Activin a拮抗剂抑制子宫内膜异位症异位病灶细胞黏附及侵袭的研究

目的:利用细胞模型研究Activin a拮抗剂对子宫内膜异位症(内异症)异位病灶间质细胞及上皮细胞黏附及侵袭的影响。方法:分离培养内异症异位病灶间质细胞及上皮细胞,分别设立Activin a单抗组(加入Activin a单抗至终浓度为0.18μg/ml)和对照组(仅加入完全培养基)。结晶紫细胞黏附实验及Transwell小室细胞侵袭实验比较Activin a单抗对异位病灶间质细胞、上皮细胞黏附和侵袭能力的影响。结果:Activin a单抗抑制异位病灶间质细胞、上皮细胞的黏附及侵袭能力。结论:Activin a拮抗剂通过抑制异位病灶细胞的黏附及侵袭可能对内异症的治疗有一定的作用。

Activin A诱导大鼠胰腺导管干细胞分化形成胰岛β细胞

【目的】研究Activin A体外定向诱导大鼠胰腺导管干细胞分化形成胰岛β细胞的作用,为移植体外新生β细胞治疗宠物犬糖尿病打下基础,同时为胰岛细胞体外再生培养的优化提供科学依据。【方法】采用培养液RPMI-1640+10%FBS+10 ng/mL EGF+1%青霉素-链霉素扩增大鼠胰腺导管干细胞至单层,空白对照组加基础培养液,诱导组在基础培养液中分别添加5、10、15和20 ng/mL Activin A,连续培养28 d。诱导培养期间于倒置显微镜下观察细胞形态变化,诱导结束后通过双硫腙(DTZ)染色、细胞免疫荧光染色、ELISA检测及实时荧光定量PCR等方法对分化形成的类胰岛细胞团功能和性状进行验证。【结果】大鼠胰腺导管干细胞经Activin A诱导分化形成球形细胞,并聚集成团(类胰岛),DTZ染色结果均呈阳性。诱导28 d后,20 ng/mL Activin A诱导组的类胰岛细胞团数量及其Insulin基因表达水平均极显著高于其他3个Activin A诱导组(P<0.01,下同),且类胰岛细胞体积最大,类胰岛细胞的Pdx1基因表达水平最高;无论在低葡萄糖(5 mmol/L)还是高葡萄糖(25 mmol/L)的刺激下,20 ng/mL Activin A诱导组类胰岛细胞的Insulin和C-peptide分泌量均极显著高于其他3个Activin A诱导组。【结论】Activin A能体外诱导大鼠胰腺导管干细胞分化形成胰岛β细胞,且以基础培养液中添加20 ng/mL Activin A的诱导效果最佳。

Wnt3a和Activin A共同促进人胚胎干细胞向限定性内胚层细胞分化

【目的】研究Wnt3a和ActivinA存限定性内胚层诱导中共同作用的最佳时间窗。【方法】在无饲养层体系培养的人胚胎于细胞中，加入25ng／mLWnt3a和100ng／mLActivinA，共同作用不同时间（1～4d），收集不同作用时间点（0，1，2，3，4，5d）细胞，对原条、内中胚层前体标记Brachyury及限定性内胚层标记Sox17分别进行免疫荧光染色，统计阳性细胞总数，计算阳性细胞率。以看家基阕β-actin作为对照，采用RTPCR方法对原肠作用基因（Goosecoid（GSC）、Mixll）及三胚层发育相关基因（内胚层基因Sox17、Foxa2，内中胚层前体基因Braarchyury，中胚层基因Flk-1，外胚层基闪Pax6，胚外内胚层基因Sox7、CDX2）的表达进行检测。【结果]Wnt3a与ActivinA共同作用1～4d，均能获得限定性内胚层细胞，其中二者共同作用1d分别能获得（78．9±7．3）％Brachyury阳性细胞和（85．2±3．8）％的Sox17阳性细胞。RTPCR结果显示，在Wnt3a与ActivinA共同作用下，原肠作用基因及三胚层发育相关基因表达的时间有差异。【结论】Wnt3a和ActivinA共同作用1d，是有效促进人胚胎干细胞向限定性内胚层细胞分化的最佳作用时间窗。

Activin A与子宫内膜异位症的相关性研究

目的探讨Activin A对子宫内膜细胞迁移、侵袭、增殖、黏附的影响及其机制。方法无菌收集正常分泌期子宫内膜,分离、培养内膜原代细胞,以0(对照)、8、16、32、64 ng/mL Activin A孵育内膜腺上皮细胞、间质细胞12 h。采用体外细胞划痕实验、Transwell侵袭实验探讨不同浓度Activin A对子宫内膜间质细胞、腺上皮细胞迁移、侵袭的影响;采用CCK8法检测16 ng/mLActivin A对子宫内膜间质细胞、腺上皮细胞增殖、黏附的影响;采用Real-Time PCR检测16 ng/mL Activin A作用下子宫内膜间质细胞、腺上皮细胞血管内皮生长因子A(VEGF-A)、VEGF-C mRNA表达变化。结果体外细胞划痕实验结果显示,不同浓度Activin A处理组子宫内膜细胞划痕14 h前后面积差与对照组比较,差异无统计学意义(P>0.05)。Transwell侵袭实验结果显示:8、16、32 ng/mL Activin A处理组子宫内膜间质细胞穿透细胞数量较对照组明显增多(P<0.05);16、32 ng/mL Activin A处理组腺上皮细胞穿透细胞数量较对照组明显增多(P<0.05);16 ng/mL Activin A处理组穿透细胞数量最多。经16 ng/mL ActivinA处理的子宫内膜细胞增殖、黏附能力与对照细胞比较,差异无统计学意义(P>0.05)。经16 ng/mL Activin A处理的子宫内膜间质细胞和腺上皮细胞内VEGF-A、VEGF-C mRNA的表达与对照细胞比较,差异无统计学意义(P>0.05)。结论 Activin A可显著增强子宫内膜细胞的侵袭力,而对细胞迁移、增殖和黏附能力无明显影响;Activin A并非通过调节内膜细胞VEGF-A、VEGF-C的表达而使内膜细胞的侵袭力增强。

Neuroprotective effects of Activin A on endoplasmic reticulum stress-mediated apoptotic and autophagic PC12 cell death

Activin A, a member of the transforming growth factor-beta superfamily, plays a neuroprotective role in multiple neurological diseases. Endoplasmic reticulum（ER） stress-mediated apoptotic and autophagic cell death is implicated in a wide range of diseases, including cerebral ischemia and neurodegenerative diseases. Thapsigargin was used to induce PC12 cell death, and Activin A was used for intervention. Our results showed that Activin A significantly inhibited morphological changes in thapsigargin-induced apoptotic cells, and the expression of apoptosis-associated proteins [cleaved-caspase-12, C/EBP homologous protein（CHOP） and cleaved-caspase-3] and biomarkers of autophagy（Beclin-1 and light chain 3）, and downregulated the expression of thapsigargin-induced ER stress-associated proteins [inositol requiring enzyme-1（IRE1）, tumor necrosis factor receptor-associated factor 2（TRAF2）, apoptosis signal-regulating kinase 1（ASK1）, c-Jun N-terminal kinase（JNK） and p38]. The inhibition of thapsigargin-induced cell death was concentration-dependent. These findings suggest that administration of Activin A protects PC12 cells against ER stress-mediated apoptotic and autophagic cell death by inhibiting the activation of the IRE1-TRAF2-ASK1-JNK/p38 cascade.

Activin A特异性对人胚胎干细胞向限定性内胚层诱导分化的促进作用

【目的】研究Activin A对人胚胎干细胞(hESCs)向限定性内胚层(DE)诱导分化的促进作用及其信号通路分子,为hESCs向DE诱导分化体系的优化提供参考。【方法】在人饲养层体系培养的hESCs中,收集100ng/mL Activin A分别诱导0,6,12,24,48,72,96,120h的细胞,用实时荧光定量RT-PCR检测原条标记Gsc和Mixl1、中内胚层共同前体标记Brachyury、内胚层标记Foxa2和Sox17、中胚层标记Flk1、外胚层基因Pax6、多能性相关基因Oct4与Nanog表达水平的变化,用细胞免疫荧光检测Brachyury和Sox17蛋白表达水平的变化。【结果】在人饲养层(HEF)培养体系上,高浓度Activin A能更快地促进中内胚层基因的表达并提高其表达水平;Brachyury和Sox17蛋白的细胞免疫荧光检测表明,Activin A诱导12和48h就可检测二者的表达明显增加,且二者的表达水平分别在诱导48和96h时达到高峰;hESCs高效分化为限定性内胚层细胞,DE细胞分化率为(81.7±5.4)%,并且体外的内胚层分化过程遵循从原条开始、经过中内胚层共同前体阶段、再到内胚层的发育过程,与体内发育规律相似。【结论】Activin A能特异性地诱导人胚胎干细胞向限定性内胚层分化,转录调控Brachyury和Sox17蛋白的表达。

Emodin prevents hypoxic-ischemic neuronal injury Involvement of the activin A pathway

Emodin, an extract of dried rhizomes and the root of the Rhizoma Polygoni Cuspidati, can protect neurons from hypoxic-ischemic brain damage. This study aimed to verify the underlying mechanism After PC12 cells had differentiated into neuron-like cells under the induction of mouse nerve growth factor, cells were subjected to oxygen-glucose deprivation and treated with emodin. Results shewed that the viability of neuron-like cells cultured under an ischemia-hypoxia environment decreased, while the expression of activin A and caspase-3 in cells increased. Emodin raised the survival rate of oxygen-glucose deprived neuron-like cells~ increased activin A expression, and decreased caspase-3 expression. Experimental findings indicate that emodin can inhibit neuronal apoptosis and alleviate the injury of nerve cells after oxygen-glucose deprivation through the activin A pathway.

Activin A prevents neuron-like PC12 cell apoptosis after oxygen-glucose deprivation

In this study, PC12 cells were induced to differentiate into neuron-like cells using nerve growth factor, and were subjected to oxygen-glucose deprivation. Cells were treated with 0, 10, 20, 30, 50, 100 ng/mL exogenous Activin A. The 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl tetrazolium bromide assay and Hoechst 33324 staining showed that the survival percentage of PC12 cells significantly decreased and the rate of apoptosis significantly increased after oxygen-glucose deprivation. Exogenous Activin A significantly increased the survival percentage of PC12 cells in a dose-dependent manner. Reverse transcription-PCR results revealed a significant increase in Activin receptor IIA, Smad3 and Smad4 mRNA levels, which are key sites in the Activin A/Smads signaling pathway, in neuron-like cells subjected to oxygen-glucose deprivation, while mRNA expression of the apoptosis-regulation gene caspase-3 decreased. Our experimental findings indicate that exogenous Activin A plays an anti-apoptotic role and protects neurons by means of activating the Activin A/Smads signaling pathway.

基于Activin A途径的大黄素神经保护机制的研究

目的明确大黄素对缺血缺氧性脑损伤中的神经元具有保护作用的内在机制。方法应用pc-12细胞经牛膝多肽刺激转化为神经元样细胞后,并制备神经元细胞的氧糖剥夺(OGD)模型,并在此基础上,给予大黄素干预,分别检测细胞的生存力;应用酶联免疫方法检测细胞培养基的Activin A的表达含量;应用Western blotting对caspase-3蛋白的检测。结果 pc-12细胞经牛膝多肽刺激后转化为类神经元样细胞,在OGD后的pc-12细胞呈明显的梯度变化;实验组细胞的生存力明显提高,其培养基的Activin A含量升高明显,caspase-3蛋白的表达与单纯应用氧糖剥夺组比较,表达降低(P<0.05)。结论大黄素可以提高缺血缺氧状态下神经元细胞的生存力,同时可促进激活素的表达上调,并通过下调caspase3表达来抑制神经凋亡,起到神经保护的作用。

Activin A maintains cerebral cortex neuronal survival and increases voltage-gated Na^+ neuronal current

Activin A, which was first described in 1986, has been shown to maintain hippocampal neuronal survival. Activin A increases intracellular free Ca2＋ via L-type Ca2＋ channels. Our previous study showed that activin A promotes neurite growth of dorsal root ganglia in embryonic chickens and inhibits nitric oxide secretion. The present study demonstrated for the first time that activin A could maintain cerebral cortex neuronal survival in vitro for a long period, and that activin A was shown to increase voltage-gated Na＋ current （/Na） in Neuro-2a cells, which was recorded by patch clamp technique. The present study revealed a novel mechanism for activin A, as well as the influence of activin A on neurons by regulating expressions of vasoactive intestine peptide and inducible nitric oxide synthase.

Analysis of bacterial spectrum,activin A,and CD64 in chronic obstructive pulmonary disease patients complicated with pulmonary infections

BACKGROUND Pulmonary infections often lead to poor prognoses in patients with chronic obstructive pulmonary disease(COPD).Activin A and CD64 play crucial pathological roles in the development of COPD.AIM To explore the bacterial spectrum via analysis of activing A levels,CD64 index,and related mechanisms in COPD patients complicated with pulmonary infection.METHODS Between March 2015 and January 2018,a total of 85 patients with COPD,who also suffered from pulmonary infections,were enrolled in this study as the pulmonary infection group.In addition,a total of 96 COPD patients,without pulmonary infection,were selected as the control group.Sputum samples of patients in the pulmonary infection group were cultivated for bacterial identification prior to administration of antibiotics.The neutrophil CD64 index was measured using flow cytometry,serum activin A levels were detected via an enzyme-linked immunosorbent assay,and activin A,Smad3,TLR4,My D88,and NFκB protein expression was analyzed by Western blotting.RESULTS Gram-negative bacteria were identified in 57.65%of the sputum samples in the pulmonary infection group.The most prevalent Gram-negative species were Pseudomonas aeruginosa and Klebsiella pneumoniae.Conversely,Gram-positive bacteria were identified in 41.18%of the sputum samples in the pulmonary infection group.The most common Gram-positive species was Streptococcus pneumoniae.Fungi were identified in 1.17%of the sputum samples in the pulmonary infection group.The CD64 index was significantly higher in the pulmonary infection group(0.91±0.38)than in the control group(0.23±0.14,P<0.001).The serum activin A levels were significantly higher in the pulmonary infection group(43.50±5.22 ng/m L),compared to the control group(34.82±4.16 ng/m L,P<0.001).The relative expression levels of activin A,Smad3,TLR4,My D88,and NFκB were all significantly higher in the pulmonary infection group,compared to the control group(all P<0.001).CONCLUSION Pulmonary infections in COPD patients are mainly caused by Streptococcus pneumoniae,Pseudomonas aeruginosa,and Klebsiella pneumoniae.Pulmonary infections can significantly increase neutrophil CD64 index and serum levels of activin A,thereby activating the activin A/Smad3 signaling pathway,which may positively regulate the TLR4/My D88/NFκB signaling pathway.

Deriving striatal projection neurons from human pluripotent stem cells with Activin A

The striatum is the main input structure of the basal ganglia and is involved in voluntary motor control,habit learning and reward processing.Medium spiny neurons（MSNs）comprise80%and 95%of striatal neurons in primates and rodents,respectively.

SMAD2介导的Activin A信号对人胚胎干细胞向限定性内胚层细胞诱导分化的促进作用

【目的】研究SMAD2在Activin A促进人胚胎干细胞向限定性内胚层诱导分化中的作用。【方法】在人胚胎干细胞中转染SMAD2基因siRNA或者过表达质粒后,收集经Activin A分别诱导0,6,12,24,48,72,96和120h的细胞各100ng/mL,采用实时定量PCR检测SMAD2与内中胚层共同前体标记Brachyury和内胚层标记Sox17的表达,进一步通过Western-blot分析Activin A诱导中SMAD2和磷酸化SMAD2(p-SMAD2)表达的变化。【结果】在Activin A诱导人胚胎干细胞向限定性内胚层细胞分化的过程中,干扰SMAD2后48h时才检测到Brachyury强表达,而单纯Activin A处理组24h就检测到强表达;Sox17的表达始终较单纯Activin A处理组明显降低,因此,干扰SMAD2直接抑制了Activin A的诱导作用。而过表达SMAD2,Brachyury和Sox17的表达水平较单纯Activin A处理组明显增加,促进了限定性内胚层的发生;并且在Activin A诱导过程中,p-SMAD2的表达水平明显提高,而SMAD2的表达没有明显改变。【结论】SMAD2作为关键因子,介导了Activin A诱导人胚胎干细胞向限定性内胚层的分化,并转录调控Brachyury和Sox17的表达;SMAD2的磷酸化,激活并介导了Activin A诱导的信号转导通路。

重组人activin A对A549细胞增殖及凋亡的影响

背景与目的:活化素(activins)是转化生长因子TGF-β超家族成员。有研究表明,活化素可以诱导多种肿瘤细胞的凋亡。本研究旨在探讨重组人activin A对人肺腺癌细胞系A549增殖及凋亡的影响。方法:体外培养A549细胞,以不同浓度activin A处理A549细胞不同时间后,用MTT法检测其生长抑制情况;流式细胞仪及Annexin V-FITC试剂盒检测activin A对A549细胞凋亡的影响;Western blot检测活化素Ⅱ型受体(ActRⅡ和ActRⅡB)的表达情况。结果:activin A能抑制A549细胞增殖,且呈剂量和时间依赖性。流式细胞仪检测结果显示,activin A能促进A549细胞凋亡。Western blot结果显示,随着activin A浓度的增加,活化素Ⅱ型受体的表达量呈浓度依赖性增加。结论:activin A能在体外抑制A549细胞的增殖并诱导其凋亡。推测是通过诱导活化素Ⅱ型受体的表达,激活其下游一系列信号转导通路,从而发挥其生物学功能。

Activin A和Lefty A对人胚胎干细胞生长的影响

目的探讨TGF-β/Activin/Nodal信号通路的相关因子Activin A和Lefty A在一定浓度范围内,对人胚胎干细胞(hESC)自我更新的影响。方法在hES3细胞株的无滋养层无血清培养体系中加入1-100ng/ml的Activin A和Lefty A。7天后,通过碱性磷酸酶染色法对hES3细胞的自我更新状态进行评估。结果 Activin A在浓度为1,3,10,30和100ng/ml时,与阴性对照(SR培养基)组相比,未分化克隆的比率从7.7%分别提高到了18.5%,46.8%,61.4%,64.4%和79.1%,差异有统计学意义(P<0.01)。Lefty A组在浓度为1,3,10,30和100ng/ml时,与阴性对照(MCM培养基)组相比,未分化克隆的比率从80.5%分别降低到了72.4%,74.6%,72.2%,69.5%和65.3%,在浓度为100ng/ml时,差异有统计学意义(P<0.05)。结论较低浓度的Activin A即能有效维持hESC的自我更新,而较高浓度的Lefty A能诱导hESC分化。该结果进一步揭示了TGF-β/Activin/Nodal信号通路及其相关因子对hESC自我更新和分化的作用特点,有待对其机制进行深入研究。

激活素Activin A对猪卵母细胞胞质成熟的影响

[目的]探讨体外成熟过程中在培养液中添加激活素Activin A对猪卵母细胞胞质成熟的影响。[方法]在TCM-199中添加不同剂量Activin A的培养猪卵母细胞40 h,以激光共聚焦显微镜观察到的着色的线粒体排布形态类型来判断卵母细胞胞质是否成熟。[结果]当Activin A剂量为100 ng/ml时,胞质中线粒体呈弥散型分布形态的卵母细胞数目极显著增加(P<0.01),同时线粒体呈圆周型排布与半周型排布的卵母细胞数目显著下降(P<0.05)。[结论]猪卵母细胞体外成熟过程中在成熟培养液中添加激活素Activin A,能促进卵母细胞胞质的成熟。

Activin A对卵巢癌细胞系OVCAR-3的作用探讨

目的检测ActivinA对卵巢癌细胞系OVCAR-3的作用及其作用途径。方法用细胞免疫组织化学方法检测OVCAR-3细胞系膜受体ActRⅡ,用10ng/mL Activin A连续作用7d绘制细胞的生长曲线,设未加Activin A为对照组。用MTT方法检测Activin A对OVCAR-3刺激作用与时间和剂量的关系。West-ern-blot法检测bcl-2蛋白表达。结果OVCAR-3细胞系膜受体ActRⅡ表达阳性,Activin A对细胞增值作用与对照组比较差异有统计学意义,Activin A促进细胞增殖有时间依赖性,在48h达高峰;但无剂量依赖性。通过Western blot检测Activin A作用48h的OVCAR-3细胞Bcl-2蛋白表达情况,发现加Activin A组Bcl-2蛋白表达明显升高,差异有统计学意义。结论Activin A对OVCAR-3细胞系有促细胞增值作用,其促细胞增殖作用途径可能是通过Bcl-2抗凋亡通路发挥作用。

Effect of Activin A on OVCAR-3 Ovarian Epithelial Cancer Cells

Objective： To investigate the proliferation effect and its pathway of activin A on Ovarian epithelial cancer cells line OVCAR-3. Methods： OVCAR-3 cells were cultured in vitro and the membrane receptor ActR II was detected by immunohistochemical method. The OVCAR-3 cells were cultured with 10 ng/ml activin A for 7 d to observe the effects. Activin A at 5, 10, 15 and 20 ng/ml was used separately to treat the OVCAR-3 cancer cells for 24, 48 and 72 h in order to draw the growth proliferation rate curve measured by MTT method. The expression of protein bcl-2 was detected by western-blot. When OVCAR-3 cells were treated with 10 ng/ml activin A and 5 lag/ml DDP for 24, 48 and 72 h, cell apoptosis could be detected by electron microscopy and flow cytometry （FCM）. Results： Positive expression of ActR II was detected. We also found that the proliferation of OVCAR-3 reached to the climax on the 5th day of culture. The experiments showed that the cells treated with activin A increased quickly and grew faster than those in control group. Moreover, OVCAR-3 cells treated with activin A for 48 h proliferated significantly greater than those treated for 24 h or 72 h （P〈0.01）. bcl-2 protein expression increased expression in activin A treated group than in control group （P〈0.05）. Conclusion： Activin A could increase the proliferation of OVCAR-3 cells which may be through bcl-2 anti-apoptosis pathway.

Synergistic Effects of Activin A and Fibroblast Growth Factor 2 in the Modulation of Insulin Expression

Diabetes is the most prevalent and serious metabolic disease, and the number of diabetic patients worldwide is increasing. The reduction of insulin biosynthes is in pancreatic E-cells is closely associated with the onset and progression of diabetes, therefore, it is important to search for ways to induce insulin-producing cells in non-E-cells. In the present study, it has been reported that activin A and a basic fibroblast growth factor 2 （ FGF2）, can synergistically increase the insulin mRNA level, in both mouse El4 striatal primary cell cultures and the hippocampal neuronal cell line HT22. Activin A and FGF2 can jointly stimulate the nuclear translocation of Smad3 and specifically activate ERK1/2. It is interesting to note that a specific inhibitor for MEK, U0126, can efficiently block the induction of an insulin promoter activity by activin A and FGF2. This indicates that activin A collaborates with FGF2, giving a signal to induce the insulin gene through selective activation of the ERK-type MAP kinase and Smad3 in mouse striatal and HT22 cells. These data suggest that activin A may act in concert with FGF2 for the development of insulin -positive neurons

Activin A通过JNK/ERK信号通路调控皮肤成纤维细胞肌动蛋白重组的研究

目的探讨Activin A对成纤维细胞微丝肌动蛋白的作用以及其信号机制。方法出生24 h内的C57BL/6乳鼠皮肤成纤维细胞分离培养。实验分为PBS组、Activin A组以及JNK特异性抑制剂SP600125组、ERK特异性抑制剂SL327组、p38特异性抑制剂SB202190组。3～5代成纤维细胞,提取细胞总蛋白,Western blot检测JNK、ERK、p38的磷酸化活性并且通过Phallotoxins染色观察成纤维细胞肌动蛋白变化。结果 Activin A可诱导成纤维细胞肌动蛋白聚集,给予JNK特异性抑制剂SP600125后,成纤维细胞肌动蛋白聚集现象完全被抑制;给予ERK特异性抑制剂SL327后,成纤维细胞肌动蛋白聚集现象减弱,但是尚未完全被抑制;而p38特异性抑制剂SB202190处理后,成纤维细胞肌动蛋白聚集未被抑制。结论 Activin A通过JNK和ERK信号通路诱导成纤维细胞肌动蛋白重组聚集。