人脐带间充质干细胞TLR4信号通路及与BV-2细胞共培养研究
发布时间：2018-07-29 06:55
【摘要】：研究背景 新生儿脑损伤目前尚无及时有效治疗手段，MSCs是目前最有希望的治疗新生儿脑损伤的细胞治疗手段。但有关MSCs治疗脑损伤仍处于研究阶段。 目的 体外分离、鉴定人脐带间充质干细胞（hWJ-MSCs），观察hWJ-MSCs在体外的扩增效率、不同扩增代数之间的生长差异，观察hWJ-MSCs在体外模拟炎性环境下的生物学表型改变，并将hWJ-MSCs与中枢神经系统主要的免疫细胞-神经小胶质细胞在炎症模型环境下进行共培养，观察两种细胞之间的相互调控作用，为进一步研究MSCs的颅内移植治疗提供研究基础。 方法 为以上目的，本研究分为以下3部分。 1. hWJ-MSCs的分离、体外扩增及鉴定 收集北京军区总医院正常足月剖宫产新生儿脐带组织5根。所有脐带组织在离体后立即放置入无菌环境保存，并在1-6h内处理完毕以保证分离细胞活力。脐带组织在无菌环境下剔除脐动、静脉，物理方法剪碎，随后用胰酶+胶原酶-Ⅱ联合消化法及贴壁法分离纯化并体外扩增hWJ-MSCs。采用流式细胞术对hWJ-MSCs细胞表面标记CD14、CD31、CD73、CD105、CD90、HLA-ABC、HLA-DR鉴定。并采用体外成软骨、成脂肪分化试剂盒观察细胞分化潜力。将两种分离方法所得细胞分别传代，观察细胞形态及细胞周期变化。 2.观察toll-like receptor4（TLRs）在hWJ-MSCs上的表达及其信号通路对hWJ-MSCs生物学功能的影响 采用流式细胞术及qRT-PCR方法检测TLR4在hWJ-MSCs上的表达情况。采用TLR4激活剂-LPS体外刺激hWJ-MSCs，在LPS刺激不同时间点观察hWJ-MSCs细胞形态变化、细胞增殖及生长周期变化。收集不同LPS刺激时间（24h、48h、72h）后的hWJ-MSCs细胞，采用trizol试剂提取总RNA，采用逆转录试剂盒及SYRB Green试剂盒检测hWJ-MSCs的细胞因子（IL-1β、IL-1α、IL-2、IL-4、IL-6、IL-8、IL-10、IL-12、IL-13、indoleamine2,3-dioxygenase [IDO]-1、TNF-α、IFN-γ、MMP-2和MMP-9）在LPS刺激条件下的表达改变情况。收集细胞同时收集细胞培养上清，观察细胞因子蛋白表达水平差异，进一步确认qRT-PCR结果。 3. hWJ-MSCs与小鼠源小胶质神经细胞系BV-2体外共培养研究 将体外分离所得的hWJ-MSCs与BV-2细胞在体外按不同比例进行共培养，观察两种细胞的共培养体系在LPS+/LPS-下不同时间点两种细胞各自的生物学变化。采用qRT-PCR检测共培养条件下hWJ-MSCs表达IL-1β、IL-6、 IL-8及MMP-2差异；同时检测BV-2细胞M2细胞表型基因Arginase1、Arginase2、Mrc1和Ym1的表达改变，观察BV-2选择性激活（M2型）标记是否受到hWJ-MSCs的影响。使用NO检测试剂盒检测共培养体系中BV-2细胞NO分泌差异，观察直接接触共培养时hWJ-MSCs对LPS诱导BV-2细胞向M2型转化的影响。 结果 1.成功分离并鉴定hWJ-MSCs 脐带组织在无菌条件下经剪成碎片贴壁培养皿生长或进一步消化获得单细胞悬液接种培养瓶，均可见长梭形类似成纤维细胞样细胞生长，细胞经扩增纯化，体外最多可成功扩增至20代。在20代之前细胞细胞可保持原代生长形态。流式细胞术鉴定细胞表面标记结果显示所得细胞CD14、CD31、HLA-DR阴性， CD73、CD105、CD90、HLA-ABC阳性，体外分化实验提示所得细胞体外诱导成软骨、成脂肪分化成功。依据贴壁生长形态、细胞表面标记及分化潜力，符合国际细胞疗法协会有关间充质干细胞的定义。提示采用贴壁法、胰酶-胶原酶联合消化法均成功获得脐带间充质干细胞。所得细胞在3-7代间经PI染色法观察细胞周期，细胞均处于G0/G1期，极少数细胞处于增殖分裂期，具有干细胞特性。未见死亡、晚期凋亡细胞。 2.鉴定TLR4为hWJ-MSCs表面的功能性受体 流式细胞术和qRT-PCR分析揭示TLR4在hWJ-MSCs上的表达。LPS体外刺激hWJ-MSCs成功引发细胞应激性反应，检测TLR4信号通路相关炎性因子（IL-1β、IL-1α、IL-6、IL-8）基因表达差异，提示所检测炎性因子基因均在刺激72h后表达明显上调，但IL-12在LPS刺激条件后均表现为明显的下调，未检测到IL-2、IL-4、IL-10、IL-13、TNF-α、IFN-γ的表达。对MMP-2和MMP-9基因表达情况的检测提示MMP-2在LPS刺激后72h出现明显上调，但MMP-9表达在LPS刺激后出现短暂（48h）的下调，，但在72h后，MMP-9表达上升至与阴性对照大致持平的水平。此外，MSC免疫抑制分子IDO1、IDO-2、IFN-β和Cox2基因表达情况的检测结果显示：IDO1和IFN-β在LPS刺激条件下出现明显上调，IDO1表达上调在72h内上调呈现为双峰，在48h检测表达为低谷。Cox2的表达同样受到LPS的诱导，但仅在LPS刺激72h后出现明显的上调；此外，在我们观察的3个时间点未发现IDO-2表达变化。该结果显示LPS可修饰hWJ-MSCs的免疫抑制功能。 3.hWJ-MSCs与BV-2体外共培养时的相互调节作用 在体外共培养条件下，hWJ-MSCs和BV-2两种细胞均为贴壁生长。在LPS刺激条件下，hWJ-MSCs可显著减弱LPS对BV-2的刺激作用；与hWJ-MSCs共培养时：如无LPS刺激，BV-2仅分泌极低量的NO；但LPS刺激条件下，hWJ-MSCs对BV-2分泌NO有明显的抑制作用，且与细胞共培养比例有关；qRT-PCR分析发现：当两种细胞比例维持在1:1时，hWJ-MSCs可明显上调BV-2细胞的M2细胞表型标记Arginase1表达；BV-2与hWJ-MSCs共培养条件下，hWJ-MSCs表达IL-1β、IL-1α、IL-6、IL-8和MMP-2明显受到LPS、BV-2细胞以及共培养细胞比例三种因素的影响：其中IL-1β表达表现为仅受到LPS的影响（LPS-上调，LPS+抑制）；MMP-2仅在LPS-共培养72h后出现上调，其他情况下均表现为抑制状态；IL-6仅在LPS+共培养24h内上调，其他时间、环境下均表现为下调；而IL-8的表达影响主要与时间有关，在共培养24h内（LPS-/LPS+）均表现为上调，而在其他时间内表现为下调。与共培养中BV-2细胞相比，hWJ-MSCs受到的调节更为复杂，与多种环境因素有关。 结论 1.贴壁法以及胰酶-胶原酶Ⅱ联合消化法均能成功分离hWJ-MSCs，体外标准培养条件下，可体外传代20代，提示hWJ-MSCs为有限传代的MSCs； 2.基因及蛋白水平检测提示MSCs表达TLR4，经LPS体外刺激模型试验进一步提示TLR4在hWJ-MSCs为一功能性受体，但与其他成体组织来源MSCs比较，hWJ-MSCs对LPS的刺激反应显迟钝，需要LPS刺激72小时才能出现一系列基因表达上调（IL-1β、IL-1α、IL-6、IL-8、IDO1、MMP-2、TLR4、CD14）和下调（I L-12、MMP-9），且LPS通过上调IDO1、MMP-2可调节hWJ-MSC免疫功能、迁移能力； 3. hWJ-MSCs可诱导BV-2向M2型转化，这一过程需要细胞间直接接触；体外hWJ-MSCs与BV-2存在相互调节作用。
[Abstract]:Research background
There is no timely and effective treatment for brain injury in newborns. MSCs is the most promising treatment for brain injury in newborns. However, MSCs treatment of brain injury is still at the stage of study.
objective
In vitro isolation and identification of human umbilical cord mesenchymal stem cells (hWJ-MSCs), the amplification efficiency of hWJ-MSCs in vitro, the growth difference between different amplification algebras, and the biological phenotypic changes of hWJ-MSCs in the simulated inflammatory environment were observed, and the main immune cell neuroglia cells of the central nervous system and the central nervous system were in the inflammatory model. Co culture was carried out in the environment to observe the interaction between the two cells, and provide a basis for further study of MSCs's intracranial transplantation.
Method
For the above purpose, this study is divided into the following 3 parts.
Isolation, in vitro amplification and identification of 1. hWJ-MSCs
5 neonates with normal term caesarean section of General Hospital of Beijing Military Region were collected. All umbilical cord tissues were stored in aseptic environment immediately after in vitro and processed in 1-6h to ensure the vitality of the isolated cells. The umbilical tissue was removed from the umbilical cord tissue under the aseptic environment, the vein, and the physical methods were cut down, and then combined with pancreatin and collagenase - II. The separation and purification and in vitro amplification of hWJ-MSCs. by flow cytometry were used to identify the surface of hWJ-MSCs cells by flow cytometry on CD14, CD31, CD73, CD105, CD90, HLA-ABC, HLA-DR. The cell differentiation potential was observed by the chondrogenic and adipose differentiation kit in vitro. The cell morphology and cell were observed by two separate methods. Periodic changes.
2. to observe the expression of Toll-like receptor4 (TLRs) on hWJ-MSCs and the effect of signal transduction pathway on hWJ-MSCs biological function.
The expression of TLR4 on hWJ-MSCs was detected by flow cytometry and qRT-PCR. TLR4 activator -LPS was used to stimulate hWJ-MSCs in vitro. The morphological changes, cell proliferation and growth cycle of hWJ-MSCs cells were observed at LPS at different time points. Take the total RNA, use the reverse transcriptase kit and the SYRB Green kit to detect the cell factors of hWJ-MSCs (IL-1 beta, IL-1 alpha, IL-2, IL-4, IL-6, IL-8, and IL-10) in the stimulus conditions. Collect cells and collect cell culture supernatant and observe cells The level of factor protein expression was further confirmed by qRT-PCR results.
Co culture of 3. hWJ-MSCs and mouse microglia cell line BV-2 in vitro
The hWJ-MSCs and BV-2 cells isolated in vitro were co cultured in different proportion in vitro, and the biological changes of two cells in the co culture system of two cells at different time points in LPS+/LPS- were observed. The difference of hWJ-MSCs expression of IL-1 beta, IL-6, IL-8 and MMP-2 under the co culture condition of qRT-PCR, and the M2 of BV-2 cell M2 were detected. The expression of cell phenotypic gene Arginase1, Arginase2, Mrc1 and Ym1 were changed, and the effect of hWJ-MSCs was observed by the selective activation of BV-2 (M2 type) markers. The difference of BV-2 cell NO secretion in the co culture system was detected by NO detection kit, and the effect of hWJ-MSCs on LPS induced transformation was observed when co culture was directly exposed to co culture.
Result
1. successful separation and identification of hWJ-MSCs
Under the aseptic condition, the cell growth of a spindle like cell like cell can be grown by the growing or further digestion of a single cell suspension culture bottle under the aseptic condition. The cells can be amplified and purify to 20 generations in vitro. The cell cells can maintain the original growth form before the 20 generation. Cell surface labeling results showed that the cells CD14, CD31 and HLA-DR were negative, CD73, CD105, CD90, HLA-ABC positive. The differentiation experiment in vitro suggested that the cells were induced into cartilage in vitro, and the adipose differentiation was successful. It was suggested that the umbilical cord mesenchymal stem cells were successfully obtained by the adherence method and the combined digestion of pancreatin collagenase and collagenase. The cell cycle was observed by PI staining between the 3-7 generations. The cells were in the G0/G1 stage, and a few cells were in the stage of proliferation and division, and they had the characteristics of stem cells. No death and late apoptotic cells were not found.
2. identification of TLR4 as a functional receptor on the surface of hWJ-MSCs
Flow cytometry and qRT-PCR analysis revealed that the expression of TLR4 on hWJ-MSCs stimulated hWJ-MSCs in vitro to trigger the cell stress response, and to detect the difference in the expression of the TLR4 signaling pathway related inflammatory factors (IL-1 beta, IL-1 a, IL-6, IL-8), suggesting that the detected inflammatory factor genes were obviously up-regulated after the stimulation of 72h, but IL-12 was in the stimulus bar. The expression of IL-2, IL-4, IL-10, IL-13, TNF-, and IFN- y was not detected. The detection of the expression of MMP-2 and MMP-9 gene indicated that MMP-2 was obviously up-regulated after LPS stimulation, but the MMP-9 expression was down down after the stimulation, but the expression rose to the same level as the negative control. In addition, the detection results of the expression of IDO1, IDO-2, IFN- beta and Cox2 genes of MSC immunosuppressive molecules showed that IDO1 and IFN- beta were up regulated under the stimulus of LPS, and the up regulation of IDO1 expression was in Shuangfeng. The expression of IDO-2 was not detected at the 3 time points observed. The results showed that LPS could modify the immunosuppressive function of hWJ-MSCs.
Interaction between 3.hWJ-MSCs and BV-2 co cultured in vitro
Under the conditions of co culture in vitro, both hWJ-MSCs and BV-2 cells were adherent growth. Under the LPS stimulation, hWJ-MSCs could significantly weaken the stimulation of LPS to BV-2; when co cultured with hWJ-MSCs, BV-2 only secreted a very low amount of NO without LPS stimulation. QRT-PCR analysis showed that when the proportion of two cells was maintained at 1:1, hWJ-MSCs could obviously increase the expression of M2 cell phenotypic marker Arginase1 expression in BV-2 cells; BV-2 and hWJ-MSCs co culture conditions, hWJ-MSCs expressed IL-1 beta, IL-1 alpha, IL-6, and three kinds of co culture cells. Influence of factors: IL-1 beta expression was only affected by LPS (LPS- up, LPS+ inhibition); MMP-2 was up-regulated only after LPS- co culture 72h, and all other cases were inhibited; IL-6 was up only in LPS+ co culture 24h, and the other time, under the environment, was down-regulation, and IL-8 expression was mainly related to time. Both in co cultured 24h (LPS-/LPS+) showed up regulation and down regulated at other times. Compared with BV-2 cells in co culture, the regulation of hWJ-MSCs was more complex and related to a variety of environmental factors.
conclusion
1. adherent wall method and pancreatin collagenase II combined digestion method can successfully separate hWJ-MSCs. Under the standard culture conditions, the 20 generation can be passed to the 20 generation, suggesting that hWJ-MSCs is the MSCs of the limited passages.
The detection of 2. gene and protein level suggested that MSCs expressed TLR4. The stimulation model test of LPS in vitro further hinted that TLR4 was a functional receptor in hWJ-MSCs, but compared with other adult tissue sources MSCs, hWJ-MSCs had a slow response to LPS, and a series of gene expression up-regulated (IL-1 beta, IL-1 alpha, IL-6, and IL-6) needed LPS stimulation for 72 hours. IDO1, MMP-2, TLR4, CD14) and down regulation (I L-12, MMP-9), and LPS can regulate the immune function and migration ability of the hWJ-MSC by upregulating IDO1.
3. hWJ-MSCs can induce BV-2 to transform into M2, which requires direct contact between cells. HWJ-MSCs and BV-2 interact in vitro.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R722.1
本文编号：2151881
[Abstract]:Research background
There is no timely and effective treatment for brain injury in newborns. MSCs is the most promising treatment for brain injury in newborns. However, MSCs treatment of brain injury is still at the stage of study.
objective
In vitro isolation and identification of human umbilical cord mesenchymal stem cells (hWJ-MSCs), the amplification efficiency of hWJ-MSCs in vitro, the growth difference between different amplification algebras, and the biological phenotypic changes of hWJ-MSCs in the simulated inflammatory environment were observed, and the main immune cell neuroglia cells of the central nervous system and the central nervous system were in the inflammatory model. Co culture was carried out in the environment to observe the interaction between the two cells, and provide a basis for further study of MSCs's intracranial transplantation.
Method
For the above purpose, this study is divided into the following 3 parts.
Isolation, in vitro amplification and identification of 1. hWJ-MSCs
5 neonates with normal term caesarean section of General Hospital of Beijing Military Region were collected. All umbilical cord tissues were stored in aseptic environment immediately after in vitro and processed in 1-6h to ensure the vitality of the isolated cells. The umbilical tissue was removed from the umbilical cord tissue under the aseptic environment, the vein, and the physical methods were cut down, and then combined with pancreatin and collagenase - II. The separation and purification and in vitro amplification of hWJ-MSCs. by flow cytometry were used to identify the surface of hWJ-MSCs cells by flow cytometry on CD14, CD31, CD73, CD105, CD90, HLA-ABC, HLA-DR. The cell differentiation potential was observed by the chondrogenic and adipose differentiation kit in vitro. The cell morphology and cell were observed by two separate methods. Periodic changes.
2. to observe the expression of Toll-like receptor4 (TLRs) on hWJ-MSCs and the effect of signal transduction pathway on hWJ-MSCs biological function.
The expression of TLR4 on hWJ-MSCs was detected by flow cytometry and qRT-PCR. TLR4 activator -LPS was used to stimulate hWJ-MSCs in vitro. The morphological changes, cell proliferation and growth cycle of hWJ-MSCs cells were observed at LPS at different time points. Take the total RNA, use the reverse transcriptase kit and the SYRB Green kit to detect the cell factors of hWJ-MSCs (IL-1 beta, IL-1 alpha, IL-2, IL-4, IL-6, IL-8, and IL-10) in the stimulus conditions. Collect cells and collect cell culture supernatant and observe cells The level of factor protein expression was further confirmed by qRT-PCR results.
Co culture of 3. hWJ-MSCs and mouse microglia cell line BV-2 in vitro
The hWJ-MSCs and BV-2 cells isolated in vitro were co cultured in different proportion in vitro, and the biological changes of two cells in the co culture system of two cells at different time points in LPS+/LPS- were observed. The difference of hWJ-MSCs expression of IL-1 beta, IL-6, IL-8 and MMP-2 under the co culture condition of qRT-PCR, and the M2 of BV-2 cell M2 were detected. The expression of cell phenotypic gene Arginase1, Arginase2, Mrc1 and Ym1 were changed, and the effect of hWJ-MSCs was observed by the selective activation of BV-2 (M2 type) markers. The difference of BV-2 cell NO secretion in the co culture system was detected by NO detection kit, and the effect of hWJ-MSCs on LPS induced transformation was observed when co culture was directly exposed to co culture.
Result
1. successful separation and identification of hWJ-MSCs
Under the aseptic condition, the cell growth of a spindle like cell like cell can be grown by the growing or further digestion of a single cell suspension culture bottle under the aseptic condition. The cells can be amplified and purify to 20 generations in vitro. The cell cells can maintain the original growth form before the 20 generation. Cell surface labeling results showed that the cells CD14, CD31 and HLA-DR were negative, CD73, CD105, CD90, HLA-ABC positive. The differentiation experiment in vitro suggested that the cells were induced into cartilage in vitro, and the adipose differentiation was successful. It was suggested that the umbilical cord mesenchymal stem cells were successfully obtained by the adherence method and the combined digestion of pancreatin collagenase and collagenase. The cell cycle was observed by PI staining between the 3-7 generations. The cells were in the G0/G1 stage, and a few cells were in the stage of proliferation and division, and they had the characteristics of stem cells. No death and late apoptotic cells were not found.
2. identification of TLR4 as a functional receptor on the surface of hWJ-MSCs
Flow cytometry and qRT-PCR analysis revealed that the expression of TLR4 on hWJ-MSCs stimulated hWJ-MSCs in vitro to trigger the cell stress response, and to detect the difference in the expression of the TLR4 signaling pathway related inflammatory factors (IL-1 beta, IL-1 a, IL-6, IL-8), suggesting that the detected inflammatory factor genes were obviously up-regulated after the stimulation of 72h, but IL-12 was in the stimulus bar. The expression of IL-2, IL-4, IL-10, IL-13, TNF-, and IFN- y was not detected. The detection of the expression of MMP-2 and MMP-9 gene indicated that MMP-2 was obviously up-regulated after LPS stimulation, but the MMP-9 expression was down down after the stimulation, but the expression rose to the same level as the negative control. In addition, the detection results of the expression of IDO1, IDO-2, IFN- beta and Cox2 genes of MSC immunosuppressive molecules showed that IDO1 and IFN- beta were up regulated under the stimulus of LPS, and the up regulation of IDO1 expression was in Shuangfeng. The expression of IDO-2 was not detected at the 3 time points observed. The results showed that LPS could modify the immunosuppressive function of hWJ-MSCs.
Interaction between 3.hWJ-MSCs and BV-2 co cultured in vitro
Under the conditions of co culture in vitro, both hWJ-MSCs and BV-2 cells were adherent growth. Under the LPS stimulation, hWJ-MSCs could significantly weaken the stimulation of LPS to BV-2; when co cultured with hWJ-MSCs, BV-2 only secreted a very low amount of NO without LPS stimulation. QRT-PCR analysis showed that when the proportion of two cells was maintained at 1:1, hWJ-MSCs could obviously increase the expression of M2 cell phenotypic marker Arginase1 expression in BV-2 cells; BV-2 and hWJ-MSCs co culture conditions, hWJ-MSCs expressed IL-1 beta, IL-1 alpha, IL-6, and three kinds of co culture cells. Influence of factors: IL-1 beta expression was only affected by LPS (LPS- up, LPS+ inhibition); MMP-2 was up-regulated only after LPS- co culture 72h, and all other cases were inhibited; IL-6 was up only in LPS+ co culture 24h, and the other time, under the environment, was down-regulation, and IL-8 expression was mainly related to time. Both in co cultured 24h (LPS-/LPS+) showed up regulation and down regulated at other times. Compared with BV-2 cells in co culture, the regulation of hWJ-MSCs was more complex and related to a variety of environmental factors.
conclusion
1. adherent wall method and pancreatin collagenase II combined digestion method can successfully separate hWJ-MSCs. Under the standard culture conditions, the 20 generation can be passed to the 20 generation, suggesting that hWJ-MSCs is the MSCs of the limited passages.
The detection of 2. gene and protein level suggested that MSCs expressed TLR4. The stimulation model test of LPS in vitro further hinted that TLR4 was a functional receptor in hWJ-MSCs, but compared with other adult tissue sources MSCs, hWJ-MSCs had a slow response to LPS, and a series of gene expression up-regulated (IL-1 beta, IL-1 alpha, IL-6, and IL-6) needed LPS stimulation for 72 hours. IDO1, MMP-2, TLR4, CD14) and down regulation (I L-12, MMP-9), and LPS can regulate the immune function and migration ability of the hWJ-MSC by upregulating IDO1.
3. hWJ-MSCs can induce BV-2 to transform into M2, which requires direct contact between cells. HWJ-MSCs and BV-2 interact in vitro.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R722.1
【共引文献】
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