自组装短肽GFS-4作为细胞三维培养及心肌梗死修复支架材料的研究EI北大核心CSCD

本研究探索自组装短肽GFS-4在心肌细胞三维培养中的应用效果及其对心肌梗死区域的组织修复作用。通过圆二色谱仪分析短肽GFS-4的二级结构,原子力显微镜检测短肽GFS-4自组装的微观形态。将GFS-4自组装形成的纳米纤维支架作为心肌细胞三维培养材料,观察心肌细胞的生长状况;建立大鼠心肌梗死模型,加入水凝胶GFS-4研究其对心肌梗死修复的效果。结果发现,GFS-4自组装形成的二级结构主要为β折叠;自组装24 h后形成致密的纳米纤维支架;心肌细胞三维培养结果表明心肌细胞在GFS-4水凝胶中生长状况良好;心肌梗死体外修复实验发现,短肽GFS-4水凝胶支架可缓解心肌梗死区域组织坏死。自组装短肽GFS-4作为新的纳米支架材料,可用于细胞三维培养和心肌梗死区域组织修复。     

自组装短肽GFS-4作为细胞三维培养及心肌梗死修复支架材料的研究

本研究探索自组装短肽GFS-4在心肌细胞三维培养中的应用效果及其对心肌梗死区域的组织修复作用。通过圆二色谱仪分析短肽GFS-4的二级结构,原子力显微镜检测短肽GFS-4自组装的微观形态。将GFS-4自组装形成的纳米纤维支架作为心肌细胞三维培养材料,观察心肌细胞的生长状况;建立大鼠心肌梗死模型,加入水凝胶GFS-4研究其对心肌梗死修复的效果。结果发现,GFS-4自组装形成的二级结构主要为β折叠;自组装24 h后形成致密的纳米纤维支架;心肌细胞三维培养结果表明心肌细胞在GFS-4水凝胶中生长状况良好;心肌梗死体外修复实验发现,短肽GFS-4水凝胶支架可缓解心肌梗死区域组织坏死。自组装短肽GFS-4作为新的纳米支架材料,可用于细胞三维培养和心肌梗死区域组织修复。     

TD—PCR技术用于肠道病毒71的检测

目的评价降落聚合酶链反应（touchdown PCR,TD—PCR）检测肠道病毒71型（Enterovirus 71,EV71）的效能和敏感性。方法根据touchdown原理设计TD-PCR程序,试验选择PCR最佳反应条件,分别与普通PCR及TD—PCR、普通Taq酶与热启动酶扩增效果进行比较．并利用10倍稀释法检验TD—PCR方法的灵敏度。琼脂糖凝胶电泳验证扩增产物,纯化后DNA产物测序验证该方法的特异性。结果TD—PCR的扩增片段条带清晰．检测效果明显优于普通PCR,使用普通Taq酶的TD—PCR比使用热启动酶有更优的性价比,测序证实了此组片段的特异性．cDNA的最低检测浓度为1．031μg／mL。结论成功建立TD—PCR检测EV71的方法,使用普通Taq酶获得理想的检测结果,为快速检测手足口病的病原体提供了可靠的手段。     

Taq聚合酶逆转录并直接扩增细胞RNA

几年来的实践表明,PCR技术是基因分析中的一种十分有用的手段。近来结合mRNA逆转录为cDNA方法,应用PCR技术分析基因转录体(RT-PCR)。作者在用PCR技术分析基因组DNA过程中,注意到时常出现相应于cDNA片段大小的扩增产物,因而作者推测,Taq聚合酶直接合成并扩增了cDNA片段。为了证明Taq聚合酶能逆转录RNA模板,从末稍血分离4μg总RNA,用Taq聚合酶进行40轮的PCR扩增,所用引物为与人β血影蛋白cDNA 0.4kb两侧互补的寡核苷酸引物。在另一反应管中,同时用1μg基因组DNA用同一引物进行PCR扩增。PCR反应产物电泳分析表明,前者可是0.4kb扩增片     

一步法直接克隆PCR产物

很多耐高温的DNA聚合酶,如Taq DNA聚合酶,因其具有非模板依赖性末端转移酶活性,因而常在PCR产物的3'末端加上一个多余的碱基,通常为3'-dA,从而使通过平端连接克隆PCR产物的效率很低。有几种方法可以解决这个问题。①在dNTP存在的情况下,用T_4DNA聚合酶除去3'末端突出的碱基。②利用具有3'-dT突出末端的载体进行克隆,这种载体有商品。③利用其它的DNA聚合酶,如pfu DNA聚合酶进行PCR得到具平头末端的PCR产物。在本文中,作者介绍了二种对Taq DNA聚合     

聚合酶链式反应非特异性扩增抑制因子的发现以及全血直接PCR检测G6PD基因外显子3—4突变

目的（1）研究消除PCR反应非特异性扩增新技术。（2）研究全血直接PCR能否用于葡萄糖6-磷酸脱氢酶基因突变检测。方法（1）将新鲜全血混合基因组DNA进行PCR扩增,看全血能否抑制基因组DNA非特异性扩增。将新鲜人血清混合基因组DNA进行PCR扩增,看新鲜人血清能否抑制基因组DNA非特异性扩增。（2）将1μl全血加入PCR反应液,98℃变性后转入PCR循环,使用Taq（Fermentas）扩增G6PD基因外显子3—4,取扩增产物50μl进行DNA双向自动测序,使用ClustalX软件将测序结果与基因库中标准G6PD基因外显子3—4序列进行比对分析。结果（1）发现新鲜全血能抑制基因组DNA非特异性扩增,进一步研究发现是新鲜人血清抑制了基因组DNA非特异性扩增。（2）全血直接PCR加上DNA测序找到一例G6PD突变g．13503A〉G。结论（1）新鲜人血清中存在PCR非特异性扩增抑制因子。（2）使用TaqDNA聚合酶（Fermentas）能进行全血直接PCR扩增,加上DNA测序可用于G6PD基因突变研究。     

利用反转录环介导等温扩增技术检测丙型肝炎病毒

鉴于丙型肝炎病毒(HCV)危害着人类健康,本研究拟用反转录环介导等温扩增(RT-LAMP)建立一种简易检测方法。收集并用FQ-PCR(金标准)检测临床样本。根据HCV保守5'非编码区设计4条通用引物,然后用Taq DNA聚合酶优化传统RT-LAMP体系,通过酶切和交叉实验以评价RT-LAMP的特异性,通过和反转录PCR(RT-PCR)同时检测倍比稀释的模板以评价RT-LAMP的灵敏度。同时判断钙黄绿素和羟基萘芬蓝(HNB)染色法是否与电泳法的结果相同。结果显示,Taq DNA聚合酶可提高扩增效率,RT-LAMP的特异性好,酶切片段与预期相一致(216bp)且只有HCV被扩增,灵敏度为10IU/tube,比RT-PCR高10倍。另外,两种染料和电泳法的检测结果相同。RT-LAMP和RT-PCR检测75例临床样本的一致性差(P0.05,Kappa=0.375),和FQ-PCR的一致性好(P0.05,Kappa=0.762)。综上所述,RT-LAMP简易、特异、灵敏,适用于基层医疗机构。     

新型手性自组装短肽R-LIFE-1的理化性质及其对外泌体的控释研究EI北大核心

本研究旨在探究手性自组装短肽R-LIFE-1对外泌体的包裹控释作用。采用原子力显微镜、透射电镜及冷冻扫描电镜等观察短肽R-LIFE-1的成胶能力及形态结构;采用光镜观察及荧光染色探究R-LIFE-1的生物相容性;采用超滤离心法提取外泌体并采用Western blot、纳米粒径追踪分析(NTA)、透射电镜检测外泌体质量;采用激光共聚焦显微镜及BCA蛋白定量探究短肽水凝胶对外泌体的控释效果。结果显示手性自组装短肽R-LIFE-1能够在离子触发下快速自组装形成稳定的纳米纤维网络膜片状结构,具有良好的生物相容性,能够负载外泌体对它进行包裹控释,提高外泌体的利用效率。本研究证明短肽R-LIFE-1可对外泌体进行控释,是一种理想的组织工程材料。     

锚定PCR技术

Saiki等于1985年首创的多聚酶链反应(PCRpolymerase chain reaction)能够将微量DNA在体外迅速进行扩增。与目的基因片段侧翼顺序(flan-king sequence)互补的引物在Taq酶及4种脱氧核苷三磷酸的作用下延伸;新合成的片段又可作为模板。如此反复进行,使目的基因迅速得的扩增。PCR技术一经问世,迅速得到广泛应用。但在用于     

降钙素基因相关肽表达载体的构建

背景：为研究降钙素基因相关肽基因治疗在骨质疏松中的应用,首先要建立高效的降钙素基因相关肽基因载体。 目的：构建降钙素基因相关肽真核表达载体pBaBb-puro-CGRP。 方法：设计引物并通过PCR扩增出降钙素基因相关肽,酶切后用T4 DNA连接酶将其与pBaBb-puro进行连接,经过转化、阳性克隆筛选后,进行酶切和测序鉴定。 结果与结论：经PCR扩增获得了含430 bp的降钙素基因相关肽基因编码序列,经过酶切、连接、转化后,挑选10个菌落进行PCR检测,筛查到8个菌落含有重组质粒,进一步行酶切和测序鉴定,结果与理论预期完全相符。提示实验成功构建了pBaBb-puro-CGRP真核表达载体。     

SASH1通过MAP2K2和MAP4K4与ERK信号通路交互作用

目的 探讨新候选抑癌基因SASH1与细胞外调节蛋白激酶(ERK)信号通路的两个关键分子MAP2K2和MAP4K4的蛋白-蛋白相互作用关系.方法 用Xho Ⅰ和HpaⅠ构建SBP-Flag-SASH1-pBABE-puro反转录病毒载体,转染HEK-293T细胞,通过嘌呤霉素筛选出稳定表达外源性SASH1基因的细胞系.Western blot检测外源性Flag-SASH1蛋白表达,利用pull-down实验、质谱技术、免疫沉淀鉴定和分析与SASH1结合的并可能调节细胞增殖、转移和凋亡等的关键蛋白.用SASH1-siRNA1和SASH1-siRNA2分别转染MDA-MB-231细胞系,以空白组和Negative-siRNA组为对照.72 h后Western blot检测SASH1的干扰效果和P-ERK1/2水平.结果 成功构建稳定表达SBP-Flag-SASH1-pBABE-puro重组质粒的HEK-293T细胞系,SASH1与MAP2K2和MAP4K4结合并发生相互作用.SASH1-siRNA有效抑制MDA-MB-231细胞SASH1蛋白表达(P＜0.05),且P-ERK1/2在SASH1抑制组表达增加.结论 MAP2K2和MAP4K4是SASH1的重要的候选结合蛋白,SASH1可能通过与其直接或间接结合串话ERK信号传导通路,进而调节细胞增殖和迁移等细胞生物学功能.     

MAP2K1 and MAP3K1 mutations in langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is now understood to be a neoplastic disease in which over 50% of cases have somatic activating mutations of BRAF. However, the extracellular signal‐related (ERK) pathway is activated in all cases including those with wild type BRAF alleles. Here, we applied a targeted massively parallel sequencing panel to 30 LCH samples to test for the presence of additional genetic alterations that might cause ERK pathway activation. In 20 BRAF wild type samples, we found 3 somatic mutations in MAP2K1 (MEK1) including C121S and C121S/G128D in the kinase domain, and 56_61QKQKVG>R, an in‐frame deletion in the N‐terminal regulatory domain. All three variant proteins constitutively phosphorylated ERK in in vitro kinase assays. The C121S/G128D and 56_61QKQKVG>R variants were resistant to the mitogen‐activated protein kinase kinase (MEK) inhibitor trametinib in vitro. Within the entire sample set, we found 3 specimens with mutations in MAP3K1 (MEKK1), including two truncation mutants, T779fs and T1481fs; T1481fs encoded an unstable and nonfunctional protein when expressed in vitro. T779fs was present in a specimen carrying BRAF V600E. The third variant was a single nucleotide substitution, E1286V, which was fully functional and is likely a germline polymorphism. These results indicate that LCH cells can harbor additional genetic alterations in the RAS‐RAF‐MEK pathway which, in the case of MAP2K1, may be responsible for ERK activation in a wild type BRAF setting. The resistance of some of these variants to trametinib may also have clinical implications for the combined use of RAF and MEK inhibitors in LCH. © 2015 Wiley Periodicals, Inc.     

Effects of Na+, K+ and Mg2+ on45Ca2+ uptake by pancreatic islets

Microdissected pancreatic islets of noninbredob/ob-mice were used to study ionic effects on the lanthanum-nondisplaceable⁴⁵Ca²⁺ uptake by islet cells. Omission of Mg²⁺ from the incubation medium had no effect, but the⁴⁵Ca²⁺ uptake was increased by omission of Na⁺ and decreased by omission of K⁺. Excess Mg²⁺ (1.2–15 mM) inhibited and excess K⁺ (4.7–25 mM) stimulated the⁴⁵Ca²⁺ uptake in a concentration-dependent manner. Stimulation of⁴⁵Ca²⁺ uptake in Na⁺-deficient islets was associated with an enhancement of the basal insulin release. Total abolishment of glucose-stimulated⁴⁵Ca²⁺ uptake in K⁺-deficient islets did not preclude a significant secretory response to glucose. It is concluded that the lanthanum-nondisplaceable⁴⁵Ca²⁺ uptake shows a partial correlation to insulin release.     

G protein modulation of K2P potassium channel TASK-2

TASK-2 (K_2P5.1) is a background K⁺ channel opened by extra- or intracellular alkalinisation that plays a role in renal bicarbonate handling, central chemoreception and cell volume regulation. Here, we present results that suggest that TASK-2 is also modulated by Gβγ subunits of heterotrimeric G protein. TASK-2 was strongly inhibited when GTP-γ-S was used as a replacement for intracellular GTP. No inhibition was present using GDP-β-S instead. Purified Gβγ introduced intracellularly also inhibited TASK-2 independently of whether GTP or GDP-β-S was present. The effects of GTP-γ-S and Gβγ subunits were abolished by neutralisation of TASK-2 C terminus double lysine residues K257–K258 or K296–K297. Use of membrane yeast two hybrid (MYTH) experiments and immunoprecipitation assays using tagged proteins gave evidence for a physical interaction between Gβ1 and Gβ2 subunits and TASK-2, in agreement with expression of these subunits in proximal tubule cells. Co-immunoprecipitation was impeded by mutating C terminus K257–K258 (but not K296–K297) to alanines. Gating by extra- or intracellular pH was unaltered in GTP-γ-S-insensitive TASK-2-K257A-K258A mutant. Shrinking TASK-2-expressing cells in hypertonic solution decreased the current to 36 % of its initial value. The same manoeuvre had a significantly diminished effect on TASK-2-K257A-K258A- or TASK-2-K296-K297-expressing cells, or in cells containing intracellular GDP-β-S. Our data are compatible with the concept that TASK-2 channels are modulated by Gβγ subunits of heterotrimeric G protein. We propose that this modulation is a novel way in which TASK-2 can be tuned to its physiological functions.     

GENETIC REGULATION OF THE EXPRESSION OF H-2K.5 ANTIGEN ON ERYTHROID CELLS BY H-2K END

Difference in the amounts of H-2K.5 antigens present on erythrocytes was observed, using quantitative absorption method, among several B10 congenic strains. However, no such variation was seen on the lymphocytes and lung cells. The variability in the amount of this antigen on the erythrocyte surface was primarily dependent on the haplotype of the H-2K end in the B10 recombinant strains examined. This suggests that the regulator of H-2 expression on erythrocytes is in this region. Further genetic analysis confirmed that this regulation functions in the cis-position. Finally, the H-2K.5 antigenic activity was expressed on the reticulocytes of all strains tested, but appeared lower in the type 2 group indicating that the regulation begins early in erythropoietic differentiation and eventually results in a complete loss of detectable activity.     

Y2K: prepare, don't panic

Y2K issues could affect anyone, so it is important for HIV-positive people to make preparations to ensure their health and security. There are wide-ranging opinions about the effects of the so-called Millennium Bug, but some planning will make any changes more manageable. Patients should have adequate supplies of food, water, and medications at home in case of shortages or production problems. Other prudent steps include keeping extra cash on hand and obtaining copies of medical records and benefit plans. Internet resources are listed.     

An ROC Study of Chest Radiographs: 2K Versus 4K High-Resolution Soft-Copy Images

Computed radiography of chest with a 4K image array was recently introduced. We performed a multiobserver study to compare the diagnostic accuracy of 2K (standard) and 4K (high quality) chest radiographs displayed on a 5-mega-pixel monitor (2K monitor). One hundred cases of posteroanterior chest radiographs (a total of 200 images) were selected by two chest radiologists. Those radiographs included pneumothorax (n = 14), nodules (n = 15), interstitial disease (n = 10), or neither abnormality (n = 61). These were interpreted by four radiologists in two separate sessions. They recorded their confidence scale for the presence or absence of abnormality. Diagnostic accuracy was determined by receiver operating characteristic (ROC) analysis for each observer. ROC analysis showed no statistically significant difference between the 2K and 4K modes for the detection of any of the different abnormalities by individual readers. Our preliminary study suggests that 2K mode would be sufficient for the detection of abnormality on chest radiograph and there is no considerable validity to incline toward the 4K mode in current picture archiving and communication system environment using 2K monitor. However, we think that additional investigation using more subtle parenchymal or rib lesion should be followed.