of fusion cell proliferation and growth of the cells in MTT.10%BPS3d group the proliferative phase inverted microscope (S) were significantly higher than those of the other two groups, while the percentage of senescent cells is significantly lower than the other two groups. To sum up, the serious burn serum contains some protective cells and promote cell Cell proliferation factor, therefore the hUCMSCs transplantation in the treatment of severe burn animal model is feasible. (3) the GFP-hUCMSCs transplanted into rats with severe burns, which can migrate to the blood circulation of burn wounds, significantly reduced inflammatory cell infiltration of the wound, wound significantly reduced proinflammatory cytokines IL-1, IL-6, TNF- and alpha level increased stimulation of anti-inflammatory factor IL-10 and TNF- alpha gene -6 (TSG-6) level. In addition, hUCMSCs also significantly increased the wound microvascular number and VEGF levels and significantly increased the wound in type I collagen, type III collagen ratio, and then accelerate the healing process of severe burn wounds. (4) compared with 10%HCS. 10%BPS3d can significantly promote the hUCMSCs rapid proliferation and significantly promote cell cycle hUCMSCs in proliferative stage from quiescence, the expression level can be significantly increased Cyclin and D significantly increased Notch signal key The expression level of Notch-1 and Hes-1mRNA and protein molecules, and give Notch a specific inhibitor of DAPT/GSI signal, can significantly reduce 10%BPS3d induced hUCMSCs proliferation and significantly reduce the content of VEGF and bFGF Notch-1 and the expression level of Hes-1 in.BPS was significantly higher than that of other cytokines and the expression of hUCMSCs, VEGF and bFGF were used and the receptor. The combined application of VEGF antibody and bFGF antibody, found that the combination of the two can be significantly reduced by 10%BPS3d induced hUCMSCs proliferation, instead of adding exogenous bFGF and VEGF could significantly induce hUCMSCs proliferation and significantly up-regulated expression of Notch-1 and Hes-1mRNA and protein. Severe burn serum levels of bFGF and VEGF is a key factor the promotion of hUCMSCs.
Conclusion hUCMSCs can obviously accelerate wound healing of rats with severe burns. Followed by bFGF and VEGF in serum of patients with severe burns in the activation of Notch signaling pathway and promote the survival and proliferation of hUCMSCs, which may also be hUCMSCs survival in severe burn rats wound microenvironment, proliferation and promote healing mechanism of function.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R644

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