Sertoli 细胞对大鼠大脑皮质神经前体细胞的诱导分化作用

目的:研究Sertoli细胞对体外培养大鼠皮质神经前体细胞生长发育的促进作用,及向多巴胺能神经元分化的诱导作用.方法:将大鼠Sertoli细胞与14 d大鼠皮质神经前体细胞体外共培养,免疫细胞化学检测神经干细胞标记物巢蛋白,神经元、星形胶质细胞和小胶质细胞标记物βⅢ-微管蛋白(βⅢ-tubulin)、胶质纤维酸性蛋白(GFAP)和半乳糖脑苷脂(GalC),及多巴胺能神经元标记物酪氨酸羟化酶(TH)的表达情况.结果:随着共培养时间的延长,神经前体细胞中GFAP阳性细胞数量逐渐减少,而β-Ⅲ tubulin 阳性细胞数增多,并出现TH阳性神经元.结论:Sertoli细胞抑制体外培养的大鼠皮质神经前体细胞的生长,但具有显著促神经元分化作用,并能够诱导与其共培养的大鼠皮质神经前体细胞分化为多巴胺能神经元.     

快速老化小鼠胸腺细胞分化与Notch信号相关基因表达关系的研究

目的：研究快速老化小鼠（Senescence-accelerated mice，SAM）增龄过程中胸腺T细胞亚群的变化与Notch 1、Presenilin 1、2（PS1、2）、HES1等Notch信号转导通路相关基因mRNA表达变化及这两种变化的关系。方法：选择出生后1—8周龄SAM，采用流式细胞技术检测胸腺细胞亚群变化、用荧光实时定量PCR的方法检测Notch1、PS1、PS2、HES1基因mRNA表达的动态变化。结果：SAM出生1-2周，胸腺中CD4^＋CD8^＋细胞所占比例较高，4周龄后逐渐降低，各周龄SAMP8与SAMR1之间无显著性差异；在CD4^＋CD8^＋。细胞比例上，SAM呈增龄性增加，1—6周龄的SAMP8与SAMR1之间无显著性差异，8周龄的SAMP8低于SAMR1；在CD4^＋CD8^＋细胞比例上，SAM呈增龄性增加，4周龄后的SAMP8均明显高于SAMR1；Notch1、PS2、HES1基因mRNA表达量呈增龄性增加，各周龄SAMP8的Notch1基因表达水平高于SAMR1，4周龄后SAMP8的PS2、HES1基因表达水平高于SAMR1；PS1基因mRNA表达量呈增龄性降低，4周龄后的SAMP8低于SAMR1。结论：Notch1、PS2、HES1的表达与胸腺CD4^＋CD8^＋细胞的分化呈正相关，PS1表达与胸腺CD4^＋CD8^＋细胞的分化呈负相关。     

大鼠大脑皮质神经前体细胞的分离培养及其增殖分化鉴定

目的:体外分离和培养大鼠大脑皮质神经前体细胞并进行增殖和分化鉴定。方法:分离2周龄大鼠皮质神经前体细胞,在含EGF、bFGF和GDNF的NSC培养基中进行体外培养,使用免疫细胞荧光染色技术对细胞的分化特性进行鉴定。结果:EGF、bFGF和GDNF可促进神经前体细胞的增殖及神经球的克隆形成,并获得了Nestin阳性的神经前体细胞,其可分化为分别表达β-Ⅲtubulin、GFAP和GalC的阳性细胞。结论:体外分离和培养的大脑皮质神经前体细胞,在含EGF、bFGF和GDNF的NSC培养基中培养,具有增殖分化的能力,有望应用于神经系统疾病的细胞移植治疗。     

大鼠Sertoli细胞与精原细胞的分离及共培养

目的 分离纯化14 d大鼠Sertoli细胞与精原细胞,用Sertoli细胞作为饲养层对大鼠精原细胞进行体外培养研究.方法 酶消化法制备大鼠睾丸组织单细胞悬液,采用牛血清白蛋白(BSA)不连续密度梯度沉降法分离Sertoli细胞和精原细胞.结果 经分离的Sertoli细胞与精原细胞的纯度分别达到92.73%和78.36%.Sertoli细胞与精原细胞共培养,可见精原细胞发生分裂增殖等行为.结论 在添加EGF、bFGF和GDNF等细胞因子的10%胎牛血清DMEM/F12培养基中,精原细胞能够存活一定时间;而Sertoli细胞表现为旺盛的生长状态,并可促进精原细胞的分裂与增殖.     

大鼠Hes1基因真核表达载体的构建及瞬时表达在神经前体细胞分化中的作用

目的 探讨Hes1基因高表达对神经前体细胞分化的影响. 方法从大鼠脑组织中提取总BNA,用RT-PCR方法获得Hes1基因的全长cDNA,插入pGEM-T-Easy克隆载体中进行序列测定,测序正确后将其亚克隆至表达载体pCDNA3.1,用脂质体将重组质粒转染原代培养的神经前体细胞,经G418筛选获得抗性细胞克隆,用RT-PCR方法鉴定Hes1基因在神经前体细胞基因组中的存在,实时荧光定量PCR检测Hes1基因在转染细胞中的过表达情况. 结果 经限制性内切酶酶切图谱分析和DNA序列测定,证实目的 基因已插入重组质粒,RT-PCR证明,经G418筛选得到的转基因神经前体细胞克隆的基因组DNA中存在Hes1基因;实时荧光定量PCR进一步证明,转基因神经前体细胞Hes1基因的mRNA高表达. 结论 构建了大鼠Hes1基因的真核表达载体,获得了高表达Hes1基因的神经前体细胞克隆.Hes1基因高表达可抑制神经前体细胞Ngn1的表达,并促进神经前体细胞向神经胶质细胞的分化.     

肝细胞生长因子促进人胚胎干细胞向神经前体细胞分化

目的:研究肝细胞生长因子(HGF)诱导人胚胎干细胞(hESCs)定向分化为神经前体细胞(NPs)的作用。方法:诱导拟胚体(EBs)生成,随机将EBs分为正常对照组、G5 supplement组、HGF组和HGF+G5 supple-ment组,悬浮培养诱导7d,转移至多聚赖氨酸/层黏连蛋白(20mg/L)包被的24孔培养板中继续培养7-10d。免疫荧光染色鉴定NPs和体外分化能力,流式细胞仪检测各组巢蛋白(nestin)阳性细胞的比例,RT-PCR检测音猥因子(Shh)对NPs的脑区标记基因表达的影响。结果:HGF+G5可诱导hESCs定向分化为NPs,HGF+G5组的nestin阳性的NPs比例(87.3%±3.9%)显著高于其它组(P0.05),NPs具有分化成神经元、少突和星形胶质细胞的能力;HGF+G5诱导时间对于NPs的分化有影响,7d时nestin+细胞比例达到最大;Shh可使NPs表达腹侧化基因,后脑标记表达上调,而前脑标记表达下调。结论:含HGF和G5的无血清神经分化体系可有效诱导hESCs神经分化,是研究神经诱导的良好体系。     

牙乳头细胞对大鼠牙髓干细胞中Notch信号的影响

目的探讨大鼠牙乳头细胞对牙髓干细胞增殖作用的影响过程中,相关牙齿发育信号通路的机制。方法原代培养大鼠牙髓干细胞和牙乳头细胞,建立牙髓干细胞和牙乳头细胞的分层共培养体系。共培养5d后,应用反转录聚合酶链反应(RT-PCR)和Western blot方法检测Notch信号通路中Notch2、Hes1 mRNA与蛋白的表达。结果 RT-PCR和Western blot结果显示牙髓干细胞和牙乳头细胞分层共培养组中Notch2、Hes1 mRNA和蛋白表达水平较单纯牙髓干细胞培养组显著增强(P<0.01)。结论牙乳头细胞促进大鼠牙髓干细胞增殖可能与牙乳头细胞促进牙髓干细胞中Notch信号分子的表达有关。     

细胞来源和培养液成分对大鼠神经前体细胞增殖和分化的影响

在成功建立体外分离培养大鼠胚胎脑和脊髓神经前体细胞(neuron precursor cells,NPCs)的基础上,本研究设计了三种培养液组合:DF/N2、DF/B27和DF/(N2+B27),观察在不同培养液成分对胚胎脑和脊髓NPCs增殖和分化的影响。结果显示:与NF/N2組和DF/B27组相比,脑来源的NPCs在DF/(N2+B27)中增殖最快、最稳定(P<0.01),而脊髓来源的NPCs在三种培养液组合中的增殖速度无明显差异。脑和胚胎15 d脊髓来源的NPCs在DF/B27和DF/(N2+B27)中分化为神经元的比例明显高于DF/N2组合(P<0.01);取自胚胎15 d的脊髓NPCs分化为神经元和少突胶质细胞的比例均显著高于胚胎16 d的NPCs(P<0.05)。以上结果提示:(1)在培养液中同时添加N2和B27不仅可以提高体外培养的NPCs的增殖速度,同时可显著增加神经元分化的比例;(2)NPCs的分化潜能可因NPCs来源(脑或脊髓)和发育阶段的不同而有差异。     

Sertoli细胞诱导大鼠胚胎中脑神经干细胞定向分化的研究

目的研究Sertoli细胞对胚胎中脑神经干细胞的营养及向多巴胺能神经元分化的诱导作用。方法将大鼠Sertoli细胞与胚胎13d大鼠中脑神经前体细胞体外共培养5、7、10、14d后,免疫荧光检测神经干细胞标记物Nestin、神经元前体细胞标记物β-Ⅲ tubulin、多巴胺能神经元标记物酪氨酸羟化酶(TH)的表达情况。结果随着共培养时间的延长,神经干细胞中nestin阳性细胞数量逐渐减少,β-Ⅲ tubulin阳性细胞数在共培养7d时较多,TH阳性的神经元在共培养10d时数量最多。而且在各时间段TH阳性细胞数均多于单独培养的神经前体细胞。结论Sertoli细胞能够诱导与其共培养的胚胎中脑神经干细胞定向分化为多巴胺能神经元。     

Olig2对胚胎干细胞向神经前体细胞分化过程中Nestin蛋白表达的影响

目的:探讨碱性螺旋-环-螺旋家族的少突胶质细胞转录因子2(Olig2)对小鼠胚胎干细胞(mESCs)向神经前体细胞(NPCs)分化过程中巢蛋白(Nestin)表达的影响。方法:将mESCs分为正常组、空载体组和Olig2转染组,用含有RA、Purmorphamine、bF GF和PDGF-AA的培养基将其诱导分化为NPCs;分别在诱导分化的第11 d和14 d,用免疫细胞化学荧光染色和蛋白免疫印迹法检测其神经前体细胞特异性标记物巢蛋白(Nestin)的表达情况。结果:经Olig2-GV218慢病毒转染后的mE SCs,Olig2蛋白的表达较正常组和空载体组明显增强;诱导分化后11 d,三组细胞Nestin蛋白表达无明显区别;14 d时,正常组和空载体组仍能检测到较高的Nestin蛋白表达,而Olig2转染组Nestin蛋白的表达明显减弱。结论:mESCs向NPCs分化过程,过表达Olig2可降低Nestin蛋白的表达。     

Gallium nitride induces neuronal differentiation markers in neural stem/precursor cells derived from rat cerebral cortex

In the present study, gallium nitride (GaN) was used as a substrate to culture neural stem/precursor cells (NSPCs), isolated from embryonic rat cerebral cortex, to examine the effect of GaN on the behavior of NSPCs in the presence of basic fibroblast growth factor (bFGF) in serum-free medium. Morphological studies showed that neurospheres maintained their initial shape and formed many long and thick processes with the fasciculate feature on GaN. Immunocytochemical characterization showed that GaN could induce the differentiation of NSPCs into neurons and astrocytes. Compared to poly-d-lysine (PDL), the most common substrate used for culturing neurons, there was considerable expression of synapsin I for differentiated neurons on GaN, suggesting GaN could induce the differentiation of NSPCs towards the mature differentiated neurons. Western blot analysis showed that the suppression of glycogen synthase kinase-3β (GSK-3β) activity was one of the effects of GaN-promoted NSPC differentiation into neurons. Finally, compared to PDL, GaN could significantly improve cell survival to reduce cell death after long-term culture. These results suggest that GaN potentially has a combination of electric characteristics suitable for developing neuron and/or NSPC chip systems.     

Favorable proliferation and differentiation capabilities of neural precursor cells derived from rat cochlear nucleus

NSCs/NPCs could be used for Sensorineural hearing loss treatment, because of the extensive capacity for self-renewal and pluripotency. In order to isolate and identify neural precursor cells (NPCs), we established a strategy to isolate and cultivate NPCs. Immunohistochemistry, immunofluorescence, Western blotting, and electron microscopy were used to characterize the cells and compare their differentiation patterns with those of olfactory bulb and olfactory epithelium NPCs. Furthermore, NPCs from the cochlear nucleus were sustained good cell viability and cloning efficiency after cryopreservation and thawing. Finally, high capacity to differentiate into astrocytes, oligodendrocytes, and neurons of NPCs was found. In conclusion, NPCs isolated from the cochlear nucleus can proliferate and differentiate into functional neurons, which offers a potential strategy for sensorineural hearing loss treatment. In addition, the storage method developed here will benefit further exploration of NPCs.     

Developmental potential of human spermatogenic cells co-cultured with Sertoli cells.

BACKGROUND: Development of an in-vitro culture system capable of supporting human early germ cell differentiation would be important for treatment of azoospermic patients. METHODS: Sertoli cells, spermatogonia and spermatocytes were isolated from testicular biopsies of 61 non-obstructive azoospermic patients, and co-cultured using Vero cell conditioned medium only or supplemented with recombinant (r)FSH or rFSH plus testosterone. Germ cell purity was checked by fluorescent in-situ hybridization (FISH) analysis. RESULTS: Best results were achieved with both hormones, which elicited 6.9% of meiosis index and 22.7% of differentiation into normal late spermatids after 2-3 weeks of culture. In-vitro matured spermatids were microinjected into oocytes to study their developmental potential. Round spermatids elicited 37.5% of fertilization and 28.6% blastocyst rates. Abnormal elongating and elongated spermatids enabled 8.3 and 27.3% fertilization rates respectively, but none achieved the blastocyst stage. Normal elongating and elongated spermatids elicited 30.5% fertilization and 42.9% of blastocyst rates. FISH analysis showed sex chromosome anomalies in all embryos, except in the case of morulae from normal late spermatids. CONCLUSIONS: Results suggest that meiosis and spermiogenesis can be resumed in vitro, with normal differentiated spermatids showing a low fertilization potential but regular rates of blastocyst formation. However, most of the embryos did not reach the morula stage and showed major sex chromosome abnormalities.     

从多潜能干细胞诱导成神经前体细胞及其分化探讨

目的探讨诱导多潜能干细胞在体外分化为神经前体细胞。方法采用N2B27作为选择培养基,多步法贴壁培养把诱导多潜能干细胞诱导为神经前体细胞。结果免疫荧光染色发现,该细胞神经干细胞的标记物巢蛋白阳性,有极个别的β-微管蛋白Ⅲ(β-Tub-Ⅲ)阳性细胞。在分化培养基内培养7d后,β-Tub-Ⅲ阳性细胞增多。结论本实验初步表明,诱导多潜能干细胞在体外可以被诱导为有一定神经特征的神经前体细胞,该细胞有望用于细胞移植治疗缺血性脑损伤。     

Discovery of two novel functional genes from differentiation of neural stem cells in the striatum of the fetal rat

Neural stem cells (NSC) are capable of differentiating into neurons and glia. However, the molecular mechanisms regulating NSC differentiation are not well understood. We have used the differential display polymerase chain reaction to analyze the differentially expressed genes of NSC from Sprague-Dawley rat striatum. Twelve differentially expressed sequence tags (ESTs) have been discovered and two of them, SHD10 and SHD11, were confirmed to be positive by reverse Northern blot techniques. Sequencing analyses showed that SHD10 shared a 94% (547/581) homology with mouse EST BI687817, but its biological function has not been reported. SHD11 shared a 91% (512/562) homology with mouse EST BG172336. It encodes an open reading frame containing 117 amino acids. Analysis of protein sequence indicated that it has a 98% homology with dendritic cell factor (gi18203393). Our research primarily discovered that these two genes are associated with differentiation of NSC. How they function in the process of differentiation needs further study.     

Amyloid precursor protein regulates differentiation of human neural stem cells

Although amyloid beta (Abeta) deposition has been a hallmark of Alzheimer's disease (AD), the absence of a phenotype in the beta amyloid precursor protein (APP) knockout mouse, tends to detract our attention away from the physiological functions of APP. Although much attention has been focused on the neurotoxicity of Abeta, many studies suggest the involvement of APP in neuroplasticity. We found that secreted amyloid precursor protein (sAPP) increased the differentiation of human neural stem cells (hNSCs) in vitro, while an antibody-recognizing APP dose-dependently inhibited these activities. With a high dose of sAPP treatment or wild-type APP gene transfection, hNSCs were differentiated into astrocytes rather than neurons. In vivo, hNSCs transplanted into APP-transgenic mouse brain exhibited glial differentiation rather than neural differentiation. Our results suggest that APP regulates neural stem cell biology in the adult brain, and that altered APP metabolism in Down syndrome or AD may have implications for the pathophysiology of these diseases.     

骨膜细胞与髓核细胞共培养向成骨方向的分化

背景：骨膜细胞已被应用于骨缺损及修复,但其可否在成骨诱导环境中与髓核细胞共培养向成骨分化,并应用于椎间隙骨性融合的相关研究报道较少。目的：分离培养髓核细胞与骨膜细胞,观察特定环境下髓核细胞的成骨潜能,以及加入骨膜细胞与其共培养后的成骨能力。方法：Ⅱ型胶原酶消化离心贴壁法分离兔髓核细胞;组织贴壁培养法分离兔骨膜细胞。采用细胞表面抗原CD90、CD105免疫荧光染色法鉴定骨膜细胞,甲苯胺蓝染色及Ⅱ型胶原免疫组织化学鉴定髓核细胞。实验分为两组：成骨诱导环境单独培养的髓核细胞为对照组,加入骨膜细胞与髓核细胞共培养为实验组。CCK8法检测细胞增殖,分别行碱性磷酸酶、茜素红染色、Ⅰ型胶原免疫组织化学染色进行成骨鉴定,Western blot检测两组细胞成骨诱导后骨桥蛋白的表达。结果与结论：骨膜细胞表面抗原CD90、CD105呈阳性,髓核细胞甲苯胺蓝染色及Ⅱ型胶原染色阳性;1,3,5,7,9 d各时间点实验组髓核细胞增殖活性显著高于对照组;诱导2周后2组细胞碱性磷酸酶染色、茜素红染色及Ⅰ型胶原免疫组织化学染色均为阳性,但实验组阳性表达高于对照组(P < 0.05);实验组骨桥蛋白表达强于对照组。髓核细胞具有成骨分化的潜能,但体外的增殖能力较低,加入骨膜细胞后,共培养细胞增殖分化能力提高。成骨诱导下骨膜细胞与髓核细胞共培养具有良好相容性,细胞相互黏附,并具有强于单独诱导髓核细胞的成骨能力。中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程