大鼠胎脑神经干细胞HPRT基因的敲除

根据已知大鼠次黄嘌呤鸟嘌呤磷酸核糖转移酶 (HypoxanthineGuaninePhosphoribosylTransferase ,HPRT)基因的外显子序列 ,从大鼠HPRT基因组DNA序列的细菌人工染色体 (BacterialArtificialChromosome ,BAC)中用酶切和PCR方法分别分离得到用于构建基因敲除载体的 3 0kb的 5′长臂 (LongArm ,LA)和 1 7kb的 3′短臂 (ShortArm ,SA) ,并分别克隆到pSL1180和pCR2 1中。进一步构建大鼠HPRT基因打靶载体———pKO HPRT ,经酶切鉴定后的大鼠HPRT基因敲除载体用NotⅠ酶切使其线性化 ,经溴乙锭、正丁醇、酚、酚 /氯仿提纯后 ,将终浓度调至 1μg μl。在FuGene 6转染试剂的作用下转染培养 2 4h的第二代大鼠胎脑神经干细胞 (RatFetalNeuralStemCells,rFNSCs)。转染后的细胞用 80 μg mlG4 18和 0 2 μmol L的Ganc全培养液筛选 ,2w后将存活细胞进行悬浮培养 ,使细胞形成球形物 ,挑选单个的球形物进行单克隆增殖 ,其中一部分细胞 (约 2～ 3× 10 3)用裂解液处理 ,取上清用于PCR检测 ,大部分细胞 (5× 10 7)用于DNA和RNA的提取 ,进行Southernbolt和RT PCR检测 ,剩余细胞冷冻保存。最后 1次实验共分离培养了 32个rFNSCs单克隆 ,其中 3个单克隆 (9 3% )经PCR、Southernbolt和RT

纳洛酮对七氟醚诱导的胎鼠神经干细胞凋亡的影响

目的探讨纳洛酮对七氟醚诱导的胎鼠神经干细胞(NSCs)凋亡的影响。方法将NSCs分为空白组、对照组和低、中、高剂量实验组。空白组给予正常培养;对照组通入含3%七氟醚的混合气体培养48 h;低、中、高剂量实验组均通入含3%七氟醚的混合气体,并分别加入含0.1,1.0和10.0μmol·L^(-1)纳洛酮溶液的DMEM-12F培养基共培养48 h。用流式细胞仪检测NSCs的凋亡情况,用蛋白质印迹法检测沉默信息调节因子1(SIRT1)、磷脂肌醇3-激酶(PI3K)、蛋白激酶B(AKT)和哺乳动物西罗莫司靶蛋白(mTOR)蛋白的表达水平。结果低、高剂量实验组和对照组、空白组的NSCs凋亡率分别为(32.25±4.83)%,(16.77±2.52)%,(43.59±6.53)%和(8.66±1.29)%;SIRT1蛋白表达水平分别为0.50±0.08,0.99±0.15,0.20±0.03和1.27±0.19;p-PI3K/PI3K蛋白相对表达水平分别为0.85±0.13,0.47±0.04,1.06±0.16和0.25±0.03;p-AKT/AKT蛋白相对表达水平分别为0.90±0.14,0.50±0.06,1.13±0.17和0.32±0.05;p-mTOR/mTOR蛋白相对表达水平分别为0.99±0.15,0.55±0.08,1.26±0.19和0.35±0.05。低、高剂量实验组的上述指标与对照组比较,差异均有统计学意义(均P<0.05)。结论纳洛酮能有效抑制七氟醚诱导的NSCs凋亡,其机制可能与上调SIRT1表达,进而抑制PI3K/AKT/mTOR信号通路激活有关。

Effects of Rat Cytomegalovirus on the Nervous System of the Early Rat Embryo

The purpose of the study was to investigate the impact of rat cytomegalovirus （RCMV） infection on the development of the nervous system in rat embryos, and to evaluate the involvement of Wnt signaling pathway key molecules and the downstream gene neurogenin 1 （Ngnl） in RCMV infected neural stem cells （NSCs）. Infection and control groups were established, each containing 20 pregnant Wistar rats. Rats in the infection group were inoculated with RCMV by intraperitoneal injection on the first day of pregnancy. Rat E20 embryos were taken to evaluate the teratogenic rate. NSCs were isolated from El3 embryos, and maintained in vitro. We found： 1） Poor fetal development was found in the infection group with low survival and high malformation rates. 2） The proliferation and differentiation of NSCs were affected. In the infection group, NSCs proliferated more slowly and had a lower neurosphere formation rate than the control. The differentiation ratio from NSCs to neurons and glial cells was significantly different from that of the control, showed by immunofluorescenee staining. 3） Ngnl mRNA expression and the nuclear p-catenin protein level were significantly lower than the control on day 2 when NSCs differentiated. 4） The Morris water maze test was performed on 4-week pups, and the infected rats were found worse in learning and memory ability. In a summary, RCMV infection caused abnormalities in the rat embryonic nervous system, significantly inhibited NSC proliferation and differentiation, and inhibited the expression of key molecules in the Wnt/β-catenin signaling pathway so as to affect NSCs differentiation. This may be an important mechanism by which RCMV causes embryonic nervous system abnormalities.

The Potential of Rat Inner Cell Mass and Fetal Neural Stem Cells to Generate Chimeras

The rat chimera is an important animal model for the study of complex human diseases. In the present study we evaluated the chimeric potential of rat inner cell masses （ICMs） and fetal neural stem （FNS） cells. In result, three rat chimeras were produced by day 5 （D5） Sprague-Dawley （SD） blastocysts injected with ICMs derived from day 6 （D6） and D5 Dark Agouti （DA） blastocysts; four rat chimeras had been generated by D5 DA blastocyst injected with D5 SD ICMs. For the requirement of gene modification, cultured rat inner cell mass cells were assessed to produce chimeras, but no chimeras were generated from injected embryos. The potential to generate chimeras from rFNS and transfected rFNS cells were tested, but no chimeric pups were produced. Only 2 of 41 fetuses derived from D5 DA blastocyst injection with SD LacZ transfected rFNS cells showed very low number of LacZ positive cells in the section. These results indicate that DA and SD rat ICMs arc able to contribute to chimeras, but their potential decreases significantly after culture in vitro （P〈0.05）, and rFNS cells only have the potential to contribute to early fetal development.

LincRNA1230 inhibits the differentiation of mouse ES cells towards neural progenitors

In vitro, mouse embryonic stem （ES） cells can differentiate into many somatic cell types, including neurons and glial cells. When cultured in serum-free medium, ES cells convert spontaneously and efficiently to a neural fate. Previous studies have shown that the neural conversion of mouse ES cells includes both the participation of neural-specific transcription factors and the regulation of epigenetic modifications. However, the intracellular mechanism underlying this intrinsic transition still re- mains to be further elucidated. Herein, we describe a long intergenic non-coding RNA, LincRNA1230, which participates in the regulation of the neural lineage specification of mouse ES cells. The ectopic forced expression of LincRNAI230 dramatically inhibited mouse ES cells from adopting a neural cell fate, while LincRNA1230 knockdown promoted the conversion of mouse ES cells towards neural progenitors. Mechanistic studies have shown that LincRNA1230 inhibits the activation of early neural genes, such as Pax6 and Soxl, through the modulation of bivalent modifications （tri-methylation of histone3 lysine4 and his- tone3 lysine27） at the promoters of these genes. The interaction of LincRNA1230 with Wdr5 blocked the localization of Wdr5 at the promoters of early neural genes, thereby inhibiting the enrichment of H3K4me3 modifications at these loci. Collectively, these findings revealed a crucial role for LincRNA1230 in the regulation of the neural differentiation of mouse ES cells.