Trop2對(duì)小鼠骨密質(zhì)來(lái)源間充質(zhì)干細(xì)胞體外增殖、分化影響的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-09-10 16:09
【摘要】：研究背景和目的 干細(xì)胞參與胚胎發(fā)育的全過(guò)程,同時(shí)也存在于一些成體器官中,是由一群未分化的、持續(xù)自我更新的細(xì)胞組成。間充質(zhì)干細(xì)胞(MSCs)最初分離白骨髓,是一群可以向骨、軟骨、脂肪及肌等分化的成體干細(xì)胞,這些細(xì)胞在多次分裂之后仍具備多向分化的能力。目前MSCs已被許多的研究者,采用不同的方法從眾多的器官組織中分離獲得。由于這些成體干細(xì)胞至今仍沒(méi)有發(fā)現(xiàn)其特定的表面標(biāo)志物,通常只能采用一系列細(xì)胞表面標(biāo)志的組合來(lái)間接進(jìn)行鑒定,為其高效的體外分離與擴(kuò)增帶來(lái)諸多不便。因此,找到真正能體現(xiàn)MSCs自我更新與分化功能的“干性標(biāo)志”,并闡明其調(diào)控途徑將有利于對(duì)其自我更新與分化的分子機(jī)制的理解,有利于MSCs基于細(xì)胞治療的臨床應(yīng)用的發(fā)展。最近的研究提示,Trop2或許是一種新的干性標(biāo)志o Trop2是隸屬于TASTCAD基因家族的細(xì)胞表面糖蛋白,最初被發(fā)現(xiàn)表達(dá)于上皮細(xì)胞,通常也過(guò)表達(dá)于一些上皮來(lái)源的腫瘤組織,與腫瘤的發(fā)生及侵襲密切相關(guān)。最近有研究發(fā)現(xiàn)Trop2還表達(dá)于“祖細(xì)胞樣的/干細(xì)胞樣的”人與小鼠的前列腺基底細(xì)胞和肝卵圓細(xì)胞上,并發(fā)現(xiàn)只有這些高表達(dá)Trop2的細(xì)胞才表現(xiàn)出自我更新和向下分化的潛能。這一現(xiàn)象表明,或許Trop2參與維系這些“干細(xì)胞樣的”細(xì)胞的自我更新。盡管目前對(duì)Trop2激活其下游信號(hào)通路的確切機(jī)制還沒(méi)有完全明確,但是其胞尾區(qū)的P工P2結(jié)合域提示我們,PI3K/AKT信號(hào)通路可能在其中起某種作用。本研究的目的是：明確是否小鼠基質(zhì)干細(xì)胞也可以合成Trop2,并探討Trop2對(duì)小鼠骨密質(zhì)來(lái)源的MSC生物學(xué)作用的影響。 研究方法 1.采用骨片法分離獲取小鼠MSCs,對(duì)其進(jìn)行免疫表型鑒定和成脂及成骨誘導(dǎo)分化。 2.通過(guò)免疫熒光染色,利用激光共聚焦顯微鏡觀察Trop2在小鼠MSCs細(xì)胞膜上的定位表達(dá)；通過(guò)Western Blot檢測(cè)小鼠骨密質(zhì)來(lái)源MSCs不同培養(yǎng)時(shí)期Trop2的表達(dá)變化。 3.建立Trop2基因敲除(Trop2 KO)小鼠模型,通過(guò)RT-PCR、Southern Blot、WesternBlot等方法分別從DNA、RNA、蛋白表達(dá)水平進(jìn)行鑒定。 4.利用流式細(xì)胞術(shù)檢測(cè)并比較Trop2基因敲除小鼠來(lái)源MSCs和野生型(WT)小鼠MSCs表型差異。 5.通過(guò)CCK-8和CFSE檢測(cè)并比較Trop2基因敲除小鼠來(lái)源MSCs和WT小鼠MSCs細(xì)胞增殖能力的差異。 6.通過(guò)RT-PCR和real-time PCR比較Trop2基因敲除小鼠來(lái)源MSCs和WT小鼠MSCs在經(jīng)過(guò)完全成脂和成骨誘導(dǎo)之后,成脂標(biāo)志基因PPAR-γ2、Adipsin和成骨標(biāo)志基因Osteocalcin、Osteopontin的定量表達(dá)差異；通過(guò)特異性的油紅“0”和茜素紅染色來(lái)判別Trop2基因敲除小鼠來(lái)源MSCs和WT小鼠MSCs在經(jīng)過(guò)完全成脂和成骨誘導(dǎo)后脂滴聚集和礦物質(zhì)沉積的差異。 7.通過(guò)流式細(xì)胞術(shù)明確Trop2基因敲除小鼠來(lái)源MSCs和WT小鼠MSCs進(jìn)入細(xì)胞周期S期細(xì)胞百分?jǐn)?shù)的差異；通過(guò)Western Blot明確在傳代培養(yǎng)過(guò)程中Trop2基因敲除小鼠來(lái)源MSCs和WT小鼠MSCs活化AKT表達(dá)比率的差異,以及AKT通路下游Cyclin D1的表達(dá)差異；利用PI3K抑制劑LY294002處理WT小鼠MSCs后明確活化AKT表達(dá)比率的差異以及其下游Cyclin D1的表達(dá)差異,并將其與Trop2基因敲除小鼠來(lái)源MSCs進(jìn)行比較,明確Trop2對(duì)AKT/Cyclin Dl通路的影響。 研究結(jié)果 1.骨片法可成功體外分離獲取小鼠MSCs,所獲取的MSCs不僅具備通過(guò)經(jīng)典方法獲取的MSCs的免疫表型特征,而且還可以向脂肪和骨誘導(dǎo)分化,是同質(zhì)性比較一致的間充質(zhì)干細(xì)胞。 2.通過(guò)激光共聚焦顯微鏡首次明確觀察到MSCs細(xì)胞膜上Trop2的定位表達(dá),通過(guò)Western Blot發(fā)現(xiàn)Trop2的表達(dá)量隨MSCs體外分裂次數(shù)的增加而減少。 3.通過(guò)RT-PCR、Southern Blot、Western Blot明確鑒定了Trop2基因敲除小鼠的構(gòu)建成功。 4.通過(guò)流式細(xì)胞術(shù)、RT-PCR、real-time PCR、Western Blot等檢測(cè)發(fā)現(xiàn),Trop2基因敲除小鼠和WT小鼠來(lái)源的MSCs在以下方面存在明顯差異： (1)通過(guò)表型觀察發(fā)現(xiàn)：與WT MSCs相比,KO MSCs在第一代時(shí)同質(zhì)性較差,CD45陽(yáng)性細(xì)胞(造血系細(xì)胞)混雜較多；隨著傳代至第4代后其同質(zhì)性漸趨一致。 (2)CCK-8和CFSE檢測(cè)發(fā)現(xiàn)：與WT MSCs相比,KO MSCs的增殖速率明顯降低,細(xì)胞倍增時(shí)間明顯延長(zhǎng)。 (3)成脂與成骨誘導(dǎo)實(shí)驗(yàn)發(fā)現(xiàn)：與WT MSCs相比,KO MSCs在相同誘導(dǎo)之后脂滴聚集減少,礦物質(zhì)沉積減少；real-time PCR檢測(cè)發(fā)現(xiàn)經(jīng)完全誘導(dǎo)后WT MSCs較來(lái)源于KO MSCs的脂肪標(biāo)志基因Adipsin、PPAR-γ2和骨標(biāo)志基因Osteocalcin、Osteopontin分別高出400%、500%、350%和100%。 (4)細(xì)胞周期：與WT MSCs相比,KO MSCs進(jìn)入細(xì)胞周期S期的細(xì)胞比率明顯降低。 (5) AKT/Cyclin D1:與WT MSCs相比,KO MSCs活化AKT的表達(dá)比率明顯降低,AKT信號(hào)通路下游細(xì)胞周期關(guān)鍵節(jié)點(diǎn)蛋白Cyclin D1的表達(dá)明顯降低；但活化AKT和Cyclin D1表達(dá)降低程度不及經(jīng)LY294002阻斷AKT通路所引起的降低程度大。 結(jié)論 1. Trop2定位表達(dá)在MSCs細(xì)胞膜。 2. Trop2的缺失可以減弱MSCs的增殖與分化能力。 3. Trop2的存在有利于維持AKT的活化狀態(tài),繼而保護(hù)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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329
本文編號(hào)：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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329
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