miR-513a-5p慢病毒载体的构建及其对人骨肉瘤细胞放疗敏感性的影响

目的:构建miR-513a-5p慢病毒过表达载体，转染人骨肉瘤细胞株，观察miR-513a-5p对人骨肉瘤细胞放疗敏感性的影响。方法:PCR法扩增人miR-513a-5p基因，克隆入pLentis-CMV-GFP-MCS-PGK-PURO载体获得重组质粒pLentis-miR513a，双酶切鉴定并测序后将正确的重组质粒和对照质粒转染293FT细胞制备慢病毒，分别转染骨肉瘤HOS和U2OS细胞，qRT-PCR法及荧光显微镜鉴定转染结果。克隆形成实验、MTT法检测miR-513a-5p高表达HOS和U2OS细胞在X射线照射下细胞存活情况。结果:双酶切及测序结果确定成功构建miR-513a-5p慢病毒载体pLentis-miR513a。qRT-PCR结果提示，转染骨肉瘤细胞株后miR-513a-5p表达显著升高。克隆形成实验结果显示miR-513a-5p高表达后骨肉瘤细胞在X射线照射下细胞增殖减慢。MTT结果提示miR-513a-5p高表达骨肉瘤细胞经X射线照射后细胞存活减少。结论:成功构建了miR-513a-5p慢病毒载体，建立了高效稳定表达miR-513a-5p的骨肉瘤细胞株，高表达miR-513a-5p能显著增加X射线照射后骨肉瘤细胞的放疗敏感性。     

Molecular simplification of lipid A structure: TLR4-modulating cationic and anionic amphiphiles

A growing body of data suggests that therapies based on Toll-like receptors (TLR) targeting, in particular TLR4, holds promise in curing autoimmune and inflammatory pathologies still lacking specific treatment, included several rare diseases. While TLR4 activators (agonists) have already found successful clinical application as vaccine adjuvants, the use of TLR4 blockers (antagonists) as antisepsis agents or as agents against inflammatory diseases (including arthritis, multiple sclerosis, neuroinflammations) and cancer is still at a preclinical phase of development. This minireview focuses on recent achievements on the development of TLR4 modulators based on lipid A structure simplification, in particular on compounds having disaccharide or monosaccharide structures. As the TLR4 activity of natural TLR4 ligands (lipopolysaccharide, LPS and its biologically active part, the lipid A) depends on both the structure of endotoxin aggregates in solution and on single-molecule interaction with MD-2 and CD14 receptors, the rational design of TLR4 modulators should in principle take into account both these factors. In the light of the most recent advances in the field, in this minireview we discuss the structure–activity relationship in simplified lipid A analogs, with cationic or anionic amphiphilic structures.     

基因间长链非编码RNA152靶向微小RNA?103a?3p对非小细胞肺癌细胞增殖和侵袭迁移的影响

目的探讨基因间长链非编码RNA 152(LINC00152)靶向调控微小RNA-103a-3p(miR-103a-3p)表达及对非小细胞肺癌(NSCLC)细胞增殖和侵袭迁移的影响。方法采用实时定量PCR(QPCR)检测正常肺上皮细胞BEAS-2B及NSCLC细胞(ANIP-973、NCI-H157、A549和NCI-H1975)的LINC00152水平。选取LINC00152水平最高的细胞分别转染LINC00152特异性小干扰RNA(si-LINC00152组)或无关序列(si-NC组),另设未转染细胞为对照组。QPCR检测LINC00152水平,活细胞计数CCK-8法、Transwell小室和划痕实验测定细胞增殖、侵袭和迁移能力,Western blotting检测基质金属蛋白酶(MMP)-2、MMP-9和第10号染色体缺失的磷酸酶及张力蛋白同源基因(PTEN)的水平;荧光素酶报告实验验证LINC00152靶向结合miR-103a-3p的能力。结果NSCLC细胞的LINC00152水平均高于BEAS-2B细胞(P<0.05),尤其是NCI-H1975细胞的最高。si-LINC00152组的LINC00152水平为0.352±0.087,低于对照组的1.058±0.219和si-NC组的1.126±0.139(P<0.05)。与si-NC组和对照组相比,si-LINC00152组NCI-H1975细胞转染48、72 h的增殖活力下降(P<0.05);si-LINC00152组的划痕愈合率和穿膜细胞数分别为(27.386±2.428)%和(78.840±5.031)个,低于si-NC组的(77.675±4.803)%和(179.208±13.264)个及对照组的(76.371±5.385)%和(174.003±15.678)个(P<0.05);与si-NC组和对照组相比,si-LINC00152组的MMP-2和MMP-9水平均降低,而PTEN水平升高(P<0.05)。对照组和si-NC组上述指标的差异无统计学意义(P>0.05)。双荧光素酶报告分析证实,miR-103a-3p模拟物降低了野生型LINC00152的荧光素酶活性(P<0.05),但对突变型无影响(P>0.05)。结论LINC00152在NSCLC细胞中高表达并发挥促癌作用,与NSCLC的迁移侵袭密切相关,LINC00152与miR-103a-3p间的相互作用在NSCLC靶向治疗中有一定潜能。     

体外转染Cyclin A2基因对原代大鼠心肌细胞增殖的影响

目的探讨体外转染细胞周期素A2(Cyclin A2)基因对原代大鼠心肌细胞增殖的影响,为心脏再生提供细胞学依据。方法 SD乳鼠12只,分离、培养、鉴定原代乳鼠心肌细胞并分为3组,实验组:转染带有强化绿色荧光蛋白(GFP)和Cyclin A2基因的重组腺病毒(Ad-Cyclin A2-GFP);空病毒组:转染不含目的基因带有GFP的重组腺病毒(Ad-Null-GFP);阴性对照组:未做转染处理,仅加入等量的培养基。利用GFP示踪技术,评估原代心肌细胞转染效率;转染后的细胞继续体外培养3~5d,利用免疫荧光技术分别检测Cyclin A2、磷酸化组蛋白H3(H3P)、心肌肌钙蛋白-T(c Tn T)。结果 1.荧光GFP示踪表明原代心肌细胞的转染效率高达(97±0.74)%;2.免疫荧光标记显示,空病毒组和对照组结果相似;心肌细胞特异性标记蛋白c Tn T分布于细胞质,原代心肌细胞纯度高达(95±0.62)%;心肌细胞Cyclin A2主要在胞核内聚集,少数分布于细胞质。H3P为核蛋白在细胞核内分布。3.转染Cyclin A2后,实验组H3P阳性率明显高于空病毒组和对照组,差异具有统计学意义(P0.05);实验组可见大量多核细胞,以双核为主,伴少量3核细胞。结论腺病毒作为转染载体对原代心肌细胞有很好的侵染效率;Cyclin A2超表达促进原代心肌细胞形成双核。     

(-)-Epigallocatechin-3-gallate,reduces corneal damage secondary from experimental grade II alkali burns in mice

Background

Since recent reports have shown that (-)-Epigallocatechin-3-gallate (EGCG) could be used for treating proliferative and inflammatory disorders, we explored its use for the management of corneal chemical burns.

m6A去甲基化酶ALKBH5与弱精症的相关性研究

目的探究N⁶-甲基腺嘌呤(m⁶A)去甲基化酶ALKBH5与弱精症的相关性。方法收集精液标本30份,包括弱精症患者与健康者精液标本各15份,密度梯度离心纯化精子,使用液相色谱与质谱联用(LC-MS/MS)检测精子RNA的m⁶A水平,使用实时荧光定量PCR法检测ALKBH5相对表达水平。结果弱精症患者m⁶A水平明显高于健康者,ALKBH5相对表达水平低于健康者,差异均有统计学意义(P<0.05)。精子活动度参数相关性分析结果显示,ALKBH5相对表达水平与前向运动精子数和非前向运动精子数呈正相关(r=0.512、0.377,P<0.05),与不动精子数呈负相关(r=-0.497,P<0.05);m⁶A与前向运动精子数和非前向运动精子数呈负相关(r=-0.442、-0.425,P<0.05),与不动精子数呈正相关(r=0.528,P<0.05)。结论在弱精症患者中,m⁶A及其去甲基化酶ALKBH5与精子活动度具有相关性。     

Anisotropic Bladder Planning Target Volume in Bladder Radiation Therapy

Purpose

This study aimed to investigate 3 planning target volume (PTV) margin expansions and determine the most appropriate volume to be used in bladder preservation therapy when using daily cone beam computed tomography (CBCT). We aimed to establish whether a smaller PTV expansion is feasible without risking geographical miss.

Secretion,blood levels and cutaneous expression of TL1A in psoriasis patients

TL1A is a TNF‐like cytokine which has been shown to co‐stimulate TH1 and TH17 responses during chronic inflammation. The expression of this novel cytokine has been investigated in inflammatory disorders like rheumatoid arthritis and inflammatory bowel disease, but little is known about expression and induction in psoriasis. Indeed, the pathogenesis in psoriasis is still not fully understood and it is speculated that cytokines other than TNF‐α are important in subsets of patients. Also, for patients with severe disease that are treated with systemic anti‐TNF‐α blockade, novel candidates to be used as disease and response biomarkers are of high interest. Here, we demonstrate TL1A expression in biopsies from psoriatic lesions. Also, we investigated spontaneous and induced TL1A secretion from PBMCs and blood levels from a cohort of psoriasis patients. Here, increased spontaneous secretion from PBMCs was observed as compared to healthy controls and a small subset of patients had highly elevated TL1A in the blood. Interestingly, activation of PBMCs with various cytokines showed a decreased sensitivity for TL1A activation in psoriasis patients compared to healthy controls.TL1A levels in blood and biopsies could not be correlated with disease activity with this patient cohort. Thus, additional large‐scale studies are warranted to investigate TL1A as a biomarker.