A rapidly inactivating Ca2+-dependent K+ current in pheochromocytoma cells (PC12) of the rat

The membrane electrical properties of undifferentiated pheochromocytoma cells of the rat (PC12) were studied using both current-and voltage-clamp techniques with the use of low-resistance blunt-tipped micropipettes (patch electrodes). In the presence of tetrodotoxin (TTX, 2–3 M), a spike-like wave form with a prominent after-hyperpolarization (AHP) was recorded following brief (< 10 ms) depolarizing current pulses. The inorganic divalent cations, Cd²⁺ (0.5 mM), Mn²⁺ (4mM), and 0 mM Ca²⁺/4 mM Mg²⁺ solution prolonged the duration, attenuated the AHP, slowed the rate of repolarization, and slightly enhanced the amplitude of this wave form. A rapidly inactivating outward current was recorded in over 70% of the cells under voltage-clamp conditions. This transient current was elicited at about ±30 mV, and was blocked by tetraethylammonium (5 mM), inorganic divalent cations (Cd²⁺, 0.5 mM; Mn²⁺, 4 mM; Ba²⁺, 3 mM), and removal of Ca²⁺ (0 mM Ca²⁺/4 mM Mg²⁺) from the local perfusion medium. In addition, 4-aminopyridine (5 mM), which blocks the transient outward K⁺ current I_A in a variety of excitable cells, did not have any appreciable effect on this rapidly inactivating current. Moreover, it was possible to elicit the current at a holding potential of ±40 mV. The reversal potential of this current was ±90 mV, and shifted positively when extracellular K⁺ concentrations were elevated. It is concluded that PC12 cells have a rapidly inactivating Ca²⁺ -dependent K⁺ current. A possible explanation for the transient nature of this current may be the presence of an effective intracellular Ca²⁺ buffering (uptake or extrusion) system.     

Phase-contrast cine MRI versus MR cisternography on the evaluation of the communication between intraventricular arachnoid cysts and neighbouring cerebrospinal fluid spaces

Introduction  The objective of this study was to evaluate the role of phase-contrast cine magnetic resonance imaging (PC-MRI) in detecting possible communications between intraventricular arachnoid cysts (IV-ACs) and cerebrospinal fluid (CSF) spaces based on MR cisternography (MRC) comparison. Materials and methods  Twenty-one patients with IV-AC were examined by PC-MRI and MRC. In order to determine the communication of IVAC with its neighbouring CSF spaces, PC-MRI was employed. The communication of IV-ACs with the ventricular system was examined on at least two anatomic planes. Precontrast images and PC-MRI were followed by the intrathecal administration of 0.5–1 ml gadopentetate dimeglumine. Early and delayed MRC were then carried out. Results of PC-MRI were compared with findings of MRC (McNemar’s test). Results  In seven IV-ACs, no communication was detected by PC-MRI. In 14 IVACs, a pulsatile CSF flow into the IV-ACs was observed. All the IV-ACs, which have been determined as non-communicating (NC) on the PC-MRI, showed NC character on MRC as well. Six cases suggesting a communication on PC-MRI showed no communication on MRC. MRC revealed eight communicating (38%) and 13 NC (62%) IV-ACs among a total of 21 cases. The sensitivity and specificity of PC-MRI imaging in demonstrating the communication between the IV-ACs and the CSF were 100% and 54%, respectively. Conclusion  PC-MRI is an effective method for evaluating NC IV-ACs. In order to decide about the management of IV-ACs, which are communicating according to the PC-MRI, the results should be confirmed with MRC if suspected jet flow is depicted.     

重组pIRES-AD7c-NTP质粒的构建表达及其对PC12细胞凋亡的影响

目的构建重组pIRES-AD7c-NTP质粒,探究其对PC12细胞凋亡的影响。方法构建pIRESAD7c-NTP重组质粒,通过脂质体体外转染至PC12细胞内,RT-PCR检测凋亡相关蛋白bcl-2和bax的mRNA表达变化含量变化。结果成功构建pIRES-AD7c-NTP重组质粒并转染至PC12细胞内;重组质粒转染组出现了明显的细胞凋亡,流式细胞仪检测出典型凋亡峰;重组质粒转染组中bcl-2表达低于对照组,bax表达高于对照组。结论 AD7c-NTP重组质粒转染组PC12细胞内bcl-2/bax表达异常,说明AD7c-NTP增加凋亡促进蛋白的表达、同时减少凋亡抑制蛋白的表达,可见AD7c-NTP可导致神经细胞发生凋亡,为后续研究提供实验基础。     

hBcl-2基因修饰的骨髓基质细胞对体外培养PC12细胞的保护作用

目的观察hBcl-2转染的骨髓基质细胞(MSCs)对体外培养PC12细胞的保护作用,为下一步hBcl-2修饰的MSCs脑内移植治疗奠定基础。方法采用密度梯度离心结合贴壁法分离、培养大鼠MSCs,对培养第3代的MSCs进行鉴定;hBcl-2用脂质体转染MSCs建立MSCs-hBcl-2基因工程细胞;用H2O2建立PC12细胞氧化应激损伤模型;MTT法检测MSCs-hBcl-2基因工程细胞上清对体外培养的PC12细胞活性的影响。结果成功培养出MSCs细胞,免疫组化结果显示培养的细胞CD44、CD71表达阳性,而CD45表达阴性;建立了有稳定高效hBcl-2表达的MSCs-hBcl-2基因工程细胞,并观察到MSCs-hBcl-2基因工程细胞较MSCs更能减轻PC12细胞的氧化损伤(P<0.05)。结论hBcl-2基因修饰的MSCs对PC12细胞的生长、生存有着重要的保护作用。     

3-(Fur-2-yl)-10-(2-phenylethyl)-[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one, a Novel Adenosine Receptor Antagonist with A(2A)-Mediated Neuroprotective Effects

In this study, compound FTBI (3-(2-furyl)-10-(2-phenylethyl)[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one) was selected from a small library of triazinobenzimidazole derivatives as a potent A(2A) adenosine receptor (AR) antagonist and tested for its neuroprotective effects against two different kinds of dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium (MPP+) and methamphetamine (METH), in rat PC12 and in human neuroblastoma SH-SY5Y cell lines. FTBI, in a concentration range corresponding to its affinity for A(2A) AR subtype, significantly increased the number of viable PC12 cells after their exposure to METH and, to a similar extent, to MPP+, as demonstrated in both trypan blue exclusion assay and in cytological staining. These neuroprotective effects were also observed with a classical A(2A) AR antagonist, ZM241385, and appeared to be completely counteracted by the AR agonist, NECA, supporting A(2A) ARs are directly involved in FTBI-mediated effects. Similarly, in human SH-SY5Y cells, FTBI was able to prevent cell toxicity induced by MPP+ and METH, showing that this A(2A) AR antagonist has a neuroprotective effect independently by the specific cell model. Altogether these results demonstrate that the A(2A) AR blockade mediates cell protection against neurotoxicity induced by dopaminergic neurotoxins in dopamine containing cells, supporting the potential use of A(2A) AR antagonists in dopaminergic degenerative diseases including Parkinson's disease.     

突变型α-核突触蛋白的自噬性降解途径及可能机制

目的 观察突变型α-核突触蛋白对PC12细胞增殖的影响和可能的降解途径,探讨其在帕金森病发病机制中的作用.方法 对转染了α-核突触蛋白(A30P)的PC12细胞进行药物干预,检测细胞的增殖活性,并采用透射电镜观察细胞超微结构改变以及自噬的特征性改变,同时检测α-核突触蛋白的表达和超氧化物歧化酶(SOD)的水平.结果 (1)Western Blot法检测α-核突触蛋白的表达:A30P+渥曼青霉素组(A30P+W组)、A30P+1-甲基4-苯基吡啶组(A30P+MPP+组)较A30P组明显增高,以A30P+W组最为明显;而A30P+雷帕霉素组(A30P+R组)条带较A30P组减低(P＜0.01);(2)不同时间点细胞培养液中SOD水平(U/ml)的测定:用MPP+处理转染了突变型α-核突触蛋白的PC12细胞后,培养液中SOD水平(A30P+MPP+组:3 h:97.49±13.8;12 h:102.7±12.7:24 h:101.5±11.8;48 h:104.3±12.4)较A30P组在各时间点显著下调(t=3.7721,P=0.0017);A30P+R组在给药12 h以后,培养液中SOD水平逐渐升高,其中在24 h(121.2±13.0)、48 h(124.3±14.1)和72 h(127.7±13.7)时与A30P+W组比较差异有统计学意义(t=2.9746,P=0.0083);突变型α-核突触蛋白激活了自噬途径,并介导了MPP+的毒性作用,自噬抑制剂渥曼青霉素可通过抑制自噬而加剧α-核突触蛋白积聚,导致细胞死亡;而自噬诱导剂雷帕霉素则可以通过诱导自噬的发生而促进α-核突触蛋白的降解和细胞生长.结论 α-核突触蛋白的异常积聚导致PC12细胞的自噬性细胞死亡,促进自噬有助于突变型α-核突触蛋白降解,对细胞具有保护作用.     

Results of Controlled Donation After Circulatory Death in a Third-Level Hospital

Objective

To investigate the characteristics and evolution of controlled donation after circulatory death (DCD) type III.

Fas/FasL在罗哌卡因致PC12细胞凋亡中的表达

目的检测Fas、FasL在罗哌卡因诱导PC12细胞凋亡中表达的变化,探讨罗哌卡因的神经毒性机制。方法采用不同浓度罗哌卡因(0.1、0.5、1、2、4 mmol/L)处理PC12细胞24 h以建立细胞的神经毒性模型,CCK-8法测定细胞活力。最终将细胞随机分为三组:0.5 mmol/L组、2mmol/L组和正常对照组。各组细胞培养24 h后用光学显微镜观察细胞形态学变化(加上1 mmol/L组),流式细胞仪检测细胞凋亡,免疫荧光检测Fas、FasL表达。结果与正常对照组比较,0.5 mmol/L组和2 mmol/L组细胞活力明显降低(P0.05),细胞形态明显异常(包括1 mmol/L组),凋亡率明显升高(P0.05),Fas、FasL表达明显增强(P0.05);与0.5 mmol/L组比较,2 mmol/L组细胞凋亡率和Fas、FasL表达明显增加(P0.05)。结论罗哌卡因可诱导PC12细胞凋亡,其机制可能与Fas/FasL上调有关。     

氧浓度非敏感性低氧诱导因子-1α表达载体的构建及其在PC12细胞系中的表达

目的 构建大鼠非氧浓度敏感性低氧诱导因子1α(HIF-1α)真核表达载体并观察其在PC12细胞系中的表达. 方法 应用RT-PCR从脊髓损伤区域的细胞总RNA中扩增HIF-1αcDNA,采用重叠延伸PCR的方法克隆获得缺失氧敏感性降解结构域(ODD)的HIF-1α突变体基因克隆,采用质粒_pEGFP-C1构建重组真核融合表达载体,将其转入培养的PC12神经细胞系中,应用所融合的绿色荧光蛋白的表达和Western blot鉴定其在细胞内的表达. 结果 通过重叠延伸PCR的方法成功扩增得到非氧浓度敏感性的HIF-1α突变体基因(HIF-1α△ODD)序列,并构建了过量表达缺失ODD的转录调控因子HIF-1α突变体的重组表达质粒()pEGFPC1-HIF-1α△ODD).将其转染PCI2细胞系后,Western blot和绿色荧光蛋白结果均表明HIF-1α△AODD蛋白能在PC12神经细胞系中正确表达. 结论 成功构建了_pEGFPC1-HIF-1α△ODD真核表达载体并在PC12细胞系中观察到融合蛋白表达.     

同源盒基因Lhx4在PC12细胞缺氧损伤保护中的作用

目的:转染并筛选Lhx4高表达的PC12细胞株,观察Lhx4基因对PC12细胞的缺氧保护作用.方法:①用RT-PCR检测PC12细胞缺氧培养后Lhx4基因的表达变化;②Lhx4基因连接至pLXIN逆转录病毒载体,收集病毒上清,感染PC12细胞,G418筛选阳性克隆,PCR鉴定,所筛选的克隆缺氧培养;③通过台盼蓝染色和培养液中乳酸脱氢酶活性的检测评价PC12细胞的存活状态.结果和结论:PC12细胞缺氧培养4 h后Lhx4基因的表达明显上调;缺氧8 h后Lhx4基因表达开始下调.所筛选的Lhx4高表达转染细胞株表现出明显的缺氧耐受性,相同的损伤条件下,细胞的存活数目及生存状态明显优于对照组.说明Lhx4基因在PC12细胞的缺氧损伤保护中具有重要的作用.