切应力对与内皮细胞联合培养的血管平滑肌细胞粘附的影响及其机制

目的　细胞粘附是细胞迁移的关键性起始步骤。研究切应力和内皮细胞(endothelial cells, ECs)对血管平滑肌细胞(vascular smooth muscle cells, VSMCs)粘附的影响及其可能的信号通路，为探讨流体切应力诱导的血管壁细胞迁移行为的机制提供一些实验依据。方法　应用VSMCs和ECs联合培养流动腔系统，对ECs面施加1.5 Pa切应力，12 h；以静止状态下，单独培养的VSMCs以及与ECs联合培养的VSMCs 为对照，应用细胞粘附实验和Western Blot技术，观察切应力对与EC联合培养的VSMCs粘附的影响及蛋白激酶B(PKB/Akt)磷酸化水平表达变化。结果　静态联合培养12 h，VSMCs的粘附能力明显增强，同时磷酸化Akt的表达平行增高。切应力作用下，明显抑制了联合培养的VSMCs粘附，同时磷酸化Akt的表达平行降低。结论　生理大小切应力明显抑制了ECs诱导的VSMCs粘附，其中Akt信号通路起了关键作用。     

活化激酶C受体1在切应力调控血管平滑肌细胞增殖中的作用

目的 探讨活化激酶C受体1（receptor for actived C kinase 1, RACK1）在内皮细胞（endothelial cells, ECs）感受切应力刺激调控血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖中的作用及其机制。方法 应用平行平板流动腔系统,对联合培养的大鼠ECs和VSMCs施加1.5 Pa正常切应力（normal shear stress, NSS）和0.5 Pa低切应力（low shear stress,LowSS）,应用BrdU ELISA方法检测VSMCs增殖水平,对蛋白质组学研究发现的力学响应分子RACK1表达以及Akt磷酸化,应用Western blot技术进行检测。静态条件下,应用RNA干扰技术特异性抑制VSMCs的RACK1表达,检测其对细胞增殖和Akt磷酸化的作用。应用ECs与VSMCs隔开培养和联合培养模型,检测ECs对VSMCs的RACK1表达和Akt磷酸化水平的影响。结果 血管差异蛋白质组学的结果发现,与NSS组相比,RACK1在LowSS组血管组织的表达水平明显升高。细胞实验结果显示,LowSS诱导了与ECs联合培养的VSMCs增殖,上调VSMCs的RACK1表达和Akt磷酸化。静态条件下,特异性抑制VSMCs的RACK1表达后,VSMCs的增殖水平和Akt磷酸化水平均显著下降。与ECs联合培养VSMCs,其RACK1表达和Akt磷酸化水平较隔开培养组均上调。结论 VSMCs的RACK1表达受细胞接触与切应力的影响,并可能通过PI3K/Akt信号通路参与LowSS诱导的VSMCs增殖的调控。探讨VSMCs增殖功能变化及其力学生物学机制对于认识动脉粥样硬化等疾病发病机理和疾病防治有重要意义。     

Normal Shear Stress and Vascular Smooth Muscle Cells Modulate Migration of Endothelial Cells Through Histone Deacetylase 6 Activation and Tubulin Acetylation

Endothelial cells (ECs) line the innermost of the blood vessel wall and are constantly subjected to shear stress imposed by blood flow. ECs were also influenced by the neighboring vascular smooth muscle cells (VSMCs). The bidirectional communication between ECs and VSMCs modulates vascular homeostasis. In this study, the involvement of histone deacetylase 6 (HDAC6) in modulating migration of ECs co-cultured with VSMCs by the normal level of laminar shear stress (NSS) was investigated. ECs was either cultured alone or co-cultured with VSMCs under static conditions or subjected to NSS of 15 dyne/cm² by using a parallel-plate co-culture flow chamber system. It was demonstrated that both NSS and VSMCs could increase EC migration. The migration level of ECs co-cultured with VSMCs under NSS was not higher than that under the static condition. The process of EC migration regulated by VSMCs and NSS was associated with the increased expression of HDAC6 and low level of acetylated tubulin. The increase in HDAC6 expression was accompanied by a time-dependent decrease in the acetylation of tubulin in ECs co-cultured with VSMCs. Inhibition of the HDAC6 by siRNA or tributyrin, an inhibitor of HDACs, induced a parallel alteration in the migration and the acetylated tubulin of ECs co-cultured with VSMCs. It was observed by immunofluorescence staining that the acetylated tubulin was distributed mostly around the cell nucleus in ECs co-cultured with VSMCs. The results suggest that the NSS may display a protective function on the vascular homeostasis by modulating EC migration to a normal level in a VSMC-dependent manner. This modulation process involves the down-regulation of acetylated tubulin which results from increased HDAC6 activity in ECs.     

切应力作用下联合培养的血管平滑肌细胞对内皮细胞F-肌动蛋白构筑的影响

目的　探讨切应力作用下联合培养的血管平滑肌细胞对内皮细胞抗应力和粘附能力的影响 ,为改进血管内皮细胞种植的组织工程学技术提供生物力学基础。　方法　应用荧光标记和激光共聚焦扫描显微镜技术 ,以静态条件下单独培养的内皮细胞、联合培养的内皮细胞以及切应力作用下单独培养的内皮细胞为对照组 ,研究了切应力作用下与血管平滑肌细胞联合培养的内皮细胞的细胞骨架F 肌动蛋白构筑的变化。　结果　静态条件下单独培养的内皮细胞的F 肌动蛋白排列松散 ,不规则 ,微丝较细 ;联合培养的内皮细胞的F 肌动蛋白微丝明显增多增粗。切应力作用下 ,与血管平滑肌细胞联合培养的内皮细胞的F 肌动蛋白发生重排 ,并形成大量沿切应力方向排列的应力纤维 ,且发生重排的时间明显早于单独培养的内皮细胞。　结论　在切应力作用和血管平滑肌细胞的影响下 ,内皮细胞F 肌动蛋白构筑的变化有利于增强内皮细胞的抗应力和粘附能力     

切应力作用下联合培养的血管平滑肌细胞对内皮细胞PDGF-B mRNA表达的影响

目的：探讨切应力作用下联合培养的血管平滑肌细胞（VSMCs）对内皮细胞（ECs)PDGF-BmRNA表达的影响，为预防血管移植物发生再狭窄提供实验资料。方法：用原位杂交和图像分析等技术，以静态条件下单独培养的ECs和联合培养的ECs为两对照组，观察切应力作用下单独培养的ECs和与VSMCs联合培养ECs的PDGF－BmRNA表达变化。结果：静态条件下联合培养ECs和PDGF－BmRNA表达水平比单独培养的ECs下降；切应力作用下，联合培养ECs的PDGF－BmRNA的表达在切应力作用1h左右有瞬时上升，6h后 下降至低于联合培养条件下的静态水平，且瞬时上升的时间点比单独培养的ECs提前。结论：切应力作用下，与VSMCs联合培养ECs的PDGF－BmRNA表达水平下降，这可能有利于抑制VSMCs的增生。     

切应力对与血管平滑肌细胞联合培养的内皮细胞微管骨架重构的影响

目的探讨切应力对与血管平滑肌细胞(VSMCs)联合培养的内皮细胞(ECs)中微管的聚集重构的影响,为阐明应力诱导血管重建的分子机制提供一些实验证据。方法应用ECs与VSMCs联合培养的平行平板流动腔系统,给ECs面施加15dyne/cm2的层流切应力,以静态条件下联合培养的ECs为对照组,用WesternBlot、免疫荧光细胞化学和图像分析等技术,研究切应力作用下与VSMCs联合培养的ECs的微管聚集的变化。结果静态联合培养组,ECs微管骨架的排列是稀疏、发散和无规律的。切应力诱导了ECs的微管的重构,,微管骨架变得有序,朝切应力的方向规律的排列。切应力能够促进ECs的微管聚集,与对照组相比,切应力作用下的ECs内多聚微管的数量增加,切应力作用3h,ECs内多聚微管的数量达到峰值,之后开始下降。结论切应力诱导和促进了EC的微管骨架发生重构(聚集)。结果提示:微管可能是机械应力刺激作用的靶标,应力可能通过它改变ECs的形态,影响细胞的黏附与迁移等功能。     

机械张应变诱导蛋白激酶B活化对血管平滑肌细胞迁移的影响

目的研究机械张应变诱导蛋白激酶B活化对大鼠血管平滑肌细胞迁移能力的影响。方法应用FX-4000T细胞应变加载系统,对大鼠血管平滑肌细胞施加牵拉幅度为15%、频率为1Hz的张应变。以Transwell和Westernblot等方法观察张应变作用下蛋白激酶B磷酸化和血管平滑肌细胞迁移能力的变化,以未加载张应变的血管平滑肌细胞为对照组。结果与对照组相比,机械张应变增加细胞中蛋白激酶B磷酸化水平,促进血管平滑肌细胞的迁移;PI3K的特异性抑制剂Wortmannin抑制张应变诱导的蛋白激酶B的磷酸化,降低了血管平滑肌细胞迁移能力。结论机械张应变通过上调蛋白激酶B磷酸化水平促进了血管平滑肌细胞迁移,提示蛋白激酶B信号通路参与机械张应变条件下血管平滑肌细胞迁移过程的信号传导。     

Rab28相关信号通路在低切应力诱导 血管平滑肌细胞迁移中的作用

目的 研究低切应力（low shear stress, LowSS）诱导的血管平滑肌细胞（vascular smooth muscle cells, VSMCs）迁移功能异常在动脉粥样硬化血管重建病理过程中的作用及其分子机制。 方法 应用双向凝胶电泳结合质谱分析的差异蛋白质组学方法,研究1.5 Pa正常切应力（normal shear stress, NSS）与0.5 Pa LowSS条件下培养血管组织的蛋白质差异表达谱。应用血管内皮细胞（endothelial cells, ECs）与VSMCs联合培养的平行平板流动腔系统,分别施加NSS和LowSS,Western blot检测ECs与VSMCs的Rab28表达水平以及VSMCs的磷酸化ERK表达水平;Transwell法检测VSMCs的迁移能力;RNA干扰和PD98059分别特异性抑制VSMCs的Rab28表达和ERK磷酸化,再观察VSMCs迁移能力变化。结果 血管差异蛋白质组学的结果发现,与NSS组相比,Rab28在LowSS组血管组织的表达水平明显升高。细胞实验结果显示,LowSS加载明显上调VSMCs的Rab28蛋白表达、VSMCs迁移和ERK磷酸化。静态条件下RNA干扰抑制单独培养VSMCs的Rab28表达,VSMCs迁移能力明显降低,但ERK磷酸化水平无明显变化;应用PD98059特异性抑制VSMCs的ERK磷酸化,VSMCs迁移能力和Rab28表达水平均明显降低。结论 LowSS可能通过上调VSMCs的ERK磷酸化水平引起Rab28表达水平增加,从而诱导VSMCs迁移。探讨Rab28及其相关信号通路在切应力调控VSMCs功能中的作用及其机制,可能为深入理解动脉粥样硬化血管重建疾病发病机制和寻找新的药物治疗靶点提供力学生物学依据。     

Steady unidirectional laminar flow inhibits monolayer formation by human and rat microvascular endothelial cells.

Endothelialization of artificial vascular grafts is rapid and complete in numerous animal models, including dogs and rats, but not in human patients. One possible explanation for this well-known, yet puzzling observation might be that monolayer formation of human endothelial cells (ECs), and of canine or rodent ECs, is affected differently by flow-induced shear stress. To begin testing this hypothesis, the authors wounded confluent monolayers of cultured rat and human ECs and exposed these cultures for 20 h to unidirectional steady laminar shear stress of 10 dyn/cm(2) induced by fluid flow perpendicular to the wound boundaries. In comparison to experimental control cultures simultaneously maintained under static (no-flow) conditions, flow-induced shear stress attenuated the monolayer formation (sheet migration) in both human and rat ECs. In brief, compared to control, the average human EC monolayer formation under shear was reduced by 33% whereas the average rat EC monolayer formation was reduced by 34%. Furthermore, the cell responses showed a dependence on fluid flow direction that differed per species. When exposed to shear stress, human EC monolayer formation was reduced by 16% in the upstream direction (opposing the direction of flow) and reduced by 50% in the downstream direction (with the direction of flow), whereas rat EC monolayer formation was reduced by 64% upstream and showed no change downstream. These findings suggest that although overall monolayer formation is inhibited by fluid-induced shear stress to the same extent in both species, there are cell type- and/or species-dependent migration responses to fluid-induced shear stress, and that different flow conditions possibly contribute to species-specific patterns of endothelialization.     

Angiogenesis-like activity of endothelial cells co-cultured with VEGF-producing smooth muscle cells

A number of strategies have been investigated to improve therapeutic vascularization of ischemic and bioengineered tissues. In these studies, we genetically modified vascular smooth muscle cells (VSMC) to promote endothelial cell proliferation, migration, and formation of microvascular networks. VSMCs were virally transduced to produce vascular endothelial growth factor (VEGF), which acts as a chemoattractant and mitogen of endothelial cells (EC). VSMCs transduced with VEGF(165) cDNA produced significant levels of the protein (2-4 ng/10(5) cell/day). The proliferation of ECs increased after exposure to VEGF-transfected SMCs or their conditioned media. The chemotactic response of ECs to the VEGF-producing cells was explored in two in vitro systems, the modified Boyden chamber assay and a 2-D fence-style migration assay, and both demonstrated increased migration of ECs in response to VEGF-transfected cells. Furthermore, endothelial cells seeded on top of the VEGF-transfected SMCs formed capillary-like structures. These results suggest that VSMCs genetically modified to produce VEGF could be a potential delivery mechanism to enhance endothelial cell migration and subsequent capillary formation, which in turn could improve vascularization of ischemic or regenerating tissue. Furthermore, this system could potentially be used as an in vitro test bed for evaluation of novel angiogenic and anti-angiogenic compounds.     

Development of an Endothelial–Smooth Muscle Cell Coculture Model Using Phenotype-Controlled Smooth Muscle Cells

A coculture of endothelial cells (ECs) and smooth muscle cells (SMCs), which mimics cellular interactions appearing in vivo, has been performed in studies on the relationship between atherogenesis and fluid shear stress conditions. Although healthy arteries in vivo consist of contractile phenotype SMCs, cultured cells used in many studies normally exhibit a synthetic phenotype. Here, we developed an EC–SMC coculture model to investigate the interactions between ECs and contractile SMCs, and examined the effect of shear stress applied to ECs on SMC phenotypes. Cultured human umbilical artery SMCs were differentiated into contractile states by arresting cell growth using a serum-free medium. Western blotting confirmed that SMC expression of contractile protein markers, α-smooth muscle actin (SMA) and calponin, increased to levels similar to those observed in arterial cells. After coculturing contractile SMCs with ECs separated by a collagen gel layer, the expression of α-SMA decreased under static conditions, indicating that the SMC phenotype tended to be synthetic by coculturing with ECs, but shear stress applied to cocultured ECs maintained the level of α-SMA expression in SMCs. The coculture model constructed in the present study will be a useful tool to investigate interactions between ECs and contractile SMCs under shear conditions.     

Endothelial cells cultured on engineered vascular grafts are able to transduce shear stress

In vitro endothelialization of small-diameter vascular prostheses confluently lined with cultured autologous endothelial cells (ECs) before clinical implantation improves their patency. Many authors have studied the effects of shear stress on ECs seeded on various substrates showing activation of mitogen-activated protein (MAP) kinases. Very few studies have reported any functional EC response to shear stress when they are seeded on vascular grafts. The purpose of this in vitro study was to investigate whether ECs were able to transduce shear stress. Human saphenous vein ECs were seeded on 6 mm fibrin-glue-coated grafts, then submitted to 15 dyn/cm(2) for 10, 30, and 120 min. Cell lysates were submitted to Western blot analysis to detect phosphorylated ERK 1/2 and p38. ERK 1/2 activation was observed at 10 min (1.6 fold) followed by a lower activation than under static conditions at 30 and 120 min. Shear stress induced a significant increase in p38 phosphorylation (2.5 fold) at 10 and 30 min, decreasing at 120 min. Thus, ECs are able to transduce shear stress in an in vitro model in closed clinical conditions, but the ERK 1/2 and p38 temporal activation profile is different. We provide new insights into the validity of the vascular tissue engineering approach.     

胰岛素通过活性氧的产生促进VEGF表达及血管平滑肌细胞迁移和增殖

 目的:探讨活性氧（reactive oxygen species, ROS）在胰岛素促进的血管平滑肌细胞迁移和增殖中的作用及分子机制。方法:采用原代培养的大鼠主动脉血管平滑肌细胞,应用DCF-DA荧光探针检测细胞内ROS的生成;应用实时定量PCR、Western blotting和ELISA法检测mRNA和蛋白的表达;应用转染报告基因的方法检测基因的转录活性;划痕法测定细胞迁移;CCK-8法测定细胞增殖。结果:胰岛素处理后血管平滑肌细胞内ROS产生明显增加。过氧化氢酶和NADPH氧化酶抑制剂二亚苯基碘鎓（DPI）明显抑制胰岛素促进的ROS生成及p-Akt、p-p70S6K1和p-ERK1/2蛋白的表达。过氧化氢酶和DPI明显降低胰岛素促进的血管内皮生长因子（vascular endothelial growth factor, VEGF）的mRNA和蛋白表达及转录激活。抑制ROS产生明显抑制胰岛素刺激的血管平滑肌细胞迁移和增殖。结论: 胰岛素通过NADPH氧化酶途径促进血管平滑肌细胞ROS产生。ROS介导了胰岛素促进的Akt/p70S6K1和ERK信号通路的激活、VEGF表达及血管平滑肌细胞的迁移和增殖。     

切应力对与血管平滑肌细胞联合培养的内皮细胞细胞外基质构筑及含量的影响

为了探讨切应力对血管平滑肌细胞联合培养的内皮细胞黏附能力的影响，为改进血管内皮细胞种植的组织工程技术提供实验基础，我们应用免疫荧光细胞化学、激光共聚焦扫描显微镜和图像分析等技术，研究了切应力作用下与血管平滑肌细胞联合培养的内皮细胞的细胞外基质中纤维粘连蛋白、层粘连蛋白和IV型胶原的构筑及含量的变化，同时以静态条件下联合培养的内皮细胞为对照组。结果表明，静态条件下，纤维粘连蛋白和层粘连蛋白在细胞内均以颗粒的形式存在于核周区，以纤丝的形式存在于细胞外，且纤丝相互交职成网状。IV型胶原主要以颗粒的形式存在于核周围，纤丝极少。切应力作用下纤维粘连蛋白形成纤丝束，并有沿切应力方向排列的趋势，层粘连蛋白、IV型胶原也形成纤维束，但排列无方向性。细胞外基质各组分的含量均有不同程度的变化。本文结果提示，切应力作用下，与血管平滑肌细胞联合培养的内皮细胞的黏附能力可能增强。     

低切应力对体外培养动脉壁细胞PDGF表达的影响

为了研究切应力对完整血管的生物学作用 ,探讨应力引起血管重建过程中生长因子的变化 ,用血管应力培养系统体外培养猪颈总动脉 ,通过免疫组织化学、抗体夹心法酶联免疫吸附测定实验 (EL ISA)和计算机图像分析等方法 ,观察了低切应力作用下动脉壁细胞 PDGF- A、PDGF- B表达变化情况。结果显示 ,低切应力作用下体外培养动脉 EC中 PDGF- B及 VSMC中 PDGF- A的合成与分泌皆增加。PDGF表达上升 ,促进了 VSMC表型转换与增殖。在低切应力引起的血管重建中 ,这一机制可能起着重要作用。     

The Effects of a Shear Flow on the Uptake of LDL and Acetylated LDL by an EC Monoculture and an EC–SMC Coculture

To elucidate the mechanisms of localized genesis and development of atherosclerosis and anastomotic intimal hyperplasia in man, a coculture of bovine aortic endothelial cells (ECs) and smooth muscle cells (SMCs) was prepared, and the effects of a shear flow on the uptake of lipoproteins by the cells was studied by incubating the EC-SMC coculture as well as an EC monoculture with a culture medium containing either DiI-LDL or DiI-Ac-LDL and subjecting to a laminar shear flow. It was found that in both the presence and absence of a shear flow that imposed the ECs an area mean shear stress of 13.3 dynes/cm2, the uptake of LDL by an EC-SMC coculture was much greater than that by an EC monoculture, whereas that of Ac-LDL was almost the same. The uptake of LDL by an EC monoculture increased slightly by being exposed to a shear flow, whereas that by an EC-SMC coculture did not. In contrast to this, the uptake of Ac-LDL by both an EC monoculture and an EC-SMC coculture decreased drastically by a shear flow, suggesting that the action of a shear flow on the uptake of Ac-LDL by vascular cells is very different from that of LDL.     

生理性周期性张应变通过激活AMPK磷酸化抑制血管平滑肌细胞迁移

目的 探讨细胞能量代谢的关键调节因子 AMP激活的蛋白激酶AMPK在血管平滑肌细胞（vascular smooth muscle cells, VSMCs）响应生理性周期性张应变力学刺激后对VSMCs迁移的影响。方法 采用 Flexcell-5000T体外细胞张应变加载系统,对大鼠原代培养的 VSMCs 施加10%幅度、1.25 Hz 频率的周期性张应变,模拟VSMCs在体内的生理性力学环境;以未加载周期性张应变的静态细胞为对照组,Western blotting 检测 VSMCs的 p-AMPK蛋白表达;划痕实验检测 VSMCs 迁移功能。结果 与静态组的细胞相比,生理性周期性张应变加载24 h后显著减少划痕愈合面积,提示生理性周期性张应变抑制VSMCs迁移;生理性周期性张应变加载3 h后,VSMCs的p-AMPK蛋白表达显著升高,而加载24 h后p-AMPK蛋白表达显著降低。在生理性周期性张应变加载条件下,孵育AMPK抑制剂可以在张应变加载3 h后显著降低 p-AMPK蛋白表达,而在张应变加载24 h后显著促进VSMCs迁移;在静态条件下孵育AMPK激活剂 AICAR 3 h后显著诱导p-AMPK蛋白表达,孵育24 h后显著抑制VSMCs迁移;提示p-AMPK蛋白表达参与调控VSMCs迁移。结论 生理性周期性张应变能通过激活p-AMPK蛋白表达,进而抑制VSMCs迁移,提示生理性周期性张应变调控VSMCs迁移对维持血管稳态具有重要意义。     

Effect of Fluid Shear Stress on Migration of Vascular Smooth Muscle Cells in Cocultured Model

Migration of smooth muscle cells (SMCs) in hyperplasia is thought to have a correlation with blood flow conditions. In this study, the effect of shear stress applied to endothelial cells (ECs) on SMC migration was examined using a newly designed EC–SMC coculture model (CM), in which bovine SMCs and ECs were separated by a collagen layer and a membrane filter. After exposing the CM to shear stresses of 0.5, 1.0, or 1.5 Pa for 48 h, the number of SMCs migrating into the collagen layer was counted. Under static conditions, the migration of SMCs in the CM increased compared with SMCs cultured alone. Shear stress of 1.5 Pa significantly suppressed the SMC migration (p < 0.05) compared with the static CM. Media conditioned with the CM exposed to shear stress of 1.0 Pa (p < 0.05) and 1.5 Pa (p < 0.005) exhibited reduction in activated matrix metalloproteinase-2 (MMP-2) compared with the static CM, as analyzed by zymography. Addition of an inhibitor of nitric oxide (NO) synthase, N ^ω-nitro-l-arginine methyle ester, to the media inhibited the effect of 1.5 Pa shear stress on SMC migration but MMP-2 activity was unaffected. These results suggest that physiological shear stress has protective roles in atherosclerogenesis.     

力-化学耦合作用在血管内皮细胞迁移中的作用及其力学生物学机制

目的 研究力学与化学因素的耦合在内皮细胞迁移过程中的作用以及其中的力学生物学机制。方法 在不同大小剪应力下分别用RTPCR、Western blot以及免疫荧光的方法检测内皮细胞CXCR1和CXCR2的表达变化;用antiIL8RA和antiIL8RB拮抗CXCR1和CXCR2,在剪应力作用下观察内皮细胞迁移情况;采用脂质体包绕法分别将Rac1及RhoA的野生型、活化型和抑制型3种质粒转染入内皮细胞,将转染了Rac1的3种质粒的细胞分别施加力学（剪应力）和化学（IL-8）刺激,对转染了RhoA的3种质粒的细胞施加化学刺激,检测以上条件下内皮细胞迁移情况。结果 CXCR1和CXCR2作为新型力学感受器参与调节内皮细胞迁移;Rac1与RhoA的高表达能促进内皮细胞迁移,反之,内皮细胞迁移被抑制。结论 IL-8Rs (CXCR1、CXCR2)、Rac1、RhoA是将力学、化学信号进行“耦合”的关键信号分子。