目的:通过沉默人永生化角质形成细胞HaCaT细胞中nicastrin（NCSTN）基因的表达，研究其下游细胞增殖及分化相关信号通路的改变。方法:将HaCaT细胞分为干扰组、阴性对照组和空白对照组：干扰组转染特异性NCSTN-siRNA，阴性对照组转染阴性对照siRNA，空白对照组转染等量转染试剂。实时PCR及Western印迹检测各组NCSTN mRNA和蛋白表达验证转染效率。运用Agilent人类全基因组表达谱芯片技术研究干扰组HaCaT细胞基因表达谱与阴性对照组之间的差异，以表达上调或者下调倍数≥ 2.0且 P ≤ 0.05为标准，将差异基因用GO分析进行富集，筛选出表达差异显著且与角质形成细胞增生分化相关的基因，用实时PCR验证结果。 结果:干扰组HaCaT细胞NCSTN mRNA及蛋白相对表达量分别为0.287 ± 0.090、0.443 ± 0.085，明显低于阴性对照组（0.969 ± 0.127、1.047 ± 0.114）以及空白对照组（1.000 ± 0.151、1.000 ± 0.111），差异均有统计学意义（ F值分别为30.787、31.139， P值均为0.001）。表达谱芯片显示，与阴性对照组相比，干扰组表达下调基因605条，上调基因444条。GO分析显示，干扰后差异表达基因富集到上皮发育、上皮细胞分化、角质形成细胞分化、角化4种生物学过程。对表达差异显著且与角质形成细胞增生分化相关的Sprouty相关蛋白2基因、表皮生长因子7基因、胰岛素样生长因子结合蛋白5基因、人Rho关联含卷曲螺旋蛋白激酶2基因和骨形成发生蛋白6基因通过实时PCR验证，验证结果与芯片结果显示的差异趋势一致。 结论:NCSTN基因功能缺失有可能通过调节其下游细胞增殖及分化相关信号通路的表达，影响角质形成细胞的正常增殖和分化。

目的:研究Sprouty在寻常型银屑病患者皮损组织中的表达及其与病情严重度的相关性。方法:2018年5 - 11月于南方医科大学皮肤病医院收集15例寻常型银屑病皮损及10例正常对照皮肤标本，采用免疫组化检测银屑病皮损与对照皮肤组织中Sprouty1、2和4蛋白的分布，Western印迹检测Sprouty1、2和4蛋白的表达，逆转录PCR（RT-PCR）检测Sprouty1、2和4 mRNA的表达。采用Pearson相关分析检验Sprouty蛋白表达量与银屑病皮损面积和严重程度指数（PASI）相关性。结果:免疫组化显示，银屑病皮损和对照皮肤均有Sprouty1、2和4表达，Sprouty1在对照皮肤颗粒层细胞的细胞膜和细胞质呈强表达，在银屑病皮损颗粒层的细胞膜少量表达，Sprouty4在银屑病组中的表达强于对照组，Sprouty2在两组中均少量表达。RT-PCR和Western印迹显示，银屑病组和对照组皮肤中均有Sprouty1 mRNA和蛋白的表达，银屑病组Sprouty1 mRNA（0.844 ± 0.169）和蛋白（0.148 ± 0.141）的表达均明显低于对照组（分别为0.972 ± 0.105和0.413 ± 0.108），两组比较，均 P < 0.05；且银屑病组Sprouty1蛋白表达量与PASI评分呈负相关（ r = -0.628， P = 0.012）。银屑病组Sprouty4蛋白和mRNA表达量分别为1.306 ± 0.283、1.727 ± 1.017，均明显高于对照组（0.584 ± 0.304和0.714 ± 0.615），差异均有统计意义（ t值分别为6.063和2.814，均 P<0.05），且银屑病组Sprouty4蛋白表达量与PASI评分呈正相关（ r = 0.812， P < 0.001）；两组均少量表达Sprouty2蛋白和mRNA，组间差异无统计学意义（均 P>0.05），且蛋白表达量与PASI评分无显著相关性（ r = 0.436， P = 0.104）。 结论:银屑病皮损中Sprouty1、4蛋白表达水平与PASI评分存在相关性，提示二者与银屑病的发病相关。

目的:检测1例以多发咖啡斑为主要临床表现的Legius综合征家系的基因突变情况，并明确其诊断。方法:收集1例以多发咖啡斑为主要临床表现的先证者及其父母和外祖父母临床资料，采集上述受试者外周血提取基因组DNA，对先证者进行全外显子组测序，确定突变位点，再在家系中对突变位点进行PCR扩增及Sanger测序进行验证，并明确其诊断。结果:先证者男，12岁，躯干可见10余处长径> 5 mm的咖啡斑，腋窝、腹股沟多发雀斑。先证者外周血基因组DNA中编码Sprouty相关EVH1功能域蛋白1的SPRED1基因第7号外显子中存在小片段杂合缺失（c.1220_1238del），引起氨基酸序列发生移码突变（p.L407fs*），先证者患病母亲检出该突变，外祖父母及父亲未检出该突变。该突变为新发突变，先证者突变遗传自母亲，突变与疾病符合共分离，确诊为Legius综合征。结论:Legius综合征与神经纤维瘤病Ⅰ型早期临床症状相近，基因检测有助于早期诊断、判断预后和制定随访计划。

目的 探讨微小RNA-602(miR-602)对神经母细胞瘤(NB)SH-SY5Y细胞生长、侵袭的影响及其可能的机制.方法 实时定量聚合酶链反应(qPCR)检测人NB细胞系SH-SY5Y和胚肾细胞系HEK-293中miR-602的表达水平.四甲基噻唑蓝(MTT)法检测miR-602对SH-SY5Y细胞生长的影响.Transwell侵袭实验检测miR-602对SH-SY5Y细胞侵袭的影响.筛选miR-602可能的靶基因,并通过荧光素酶报告基因系统结合qPCR技术验证miR-602对靶基因的调控作用.结果 将健康者胚肾HEK-293细胞的表达量标化为1,人NB细胞系SH-SY5Y中miR-602的相对表达水平为3.83±0.85,差异有统计学意义(P＜0.01);MTT实验结果显示,miR-602 mimics 组细胞相对活性(1.20±0.05)高于 NC mimics 组(1.00±0.01),而 miR-602 in-hibitor 组细胞相对活性为0.76±0.04低于NC inhibitor组(1.00±0.01),差异均 有统计学意义(P＜0.05);Transwell侵袭实验结果显示,miR-602 mimics组穿膜细胞数[(193.33±8.02)个]高于NC mimics组[(97.33±20.03)个],而 miR-602 inhibitor 组穿膜细胞数[(62.01±11.79)个]低于 NC inhibitor 组[(132.33±11.24)个],差异均有统计学意义(P＜0.05);qPCR结果显示,在 miR-602 mimics组重组人Sprouty相关EVH1域含蛋白1(SPRED1)mRNA表达水平较对照NC mimics组相对变化倍数为0.56±0.08,miR-602 inhibitor 组 SPRED1 mRNA 表达水平较对照 NC inhibitor 组相对变化倍数为 4.16±0.91,差异有统计学意义(P＜0.01).结论 miR-602在SH-SY5Y细胞中高表达,可能通过调控SPRED1促进SH-SY5Y细胞生长和侵袭.

Long noncoding RNAs(lncRNAs)play a crucial regulatory role in the development and progression of multiple cancers.However,the potential mechanism by which lncRNAs affect the recurrence and metastasis of ovarian cancer remains unclear.In the current study,the lncRNA LOC646029 was markedly downregulated in metastatic ovarian tumors compared with primary tumors.Gain-and loss-of-function assays demonstrated that LOC646029 inhibits the proliferation,invasiveness,and metastasis of ovarian cancer cells in vivo and in vitro.Moreover,the downregulation of LOC646029 in metastatic ovarian tumors was strongly correlated with poor prognosis.Mechanistically,LOC646029 served as a miR-627-3p sponge to promote the expression of Sprouty-related EVH1 domain-containing protein 1,which is necessary for suppressing tumor metastasis and inhibiting KRAS signaling.Collectively,our results demonstrated that LOC646029 is involved in the progression and metastasis of ovarian cancer,which may be a potential prognostic biomarker.

目的 探讨快速发育生长因子同源蛋白2 抗体(Sprouty2)蛋白调节去势抵抗性前列腺癌细胞增殖、迁移和凋亡的机制.方法 根据试验目的将人去势抵抗性前列腺癌细胞分为Sprouty2 蛋白高表达组、阴性对照组、空白对照组.Sprouty2 蛋白高表达组通过siRNA技术将小激活RNA(saRNA)转入人去势抵抗性前列腺癌细胞中进行干预;阴性对照组通过siRNA技术将空载体质粒转入人去势抵抗性前列腺癌细胞中进行干预;空白对照组进行常规培养;取对数生长期细胞用于后续实验.噻唑蓝(MTT)测定细胞增殖情况;划痕实验测定细胞迁移能力;Annexin V-FITC和PI双染色测定细胞凋亡情况.Western blot测定 Myc促癌基因(c-Myc)、SRY盒转录因子 4(Sox4)、E-box结合同源盒 1(ZEB1)蛋白、E-钙黏蛋白、波形蛋白表达.结果 Sprouty2 蛋白免疫细胞百分比、Sprouty2 蛋白免疫细胞荧光强度在各组间比较差异均具有统计学意义(P<0.05).细胞增殖活力在阴性对照组与空白对照组之间比较差异无统计学意义(P>0.05);Sprouty2 蛋白高表达组细胞迁移率明显低于阴性对照组和空白对照组,细胞凋亡率明显高于阴性对照组和空白对照组(P<0.01);细胞迁移率和细胞凋亡率在阴性对照组与空白对照组之间比较差异无统计学意义(P>0.05).E-钙黏蛋白表达水平明显高于阴性对照组和空白对照组(P<0.01);c-Myc、Sox4、ZEB1 蛋白、E-钙黏蛋白、波形蛋白表达水平在阴性对照组与空白对照组之间比较差异无统计学意义(P>0.05).结论 Sprouty2 蛋白高表达可能通过下调c-Myc、Sox4、ZEB1 蛋白、波形蛋白表达,上调Sprouty2 蛋白表达抑制去势抵抗性前列腺癌细胞的增殖、迁移和凋亡.

目的:研究远隔缺血预适应(RIPC)对脑缺血模型大鼠的保护作用及分子机制.方法:18只成年雄性SD大鼠随机分为3组:假手术组(sham)、缺血再灌注组(MCAO/R)组、RIPC+MCAO/R组;术前利用间断夹闭双侧股动脉的方法给予大鼠RIPC处理,利用大脑中动脉栓塞法(MCAO)制备大鼠缺血性脑卒中模型,利用转棒实验检测大鼠运动功能,利用TUNEL染色检测缺血区细胞凋亡,利用real time RT-PCR检测大脑缺血区皮质中miR-21-5p及SPRY1和程序性细胞死亡因子4(PDCD4)mRNA的表达.结果:与MCAO/R组大鼠相比,RIPC处理组大鼠运动功能有所改善,皮质细胞凋亡减少.miR-21-5p表达增加,而SPRY1和PDCD4 mRNA表达下调(P＜0.05).结论:RIPC处理对减轻缺血性脑卒中大鼠miR-21-5p表达上调,后者通过抑制靶分子SPRY1和PDCD4的表达抑制细胞凋亡.

Testicular microlithiasis (TM) is one of the symptoms of testicular dysgenesis syndrome (TDS).TM is particularly interesting as an informative marker of testicular germ cell tumors (TGCTs).KIT ligand gene (KITLG),BCL2 antagonist/killer 1 (BAK1),and sprouty RTK signaling antagonist 4 (SPRY4) genes are associated with a high risk of TGCTs,whereas bone morphogenetic protein 7 gene (BMP7),transforming growth factor beta receptor 3 gene (TGFBR3),and homeobox D cluster genes (HOXD) are related to TDS.Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis,we investigated allele and genotype frequencies for KITLG (rs995030,rs1508595),SPRY4 (rs4624820,rs6897876),BAK1 (rs210138),BMP7 (rs388286),TGFBR3 (rs12082710),and HOXD (rs17198432) in 142 TGCT patients,137 TM patients,and 153 fertile men (control group).We found significant differences in the KITLG GG_rs995030 genotype in TM (P=0.01) and TGCT patients (P=0.0005) compared with the control.We also revealed strong associations between KITLG_rs1508595 and TM (G allele,P=0.003;GG genotype,P=0.01) and between KITLG_rs1508595 and TGCTs (G allele,P=0.0001;GG genotype,P =0.0007).Moreover,there was a significant difference in BMP7_rs388286 between the TGCT group and the control (T allele,P =0.00004;TT genotype,P =0.00006) and between the TM group and the control (T allele,P=0.04).HOXDalso demonstrated a strong association with TGCTs (rs17198432 A allele,P =0.0001;AA genotype,P =0.001).Furthermore,significant differences were found between the TGCT group and the control in the BAK1_rs210138 G allele (P=0.03) and the GG genotype (P =0.01).KITLG and BMP7genes,associated with the development of TGCTs,may also be related to TM.In summary,the KITLG GG_rs995030,GG_rs1508595,BMP7 TT_rs388286,HOXD AA_rs17198432,and BAK1 GG_rs210138 genotypes were associated with a high risk of TGCT development.

Objective Arsenic is a metalloid environmental carcinogen involved in the occurrence and development of many cancers.miRNA-21 plays a crucial role in arsenic-induced carcinogenesis.We aimed to elucidate the mechanism by which miRNA-21 influences arsenic-induced cancer.Methods We used meta-analysis of published studies to determine how arsenic induces cancerous cells through miRNA-21.Results Low-dose arsenic exposure (≤ 5 μmol/L) can increase miRNA-21 and phosphorylated signal transducter and activator of transcription 3 (pSTAT3) expression,and decrease programmed cell death protein 4 (PDCD4) and protein sprouty homolog 1 (Spry1) expression.High-dose arsenic exposure (＞ 5 μmol/L),can increase miRNA-21 expression,and decrease Spry1 and E-cadherin expression.Short-term arsenic exposure (≤ 24 h) can increase miRNA-21 and pSTAT3 expression,and decrease PDCD4 expression.Moreover,long-term arsenic exposure (＞ 24 h) can increase the miRNA-21,STAT3,and pSTAT3 expression,and decrease PDCD4 expression.We found that activation of miRNA-21 and pSTAT3 were most pronounced following long-term arsenic exposure at low doses,and the effects on PDCD4 expression were most pronounced following short-term arsenic exposure at low doses.miRNA-21 inhibitors increased the expression of tumor suppressor genes PDCD4,PTEN,and Spry1 and miRNA-21-mimics suppressed the expression of these tumor suppressor genes.Conclusion Arsenic can cause cancer by activating miRNA-21 and inhibiting the expression of PDCD4,PTEN,and Spry1.

目的 探讨FOXO3a与SPROUTY2(SPRY2)在三阴型乳腺癌(triple negative breast cancer,TNBC)中的表达及其相关性.方法 采用免疫组化EnVision两步法检测TNBC组织和正常乳腺组织中FOXO3a与SPRY2的表达,并分析两者与TNBC临床病理特征的关系.结果 TNBC组织中FOXO3a与SPRY2表达显著低于正常乳腺组织(P<0.01).秩和检验结果显示,TNBC中FOXO3a与SPRY2的表达与组织学分级及淋巴结转移呈负相关.Spearman相关性分析结果显示,TNBC中FOXO3a与SPRY2的表达呈正相关(P<0.001).结论 TNBC中FOXO3a与SPRY2的表达呈正相关,两者在TNBC中起抑制作用,可能存在调控关系,从而抑制淋巴结转移.FOXO3a与SPRY2可作为新的判断预后的重要因子,并为临床基因靶向治疗提供新的理论依据.