构建具有神经支配的工程化心肌组织的初步研究

探索神经生长因子诱导的交感神经元样PC12细胞作为组织工程化心肌组织神经支配研究模型的可行性。用含0.04?TA的0.25%胰酶分离新生大鼠原代心肌细胞,然后与NGF诱导的交感神经元样PC12细胞在液态的Ι型胶原中共培养,通过光学显微镜观察、常规H.E.染色、免疫组织化学染色和透射电镜观察等对其进行评价。在三维共培养模型中,NGF诱导的交感神经元样PC12细胞长出神经突起,突起及其上的膨体能够到达跳动的心肌细胞表面,神经突起随心肌细胞一起跳动。说明采用神经元样PC12细胞作为组织工程化心肌神经支配的模型是可行的,神经细胞与心肌细胞可能有支配关系。     

5-氮胞苷诱导体外培养的胚胎干细胞向心肌样细胞分化

目的研究5-氮胞苷(5-aza)体外诱导胚胎干细胞分化为心肌细胞的可行性。方法将P19细胞接种于培养板中或铺有琼脂的培养板中,5μmol/L5-aza培养7d后用不含5-aza的培养基继续培养。倒置显微镜观察细胞跳动情况,用免疫细胞化学、RT-PCR检测及电镜观察等方法鉴定细胞分化。结果5-aza暴露结合悬浮培养可诱导P19细胞分化为有节律跳动的心肌细胞。分化的细胞表达心肌特异的GATA-4、α-MHC mRNA及α-sarcomeric actin、cTnT蛋白,同时透射电镜观察到细胞质内有明显的肌丝。结论5-aza在体外可诱导胚胎干细胞向心肌样细胞分化,悬浮培养有助于细胞的心肌化过程。     

交感神经节与心肌细胞联合培养的光镜和扫描电镜观察

目的：为了研究体外培养中交感神经纤维或神经终末与心肌细胞之间的关系。方法：将新生大鼠的交感神经节与心肌细胞进行联合培养,用相差显微镜观察神经元的生长,用扫描电子显微镜观察神经肌肉连接的形成。结果：交感神经节与心肌细胞联合培养７２￣９６小时,在相差显微镜下可观察到神经突起终止于搏动的心肌细胞表面。在扫描电子显微镜下可观察到神经纤维粗细不一,在心肌细胞表面交织成网状。有的神经纤维末端膨大并贴于心肌细胞     

迷走神经结状神经节与心肌细胞联合培养的活细胞观察

用新生大鼠的连走神经结状神经节与心肌细胞进行联合培养。在相差显微镜下观察了神经元的生长以及神经一肌肉连接的形成．结状神经书与心肌细胞联合培养72～96h可观察到神经突起的终未终止于搏动的心肌细胞表面．神经节组织块周围有许多突起在心肌细胞表面相互交织成网状．交叉的突起相互粘连在一起，终止于抢动的心肌细胞表面的一个突起移动可牵动邻近的交叉突起同．本研究首次建立了在体外培养中具有结状神经节神经支配的心肌细胞模型．     

交感神经节在共同培养的心肌细胞诱导下神经元的生长和迁移

目的：探讨联合培养中大鼠心肌细胞对交感神经节神经元的生长和迁移的影响。方法：在体外建立大鼠交感神经节与心肌细胞联合培养的模型。用倒置相差显微镜观察交感神经节与心肌细胞联合培养不同时间的活细胞生长状况,计数神经纤维束的数目。并用Holmes还原银染色法计数神经元迁移的数目。结果：联合培养中由交感神经节组织块长出的神经纤维束数目、直径大于5μm的神经纤维束的数目以及由交感神经节组织块迁出的神经元的数目均较单纯交感神经节组织块培养的明显增加。结论：联合培养中分散的心肌细胞诱导培养的交感神经节神经元神经突起的生长和神经元向周围的迁移。     

成熟心肌细胞诱导胚胎干细胞定向分化为心肌样细胞

目的： 拟证实成熟心肌细胞对胚胎干细胞（ESCs）定向分化的诱导作用。方法：分离培养SD大鼠乳鼠心肌细胞,以DAPI（4’6-联眯-2-苯基吲哚）染核作为细胞标记。取昆明小鼠3.5-4 d的囊胚培养后分离出ESCs,以1和（或）2代的小鼠ESCs细胞团与心肌细胞共培养。录像动态观察ESCs向心肌细胞分化的情况;分别于共培养后3、7、14 d对ESCs行肌钙蛋白T（cTnT）免疫荧光染色检测。结果：1代或2代的ESCs和/或细胞团与心肌细胞共培养约7 d左右出现成节律搏动的心肌样细胞;最好实验批次,可计数到1/4以上的ESCs和/或ESCs细胞团出现节律性搏动。心肌细胞共培养体系中添加0.6% DMSO时,节律搏动的心肌样细胞分化率未见提高。心肌细胞诱导组,记录已搏动和未搏动ESCs诱导分化后心肌样细胞cTnT蛋白荧光染色阳性;ESCs与心肌成纤维细胞共培养诱导组,分化的细胞cTnT蛋白荧光染阴性;0.6% DMSO诱导组约3％的细胞cTnT蛋白荧光染色阳性;DMSO联合心肌细胞诱导组,结果与单纯心肌细胞诱导组相似。结论：未经建系操作的ESCs可直接诱导分化为节律搏动的心肌细胞;成熟心肌细胞与ESCs共培养状态下,成熟心肌细胞是ESCs定向分化较强的诱导因素。     

正常和损伤坐骨神经及其提取液对PC12细胞和颈上神经节促神经突起生长的作用

本文一方面用大鼠正常和损伤坐骨神经与新生大鼠颈上节联合培养,观察坐骨神经对颈上节神经元突起生长的诱导作用;另一方面制备正常和损伤坐骨神经提取液,以观察不同浓度提取液对PC12细胞的促突起生长作用。结果表明,正常和损伤坐骨神经都含有对颈上节交感神经元起作用的促神经突起生长因子(neurite-promoting factors,NPFs),损伤神经的NPFs活性显著高于正常神经,损伤1～41天,坐骨神经的NPFs活性对颈上节的差别不大。正常和损伤坐骨神经提取液对PC12细胞亦有促突起生长作用,以损伤第4天神经提取液对PC12细胞的作用最大,这种作用从神经损伤后12小时持续至42天。此外,发现PC12细胞突起生长的速度与损伤神经提取液浓度成正相关。实验分析提示损伤神经所含NPFs可能不是单一的,估计有两种或两种以上,这些活性因子很可能是Schwann细胞产生和分泌的。     

与心肌细胞共培养的小鼠胚胎干细胞向心肌样细胞分化

目的 探讨心肌细胞促进胚胎干细胞（ESCs）分化为心肌样细胞的诱导作用。方法 收集小鼠3.5d胚龄的囊胚,将其培养在小鼠胚胎成纤维细胞饲养层上,4～5d后取内细胞团接种在饲养层上分离培养出ESCs。取3～5代ESCs,先将ESCs悬浮培养形成2～3d的拟胚体(EBs),再与新生大鼠心肌细胞共培养诱导向心肌细胞分化,相差显微镜下观察分化细胞的形态学变化,免疫细胞荧光技术检测心肌细胞特异性肌钙蛋白T(TnT)、α－肌动蛋白（α-Actin）的表达。结果 诱导第3天起可见自发性、有节律跳动的拟胚体出现,12d时第2组约有93%的拟胚体出现节律性收缩,显著高于其他各组,均表达心肌细胞特异性蛋白cTnT、а-actin,心肌细胞直接接触诱导组其分化比率达56.5%,高于其他各组,并且分化的细胞形态较单一。结论 心肌细胞和心肌细胞裂解液均可诱导ESCs向心肌细胞定向分化,且心肌细胞的诱导作用强于心肌细胞裂解液。     

诱导间充质干细胞向心肌样细胞的分化*

背景：目前很多体内外实验都已表明间充质干细胞具有向心肌样细胞或心肌细胞分化的能力,这使间充质干细胞治疗心脏疾病成为可能。 目的：比较诱导间充质干细胞向心肌样细胞分化实验方法的异同及存在的问题。 方法：以“mesenchymal stem cells,cardiomyocyte, differentiation;间充质干细胞,心肌细胞,诱导;分化”为检索词,应用计算机检索2000-12/2010-12 PubMed 数据库与万方数据库,与间充质干细胞诱导分化为心肌细胞相关的文章。 结果与结论：间充质干细胞向心肌细胞分化的方法主要有：①心肌细胞条件培养液：间充质干细胞在体外经药物诱导或模拟体内心肌微环境能定向诱导分化为心肌样细胞。②与希望诱导成的目的细胞共同培养：间充质干细胞可经药物诱导诱导分化为心肌细胞,也可不经任何诱导在心肌微环境中分化为心肌细胞。虽然间充质干细胞在体外心脏病理条件下可分化为心肌细胞,使间充质干细胞治疗心脏疾病成为可能,但其研究有待进一步完善。关键词：诱导;分化;间充质干细胞;心肌样细胞;干细胞 doi:10.3969/j.issn.1673-8225.2012.19.035     

银杏内酯B对高胆固醇诱导的PC12细胞胆固醇含量的影响

目的: 基于体外血脑屏障环境下观察银杏内酯B(ginkgolide B,GB)对高胆固醇诱导PC12细胞胆固醇含量的影响。方法: 利用Transwell共培养脑微血管内皮细胞和星形胶质细胞建立体外血脑屏障,并与PC12细胞共培养。在Transwell内皮细胞侧添加40 μmol/L胆固醇,并选择不同浓度银杏内酯B分别干预上述模型。观察PC12细胞的形态学变化,通过MTT法筛选银杏内酯B的干预浓度,用HPLC法检测PC12细胞内胆固醇含量的变化。结果: 倒置相差显微镜观察发现PC12细胞大多呈圆形、短梭形或三角形,胞浆伸展,细胞两极有短突起,细胞折光性好,胞核可见。通过MTT确定的银杏内酯B干预剂量分别为低剂量1 μmol/L、中剂量5 μmol/L和高剂量25 μmol/L。HPLC法检测显示模型组PC12细胞内胆固醇含量升高,与对照组相比差异显著 (P<0.01)。银杏内酯B干预上述模型16 h后,25 μmol/L GB降胆固醇的效果最佳,与模型组相比差异显著(P<0.05),25 μmol/L GB与阳性对照药辛伐他汀相比差异无显著(P>0.05)。结论: 中药银杏叶有效单体GB可以降低体外血脑屏障环境下PC12细胞内胆固醇含量。     

P53和P21在PC12细胞分化中的作用

目的探讨P53及其下游P21蛋白在PC12细胞分化中的可能作用机制。方法用反转录病毒质粒pBabe-P53/m175转染未分化PC12细胞,经筛选后建立野生型P53蛋白功能丧失的细胞系PC12(P53/m175);利用倒置相差显微镜、流式细胞术及Western blot等方法,比较两种细胞在神经生长因子(NGF)作用后,细胞分化表型、细胞周期及相关蛋白表达的改变。结果NGF作用于正常PC12细胞组,P53/P21蛋白表达持续升高,细胞G1期阻滞出现的时间与蛋白表达开始增加的时间相一致;而在NGF作用的PC12(P53/m175)细胞组,细胞不能表达P21蛋白,且细胞周期G1期阻滞的程度也出现显著下降。NGF作用下,两组细胞都能观察到神经突起的生长,表现出明显的分化表型。结论PC12细胞经NGF作用后出现P53及P21蛋白持续表达增加,主要介导了细胞分化过程中细胞周期G1期阻滞这一特征的出现,但并不是导致神经突起进行性生长这一特征的必需条件。     

JNK信号转导通路在NGF抗6-OHDA诱导PC12细胞凋亡中的作用

目的： 以6-羟基多巴胺(6-OHDA)作用于大鼠肾上腺嗜铬细胞瘤细胞(PC12 cells)以诱导其凋亡,然后在其中分别加入神经生长因子（NGF）及c-Jun氨基端激酶（JNK）阻断剂SP600125,研究在加入NGF后JNK的活性与凋亡的关系。方法: 实验分为对照组、6-OHDA组、NGF组、6-OHDA+NGF组、6-OHDA+JNK阻断剂SP600125组,以流式细胞分析法检测各组PC12细胞的凋亡率,以免疫印迹（Western blotting）法检测各组PC12细胞JNK的活化情况。结果: 6-OHDA导致PC12细胞凋亡,JNK1活性提高;预孵SP600125或NGF15min后再加入6-OHDA则PC12细胞凋亡率及JNK1活性均降低。结论: JNK1参与了6-OHDA致PC12细胞凋亡作用,NGF抗6-OHDA所诱导的PC12细胞凋亡作用与其抑制JNK的活化有关。     

Release of interleukin-6 via the regulated secretory pathway in PC12 cells

A growing body of evidence suggests that diverse growth factors such as neurotrophins (NTs), insulin-like growth factor-1 (IGF-1), and glial cell line-derived neurotrophic factor (GDNF) can be released via the regulated secretory pathway in neuronal cells, possibly representing a mechanism for preferentially supplying these growth factors to active synapses. Here we investigated whether interleukin-6 (IL-6), a member of the family of neuropoietic cytokines, can be released via stimulus-coupled secretion as well. IL-6 was expressed in PC12 cells, a neuronal model cell line that is frequently used for the study of vesicle release and trafficking. Regulated secretion of this cytokine was induced by 0.5 mM ATP and treatment with epidermal growth factor (EGF) and nerve growth factor (NGF). Release induced by 0.5 mM ATP but not by NGF or EGF depended on the presence of extracellular Ca(++). Furthermore, IL-6 colocalized with the dense core vesicle (DCV)-marker secretogranin-II (Sg-II) in transfected PC12 cells. Our data suggest that the neuropoietic cytokine IL-6 can be sorted to the regulated secretory pathway in neuronal cells and indicate a potential role for this cytokine in synaptic plasticity.     

Simultaneous visualization of the binding of nerve growth factor and epidermal growth factor to single rat pheochromocytoma (PC12) cells through indirect immunohistofluorescence

The rat pheochromocytoma cell line, PC12, which has receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF), was used to develop a technique for the simultaneous visualization of separate growth factor receptors by indirect immunohistofluorescence. The cells were incubated with saturating concentrations of nerve growth factor and epidermal growth factor. After fixation, the cells were treated with anti-NGF sheep antiserum and then with antisheep rabbit IgG conjugated with fluorescein; they also were treated with anti-EGF rabbit antiserum and then with anti-rabbit sheep IgG conjugated with rhodamine. Fluorescence microscopy showed that a single PC12 cell bound both NGF and EGF. The fluorescence due to EGF binding was reduced when the cells were grown in the presence of NGF. A similar reduction of fluorescence was observed after addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Both manipulations are known to reduce the specific binding of 125I-EGF to these cells. Subclones of PC12 cells, NR11 and NR20, reported not to have NGF receptors, did not demonstrate NGF binding when tested with this indirect immunohistofluorescence method. Thus, the binding of growth factors which is demonstrable by indirect immunohistofluorescence method seems to reflect the presence of the specific cell surface receptors for both peptides on individual PC12 cells.     

Neurite outgrowth of PC12 mutant cells induced by orange oil and d-limonene via the p38 MAPK pathway

We studied the effects of natural essential oil on neurite outgrowth in PC12m3 neuronal cells to elucidate the mechanism underlying the action of the oils used in aromatherapy. Neurite outgrowth can be induced by nerve growth factor (NGF), where ERK and p38 MAPK among MAPK pathways play important roles in activating intracellular signal transduction. In this study, we investigated whether d-limonene, the major component of essential oils from oranges, can promote neurite outgrowth in PC12m3 cells, in which neurite outgrowth can be induced by various physical stimulations. We also examined by which pathways, the ERK, p38 MAPK or JNK pathway, d-limonene acts on PC12m3 cells. Our results showed that neurite outgrowth can be induced when the cells are treated with d-limonene. After treatment with d-limonene, we observed that p38 MAPK is strongly activated in PC12m3 cells, while ERK is weakly activated. In contrast, JNK shows little activity. A study using an inhibitor of p38 MAPK revealed that neurite outgrowth in PC12m3 cells is induced via the activation of p38 MAPK by d-limonene. The results thus indicate that d-limonene may promote neural cell differentiation mainly via activation of the p38 MAPK pathway.     

Nerve growth factor decreases potassium currents and alters repetitive firing in rat sympathetic neurons

The sympathetic nervous system is an essential regulator of the cardiovascular system and interactions with target tissue regulate sympathetic neuronal properties. The heart produces nerve growth factor (NGF), which promotes sympathetic noradrenergic innervation of cardiac tissue and affects sympathetic synaptic strength. Neurotrophins, including NGF, are important modulators of synaptic plasticity and membrane electrical properties. Here we show that acute application of NGF causes a change in the repetitive firing pattern of cultured sympathetic neurons of the rat superior cervical ganglion. Neurons fire fewer action potentials in NGF, but with increased frequency, demonstrating an NGF-dependent change from a tonic to a phasic firing pattern. Additionally, NGF decreases the spike time variance, making spikes more tightly time locked to stimulus onset. NGF causes a decrease in the amplitude of both calcium-dependent and -independent potassium currents, and inhibition of calcium-dependent potassium currents using CdCl(2) reproduces some, but not all, of the firing properties induced by NGF. This study suggests that NGF release from cardiac tissue may act to modulate the repetitive firing properties of sympathetic neurons to tune their output to meet the physiological needs of the organism.     

Glial cell line-derived neurotrophic factor (GDNF) enhances sympathetic neurite growth in rat hearts at early developmental stages

Molecular signaling of sympathetic innervation of myocardium is an unresolved issue. The purpose of this study was to investigate the effect of neurotrophic factors on sympathetic neurite growth towards cardiomyocytes. Cardiomyocytes (CMs) and sympathetic neurons (SNs) were isolated from neonatal rat hearts and superior cervical ganglia, and were co-cultured, either in a random or localized way. Neurite growth from SNs toward CMs was assessed by immunohistochemistry for neurofilament M and α-actinin in response to neurotrophic factors-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) and a chemical repellent, semaphorin 3A. As a result, GDNF as well as NGF and BDNF stimulated neurite growth. GDNF enhanced neurite outgrowth even under the NGF-depleted culture condition, excluding an indirect effect of GDNF via NGF. Quantification of mRNA and protein by real-time PCR and immunohistochemistry at different developmental stages revealed that GDNF is abundantly expressed in the hearts of embryos and neonates, but not in adult hearts. GDNF plays an important role in inducing cardiac sympathetic innervation at the early developmental stages. A possible role in (re)innervation of injured or transplanted or cultured and transplanted myocardium may deserve investigation.     

前列腺凋亡反应基因4反义寡核苷酸抑制谷氨酸诱导的PC12细胞内钙离子浓度上调及其抗凋亡意义

目的　研究前列腺凋亡反应基因4(par -4)反义寡核苷酸拮抗谷氨酸对PC12细胞内游离钙离子浓度的上调作用及其抗凋亡意义。方法　脂质体介导转染par -4反义寡核苷酸。谷氨酸诱导PC12细胞凋亡。Hoechest33258 /碘化丙啶荧光染色观察细胞凋亡形态,流式细胞术分析凋亡百分率。Fura 2 /AM荧光染色结合激光共聚焦显微镜测定细胞内游离钙离子浓度。逆转录聚合酶链反应(RT- PCR)测定钙依赖性蛋白酶Calpain10的mRNA水平。Western印迹测定par -4蛋白表达量。结果与正常对照组(25 .6±4.1 )相比,谷氨酸诱导PC12细胞中par- 4蛋白表达上调( 90. 0±3 2,P<0. 01),par- 4反义寡核苷酸拮抗其上调( 52 .3±5 .0,P<0 .01 );谷氨酸诱导凋亡组凋亡百分率为53%, par -4反义寡核苷酸拮抗谷氨酸诱导的PC12细胞凋亡(31%,P<0. 01);谷氨酸诱导PC12细胞内游离钙离子浓度上调,par- 4反义寡核苷酸拮抗其上调(荧光强度比值分别为167 .9±32 .4、228. 8±36. 8,P<0 .01);谷氨酸诱导PC12细胞内钙依赖性蛋白酶Calpain10mRNA水平上调( 46 .3±3 .7 ),par- 4反义寡核苷酸抑制其上调(34 8±2 1,P<0 01 )。结论　par -4反义寡核苷酸拮抗谷氨酸诱导的PC12细胞凋亡,其机制可能与抑制细胞内游离钙离子浓度上调和抑制Calpain10基因转录有关。     

SHP-2通过ERK和JNK通路调节PC12细胞应对NGF的细胞生存和凋亡

目的：探讨蛋白酪氨酸磷酸酶SHP-2在神经生长因子(NGF)作用下大鼠嗜铬细胞瘤PC12细胞生存及NGF撤除后细胞凋亡的过程中的作用及机制。方法：SHP-2 抑制剂NSC87877作用于PC12细胞, MTT测定PC12细胞存活率;流式细胞仪检测细胞凋亡率。将pIRES-GFP空载体、pIRES-GFP-SHP-2野生型和pIRES-GFP-SHP-2C459S突变体通过脂质体方法转染PC12细胞;加入NGF作用１h和撤除NGF 5 h后分别用Western blotting方法检测细胞外信号调节蛋白激酶(ERK)、磷酸化ERK（p-ERK）、c-Jun氨基末端激酶(JNK)及磷酸化JNK (p-JNK)表达变化。结果：MTT和流式细胞仪检测表明SHP-2 可以促进PC12细胞生存,抑制细胞凋亡。Western blotting结果显示无SHP-2抑制剂组和转染pIRES-SHP-2野生型组p-ERK表达在加入NGF的过程中升高;撤除NGF后,各组p-ERK表达均降低,pIRES-GFP-SHP-2C459S突变体组和pIRES-GFP组p-ERK表达明显低于pIRES-GFP-SHP-2野生型组, NGF去除+ SHP-2抑制剂组的表达水平明显低于NGF去除对照组; NGF去除+SHP-2抑制剂组p-JNK表达高于NGF去除对照组;pIRES-GFP-SHP-2C459S突变体组高于pIRES-GFP空载体组,pIRES-GFP-SHP-2野生型组低于pIRES-GFP空载体组。结论：SHP-2可能通过对ERK的正向激活,抑制JNK的激活,增强NGF作用下PC12细胞生存及抑制NGF撤除后所致细胞凋亡,从而在NGF的信号转导中起到一个中心环节的作用。