酒精对特发性股骨头坏死骨髓成骨微环境作用机制研究
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摘要
研究背景
股骨头缺血性坏死(osteonecrosis of the femoral head, ONFH)是由股骨头血供中断或骨细胞变性导致相关活力成分(骨细胞、骨髓造血细胞及脂肪细胞)死亡及随后的修复而导致股骨头结构改变、股骨头塌陷、关节功能障碍的一种疾病。它不是一个单一的过程,而是遗传因素与一个或多个危险因素综合作用的结果。它是骨科临床中常见疾病之一,是由不同原因导致的股骨头骨髓造血细胞、骨髓脂肪细胞及骨细胞变性坏死的一种疾病。
股骨头缺血性坏死主要分为两大类,包括创伤性股骨头坏死(traumatic osteonecrosis of femoral haead TONFH)和非创伤性股骨头坏死(non.traumatic osteonecrosis of femoralhead,NONFH)。在髋关节疾病中,它已经代替了髋关节结核的位置,跃居首位。股骨头缺血性坏死病程缓慢,病因复杂,治疗困难,致残率高,长期以来困扰着国内外学者,被医学界称为“不死的癌症”。目前临床上缺乏有效的早期筛查和检测手段,所以早期发现和诊断股骨头坏死从而进行药物或手术等有效干预非常欠缺,很多患者直到出现无法忍受疼痛,髋关节活动受限才来就诊,但此时大都已经出现了股骨头塌陷变形、骨性关节炎等晚期病变,只能施行人工髋关节置换手术,最终导致患者生活质量下降以及国家家庭个人的经济负担增加。因此,明确股骨头坏死的原因,弄清其发病机制,是临床和实验室专家一直努力研究的方向。
特发性股骨头缺血性坏死(Idiopathic femoral head necrosis,IFHN)主要包括激素性和酒精性的股骨头缺血坏死,约占股骨头缺血坏死病例的40%,临床上以酒精性股骨头缺血坏死常见。长期大量饮酒可引起股骨头髓内脂肪细胞大量增殖,脂肪细胞肥大,使相对密闭的骨髓内压力明显增高,微小血管受压变细,血流受阻。IFHN是一种常见致残性疾病,发病率较高,近年来呈逐渐上升趋势。如果早期得不到及时有效的处理,80%以上的患者将最终导致坏死性骨关节病的发生,甚至致残。
目前对于IFHN的发病机制尚不清楚,国内外尚无定论。股骨头缺血性坏死仍是医学界的一大难题,了解IFHN发病机制,对探索该病有效的预防、早期诊断和治疗方法有重要的临床意义。近年来,国内外学者对股骨头坏死的发病机制进行了深入研究,提出多种学说,包括：脂质代谢紊乱学说、血液循环的机械损伤学说、血管内凝血学说、骨内压增高学说、细胞毒性与细胞损伤学说、骨髓间充质干细胞成骨与成脂分化学说等。在目前诸多观点中,脂质代谢紊乱学说是一种相对成熟的观点。IFHN的发病机理极其复杂,髓内脂肪细胞增殖肥大继发脂肪栓塞在股骨头坏死发生发展过程中的作用逐渐受到重视。当前的相关研究认为脂肪细胞的生成在骨质减少性疾病的发生发展过程中起着非常重要的作用,“脂质学说”已成为目前IFHN发病的研究热点领域。
骨质疏松症是以破骨细胞对骨的吸收和成骨细胞成骨之间的平衡被打破造成的骨量减低为特点。因此,成骨细胞骨形成减少可导致骨质疏松症的进一步发展,同时,骨细胞凋亡率可以调节骨细胞的形成。在成年人中,负责骨形成的成骨细胞来自骨髓间充质干细胞(BMSCs)。通过几项研究的大量数据表明,成骨细胞和成脂肪细胞均有着共同的多能骨髓间充质干细胞,而成脂肪细胞和成骨细胞之间是相互作用的关系,即成脂细胞分化越来越多的同时,成骨细胞的形成则越来越少,反之亦然。
过量饮酒的习惯通常被认为是骨质疏松的主要原因并且可以增加发生骨折的风险。临床和实验研究目前证实,酒精引起的骨质疏松主要是由于抑制了新骨的形成,而对骨质的吸收方面影响较少。在成年人中,来自于骨髓中的骨髓间充质干细胞(BMSC)的成骨细胞主要负责骨的形成。这些骨髓间充质干细胞分化成骨细胞的能力在维持成人骨骼起中着关键的作用。
骨髓和其他组织中都可以有BMSCs的存在,它具有典型干细胞的特征,在不同的特定条件下它可以分化为不同的间充质组织,如再生骨、软骨、肌肉、脂肪等。它的分化环境主要在体内骨松质骨小梁及骨髓内。在受到外界刺激下,体内的成骨微环境中的细胞信号传导系统开始作用,BMSCs开始增殖分化,从原代细胞到成骨细胞逐步分化,最后参与完成体内组织的更新和创伤的修复,特别是在骨组织损伤后修复中,BMSCS发挥着尤为重要的作用。即使在体外,成骨细胞或成脂细胞诱导BMSCs分化的机制也涉及好几个方面。由于人体股骨头周围的特殊结构,头颈局部骨膜结构的缺乏从而导致使得骨膜内成骨不可能在股骨头部位产生,再加上该处软骨的成骨能力又非常弱,这些使股骨头部位的成骨机制被BMSCs成骨占据主要地位。相关研究表明,BMSCs可在酒精的诱导下大量成脂分化,而成骨分化受抑制,揭示了酒精性ONFH的新的发病机理。相关实验研究表明,成熟的成骨细胞可以通过细胞间的直接接触作用促进骨髓基质干细胞增殖。大量的临床及实验研究证实成骨细胞具有分泌作用,已经被证实的可分泌的生长因子有碱性成纤维细胞生长因子(b-FGF),胰岛素样生长因子(IGF),血小板衍生生长因子(PDGF),骨形成蛋白(BMP)等,它们被认为是BMSCs骨分化诱导因子。BMSCs表面的受体与这些生长因子结合后,细胞内信号系统被受体介导作用改变,从而激发BMSCs的特异性成骨转录因子Osf2/Cbfq1或Cbfq1/Runx2,使BMSCs的增殖及成骨分化加速。其中BMP和转化生长因子(TGF)对BMSCs分别起着骨诱导作用和增殖作用。
已有研究证实骨髓间充质干细胞具有分化为多种细胞的潜能,并可以分化成一种或多种细胞系。在其分化的多种细胞系中,成脂分化与成骨分化的关系最为密切：多项研究已经表明,成骨细胞和脂肪细胞具有共同祖母细胞-骨髓间充质干细胞。研究证实骨髓间充质干细胞的成脂分化与成骨分化的途径之间存在一种对立关系。已有证据显示成骨细胞和脂肪细胞之间在很大程度上是可以相互转换的,在成脂分化及成骨分化时两者相互影响,这可能为通过抑制脂肪细胞分化来防治骨质疏松症或其它骨疾病提供依据。因此,在这些过程中有关的信号通路被认为是治疗骨质减少性疾病时的目标所在。
酒精摄入后,在体内分解随着血液流经整个身体并穿过细胞膜进入细胞内,进而几乎影响所有细胞的正常生理活动。大量摄入酒精可以促进成脂细胞分化,抑制成骨。此外,慢性少量和大量饮酒同样会导致骨吸收增强而抑制骨形成。因此,酒精被认为是造成骨质疏松症的主要危险因素。酒精和激素是有力的IFHN发病因素,但它们具体是通过什么途径导致疾病的发生?抑或是多种途径联合起作用?抑或是另存在新的发病过程?目前尚无明确答案。阐明IFHN的发病机制,并据此寻找治疗的靶点仍是目前研究的一项重要任务。
微环境指细胞周围近距离作用和调控细胞兴奋或抑制活动的微量理化因素,以及该细胞在进化和分化过程中形成且对相关能量敏感的特殊结构和受体等在细胞局部组成的机能环境。许多研究表明激素和细胞分泌的各种因子对成骨细胞、骨髓间充质干细胞、造血干细胞群和体内骨重建的作用是极其复杂的,可以是交叉重叠的、协同的,也可以是拮抗的、短暂的。多种全身性的和局部的信号被网络中的细胞接受、传导、整合,然后被进一步释放刺激网络的其他成员,从而使信号逐级放大。在此过程中不仅把循环、局部的信号与骨微环境内的细胞联系起来,而且本身也参与了生理活动的调节。因此,骨髓内微环境的稳定以及骨吸收和骨形成之间的偶联,可能正是此调控网络通过骨髓内多种细胞之间的信号联系动态调节的结果。
一些研究数据表明,酒精通过上调过氧化酶基因的表达激活受体Y(PPARy),使得骨髓间充质干细胞由成骨分化向成脂肪细胞分化转换,但相关具体的转换机制目前并不清楚。
本项目通过对酒精作用下骨髓间充质干细胞分化方向、成骨细胞的成骨能力、脂肪细胞功能是否存在调节作用,以及脂肪细胞+骨髓间充质干细胞、脂肪细胞+成骨细胞共培养体系的影响进行研究。观察成骨与成脂分化的关键基因PPARy与cbfal在不同条件下的表达,检测成骨相关的指标ALP、OCN、I型胶原及脂肪细胞LPL。探讨致病因素的作用途径,及对髓内细胞池中骨髓间充质干细胞、成骨细胞、脂肪细胞的影响,脂肪细胞受到致病因子作用后是否会产生局部调节效应,以进一步阐明骨髓内成骨微环境在IFHN发病过程中的作用与可能机制,并为寻找可能的治疗靶点提供支持。
目的
1阐明IFHN发病过程中致病因素对髓内细胞池中骨髓间充质干细胞成脂与成骨分化影响所造成的脂质异常；
2探讨成骨细胞与脂肪细胞受酒精作用后的功能改变；
3探讨脂肪细胞功能改变对成骨细胞及骨髓间充质干细胞的调节作用；
4分析酒精对脂肪细胞+骨髓间充质干细胞,脂肪细胞+成骨细胞共培养体系的影响。
方法
1体外分离、培养新西兰白兔骨髓间充质干细胞、脂肪细胞及成骨细胞。作骨髓间充质干细胞多分化潜能鉴定以及骨髓成骨细胞及脂肪细胞的表型鉴定。
2应用酒精作用于骨髓间充质干细胞、脂肪细胞及成骨细胞,观察酒精对骨髓间充质干细胞的分化,以及脂肪细胞、成骨细胞的功能影响。
3用酒精作用于脂肪细胞后,制备条件培养基,然后作用于骨髓间充质干细胞与髓内成骨细胞,观察脂肪细胞受到影响后对二者的作用。
4将脂肪细胞+骨髓间充质干细胞,脂肪细胞+成骨细胞作共培养,观察共培养体系中的细胞分化与成骨指标的改变情况,以及在酒精作用下二共培养体系的细胞分化与成骨指标改变情况。
5检测细胞内质网的活性及与之相关的基因的变化,采用RT-PCR和免疫印迹法实时定量分析脂肪细胞和成骨细胞的标志物,进行油红O染色,并对形成的脂肪细胞进行量化,对成骨细胞的生物标志物碱性磷酸酶及Ⅰ型胶原和骨钙素进行检测,同时还进行半胱氨酸蛋白酶3的活性测定,进而评估骨髓间充质干细胞的凋亡程度。
结果
培养12天后对两组细胞进行ALP染色,显示实验组细胞质内ALP染色阳性反应明显,对照组染色显色弱,实验组细胞钙化结节数量多且较大,对照组也有钙结节形成,但较小。实验组标准钙化结节计数显著高于对照组,差异具有统计学意义(P<0.05),加入0.15mol/L酒精干预后,成骨细胞发生皱缩,AO/EB染色出现凋亡细胞。取生长对数期的兔骨髓间充质干细胞进行诱导培养,当汇合度达到70-80%的加成骨诱导培养基,进行诱导培养。随着时间延长,实验组成骨细胞数量明显多于对照组。在3、6、9天的每张爬片各取4个低倍视野进行钙结节成骨数量计数,实验组分别为(200.83±24.73)、(1112.00±77.56)、(1199.00±44.91)个/cm2；对照组分别是(99.75±10.15)、(790.00±94.13)、(1059.00±26.39)个/cm2(P<0.001)
随着时间延长,实验组脂肪细胞数量明显多于对照组,两组小脂滴均逐渐增多、增大,但实验组更明显。在4、6、8、10d的髓内脂肪细胞数量实验组分别为(200.83±24.78)、(1102.00±76.21)、(1160.00±28.83)、(1199.00±44.51)个/cm2；对照组分别是(99.75±10.67)、(790.00±94.63)、(1000.00±41.30)、(1059.00±26.54)个/cm2,脂肪细胞数量随酒精作用的时间延长而增多(P<0.001)。研究发现,加入了酒精的培养皿中检测到脂肪细胞标记物(PPARγ2和AP2)数量增加、成骨标记(Osf2/Cbfa1)数量减少,而表现为脂滴蓄积的现象,因此,长期接触酒精能够使骨髓间充质干细胞成脂细胞分化增强,成骨分化受到抑制。
结论
通过密度梯度离心贴壁法所获得的骨髓间充质干细胞在常规培养及诱导培养时均可表现出较强的增殖能力及成骨、成脂分化潜能,足以表明骨髓间充质干细胞能够为骨组织工程的研究提供可靠种子细胞来源；酒精抑制骨髓间充质干细胞的成骨分化并能促进其向成脂细胞分化；酒精能够诱导成骨细胞的凋亡,促进骨髓内脂肪细胞的增殖肥大。长期过量饮酒会导致股骨头髓内脂肪细胞快速大量增殖,同时脂肪细胞较正常肥大,从而使相对封闭的骨髓内压力显著增高,微小血管因受压而变细,最终导致血流受阻或者中断,从而形成股骨头缺血性坏死。
主要创新点
1、本研究提出酒精能够促进髓内脂肪细胞增殖肥大,同时可以抑制骨髓间充质干细胞的成骨细胞分化；
2、本研究提出酒精作用于脂肪细胞后的代谢产物对骨髓间充质干细胞及骨细胞的增殖均具有一定的抑制作用；
3、本研究建立了脂肪细胞+骨髓间充质干细胞,脂肪细胞+成骨细胞共培养体系。
Background
Osteonecrosis of the femoral head (ONFH) is a disease which was caused by the changes of the femoral head structure, collapsed of the femoral head, joint dysfunction after dynamic components death and subsequent repair by the femoral blood supply interruption or bone cell degeneration. It is not a single process, but the results of genetic factors and one or more risk factors role. It was a pathologic process caused by multi—pathogenic factors that damaged the blood supply of the femoral head, which led to the death ofinyeloid element,adipocyte and osteocytes.
Osteonecrosis of the femoral head was a pathologic process caused by multi—pathogenic factors that damaged the blood supply of the femoral head, which led to the death ofinyeloid element,adipocyte and osteocytes. It is one of the common clinic diseases and mainly divided into two groups:traumatic osteonecrosis of femoral head(TONFH) and non—traumatic osteonecrosis of femoral head(NONFH). Some data indicates that ONFH has replaced the hip tuberculosis as the first place in hip diseases. Osteonecrosis of the femoral head have troubled domestic and foreign scholars for a long time.it is also called"the deadless cancer". But now, due to a lack of effective early screening and detection methods, it is rarely detected and intervented in early time. So most patients have a feel of hip'pain and limitation of hip'motion for a long time, then, think about the possibility of osteonecrosis of the femoral head. At this time, these patients had to be operated with THA because of the collapse and defomation of femoral head and osteoarthritis. It is the clinical and laboratory experts' task to make clear its pathogenesis, search valuable biomarkers for early screening and treatment.
Idiopathic femoral head necrosis (IFHN) account40%of osteonecrosis of the femoral head, including hormones and alcoholic osteonecrosis,the alcoholic osteonecrosis is more. Long-term and excessive quantities of alcoholy can make a lot of femoral intramedullary fat cells proliferated and fat cells hypertrophia,so that a relatively confined intramedullary pressure is significantly increased, tiny blood vessels become thin, blood flow is blocked. IFHN is a common disabling disease.It has a high incidence and show a gradual upward trend in recent years. If the lack of timely and effective early treatment, more than80%of patients will eventually lead to necrosis of bone and joint disease, or disability.
At present, the pathogenesis of IFHN is not yet clear.There are not any similar reports at home and abroad. Osteonecrosis of the femoral head is still a major problem in the medical profession.There is an important clinical significance in the aspects of the search for effective disease prevention, early diagnosis and treatment by understanding the pathogenesis of IFHN. In recent years, scholars make more and more depth research about the pathogenesis of osteonecrosis.They proposed a variety of theories including the doctrine of lipid metabolism, the doctrine of mechanical damage to the blood circulation, intravascular coagulation theory, theory of increased pressure within the bone, cytotoxicity and cell damage theory, bone marrow mesenchymal stem cells into bone and adipogenic differentiation doctrine and so on. The doctrine of lipid metabolism was more mature than others. The pathogenesis of IFHN is extremely complex. Proliferation and hypertrophy of intramedullary fat cells, fat embolism were taken more attention in the development of osteonecrosis. The current study suggests that" the generation of fat cells," is an important mechanism for the development of osteopenia of the disease,"lipid theory" has become a hot area of IFHN disease.
Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and thus rate of apoptosis of osteoporosis is responsible for the regulation of bone formation. In adults, the osteoblast, which is responsible for bone formation, is derived from multipotential mesenchymal stem cells (MSC) in bone marrow (BMSCs). Several studies have provided substantial evidence that osteoblasts and adipocytes share common progenitor:multipotential BMSCs, and the relationship between adipogenesis and osteogenesis is reciprocal with increasing adipogenesis accompanied by decreasing osteogenesis and vice versa.
Habitual consumption of excessive quantities of alcohol is commonly recognized as a major factor for osteopaenia and increased incidence of bone fracture in alcoholics. The current consensus of both clinical and experimental studies is that alcohol-induced bone loss is mainly be due to the suppression of new bone formation with only relatively small changes (increase or decrease) occur in bone resorption. In adults, the osteoblast, which is responsible for bone formation, is derived from multipotential mesenchymal stem cells in bone marrow (BMSC). The capacity and dynamics of these BMSCs to differentiate into osteoblasts plays a critical role in the cellular processes involved in the maintenance of the adult human skeleton.
BMSCs can exist in the bone marrow and other tissues, it has the typical characteristics of stem cells, under different conditions which can differentiate into a specific type of mesenchymal tissue, such as the regeneration of bone, cartilage, muscle and fat. Its main differentiation environment are spongy trabecular bone and bone marrow. Subjected to external stimulation, cell signaling systems of osteogenic microenvironment began to work, BMSCs began to proliferate and differentiate, from primary cells to osteoblasts, involved in updating tissue and repairing wounds,particularly in repairing the bone tissue injury, BMSCS plays a particularly important role. Even in vitro, mechanism of osteoblasts or adipogenic induced BMSCs differentiation is also involved in several respects. Osteogenesis in the periosteum can not be produced in the femoral head because of the special structure of human femoral head around that femoral head and neck lacks of periosteal structure, coupled with the ability of cartilage into bone was very weak in this place which made osteogenesis of BMSCS has the dominant position in osteogenic mechanisms of the femoral bone. Research shows that BMSCS can be a lot of adipogenic differentiation in the induction of alcohol, however, osteogenic differentiation was inhibited which revealed a new pathogenesis of alcoholic ONFH. Relevant experiments studies had shown that mature osteoblasts can promote the proliferation of bone marrow stromal cells through direct contact with the role of cells. Large number of clinical and experimental studies had confirmed that osteoblasts have secretory action,they could secrete b-FGF, IGF, PDGF, BMP and so on. They were considered as BMSCs bone differentiation-inducing factor.The surface receptor of BMSCs after combined with these growth factors, intracellular signaling receptor mediated effect was changed which to stimulate osteoblast-specific transcription factor Osf2/Cbfql or Cbfql/Runx2, then the proliferation and osteogenic differentiation of BMSCs was accelerated.BMP and TGF plays on BMSCs were induced bone and proliferation.
The BMSCs have been proven to differentiate into multiple lineages and generate progenitors committed to one or more cell lines. Among these multiple lineages, those of adipogenesis and osteogenesis are the most closely related:Several studies have provided substantial evidence that osteoblasts and adipocytes share a common progenitor:multipotential BMSCs. An inverse relationship has been demonstrated between osteogenesis and adipogenesis suggesting the possible reciprocal relationship between the two differentiation pathways. There has been evidence for the differentiation switching between these two cell lineages, suggesting a large degree of plasticity between osteoblasts and adipocytes. Because the relationship between adipogenesis and osteogenesis is reciprocal and the adipocytic and osteogenic cells share a common lineage, it is possible that inhibition of adipogenesis may provide an approach to prevent or treat osteoporosis or other bone diseases. Thus, the signal transduction pathways implicated in these processes are evaluated as potential targets for therapeutic intervention of osteopenic disorders.
After consumption, alcohol is readily distributed throughout the body in the blood stream and crosses biological membranes, affecting virtually all biological processes inside the cell. Excessive alcohol consumption substantially promotes adipogenesis and inhibits osteogenesis. Moreover, both chronic and binge consumptions of ethanol are known to cause suppression of bone formation and enhancement of bone resorption. Thus, alcohol is considered a major risk factor for osteoporosis, a disease particularly prevalent in women worldwide. Alcohol and hormones are powerful factors of the pathogenesis of IFHN, but what channels how do they through to make the disease? Or a variety of ways to jointly work? or a new disease process? There is no clear answer. To find therapeutic targets according to elucidate the pathogenesis IFHN remains an important task for the present study.
Microenvironment is a functional environment which was composed including two aspects:microamount physical and chemical factors of the surrounding cells which can excite or inhibit the action of cells by Close-up action and regulation,and special structures and receptors which are formed in the process of evolution and differentiation of the cell. Many studies have shown that the role of hormones and partial adjustment factors on osteoblasts, BMSC, hematopoietic stem cells and the role of vivo bone reconstruction is extremely complex which can be overlapping, collaborative, antagonistic or short-lived. Many kinds of systemic and local signal was received,conducted and integrated by the cell in the net, and further stimulate the release of other members of the network, so that a signal amplification cascade. In this process. not only the cycles of the local cell signal within the bone microenvironment link, but is also involved in the regulation of its physiological activities. Thus, the coupling within the bone marrow microenvironment and stability between bone resorption and bone formation, perhaps the result of this regulatory network via signal contact between cells in the bone marrow variety of dynamic regulation.
Several studies have provided evidence that alcohol shifts lineage commitment and differentiation of BMSCs from osteogenesis to adipogenesis by upregulation of gene expression for peroxisome proliferator-activated receptor gamma (PPARy); however, the molecular mechanism underlying the reciprocal relationship is not yet well understood.
Through this project,we make a research about whether the existence of regulation of the the differentiation direction of BMSC by the influence of alcohol, the ability of osteoblasts and fat cell function, as well as the system of fat cells+BMSC, fat cells+osteoblast cells. We observed the expression of PPARy and cbfal which are the key gene of adipogenic Osteogenic differentiation differentiation under different conditions, detect osteogenesis-related indicators,such as ALP, OCN, I collagen and fat cells LPL. Explore the pathways of the risk factors, and its influence to intramedullary BMSC cells, osteoblasts, adipocytes. Whether it will produce local effects after the fat cells are effected by causative factors, further clarify the contribution and mechanisms of the bone microenvironment of the bone marrow in the pathogenesis of IFHN.
Objective
1To elucidate lipid abnormalities which was caused by the risk factors effected the BMSC adipogenic and osteogenic differentiation of the Intramedullary cell pool in the pathogenesis of IFHN.
2To discuss functional changes of osteoblasts and fat cells afer effectd
3To investigate the adjustment of fat cells' changes to osteoblasts and BMSC
4To analyse the effection of alcohol on co-culture system which was Consisted of fat cells+BMSC and fat cells+osteoblasts
Methods
1We isolated and cultured marrow BMSC, fat cells and osteoblasts from the New Zealand rabbits;made an identification about BMSC differentiation potential,the surface shape of marrow osteoblasts and fat cellcells.
2To observe the effection to the differentiation of BMSC, function of fat cells and osteoblasts after applicating of the role of alcohol in BMSC, fat cells and osteoblasts.
3After alcohol acted on fat cells, we Preparated the conditioned medium,then acted on intramedullary osteoblasts and BMSC,observed the contribution after the fat cells was effected.
4We made a co-culture system which was Consisted of fat cells+BMSC and fat cells+osteoblasts, observed the circumstances change of cell differentiation and osteogenic index in the co-culture system and the change after acted by alcohol.
5We measured changes of genes related to endoplasmic reticulum (ER) stress, adipogenic markers and osteogenic markers using quantitative real-time RT-PCR and Western blot analysis. We performed Alizarin red staining for osteogenesis. We also conducted assays for osteogenic biomarkers alkaline phosphatase, collagen-I and osteocalcin.We also conducted caspase3activity assay to assess BMSC apoptosis.
Results
After12days of culture, ALP staining was performed on two groups of cells, the experimental group showed that the cytoplasmic ALP staining was positive, the control group, experimental group cells staining, alizarin red staining after calcified nodules number and larger, control group with a small amount of calcium nodules, but relatively small, experimental group standard calcium nodule count significantly higher than the control group differences were statistically significant (P<0.05). We trained the BMSCs of rabbit in logarithmic growth phase for induction, when the confluence reached70-80%, added osteogenic induction medium. With time, the number of osteoblasts of the experimental group was significantly more than the control group. The number of Calcium nodules in experimental groups of3,6,9d, respectively (200.83±24.73)、(1112.00±77.56)、(1199.00±44.91)个/cm2; control group, respectively.(99.75±10.15)、(790.00±94.13)、(1059.00±26.39)个/cm2(P<0.001).
With time, the number of fat cells of the experimental group was significantly more than the control group. Two groups of small lipid droplets are gradually increasing and enlarging, but the experimental group was significant. The number of experimental groups in the the intramedullary fat cells of4,6,8,10d, respectively (200.83±24.78)、(1102.00±76.21)、(1160.00±28.83)、(1199.00±44.51)/cm2; control group, respectively.(99.75±10.67)、(790.00±94.63)、(1000.00±41.30)、(1059.00±26.54)/cm2, the number of fat cells increased with time of the influence of alcohol (P<0.001). We showed here that chronic exposure of BMSCs to alcohol induced adipogenesis and disrupted osteogenesis as indicated by upregulation of adipogenic markers (PPARy2and aP2), downregulation of osteogenic markers (Osf2/Cbfal), and accumulation of lipid droplets. Long-term excessive drinking could lead to femoral intramedullary fat cells rapidly proliferate, meanwhile,the fat cells were hypertrophy than normal, so that the relatively closed intramedullary pressure increased significantly, tiny blood vessels become thinner due to pressure, eventually leading to disruption or interruption of blood flow, thereby forming osteonecrosis of the femoral head.
Conclusions
By density gradient centrifugation and differential velocity adherent method the obtained BMSCs in conventional culture or inducing culture can be showed osteogenic potential of bone induction ability cultivation.lt shows that BMSCs can provide a reliable source of seed cells for bone tissue engineering research. Alcohol can restrain BMSC into osteoblasts and promote them into adipocytes; Alcohol can cause the osteoblasts to death,promote the intramedullary fat cells to increase and enlarge.
Innovation
1.This study proposes that alcohol can promote intramedullary fat cells proliferation and hypertrophy and suppress osteogenic differentiation potential of BMSCs
2.This study presents that the influence of alcohol on the metabolites of fat cells have certain extent to the proliferation of mesenchymal stem cells and bone marrow cells.
3. This study established the fat cells+bone marrow mesenchymal stem cells, adipocytes+osteoblasts co-culture system.
股骨头缺血性坏死(osteonecrosis of the femoral head, ONFH)是由股骨头血供中断或骨细胞变性导致相关活力成分(骨细胞、骨髓造血细胞及脂肪细胞)死亡及随后的修复而导致股骨头结构改变、股骨头塌陷、关节功能障碍的一种疾病。它不是一个单一的过程,而是遗传因素与一个或多个危险因素综合作用的结果。它是骨科临床中常见疾病之一,是由不同原因导致的股骨头骨髓造血细胞、骨髓脂肪细胞及骨细胞变性坏死的一种疾病。
股骨头缺血性坏死主要分为两大类,包括创伤性股骨头坏死(traumatic osteonecrosis of femoral haead TONFH)和非创伤性股骨头坏死(non.traumatic osteonecrosis of femoralhead,NONFH)。在髋关节疾病中,它已经代替了髋关节结核的位置,跃居首位。股骨头缺血性坏死病程缓慢,病因复杂,治疗困难,致残率高,长期以来困扰着国内外学者,被医学界称为“不死的癌症”。目前临床上缺乏有效的早期筛查和检测手段,所以早期发现和诊断股骨头坏死从而进行药物或手术等有效干预非常欠缺,很多患者直到出现无法忍受疼痛,髋关节活动受限才来就诊,但此时大都已经出现了股骨头塌陷变形、骨性关节炎等晚期病变,只能施行人工髋关节置换手术,最终导致患者生活质量下降以及国家家庭个人的经济负担增加。因此,明确股骨头坏死的原因,弄清其发病机制,是临床和实验室专家一直努力研究的方向。
特发性股骨头缺血性坏死(Idiopathic femoral head necrosis,IFHN)主要包括激素性和酒精性的股骨头缺血坏死,约占股骨头缺血坏死病例的40%,临床上以酒精性股骨头缺血坏死常见。长期大量饮酒可引起股骨头髓内脂肪细胞大量增殖,脂肪细胞肥大,使相对密闭的骨髓内压力明显增高,微小血管受压变细,血流受阻。IFHN是一种常见致残性疾病,发病率较高,近年来呈逐渐上升趋势。如果早期得不到及时有效的处理,80%以上的患者将最终导致坏死性骨关节病的发生,甚至致残。
目前对于IFHN的发病机制尚不清楚,国内外尚无定论。股骨头缺血性坏死仍是医学界的一大难题,了解IFHN发病机制,对探索该病有效的预防、早期诊断和治疗方法有重要的临床意义。近年来,国内外学者对股骨头坏死的发病机制进行了深入研究,提出多种学说,包括：脂质代谢紊乱学说、血液循环的机械损伤学说、血管内凝血学说、骨内压增高学说、细胞毒性与细胞损伤学说、骨髓间充质干细胞成骨与成脂分化学说等。在目前诸多观点中,脂质代谢紊乱学说是一种相对成熟的观点。IFHN的发病机理极其复杂,髓内脂肪细胞增殖肥大继发脂肪栓塞在股骨头坏死发生发展过程中的作用逐渐受到重视。当前的相关研究认为脂肪细胞的生成在骨质减少性疾病的发生发展过程中起着非常重要的作用,“脂质学说”已成为目前IFHN发病的研究热点领域。
骨质疏松症是以破骨细胞对骨的吸收和成骨细胞成骨之间的平衡被打破造成的骨量减低为特点。因此,成骨细胞骨形成减少可导致骨质疏松症的进一步发展,同时,骨细胞凋亡率可以调节骨细胞的形成。在成年人中,负责骨形成的成骨细胞来自骨髓间充质干细胞(BMSCs)。通过几项研究的大量数据表明,成骨细胞和成脂肪细胞均有着共同的多能骨髓间充质干细胞,而成脂肪细胞和成骨细胞之间是相互作用的关系,即成脂细胞分化越来越多的同时,成骨细胞的形成则越来越少,反之亦然。
过量饮酒的习惯通常被认为是骨质疏松的主要原因并且可以增加发生骨折的风险。临床和实验研究目前证实,酒精引起的骨质疏松主要是由于抑制了新骨的形成,而对骨质的吸收方面影响较少。在成年人中,来自于骨髓中的骨髓间充质干细胞(BMSC)的成骨细胞主要负责骨的形成。这些骨髓间充质干细胞分化成骨细胞的能力在维持成人骨骼起中着关键的作用。
骨髓和其他组织中都可以有BMSCs的存在,它具有典型干细胞的特征,在不同的特定条件下它可以分化为不同的间充质组织,如再生骨、软骨、肌肉、脂肪等。它的分化环境主要在体内骨松质骨小梁及骨髓内。在受到外界刺激下,体内的成骨微环境中的细胞信号传导系统开始作用,BMSCs开始增殖分化,从原代细胞到成骨细胞逐步分化,最后参与完成体内组织的更新和创伤的修复,特别是在骨组织损伤后修复中,BMSCS发挥着尤为重要的作用。即使在体外,成骨细胞或成脂细胞诱导BMSCs分化的机制也涉及好几个方面。由于人体股骨头周围的特殊结构,头颈局部骨膜结构的缺乏从而导致使得骨膜内成骨不可能在股骨头部位产生,再加上该处软骨的成骨能力又非常弱,这些使股骨头部位的成骨机制被BMSCs成骨占据主要地位。相关研究表明,BMSCs可在酒精的诱导下大量成脂分化,而成骨分化受抑制,揭示了酒精性ONFH的新的发病机理。相关实验研究表明,成熟的成骨细胞可以通过细胞间的直接接触作用促进骨髓基质干细胞增殖。大量的临床及实验研究证实成骨细胞具有分泌作用,已经被证实的可分泌的生长因子有碱性成纤维细胞生长因子(b-FGF),胰岛素样生长因子(IGF),血小板衍生生长因子(PDGF),骨形成蛋白(BMP)等,它们被认为是BMSCs骨分化诱导因子。BMSCs表面的受体与这些生长因子结合后,细胞内信号系统被受体介导作用改变,从而激发BMSCs的特异性成骨转录因子Osf2/Cbfq1或Cbfq1/Runx2,使BMSCs的增殖及成骨分化加速。其中BMP和转化生长因子(TGF)对BMSCs分别起着骨诱导作用和增殖作用。
已有研究证实骨髓间充质干细胞具有分化为多种细胞的潜能,并可以分化成一种或多种细胞系。在其分化的多种细胞系中,成脂分化与成骨分化的关系最为密切：多项研究已经表明,成骨细胞和脂肪细胞具有共同祖母细胞-骨髓间充质干细胞。研究证实骨髓间充质干细胞的成脂分化与成骨分化的途径之间存在一种对立关系。已有证据显示成骨细胞和脂肪细胞之间在很大程度上是可以相互转换的,在成脂分化及成骨分化时两者相互影响,这可能为通过抑制脂肪细胞分化来防治骨质疏松症或其它骨疾病提供依据。因此,在这些过程中有关的信号通路被认为是治疗骨质减少性疾病时的目标所在。
酒精摄入后,在体内分解随着血液流经整个身体并穿过细胞膜进入细胞内,进而几乎影响所有细胞的正常生理活动。大量摄入酒精可以促进成脂细胞分化,抑制成骨。此外,慢性少量和大量饮酒同样会导致骨吸收增强而抑制骨形成。因此,酒精被认为是造成骨质疏松症的主要危险因素。酒精和激素是有力的IFHN发病因素,但它们具体是通过什么途径导致疾病的发生?抑或是多种途径联合起作用?抑或是另存在新的发病过程?目前尚无明确答案。阐明IFHN的发病机制,并据此寻找治疗的靶点仍是目前研究的一项重要任务。
微环境指细胞周围近距离作用和调控细胞兴奋或抑制活动的微量理化因素,以及该细胞在进化和分化过程中形成且对相关能量敏感的特殊结构和受体等在细胞局部组成的机能环境。许多研究表明激素和细胞分泌的各种因子对成骨细胞、骨髓间充质干细胞、造血干细胞群和体内骨重建的作用是极其复杂的,可以是交叉重叠的、协同的,也可以是拮抗的、短暂的。多种全身性的和局部的信号被网络中的细胞接受、传导、整合,然后被进一步释放刺激网络的其他成员,从而使信号逐级放大。在此过程中不仅把循环、局部的信号与骨微环境内的细胞联系起来,而且本身也参与了生理活动的调节。因此,骨髓内微环境的稳定以及骨吸收和骨形成之间的偶联,可能正是此调控网络通过骨髓内多种细胞之间的信号联系动态调节的结果。
一些研究数据表明,酒精通过上调过氧化酶基因的表达激活受体Y(PPARy),使得骨髓间充质干细胞由成骨分化向成脂肪细胞分化转换,但相关具体的转换机制目前并不清楚。
本项目通过对酒精作用下骨髓间充质干细胞分化方向、成骨细胞的成骨能力、脂肪细胞功能是否存在调节作用,以及脂肪细胞+骨髓间充质干细胞、脂肪细胞+成骨细胞共培养体系的影响进行研究。观察成骨与成脂分化的关键基因PPARy与cbfal在不同条件下的表达,检测成骨相关的指标ALP、OCN、I型胶原及脂肪细胞LPL。探讨致病因素的作用途径,及对髓内细胞池中骨髓间充质干细胞、成骨细胞、脂肪细胞的影响,脂肪细胞受到致病因子作用后是否会产生局部调节效应,以进一步阐明骨髓内成骨微环境在IFHN发病过程中的作用与可能机制,并为寻找可能的治疗靶点提供支持。
目的
1阐明IFHN发病过程中致病因素对髓内细胞池中骨髓间充质干细胞成脂与成骨分化影响所造成的脂质异常；
2探讨成骨细胞与脂肪细胞受酒精作用后的功能改变；
3探讨脂肪细胞功能改变对成骨细胞及骨髓间充质干细胞的调节作用；
4分析酒精对脂肪细胞+骨髓间充质干细胞,脂肪细胞+成骨细胞共培养体系的影响。
方法
1体外分离、培养新西兰白兔骨髓间充质干细胞、脂肪细胞及成骨细胞。作骨髓间充质干细胞多分化潜能鉴定以及骨髓成骨细胞及脂肪细胞的表型鉴定。
2应用酒精作用于骨髓间充质干细胞、脂肪细胞及成骨细胞,观察酒精对骨髓间充质干细胞的分化,以及脂肪细胞、成骨细胞的功能影响。
3用酒精作用于脂肪细胞后,制备条件培养基,然后作用于骨髓间充质干细胞与髓内成骨细胞,观察脂肪细胞受到影响后对二者的作用。
4将脂肪细胞+骨髓间充质干细胞,脂肪细胞+成骨细胞作共培养,观察共培养体系中的细胞分化与成骨指标的改变情况,以及在酒精作用下二共培养体系的细胞分化与成骨指标改变情况。
5检测细胞内质网的活性及与之相关的基因的变化,采用RT-PCR和免疫印迹法实时定量分析脂肪细胞和成骨细胞的标志物,进行油红O染色,并对形成的脂肪细胞进行量化,对成骨细胞的生物标志物碱性磷酸酶及Ⅰ型胶原和骨钙素进行检测,同时还进行半胱氨酸蛋白酶3的活性测定,进而评估骨髓间充质干细胞的凋亡程度。
结果
培养12天后对两组细胞进行ALP染色,显示实验组细胞质内ALP染色阳性反应明显,对照组染色显色弱,实验组细胞钙化结节数量多且较大,对照组也有钙结节形成,但较小。实验组标准钙化结节计数显著高于对照组,差异具有统计学意义(P<0.05),加入0.15mol/L酒精干预后,成骨细胞发生皱缩,AO/EB染色出现凋亡细胞。取生长对数期的兔骨髓间充质干细胞进行诱导培养,当汇合度达到70-80%的加成骨诱导培养基,进行诱导培养。随着时间延长,实验组成骨细胞数量明显多于对照组。在3、6、9天的每张爬片各取4个低倍视野进行钙结节成骨数量计数,实验组分别为(200.83±24.73)、(1112.00±77.56)、(1199.00±44.91)个/cm2；对照组分别是(99.75±10.15)、(790.00±94.13)、(1059.00±26.39)个/cm2(P<0.001)
随着时间延长,实验组脂肪细胞数量明显多于对照组,两组小脂滴均逐渐增多、增大,但实验组更明显。在4、6、8、10d的髓内脂肪细胞数量实验组分别为(200.83±24.78)、(1102.00±76.21)、(1160.00±28.83)、(1199.00±44.51)个/cm2；对照组分别是(99.75±10.67)、(790.00±94.63)、(1000.00±41.30)、(1059.00±26.54)个/cm2,脂肪细胞数量随酒精作用的时间延长而增多(P<0.001)。研究发现,加入了酒精的培养皿中检测到脂肪细胞标记物(PPARγ2和AP2)数量增加、成骨标记(Osf2/Cbfa1)数量减少,而表现为脂滴蓄积的现象,因此,长期接触酒精能够使骨髓间充质干细胞成脂细胞分化增强,成骨分化受到抑制。
结论
通过密度梯度离心贴壁法所获得的骨髓间充质干细胞在常规培养及诱导培养时均可表现出较强的增殖能力及成骨、成脂分化潜能,足以表明骨髓间充质干细胞能够为骨组织工程的研究提供可靠种子细胞来源；酒精抑制骨髓间充质干细胞的成骨分化并能促进其向成脂细胞分化；酒精能够诱导成骨细胞的凋亡,促进骨髓内脂肪细胞的增殖肥大。长期过量饮酒会导致股骨头髓内脂肪细胞快速大量增殖,同时脂肪细胞较正常肥大,从而使相对封闭的骨髓内压力显著增高,微小血管因受压而变细,最终导致血流受阻或者中断,从而形成股骨头缺血性坏死。
主要创新点
1、本研究提出酒精能够促进髓内脂肪细胞增殖肥大,同时可以抑制骨髓间充质干细胞的成骨细胞分化；
2、本研究提出酒精作用于脂肪细胞后的代谢产物对骨髓间充质干细胞及骨细胞的增殖均具有一定的抑制作用；
3、本研究建立了脂肪细胞+骨髓间充质干细胞,脂肪细胞+成骨细胞共培养体系。
Background
Osteonecrosis of the femoral head (ONFH) is a disease which was caused by the changes of the femoral head structure, collapsed of the femoral head, joint dysfunction after dynamic components death and subsequent repair by the femoral blood supply interruption or bone cell degeneration. It is not a single process, but the results of genetic factors and one or more risk factors role. It was a pathologic process caused by multi—pathogenic factors that damaged the blood supply of the femoral head, which led to the death ofinyeloid element,adipocyte and osteocytes.
Osteonecrosis of the femoral head was a pathologic process caused by multi—pathogenic factors that damaged the blood supply of the femoral head, which led to the death ofinyeloid element,adipocyte and osteocytes. It is one of the common clinic diseases and mainly divided into two groups:traumatic osteonecrosis of femoral head(TONFH) and non—traumatic osteonecrosis of femoral head(NONFH). Some data indicates that ONFH has replaced the hip tuberculosis as the first place in hip diseases. Osteonecrosis of the femoral head have troubled domestic and foreign scholars for a long time.it is also called"the deadless cancer". But now, due to a lack of effective early screening and detection methods, it is rarely detected and intervented in early time. So most patients have a feel of hip'pain and limitation of hip'motion for a long time, then, think about the possibility of osteonecrosis of the femoral head. At this time, these patients had to be operated with THA because of the collapse and defomation of femoral head and osteoarthritis. It is the clinical and laboratory experts' task to make clear its pathogenesis, search valuable biomarkers for early screening and treatment.
Idiopathic femoral head necrosis (IFHN) account40%of osteonecrosis of the femoral head, including hormones and alcoholic osteonecrosis,the alcoholic osteonecrosis is more. Long-term and excessive quantities of alcoholy can make a lot of femoral intramedullary fat cells proliferated and fat cells hypertrophia,so that a relatively confined intramedullary pressure is significantly increased, tiny blood vessels become thin, blood flow is blocked. IFHN is a common disabling disease.It has a high incidence and show a gradual upward trend in recent years. If the lack of timely and effective early treatment, more than80%of patients will eventually lead to necrosis of bone and joint disease, or disability.
At present, the pathogenesis of IFHN is not yet clear.There are not any similar reports at home and abroad. Osteonecrosis of the femoral head is still a major problem in the medical profession.There is an important clinical significance in the aspects of the search for effective disease prevention, early diagnosis and treatment by understanding the pathogenesis of IFHN. In recent years, scholars make more and more depth research about the pathogenesis of osteonecrosis.They proposed a variety of theories including the doctrine of lipid metabolism, the doctrine of mechanical damage to the blood circulation, intravascular coagulation theory, theory of increased pressure within the bone, cytotoxicity and cell damage theory, bone marrow mesenchymal stem cells into bone and adipogenic differentiation doctrine and so on. The doctrine of lipid metabolism was more mature than others. The pathogenesis of IFHN is extremely complex. Proliferation and hypertrophy of intramedullary fat cells, fat embolism were taken more attention in the development of osteonecrosis. The current study suggests that" the generation of fat cells," is an important mechanism for the development of osteopenia of the disease,"lipid theory" has become a hot area of IFHN disease.
Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and thus rate of apoptosis of osteoporosis is responsible for the regulation of bone formation. In adults, the osteoblast, which is responsible for bone formation, is derived from multipotential mesenchymal stem cells (MSC) in bone marrow (BMSCs). Several studies have provided substantial evidence that osteoblasts and adipocytes share common progenitor:multipotential BMSCs, and the relationship between adipogenesis and osteogenesis is reciprocal with increasing adipogenesis accompanied by decreasing osteogenesis and vice versa.
Habitual consumption of excessive quantities of alcohol is commonly recognized as a major factor for osteopaenia and increased incidence of bone fracture in alcoholics. The current consensus of both clinical and experimental studies is that alcohol-induced bone loss is mainly be due to the suppression of new bone formation with only relatively small changes (increase or decrease) occur in bone resorption. In adults, the osteoblast, which is responsible for bone formation, is derived from multipotential mesenchymal stem cells in bone marrow (BMSC). The capacity and dynamics of these BMSCs to differentiate into osteoblasts plays a critical role in the cellular processes involved in the maintenance of the adult human skeleton.
BMSCs can exist in the bone marrow and other tissues, it has the typical characteristics of stem cells, under different conditions which can differentiate into a specific type of mesenchymal tissue, such as the regeneration of bone, cartilage, muscle and fat. Its main differentiation environment are spongy trabecular bone and bone marrow. Subjected to external stimulation, cell signaling systems of osteogenic microenvironment began to work, BMSCs began to proliferate and differentiate, from primary cells to osteoblasts, involved in updating tissue and repairing wounds,particularly in repairing the bone tissue injury, BMSCS plays a particularly important role. Even in vitro, mechanism of osteoblasts or adipogenic induced BMSCs differentiation is also involved in several respects. Osteogenesis in the periosteum can not be produced in the femoral head because of the special structure of human femoral head around that femoral head and neck lacks of periosteal structure, coupled with the ability of cartilage into bone was very weak in this place which made osteogenesis of BMSCS has the dominant position in osteogenic mechanisms of the femoral bone. Research shows that BMSCS can be a lot of adipogenic differentiation in the induction of alcohol, however, osteogenic differentiation was inhibited which revealed a new pathogenesis of alcoholic ONFH. Relevant experiments studies had shown that mature osteoblasts can promote the proliferation of bone marrow stromal cells through direct contact with the role of cells. Large number of clinical and experimental studies had confirmed that osteoblasts have secretory action,they could secrete b-FGF, IGF, PDGF, BMP and so on. They were considered as BMSCs bone differentiation-inducing factor.The surface receptor of BMSCs after combined with these growth factors, intracellular signaling receptor mediated effect was changed which to stimulate osteoblast-specific transcription factor Osf2/Cbfql or Cbfql/Runx2, then the proliferation and osteogenic differentiation of BMSCs was accelerated.BMP and TGF plays on BMSCs were induced bone and proliferation.
The BMSCs have been proven to differentiate into multiple lineages and generate progenitors committed to one or more cell lines. Among these multiple lineages, those of adipogenesis and osteogenesis are the most closely related:Several studies have provided substantial evidence that osteoblasts and adipocytes share a common progenitor:multipotential BMSCs. An inverse relationship has been demonstrated between osteogenesis and adipogenesis suggesting the possible reciprocal relationship between the two differentiation pathways. There has been evidence for the differentiation switching between these two cell lineages, suggesting a large degree of plasticity between osteoblasts and adipocytes. Because the relationship between adipogenesis and osteogenesis is reciprocal and the adipocytic and osteogenic cells share a common lineage, it is possible that inhibition of adipogenesis may provide an approach to prevent or treat osteoporosis or other bone diseases. Thus, the signal transduction pathways implicated in these processes are evaluated as potential targets for therapeutic intervention of osteopenic disorders.
After consumption, alcohol is readily distributed throughout the body in the blood stream and crosses biological membranes, affecting virtually all biological processes inside the cell. Excessive alcohol consumption substantially promotes adipogenesis and inhibits osteogenesis. Moreover, both chronic and binge consumptions of ethanol are known to cause suppression of bone formation and enhancement of bone resorption. Thus, alcohol is considered a major risk factor for osteoporosis, a disease particularly prevalent in women worldwide. Alcohol and hormones are powerful factors of the pathogenesis of IFHN, but what channels how do they through to make the disease? Or a variety of ways to jointly work? or a new disease process? There is no clear answer. To find therapeutic targets according to elucidate the pathogenesis IFHN remains an important task for the present study.
Microenvironment is a functional environment which was composed including two aspects:microamount physical and chemical factors of the surrounding cells which can excite or inhibit the action of cells by Close-up action and regulation,and special structures and receptors which are formed in the process of evolution and differentiation of the cell. Many studies have shown that the role of hormones and partial adjustment factors on osteoblasts, BMSC, hematopoietic stem cells and the role of vivo bone reconstruction is extremely complex which can be overlapping, collaborative, antagonistic or short-lived. Many kinds of systemic and local signal was received,conducted and integrated by the cell in the net, and further stimulate the release of other members of the network, so that a signal amplification cascade. In this process. not only the cycles of the local cell signal within the bone microenvironment link, but is also involved in the regulation of its physiological activities. Thus, the coupling within the bone marrow microenvironment and stability between bone resorption and bone formation, perhaps the result of this regulatory network via signal contact between cells in the bone marrow variety of dynamic regulation.
Several studies have provided evidence that alcohol shifts lineage commitment and differentiation of BMSCs from osteogenesis to adipogenesis by upregulation of gene expression for peroxisome proliferator-activated receptor gamma (PPARy); however, the molecular mechanism underlying the reciprocal relationship is not yet well understood.
Through this project,we make a research about whether the existence of regulation of the the differentiation direction of BMSC by the influence of alcohol, the ability of osteoblasts and fat cell function, as well as the system of fat cells+BMSC, fat cells+osteoblast cells. We observed the expression of PPARy and cbfal which are the key gene of adipogenic Osteogenic differentiation differentiation under different conditions, detect osteogenesis-related indicators,such as ALP, OCN, I collagen and fat cells LPL. Explore the pathways of the risk factors, and its influence to intramedullary BMSC cells, osteoblasts, adipocytes. Whether it will produce local effects after the fat cells are effected by causative factors, further clarify the contribution and mechanisms of the bone microenvironment of the bone marrow in the pathogenesis of IFHN.
Objective
1To elucidate lipid abnormalities which was caused by the risk factors effected the BMSC adipogenic and osteogenic differentiation of the Intramedullary cell pool in the pathogenesis of IFHN.
2To discuss functional changes of osteoblasts and fat cells afer effectd
3To investigate the adjustment of fat cells' changes to osteoblasts and BMSC
4To analyse the effection of alcohol on co-culture system which was Consisted of fat cells+BMSC and fat cells+osteoblasts
Methods
1We isolated and cultured marrow BMSC, fat cells and osteoblasts from the New Zealand rabbits;made an identification about BMSC differentiation potential,the surface shape of marrow osteoblasts and fat cellcells.
2To observe the effection to the differentiation of BMSC, function of fat cells and osteoblasts after applicating of the role of alcohol in BMSC, fat cells and osteoblasts.
3After alcohol acted on fat cells, we Preparated the conditioned medium,then acted on intramedullary osteoblasts and BMSC,observed the contribution after the fat cells was effected.
4We made a co-culture system which was Consisted of fat cells+BMSC and fat cells+osteoblasts, observed the circumstances change of cell differentiation and osteogenic index in the co-culture system and the change after acted by alcohol.
5We measured changes of genes related to endoplasmic reticulum (ER) stress, adipogenic markers and osteogenic markers using quantitative real-time RT-PCR and Western blot analysis. We performed Alizarin red staining for osteogenesis. We also conducted assays for osteogenic biomarkers alkaline phosphatase, collagen-I and osteocalcin.We also conducted caspase3activity assay to assess BMSC apoptosis.
Results
After12days of culture, ALP staining was performed on two groups of cells, the experimental group showed that the cytoplasmic ALP staining was positive, the control group, experimental group cells staining, alizarin red staining after calcified nodules number and larger, control group with a small amount of calcium nodules, but relatively small, experimental group standard calcium nodule count significantly higher than the control group differences were statistically significant (P<0.05). We trained the BMSCs of rabbit in logarithmic growth phase for induction, when the confluence reached70-80%, added osteogenic induction medium. With time, the number of osteoblasts of the experimental group was significantly more than the control group. The number of Calcium nodules in experimental groups of3,6,9d, respectively (200.83±24.73)、(1112.00±77.56)、(1199.00±44.91)个/cm2; control group, respectively.(99.75±10.15)、(790.00±94.13)、(1059.00±26.39)个/cm2(P<0.001).
With time, the number of fat cells of the experimental group was significantly more than the control group. Two groups of small lipid droplets are gradually increasing and enlarging, but the experimental group was significant. The number of experimental groups in the the intramedullary fat cells of4,6,8,10d, respectively (200.83±24.78)、(1102.00±76.21)、(1160.00±28.83)、(1199.00±44.51)/cm2; control group, respectively.(99.75±10.67)、(790.00±94.63)、(1000.00±41.30)、(1059.00±26.54)/cm2, the number of fat cells increased with time of the influence of alcohol (P<0.001). We showed here that chronic exposure of BMSCs to alcohol induced adipogenesis and disrupted osteogenesis as indicated by upregulation of adipogenic markers (PPARy2and aP2), downregulation of osteogenic markers (Osf2/Cbfal), and accumulation of lipid droplets. Long-term excessive drinking could lead to femoral intramedullary fat cells rapidly proliferate, meanwhile,the fat cells were hypertrophy than normal, so that the relatively closed intramedullary pressure increased significantly, tiny blood vessels become thinner due to pressure, eventually leading to disruption or interruption of blood flow, thereby forming osteonecrosis of the femoral head.
Conclusions
By density gradient centrifugation and differential velocity adherent method the obtained BMSCs in conventional culture or inducing culture can be showed osteogenic potential of bone induction ability cultivation.lt shows that BMSCs can provide a reliable source of seed cells for bone tissue engineering research. Alcohol can restrain BMSC into osteoblasts and promote them into adipocytes; Alcohol can cause the osteoblasts to death,promote the intramedullary fat cells to increase and enlarge.
Innovation
1.This study proposes that alcohol can promote intramedullary fat cells proliferation and hypertrophy and suppress osteogenic differentiation potential of BMSCs
2.This study presents that the influence of alcohol on the metabolites of fat cells have certain extent to the proliferation of mesenchymal stem cells and bone marrow cells.
3. This study established the fat cells+bone marrow mesenchymal stem cells, adipocytes+osteoblasts co-culture system.
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