应用荧光共振能量转移（FRET）法研究瘦素及外力刺激对于软骨细胞骨架调控蛋白RhoA的作用
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摘要
研究背景与目的:骨关节炎(OA)是骨科的常见疾病之一,是一种退行性关节疾病,其病因尚不明了。其中,长期超负荷的外力作用是较为公认的OA危险因素之一,因而在体重超重人群中,OA的发病率显著增加。瘦素(leptin)是一种白色脂肪组织分泌的小分子量蛋白,在体重超重人群中其血清含量往往升高。2003年Dumond等人发现OA患者关节液中瘦素(leptin)含量明显高于正常人,提示瘦素可能在OA的发病过程中起到某种作用。RhoA蛋白是调控细胞骨架的重要分子之一,OA软骨细胞与正常软骨细胞的显著差异之一就是细胞骨架含量的明显升高。本文拟通过荧光共振能量转移(FRET)法,研究活细胞条件下瘦素及外力刺激对于正常软骨细胞RhoA蛋白的作用,分别探讨瘦素、外力对于软骨细胞细胞骨架的影响,为OA的病因研究提供参考。
研究方法:以编码YFP-RhoA-PKN-CFP融合蛋白的pRaichu-1237x质粒电转染正常人软骨细胞。观察在200ng/ml瘦素刺激下,RhoA蛋白的FRET信号改变。另外,应用原子力显微镜(AFM)作为施力工具,观察在AFM的轻敲模式与接触模式作用下,RhoA蛋白的FRET信号改变。
研究结果:在200ng/ml的瘦素作用下,软骨细胞伪足迅速回缩,FRET信号逐渐减弱消失,直至最终细胞皱缩,形成许多细胞膜周围的小泡。
软骨细胞在接受AFM轻敲模式刺激后逐渐出现胞质回缩,FRET信号明显增强,以接触探针刺激处最为明显。
软骨细胞在接受AFM接触模式刺激后,细胞形态无明显改变,FRET信号明显增强,以接触探针刺激处最为明显。
研究结论:
(1)以大剂量瘦素(200ng/ml)刺激软骨细胞,不但不能激活RhoA蛋白,反而会使RhoA蛋白的FRET信号明显减弱,并导致软骨细胞逐渐皱缩,最终在细胞膜表面出现类似凋亡小体的小泡,提示大剂量的瘦素可能诱导软骨细胞的凋亡。
(2)AFM作为施力工具可以直接给予体外培养的软骨细胞以力学刺激。以AFM的轻敲模式和接触模式作用于软骨细胞可以分别模拟垂直方向的压力和水平方向的剪切力。软骨细胞在AFM的外力作用下均发生了较强的FRET现象。细胞对于垂直方向的压力更为敏感,FRET信号更强,并出现了胞质回缩。提示外力作用可以显著激活细胞骨架的调控蛋白RhoA。
Background:Osteoarthritis is a kind of degenerative disease,which represents one of the most frequent diseases encountered in the clinics.However,the pathophysiology of OA is still unclear.It is believed that obesity represents one of the significant risk factors for OA.Many studies have demonstrated a clear link between obesity and OA.Leptin is a small polypeptide excreted by white adipose tissue, whose circulating concentrations are proportional to the degree of adiposity.In 2003, Dumond et al.found that leptin was strongly overexpressed in human OA cartilage and synovial fluid,which indicates leptin may play a key role in the pathophysiology of OA.RhoA is a significant molecular switch,which regulates a wide spectrum of cellular functions such as remodeling of the actin cytoskeleton.Many studies showed that OA chondrocytes have an elevated level of cytoskeletal component.Our research hypothesizes that leptin and external force may affect the activity of RhoA in normal chondrocytes,consequently induce some changes of the cytoskeleton.We believe that fluorescent resonance energy transfer(FRET) could be a proper method to demonstrate the changes of RhoA in a single living chondrocyte.
Methods:pRaichu-1237x vector,encoding YFP-RhoA-PKN-CFP fusion protein, was transfected into normal chondrocytes via Human Chondrocyte Nucleofector~(TM) Kit(Amaxa Corporation,Germany).Chondrocytes were incubated with 200ng/ml leptin for 30 minutes.The FRET signal of RhoA was recorded during the incubation. Besides,we also used AFM as an apparatus to apply external force to the chondrocytes,with the purpose of observing the change of RhoA FRET signal.
Results:During the incubation,we observed a pseudopodium contraction and cytoplasm streaming.The FRET signal gradually vanished.The whole chondrocyte collapsed in the end.Many small bubbles could be found around the plasma membrane.
After applying external force to the chondrocytes by AFM,we observed an obvious enhancement of the RhoA FRET signal.Cell accepted a tapping mode of external force also appeared an obvious pseudopodium contraction,whereas cell accepted a contact mode of external force did not show any change of the outline.
Conclusions:1.Incubating chondrocytes with high-dose leptin(200ng/ml) could not activate RhoA,but lead to FRET signal vanishing.Cell collapse and small bubbles around the membrane may indicate cell apoptosis.2.AFM has been proved to be a successful method to apply external force to the living cells.Tapping mode and contact mode could simulate pressure and shearing force separately.It seemed that chondrocytes were more sensitive to the tapping mode,which induced stronger RhoA FRET signal and more visible change of the cell outline.All the evidences show that external force could obviously activate RhoA
研究方法:以编码YFP-RhoA-PKN-CFP融合蛋白的pRaichu-1237x质粒电转染正常人软骨细胞。观察在200ng/ml瘦素刺激下,RhoA蛋白的FRET信号改变。另外,应用原子力显微镜(AFM)作为施力工具,观察在AFM的轻敲模式与接触模式作用下,RhoA蛋白的FRET信号改变。
研究结果:在200ng/ml的瘦素作用下,软骨细胞伪足迅速回缩,FRET信号逐渐减弱消失,直至最终细胞皱缩,形成许多细胞膜周围的小泡。
软骨细胞在接受AFM轻敲模式刺激后逐渐出现胞质回缩,FRET信号明显增强,以接触探针刺激处最为明显。
软骨细胞在接受AFM接触模式刺激后,细胞形态无明显改变,FRET信号明显增强,以接触探针刺激处最为明显。
研究结论:
(1)以大剂量瘦素(200ng/ml)刺激软骨细胞,不但不能激活RhoA蛋白,反而会使RhoA蛋白的FRET信号明显减弱,并导致软骨细胞逐渐皱缩,最终在细胞膜表面出现类似凋亡小体的小泡,提示大剂量的瘦素可能诱导软骨细胞的凋亡。
(2)AFM作为施力工具可以直接给予体外培养的软骨细胞以力学刺激。以AFM的轻敲模式和接触模式作用于软骨细胞可以分别模拟垂直方向的压力和水平方向的剪切力。软骨细胞在AFM的外力作用下均发生了较强的FRET现象。细胞对于垂直方向的压力更为敏感,FRET信号更强,并出现了胞质回缩。提示外力作用可以显著激活细胞骨架的调控蛋白RhoA。
Background:Osteoarthritis is a kind of degenerative disease,which represents one of the most frequent diseases encountered in the clinics.However,the pathophysiology of OA is still unclear.It is believed that obesity represents one of the significant risk factors for OA.Many studies have demonstrated a clear link between obesity and OA.Leptin is a small polypeptide excreted by white adipose tissue, whose circulating concentrations are proportional to the degree of adiposity.In 2003, Dumond et al.found that leptin was strongly overexpressed in human OA cartilage and synovial fluid,which indicates leptin may play a key role in the pathophysiology of OA.RhoA is a significant molecular switch,which regulates a wide spectrum of cellular functions such as remodeling of the actin cytoskeleton.Many studies showed that OA chondrocytes have an elevated level of cytoskeletal component.Our research hypothesizes that leptin and external force may affect the activity of RhoA in normal chondrocytes,consequently induce some changes of the cytoskeleton.We believe that fluorescent resonance energy transfer(FRET) could be a proper method to demonstrate the changes of RhoA in a single living chondrocyte.
Methods:pRaichu-1237x vector,encoding YFP-RhoA-PKN-CFP fusion protein, was transfected into normal chondrocytes via Human Chondrocyte Nucleofector~(TM) Kit(Amaxa Corporation,Germany).Chondrocytes were incubated with 200ng/ml leptin for 30 minutes.The FRET signal of RhoA was recorded during the incubation. Besides,we also used AFM as an apparatus to apply external force to the chondrocytes,with the purpose of observing the change of RhoA FRET signal.
Results:During the incubation,we observed a pseudopodium contraction and cytoplasm streaming.The FRET signal gradually vanished.The whole chondrocyte collapsed in the end.Many small bubbles could be found around the plasma membrane.
After applying external force to the chondrocytes by AFM,we observed an obvious enhancement of the RhoA FRET signal.Cell accepted a tapping mode of external force also appeared an obvious pseudopodium contraction,whereas cell accepted a contact mode of external force did not show any change of the outline.
Conclusions:1.Incubating chondrocytes with high-dose leptin(200ng/ml) could not activate RhoA,but lead to FRET signal vanishing.Cell collapse and small bubbles around the membrane may indicate cell apoptosis.2.AFM has been proved to be a successful method to apply external force to the living cells.Tapping mode and contact mode could simulate pressure and shearing force separately.It seemed that chondrocytes were more sensitive to the tapping mode,which induced stronger RhoA FRET signal and more visible change of the cell outline.All the evidences show that external force could obviously activate RhoA
引文
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[2] Zigang Ge, Yang Hu, Boon Chin Heng et al. Osteoarthritis and Therapy. Arthritis Rheum 2006; 55(3):493-500.
[3] Kuettner KE, Goldberg VM. Introduction. In: Kuettner KE, Goldberg VM, editors. Osteoarthritic disorders. Rosemont (IL): American Academy of Orthopaedic surgeons; 1995. p.xxi - xxv.
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