昆明小鼠胚胎干细胞的体外分离培养

利用昆明小鼠13.5dpc的胚胎制备小鼠胚胎成纤维细胞,并以小鼠胚胎成纤维细胞作为饲养层;收集3.5dICR小鼠的囊胚和桑椹胚进行体外培养,筛选纯化ES细胞集落,使其稳定传代后,对其形态学和生物学性状进行初步鉴定,获得阳性细胞集落。     

胚胎干细胞的分离与培养

如何分离胚胎干细胞，并在体外进行分化抑制培养，仍是探索性很强的研究课题，本文简述了胚胎干细胞分离培养方法的主要进展与趋势。     

神经干细胞分离培养方法的介绍

在成体的许多组织中发现了多能干细胞,这些干细胞可以进行自我复制,参与组织的正常修复。神经干细胞在体外能分化为神经元、星形胶质细胞和少突胶质细胞,并具有多向分化潜能。成体神经干细胞和胚胎干细胞都能分化成成体神经系统中的各种神经细胞。神经干细胞具有自我更新能力,因此神经干细胞可以应用于神经损伤或者神经疾病的修复。本文概述了神经干细胞体外分离培养的方法及其生长影响因子。     

昆明白小鼠胚胎干细胞分离与体外培养

为探索昆明白小鼠胚胎干细胞建系方法,将受孕4.5天的昆明白小鼠囊胚用免疫手术法去除滋胚层,然后将内细胞团(ICM)接种于胎鼠成纤维细胞饲养层上培养,形成的胚胎干细胞样集落用胰蛋白酶-EDTA消化法传代,培养后进行相差显微镜观察及碱性磷酸酶染色。结果饲养层上生长的ICM细胞呈典型的ES样细胞集落,传至第8代碱性磷酸酶染色呈强阳性。实验表明免疫手术法适用于昆明白小鼠ES细胞建系,获得的细胞集落具有ES细胞的主要生物学性状。     

小鼠卵巢生殖干细胞分离培养鉴定

通过比较不同酶分离方法获得小鼠（Mus musculus）卵巢生殖干细胞的数量以及不同饲养层培养卵巢生殖干细胞的生长状态,筛选出最佳酶分离方法和饲养层,以期建立更优化的培养体系,并对优化培养体系长期培养的卵巢生殖干细胞进行功能鉴定。选取5日龄C57BL/6雌性乳鼠卵巢,机械法制备卵巢组织碎片后,分别采用传统两步酶法（胶原酶和胰蛋白酶）和改良两步酶法（胶原酶、DNA酶、透明质酸酶、分离酶Ⅱ和胰蛋白酶）分离卵巢生殖干细胞,差速贴壁法纯化后,观察不同酶消化方法对卵巢生殖干细胞数量的影响;将分离得到的卵巢生殖干细胞分别培养于小鼠胚胎成纤维细胞STO系[sandosinbred mice(SIM)embryo-derived thioguanine and ouabain-resistant]、多聚赖氨酸以及明胶3种不同的饲养层,观察卵巢生殖干细胞的生长增殖状态;将传至第6代的卵巢生殖干细胞用碱性磷酸酶检测法和免疫荧光法对卵巢生殖干细胞标志物MVH、Oct4、C-kit和增殖标记物Brdu进行鉴定。结果发现两种酶分离方法均可从卵巢中分离得到卵巢生殖干细胞,其中传统两步酶法获得的细胞经差速贴壁法纯...     

小鼠胚胎神经干细胞的分离培养及其鉴定

且的探索小鼠胚胎神经干细胞的体外培养方法,并获取高纯度的神经干细胞,为神经干细胞的深入研究提供实验材料。方法无菌条件下分离E15天小鼠胚脑皮质,制成单细胞悬液,在bFGF和B27存在的培养基中培养扩增,通过免疫细胞化学染色鉴定神经干细胞及其子代细胞的分化方向。结果培养的部分细胞在B27和bFGF存在的无血清培养基中可以在体外分裂增殖,同时表达神经干细胞特异性抗原nestin,并在撤出B27和bFGF的有血清培养基中向神经细胞和胶质细胞分化。结论小鼠胚脑皮质存在具有多向分化潜能的神经干细胞,这些细胞可以在体外稳定培养、传代并自然分化,为细胞替代治疗提供了理想的细胞来源。     

小鼠表皮干细胞的分离与培养

目的:探讨体外分离和培养小鼠表皮干细胞和分析表皮干细胞克隆形成能力的方法。方法:采用中性蛋白酶和胰酶消化新生小鼠表皮基底层细胞,将细胞直接接种在细胞瓶中,在无滋养层条件下培育;利用表皮干细胞标记物K15和α6整联蛋白进行免疫荧光鉴定;以小鼠胚胎成纤维细胞作为滋养层与成年小鼠角质细胞共培养,进而分析表皮干细胞的克隆形成能力。结果:新生小鼠表皮干细胞克隆在培养2～3 d后开始形成,细胞核质较小,细胞呈小而圆的形态特征;传代后的细胞可以被K15和α6整联蛋白特异性标记。结论:利用该方法能够实现对小鼠表皮干细胞的体外培养和传代。     

小鼠胚胎干细胞的培养

目的:建立小鼠胚胎干细胞(embryonic stem cells,ES)的培养方法。方法:制备G418抗性的原代小鼠胚胎成纤维细胞,经丝裂霉素C处理后成滋养层细胞,将小鼠胚胎干细胞复苏后,应用含白血病抑制因子的ES细胞培养液,培养小鼠ES细胞,观察集落的生长情况,并在光镜下观察细胞形态。结果:小鼠胚胎成纤维细胞生长良好,ES细胞呈克隆状生长,且保持未分化状态。结论:建立了小鼠胚胎干细胞培养的有效方法,为下一步基因打靶奠定基础。     

小鼠类胚胎干细胞分离培养及用于制作嵌合体小鼠的实验研究

目的探讨分离小鼠囊胚内细胞群类胚胎干细胞及用于制作嵌合体小鼠的方法及应用价值。方法分离3.5d小鼠囊胚内细胞群的类胚胎干细胞作为供体细胞,通过显微注射方法将分离的类胚胎干细胞注射到供体小鼠的囊胚腔中,再将注射后的囊胚移植到假孕雌鼠的子宫中制作嵌合体小鼠。结果分离36枚囊胚的内细胞群类胚胎干细胞,注射256只昆明小鼠囊胚中,移植32只假孕雌鼠子宫中,获产崽2窝,共12只,其中2只获毛色嵌合体小鼠。结论采用该技术分离所获得的类胚胎干细胞作为供体细胞制作嵌合体小鼠获得成功,该方法为ES细胞介导的转基因动物制作增添了一条新的途径,在同种不同品系的动物改良及遗传病基因治疗中有一定的应用价值,尤其是对未能建立ES细胞系的大动物的遗传工程操作具有一定意义。     

小鼠精原干细胞冻存后体外培养

目的:研究冷冻后精原干细胞体外培养时的生物学行为.方法:体外培养冻存后的6日龄小鼠生精上皮细胞,并利用碱性磷酸酶活性及细胞形态,检测其中的精原干细胞.结果:当有BRL饲养层时,冻存后的精原细胞在贴壁、存活及增殖等生物学行为方面与新分离的精原细胞均无明显不同.培养25～30 d,培养体系中仍保留有少量精原干细胞及其最初几代分化细胞.结论:冷冻保存后的精原干细胞能在BRL细胞饲养层上正常地贴壁、生长和分裂.     

SD大鼠和Beagle犬大唾液腺的形态学观察

目的-研究及观察SD大鼠和Beagle犬大唾液腺正常比较组织学.方法-SD大鼠和Beagle犬三对大唾液腺剖取后进行石蜡切片、HE染色和PAS染色,光学显微镜观察.结果-SD大鼠腮腺是纯浆液腺,Beagle犬腮腺属混合腺,以浆液性腺泡为主,偶见小的粘液细胞群.SD大鼠的下颌下腺属于以浆液腺泡为主的混合腺,Beagle犬的下颌下腺属于以粘液腺泡为主的混合腺.SD大鼠与Beagle犬的舌下腺均为粘液性腺泡为主的混合腺.Beagle犬的眶腺亦是以纯粘液性腺泡为主的混合腺结构.     

肿瘤干细胞分离研究进展

癌症是严重威胁人类生命健康的疾病之一,传统治疗方法未能彻底根除癌症。究其原因是癌症的发病机制复杂,至今尚未完全认识。肿瘤干细胞理论的提出为肿瘤的发生、发展乃至术后复发提供了新的研究思路,同时也为癌症的治疗带来了新的曙光。但由于肿瘤干细胞含量极低、分离困难,给肿瘤干细胞的研究带来诸多不便。目前常用的分离方法为无血清培养法与流式分选法,但二者均存在一定的缺陷,因此亟须建立一种新型有效地分离方法。肿瘤干细胞与普通干细胞有许多相似的信号通路,因此可利用干细胞的微环境筛选分离分化程度较低的肿瘤干细胞。水凝胶由于其独特的性质,目前广泛用于细胞的三维培养。有鉴于此,科研工作者利用水凝胶模拟普通干细胞生长的力学环境筛选、富集肿瘤干细胞。本文就肿瘤干细胞目前常用分离及鉴定方法,同时结合国内外最新的肿瘤干细胞分离方法进行综述。     

Coexpression of Vimentin, Cytokeratin and S-100 In Monomorphic Adenoma of Salivary Gland; Value of Marker Studies In the Differential Diagnosis of Salivary Gland Tumours

An unusual coexpression of glial fibrillary acid protein (GFAP), keratin and vimentin occurs in pleomorphic adenoma of salivary gland. We designed this study to see if coexpression of the markers was also present in monomorphic adenoma of the salivary gland and whether monomorphic adenoma could be distinguished from other salivary gland tumours by marker studies. Immunocytochemical markers were used on fine needle aspiration samples from four cases of monomorphic adenoma, two cases of oncocytic adenoma, three cases of adenoid cystic carcinoma and four cases of pleomorphic adenoma. While positivity for cytokeratin, vimentin and S-100 was consistently found in all cases of monomorphic adenoma, only cytokeratin was expressed in adenoid cystic carcinoma. In pleomorphic adenoma, GFAP, cytokeratin and vimentin were coexpressed while in cases of oncocytic adenoma none of the markers was localized. Thus, it appears that by using a combination of GFAP, cytokeratin, vimentin and S-100 a distinction between these neoplasms may be possible. However, a larger study is needed to establish the usefulness of this approach.     

实验室果蝇(Drosophil melanogaster)唾腺染色体制备前期技术优化

经过多年的实践积累,从果蝇(Drosophil melanogaster)品系的选择、培养基的配制和果蝇的饲养等方面摸索出了一套改进的制备果蝇唾腺染色体的经验方法。     

miR-26a修饰的人牙周膜干细胞成骨分化能力的研究

目的:研究人牙周膜干细胞(hPDLSCs)在转染miR-26a后成骨分化的促进效果。方法:从因正畸拔除的无龋坏、无牙周疾病离体牙的牙根中部牙周膜组织中分离、胶原酶消化,进行人牙周膜干细胞的体外培养。使用脂质体2000进行miRNA转染,采用激光共聚焦观察转染效果;MTT方法检测转染后的细胞活力;成骨诱导后使用实时定量PCR技术检测miRNA修饰的hPDLSCs成骨分化相关基因的表达;采用BCIP/NBT、天狼星红、茜素红S分别对碱性磷酸酶、胶原以及钙化进行染色观察。结果:采用脂质体2000能够成功转染hPDLSCs,且转染效率较高;转染后的细胞活力有所下降,但仍在80%以上;转染后的细胞在经成骨诱导分化过程中骨钙素(OCN)和骨桥蛋白(OPN)基因表达显著上调,同时碱性磷酸酶活性、胶原分泌以及钙化能力均得到显著提升。结论:miR-26a可以用于修饰hPDLSCs以提高其成骨分化能力。     

小鼠骨髓间充质干细胞的分离、培养及鉴定

目的建立一种从小鼠骨髓中分离培养间充质干细胞（MSCs）的高效方法。方法采取贴壁细胞分离法分离和纯化小鼠骨髓间充质干细胞（mMSCs）,检测mMSCs在不同诱导条件下向成骨细胞及脂肪细胞分化能力,用流式细胞术及显微镜分别检测mMSCs纯度和形态特征。结果mMSCs贴壁生长后形态较均一,细胞形态呈成纤维细胞样,流式细胞术检测：CD45、CD11b、CD44及CD29分别为（3.34）%、（2.41）%、（98.46）%及（99.36）%。第4代mMSCs经诱导后可向成骨细胞和脂肪细胞分化。结论通过贴壁培养可以从小鼠骨髓中分离培养出高纯度mMSCs,该方法效率高,稳定性好。     

BMP7对小鼠诱导多能干细胞骨向分化作用的研究

目的:探讨骨形态蛋白(bone morphogenetic protein,BMP)超家族成员之一BMP7在小鼠诱导多能干细胞(Induced pluripotent stem cells,iPS)骨向分化过程中的作用。方法:本试验分成三组,分别是自发分化组,骨诱导组和添加BMP7的骨诱导组。每天观察各组细胞形态学特征及生长状况的差异,在诱导第14天通过茜素红染色检测基质矿化情况,判断BMP7在体外骨诱导条件下对小鼠iPS细胞骨向分化过程所发挥的作用。结果:完成了小鼠iPS细胞的培养鉴定,并诱导形成理想状态的拟胚体(Embryoid body,EB)用于分化接种。结果发现,添加BMP7的骨诱导组细胞的矿化结节阳性率明显增加。结论:BMP7在诱导小鼠iPS细胞骨向分化过程中起促进作用,而对非骨向分化的细胞无成骨促进作用。     

Advanced Glycation End Products Increase Salivary Gland Hypofunction in d-Galactose-Induced Aging Rats and Its Prevention by Physical Exercise

A declined salivary gland function is commonly observed in elderly people. Advanced glycation end products (AGEs) are believed to contribute to the pathogenesis of aging. Although physical exercise is shown to increase various organ functions in human and experimental models, it is not known whether it has a similar effect in the salivary glands. In the present study, we evaluated the AGEs burden in the salivary gland in the aging process and the protective effect of physical exercise on age-related salivary hypofunction. To accelerate the aging process, rats were peritoneally injected with D-galactose for 6 weeks. Young control rats and d-galactose-induced aging rats in the old group were not exercised. The rats in the physical exercise group ran on a treadmill (12 m/min, 60 min/day, 3 days/week for 6 weeks). The results showed that the salivary flow rate and total protein levels in the saliva of the d-galactose-induced aging rats were reduced compared to those of the young control rats. Circulating AGEs in serum and secreted AGEs in saliva increased with d-galactose-induced aging. AGEs also accumulated in the salivary glands of these aging rats. The salivary gland of aging rats showed increased reactive oxygen species (ROS) generation, loss of acinar cells, and apoptosis compared to young control mice. However, physical exercise suppressed all of these age-related salivary changes. Overall, physical exercise could provide a beneficial option for age-related salivary hypofunction.     

Separation of Monomerizing and Lysozyme Activities of Destabilase from Medicinal Leech Salivary Gland Secretion

Destabilase, endo--(-Glu)-Lys-isopeptidase, was prepared from the salivary gland secretion of the medicinal leech (Hirudo medicinalis). The secretion prepared by the known method of Rigbi et al. (1987) (secretion-K) lacks the destabilase-characteristic highly specific isopeptidase activity (the D-dimer-monomerizing activity) because of its degradation by proteolytic activity (the substrate of Glp-Ala-Ala-Leu-pNA) due to contamination with leech intestinal channel contents. Therefore, we have elaborated a new technique for preparation of a true leech secretion (secretion-I). This secretion is characterized by the complete absence of the leech intestinal channel contents and has no proteolytic activity. For the first time the destabilase-specific D-dimer-monomerizing and lysozyme activities were separated by fractionation of secretion-I by HPLC gel filtration through Superose S-12. For the purified destabilase preparation, these activities were separated by reversed-phase chromatography in an acetonitrile gradient (0-60%) in the presence of 0.1% trifluoroacetic acid. The monomerizing activity of destabilase is responsible for the ability of secretion-I to dissolve stabilized fibrin via isopeptidolysis of - and - fibrin chains bound by -(-Glu)-Lys-isopeptide bonds.     

Protein Profiling of the Medicinal Leech Salivary Gland Secretion by Proteomic Analytical Methods

Protein diversity of the high molecular weight fraction (molecular mass > 500 daltons) of salivary grand secretion of the medicinal leech Hirudo medicinalis has been demonstrated using methods of proteomic analysis. One-dimensional (1D) electrophoresis revealed the presence of more than 60 bands corresponding to molecular masses ranging from 11 to 483 kD. 2D-electrophoresis revealed more than 100 specific protein spots differing in molecular masses and pI values. SELDI-mass spectrometry analysis using the ProteinChip. System based on chromatography surfaces of strong anion or weak cation exchanger detected 45 individual compounds of molecular masses ranged from 1.964 to 66.5 kD. Comparison of SELDI-MS data with protein databases revealed eight known proteins from the medicinal leech. Other masses detected by proteomic analytical methods may be related to both modifications of known proteins and unknown biologically active components of leech saliva secretion.