本文關(guān)鍵詞：骨重塑在造血干細(xì)胞動(dòng)員中的機(jī)制及應(yīng)用研究　出處：《中國(guó)協(xié)和醫(yī)科大學(xué)》2010年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 成骨細(xì)胞 破骨細(xì)胞 造血干細(xì)胞動(dòng)員 干祖細(xì)胞動(dòng)員 甲狀旁腺激素 NF-KB配體的受體

【摘要】： 目的骨髓中大多數(shù)造血干細(xì)胞位于骨組織構(gòu)成的骨龕中。成骨細(xì)胞是骨內(nèi)膜表面的內(nèi)襯細(xì)胞,生理?xiàng)l件下HSC及移植后歸巢至骨髓的HSC與之密切接觸,這種解剖定位提示成骨細(xì)胞可能調(diào)節(jié)HSC的功能。這一觀點(diǎn)首先在成骨細(xì)胞和HSC的體外共培養(yǎng)中得到證實(shí),隨即一系列的研究幾乎同時(shí)確定：成骨細(xì)胞是HSC龕中一關(guān)鍵組成成分,目前人們將之命名為‘成骨細(xì)胞龕’或‘骨內(nèi)膜龕’。通過調(diào)節(jié)‘骨內(nèi)膜龕’的大小,可以控制HSC的數(shù)量,維持HSC的穩(wěn)定狀態(tài)。本研究以干細(xì)胞動(dòng)員中的成骨細(xì)胞及破骨細(xì)胞為研究對(duì)象,觀察干細(xì)胞動(dòng)員過程中成破骨細(xì)胞數(shù)量和功能的變化及其與動(dòng)員的關(guān)系,進(jìn)一步研究造血干細(xì)胞動(dòng)員的機(jī)制。 方法利用流式細(xì)胞術(shù)檢測(cè)動(dòng)員0、3、5天小鼠外周血干細(xì)胞數(shù)量,評(píng)價(jià)動(dòng)員效果；通過實(shí)時(shí)定量PCR的方法比較動(dòng)員0、3、5天小鼠成骨細(xì)胞特異性基因OCN.OPN.SDF-1.SCF的水平；并通過免疫組化及流式細(xì)胞術(shù)比較動(dòng)員0、3、5天人類及小鼠成骨細(xì)胞數(shù)量功能的差異；利用細(xì)胞化學(xué)TRACP染色的方法比較動(dòng)員0、3、5天小鼠破骨細(xì)胞數(shù)量和功能的差異；利用連續(xù)切片標(biāo)記Caspase3的方法檢測(cè)人類及小鼠成骨細(xì)胞的凋亡情況;利用ELISA的方法檢測(cè)人類及小鼠循環(huán)中OCN. TRAP-5b蛋白水平反應(yīng)成破骨細(xì)胞活性；共培養(yǎng)小鼠成骨細(xì)胞和小鼠骨髓有核細(xì)胞,檢測(cè)成骨細(xì)胞活性。 結(jié)果 1.短期應(yīng)用G-CSF可導(dǎo)致人類和小鼠動(dòng)員模型中干骺端成骨細(xì)胞數(shù)量減少活性下降：動(dòng)員前小鼠成骨細(xì)胞OCNmRNA水平為動(dòng)員第三天的27±6倍(P＜0.01)；且動(dòng)員后第3天骨內(nèi)膜成骨細(xì)胞數(shù)量明顯減少,形態(tài)由立方形變?yōu)樗笮�；CD45-Ter119-OPN成骨細(xì)胞數(shù)量在動(dòng)員后3天及5天明顯減少(動(dòng)員前,4085±135cells／femur;動(dòng)員后3天,1118±80 cells／femur:動(dòng)員后5天,1032±55cell s/femur；P=0.02);動(dòng)員3天和5天成骨細(xì)胞活性明顯下降,OCN水平分別為59.44±3.16 ng/ml(動(dòng)員前),39.21±4.49 ng/ml(動(dòng)員后第3天)42.36±2.23 ng/ml(動(dòng)員后第5天),P＜0.01。進(jìn)一步檢測(cè)正常供者和自體移植患者標(biāo)本同樣顯示動(dòng)員后成骨細(xì)胞數(shù)量減少并伴有活性下降。然而在小鼠G-CSF動(dòng)員第3天外周血LSK細(xì)胞比例并未明顯增加,動(dòng)員前、動(dòng)員3天及動(dòng)員5天分別為0.40±0.07%、0.55±0.05%和2.9±0.32%,因此成骨細(xì)胞的變化發(fā)生于動(dòng)員之前。 2.成骨細(xì)胞數(shù)量減少導(dǎo)致SDF-1、SCF、OPN等蛋白表達(dá)的減少,動(dòng)員前小鼠成骨細(xì)胞SDF-1 mRNA的表達(dá)水平為動(dòng)員后3天的3.44±0.3倍,進(jìn)而引起動(dòng)員的發(fā)生。 3.G-CSF誘導(dǎo)的成骨細(xì)胞數(shù)量減少功能下降部分是由于動(dòng)員過程中成骨細(xì)胞發(fā)生了凋亡,但成骨細(xì)胞的分化并未受到抑制,供者血清DKK1水平在動(dòng)員前和動(dòng)員后5天并無明顯差異,13621.13±1081.99 pg/ml和10079.83±2055.82pg/ml,P=0.22。 4.共培養(yǎng)成骨細(xì)胞和骨髓有核細(xì)胞發(fā)現(xiàn),加或不加G-CSF兩組成骨細(xì)胞OCNmRNA表達(dá)情況無明顯差別,P=0.69。G-CSF通過間接途徑抑制成骨細(xì)胞。 5.動(dòng)員后3天供者(動(dòng)員前5.04±0.43 U／L,動(dòng)員后3天3.45±0.37 U／L,P=0.03)及小鼠(動(dòng)員前5.43±1.2 U／L,動(dòng)員后3天4.04±0.86 U／L,P=0.47)血清TRAP-5b水平稍有下降,隨后血清TRAP-5b水平明顯上升(供者動(dòng)員前為5.04±0.43 U／L,動(dòng)員后5天為6.87±0.57U／L,P=0.04；小鼠動(dòng)員前5.43±1.2U／L,小鼠動(dòng)員后5天13.06±1.65,P=0.02)。動(dòng)員過程中破骨細(xì)胞的活性逐漸增強(qiáng)。 結(jié)論動(dòng)員過程中成骨細(xì)胞數(shù)量活性的下降導(dǎo)致了動(dòng)員的發(fā)生,并伴隨有破骨細(xì)胞的活化。 目的造血干細(xì)胞移植不僅是惡性血液病、嚴(yán)重免疫系統(tǒng)疾病及部分實(shí)體腫瘤主要甚至唯一的治愈手段,而且隨著對(duì)干細(xì)胞及其細(xì)胞和組織工程研究的深入,正逐漸應(yīng)用到心腦血管疾病、神經(jīng)系統(tǒng)疾病等領(lǐng)域。 我們?cè)诘谝徊糠值难芯恐凶C明了骨重塑在造血干細(xì)胞動(dòng)員過程中發(fā)揮了重要的作用,在第二部分的研究中我們用6種小鼠模型模擬臨床上的自體干細(xì)胞移植過程,研究應(yīng)用甲狀旁腺激素(PTH)或NF-KB配體的受體(RANKL)靶向于成骨細(xì)胞或破骨細(xì)胞是否能夠增加干細(xì)胞數(shù)量,保護(hù)干細(xì)胞造血重建的功能。 方法利用細(xì)胞毒藥物CTX處理小鼠,建立貧動(dòng)員模型,進(jìn)行造血干祖細(xì)胞培養(yǎng),比較正常動(dòng)員和貧動(dòng)員小鼠模型中干細(xì)胞功能,并利用流式計(jì)數(shù)、RQ-PCR、ELISA的方法檢測(cè)貧動(dòng)員小鼠成骨細(xì)胞數(shù)量和功能。模擬臨床自體移植過程,用PTH、RANKL干預(yù)貧動(dòng)員小鼠(具體分組見材料與方法圖1),利用競(jìng)爭(zhēng)性移植模型(CRA)檢測(cè)不同用藥組小鼠干細(xì)胞移植16周后外周血中CD45.2陽性細(xì)胞比例。 結(jié)果 1.多次細(xì)胞毒藥物化療嚴(yán)重影響了成骨細(xì)胞和造血干細(xì)胞功能：自體干細(xì)胞移植患者化療后血清OCN水平明顯下降(化療前：22.19±1.08 ng/mL和化療后：16.08±2.12 ng/mL,P=0.01),小鼠模型中同樣證明了上述觀點(diǎn),成骨細(xì)胞數(shù)量在多次應(yīng)用細(xì)胞毒藥物小鼠中明顯減少,功能下降。且多次應(yīng)用細(xì)胞毒嚴(yán)重影響了造血干祖細(xì)胞功能。CTLs/Gs組小鼠集落形成實(shí)驗(yàn)結(jié)果分別為21.16±1.35U,和13.00±1.71U,正常未用藥小鼠為29.17±1.22U,三者相比P=0.01。 2.骨髓CRA結(jié)果顯示給予G-CSF支持的小鼠干細(xì)胞功能明顯下降(G組比CTL組,P=0.01)。然而,應(yīng)用PTH后可顯著改善該組小鼠干細(xì)胞造血重建的功能(PTH組比G組,P＜0.01;PTH組比CTL組,P＜0.05)。外周血CRA結(jié)果同樣證明應(yīng)用G-CSF支持治療的G組小鼠僅有極少數(shù)周血干細(xì)胞能進(jìn)行造血重建(G組比CTL組,P＜0.05)。而應(yīng)用PTH的P+G組小鼠外周血干細(xì)胞造血重建的能力明顯增加(P+G組比G組,P＜0.01;P+G組比CTL組,P＜0.05)。與CTL組或G組相比,P+R及P+R+G組周血干細(xì)胞同樣顯示出了更強(qiáng)的林髓系造血重建能力。RANKL同G-CSF一樣可有效地動(dòng)員骨髓中的造血干細(xì)胞(P+G組比P+R組,P＞0.05)因此,應(yīng)用PTH和RANKL可增加小鼠模型中動(dòng)員至外周血中的造血干細(xì)胞數(shù)量并且保護(hù)多次應(yīng)用細(xì)胞毒藥物后造血干細(xì)胞功能。 結(jié)論靶向于骨龕的藥物可有效地改善多次應(yīng)用細(xì)胞毒藥物,尤其是聯(lián)用G-CSF日寸干細(xì)胞治療的效果。
[Abstract]:The majority of hematopoietic stem cells in the bone marrow to the niche structure of the bone tissue. Osteoblasts are the cells lining the endosteal surface, under physiological conditions and HSC after transplantation of bone marrow HSC homing to close contact with them, the anatomical locations suggest the osteoblast may regulate the function of HSC. The first point in the bone cells and HSC cultured in vitro were confirmed, then a series of studies identified almost at the same time: osteoblast is a key component in the HSC niche, at present people named the 'osteoblast niche' or '. By adjusting the endosteal niches'' niche endosteal size, we can control the number. "HSC, maintain the stable state of HSC. In this study, stem cell mobilization in osteoblasts and osteoclasts as the research object, observe the mobilization of stem cells in the process of change as the number and function of osteoclasts and mobilization, into the One step is to study the mechanism of hematopoietic stem cell mobilization.
Methods using flow cytometry to detect the mobilization of peripheral blood stem cells in mice of 0,3,5 days, the mobilization effect evaluation; through real-time quantitative PCR method to compare the mobilization of 0,3,5 days of mouse osteoblast specific gene OCN.OPN.SDF-1.SCF levels; and by immunohistochemistry and flow cytometry compared mobilization 0,3,5 days of mice and humans into different bone cells the number of function; difference method using cytochemical TRACP staining compared mobilization 0,3,5 days mouse osteoclast number and function of bone cells; detection of mice and humans into apoptosis of bone cells using the method of serial sections labeled with Caspase3; the levels of OCN. TRAP-5b protein in human and mouse cycle reaction using ELISA method into osteoclast activity; bone cells and mouse bone marrow mononuclear cells into mice were cultured to detect the osteoblast activity.
Result
1. short term application of G-CSF can lead to the mobilization of human and mouse end of reducing the number of osteoblast activity decreased diaphysis model: mobilization of mouse osteoblast OCNmRNA level for the mobilization of third days of 27 + 6 times (P < 0.01); and the third day after the mobilization of endosteal osteoblasts decrease by cubic shape deformation spindle; CD45-Ter119-OPN the number of osteoblasts in the mobilization after 3 days and 5 days was significantly reduced (before mobilization, 4085 + 135cells / femur; mobilization after 3 days, 1118 + 80 cells / femur: mobilization after 5 days, 1032 + 55cell s/femur; P=0.02); 3 days and 5 days of bone cell activity decreased mobilization OCN, ng/ml levels were 59.44 + 3.16, 39.21 + (mobilization before) 4.49 ng/ml (third days after the mobilization of 42.36 + 2.23 (ng/ml) fifth days after mobilization), P < 0.01. for further detection of normal specimens from patients with autologous transplantation and also showed that after mobilization as the number of cells with reduced bone Activity decreased. However, the proportion of LSK cells in peripheral blood did not increase significantly after mobilization of G-CSF for third days. Mobilization for 3 days and mobilization for 5 days were 0.40 0.40, 0.07%, 0.55 + 0.05% and 2.9 0.32%, respectively, so the change of osteoblasts occurred before mobilization.
2., the number of osteoblasts decreased, resulting in a decrease in protein expression of SDF-1, SCF and OPN. The expression level of SDF-1 mRNA in rat osteoblasts before mobilization was 3.44 + 0.3 times on the 3 day after mobilization, which led to mobilization.
3.G-CSF induced by reducing the number of osteoblast function decline is due in part to the mobilization process of osteoblast apoptosis, but the osteoblast differentiation was not inhibited, the level of serum DKK1 in donors before mobilization and mobilization after 5 days there was no significant difference, 13621.13 + 1081.99 and 10079.83 + pg/ml, 2055.82pg/ml, P=0.22.
4. co culture of osteoblasts and bone marrow nucleated cells revealed that there was no significant difference in OCNmRNA expression between G-CSF cells and bone marrow cells, and P=0.69.G-CSF inhibited osteoblasts indirectly through P=0.69.G-CSF.
For 3 days and 5. after mobilization (mobilization 5.04 + 0.43 U / L, 3 days after the mobilization of 3.45 + 0.37 U / L, P=0.03) and mice (5.43 mobilization + 1.2 U / L, 3 days after the mobilization of 4.04 + 0.86 U / L, P=0.47) level of serum TRAP-5b decreased slightly, then serum the level of TRAP-5b increased significantly (donors before mobilization of 5.04 + 0.43 U / L, 5 days after the mobilization of 6.87 + 0.57U / L, P=0.04; mice mobilization 5.43 + 1.2U / L mice after 5 days, the mobilization of 13.06 + 1.65, P=0.02). In the process of mobilization of osteoclast activity gradually increased.
Conclusion the decrease of the number of osteoblasts in the mobilization process leads to the mobilization and the activation of osteoclast.
The purpose of hematopoietic stem cell transplantation is not only the malignant blood disease, serious diseases and some solid tumor immune system mainly or only cure, and with the stem cells and cell and tissue engineering research, is gradually applied to the field of cardiovascular and cerebrovascular diseases, diseases of the nervous system.
We show that bone remodeling in hematopoietic stem cell mobilization plays an important role in the process of the first part of the study, in the second part of the study we used 6 mouse model to simulate clinical autologous stem cell transplantation, application of parathyroid hormone (PTH) or NF-KB ligand receptor (RANKL) targeted to whether osteoblasts or osteoclasts can increase the number of stem cells, stem cells protect hematopoietic reconstruction function.
Cytotoxic drugs in mice treated with CTX, using the method of establishment, lean mobilization model, hematopoietic stem progenitor cells, stem cell mobilization and mobilization function of normal lean mouse model, and using flow cytometry counting, RQ-PCR, ELISA to detect poor mobilization of mouse osteoblast number and function. PTH used to simulate the process of clinical transplantation RANKL, lean mice (specific mobilization intervention group see materials and methods in Figure 1), using competitive transplantation model (CRA) detection of different treatment group mice transplantation of stem cell proportion in peripheral blood of CD45.2 positive cells after 16 weeks.
Result
1. times of cytotoxic chemotherapy affect osteoblasts and hematopoietic stem cell function: autologous stem cell transplantation after chemotherapy in patients with serum OCN levels were significantly decreased (22.19 + 1.08 ng/mL before chemotherapy and after chemotherapy: 16.08 + 2.12 ng/mL, P=0.01), mouse model also proves the point, the number of osteoblasts obviously reduce the repeated application of cytotoxic drugs in mice and multiple function decline. Ofcytotoxic seriously affected the function of hematopoietic stem and progenitor cells in.CTLs/Gs mice colony formation experiment results were 21.16 + 1.35U and 13 + 1.71U, normal untreated mice was 29.17 + 1.22U, compared to three P=0.01.
2.楠ㄩ珦CRA緇撴灉鏄劇ず緇欎簣G-CSF鏀寔鐨勫皬榧犲共緇嗚優(yōu)鍔熻兘鏄庢樉涓嬮檷(G緇勬瘮CTL緇，

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