Altered Regulation of mRNA and miRNA Expression in Epithelial and Stromal Tissue of Keratoconus Corneas

PurposeEvaluation of mRNA and microRNA (miRNA) expression in epithelium and stroma of patients with keratoconus.MethodsThe epithelium and stroma of eight corneas of eight patients with keratoconus and eight corneas of eight non-keratoconus healthy controls were studied separately. RNA was extracted, and mRNA and miRNA analyses were performed using microarrays. Differentially expressed mRNAs and miRNAs in epithelial and stromal keratoconus samples compared to healthy controls were identified. Selected genes and miRNAs were further validated using RT-qPCR.ResultsWe discovered 170 epithelial and 1498 stromal deregulated protein-coding mRNAs in KC samples. In addition, in epithelial samples 180 miRNAs and in stromal samples 379 miRNAs were significantly deregulated more than twofold compared to controls. Pathway analysis revealed enrichment of metabolic and axon guidance pathways for epithelial cells and enrichment of metabolic, mitogen-activated protein kinase (MAPK), and focal adhesion pathways for stromal cells.ConclusionsThis study demonstrates significant differences in the expression and regulation of mRNAs and miRNAs in the epithelium and stroma of Patients with KC. Also, in addition to the well-known target candidates, we were able to identify further genes and miRNAs that may be associated with keratoconus. Signaling pathways influencing metabolic changes and cell contacts are affected in epithelial and stromal cells of patients with keratoconus.     

PAX2在肾小管上皮细胞转分化中的作用

目的 揭示核转录因子（PAX2）在肾小管上皮细胞转分化过程中的可能作用。方法 38例病例组肾组织按间质病变程度分为轻、中、重3组,其中轻度病变15例,中度病变11例,重度病变12例;对照组为5例外伤后肾切除组织。采用免疫组化法评定肾组织PAX2的表达率和肾小管上皮细胞转分化率。对肾间质病变轻、中、重3组组内肾小管上皮细胞转分化率、PAX2表达率作方差分析,对PAX2表达量与肾小管上皮细胞转分化率作相关分析。结果 正常对照组肾小管上皮细胞只表达角蛋白（CK）,不表达平滑肌肌动蛋白（α-SMA）及PAX2。病例组肾间质病变轻、中、重3组肾小管上皮细胞均不同程度表达α-SMA及PAX2;不同肾间质病变组,肾小管上皮细胞转分化率及PAX2表达量差异均有统计学意义。相关分析表明,PAX2表达量和肾小管上皮细胞转分化率呈正相关（r=0．886,P〈0．05）。结论 PAX2回归表达可能导致肾小管上皮细胞转分化。     

The spatial organization of olfactory nerve projections

The spatial organization of olfactory nerve projections was examined in the rat. The pathway was traced by orthograde transport of HRP following nasal lavage and by retrograde transport of HRP following injections into the olfactory bulb. The results indicated that there was a broad relationship between the epithelium and the olfactory bulb. Specific regions of the olfactory bulb received input from a large region of the epithelium. However, there was evidence that olfactory nerve terminations were not uniformly dense across their terminal fields. The results suggest that there may be finer sorting of olfactory nerves based on functional specificity.     

内向矫正性钾通道Kir4.1和Kir7.1在大鼠睫状体的分布

目的:在蛋白水平探讨Kir4.1和Kir7.1在大鼠睫状体的分布情况。方法:用间接免疫荧光组织化学和免疫荧光双标记在冰冻切片上检测Kir4.1和Kir7.1在大鼠睫状体的分布和定位。结果:Kir4.1蛋白分布于睫状体的色素上皮细胞和无色素上皮细胞的胞质和胞膜,内层的无色素上皮细胞免疫活性较强,免疫双标显示Kir4.1蛋白与谷氨酰胺合成酶蛋白重叠分布,Kir4.1蛋白免疫活性在睫状体的扁平部和睫状突上无明显差异。Kir7.1蛋白分布于睫状体的无色素上皮细胞和睫状肌,与特异性标记睫状上皮的Ezrin蛋白双标显示Kir7.1分布在无色素上皮的基底面,其免疫活性在睫状突强表达,在睫状体扁平部弱表达。结论:Kir4.1和Kir7.1均分布于大鼠睫状体的无色素上皮的基底面,可能与房水的生成有重要关系。睫状肌的Kir7.1分布,与调节睫状肌的舒缩、参与房水的排出密切相关。     

Innate and Adaptive Immune Responses in the Upper Respiratory Tract and the Infectivity of SARS-CoV-2

Increasing evidence shows the nasal epithelium to be the initial site of SARS-CoV-2 infection, and that early and effective immune responses in the upper respiratory tract (URT) limit and eliminate the infection in the URT, thereby preventing infection of the lower respiratory tract and the development of severe COVID-19. SARS-CoV-2 interferes with innate immunity signaling and evolves mutants that can reduce antibody-mediated immunity in the URT. Recent genetic and immunological advances in understanding innate immunity to SARS-CoV-2 in the URT, and the ability of prior infections as well as currently available injectable and potential intranasal COVID-19 vaccines to generate anamnestic adaptive immunity in the URT, are reviewed. It is suggested that the more detailed investigation of URT immune responses to all types of COVID-19 vaccines, and the development of safe and effective COVID-19 vaccines for intranasal administration, are important needs.     

细胞外基质蛋白和转化生长因子β2共同作用诱导人视网膜色素上皮细胞向肌纤维母细胞转化

目的 了解转化生长因子β2（TGFβ2）和细胞外基质（ECM）调节人胚胎视网膜色素上皮（hfRPE）细胞向肌纤维母细胞分化的作用，确定这种调节的信号传导机制。 方法 hfRPE细胞培养于由ECM包被或未包被的培养皿上，培养液中加入或不加入TGFβ2，用免疫组织化学、流式细胞仪、蛋白印迹技术检测α平滑肌机动蛋白（α-SMA）的表达。在培养液中分别加入calphostin C, 染料木黄铜（genistein）, PD98059和渥曼青霉素（Wortmannin），测定α-SMA的表达。 结果 hfRPE细胞培养于ECM包被的培养板上，培养液中加入TGFβ2后，出现明显的形态学改变，包括细胞伸展变长，可见肌动蛋白微丝的形态。流式细胞仪及免疫细胞化学检测结果显示，培养液中加入TGFβ2可明显增加α-SMA的表达，并与剂量成正相关。当TGFβ2 的刺激浓度为50 ng/ml时，与培养于未包被的培养板上的hfRPE细胞相比，用纤维连接蛋白（FN）包被培养板后，〖JP1〗总的平均荧光强度（TMFI）增加（38.01±1.14）%（P<0.05）。蛋白印迹技术检测显示同样的结果。而上述效应可以大部分被蛋白激酶C（PKC）通路抑制剂calphostin C（10 nmol/L）抑制（P<0.01）。 结论 TGFβ2可诱导hfRPE细胞向肌纤维母细胞转化，并与其剂量成正相关，这种作用可被FN加强。TGFβ2和ECM的这种调节作用可能是通过PKC细胞内信号传导通路而发挥作用 。 (中华眼底病杂志, 2006, 22: 328-332)     

中国人视网膜色素变性1基因突变频率及其与视网膜色素变性的关联分析

目的 研究常染色体显性遗传视网膜色素变性(ADRP)患者和散发视网膜色素变性（RP）患者RP1基因突变频率及特征，并探讨它们在RP发病机制中潜在的作用。 方法 运用聚合酶链反应和直接测序方法，对7个ADRP家系的55例成员、散发RP患者30例及75名健康成年人进行了RP1基因全编码区和邻近剪切位点的内含子区域序列突变的检测。运用单因素分析、多因素Logistic回归分析研究RP1基因突变位点对RP的作用。 结果 ADRP家系成员和散发的RP患者在RP1基因的第四外显子上均检测出852、872、921和939共4个变异位点。在ADRP家系和散发的RP患者中RP1基因的R872H位点改变与RP之间存在显著相关性(χ²=4.469，P=0.03)。P903L位点的改变仅在家系成员中检出，散发病例及正常人中均未检测出。 结论 RP1基因R872H位点多态性可增高RP的危险性，具有潜在的致病性，考虑为家系和散发RP患者的易感基因。P903L位点改变是否为致病基因有待于进一步证实。     

槲皮素对培养人视网膜色素上皮细胞增生及DNA合成的影响

目的研究槲皮素(quercetin, QUE)对培养人视网膜色素上皮(retinal pigment epithelium, RPE)细胞增生和DNA合成以及对表皮生长因子(epidermal growth factor, EGF)促RPE细胞增生和DNA合成作用的影响。方法用细胞计数和3H-胸腺嘧啶核苷(3H-thymidine, 3H-TdR)掺入方法,观察不同浓度(200、100、50、1μmol/L)的QUE和最大浓度的QUE(200μmol/L)在不同作用时间（24-168小时）,分别单独或与EGF共同作用时,对RPE细胞增生及DNA合成的影响,排除着色的死细胞,用活细胞计数法判断药物的细胞毒性作用。结果QUE 200μmol/L具有最强的抑制效应,48小时时抑制效应已明显出现,96小时时抑制达到高峰。与QUE单独作用相比,QUE对EGF的促RPE细胞增生效应能产生更强的抑制。QUE无细胞毒性作用,各实验组细胞活力均在85.00%以上。结论QUE以剂量依赖和时间依赖的方式抑制RPE细胞的增生,尤其是由EGF刺激的增生,并且对培养的RPE细胞无细胞毒作用。(中华眼底病杂志,1999,15:27-29)     

玻璃体液对培养人视网膜微血管内皮细胞和色素上皮细胞增生的影响

目的 探讨玻璃体液对培养的人视网膜微血管内皮细胞(human retinal capillary endothelialcells,HRCECs)和色素上皮(retinal pigmental epithelial cells,RPE)细胞增生的影响.方法 原代培养HRCECs和RPE细胞,鉴定并传至第3代,再分别培养在1:8、1:4、1:2(玻璃体液在总培养液中的体积比)的人玻璃体条件培养液(vitreous-conditioned medium,VCM)中,其中VCM分为有无血清2组,在不同作用时间(24～72h),采用四唑盐(tetrazolium,MTT)比色法检测VCM对人HRCECs和RPE细胞增生的影响.结果 在有血清时.与对照组比较,1:4、1:2的VCM在培养的各时间段对HRCECs的抑增生作用差异有显著性意义(P<0.05),而对RPE细胞和混合细胞(HRCECs和RPE混合1:1)的促增生作用差异有统计学意义(P<0.01).在无血清时与对照组比较,1:4VCM组在培养60、72h时以及1:2VCM组在培养24h,RPE细胞的增生差异有统计学意义(P<0.05),而1:2VCM组在培养48、印、72h时RPE细胞的增生差异有非常统计学意义(P<0.01);1:2VCM组在培养各时间段混合细胞的增生差异有统计学意义(P<0.05),均表现为促增生效应.结论 一定体积分数的人VCM抑制HRCECs的增生,但明显促进人RPE细胞以及混合细胞的增生,提示玻璃体中RPE细胞浸润是加重外伤增生性玻璃体视网膜病变和眼内血管增生性疾病的高危因素.