维甲酸对人RPE细胞胞间通讯功能和Cx43表达的影响

目的观察培养的人视网膜色素上皮（RPE）细胞中连接蛋白Cx43的表达特点，以及维甲酸（RA）对人RPE细胞生长和细胞间缝隙连接功能、细胞间通道蛋白Cx43表达的影响及其相互关系。方法免疫组织化学方法观察缝隙连接蛋白（Cx43）在培养的人RPE细胞中的表达特点；不同浓度的RA作用于培养的第4代人的色素上皮细胞，用MTT方法观察RA对体外培养人RPE细胞生长的抑制；用LY染料传输方法测定细胞间隙连接功能，观察这三组不同浓度的RA对人RPE细胞缝隙连接功能的影响；免疫组织化学方法鉴别不同浓度RA影响后的缝隙连接蛋白Cx43的表达，用计算机灰度测量的方法评估表达的强度并作统计学分析。结果免疫组织化学染色显示体外培养的人RPE细胞表达缝隙连接蛋白Cx43，表达的部位主要在细胞浆和细胞膜上；RA对人RPE细胞的生长有明显的抑制作用，其抑制作用呈剂量依赖性；LY染料传输试验表明在RA的作用下RPE细胞间通讯功能明显增强；经计算机图像分析表明RA使连接蛋白Cx43表达增强，增强的程度与RA的浓度呈正相关。结论体外培养的人RPE细胞可以表达Cx43蛋白，RA抑制培养的色素上皮细胞的生长，提高细胞间隙连接通讯功能，上调Cx43蛋白在人RPE细胞中的表达。     

Prolyl Isomerase Pin1 Expression in the Spinal Motor Neurons of Patients With Sporadic Amyotrophic Lateral Sclerosis

Background and PurposeAmyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Selective deficiency of edited adenosine deaminase acting on RNA 2 (ADAR2), a key molecule in the acquisition of Ca²⁺ resistance in motor neurons, has been reported in sporadic ALS (sALS) spinal motor neurons. Since ADAR2 activity is positively regulated by prolyl isomerase Protein never in mitosis gene A interacting-1 (Pin1), a known phosphorylation-dependent peptidyl-prolyl cis/trans isomerase, we investigated Pin1 expression in spinal motor neurons in sALS.MethodsSpecimens of the spinal cord were obtained from the lumbar region in eight sALS patients and age-matched five controls after postmortem examinations. The specimens were double stained with anti-Pin1 and anti-TAR DNA-binding protein of 43 kDa (TDP-43) antibodies, and examined under a fluorescence microscope.ResultsThis study analyzed 254 and 422 spinal motor neurons from 8 sALS patients and 5 control subjects, respectively. The frequency of motor neurons with high cytoplasmic Pin1 expression from the spinal cord did not differ significantly between sALS specimens without cytoplasmic TDP-43 inclusions and control specimens. However, in sALS specimens, neurons for which the Pin1 immunoluminescence intensity in the cytoplasm was at least twice that in the background were more common in specimens with cytoplasmic TDP-43 inclusions (p<0.05 in χ² test).ConclusionsIn sALS, neurons with higher expression levels of Pin1 levels had more TDP-43 inclusions. Despite the feedback mechanism between Pin1 and ADAR2 being unclear, since Pin1 positively regulates ADAR2, our results suggest that higher Pin1 expression levels in motor neurons with TDP-43 pathology from sALS patients represent a compensatory mechanism.     

APP17肽对缺氧缺血性脑病新生鼠皮质区GAP-43表达的影响

目的：观察APP（β—amyloid precursop protein,APP）17肽（APP695—319—335）肽段对缺氧缺血性脑病新生鼠皮质区GAP-43表达的影响。方法：将生后7d的新生大鼠随机分为假手术组、脑病组和APP17肽治疗组。采用一侧颈动脉结扎,然后放在缺氧仓内2h的方法制备缺氧缺血性脑病模型。应用免疫组化方法观察各组大鼠不同时期脑皮质GAP-43表达的变化。结果：免疫组化结果显示,脑病组较假手术组小鼠皮质区GAP-43免疫阳性细胞增加（P〈0．01）．治疗组较脑病组GAP-43免疫阳性细胞明显增多（P〈0．01）。结论：缺氧缺血性脑损伤后,皮质区GAP-43表达和合成增加,GAP-43的表达增加可能与神经元再生和轴突重塑有关,是脑缺氧缺血后神经细胞内源性代偿机制之一。APP17肽可能通过调节GAP-43的表达来增加神经细胞之间的突触联系,促进神经的生长起到营养神经的作用,从而改善缺氧缺血性脑病小鼠的预后。     

扶芳藤合剂对兔病态窦房结综合征Cx45、Cx43及Cx40蛋白表达的影响

目的观察复方扶芳藤液对兔病态窦房结综合征模型心率（HR） 及窦房结电生理、及心脏窦房结组织Cx45、Cx43及Cx40蛋白表达的影响,并探讨其作用机制.方法将40只清洁级健康日本大耳白兔采用随机、平行对照的方法,按随机数字表分为空白对照组、模型组、心宝丸组及复方扶芳藤液组（中药组）高、低剂量组,每组各 8 只.造模成功后空白对照组、模型组分别予生理盐水15 mL/d,心宝丸组予心宝丸悬浮液,中药组高、低剂量组分别予复方扶芳藤液连续灌胃14 d.取各组兔上下腔静脉之间的窦房结组织,常规切片,免疫组化检测窦房结组织Cx45、Cx43及Cx40蛋白表达.结果大剂量组窦房结组织Cx45、Cx43及Cx40蛋白表达明显高于模型组（P＜0.05）;小剂量组干预后窦房结组织Cx45、Cx43及Cx40蛋白表达较模型组增高,其中,Cx40较模型明显增高（P＜0.05）.结论复方扶芳藤可上调损伤兔窦房结组织Cx45、Cx43及Cx40蛋白的表达,可能是治疗病态窦房结综合征的机制之一.     

生长相关蛋白GAP-43多肽抗体的制备

目的对生长相关蛋白-43（growth associated proteins-43,GAP-43）进行抗原表位分析并制备兔多克隆抗体。方法通过对GAP-43 cDNA序列及氨基酸序列的结构进行生物信息分析,依据蛋白质的二级结构、亲水性、疏水性、抗原性及理化特性,经过同源性检索后,综合考虑抗体设计的其他因素,选出具有免疫活性的抗原决定簇多肽片段,采用有机固相多肽合成法合成了GAP-43的多肽片段,并与载体蛋白血蓝蛋白（KLH）偶联制备成抗原,免疫新西兰家兔。结果 ELISA测定GAP-43抗体效价为1∶32 000;Western blot检测结果显示：在分子量25kDa出现单一条带;免疫组织化学法检测显示：GAP-43蛋白在小鼠海马神经元中有表达;免疫细胞化学法检测显示：GAP-43蛋白在人神经母细胞瘤株SH-SY5Y中存在表达。结论利用生物信息学软件比较准确地预测GAP-43的抗原决定簇,并成功制备了高效价、高特异性的多肽抗体。     

模拟肽Gap27抑制缝隙连接蛋白43在帕金森病小鼠模型中的作用

目的探索在6-羟基多巴胺(6-hydroxydopamine，6-OHDA)诱导的帕金森病(Parkinson’s disease，PD)小鼠模型中，应用缝隙连接蛋白43(connexin 43，Cx43)选择性抑制剂模拟肽Gap27能否改善多巴胺神经元死亡以及对Cx43表达的影响。方法将18只C57BL/6小鼠随机分为对照组、6-OHDA组与6-OHDA+Gap27组，每组6只，进行双侧黑质脑立体定位注射。对照组注射抗坏血酸盐溶液，6-OHDA组注射6-OHDA溶液，6-OHDA+Gap27组注射6-OHDA和Gap27混合溶液，用免疫组织化学法对多巴胺神经元标志物酪氨酸羟化酶(tyrosine hydroxylase，TH)染色检测多巴胺神经元数量，实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction，qRT-PCR)检测Cx43信使核糖核酸(messenger ribonucleic acid，mRNA)表达，免疫荧光染色检测Cx43蛋白分布，Western blot法检测小鼠中脑Cx43蛋白及Cx43的第368位点丝氨酸磷酸化(Cx43 phosphorylation at serine 368，Cx43-ps368)蛋白含量。结果注射6-OHDA后，小鼠出现黑质多巴胺神经元大量死亡，6-OHDA组TH阳性神经元数量降为对照组的27.7%±0.02%(P < 0.01)，模拟肽Gap27的使用减少了多巴胺神经元死亡数量，6-OHDA+Gap27组TH阳性神经元数量为6-OHDA组的(1.64±0.16)倍(P < 0.05)；此外，6-OHDA引起Cx43蛋白含量增加，Cx43-ps368蛋白含量降低。Gap27减弱了6-OHDA引起的Cx43蛋白与Cx43-ps368蛋白含量变化，6-OHDA组中脑总Cx43蛋白含量为6-OHDA+Gap27组的(1.44±0.07)倍(P < 0.05)，为对照组的(1.68±0.07)倍(P < 0.01)，且6-OHDA组Cx43-ps368蛋白含量及占总Cx43蛋白比例显著低于6-OHDA+Gap27组(P＜0.05)。结论模拟肽Gap27在6-OHDA诱导的小鼠模型中可减少黑质多巴胺神经元死亡从而发挥神经保护作用，6-OHDA引起的Cx43蛋白过表达对多巴胺神经元可能存在神经毒性，而降低Cx43蛋白水平及维持Cx43-ps368蛋白水平可能是Gap27发挥保护作用的机制。     

大鼠脊髓损伤后坐骨神经电生理学以及损伤脊髓GAP-43蛋白表达的变化

目的：研究大鼠脊髓损伤后不同时间点坐骨神经兴奋阈值、动作电位（AP）幅度和传导速度,以及损伤脊髓GAP-43蛋白表达的改变。方法：采用改良的Allen′s重物坠落致大鼠脊髓损伤实验模型、电生理学和免疫组织化学染色。结果：大鼠SCI后1、3、7d时,坐骨神经兴奋阈值升高,AP幅度和传导速度降低,呈时间依赖性改变;SCI后14d,AP产生阈值、幅度和传导速度较SCI第7d有所恢复。正常大鼠脊髓灰质神经元的胞浆和轴突中有少量GAP-43蛋白表达;SCI后第1、3、5、7d时损伤脊髓周围区灰质GAP-43蛋白表达逐渐增强;在SCI后第14d其阳性表达开始减弱。结论：大鼠SCI后能够导致坐骨神经电生理学兴奋性和传导性的降低,以及神经结构重构蛋白GAP-43表达增加。     

Exosomes Derived from Glioma Cells under Hypoxia Promote Angiogenesis through Up-regulated Exosomal Connexin 43

Glioblastoma multiform (GBM) is a highly aggressive primary brain tumor. Exosomes derived from glioma cells under a hypoxic microenvironment play an important role in tumor biology including metastasis, angiogenesis and chemoresistance. However, the underlying mechanisms remain to be elucidated. In this study, we aimed to explore the role of connexin 43 on exosomal uptake and angiogenesis in glioma under hypoxia. U251 cells were exposed to 3% oxygen to achieve hypoxia, and the expression levels of HIF-1α and Cx43, involved in the colony formation and proliferation of cells were assessed. Exosomes were isolated by differential velocity centrifugation from U251 cells under normoxia and hypoxia (Nor-Exos and Hypo-Exos), respectively. Immunofluorescence staining, along with assays for CCK-8, tube formation and wound healing along with a transwell assay were conducted to profile exosomal uptake, proliferation, tube formation, migration and invasion of HUVECs, respectively. Our results revealed that Hypoxia significantly up-regulated the expression of HIF-1α in U251 cells as well as promoting proliferation and colony number. Hypoxia also increased the level of Cx43 in U251 cells and in the exosomes secreted. The uptake of Dio-stained Hypo-Exos by HUVECs was greater than that of Nor-Exos, and inhibition of Cx43 by ^37,43gap27 or lenti-Cx43-shRNA efficiently prevented the uptake of Hypo-Exos by recipient endothelial cells. In addition, the proliferation and total loops of HUVECs were remarkably increased at 24 h, 48 h, and 10 h after Hypo-Exos, respectively. Notably, ^37,43gap27, a specific Cx-mimetic peptide blocker of Cx37 and Cx43, efficiently alleviated Hypo-Exos-induced proliferation and tube formation by HUVECs. Finally, ^37,43gap27 also significantly attenuated Hypo-Exos-induced migration and invasion of HUVECs. These findings demonstrate that exosomal Cx43 contributes to glioma angiogenesis mediated by Hypo-Exos, and suggests that exosomal Cx43 might serve as a potential therapeutic target for glioblastoma.