Trop2对小鼠骨密质来源间充质干细胞体外增殖、分化影响的实验研究
发布时间：2018-09-10 16:09
【摘要】：研究背景和目的 干细胞参与胚胎发育的全过程,同时也存在于一些成体器官中,是由一群未分化的、持续自我更新的细胞组成。间充质干细胞(MSCs)最初分离白骨髓,是一群可以向骨、软骨、脂肪及肌等分化的成体干细胞,这些细胞在多次分裂之后仍具备多向分化的能力。目前MSCs已被许多的研究者,采用不同的方法从众多的器官组织中分离获得。由于这些成体干细胞至今仍没有发现其特定的表面标志物,通常只能采用一系列细胞表面标志的组合来间接进行鉴定,为其高效的体外分离与扩增带来诸多不便。因此,找到真正能体现MSCs自我更新与分化功能的“干性标志”,并阐明其调控途径将有利于对其自我更新与分化的分子机制的理解,有利于MSCs基于细胞治疗的临床应用的发展。最近的研究提示,Trop2或许是一种新的干性标志o Trop2是隶属于TASTCAD基因家族的细胞表面糖蛋白,最初被发现表达于上皮细胞,通常也过表达于一些上皮来源的肿瘤组织,与肿瘤的发生及侵袭密切相关。最近有研究发现Trop2还表达于“祖细胞样的/干细胞样的”人与小鼠的前列腺基底细胞和肝卵圆细胞上,并发现只有这些高表达Trop2的细胞才表现出自我更新和向下分化的潜能。这一现象表明,或许Trop2参与维系这些“干细胞样的”细胞的自我更新。尽管目前对Trop2激活其下游信号通路的确切机制还没有完全明确,但是其胞尾区的P工P2结合域提示我们,PI3K/AKT信号通路可能在其中起某种作用。本研究的目的是：明确是否小鼠基质干细胞也可以合成Trop2,并探讨Trop2对小鼠骨密质来源的MSC生物学作用的影响。 研究方法 1.采用骨片法分离获取小鼠MSCs,对其进行免疫表型鉴定和成脂及成骨诱导分化。 2.通过免疫荧光染色,利用激光共聚焦显微镜观察Trop2在小鼠MSCs细胞膜上的定位表达；通过Western Blot检测小鼠骨密质来源MSCs不同培养时期Trop2的表达变化。 3.建立Trop2基因敲除(Trop2 KO)小鼠模型,通过RT-PCR、Southern Blot、WesternBlot等方法分别从DNA、RNA、蛋白表达水平进行鉴定。 4.利用流式细胞术检测并比较Trop2基因敲除小鼠来源MSCs和野生型(WT)小鼠MSCs表型差异。 5.通过CCK-8和CFSE检测并比较Trop2基因敲除小鼠来源MSCs和WT小鼠MSCs细胞增殖能力的差异。 6.通过RT-PCR和real-time PCR比较Trop2基因敲除小鼠来源MSCs和WT小鼠MSCs在经过完全成脂和成骨诱导之后,成脂标志基因PPAR-γ2、Adipsin和成骨标志基因Osteocalcin、Osteopontin的定量表达差异；通过特异性的油红“0”和茜素红染色来判别Trop2基因敲除小鼠来源MSCs和WT小鼠MSCs在经过完全成脂和成骨诱导后脂滴聚集和矿物质沉积的差异。 7.通过流式细胞术明确Trop2基因敲除小鼠来源MSCs和WT小鼠MSCs进入细胞周期S期细胞百分数的差异；通过Western Blot明确在传代培养过程中Trop2基因敲除小鼠来源MSCs和WT小鼠MSCs活化AKT表达比率的差异,以及AKT通路下游Cyclin D1的表达差异；利用PI3K抑制剂LY294002处理WT小鼠MSCs后明确活化AKT表达比率的差异以及其下游Cyclin D1的表达差异,并将其与Trop2基因敲除小鼠来源MSCs进行比较,明确Trop2对AKT/Cyclin Dl通路的影响。 研究结果 1.骨片法可成功体外分离获取小鼠MSCs,所获取的MSCs不仅具备通过经典方法获取的MSCs的免疫表型特征,而且还可以向脂肪和骨诱导分化,是同质性比较一致的间充质干细胞。 2.通过激光共聚焦显微镜首次明确观察到MSCs细胞膜上Trop2的定位表达,通过Western Blot发现Trop2的表达量随MSCs体外分裂次数的增加而减少。 3.通过RT-PCR、Southern Blot、Western Blot明确鉴定了Trop2基因敲除小鼠的构建成功。 4.通过流式细胞术、RT-PCR、real-time PCR、Western Blot等检测发现,Trop2基因敲除小鼠和WT小鼠来源的MSCs在以下方面存在明显差异： (1)通过表型观察发现：与WT MSCs相比,KO MSCs在第一代时同质性较差,CD45阳性细胞(造血系细胞)混杂较多；随着传代至第4代后其同质性渐趋一致。 (2)CCK-8和CFSE检测发现：与WT MSCs相比,KO MSCs的增殖速率明显降低,细胞倍增时间明显延长。 (3)成脂与成骨诱导实验发现：与WT MSCs相比,KO MSCs在相同诱导之后脂滴聚集减少,矿物质沉积减少；real-time PCR检测发现经完全诱导后WT MSCs较来源于KO MSCs的脂肪标志基因Adipsin、PPAR-γ2和骨标志基因Osteocalcin、Osteopontin分别高出400%、500%、350%和100%。 (4)细胞周期：与WT MSCs相比,KO MSCs进入细胞周期S期的细胞比率明显降低。 (5) AKT/Cyclin D1:与WT MSCs相比,KO MSCs活化AKT的表达比率明显降低,AKT信号通路下游细胞周期关键节点蛋白Cyclin D1的表达明显降低；但活化AKT和Cyclin D1表达降低程度不及经LY294002阻断AKT通路所引起的降低程度大。 结论 1. Trop2定位表达在MSCs细胞膜。 2. Trop2的缺失可以减弱MSCs的增殖与分化能力。 3. Trop2的存在有利于维持AKT的活化状态,继而保护其下游的Cyclin D1不被降解而失活；Trop2的缺失部分抑制了AKT/Cyclin D1通路的活化。
[Abstract]:Research background and purpose
Mesenchymal stem cells (MSCs) originally isolated from white bone marrow are a group of adult stem cells that can differentiate into bone, cartilage, fat and muscle cells. These cells remain abundant after multiple divisions. At present, MSCs have been isolated from many organs and tissues by different methods by many researchers. Since these adult stem cells have not yet found their specific surface markers, they can only be identified indirectly by a series of combination of cell surface markers for efficient in vitro isolation and identification. Therefore, finding a "dry marker" that truly reflects the function of self-renewal and differentiation of MSCs and clarifying its regulatory pathways will be conducive to understanding the molecular mechanism of self-renewal and differentiation of MSCs and the development of clinical application of cell-based therapy. The dry marker o Trop2 is a cell surface glycoprotein belonging to the TASTCAD gene family. It was initially found to be expressed in epithelial cells and is often over-expressed in some epithelial-derived tumor tissues, which is closely related to the occurrence and invasion of tumors. This suggests that Trop2 may be involved in maintaining the self-renewal of these "stem-like" cells, although the exact mechanism by which Trop2 activates its downstream signaling pathway is not yet known. The aim of this study was to determine whether mouse mesenchymal stem cells could also synthesize Trop2 and to investigate the effect of Trop2 on the biological function of MSC derived from bone compact matter in mice.
research method
1. MSCs of mice were isolated and harvested by bone slice method. Immunophenotypic identification, adipogenesis and osteogenic differentiation were carried out.
2. The localized expression of Trop2 on MSCs cell membrane was observed by immunofluorescence staining and laser confocal microscopy. The expression of Trop2 in MSCs derived from mouse bone compact was detected by Western Blot.
3. The Trop2 knockout (Trop2 KO) mouse model was established and identified by RT-PCR, Southern Blot and Western Blot.
4. The phenotypic differences of MSCs from Trop2 knockout mice and wild type (WT) mice were detected and compared by flow cytometry.
5. The proliferation of MSCs from Trop2 knockout mice and WT mice was detected by CCK-8 and CFSE.
6. The quantitative expression of PPAR-gamma 2, Adipsin, Osteocalcin and Osteopontin in MSCs derived from Trop2 knockout mice and MSCs derived from WT mice were compared by RT-PCR and real-time PCR after complete adipogenesis and osteogenesis induction. Differences in lipid droplet aggregation and mineral deposition between MSCs from knockout mice and MSCs from WT mice after complete adipogenesis and osteogenesis induction.
7. The percentage of MSCs from Trop2 knockout mice and that from WT mice entering S phase of cell cycle was determined by flow cytometry; the percentage of activated AKT in MSCs from Trop2 knockout mice and MSCs from WT mice was determined by Western Blot; and the expression of Cyclin D1 in downstream of AKT pathway was also determined by Western Blot. After treatment of MSCs in WT mice with PI3K inhibitor LY294002, the expression ratio of activated AKT and the expression of Cyclin D1 in the downstream of MSCs were determined, and the effects of Trop2 on the AKT/Cyclin Dl pathway were compared with that of MSCs from Trop2 knockout mice.
Research results
1. MSCs of mice can be successfully isolated and obtained by bone slice method in vitro. The obtained MSCs not only have the immunophenotypic characteristics of MSCs obtained by classical methods, but also can be induced to differentiate into adipose tissue and bone. MSCs are homogeneous mesenchymal stem cells.
2. The localized expression of Trop 2 on MSCs cell membrane was observed by confocal laser microscopy for the first time. The expression of Trop 2 decreased with the increase of the number of division of MSCs in vitro.
3. through RT-PCR, Southern Blot and Western Blot, the success of construction of Trop2 knockout mice was clearly identified.
4. Flow cytometry, RT-PCR, real-time PCR, Western Blot and other tests showed that there were significant differences between MSCs from Trop2 knockout mice and WT mice in the following aspects:
(1) Compared with WT-MSCs, KO-MSCs had poorer homogeneity in the first generation and more CD45 positive cells (hematopoietic cells) were mixed in the first generation.
(2) CCK-8 and CFSE assay showed that the proliferation rate of KO-MSCs was significantly lower and the doubling time was significantly longer than that of WT-MSCs.
(3) Compared with WT MSCs, KO MSCs showed less lipid droplet aggregation and less mineral deposition after the same induction. Real-time PCR detection showed that WT MSCs were 400%, 500%, 350% higher than Adipsin, PPAR-gamma 2 and Osteocalcin, Osteopontin, respectively. % and 100%.
(4) cell cycle: compared with WT MSCs, the ratio of KO MSCs into cell cycle S phase decreased significantly.
(5) AKT/Cyclin D1: Compared with WT MSCs, the expression ratio of AKT activated by KO MSCs was significantly decreased, and the expression of Cyclin D1, the key node protein downstream of AKT signaling pathway, was significantly decreased, but the expression of AKT and Cyclin D1 decreased less than that of AKT blocked by LY294002.
conclusion
1. Trop2 was localized in MSCs cell membrane.
The deletion of 2. Trop2 can attenuate the proliferation and differentiation of MSCs.
3. The presence of Trop2 helps to maintain the activation of AKT, and consequently protects its downstream Cyclin D1 from being degraded and inactivated. The deletion of Trop2 partially inhibits the activation of AKT/Cyclin D1 pathway.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329
本文编号：2234947
[Abstract]:Research background and purpose
Mesenchymal stem cells (MSCs) originally isolated from white bone marrow are a group of adult stem cells that can differentiate into bone, cartilage, fat and muscle cells. These cells remain abundant after multiple divisions. At present, MSCs have been isolated from many organs and tissues by different methods by many researchers. Since these adult stem cells have not yet found their specific surface markers, they can only be identified indirectly by a series of combination of cell surface markers for efficient in vitro isolation and identification. Therefore, finding a "dry marker" that truly reflects the function of self-renewal and differentiation of MSCs and clarifying its regulatory pathways will be conducive to understanding the molecular mechanism of self-renewal and differentiation of MSCs and the development of clinical application of cell-based therapy. The dry marker o Trop2 is a cell surface glycoprotein belonging to the TASTCAD gene family. It was initially found to be expressed in epithelial cells and is often over-expressed in some epithelial-derived tumor tissues, which is closely related to the occurrence and invasion of tumors. This suggests that Trop2 may be involved in maintaining the self-renewal of these "stem-like" cells, although the exact mechanism by which Trop2 activates its downstream signaling pathway is not yet known. The aim of this study was to determine whether mouse mesenchymal stem cells could also synthesize Trop2 and to investigate the effect of Trop2 on the biological function of MSC derived from bone compact matter in mice.
research method
1. MSCs of mice were isolated and harvested by bone slice method. Immunophenotypic identification, adipogenesis and osteogenic differentiation were carried out.
2. The localized expression of Trop2 on MSCs cell membrane was observed by immunofluorescence staining and laser confocal microscopy. The expression of Trop2 in MSCs derived from mouse bone compact was detected by Western Blot.
3. The Trop2 knockout (Trop2 KO) mouse model was established and identified by RT-PCR, Southern Blot and Western Blot.
4. The phenotypic differences of MSCs from Trop2 knockout mice and wild type (WT) mice were detected and compared by flow cytometry.
5. The proliferation of MSCs from Trop2 knockout mice and WT mice was detected by CCK-8 and CFSE.
6. The quantitative expression of PPAR-gamma 2, Adipsin, Osteocalcin and Osteopontin in MSCs derived from Trop2 knockout mice and MSCs derived from WT mice were compared by RT-PCR and real-time PCR after complete adipogenesis and osteogenesis induction. Differences in lipid droplet aggregation and mineral deposition between MSCs from knockout mice and MSCs from WT mice after complete adipogenesis and osteogenesis induction.
7. The percentage of MSCs from Trop2 knockout mice and that from WT mice entering S phase of cell cycle was determined by flow cytometry; the percentage of activated AKT in MSCs from Trop2 knockout mice and MSCs from WT mice was determined by Western Blot; and the expression of Cyclin D1 in downstream of AKT pathway was also determined by Western Blot. After treatment of MSCs in WT mice with PI3K inhibitor LY294002, the expression ratio of activated AKT and the expression of Cyclin D1 in the downstream of MSCs were determined, and the effects of Trop2 on the AKT/Cyclin Dl pathway were compared with that of MSCs from Trop2 knockout mice.
Research results
1. MSCs of mice can be successfully isolated and obtained by bone slice method in vitro. The obtained MSCs not only have the immunophenotypic characteristics of MSCs obtained by classical methods, but also can be induced to differentiate into adipose tissue and bone. MSCs are homogeneous mesenchymal stem cells.
2. The localized expression of Trop 2 on MSCs cell membrane was observed by confocal laser microscopy for the first time. The expression of Trop 2 decreased with the increase of the number of division of MSCs in vitro.
3. through RT-PCR, Southern Blot and Western Blot, the success of construction of Trop2 knockout mice was clearly identified.
4. Flow cytometry, RT-PCR, real-time PCR, Western Blot and other tests showed that there were significant differences between MSCs from Trop2 knockout mice and WT mice in the following aspects:
(1) Compared with WT-MSCs, KO-MSCs had poorer homogeneity in the first generation and more CD45 positive cells (hematopoietic cells) were mixed in the first generation.
(2) CCK-8 and CFSE assay showed that the proliferation rate of KO-MSCs was significantly lower and the doubling time was significantly longer than that of WT-MSCs.
(3) Compared with WT MSCs, KO MSCs showed less lipid droplet aggregation and less mineral deposition after the same induction. Real-time PCR detection showed that WT MSCs were 400%, 500%, 350% higher than Adipsin, PPAR-gamma 2 and Osteocalcin, Osteopontin, respectively. % and 100%.
(4) cell cycle: compared with WT MSCs, the ratio of KO MSCs into cell cycle S phase decreased significantly.
(5) AKT/Cyclin D1: Compared with WT MSCs, the expression ratio of AKT activated by KO MSCs was significantly decreased, and the expression of Cyclin D1, the key node protein downstream of AKT signaling pathway, was significantly decreased, but the expression of AKT and Cyclin D1 decreased less than that of AKT blocked by LY294002.
conclusion
1. Trop2 was localized in MSCs cell membrane.
The deletion of 2. Trop2 can attenuate the proliferation and differentiation of MSCs.
3. The presence of Trop2 helps to maintain the activation of AKT, and consequently protects its downstream Cyclin D1 from being degraded and inactivated. The deletion of Trop2 partially inhibits the activation of AKT/Cyclin D1 pathway.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329
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