维甲酸对新生大鼠高氧肺损伤的干预作用

目的探讨高氧暴露新生大鼠肺组织结构的变化和连接蛋白Connexin26(Cx26)的表达情况及维甲酸对其影响。方法24只3日龄SD大鼠随机分为3组:高氧暴露维甲酸组(维甲酸组)、高氧暴露安慰组(高氧组)、空气暴露对照组(对照组);采用950mL/L氧体积分数条件下制作新生大鼠高氧肺损伤模型;观察大鼠一般情况,用HE染色法观察肺组织结构变化及辐射状肺泡计数(RAC),免疫组织化学检测Cx26的表达。结果体质量、RAC等方面与空气组相比,高氧组、维甲酸组均有显著差异[(13.53±1.27)、(13.38±1.29)、(17.37±0.89)g,F=30.19P=0;(11.15±1.33)、(12.49±1.47)、(13.94±0.98),F=9.59P=0];Cx26蛋白在空气组表达比较局限,高氧组弥散表达,而维甲酸组更多表达;阳性细胞分别为(13.68±1.28)%、(17.82±1.72)%、(19.69±1.77)%,F=29.36P=0。结论维甲酸对新生大鼠高氧肺损伤具有保护作用,可能与间隙连接蛋白Cx26表达的改变有密切的关系。     

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.     

Activating Connexin43 gap junctions primes adipose tissue for therapeutic intervention

Adipose tissue is a promising target for treating obesity and metabolic diseases. However, pharmacological agents usually fail to effectively engage adipocytes due to their extraordinarily large size and insufficient vascularization, especially in obese subjects. We have previously shown that during cold exposure, connexin43 (Cx43) gap junctions are induced and activated to connect neighboring adipocytes to share limited sympathetic neuronal input amongst multiple cells. We reason the same mechanism may be leveraged to improve the efficacy of various pharmacological agents that target adipose tissue. Using an adipose tissue-specific Cx43 overexpression mouse model, we demonstrate effectiveness in connecting adipocytes to augment metabolic efficacy of the β₃-adrenergic receptor agonist Mirabegron and FGF21. Additionally, combing those molecules with the Cx43 gap junction channel activator danegaptide shows a similar enhanced efficacy. In light of these findings, we propose a model in which connecting adipocytes via Cx43 gap junction channels primes adipose tissue to pharmacological agents designed to engage it. Thus, Cx43 gap junction activators hold great potential for combination with additional agents targeting adipose tissue.     

Cx43在骨肉瘤组织中的表达及临床意义

目的探讨细胞间隙连接蛋白43(Cx43)在骨肉瘤组织中的表达及其临床意义。方法收集2007年2月至2013年2月中国人民解放军第251医院收治的骨肉瘤患者70例,采用免疫组织化学(EnVisionTM)法分别检测70例骨肉瘤组织及30例骨肉瘤旁组织中Cx43的表达情况。结果Cx43在骨肉瘤组织、骨肉瘤旁组织中阳性表达率分别为14.28%、86.67%,比较差异有统计学意义(χ2=47.75,P<0.01)。在临床病理因素中,Cx43的表达与年龄、性别、组织学类型无关(P>0.05),但与肿瘤大小、临床分期、肺转移、Ki-67有关,差异有统计学意义(P<0.05)。结论 Cx43在骨肉瘤组织中呈现低表达,其低表达在一定程度上可反映骨肉瘤的生物学行为。