瘦素在人腰椎间盘髓核退变中的作用及机制研究

【作者】 李政；

【导师】 沈建雄；

【作者基本信息】 北京协和医学院 ， 骨科， 2013， 博士

【摘要】 研究背景与目的下腰疼(Low back pain, LBP)是最常见的肌肉骨骼系统疾病,年平均发病率可高达30%,据报道大于70%的人在一生中会有下腰痛的症状。椎间盘退行性变(Intervertebral disc degeneration, IDD)是导致下腰痛以及急性椎间盘源性放射痛的最常见病因。随着社会人口老龄化进程加速,IDD患者的数量不断增加。IDD的病因及发病机制目前尚不清楚,可能的相关因素包括物理负重、损伤、震动、感染以、吸烟以及某些遗传影响等。IDD的病因复杂,其确切发病原因目前仍不清楚,目前的研究进展表明：肥胖可显著地致腰痛增加,体重指数BMI≥25kg/m2的与BMI<25kg/m2相比较,IDD发生率要高4.3倍,因此肥胖是IDD的独立发病因素之一。瘦素(1eptin)是一种分子量为16kDa的小分子蛋白,主要由白色脂肪组织分泌,能调整食欲并促进脂肪代谢,引起体重下降。文献报道,循环中瘦素的浓度与肥胖成正比,即肥胖症体内瘦素水平表达增高。近年来的研究发现,瘦素不仅参与脂肪代谢,还参与诸如免疫调节、造血、卵巢功能、肿瘤发生、转移、细胞增殖及炎症反应等多种生理病理过程,并在其中发挥着重要的负作用。已有报道,在骨关节炎患者的关节液、骨赘以及关节软骨中检测到瘦素的表达。还有研究已经证实体外培养的人椎间盘细胞能够自然分泌达到检测水平含量的Leptin蛋白。研究还报道在突出的腰椎间盘髓核细胞中检测至Leptin以及其特异性受体,并进一步证实leptin能在体外刺激椎间盘细胞增殖。这些研究成果为研究肥胖及瘦素引起腰椎间盘退行性病变的机制提供了理论依据,但瘦素是如何引起腰椎间盘退变的作用方式及其具体机制,还不明确,迄今未见有论文发表。本研究的目的为阐述瘦素在椎间盘退变中的作用及其机制。研究方法我们收集腰椎间盘髓核组织,在体外分离培养人髓核细胞并利用Real-time RT-PCR和免疫荧光染色对髓核细胞进行鉴定。以不同浓度的瘦素作用于体外培养的人髓核细胞,CCK8检测髓核细胞的增殖能力。观察在10ng/ml瘦素刺激下,髓核细胞随着时间增殖能力的变化。应用Western blot法检测髓核细胞中的p-AKT、p-ERK1/2、p-STAT3的表达水平,以Real-time RT-PCR、Western blot和免疫荧光染色观察髓核细胞cyclin D1、 PCNA和Ki-67的表达。应用JAK、P13K和MEK信号通路抑制剂AG490, Wortmannin和U0126来观察髓核细胞的增殖能力、cyclin D1、 PCNA和Ki-67的表达是否被抑制。从人的髓核组织中分离提取RNA和蛋白,Real-time RT-PCR和Western blot检测髓核组织中OBRa和OBRb的表达水平,并结合临床资料进行相关性分析。瘦素作用于体外髓核细胞后,以Real-time RT-PCR、 Western blot和免疫荧光染色观察髓核细胞的骨架蛋白F-actin、β-actin、β-tubulin和vimentin的表达变化情况。以编码YFP-RhoA-PKN-CFP融合蛋白的pRaichu-1237x质粒电转染人髓核细胞。观察在10ng/ml瘦素刺激下,RhoA蛋白的FRET信号改变。应用Western blot法检测p-LIMK1和p-Cofilin的表达变化情况,并应用免疫荧光染色观察瘦素刺激后髓核细胞内F-actin的变化情况。以RhoA和ROCK信号通路抑制剂C3exoenzyme和Y-27632观察瘦素引起的p-LIMK1、p-Cofilin和F-actin的表达变化是否被抑制。以Real-time RT-PCR和Western blot法观察瘦素刺激髓核细胞后细胞外基质Collagen I、Collagen II、MMP-2和MMP-14的表达水平,并以Western blot法检测p-JAK、p-p38、p-MEK的表达水平。研究结果我们在体外成功分离培养并鉴定了人髓核细胞,并证实CA21、cytokeratin19在髓核细胞中呈高表达,而IBSP、FBN1呈低表达。瘦素通过激活p-AKT、 p-ERK1/2、p-STAT3信号通路促进髓核细胞增殖并诱导cyclin D1、PCNA和Ki-67的表达,这些效应能被JAK、PI3K和MEK信号通路抑制剂AG490, Wortmannin和U0126所抑制。OBRa和OBRb的mRNA和蛋白在髓核组织中均有表达,且个体表达差异较大。OBRb和OBRa的表达量与年龄、疾病的类型、腰痛的时间、性别无相关性,但OBRb的表达量与体重指数呈正相关性。髓核细胞在瘦素刺激后,胞浆中F-actin微丝明显粗长,整个细胞的F-actin表达量及荧光强度明显增加,β-actin的表达量增加。Vimentin的结构无明显变化,其表达量明显上升,而β-tubulin的表达无明显变化。在lOng/ml的瘦素作用下,髓核细胞逐渐回缩伪足,FRET信号逐渐增强,并于刺激后5min时达到峰值。瘦素能刺激髓核细胞的p-LIMk1和p-confilin-2磷酸化并使其骨架蛋白F-actin重塑,这些效应能被RhoA和ROCK信号通路抑制剂C3exoenzyme和Y-27632所抑制。瘦素能激活髓核细胞p-JAK、p-p38、p-MEK的表达,并上调Collagen I、MMP-2和MMP-14的表达并抑制Collagen II的表达。结论本研究对瘦素在人椎间盘退变中的作用及分子机制进行了研究。我们的研究表明,瘦素受体OBRa和OBRb在椎间盘髓核组织中均有表达,且OBRb的表达量与体重指数呈一定的正相关性。瘦素通过激活JAK、PI3K和MEK信号通路促进髓核细胞增殖。瘦素刺激髓核细胞引起骨架蛋白F-actin、p-acth、β-tubulin和vimentin的表达发生变化。瘦素通过激活RhoA/ROCK和LMK1/Cofilin信号通路导致骨架蛋白F-actin重塑。瘦素能激活Collagen I、MMP-2和MMP-14的表达而抑制Collagen Ⅱ的表达。更多还原

【Abstract】 Background and purposeLow back pain (Low back pain, LBP) is one of the most common disorders of the musculoskeletal system, with an average annual incidence rate of about30%and a lifetime prevalence of about70%. Intervertebral disc degeneration (IDD) is considered to be a major cause of LBP and acute discogenic pain. With accelerating aging process of the social population, the number of patients with IDD is increasing. Nevertheless, the exact etiology and pathogenesis of the disc degeneration remain unknown. The possible etiological factors included the body weight, injury, infection, smoking, and genetic predisposition. Research showed that obesity was an established risk factor for Low back pain. The incidence of IDD in person with body mass index, BMI≥25kg/m2was4.3times higher, than person with BMI≤25kg/m2. Obesity is one of the independent causes of IDD. Leptin (16kDa) is a small molecule protein secreted mainly by adipose tissues. It can cause weight loss by adjusting appetite and promoting fat metabolism. Study showed that the serum leptin levels were positively associated with body weight, namely the expression of leptin in obese person increased. Several recent studies indicated that leptin could not only involve in the metabolism of fat but also in other physiological and pathological processes, such as immune regulation, thermogenesis, ovarian function, tumorigenesis, metastasis, proliferation and inflammatory cell reaction. Study showed that the expression of leptin was detected in synovial fluid, osteophyte and articular cartilage in patients with osteoarthritis. And studies have confirmed that the cultured human intervertebral disc cells can naturally secreted detectable levels of Leptin. Leptin and its specific receptor were detected in NP cells of prolapsed lumbar intervertebral disc. Study confirmed that leptin could stimulate proliferation of disc cells in vitro. These researches provide the basis for further study to investigate the mechanism of leptin in the pathogenesis of IDD. However, the role of leptin in the pathogenesis of disc degeneration is not yet clear. There were no published papers before. The purpose of this study was to investigate the role of leptin in IDD and its mechanism.MethodsThe human NP cells were dissected from patient disc specimens and then cultured in vitro. The levels of leptin were measured by real-time PCR and immunofluorescence. The proliferation of NP cells was detected by CCK8method after being plated and treated in different concentrations of leptin. We also observed the proliferation ability of NP cells with changes in time when treated with10ng/ml of Leptin. The expression of p-AKT, p-ERK1/2, p-STAT3in NP cells were detected by Western blot. The expression of cyclin D1, PCNA and Ki-67were detected by real-time RT-PCR, Western blot and immunofluorescence staining. We also observed whether the proliferation ability of NP cells, as well as the expression of cyclin D1, PCNA and Ki-67was inhibited after being traeted by AG490, Wortmannin and U0126, which were the signal pathway inhibitors of JAK, PI3K and MEK pathway. RNA and protein were extracted and separated from NP cells, in which the expression level of OBRa and OBRb were detected by Real-time RT-PCR and Western blot. We also conduct the correlation analysis with clinical data. Immunofluorescence and real-time PCR and Western blot were performed to investigate the effect of leptin on cytoskeleton protein, such as F-actin, β-actin, β-tubulin and vimentin. We transfected pRaichu-1237x plasmid, which encodes YFP-RhoA-PKN-CFP fusion protein into human nucleus pulposus cells and observe the changes of FRET signal of RhoA protein in10ng/ml Leptin. Expression of p-LIMK1and p-Cofilin was detected by Western blot method, and the change of F-actin by leptin stimulation in NP cells was detected immunofluorescence staining. Using RhoA einhibitor C3exoenzyme or ROCK inhibitor Y-27632, we observed the changes in p-LIMK1、p-Cofilin和F-actin. After stimulated by leptin, the expression of extracellular matrix of Collagen I, Collagen II, MMP-2and MMP-14were detected by real-time RT-PCR and Western blot method and the expression of p-JAK, p-p38, p-MEK was detected by Western blot method.ResultsWe successfully isolated and cultured human NP cells in vitro. The expression of CA21, cytokeratin19was significantly higher and the expression of IBSP, FBN1was significantly lower in NP cells. Leptin can promote the proliferation of NP cells and induce the expression of cyclin and D1, PCNA and Ki-67through the activation of p-AKT, p-ERK1/2, p-STAT3signaling pathway. These effects can be inhibited by AG490Wortmannin and U0126JAK, which were the JAK, PI3K and MEK signal pathway inhibitor respectively. Both the mRNA and protein of OBRa and OBRb were expressed in NP tissues, and the expression was individually different. OBRb expression was correlated with patients’body weight, but no correlation was found between the expression of OBRb and OBRa with age, disease types, duaration of low back pain and sex. After leptin stimulation, the cytosolic F-actin microfilaments were thicker and longer and the expression of F-actin and fluorescence intensity increased significantly. Moreover, the expression of β-actin also increased. The expression of Vimentin increased significantly without obvious change in the structure. The expression of β-tubulin had no obvious change. In10ng/ml of leptin, NP cells gradually retracted their pseudopodia and the FRET signal also increased gradually. The peak was reached at5min after the stimulation. Leptin can stimulate the phosphorylation of p-LIMkl and p-confilin-2and remodeling of the skeleton protein F-actin in NP cells. These effects can be inhibited by C3exoenzyme and Y-27632, which were the RhoA and ROCK signal pathway inhibitor. Leptin can induce the expression of p-JAK, p-p38, p-MEK and up-regulate the expression of Collagenl, MMP-2and MMP-14, while inhibiting the expression of Collagen2.ConclusionsThis study investigated the effects of leptin on human ICD and its possible molecular mechanism. Our study shows that both OBRa and OBRb were expressed in NP tissues, and the expression was individually different. OBRb expression was correlated with BMI. Leptin can promote the proliferation of NP cells through activation of JAK, PI3K and MEK signal pathway. Leptin can change the expression of skeleton protein F-actin, β-actin,β-tubulin and vimentin. Leptin remodel the skeleton protein F-actin through the activation of RhoA/ROCK and LIMK1/Cofilin signal pathway. Leptin can induce the expression of Collagenl, MMP-2MMP-14, while inhibiting the expression of Collagen2.更多还原