全反式维甲酸对小鼠胚胎不同阶段神经干细胞诱导作用研究

目的:探讨全反式维甲酸对小鼠胚胎不同阶段神经干细胞(NSCs)的诱导分化情况.方法:分离孕12.5d及15.5d的胚胎小鼠脑皮质.取第3代NSCs,用含1μmol·L-1的全反式维甲酸在体外诱导小鼠胚胎不同阶段的NSCs.诱导5d后,通过神经元微管相关蛋白2(MAP2)免疫荧光染色和Westernblots检测NSCs分化为神经元的比例.结果:与对照组相比,全反式维甲酸可明显提高神经元分化的比例.E12.5干细胞和E15.5胚胎干细胞分化为神经元的比例分别为30%±1.47%和16.21%±1.36%.结论全反式维甲酸具有显著的促神经干细胞分化成神经元的效应,并对胚胎不同阶段神经干细胞的诱导作用有所不同.     

人和小鼠神经干细胞的体外培养的分化研究

首次克隆了小鼠神经元标志性微管蛋白βⅢ基因，从核苷酸序列推导出小鼠与人两者之间在其羧基端有相同的EAQGPK六肽，进一步证实用抗人微管蛋白βⅢ单抗可检测小鼠神经干细胞分化成的神经元细胞，免疫组化鉴定显示小鼠神经干细胞在体积分数为1％胎牛血清（FBS）诱导下，可分化成神经元，星形胶质细胞，少突胶质细胞，同时培养了13周龄胎儿脑来源的人类神经干细胞，用特异性的抗人nestin抗体鉴定，全部为阳性细胞，但它们经诱导分化产生较不同寻常的细胞分化细胞和分化程度，在生长因子减半和1％FBS诱导条件下可分化为神经元和星形胶质细胞，而无少突胶质细胞分化，NF单抗检测证实为早期分化的神经元。     

人胚胎脑神经干细胞分离纯化及体外培养的研究

为了探讨人胚神经干细胞体外培养条件下的生物学特性,为其应用于临床治疗奠定基础.取胎龄16周的人流产胚胎,胰酶消化结合机械法分离成单细胞悬液,以2×106个细胞/mL接种到含hEGF和h-bFGF的DMEM/F12、N2培养基进行体外培养;观察细胞生长情况,用10% FBS诱导神经干细胞球分化,免疫细胞化学鉴定. 结果显示从人胚大脑分离出的细胞经悬浮培养可以形成细胞球,表达Nestin蛋白.经诱导分化后具有表达神经元,神经胶质细胞的特异性抗原. 说明人胚神经干细胞在体外可以稳定生长,并能分化成为神经原及胶质细胞.     

人骨髓间充质干细胞体外扩增和向神经细胞定向诱导分化的研究

骨髓间充质干细胞(MSC)是一类存在于骨髓中的具有多向分化潜能的干细胞,在体外不仅可以分化为间充质类细胞,而且可以分化为非间充质类细胞.研究了人骨髓间充质干细胞的体外分离、扩增和向神经细胞的定向诱导分化条件.从骨髓中分离MSC,用MesenCult培养基进行纯化和扩增培养.每扩增一代,细胞数量增加约2～3倍,在体外扩增12代后扩增约4.6×10 4 倍;诱导不同扩增代数的MSC向神经细胞分化,诱导后的细胞平均有80%以上呈现典型的神经元样表型.免疫组化法检测发现,神经元样细胞强表达神经丝蛋白和神经元特异性烯醇化酶,组织化学法检测观察到神经元特有结构尼氏体,表明MSC在体外具有向神经细胞分化的潜能.     

缺氧后小鼠脑匀浆提取液对儿童MSCs分化为神经细胞的影响

目的:观察缺氧后不同时段小鼠脑匀浆提取液对儿童骨髓间充质干细胞(MSCs)分化为神经元样细胞的影响,为筛选MSCs的最佳移植时间提供依据.方法:制作缺氧小鼠模型;并制取缺氧不同时间段脑匀浆提取液;诱导儿童MSCs;比较MSCs分化为神经元样细胞的阳性率.结果:缺氧后1、4,7、10、13 d小鼠脑匀浆提取液均可将MSCs诱导成神经元样细胞,经免疫学鉴定NES染色强阳性;但各组阳性率不同,以缺氧10 d后阳性率(21.02±3.38)%最高,与其他各组比较有统计学差异(P<0.05).结论:缺氧后小鼠脑匀浆提取液可诱导儿童MSCs分化为神经元样细胞,缺氧后10 d的分化率最高.     

人胚神经干细胞的体外培养

目的　探讨人胚神经干细胞体外培养的条件和分化情况 ,以摸索出一种切实可行的能获得较纯且多潜能人胚神经干细胞的方法 .方法　采用取 3月龄人胎脑 ,用胰蛋白酶消化法分离单个细胞 ,部分冻存 ,另一部分进行细胞培养 ,加EGF ,bFGF刺激生长 ,有限稀释法获得单细胞克隆 ,血清诱导分化 ,并用免疫组化方法进行鉴定 .结果　EGF和bFGF同时存在于无血清培养基中 ,有大量神经干细胞团生成 ,含血清培养基则诱导神经干细胞分化成为神经元、星型胶质细胞、少突胶质细胞 .结论　神经干细胞的存活和分裂有赖于EGF和bFGF的共同作用 .经冻存后的胎脑细胞同样能分离培养出有活性的神经干细胞 .     

纹状体神经干细胞的分离培养及其鉴定

胎脑内存在有能分化为神经元和神经胶质细胞的神经干细胞 .在表皮生长因子 (EGF)或碱性成纤维细胞生长因子 (b FGF)存在的条件下 ,从 14d胎鼠纹状体分离培养神经干细胞 ,并通过间接免疫荧光细胞化学法对其鉴定 ,发现大量呈 Nestin抗原阳性的干细胞团的形成 ,EGF和 b FGF对神经干细胞的增殖及分化能产生一定的影响 ,但它们对增殖及分化的影响是有差别的 .     

大鼠胚胎神经干细胞的培养和鉴定

目的　体外培养和鉴定神经干细胞 .方法　从胎鼠前脑取出脑组织 ,采用酶消化、机械吹打、对倍稀释成单个细胞 ,经无血清培养获得单细胞克隆 ,由免疫细胞化学方法鉴定分离的神经干细胞 .结果　从胎鼠脑中分离的细胞具有连续传代形成克隆的能力 ,表达神经干细胞蛋白 (nestin) ,并能诱导分化成神经元和神经胶质细胞 .结论　分离的细胞是神经干细胞 .     

三七总皂苷对新生大鼠海马神经干细胞活性影响及促分化作用

目的研究三七总皂苷对海马神经干细胞活性的影响和分化作用。方法体外培养海马神经干细胞,分别接种于96孔板和12孔板,96孔板细胞按三七总皂苷不同浓度梯度和同一浓度的不同时间点进行干预,应用MTT法检测海马神经干细胞的OD值,观察三七总皂苷对海马神经干细胞活性的影响;12细胞孔板分为对照组和给药组,应用免疫荧光染色方法检测神经元新生特异抗原（Tuj-1）和胶质细胞新生抗原（Vimentin）的表达,以观察三七总皂苷对海马神经干细胞分化的影响。结果（1）一定浓度范围内三七总皂苷能增强海马神经干细胞活性;（2）三七总皂苷能促进海马神经干细胞向神经元和胶质细胞方向分化。结论三七总皂苷能增强海马神经干细胞的活性并能促进海马神经干细胞分化。     

三七总皂苷对新生大鼠海马神经干细胞活性影响及促分化作用

目的研究三七总皂苷对海马神经干细胞活性的影响和分化作用。方法体外培养海马神经干细胞,分别接种于96孔板和12孔板,96孔板细胞按三七总皂苷不同浓度梯度和同一浓度的不同时间点进行干预,应用MTT法检测海马神经干细胞的OD值,观察三七总皂苷对海马神经干细胞活性的影响;12细胞孔板分为对照组和给药组,应用免疫荧光染色方法检测神经元新生特异抗原(Tuj-1)和胶质细胞新生抗原(Vimentin)的表达,以观察三七总皂苷对海马神经干细胞分化的影响。结果(1)一定浓度范围内三七总皂苷能增强海马神经干细胞活性;(2)三七总皂苷能促进海马神经干细胞向神经元和胶质细胞方向分化。结论三七总皂苷能增强海马神经干细胞的活性并能促进海马神经干细胞分化。     

大鼠胚胎神经干细胞体外培养及诱导分化

神经干细胞在理论研究和临床应用上有着广泛的前景.本文主要在体外分离培养SD大鼠胚胎前脑的神经干细胞,并分别用去除生长因子或添加全反式维甲酸(ATRA)两种方法诱导分化.免疫荧光染色技术分别检测细胞巢蛋白(Nestin)的表达及分化后β微管蛋白Ⅲ(β-Ⅲtubulin)、胶质纤维酸性蛋白(GFAP)的表达,计算分化率.结果显示:细胞生长状态良好,呈Nestin表达阳性.分化后可获得β-Ⅲtubulin及GFAP表达阳性的细胞,其中ATRA诱导方法获得β-Ⅲtubulin阳性细胞较多.     

丹酚酸B对骨髓间充质干细胞相关因子表达的影响

目的体外分离、培养和纯化大鼠骨髓间充质干细胞(MSCs),并研究中药单体丹酚酸B对MSCs血管内皮生长因子(VEGF)、干细胞因子(SCF)、心肌特异性转录因子NKX2.5和GATA-4表达的影响。方法分别用含0.03、0.3、3、30μg/mL丹酚酸B的低糖DMEM培养液培养MSCs,RT-PCR法检测培养24h后,观察VEGF、SCF、NKX2.5和GATA-4mRNA的表达。结果丹酚酸B可明显促进VEGF、NKX2.5和GATA-4mRNA的表达(与空白对照组比较,P<0.01,P<0.05)。结论丹酚酸B可促进MSCs的VEGF的分泌及NKX2.5、GATA-4的表达,为进一步深入探讨其诱导分化打下了坚实的基础。     

Advances in tissue engineering

Tissue engineering is a newly developed specialty involved in the construction of tissues and organs either in vitro or in vivo. Tremendous progress has been achieved over the past decade in tisse construction as well as in other related areas, such as bone marrow stromal cells, embryonic stem cells and tissue progenitor cells. In our laboratory, tissues of full-thickness skin, bone, cartilage and tendon have been successfully engineered, and the engineered tissues have repaired full-thickness skin wound, cranial bone defects, articular cartilage defects and tendon defects in animals. In basic research areas, bone marrow stromal cells have been induced and transformed into osteoblasts and chondrocytes in vitro. Mouse embryo stem cell lines we established have differentiated into neuron precursor, cardiac muscle cells and epithelial cells. Genetic modifications of seed cells for promoting cell proliferation, delaying cell aging and inducing immune tolerance have also been investigated.     

Derivation of androgenetic embryonic stem cells from m-carboxycinnamic acid bishydroxamide (CBHA) treated androgenetic embryos

The androgenetic embyronic stem (aES) cells are useful models in studying the effects of imprinted genes on pluripotency maintaining and embryo development. The expression patterns of imprinted genes are significantly different between uniparental derived aES cells and zygote-derived embryonic stem (ES) cells, therefore, the imprinting related cell pluripotency needs further exploitation. Several approaches have been applied in generation of androgenetic embryos and derivation of aES cell lines. Here, we describe a method to generate androgenetic embryos by injecting two mature sperms into one enucleated oocyte. Then these androgenetic embryos were treated with a histone deacetylase inhibitor: m-carboxycinnamic acid bishydroxamide (CBHA). Further, aES cell lines were successfully derived from these treated androgenetic embryos at blastocyst stage. The CBHA could improve not only the quality of androgenetic embryos, but also the efficiencies of aES (CaES) cells derivation and chimeric mice generation. The imprinted gene expression pattern in the CBHA treated embryo-derived aES (CaES) cells was also highly similar to that of zygote-derived ES cells.     

Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres

The most basic objection to human embryonic stem (ES) cell research is rooted in the fact that ES cell derivation deprives embryos of any further potential to develop into a complete human being. ES cell lines are conventionally isolated from the inner cell mass of blastocysts and, in a few instances, from cleavage stage embryos. So far, there have been no reports in the literature of stem cell lines derived using an approach that does not require embryo destruction. Here we report an alternative method of establishing ES cell lines-using a technique of single-cell embryo biopsy similar to that used in pre-implantation genetic diagnosis of genetic defects-that does not interfere with the developmental potential of embryos. Five putative ES and seven trophoblast stem (TS) cell lines were produced from single blastomeres, which maintained normal karyotype and markers of pluripotency or TS cells for up to more than 50 passages. The ES cells differentiated into derivatives of all three germ layers in vitro and in teratomas, and showed germ line transmission. Single-blastomere-biopsied embryos developed to term without a reduction in their developmental capacity. The ability to generate human ES cells without the destruction of ex utero embryos would reduce or eliminate the ethical concerns of many.     

Simultaneous detection of seven phenolic acids in Danshen injection using HPLC with ultraviolet detector

A high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) detector had been developed for simultaneous quantification of danshensu, protocatechuic aldehyde, caffeic acid, salvianolic acid D, rosmarinic acid, salvianolic acid B and salvianolic acid A in Danshen injection. According to the UV spectra of these components, three detection wavelengths have been selected as follows: 280 nm for danshensu and protocatechuic aldehyde, 326 nm for caffeic acid, salvianolic acid D and rosmarinic acid, 286 nm for salvianolic acid B and salvianolic acid A. The limit of detection (LOD) was improved to be in the range of 0.008~0.160 μg/ml. Moreover, excellent linear behavior over the investigated concentration range was observed, with R>0.999 for all the analytes.     

Advances in stem cell research

In.1998,biologists Thomson and Gearhart successfully derived stem cells from human embryos. One year later,several researchers discovered that adult stem cells still retain the ability to be differentiated into unrelated types of cells. Advances in stem cell research open a promising derection for applied medical science. Moreover,it may also force scientists to reconsider the fundamental theory about how cells grow up. Stem cell research was considered by Science as the top of the ten breakthroughs of science of the year^[1]. This paper gives a survey of recent advances in stem cell research.     

NGF诱导PC12细胞神经元样细胞分化

目的:建立PC12细胞的神经元样细胞分化模型,并探讨ERK蛋白在PC12细胞神经元样细胞分化中的可能机制.方法:以10、20、50 g/L的NGF(NGF溶于PBS,培养基中PBS的终浓度不超过2%)培养PC12细胞,应用倒置相差显微镜、显微镜测微尺及流式细胞仪鉴定PC12细胞的分化,以确定NGF使用剂量.运用免疫印迹检测不同浓度、不同作用时间时ERK蛋白在PC12细胞中的表达,并进行统计学分析.结果:随NGF剂量的升高,PC12细胞的体积、最长突起长度和突起数目均会增大或增多。统计学分析证明50 g/L的NFG作用48 h足以诱导PC12细胞的交感神经样改变。尽管ERK总蛋白水平在NFG作用前后无明显改变,但在NFG作用5 min后磷酸化的ERK蛋白水平即显著升高,达到峰值,并持续约1 h.50 g/L的NFG作用于PC12细胞48 h可使细胞发生明显的G1期阻滞.结论:PC12细胞可在NGF作用下出现交感神经样改变,并且细胞的分化程度依赖于NGF的使用剂量和作用时间.在NGF诱导的PC12细胞神经元样分化中ERK蛋白的磷酸化对NGF呈现剂量和时间依赖性,提示ERK蛋白在分化的早期发挥重要作用.     

丹酚酸B的电化学行为及其测定

研究了丹酚酸B在玻碳电极上的电化学行为,发现丹酚酸B在裸玻碳电极上有一对可逆性较好的氧化还原峰.在最佳实验条件下,氧化峰电流与丹酚酸B的浓度在1.95×10^-7～3.90×10^-6mol/L范围内呈良好的线性关系,相关系数为0.9950,检出限为1.30×10^-7mol/L（信号与噪声比S/N=3）.配10份相同浓度溶液,连续测定,平均峰电流为6.96×10^-6A,所得结果的相对标准偏差（RSD）为3.2%,表明测定结果重现性良好.将该方法用于复方丹参片中丹酚酸B的含量测定,所得平均回收率为101.1%.该方法简单、快速,所需仪器简单,样品不需预处理,可用于实际样品中丹酚酸B的测定.