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。     

Subtle sex differences in vasopressin mRNA expression in the embryonic mouse brain

Arginine vasopressin (AVP) is a neuropeptide which acts centrally to modulate numerous social behaviors. One receptor subtype through which these effects occur is the AVP 1a receptor (AVPR1A). The modulatory effects of Avp via the AVPR1A varies by species as well as sex, since both AVP and the AVPR1A tend to be expressed more prominently in males. Beyond these neuromodulatory effects there are also indications that the AVP system may play a role in early development to, in part, organize sex‐specific neural circuitry that is important to sexually dimorphic social behaviors in adulthood. However, to date, AVP's role in early development is poorly understood, particularly with respect to its differential effect on males and females. In order to determine the timing and distribution of the AVP system in early brain development, we examined the brains of male and female C57BL/6J mice between embryonic day (E) 12.5 and postnatal day (P) 2 and quantified Avp and Avpr1a mRNA using qPCR and AVPR1A protein using receptor autoradiography. The mRNA for Avp was measurable in males and females starting at E14.5, with males producing more than females, while Avpr1a mRNA was found as early as E12.5, with no difference in expression between sexes. AVPR1A binding was observed in both sexes starting at E16.5, and while there were no observed sex differences, binding density and the number of neuroanatomical areas did increase over time. These data are significant as they provide the first whole‐brain characterization of the vasopressin system in the embryonic mouse. Further, these findings are consistent with data from other species, that have documented a sex difference in the vasopressin system during early brain formation.     

SIRT3 regulation of mitochondrial oxidative stress

Mitochondria play a central role in the production of reactive oxygen species as byproducts of metabolism and energy production. In order to protect cellular structures from oxidative stress-induced damage, cells have evolved elegant mechanisms for mitochondrial ROS detoxification. The mitochondrial sirtuin, SIRT3, is emerging as a pivotal regulator of oxidative stress by deacetylation of substrates involved in both ROS production and detoxification. This review will summarize recent findings on the regulation of mitochondrial ROS homeostasis by SIRT3.     

p53基因在亚砷酸钠致人胚胎肺成纤维细胞凋亡中的作用

目的 探讨p53、Bax、bcl-2基因在亚砷酸钠(NaAs02)致人胚胎肺成纤维细胞(HELF)凋亡中的作用.方法 分别取转染了p53质粒HELF细胞(p53组)、转染了PC质粒的HELF细胞(PC组)、常规培养的HELF细胞(正常组),在6孔板中培养48 h后,分别加入0、3、9、15 mmoL/L NaAsO2溶液,培养24 h后,用real-time PCR法检测p53、Bax、bcl-2 mRNA基因表达水平,采用免疫组织化学SABC法检测p53、Bax、bcl-2基因蛋白表达水平;用流式细胞仪检测细胞凋亡情况.结果 p53组细胞p53基因mRNA表达水平(0.51±0.29)低于PC组及正常组[(1.32±0.26),(1.00±0.20),P均<0.05],p53组p53基因蛋白表达水平[(4.10±1.20)%]低于PC组和正常组[(8.00±1.63)%、(7.90±1.79)%,P均<0.05];p53组、PC组、正常组在染砷0、3、9、15 mmol/L时,细胞凋亡率[(0.57±0.28)%、(22.91±4.86)%、(40.05±3.93)%、(44.87±3.58)%,(0.65±0.24)%、(14.09±3.49)%、(20.31±3.66)%、(32.42±3.63)%,(0.56±0.25)%、(12.14±3.70)%、(19.61±3.63)%、(30.43±2.83)%]、Bax mRNA表达水平[(12.73±3.96)、(25.12±6.42)、(104.96±26.77)、(154.04±3052),(14.63±3.57)、(36.75±3.67)、(272.26±66.11)、(846.12±243.36),(14.75±5.65)、(37.22±11.27)、(278.51±37.42)、(861.67±369.29)]、Bax蛋白表达水平[(15.07±0.83)%、(23.79±3.99)%、(3851±158)%、(53.86±124)%,(15.43±1.45)%、(36.11±1.37)%、(56.86±1.97)%、(76.09±2.01)%,(15.20±1.03)%、(35.25±1.09)%、(55.56±2.17)%、(74.48±2.85)%]均随着染毒剂量的增加而增高(P均<0.05),而bcl-2 mRNA表达水平[(443.00±244.47)、(156.79±53.18)、(62.13±13.66)、(23.10±6.44),(420.55±110.77)、(48.15±10.02)、(14.91±6.53)、(7.54±2.62),(577.75±123.22)、(49.68±10.11)、(12.41±1.28)、(7.22±1.89)]、bcl-2蛋白表达水平[(47.20±3.77)%、(41.80±2.94)%、(36.00±2.36)%、(29.00±2.91)%,(45.90±4.15)%、(35.70±2.77)%、(29.80±2.78)%、(24.80±2.66)%,(46.70±3.47)%、(36.20±2.90)%、(30.10±3.21)%、(25.10±2.28)%]均随着染毒剂量的增加而降低(P均<0.05);在染砷3、9、15 mmol/L时,p53组的细胞凋亡率、bcl-2 mRNA表达水平、bcl-2蛋白表达水平均高于同一染砷剂量的正常组和PC组(P均<0.05),而Bax mRNA表达水平、Bax基因蛋白表达水平均低于同一染砷剂量的正常组和PC组(P均<0.05).结论 p53基因减少了NaAsO2致HELF的凋亡,可能是通过改变部分凋亡途径实现.     

昆明小鼠胚胎干细胞向心肌细胞的分化

目的饲养层制作及复苏培养昆明小鼠胚胎干细胞,探索体外诱导胚胎干细胞向心肌细胞分化。方法饲养层制作,复苏及体外培养胚胎干细胞,采用一步法消化贴壁胚胎干细胞,悬浮培养5d,形成拟胚体(EB),待EB贴壁后,加入淫羊藿苷(Icraiin,ICA)诱导液对其诱导并每天观察,免疫荧光检测心肌细胞特异性肌钙蛋白T(cTnT)和心室肌球蛋白轻链(MLC-2v)的表达。结果胚胎干细胞悬浮聚集5d,形成类似球状的拟胚体,将拟胚体贴壁诱导8d,发现拟胚体中出现跳动,诱导后10d拟胚体跳动率达65%,显著高于空白对照组和阴性对照组,一个分化拟胚体中一般出现1-3个跳动点,节律约为50-80times/min,在诱导10d时跳动的合胞体,免疫荧光表明cTnT和MLC-2v表达为阳性。结论胚胎干细胞经悬浮聚集培养5d后经10-7mol/L淫羊藿苷诱导,得到了可以跳动的心肌细胞团。     

Application of hiPSCs in tooth regeneration via cellular modulation

BackgroundInduced pluripotent stem cell (iPSC)-based technology provides limitless resources for customized development of organs without any ethical concerns. In theory, iPSCs generated from terminally differentiated cells can be induced to further differentiate into all types of organs that are derived from the embryonic germ layers. Since iPSC reprogramming technology is relatively new, extensive efforts by the researchers have been put together to optimize the protocols to establish in vitro differentiation of human iPSCs (hiPSCs) into various desirable cell types/organs.HighlightsIn the present study, we review the potential application of iPSCs as an efficient alternative to primary cells for modulating signal molecules. Furthermore, an efficient culture system that promotes the differentiation of cell lineages and tissue formation has been reviewed. We also summarize the recent studies wherein tissue engineering of the three germ layers has been explored. Particularly, we focus on the current research strategies for iPSC-based tooth regeneration via molecular modulation.ConclusionIn recent decades, robust knowledge regarding the hiPSC-based regenerative therapy has been accumulated, especially focusing on cellular modulation. This review provides the optimization of the procedures designed to regenerate specific organs.     

Morphological and GABA-immunoreactive development of the embryonic chick telencephalon

The development of neurons utilizing γ-aminobutyric acid (GABAergic neurons) in prosencephalon and telencephalon from chicken embryonic days 4–14 (E4–E14) was studied by means of immunohistochemistry. Furthermore, routine histology and transmission electron microscopy, respectively, were performed in order to study the morphological development in the designated area. The main finding is that development of GABAergic neurons in the chick telencephalon is rapid; the GABA neurons are appearing in bulk at day 8, being “overexpressed” at days 10–11, decreasing in numbers thereafter and achieving mature morphology on day 14, which is considerably faster than in the rodent.Morphological analysis revealed that the prosencephalon mainly consisted of a thin layer of undifferentiated neuroblasts in the E4 embryo. By E6, the prosencephalon had increased in thickness and occasional cells outside the neuroepithelium showed a more mature morphology with a few cells weakly staining positive for GABA. At E8, the prospective granular and subventricular layers had developed. At E14, the appearance of the telencephalon is approximating that of the adult since both ependymal cells and morphologically mature neurons can be seen.     

人类性别遗传与精子的类型无关的实验研究

目的通过实验观察LY1014中药混合物对人类受精卵和胚胎干细胞性别发育趋势的影响,并探讨了人类性别遗传与精子之间究竟存在怎样的关系及其人类性别遗传的基本原理。方法按照中医中药的原则,筛选出有效的多种中药,通过水煎并浓缩制成LY1014中药混合物小丸或其他剂型,并按实验设计的要求给准备怀孕的妇女服用,得到婴儿出生的结果和比例,进而利用顺向思维和逆向思维的方法对实验结果进行分析。结果参加实验的50名妇女都产下了男孩,与人类自然对照组相比,实验组具有非常显著性差异（P〈0．001）。结论LY1014中药混合物能够诱导原本向卵巢细胞方向发育的胚胎干细胞向睾丸细胞进行基因表达,朝男性性别方向发育;根据实验结果可以明确推断出：人类性别遗传与精子的类型是没有关系的,精子是唯一的,没有X精子和Y精子之分,而且,携带唯一的一个男性性别基因,因此也没有X染色体和Y染色体之分;同时,利用逆向思维的方法,推断出卵细胞也是携带有一个女性性别遗传基因的,而且,男女性别之间存在着男女性别基因之间的相互转换关系。     

祛瘀解毒颗粒治疗子宫内膜异位症患者卵泡液对小鼠胚胎发育的影响

目的 观察经祛瘀解毒颗粒治疗后子宫内膜异位症（endometriosis,EM）患者卵泡液对小鼠胚胎发育的影响。 方法 在常规培养液中分别加入治疗组EM患者卵泡液（祛瘀解毒颗粒）、对照组EM患者卵泡液及空白组（因输卵管因素行体外受精－胚胎移植）患者卵泡液,20只小鼠超排卵共收集189枚2 细胞鼠胚分别放入各组培养液进行培养,其中治疗组培养液70枚,对照组培养液60枚,空白组培养液59枚,观察和检测2 细胞鼠胚在体外发育至8细胞期、桑葚胚期和囊胚期的比率,及早期鼠胚优质胚胎数目和优质胚胎率。 结果 治疗组培养液有53枚鼠胚（75.71%,53/70）发育到8细胞期,48枚（68.57%,48/70）发育到桑葚胚期,45枚（64.28%,45/70）发育到囊胚期,与对照组培养液(56.67%,34/60、48.33%,29/60、35.00%,21/60)比较差异有统计学意义(P＜0.05)。治疗组有61枚优质胚胎（87.14%）,对照组为44枚（73.33%）,两组比较差异有统计学意义（P＜0.05）。 结论 EM患者卵泡液对早期鼠胚存在毒性作用,经祛瘀解毒颗粒治疗后EM患者卵泡液可提高早期鼠胚培养质量。