阿魏酸钠对大鼠纤维化心肌组织中结缔组织生长因子表达的影响

目的探讨阿魏酸钠(Sodium ferulate,SF)对大鼠纤维化心肌组织中结缔组织生长因子(Connective tissue growth factor,CTGF)表达的影响。方法一次性皮下注射盐酸异丙肾上腺素(Isoproterenol,Iso)15mg/kg体重,复制Wistar大鼠心肌缺血性坏死模型,于不同时间点脱臼处死大鼠,取心脏,采用半定量RT-PCR法检测大鼠组织中CTGF基因mRNA的转录水平,免疫组化法检测CTGF蛋白的表达水平。再次复制大鼠心肌缺血坏死模型,同时注射SF进行同步干预,3周后检测大鼠心肌组织中CTGF基因mRNA的转录水平及蛋白的表达水平。结果注射Iso后24h,CTGF基因mRNA的转录水平达高峰,较正常对照组明显增加,3周时仍高于正常对照组;CTGF蛋白的表达随纤维化病变程度的加重而增加。SF干预后,CTGF基因mRNA的转录水平及蛋白的表达水平均明显下降。结论 CTGF的表达与大鼠心肌纤维化(Myocardial fibrosis,MF)程度密切相关,提示CTGF可能在MF中起重要作用;SF对CTGF的表达具有抑制作用。     

靶向高迁移率族蛋白A1基因RNA干扰真核表达载体的构建

目的构建针对人高迁移率族蛋白A1(HMGA1)基因的RNA干扰真核表达载体,为研究HMGA1基因在肿瘤细胞中的作用奠定实验基础。方法设计合成特异性针对人HMGA1基因的寡核苷酸序列,梯度退火后,与pU6mRFP载体连接,构建重组载体,转染人肝癌细胞SMMC-7721。通过激光共聚焦显微镜观察红色荧光,估测转染效率,RT-PCR法检测转染细胞HMGA1mRNA水平,流式细胞仪检测细胞凋亡和细胞周期。结果DNA测序证实,成功构建了特异性HMGA1siRNA真核表达载体,转染后72h,转染效率为40%左右。所构建的载体能够特异性沉默转染细胞HMGA1基因的表达。转染细胞HMGA1基因沉默后,细胞凋亡率(27·86%±2·44%)明显高于空载体对照组和SMMC-7721对照组(分别为2·82%±2·39%和2·04%±0·70%),G0-G1期细胞百分数(77·73%±1·78%)明显高于空载体对照组和SMMC-7721对照组(分别为42·19%±3·28%和39·23%±3·63%),G2-S期细胞百分数(22·27%±1·78%)明显低于空载体对照组和SMMC-7721对照组(分别为57·81%±3·28%和60·77%±3·63%)。结论成功构建了HMGA1的RNA干扰真核表达载体。     

脊髓灰质炎病毒冻干保护剂的筛选

目的筛选脊髓灰质炎病毒冻干保护剂。方法将不同配方的冻干保护剂与脊髓灰质炎病毒(SabinⅠ型、SabinⅡ型及中Ⅲ2型)混合,进行冻干,并检测冻干前后病毒感染性滴度及冻干后的热稳定性。结果经检测发现,第Ⅷ组配方(海藻糖8%、人白蛋白1%、山梨醇5%、明胶0·5%和甘氨酸1%)在脊髓灰质炎病毒的冻干过程中保护作用较好。冻干前后感染性滴度下降在0·5LgCCID50/ml以内,且热稳定性好,37℃放置1周后SabinⅠ型、SabinⅡ型及中Ⅲ2型病毒的感染性滴度平均下降分别为0·2、0·3和0·4LgCCID50/ml,而相应对照组的感染性滴度平均下降分别为1·1、1·1和1·3LgCCID50/ml,二者差异有显著意义(P<0·05)。结论含有海藻糖、人白蛋白、山梨醇、明胶和甘氨酸的保护剂对脊髓灰质炎病毒冻干具有良好的保护效果。     

矿物3CaO·3AI2O3·BaSO4高温稳定性研究

矿物3CaO·3Al2O3·BaSO4是一种新型胶凝矿物, 而关于其高温稳定性的研究却有多种结论.本工作综合运用IR, TG-DT A,EPMA,XRD和QXDA等多种测试方法, 系统研究了3CaO·3Al2O3·BaSO4 的高温稳定性问题.研究表明, 在温度低于1 350 ℃的条件下, 矿物3CaO·3Al2O3·BaSO4的晶相结构稳定; 而温度高于1 360 ℃时, 3CaO·3Al2O3·BaSO 4发生分解并形成新的晶相.3CaO·3Al2O3·BaSO4的分解方程式可表述为: 3CaO·3Al2O3·BaSO4→CaO·Al2O3+12CaO·7Al2O3+Ba O·Al2O3+SO2↑+O2↑鉴于矿物BaO·Al2O3的形成机理与该问题紧密相关, 本文也讨论了BaO·Al2 O3的形成机理.BaO·Al2O3的形成机理有两种:温度低于1 350 ℃时, BaO·Al2O 3由原料组分发生反应而形成的; 而温度大于1 360 ℃时, 该矿物是由3CaO·3Al2O 3·BaSO4分解形成的.     

C_(11)A_7·CaF_2、C_4A_3提高粉煤灰水泥强度机理的探讨

探讨了适量C_(11)A_7·CaF_2、C_4A_8显著提高粉煤灰水泥早期强度的机理。研究发现,适量C_(11)A_7·CaF_2、C_4A_8和石膏的掺入,使得在水泥水化早期就有较多的钙矾石生成,且A矿水化得以加速,所生成的钙矾石既形成网络结构又填补了水泥石中的孔洞和裂隙,以致粉煤灰水泥强度提高。实验表明,C_(11)A_7·CaF_2或C_4A_2的掺量为5wt％左右时,水泥凝结正常,强度提高幅度较大,可使粉煤灰水泥强度提高约1个标号,而当C_(11)A_7·CaF_2或C_4A_8掺量达10wt％以上时,水泥凝结急剧加速,水泥强度下降,掺入0.2wt％—0.3wt％柠檬酸可使水泥正常凝结。     

shRNA表达载体对耐阿霉素的人红白血病细胞株P-gp表达的抑制作用

目的探讨靶向mdr1基因的shRNA表达载体对耐阿霉素的人红白血病细胞(K562/ADM)P-gp表达的抑制作用。方法合成靶向mdr1基因的短发夹shRNA表达载体,转染K562/ADM细胞。利用RT-PCR检测转染细胞中mdr1基因mRNA,Westernblot、免疫细胞化学和流式细胞术分别检测P-gp的表达。结果在瞬时转染pSilencerTM3·1-H1neomdr1-A和mdr1-BshRNA表达载体的K562/ADM细胞中,mdr1基因mRNA转录分别减少到39·1%(P<0·05)和30·8%(P<0·01)。Westernblot、免疫细胞化学和流式细胞术检测显示P-gp表达被明显而特异地抑制。结论靶向mdr1基因的shRNA表达载体能够特异而有效地抑制耐药的人红白血病细胞中的P-gp表达。     

5-溴-PADAP-阴离子表面活性剂分光光度法测定微量锆的研究

5-溴-PADAP是目前测定多种金属元素的高灵敏显色剂。据文献[1]报道,在弱酸性介质(pH 4.8)中,该试剂与锆(Ⅳ)可以形成一蓝紫色络合物,其最大吸收峰位于583nm,摩尔吸光系数为1.35×10~5(ι·mol~(-1)·cm~(-1))。虽然此显色体系具有较高的灵敏度,但是,由于显色酸度过低,方法不可能获得满意的选择性和稳定性。我们发现,在阴离子表面活性剂胶束的存在下,上述显色反应的吸光性质和反应条件都发生了明显的变化。当酸度条件基本维持不变时,即在pH=3.0～5.0的介质中,络合物的吸收峰由583nm移至634nm,摩尔吸光率则激增至3.15×10~5(ι·mol~(-1)·cm~(-1))。当显色酸度移至0.1～0.3N HNO_3介质中时,二元络合物的摩尔吸光率下降至2.9×10~4(ι·mol~(-1)·cm~(-1)),而胶束络合物的吸收峰却移至645nm,摩尔吸光率仍可达到1.15×10~5(ι·mol~(-1)·cm~(-1))。显然,在此条件下,显色体系的选择性和稳定性都得到了保证。     

两种新N—乙酸取代氮杂冠醚及其配合物的合成与性质的研究

以N-取代氮芥、水杨醛、二胺和溴乙酸为原料可以合成一系列的新N-乙酸取代氮杂冠醚。现合成的是其中两种H_2L~1和HL~2,并在无水甲醇中制得固体配合物CuL~1·2HClO_4·1.5H_2O、LaNO_3L~1·2HNO_3·6H_2O、EuNO_3L~1·HNO_3·7H_2O、CuClO_4L~2·HClO_4·1.5H_2O 和 CuNO_3L~2·HNO_3·2.5H_2O。用元素分析、IR和~1HNMR、UV-VIS光谱等方法研究了其组成和性质。     

流行性乙型脑炎病毒在二倍体细胞上的适应性

目的研究流行性乙型脑炎病毒在二倍体(2BS)细胞上的传代适应性,确定在2BS细胞上培养乙脑病毒的条件和方法。方法通过病毒毒力和免疫原性检测,观察在不同培养条件下乙脑病毒在2BS细胞上的增殖情况。结果pH值在7·4～7·6之间、人血白蛋白含量为0·2%、病毒稀释度为1:1000时,病毒维持液培养的病毒滴度最高。在2BS细胞上传50代的病毒滴度均在7·0logLD50以上,且差异无显著意义。连续传代后病毒的免疫原性下降。结论建议用于建立病毒种子库的流行性乙型脑炎病毒,在2BS细胞上传代不应超过3代。     

硫酸钙-硫酸钠-铬酸钙-铬酸钠-水体系中复盐的生成研究

为配合化工产品新工艺的开发,研究了25～70 ℃硫酸钙-硫酸钠-铬酸钙-铬酸钠-水体系中复盐的生成规律.摇床实验及X射线衍射分析发现,将固体铬酸钙加入不同浓度的硫酸钠和铬酸钠溶液中时,除生成CaSO4·2H2O外,还可能生成4种复盐沉淀,即CaSO4·Na2SO4,CaSO4·Na2SO4·4H2O,CaSO4·2Na2SO4·2H2O和5CaSO4·Na2SO4·3H2O.当温度和溶液浓度发生变化时,复盐的形式会相应变化,低温稀溶液中只有CaSO4·2H2O沉淀,没有复盐沉淀析出,而高温浓溶液中,4种复盐都会析出.     

Molecular,Cellular and Functional Changes in the Retinas of Young Adult Mice Lacking the Voltage-Gated K+ Channel Subunits Kv8.2 and K2.1

Cone Dystrophy with Supernormal Rod Response (CDSRR) is a rare autosomal recessive disorder leading to severe visual impairment in humans, but little is known about its unique pathophysiology. We have previously shown that CDSRR is caused by mutations in the KCNV2 (Potassium Voltage-Gated Channel Modifier Subfamily V Member 2) gene encoding the Kv8.2 subunit, a modulatory subunit of voltage-gated potassium (Kv) channels. In a recent study, we validated a novel mouse model of Kv8.2 deficiency at a late stage of the disease and showed that it replicates the human electroretinogram (ERG) phenotype. In this current study, we focused our investigation on young adult retinas to look for early markers of disease and evaluate their effect on retinal morphology, electrophysiology and immune response in both the Kv8.2 knockout (KO) mouse and in the Kv2.1 KO mouse, the obligate partner of Kv8.2 in functional retinal Kv channels. By evaluating the severity of retinal dystrophy in these KO models, we demonstrated that retinas of Kv KO mice have significantly higher apoptotic cells, a thinner outer nuclear cell layer and increased activated microglia cells in the subretinal space. Our results indicate that in the murine retina, the loss of Kv8.2 subunits contributes to early cellular and physiological changes leading to retinal dysfunction. These results could have potential implications in the early management of CDSRR despite its relatively nonprogressive nature in humans.     

Voltage-Gated Potassium Channel Kv1.3 Is Highly Expressed in Human Osteosarcoma and Promotes Osteosarcoma Growth

Deregulation of voltage-gated potassium channel subunit Kv1.3 has been reported in many tumors. Kv1.3 promotes tumorigenesis by enhancing cell proliferation while suppressing apoptosis. However, the expression and function of Kv1.3 in osteosarcoma are unknown. In the present study, we detected the expression of Kv1.3 in human osteosarcoma cells and tissues by RT-PCR, Western blot and immunohistochemistry. We further examined cell proliferation and apoptosis in osteosarcoma MG-63 cells and xenografts following knockdown of Kv1.3 by short hairpin RNA (shRNA). We found that Kv1.3 was upregulated in human osteosarcoma. Knockdown of Kv1.3 significantly suppressed cell proliferation and increased apoptosis as demonstrated by enhanced cleavage of poly (ADP-ribose) polymerase (PARP) and the activation of Caspase-3/7. Furthermore, adenovirus delivered shRNA targeting Kv1.3 significantly inhibited the growth of MG-63 xenografts. Taken together, our results suggest that Kv1.3 is a novel molecular target for osterosarcoma therapy.     

Identification of selective inhibitors of the potassium channel kv1.1-1.2((3)) by high-throughput virtual screening and automated patch clamp

Two voltage-dependent potassium channels, Kv1.1 (KCNA1) and Kv1.2 (KCNA2), are found to co-localize at the juxtaparanodal region of axons throughout the nervous system and are known to co-assemble in heteromultimeric channels, most likely in the form of the concatemer Kv1.1-1.2((3)) . Loss of the myelin sheath, as is observed in multiple sclerosis, uncovers the juxtaparanodal region of nodes of Ranvier in myelinated axons leading to potassium conductance, resulting in loss of nerve conduction. The selective blocking of these Kv channels is therefore a promising approach to restore nerve conduction and function. In the present study, we searched for novel inhibitors of Kv1.1-1.2((3)) by combining a virtual screening protocol and electrophysiological measurements on a concatemer Kv1.1-1.2((3)) stably expressed in Chinese hamster ovary K1 (CHO-K1) cells. The combined use of four popular virtual screening approaches (eHiTS, FlexX, Glide, and Autodock-Vina) led to the identification of several compounds as potential inhibitors of the Kv1.1-1.2((3)) channel. From 89 electrophysiologically evaluated compounds, 14 novel compounds were found to inhibit the current carried by Kv1.1-1.2((3)) channels by more than 80?% at 10?μM. Accordingly, the IC(50) values calculated from concentration-response curve titrations ranged from 0.6 to 6?μM. Two of these compounds exhibited at least 30-fold higher potency in inhibition of Kv1.1-1.2((3)) than they showed in inhibition of a set of cardiac ion channels (hERG, Nav1.5, and Cav1.2), resulting in a profile of selectivity and cardiac safety. The results presented herein provide a promising basis for the development of novel selective ion channel inhibitors, with a dramatically lower demand in terms of experimental time, effort, and cost than a sole high-throughput screening approach of large compound libraries.     

Potassium Channels Kv1.3 and Kir2.1 But Not Kv1.5 Contribute to BV2 Cell Line and Primary Microglial Migration

(1) Background: As membrane channels contribute to different cell functions, understanding the underlying mechanisms becomes extremely important. A large number of neuronal channels have been investigated, however, less studied are the channels expressed in the glia population, particularly in microglia. In the present study, we focused on the function of the Kv1.3, Kv1.5 and Kir2.1 potassium channels expressed in both BV2 cells and primary microglia cultures, which may impact the cellular migration process. (2) Methods: Using an immunocytochemical approach, we were able to show the presence of the investigated channels in BV2 microglial cells, record their currents using a patch clamp and their role in cell migration using the scratch assay. The migration of the primary microglial cells in culture was assessed using cell culture inserts. (3) Results: By blocking each potassium channel, we showed that Kv1.3 and Kir2.1 but not Kv1.5 are essential for BV2 cell migration. Further, primary microglial cultures were obtained from a line of transgenic CX3CR1-eGFP mice that express fluorescent labeled microglia. The mice were subjected to a spared nerve injury model of pain and we found that microglia motility in an 8 µm insert was reduced 2 days after spared nerve injury (SNI) compared with sham conditions. Additional investigations showed a further impact on cell motility by specifically blocking Kv1.3 and Kir2.1 but not Kv1.5; (4) Conclusions: Our study highlights the importance of the Kv1.3 and Kir2.1 but not Kv1.5 potassium channels on microglia migration both in BV2 and primary cell cultures.     

The Role of Sulfur Dioxide in the Regulation of Mitochondrion-Related Cardiomyocyte Apoptosis in Rats with Isopropylarterenol-Induced Myocardial Injury

The authors investigated the regulatory effects of sulfur dioxide (SO₂) on myocardial injury induced by isopropylarterenol (ISO) hydrochloride and its mechanisms. Wistar rats were divided into four groups: control group, ISO group, ISO plus SO₂ group, and SO₂ only group. Cardiac function was measured and cardiomyocyte apoptosis was detected. Bcl-2, bax and cytochrome c (cytc) expressions, and caspase-9 and caspase-3 activities in the left ventricular tissues were examined in the rats. The opening status of myocardial mitochondrial permeability transition pore (MPTP) and membrane potential were analyzed. The results showed that ISO-treated rats developed heart dysfunction and cardiac injury. Furthermore, cardiomyocyte apoptosis in the left ventricular tissues was augmented, left ventricular tissue bcl-2 expression was down-regulated, bax expression was up-regulated, mitochondrial membrane potential was significantly reduced, MPTP opened, cytc release from mitochondrion into cytoplasm was significantly increased, and both caspase-9 and caspase-3 activities were increased. Administration of an SO₂ donor, however, markedly improved heart function and relieved myocardial injury of the ISO-treated rats; it lessened cardiomyocyte apoptosis, up-regulated myocardial bcl-2, down-regulated bax expression, stimulated mitochondrial membrane potential, closed MPTP, and reduced cytc release as well as caspase-9 and caspase-3 activities in the left ventricular tissue. Hence, SO₂ attenuated myocardial injury in association with the inhibition of apoptosis in myocardial tissues, and the bcl-2/cytc/caspase-9/caspase-3 pathway was possibly involved in this process.     

Contribution of K2P Potassium Channels to Cardiac Physiology and Pathophysiology

Years before the first two-pore domain potassium channel (K2P) was cloned, certain ion channels had already been demonstrated to be present in the heart with characteristics and properties usually attributed to the TREK channels (a subfamily of K2P channels). K2P channels were later detected in cardiac tissue by RT-PCR, although the distribution of the different K2P subfamilies in the heart seems to depend on the species analyzed. In order to collect relevant information in this regard, we focus here on the TWIK, TASK and TREK cardiac channels, their putative roles in cardiac physiology and their implication in coronary pathologies. Most of the RNA expression data and electrophysiological recordings available to date support the presence of these different K2P subfamilies in distinct cardiac cells. Likewise, we show how these channels may be involved in certain pathologies, such as atrial fibrillation, long QT syndrome and Brugada syndrome.     

A Novel KCNA2 Variant in a Patient with Non-Progressive Congenital Ataxia and Epilepsy: Functional Characterization and Sensitivity to 4-Aminopyridine

Kv1.2 channels, encoded by the KCNA2 gene, are localized in the central and peripheral nervous system, where they regulate neuronal excitability. Recently, heterozygous mutations in KCNA2 have been associated with a spectrum of symptoms extending from epileptic encephalopathy, intellectual disability, and cerebellar ataxia. Patients are treated with a combination of antiepileptic drugs and 4-aminopyridine (4-AP) has been recently trialed in specific cases. We identified a novel variant in KCNA2, E236K, in a Serbian proband with non-progressive congenital ataxia and early onset epilepsy, treated with sodium valproate. To ascertain the pathogenicity of E236K mutation and to verify its sensitivity to 4-AP, we transfected HEK 293 cells with Kv1.2 WT or E236K cDNAs and recorded potassium currents through the whole-cell patch-clamp. In silico analysis supported the electrophysiological data. E236K channels showed voltage-dependent activation shifted towards negative potentials and slower kinetics of deactivation and activation compared with Kv1.2 WT. Heteromeric Kv1.2 WT+E236K channels, resembling the condition of the heterozygous patient, confirmed a mixed gain- and loss-of-function (GoF/LoF) biophysical phenotype. 4-AP inhibited both Kv1.2 and E236K channels with similar potency. Homology modeling studies of mutant channels suggested a reduced interaction between the residue K236 in the S2 segment and the gating charges at S4. Overall, the biophysical phenotype of E236K channels correlates with the mild end of the clinical spectrum reported in patients with GoF/LoF defects. The response to 4-AP corroborates existing evidence that KCNA2-disorders could benefit from variant-tailored therapeutic approaches, based on functional studies.     

Designing a C84 fullerene as a specific voltage-gated sodium channel blocker

Fullerene derivatives demonstrate considerable potential for numerous biological applications, such as the effective inhibition of HIV protease. Recently, they were identified for their ability to indiscriminately block biological ion channels. A fullerene derivative which specifically blocks a particular ion channel could lead to a new set of drug leads for the treatment of various ion channel-related diseases. Here, we demonstrate their extraordinary potential by designing a fullerene which mimics some of the functions of μ-conotoxin, a peptide derived from cone snail venom which potently binds to the bacterial voltage-gated sodium channel (Na_vAb). We show, using molecular dynamics simulations, that the C₈₄ fullerene with six lysine derivatives uniformly attached to its surface is selective to Na_vAb over a voltage-gated potassium channel (Kv1.3). The side chain of one of the lysine residues protrudes into the selectivity filter of the channel, while the methionine residues located just outside of the channel form hydrophobic contacts with the carbon atoms of the fullerene. The modified C₈₄ fullerene strongly binds to the Na_vAb channel with an affinity of 46 nM but binds weakly to Kv1.3 with an affinity of 3 mM. This potent blocker of Na_vAb may serve as a structural template from which potent compounds can be designed for the targeting of mammalian Nav channels. There is a genuine need to target mammalian Nav channels as a form of treatment of various diseases which have been linked to their malfunction, such as epilepsy and chronic pain.     

Tetradecylthioacetic Acid Increases Fat Metabolism and Improves Cardiac Function in Experimental Heart Failure

Changes in myocardial metabolism, including a shift from fatty acid to glucose utilization and changes in fatty acid availability and composition are characteristics of heart failure development. Tetradecylthioacetic acid (TTA) is a fatty acid analogue lacking the ability to undergo mitochondrial β-oxidation. TTA promotes hepatic proliferation of mitochondria and peroxisomes and also decreases serum triglycerides and cholesterol in animals. We investigated the effect of TTA, in combination with a high-fat or regular diet, in a rat model of post-myocardial infarction heart failure. TTA had a beneficial effect on cardiac function in post-myocardial infarction heart failure without affecting myocardial remodeling. These effects of TTA on myocardial function were accompanied by decreased free fatty acids in plasma, increased myocardial proportion of n-3 polyunsaturated fatty acids (PUFA) and a decreased proportion of n-6 PUFA. Myocardial enzyme gene expression during TTA treatment suggested that the increase in n-3 PUFA could reflect increased n-3 PUFA synthesis and inadequately increased n-3 PUFA β-oxidation. Based on our data, it is unlikely that the changes are secondary to alterations in other tissues as plasma and liver showed an opposite pattern with decreased n-3 PUFA during TTA treatment. The present study suggests that TTA may improve myocardial function in heart failure, potentially involving its ability to decrease the availability of FFA and increase the myocardial proportion of n-3 PUFA.