【作者】 韩京；

【导师】 粟永萍；

【作者基本信息】 第三军医大学 ， 军事预防医学， 2009， 博士

【摘要】 早期的解剖学研究发现骨髓内存在交感神经支配,并且通过对骨髓中交感神经功能研究证实交感神经还可以调节骨髓的功能,例如通过电刺激交感神经,导致骨髓内血管收缩,红细胞、白细胞和幼红细胞进入血循环增加。进一步研究显示交感神经对骨髓细胞功能的调控是通过骨髓细胞上肾上腺素能受体(adrenergic receptor,AR)介导的。通过功能检测与放射性配体结合研究已证实骨髓细胞表达有功能的α,β-AR。在体和体外给予肾上腺素能受体激动剂能够促进骨髓细胞增殖和粒细胞生成,并成剂量效应关系;给予β-AR激动剂异丙肾上腺素(isoproterenol,Iso)可以通过激活骨细胞中β-AR促进成骨细胞增殖与分化。此外,有文献报道交感神经递质去甲肾上腺素(norepinephrine,NE)和多巴胺(dopamine,DA)能够提高人骨髓中CD34+细胞的迁移效率提示交感神经还参与了对干细胞动员的调节。Katayama等报道了交感神经系统还能够调节骨髓造血干(祖)细胞(hematopoietic stem and progenitor cell,HSPC)从骨髓中的动员和归巢,研究发现在β2肾上腺素能受体激动剂作用下促进了粒细胞集落刺激因子(granulocyte colony-stimulating factor,G-CSF)诱导的造血干细胞动员,证实交感神经通过AR介导了对干细胞动员的调节。这些研究打破了过去传统认为的神经系统、骨骼系统和造血系统各系统之间是相互孤立,独立起作用的观点;揭示出干细胞维持和活化过程的复杂性,这几大系统必须相互协同,共同作用来维持正常的造血功能。Katayama在研究中指出,NE一方面通过CXCR4/CXCL12趋化因子受体轴直接作用于HSPC,参与对HSPC动员和归巢的调控。另一方面,在NE刺激下G-CSF诱导的HSPC动员还需要有其它细胞信号协同作用调控HSPC动员和归巢,这些细胞信号可能与HSPC所在的干细胞壁龛,及G-CSF影响的干细胞壁龛内可溶性因子有关,但其具体机制仍不清楚,从而提出了一个更复杂的干细胞壁龛概念。干细胞壁龛,即存在于器官或组织中的可以维持干细胞的自我更新及避免分化的微环境,包括壁龛细胞、细胞外基质(extracellular matrix,ECM)和来源于壁龛细胞的可溶性因子。干细胞壁龛为干细胞提供一个保护性微环境,来源于壁龛细胞的可溶性因子可以通过与干细胞发生直接或间接的作用,从而调控干细胞;壁龛信号的变化可引起干细胞命运的改变,其中分泌的生长因子和细胞因子与干细胞的增殖分化密切相关。有研究报道骨髓基质微环境能够对黏附在其中的祖细胞发挥选择性的抑制调节作用,抑制祖细胞的分化从而促进其增殖潜能。基于上述这些研究有研究者提出“脑-骨髓-血液轴”观点,认为在“脑-骨髓-血液轴”中,一部分信号直接传递给了HSPC池,另一部分信号则通过对支持营养干细胞池的基质细胞间接作用来实现的。因此,我们猜测交感神经递质NE作用于骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),通过BMSCs细胞将信号传递给HSPC从而间接影响到HSPC动员和归巢。BMSCs是位于干细胞壁龛中的造血微环境的主要组成成分之一,既能够分泌种类众多的细胞因子和生长因子,对HSPC的增殖、凋亡和归巢起重要调控作用。同时还高表达细胞外基质蛋白,如纤维黏接素、胶原、蛋白聚糖,还能表达基质-细胞、细胞-细胞等相互作用的反受体,对骨、骨髓中细胞外基质构建起着重要作用。有研究证实受照射小鼠给予肾上腺素能受体激动剂可促进骨髓细胞的增殖,提示儿茶酚胺类神经递质能够通过刺激骨髓细胞增殖为放射损伤后的骨髓细胞提供保护作用。因此,研究交感神经递质对BMSCs的调控作用意义重大。一方面,阐明了交感神经对HSPC动员和归巢的间接调控机制,另一方面为在严重创伤和放射复合伤造成的骨髓损伤情况下,通过神经递质对BMSCs的调控机制来改善骨髓造血微环境,重建造血功能,提供新的思路和救治方法。因此,本课题首先分离培养大鼠骨髓BMSCs,并从细胞形态、标志性CD分子及分化潜能三个方面对分离细胞进行鉴定,以证实分离培养方法稳定可靠。交感神经递质通过肾上腺素能受体发挥其重要的生物学功能。BMSCs是否表达AR是交感神经递质发挥调控作用的重要前提。其次检测了分离培养BMSCs细胞肾上腺素能受体表达情况,结果显示BMSCs细胞仅有α1-ARs mRNA表达,而不表达α2-ARs及β1,β2,β3-AR mRNA。α1-ARs在BMSCs细胞表达是否具有功能?交感神经递质能否通过α1-ARs对BMSCs发挥调控作用?因此,在第二部分研究中我们将不同的化学激动剂,拮抗剂药物加入正常的BMSCs细胞中,阻断其正常的信号通路观察细胞增殖及相关基因的表达情况,以证实NE对BMSCs调控作用并探讨其具体调控机制。结果显示在α-AR激动剂NE作用下促进了BMSCs细胞增殖;在β-AR激动剂Iso作用下,BMSCs细胞增殖明显变化。NE对BMSCs细胞发挥促增殖作用的机制是通过首先激活α1-ARs,然后经过Ca2+、PKC和ERK1/2信号传导途径来实现的。NE能够促进BMSCs细胞增殖,对BMSCs细胞分化是否有调控作用呢?BMSCs作为造血微环境的主要组成成分之一,能够分泌种类众多的细胞因子和生长因子,以及黏附分子和细胞外基质,NE又能否影响BMSCs分泌细胞因子呢?故设计第三部分实验,在此部分研究中对NE作用下BMSCs诱导形成成骨、成脂细胞的细胞分化率进行检测,并采用定量RT-PCR和ELISA方法检测NE对BMSCs分泌的细胞因子表达的影响。结果显示NE对BMSCs向成骨和脂肪细胞分化无明显影响,但对BMSCs分泌细胞因子有促进作用。通过以上三部分的实验研究与分析,获得以下主要结果:1、在本实验中选取密度梯度离心法和自然贴壁法相结合的方法分离BMSCs,并从细胞形态、标志性CD分子及分化潜能三个方面对分离细胞进行鉴定。鉴定结果显示:分离细胞呈旋涡状生长,形态为成纤维细胞样的贴壁细胞;并阳性表达BMSCs的标志分子:CD44、CD73、CD90和CD29,不表达造血干细胞的标志分子:CD31和CD45;具有向成骨细胞和脂肪细胞分化的能力。证实在本实验随室条件下所分离培养的BMSCs是骨髓中区别造血干细胞的一群处于未分化状态的干细胞。2、用RT-PCR方法检测第3代BMSCs细胞肾上腺素能受体表达情况。BMSCs细胞表达α1A-,α1B-,α1D-AR mRNA,不表达α2-ARs mRNA及β1,β2,β3-AR mRNA。3、用3H-TdR掺入实验检测不同浓度NE(10-7-10-4M)作用8h及10-5M NE作用不同时间对BMSCs细胞增殖的影响。结果显示10-7、10-6、10-5、10-4M NE作用8h后均促进了BMSCs细胞的增殖,比对照组分别增加5.12%、10.06%、37.3%、25.28%,在10-5M NE作用下BMSCs细胞增殖最为显著。在α-AR拮抗剂-酚妥拉明(phentolamine,PH)作用下,BMSCs细胞增殖比在NE作用下减少了70.7%。说明NE能够促进BMSCs细胞增殖,PH则通过抑制α-AR阻断NE诱导的BMSCs细胞增殖。4、在不同浓度Iso(10-7-10-4M)作用下,BMSCs细胞增殖进行了检测。结果显示10-7、10-6、10-4M Iso作用8h后BMSCs细胞增殖与对照组比较分别减少了1.68%、1.3%、2.6%;10-5M Iso作用8h后BMSCs细胞增殖比对照组增加了0.6%,各实验组与对照组之间均没有统计学差异。此结果说明不同浓度Iso对BMSCs细胞增殖均无促进作用。5、用定量RT-PCR检测BMSCs细胞α1-AR亚型mRNA表达情况。结果显示正常组BMSCs细胞的α1A-AR,α1B-AR,α1D-AR mRNA表达维持在较低水平。在10-5M NE作用下,α1A-AR,α1B-AR,α1D-AR mRNA表达分别增加了76.54%,59.8%,113.32%,与正常对照组均差异显著(P < 0.05)。此结果说明NE对BMSCs细胞促增殖作用是通过激活α1-AR来实现的。6、激光共聚焦检测在10-5M NE作用下α1-AR下游Ca2+,PKC信号通路激活情况。结果显示在10-5M NE作用下,BMSCs细胞质内Ca2+流浓度显著升高,10-6M PH组没有观察到Ca2+流浓度的显著变化。结果提示NE能够激活Ca2+流的快速流动,使胞质内Ca2+流浓度显著升高,PH则可以阻断NE对Ca2+的激活作用。对照组BMSCs中PKC蛋白在胞质中表达,加入NE10min后观察到PKC蛋白从胞质表达转移到胞膜表达,PH预处理组PKC蛋白仅在胞质表达,在胞膜中没有观察到其表达。免疫印迹实验结果与免疫组化观察到的结果相一致。此结果说明NE可以直接激活PKC使其发生膜转位,PH则可以抑制NE诱导的PKC激活,不使其发生膜转位。十字孢碱(PKC抑制剂,10-7M)对NE刺激的BMSCs增殖检测结果显示十字孢碱预处理组较NE组BMSCs增殖减弱了59.7%,与NE组差异显著(P < 0.05)。结果提示十字孢碱可以直接抑制PKC激活从而抑制NE对BMSCs促增殖作用。说明NE是通过激活PKC对BMSCs产生促增殖作用。7、10-5M NE刺激BMSCs后ERK1/2磷酸化增加2倍(P < 0.05),PH使其增加减少62.97%(P < 0.05)。此结果说明NE可以直接刺激ERK1/2磷酸化,PH则抑制了NE引起的ERK1/2磷酸化。PD98059(ERK1/2抑制剂,10-5M)对NE刺激BMSCs细胞增殖的影响结果显示NE刺激BMSCs细胞增殖较正常组增加80%(P < 0.05),PD98059使BMSCs细胞增殖减少83.2%(P < 0.05)。结果说明PD98059可以通过抑制ERK1/2激活来抑制NE诱导的BMSCs细胞增殖,提示NE对BMSCs细胞的增殖作用经过ERK1/2信号途径。在十字孢碱作用下BMSCs细胞ERK1/2磷酸化水平减少116.67%,与NE组和对照组均有显著性差异(P < 0.05)。此结果说明十字孢碱可以通过抑制PKC激活来抑制NE诱导的ERK1/2磷酸化,提示先激活PKC,然后引起ERK1/2磷酸化,PKC是ERK1/2的上游信号通路。8、通过对BMSCs诱导形成成骨、成脂细胞的细胞分化率检测,证实NE对BMSCs分化的调控。结果显示,在10-5M NE作用下,BMSCs细胞均可诱导为成骨细胞与脂肪细胞;对照组与10-5M NE组两组细胞在形态上未见显著差别。对照组成骨细胞分化率为14.32%,NE组分化率16.78%,两组之间没有统计学差异。对照组成脂细胞分化率为26.62%,NE组分化率25.15%,两组之间没有统计学差异。9、对BMSCs细胞分泌表达细胞因子进行检测的结果显示,10-5M NE作用于BMSCs细胞后,IL-6、SCF、LIF和VEGF mRNA水平明显高于对照组,分别是对照组的5.79,3.08,1.75和5.99倍,与对照组之间均有显著性差异(P<0.05)。CXCL12 mRNA表达水平与对照组之间无显著性差异。在10-5M NE作用下BMSCs细胞分泌细胞因子IL-6、SCF和VEGF蛋白含量分别是对照组的1.24,1.3和2.04倍,与对照组比较均有显著性差异(P<0.05)。提示NE对BMSCs分泌细胞因子在蛋白水平和转录水平均有调控作用,可以促进BMSCs细胞因子的分泌。但是对BMSCs分泌趋化因子CXCL12的转录水平没有调控作用。研究结论如下:1、本实验室条件下,可以稳定的分离培养出大鼠BMSCs,并通过对分离细胞的形态学,细胞表型和诱导分化能力鉴定,证明是具有高度自我更新能力和多向分化潜能的成体干细胞。2、分离BMSCs细胞不仅表达α1-ARs mRNA,而且在NE诱导的BMSCs细胞增殖过程中发挥调控作用。3、分离BMSCs细胞不表达β-ARs mRNA,并且β-AR激动剂Iso对BMSCs细胞增殖无影响,提示Iso对BMSCs细胞增殖无调控作用与其不表达β-ARs有关。4、NE可以促进BMSCs细胞增殖。NE对BMSCs细胞促增殖作用是通过首先激活α1-ARs,然后经过Ca2+、PKC和ERK1/2信号传导途径来实现的。NE对BMSCs诱导形成成骨,脂肪细胞的细胞分化率没有影响,说明NE对BMSCs向成骨和脂肪细胞分化不产生调控作用。5、NE能够调控BMSCs细胞因子分泌,可以促进BMSCs表达并分泌IL-6、VEGF、LIF及SCF细胞因子;但是对趋化因子CXCL12表达无调控作用。本研究证实交感神经递质NE能够促进BMSCs细胞增殖,并阐明NE促进BMSCs细胞增殖效应的调控机制是通过首先激活α1-ARs,然后经过Ca2+、PKC和ERK1/2信号传导途径实现的。观察了在NE作用下BMSCs分泌细胞因子IL-6、VEGF、LIF、SCF及趋化因子CXCL12表达的变化特点。这些结果阐明了交感神经系统对HSPC动员和归巢的间接调控机制,丰富了交感神经递质对骨髓细胞功能调控的理论认识,为在严重创伤和放射复合伤造成的骨髓损伤情况下,通过神经递质对BMSCs的调控机制来改善骨髓造血微环境,重建造血功能,提供新的思路和救治方法。更多还原

【Abstract】 The sympathetic nervous system (SNS) has been shown to histologically innervate bone marrow (BM) cells. NA nerves regulated bone marrow cell’s function. Sympathetic nerve induced vessel contraction, increased red blood cell, leucocyte, normoblast into blood circulation under electrical stimulation. The SNS regulate bone marrow cells function through adrenergic receptor. Functional assays and radioligand binding studies indicate that bone marrow cells express functionalα-adrenergic receptor (AR).Adding isoproterenol to murine bone marrow cultures increases cellular proliferation and granulopoiesis dose-dependently in vitro and in vivo. The SNS regulate hematopoietic stem and progenitor cell mobilization (HSPC) through AR.NE and dopamine (DA) improved efficiency of CD34+ migration. Granulocyte colony-stimulating factor (G-CSF) induced HSPC mobilization throughβ-AR agonist isoproterenol.Katayama et al. reported a new regulatory axis for the mobilization of hematopoietic stem cells that links these cells to the nervous system and bone in an unanticipated way. This suggested an even more complex perspective of stem cells mobilization and homing. There are close relation between SNS, skeletal system and hematopoietic system. These systems must be in coordination with each other to keep haematopoiesis function.Katayama et al. indicated G-CSF dependent mobilization of HSPC through adrenergic stimulation is likely to require other cell communication mechanisms and given the newly identified non-OB stem cell niches and soluble factors. Stem cell niche is a microenvironment that maintains its self-renewal but not differentiation. Stem cell niche include niche cell, extracellular matrix (ECM), and soluble factors.They provide a protective environment through soluble factors and stem cells interaction directly and indirectly to regulate stem cells. Growth factors and cytokines are crucial cellular components of the stem cell proliferation. Zipori and Sasson et al. reported that stem cell microenvironment regulated progenitor cell differentiation. These findings support the presence of a ‘‘brain-bone-blood triad’’. Within the brain-bone-blood axis, some signals are delivered to the HSPC pool directly, while others are exerted indirectly via an impact on niche-supporting stromal cells. We hypothesis that NE regulate HSPC mobilization and homing through BMSCs.Bone marrow mesenchymal stem cells (BMSCs) located in stem cell niche and are one of components of the BM microenvironment. BMSCs are a vital source of growth factors and regulatory adhesion molecules and are thought to be the key in maintaining HSPC proliferation, homing and apoptosis. BMSCs highly expressed extracellular matrix protein such as collogen, proteoglycans, chemokine receptor which play an important role in constructon of extracellular matrix. If our hypothesis were demonstrated, our result were important to illuminate the indirectly mechanism of HSPC mobilization and homing.Our result provide a new method to improve bone marrow microenvironment an rebuild function of haemopoiesis.Adding isoproterenol to irradiated murine bone marrow cultures increases cellular proliferation and granulopoiesis dose-dependently, this suggest that sympathetic nerve neurotransmitter provide protection through bone marrow cells proliferation from irradiation injury. Whether norepinephrine relatedα1-AR activation exists in BMSCs and what biological effect it may cause remains unknown. Explores the sympathetic nervous system regulation on BMSCs function and its mechanism, provides the new treatment and the method for the clinical wound tissue repair and the BMSCs transplantation.Isolated and cultured rat BMSCs is the premise which we carry on our research. First, we established the method of isolated and cultured rat BMSCs and identified them from the cell appearance, the symbolic CD member and the differentiation potency in first part. Neurotransmitter plays its biological function through adrenergic receptor. Second, we examined the expression of of AR in rat BMSCs in vitro. Our result suggested thatα1A-,α1B-, andα1D-adrenergic receptor mRNA notα2-ARs,β1,β2,β3-AR mRNA were expressed in BMSCs in vitro. Whether NE relatedα1-AR activation exists in BMSCs and what biological effect it may cause remains unknown. In second part we examined the effect ofα-AR agonist NE andβ-AR agonist Iso on BMSCs proliferation to demonstrate regulatory mechanism. NE treatment increased [3H] thymidine incorporation in a dose-dependent manner. Iso didn’t have the stimulatory effect on cell proliferation. NE stimulated DNA synthesis viaα1-adrenergic receptors and downstream Ca2+/PKC and ERK1/2 activation in BMSCs.Whether NE regulated BMSCs differentiation and cytokines and chemokines secretion as a vital source of growth factors and regulatory adhesion molecules? In third part we examined the effect of NE on BMSCs differentiation in cell differentiation rate. Then we used RT-PCR and ELISA methods to examine the change of growth factors and chemotatic factor in transcriptional level and protein level. Our result showed that there were no effct of NE on BMSCs differentiated into adipocyte and osteoblast .NE upregulated cytokines secretion in BMSCs.Main results were as follow:1. In our experiment we used density gradient centrifugation and natural adherence methods to isolate BMSCs. In order to identify cultured BMSCs, the immunophenotype and differentiation potential of the cells was determined. When maintained in Stromal Medium, the BMSCs showed fibroblast-like morphology and formed typical colony-forming-units (CFU). Flow cytometry analysis demonstrated that rBMSCs expressed stromal-associated surface antigens CD29, CD44, CD73 and CD90; however, they did not express CD31 or CD45. Cells had multipotential differentiation (to differentiate into osteocytes, adipocytes). Our result demonstrated that cells we isolated were BMSCs.2. We examined the expression of of AR in BMSCs. Our result suggested thatα1A-,α1B-, andα1D-adrenergic receptor mRNA notα2-ARs,β1,β2,β3-AR mRNA were expressed in BMSCs in vitro. Our result was consistent with early report.3. To investigate whether norepinephrine induced BMSCs proliferation, [3H] thymidine incorporation into DNA of BMSCs was tested in the presence of various concentrations of norepinephrine (10-7-10-4M) for 8h. Norepinephrine treatment (10-7-10-4M) for 8 h increased 5.12%、10.06%、37.3%、25.28% than control group. The maximal stimulatory effect was observed at 10-5M of norepinephrine. To determine whether the effect of norepinephrine on cell proliferation was mediated throughα-AR dependent signaling, BMSCs were pretreated with phentolamine,anα-adrenergic receptor antagonist. This stimulatory effect of norepinephrine on cell proliferation was blocked by co-incubation with phentolamine.4. To investigate whether Iso induced, [3H] thymidine incorporation into DNA of BMSCs was tested in the presence of. We observed the effects of various concentrations of Iso (10-7-10-4M) on BMSCs proliferation for 8h.Our result showed that there were no promoted effect on BMSCs proliferation.5. Our result showed that BMSCs maintained in basal medium had low levels ofα1A-,α1B-, andα1D-AR mRNA expression as assessed by real-time RT-PCR. NE upregulatedα1A-,α1B-, andα1D-AR mRNA expression 76.54%,59.8%,113.32% than control group.of different AR subtypes in BMSCs.α1D-AR mRNA expression upregulated significantly. Our result showed that NE stimulates DNA synthesis viaα1-adrenergic receptors.6. To investigate whether NE induced BMSCs proliferation viaα1-adrenergic receptors and downstream Ca2+/PKC, Ca2+/PKC were tested by confocal microscopy. NE significantly increased [Ca2+]i concentration. The NE-induced increase in [Ca2+]i was completely blocked by pretreatment with phentolamine. The elevation of intracellular Ca2+ is necessary for PKC activation. We investigated the effect of NE on PKC activation using immunofluorescent staining. NE stimulation for 10 min induced PKC translocation to the cell membrane, which was blocked by pretreatment with phentolamine for 30 min. These results were consistent with the results of Western blot analysis. In addition, we examined the effect of staurosporine (10-6M) on NE-induced increases in [3H] thymidine incorporation in BMSCs. As shown in our result, pretreatment with staurosporine inhibited the increase in [3H] thymidine incorporation induced by norepinephrine. These findings suggest that the PKC pathway is involved in NE-induced increases in [3H] thymidine incorporation.7. We then examined the effect ofα1-adrenoceptor stimulation on ERK1/2 by Western blot, using an antibody specific to phosphorylated ERK1/2 at various time points. In BMSCs, ERK1/2 phosphorylation peaked 10 min following NE treatment, and phentolamine pretreatment inhibited NE-induced ERK1/2 phosphorylation. In order to determine whether PKC is involved in ERK1/2 activation, Western blot analysis was performed. Staurosporine (10-6M) inhibited NE-induced phosphorylation of ERK1/2. In cells subjected to pharmacological inhibition of PD98059 (10-5M), cell proliferation induced by norepinephrine was decreased.8. We examined the effect of NE on BMSCs differentiation in cell differentiation rate.Our result showed that there was no statistics difference in cell differentiation rate between control group and NE group in adipocyte and osteoblast.9. We examined the effect of NE on cytokines secretion by RT-PCR and ELISA. Our result showed that IL-6, SCF, LIF, VEGF mRNA expression increased 5.79, 3.08, 1.75 and 5.99 times in 10-5M NE group compared with control group. CXCL12 mRNA expression had no significant difference between NE group and control group.Our result showed NE regulated cytokines secretion and no effect on CXCL12 mRNA expression in BMSCs. Protein level of IL-6, SCF, VEGF upregulated 1.24, 1.3 and 2.04 times in NE group.Our result suggested NE promoted cytokines secretion.Conclusion:1. The method of isolated and cultured rat BMSCs were established successfully. Our result suggested isolated and cultured rat BMSCs were multipotential cells by identified them from the cell appearance, the symbolic CD member and the differentiation potency.2. BMSCs expressed functionalα1-ARs mRNA and regulated NE induced BMSCs proliferation.3. Iso had no effect on BMSCs proliferation and didn’t expressedβ-ARs mRNA.These result demonstrate that BMSCs didn’t express functionalβ-ARs mRNA.4. NE regultated BMSCs proliferation but not differentiation.NE stimulated DNA synthesis viaα1-adrenergic receptors and downstream Ca2+/PKC and ERK1/2 activation in BMSCs. There were no effect of NE on BMSCs differentiate into osteocytes and adipocytes.5. NE upregulated IL-6, SCF, LIF, VEGF mRNA and protein level expression in BMSCs.NE had no effect on CXCL12 mRNA expression. Our research demonstrated that NE stimulated BMSCs proliferation viaα1-ARs and downstream Ca2+/PKC and ERK1/2 activation .We discoverd the effect of NE on cytokines secretion first. Our result demonstrated signals from SNS are delivered to the HSPC pool directly, while others are exerted indirectly via an impact on niche-supporting BMSCs.These results provide a new recognition of sympathetic nerve neutotransmitter in bone marrow cells proliferation.更多还原