人胚胎肝上皮样细胞的体外培养

用胶原酶直接消化人胚肝组织小块,可以简便、快速地获得大量活率较高和较纯的肝上皮样细胞。在低钙、低血清浓度加有多种因素的培养液中,胚肝上皮样细胞可以体外培养一月以上。原代培养的胚肝上皮样细胞一次传代后,仍可保持一定的生长能力。表皮生长因子、霍乱毒素、转铁蛋白及肝细胞生长因子等均对培养的胚肝上皮样细胞具有一定的生长刺激作用。本文还用r-GT染色、AFP和白蛋白测定,以及~3H-TdR掺入等指标观察了培养过程中胚肝上皮样细胞的生长、增殖和生物学功能。人胚肝上皮样细胞的较长期体外培养,可望成为研究肝细胞生长、分化及癌变的一个有用的实验模型。     

基于小分子筛选的嗅上皮类器官培养体系的建立

嗅上皮接收和传导气味信号是嗅觉系统的重要组成部分。嗅上皮的损伤在通常情况下可自发恢复,但特定疾病或衰老造成的嗅上皮损伤会引起嗅觉功能减退和嗅觉障碍。嗅上皮主要由基底细胞、支持细胞以及嗅感觉神经元组成。为了在体外建立包含多种细胞类型的嗅上皮类器官,本研究采用3D细胞培养技术,通过筛选小分子药物,构建了包含多种细胞类型的嗅上皮类器官模型,包含水平基底样细胞、球形基底样细胞、支持样细胞和嗅感觉神经元样细胞多种细胞类型。类器官培养体系中多种生长因子和小分子化合物在细胞增殖速度、细胞组成以及不同细胞类型标志基因的表达水平等方面对类器官产生影响。Wnt信号通路激活剂CHIR-99021能够提高嗅上皮类器官的成克隆率和增殖速度且有利于提高嗅上皮类器官中嗅感觉神经元样细胞标志基因的表达水平;培养体系的任一因子均能提高类器官中cKit阳性的球形基底样细胞克隆比例;表皮生长因子(epidermal growth factor,EGF)和维生素C均有利于类器官中水平基底样细胞标志基因的表达。本研究建立的嗅上皮类器官系统模拟了嗅上皮干细胞分化产生多种嗅上皮细胞类型的过程,为研究嗅上皮组织损伤再生、嗅觉障碍病理...     

胰腺导管单克隆上皮样干细胞的多分化潜能

体外诱导源于成年大鼠的胰腺导管单克隆上皮样干细胞分化形成胰岛、神经、脂肪及成骨细胞,探讨干细胞的多分化潜能。扩增培养源于成年大鼠的胰腺导管单克隆上皮样干细胞,采用不同的诱导液体外诱导其向胰岛、神经、脂肪及成骨细胞分化,并通过DTZ染色、糖刺激试验、免疫荧光反应、油红O染色、茜素红染色或Vonkossa染色的方法对分化细胞进行检测。结果显示,体外诱导培养干细胞分化形成类胰岛,DTZ染色阳性,糖刺激分泌胰岛素、C-肽;分化形成类神经细胞,表达神经元特异性烯醇化酶;分化形成类脂肪细胞,油红O染色阳性;分化形成类成骨细胞,其分泌物呈岛状矿化结节,茜素红和Vonkossa染色阳性。这表明,该源于成年大鼠的胰腺导管上皮样干细胞系具有多分化潜能。     

肾上腺皮质细胞的单层培养方法

目的: 体外单层培养大鼠肾上腺皮质细胞,以便为肾上腺皮质细胞的有关实验提供必要条件.方法: 采用胶原酶Ⅱ、透明质酸酶消化等步骤分离Wistar大鼠的肾上腺皮质细胞;分离的细胞于含15% NBS的DMEM培养基、37℃、5% CO2的环境中培养,3β-羟基类固醇脱氢酶特异染色鉴定.结果: 培养的肾上腺皮质细胞呈梭形,胞体较大、有少量突起,3β-羟基类固醇脱氢酶特异染色后胞体显著着蓝色.肾上腺皮质细胞不呈典型的"S"形增殖趋势,适于作原代培养.上皮样细胞和成纤维样细胞同时存在.结论: 通过细胞形态观察及特异酶染色鉴定,本文培养的细胞为大鼠肾上腺皮质细胞.     

诱导胚胎干细胞向角膜上皮细胞分化的实验研究

探索胚胎干细胞在表层角膜缘基质诱导下向角膜上皮细胞分化的可能性.体外培养带GFP标记的ES-D3细胞,并利用视黄酸进行预诱导,然后将预诱导后的细胞接种在表层角膜缘基质上,细胞融合形成单层后,随机分为3组进行研究:第1组传代后直接进行检测;第2组在气-液界面上培养10天,然后植入裸鼠皮下以进行体内诱导;第3组作为对照组,不给予GFP-ES-D3细胞特殊诱导条件,细胞自由分化.诱导分化的细胞植入裸鼠皮下体2周后没有畸胎瘤形成.诱导分化的细胞呈现上皮样外观,体内诱导组和体外诱导组免疫组织化学染色均检测到CK3,P63和PCNA表达阳性,电子显微镜检查可见两组细胞表面都有微绒毛和细胞间紧密连接形成.实验对照组部分细胞脱落和死亡,大部分表现神经样细胞的树突样外观,小部分未死亡的贴壁细胞呈多态性,这些结果表明胚胎干细胞在特定条件下经表层角膜基质诱导能够分化为角膜上皮细胞.胚胎干细胞诱导分化有可能为眼表重建和组织工程化角膜的构建提供上皮种子细胞.     

诱导胚胎干细胞向角膜上皮细胞分化的实验研究

探索胚胎干细胞在表层角膜缘基质诱导下向角膜上皮细胞分化的可能性. 体外培养带GFP标记的 ES-D3细胞, 并利用视黄酸进行预诱导, 然后将预诱导后的细胞接种在表层角膜缘基质上, 细胞融合形成单层后, 随机分为3组进行研究: 第1组传代后直接进行检测; 第2组在 气-液界面上培养10天, 然后植入裸鼠皮下以进行体内诱导; 第3组作为对照组, 不给予GFP-ES-D3细胞特殊诱导条件, 细胞自由分化. 诱导分化的细胞植入裸鼠皮下体2周后没有畸胎瘤形成. 诱导分化的细胞呈现上皮样外观, 体内诱导组和体外诱导组免疫组织化学染色均检测到CK3, P63和PCNA表达阳性, 电子显微镜检查可见两组细胞表面都有微绒毛和细胞间紧密连接形成. 实验对照组部分细胞脱落和死亡, 大部分表现神经样细胞的树突样外观, 小部分未死亡的贴壁细胞呈多态性, 这些结果表明胚胎干细胞在特定条件下经表层角膜基质诱导能够分化为角膜上皮细胞. 胚胎干细胞诱导分化有可能为眼表重建和组织工程化角膜的构建提供上皮种子细胞.     

胚胎干细胞体外分化心肌细胞的研究

胚胎干细胞是从动物胚胎内细胞团分离的具有全能性的细胞.研究证明分离的小鼠胚胎干细胞在体外可以分化成心肌细胞,这一发现为小鼠胚胎干细胞向各种特化心脏细胞(动脉样细胞、血管样细胞、窦样细胞、心室样细胞、蒲肯野氏样细胞)分化提供了依据,使基因功能的研究在体外成为可能.1998年末人类ES细胞的成功培养奠定了心脏细胞的再生性治疗的战略基础.主要综述了目前ES细胞体外分化心肌细胞的进程,讨论了此进程对心脏基因研究的促进作用.     

牦牛乳腺上皮细胞系的建立与生物学特性

本研究旨在建立牦牛乳腺上皮细胞体外培养体系。采用胶原酶消化法成功地建立了牦牛乳腺上皮细胞系(YMEC),通过免疫细胞化学、超微结构观察和RT-PCR 法对YMEC 细胞进行了鉴定,并研究了其形态、活力、生长曲线以及核型等生物学特性。结果表明,YMEC 细胞染色体2n = 60,群体倍增时间为45 ～ 48 h,持续培养25 代后出现细胞分化;细胞呈典型的“铺路石样”形态,其表面有丰富的微绒毛,细胞质内含丰富的线粒体和粗面内质网。污染检测结果为阴性。在激素诱导培养时,检测到了β - 酪蛋白mRNA 的表达。表明本研究成功建立了保留泌乳功能的牦牛乳腺上皮细胞系,为研究牦牛乳腺上皮细胞的功能提供了理想的工具。     

神经上皮干细胞的分离培养及其体外分化特性的观察

目的探讨大鼠胚胎神经管神经上皮干细胞的分离培养条件,并观察其在体外的分化特性.方法采用显微解剖、机械吹打、无血清悬浮培养方法分离培养神经上皮干细胞,采用巢蛋白(nestin)免疫细胞化学染色技术检测神经上皮干细胞,用NSE和GFAP免疫组化染色检测并计数神经细胞和神经胶质细胞.结果大鼠胚胎神经管神经上皮干细胞在无血清培养基中可形成大量呈nestin抗原阳性细胞构成的神经球,经传代有血清培养后分化为NSE阳性和GFAP阳性细胞,其中NSE阳性细胞占细胞总数的47.7%,GFAP阳性细胞占细胞总数的39.8%.结论胎鼠神经管神经上皮干细胞在无血清培养中可增殖和传代,在有血清培养中可分化为神经细胞和神经胶质细胞,两者之比为47.7∶39.8.     

改良的高纯度人早孕绒毛膜滋养层细胞培养方法

目的培养纯度较高的人早孕绒毛膜滋养层细胞,为研究胎盘绒毛在妊娠期间的作用及其机制提供细胞学基础。方法胰蛋白酶-DNA酶消化法分离培养绒毛膜滋养层细胞,再运用流式细胞仪细胞分选获得高表达HLA-G的人早孕绒毛膜滋养层细胞。流式细胞术检测分选前后原代培养细胞HLA-G的表达率,相差显微镜观察滋养层细胞形态学特点,免疫荧光显微镜鉴定细胞来源,台盼蓝染色检测细胞活力。结果通过流式细胞检测,分选前的原代培养细胞体系中HLA-G的表达率为86.5%,经过分选带有PE荧光/HLA-G阳性表达的原代培养细胞后,其纯度可达98.0%。倒置相差显微镜下,可见细胞为上皮样细胞形态,呈片状铺展生长。细胞角蛋白染色阳性,波形蛋白染色阴性,表明细胞性质为上皮来源的绒毛膜滋养层细胞。台盼兰排斥试验检测细胞活力,细胞活力良好,存活率超过92%。结论该方法可以有效获得高纯度的,具有生物学活性的人早孕绒毛膜层细胞,为在体外研究生理妊娠及病理妊娠中滋养细胞的作用提供了一种改良的技术手段。     

大鼠颌下腺及其离体培养细胞脱氢表雄酮（DHEA）的免疫组织化学定位

用免疫组织化学ABC法,研究了颌下腺及无血清培养的颌下腺上皮细胞DHEA的定位,结果显示,大鼠颌下腺的浆液性腺泡的上皮细胞及各级导管上皮细胞均呈DHEA免疫反应阳性,无血清培养腺上皮细胞也呈DHEA免疫反应阳性,阳性物质分布于胞质,胞核呈阴性反应,此结果提示：大鼠颌下腺能自身合成DHEA,DHEA对消化功能可能具有重要的调节作用。     

小鼠胚胎发育过程中表皮生长因子受体的免疫荧光定位研究

用间接免疫荧光法研究了第12—19天小鼠胚胎的表皮生长因子受体(EGFR)在各种组织中的分布。结果表明,特异性荧光出现在EGFR阳性细胞的细胞膜上。第14.5天鼠胚鼻粘膜上皮首先显示很强的EGFR特异性荧光,此后荧光稍为减弱,直至第19天后消失。消化系统中,舌味觉上皮在第16天、胃粘膜上皮在第17—18天间、十二指肠上皮在第17.5—18.5天、直肠粘膜上皮在第15.5—16天和肛管粘膜上皮在第15天均显示强特异性荧光。肝细胞从第14.5天起有弱阳性反应,随胎龄增大强度缓慢地增强。此外,在胚胎发育不同时期,还看到若干组织呈现阳性反应,包括膀胱粘膜变移上皮、眼睑原基、腺垂体上皮、舌下腺腺泡细胞、附属腺上皮细胞、胰岛细胞、颌下腺腺上皮和导管上皮细胞、降主动脉内皮以及卵巢髓质部富含血管的疏松结缔组织中的成纤维细胞等。     

大鼠颌下腺促性腺激素释放激素及其mRNA的免疫组织化学与原位杂交研究

用免疫组织化学及原位杂交法,研究了促性腺激素释放激素及其ｍＲＮＡ在大鼠颌下腺的分布。结果显示,大鼠颌下腺的浆液性腺泡的上皮细胞,各级导管的上皮细胞及副交感神经节细胞均呈促性腺激素释放激素免疫反应阳性,阳性反应物质分布在胞质,胞核呈阴性反应。颌下腺的浆液性腺泡上皮细胞,各级导管上皮细胞同样被检测到很强的促性腺激素释放激素ｍＲＮＡ杂交信号。以上结果提示,大鼠颌下腺能自身合成促性腺激素释放激素,促性腺激素释放激素对消化功能可能有重要调节作用。     

大白鼠颌下腺促性腺激素释放激素受体(GnRHR)mRNA的RT-PCR和原位杂交的研究

本实验采用RT－PCR法探讨大白鼠颌下腺是否存在GnRH受体mRNA,并用原位杂交法对其细胞定位进行了研究。结果显示RT－PCR可扩增出大白鼠颌下腺GnRH受体mRNA的特异性片段,其碱基数与设计的一致,原位杂交发现颌下腺浆液性腺泡上皮细胞、颗粒曲管、排泄管及分泌管上皮细胞内有GnRH受体mRNA的杂交信号,信号物质分布于胸质内,胞核阴性。上述结果表明大白鼠颌下腺能合成GnRH受体,颌下腺产生的GnRH可作用于颌上腺的靶细胞,参与颌下腺生理功能的调节。     

Synthesis and secretion of nerve growth factor by mouse astroglial cells in culture

Astroglial cells cultured from the mouse brain have been found to synthesize and secrete a material(s) with nerve growth factor-like immunoreactivity (NGF-LI) into their culture medium. A material(s) with NGF-LI showed identical properties to those of beta NGF purified from the mouse submaxillary gland in immunoreactivity, molecular weight, isoelectric point, and neurite outgrowth stimulatory activity. These results indicate that astroglial cells cultured from mouse brain are able to synthesize and secrete beta NGF in culture.     

A study on the localization and distribution of GnRH and its receptor in rat submaxillary glands by immunohistochemical,in situ hybridization and RT-PCR

We investigated the rat submaxillary gland for the presence of GnRH and GnRH receptors, the localization and colocalization of GnRH, GnRH receptor and their mRNA, and studied the sequence of GnRH receptor complementary DNA (cDNA) by immunohistochemistry, in situ hybridization and RT-PCR. The results showed that GnRH and GnRH receptor immunoreactive materials were colocalized in the epithelial cells of the serous acinus and glandular duct. The GnRH and GnRH receptor mRNA hybridization signals were detected in the above cells. The sequence obtained from the RT-PCR product was identical to the published cDNA sequence of GnRH receptor in the rat pituitary. The results suggested that the rat submaxillary gland was capable of synthesizing GnRH and GnRH receptors. GnRH may be involved in the functional regulation of the submaxillary gland through autocrine or paracrine activity.     

Localization and in situ Quantification of 5-hydroxytryptamine and its Receptor in Rat Submaxillary Gland

5-Hydroxytryptamine (5-HT) and its receptor have been localized and quantified in the submaxillary gland of rats of various ages, using immunohistochemistry, in situ hybridization and in situ quantification. In male rats, the epithelial cells of serous acini, intercalated ducts, secretary tubes and excretory ducts all showed 5-HT and 5-HT receptor (5-HTR) immunoreactivity. Both 5-HT and 5-HTR reactive sites were found in the same cells of adjacent sections. 5-HT1A receptor mRNA hybridized signals could be detected in cytoplasm of these cells. The parasympathetic ganglia cells and endothelial cells of small vessels also showed 5-HT and 5-HTR immunoreactivity in the cytoplasm. However, in female rats, only the epithelial cells in excretory tubes showed 5-HT and 5-HTR immunoreactivity. The immunoreactivity was present in the same cells of adjacent sections. The relative content of 5-HT and its receptor increased during the first 60 postnatal days but remained constant from day 60 to day 90 postnatum. These results suggest that the submaxillary gland of rats possess autocrine 5-HT, which may regulate the function and development of the gland.     

EFFECTS OF ADENOSINE AND EPIDERMAL GROWTH FACTOR ON THE GROWTH OF MOUSE MAMMARY EPITHELIAL CELLS

The objective of this study was to determine if adenosine alters growth of mammary epithelium. Mouse mammary epithelial cells (NMuMG) were cultured in DMEM supplemented with 10% fetal calf serum. After serum starvation for 24h, EGF (0–100ng/ml) and/or adenosine (0–100μm ) was added. Adenosine at concentrations of 1, 10 or 100μm increased DNA synthesis significantly, when compared to control. Addition of epidermal growth factor (EGF) (10ng/ml) into 1 or 10μm adenosine showed the interaction in DNA synthesis between EGF and adenosine. A similar result was observed when 100μm adenosine added to various concentrations of EGF (0–100ng/ml). In the second mammary gland (thoracic) organ culture studies, mammary development scores were increased by adenosine (100μm ), EGF (100ng/ml) and adenosine plus EGF. These results indicate that the purine nucleoside adenosine stimulates mammary epithelial cell growth and interacts with EGF in DNA synthesis of mouse mammary epithelial cells.     

Expression and localization of immunoreactive-sialomucin complex (Muc4) in salivary glands

Sialomucin Complex (SMC; Muc4) is a heterodimeric glycoprotein consisting of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. Northern blot and immunoblot analyses demonstrated the presence of SMC/Muc4 in submaxillary, sublingual and parotid salivary glands of the rat. Immunocytochemical staining of SMC using monoclonal antisera raised against ASGP-2 and glycosylated ASGP-1 on paraffin-embedded sections of parotid, submaxillary and sublingual tissues was performed to examine the localization of the mucin in the major rat salivary glands. Histological and immunocytochemical staining of cell markers showed that the salivary glands consisted of varying numbers of serous and mucous acini which are drained by ducts. Parotid glands were composed almost entirely of serous acini, sublingual glands were mainly mucous in composition and a mixture of serous and mucous acini were present in submaxillary glands. Since immunoreactive (ir)-SMC was specifically localized to the serous cells, staining was most abundant in parotid glands, intermediate levels in submaxillary glands and least in sublingual glands. Ir-SMC in sublingual glands was localized to caps of cells around mucous acini, known as serous demilunes, which are also present in submaxillary glands. Immunocytochemical staining of SMC in human parotid glands was localized to epithelial cells of serous acini and ducts. However, the staining pattern of epithelial cells was heterogeneous, with ir-SMC present in some acinar and ductal epithelial cells but not in others. This report provides a map of normal ir-SMC/Muc4 distribution in parotid, submaxillary and sublingual glands which can be used for the study of SMC/Muc4 expression in salivary gland tumors.     

小鼠颌下腺降钙素基因相关肽的免疫组织化学定位

本文用免疫组织化学ＡＢＣ法,对小鼠颌下腺中降钙素基因相关肽（ＣａｌｃｉｔｏｎｉｃＧｅｎｅ－ＲｅｌａｔｅｄＰｅｐｔｉｄｅ,ＣＧＲＰ）的分布进行了观察。结果表明：小鼠颌小腔内有降钙素基因相关肽免疫反应神经的分布,它们主要分布于小叶间结缔组织中。此外,颗粒由管细胞也呈降钙素基因相关肽免疫反应阳性。提示降钙素基因相关肽可能参与颌下腺的分泌活动及血液循环等生理调节。