自身免疫调节因子在小鼠胚胎干细胞向胸腺上皮祖细胞分化中的表达

文题释义：自身免疫调节因子：基因分析显示由于单基因突变引起一种自身免疫病,此基因则被命名为自身免疫调节因子即AIRE基因。AIRE基因的突变或者缺失会造成胸腺内自身组织特异性抗原转录缺失,影响阴性选择,从而致使自身反应性T细胞逃逸外周,引起自身免疫反应。AIRE基因一直是免疫学相关研究中的热点。胸腺上皮细胞：胸腺上皮细胞分为髓质胸腺上皮细胞和皮质胸腺上皮细胞,二者均来源于胸腺上皮祖细胞,据研究报道髓质胸腺上皮细胞表达的AIRE基因调控着胸腺内的阴性选择,但是由于胸腺上皮祖细胞和胸腺上皮细胞不易分离且数量少,其应用和研究一直受限。该实验在体外将胚胎干细胞分化为胸腺上皮祖细胞,可以为相关研究提供细胞来源。  摘要背景：自身免疫性疾病主要是由于胸腺内自身组织特异性抗原持续表达缺失而引起强烈免疫应答的一类疾病,而胸腺功能减退和胸腺内组织特异性抗原的不稳定表达会限制治疗效果。胸腺组织主要由胸腺上皮细胞组成,但胸腺内成熟胸腺上皮细胞和胸腺上皮祖细胞有限的数量来源极大限制了相关研究。目的：研究小鼠胚胎干细胞向胸腺上皮祖细胞分化过程中自身免疫调节因子的表达变化。方法：采用两步分化方法定向诱导小鼠胚胎干细胞分化为内胚层再分化为胸腺上皮祖细胞,分别收集定向诱导分化第0,3,13天细胞,采用细胞免疫荧光、流式细胞术、Western blot、Real-Time PCR 检测相关基因及蛋白的表达变化。结果与结论：①诱导分化第0天,免疫荧光检测OCT4、SSEA1表达阳性;诱导分化第3天,免疫荧光检测SOX17、FoxA2呈双阳性表达;诱导分化第13天,流式细胞术检测EpCAM1、K5、K8表达阳性;②Real-time PCR检测小鼠胚胎干细胞定向分化过程中PAX1、PAX9、FOXN1、PLET1基因表达逐渐增高;③Real-time PCR检测分化第0,3,13天AIRE基因表达升高,INS2、GAD67基因表达也升高;④Western blot检测分化第0,3,13天AIRE蛋白表达降低,胰岛素蛋白、GAD67蛋白均无表达;⑤结果表明,小鼠胚胎干细胞成功分化为胸腺上皮祖细胞,且分化而来的胸腺上皮祖细胞中AIRE基因表达很高,促进了INS2、GAD67基因的表达,为细胞移植治疗自身免疫性疾病提供评价依据。ORCID: 0000-0001-5253-3085(胡蓉) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Dual expression of Epstein-Barr virus,latent membrane protein-1 and human papillomavirus-16 E6 transform primary mouse embryonic fibroblasts through NF-κB signaling

The prevalence of Epstein-Barr virus (EBV) and high-risk human papilloma virus (HPV) infections in patients with oral cancer in Okinawa, southwest islands of Japan, has led to the hypothesis that carcinogenesis is related to EBV and HPV co-infection. To explore the mechanisms of transformation induced by EBV and HPV co-infection, we analyzed the transformation of primary mouse embryonic fibroblasts (MEFs) expressing EBV and HPV-16 genes, alone or in combination. Expression of EBV latent membrane protein-1 (LMP-1) alone or in combination with HPV-16 E6 increased cell proliferation and decreased apoptosis, whereas single expression of EBV nuclear antigen-1 (EBNA-1), or HPV-16 E6 did not. Co-expression of LMP-1 and E6 induced anchorage-independent growth and tumor formation in nude mice, whereas expression of LMP-1 alone did not. Although the singular expression of these viral genes showed increased DNA damage and DNA damage response (DDR), co-expression of LMP-1 and E6 did not induce DDR, which is frequently seen in cancer cells. Furthermore, co-expression of LMP-1 with E6 increased NF-κB signaling, and the knockdown of LMP-1 or E6 in co-expressing cells decreased cell proliferation, anchorage independent growth, and NF-κB activation. These data suggested that expression of individual viral genes is insufficient for inducing transformation and that co-expression of LMP-1 and E6, which is associated with suppression of DDR and increased NF-κB activity, lead to transformation. Our findings demonstrate the synergistic effect by the interaction of oncogenes from different viruses on the transformation of primary MEFs.     

人Kv1.5通道基因转染人胚胎肾细胞的研究

目的建立表达人Kv1.5通道的细胞模型。方法采用Lipofactamine 2000脂质体将人Kv1.5通道基因转染人胚胎肾细胞(HEK293),通过荧光显微镜和全细胞膜片钳技术观测人Kv1.5通道基因的表达。结果pcDNAHKv1.5真核表达载体转染HEK293细胞2d后,荧光显微镜可观察到人Kv1.5通道表达;3d后全细胞膜片钳技术记录到人Kv1.5通道基因编码的通道电流。结论pcDNAHKv1.5真核表达载体转染HEK293细胞为人Kv1.5通道的研究提供了良好的真核细胞表达系统和细胞模型。     

超排卵治疗过程中卵泡液促性腺激素、雌二醇和胰岛素样生长因子水平检测

目的: 检测超排卵治疗过程中卵泡液内某些激素以及生长因子的水平,探讨它们与受精后胚胎发育潜能的关系.方法:接受辅助生殖技术治疗的患者,根据胚胎发育状况及治疗结局分为妊娠组、正常未孕组和胚胎质量较差组,超促排卵后在超声引导下经阴道穿刺获得卵泡液,用放射免疫法测定卵泡液中促卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E2)、孕酮(P)、生长激素(GH)以及胰岛素样生长因子1(IGF-1)的水平,比较其与不同胚胎发育状况及治疗结局之间的关系.结果:妊娠组及胚胎发育正常但未妊娠组E2水平明显高于胚胎质量较差组(P＜0.05);妊娠组IGF-1水平明显高于胚胎质量较差组(P＜0.05),正常未孕组居于2者之间,但差异无统计学意义;FSH、LH、P及GH水平在以上3组中差异无统计学意义.结论:超促排卵时卵泡液中E2及IGF-1可能与卵子受精后胚胎发育的潜能有关,测定其水平有助于评估辅助生殖技术治疗后的结局.     

血清癌胚抗原在转移性肺腺癌患者冷冻治疗中的疗效评估及预后分析

目的探讨转移性肺腺癌患者冷冻治疗前后血清癌胚抗原（CEA）变化,及CEA在转移性肺腺癌患者预后中的意义。方法选择56例肺腺癌患者,47例接受肺内肿瘤的氩氦刀冷冻综合治疗,9例接受肺外肿瘤氩氦刀冷冻姑息治疗。检测患者冷冻治疗前后血清CEA水平,同时回访所有入组患者,统计患者总体生存期。结果59％转移性肺腺癌患者血清CEA水平在治疗前异常。综合治疗组冷冻术后血清CEA水平显著低于冷冻术前血清CEA水平（P=0．0033）。接受综合疗法的肺腺癌患者中,术前血清CEA水平正常患者的中位生存时间为26个月,术前血清CEA水平异常患者中位生存时间为12个月,术前血清CEA水平正常患者具有较长的总体生存期。结论对于转移性非小细胞肺腺癌冷冻治疗患者,血清CEA检测在患者治疗效果及预后评价上具有重要意义。     

Mild Choline Deficiency and MTHFD1 Synthetase Deficiency Interact to Increase Incidence of Developmental Delays and Defects in Mice

Folate and choline are interconnected metabolically. The MTHFD1 R653Q SNP is a risk factor for birth defects and there are concerns that choline deficiency may interact with this SNP and exacerbate health risks. 80–90% of women do not meet the Adequate Intake (AI) for choline. The objective of this study was to assess the effects of choline deficiency on maternal one-carbon metabolism and reproductive outcomes in the MTHFD1-synthetase deficient mouse (Mthfd1S), a model for MTHFD1 R653Q. Mthfd1S^+/+ and Mthfd1S^+/− females were fed control (CD) or choline-deficient diets (ChDD; 1/3 the amount of choline) before mating and during pregnancy. Embryos were evaluated for delays and defects at 10.5 days gestation. Choline metabolites were measured in the maternal liver, and total folate measured in maternal plasma and liver. ChDD significantly decreased choline, betaine, phosphocholine, and dimethylglycine in maternal liver (p < 0.05, ANOVA), and altered phosphatidylcholine metabolism. Maternal and embryonic genotype, and diet-genotype interactions had significant effects on defect incidence. Mild choline deficiency and Mthfd1S^+/− genotype alter maternal one-carbon metabolism and increase incidence of developmental defects. Further study is required to determine if low choline intakes contribute to developmental defects in humans, particularly in 653QQ women.     

人胚胎干细胞建系、培养及体外诱导分化研究现状

胚胎干细胞具有体外无限增殖和分化成三胚层细胞的潜能,它已被视为治疗多种疾病的一种新兴策略。目前胚胎干细胞常规的建系和培养技术已很成熟,并有一套国际公认的鉴定标准。但常规方法存在异源病原体污染的可能,急需研究适于标准化、无动物源性污染及可大量培养胚胎干细胞的培养体系。在现阶段,通过不同的体外诱导途径可将胚胎干细胞诱导成为胚外和三胚层来源的各种细胞,但定向分化的问题仍亟需解决。     

RNA干扰抑制BUB1基因表达对染色体分离的影响

目的：研究 BUB1基因表达调控对染色体分离的影响。方法：用定量 RT-PCR 比较 RNA 干扰前后的胚胎细胞内源性基因 BUB1的 mRNA 水平,同时对染色体数目异常率进行比较分析。结果：干扰后 BUB1基因 mRNA 拷贝数/GAPDH 基因 mRNA 拷贝数的均值为干扰前的21．80%,染色体数目异常率均值由干扰前 5．02%上升到29．61%。经统计分析,RNA 干扰前后 BUB1基因 mRNA 水平和染色体数目异常率有显著差异。结论：BUB1基因表达的降低会导致染色体分离的异常,本研究为染色体分离相关蛋白功能研究建立了新的研究手段和实验评价体系。