兔口腔黏膜上皮干细胞分化为角膜上皮样细胞的体外研究

目的以羊膜做载体将兔口腔黏膜上皮干细胞诱导为角膜上皮样细胞,探讨口腔黏膜上皮细胞作为种子细胞体外培养构建组织工程角膜的技术方法,为眼表重建提供材料。方法用Ⅳ型胶原黏附法分离纯化口腔黏膜上皮干细胞,对体外培养的口腔黏膜干细胞进行免疫表型鉴定。将筛选后获得的口腔黏膜上皮干细胞种植在去上皮羊膜表面体外培养,待细胞融合成单层后置入插入式培养皿中进行气液界面培养,促进细胞分化形成复层。培养数日后进行苏木素-伊红(HE)染色和免疫组织化学、光镜及透射电镜检测,观察羊膜-复层上皮组织结构,免疫荧光检测干细胞特异性标志物P63以及角膜上皮细胞标志物角蛋白3(K3)并与角膜上皮组织比较。结果体外诱导培养数天后,羊膜载体的口腔黏膜上皮细胞形成复层,在组织形态及生物学特性上与角膜上皮相似。并且组织细胞P63、K3表达明显,角膜特异性生物学标志物免疫组织化学染色呈阳性。结论在本实验的培养条件下,获得的口腔黏膜上皮干细胞可在体外培养扩增,呈克隆性生长,具有很强的增殖能力。口腔黏膜上皮干细胞体外培养可构建类角膜上皮。     

皮肤角质形成细胞的无血清培养方法

目的探索建立少量成年自体皮肤角质形成细胞体外无血清培养体系,为自体组织工程皮肤的构建与移植奠定物质基础。方法:无菌条件下,取2cm×2cm 兔耳皮肤组织块,Dispase 消化,分离真表皮,表皮以胰蛋白酶+EDTA 消化获得角质形成细胞,以含钙和不含钙的角质形成细胞培养液(K-SFM),按不同细胞密度接种于24孔板,观察细胞生长状况;免疫组化鉴定,MTT 法测定不同血清浓度对角质形成细胞增殖分化的影响。结果:在适当的 Ca~(2+)浓度下,少量自体角质形成细胞能够在无血清培养液 k-SFM 中培养扩增,最多可传4～6代。添加血清后可明显加速细胞分化。结论:无血清培养体系适用于少量成年自体角质形成细胞的体外连续培养扩增,培养的细胞可用于自体组织工程皮肤的构建。     

成人骨髓基质干细胞体外诱导成骨细胞

目的建立成人骨髓基质干细胞(BMSCs)分离、扩增以及诱导和分化为成骨细胞的体外培养方法,为骨组织工程选择理想的种子细胞来源。方法抽取健康成人骨髓组织,用Percoll分离液分离出骨髓中的单个核细胞,在含体积分数为10％小牛血清的高糖：DMEM培养液中,置于37℃、含体积分数为5％的CO2湿化空气孵箱中培养,通过传代培养扩增BMSCs,传三代时改用含地塞米松、β-甘油磷酸和维生素C的条件培养基培养,用倒置显微镜、HE染色观察增殖和分化情况,并测定碱性磷酸酶活性和钙结节形成能力。结果体外培养的成骨细胞生长良好,表现出与典型的成骨细胞相似的形态特征和生物学特性。结论所建立的成人BMSCs分离、扩增以及诱导和分化为成骨细胞的体外培养方法稳定和实用,可作为骨组织工程种子细胞来源的常规方法之一。     

舌鳞癌组织中肿瘤干细胞的分离培养及PIWIL4对其的影响

目的：从人舌鳞癌组织中分离培养肿瘤干细胞细胞（CSC）,鉴定其生物学特性,并研究PIWIL4在CSC中的表达和意义。方法收集9例不同临床分期舌鳞癌患者组织标本,通过酶消化和原代培养相结合等方法处理,采用无血清悬浮培养法分离培养获得含CSC的悬浮细胞球,流式细胞仪检测细胞表面分子标志CD133和CD44的表达,免疫磁珠分选系统分离CD133＋CD44＋细胞;采用半定量逆转录聚合酶链反应检测PIWIL4 mRNA在CSC中的表达;裸鼠皮下接种CSC,观察其成瘤能力。结果成功的从人舌鳞癌组织分离培养获得可悬浮生长、稳定传代的CSC,CSC高表达CD133和CD44,裸鼠皮下接种1×10^4、1×10^5个CSC均可全部成瘤,PIWIL4 mRNA在CSC的表达也显著高于正常舌组织细胞。结论采用无血清悬浮培养法成功从人舌组织中分离获得CSC,具有肿瘤干细胞特性,能高度表达PIWIL4,为靶向肿瘤干细胞的治疗提供了实验依据。     

胎鼠脑神经干细胞的分离、培养和鉴定

目的 研究胎鼠脑皮质神经干细胞(NSCs)的分离、培养及鉴定方法。方法 从孕15d胎鼠的大脑皮层和海马区脑组织中获取NSCs,在含有B27、表皮生长因子(EGF)和碱性成纤维生长因子(bFGF)的DMEM/F12无血清培养液中培养;传代后用5%胎牛血清培养液诱导NSCs分化。结果 体外分离培养的NSCs在无血清培养液中形成大量的神经球。经3-5代传代的细胞生长稳定。经巢蛋白染色鉴定,大部分为阳性细胞,神经细胞球经胎牛血清培养液贴壁培养后可分化为神经元特异烯醇化酶、胶质纤维酸性蛋白和半乳糖脑苷脂表达阳性的细胞。结论 从孕15d胎鼠大脑皮质和海马组织分离出的NSCs具有自我更新能力和多向分化潜能,其在5%胎牛血清培养液中具有向神经元和神经胶质细胞分化的潜能。     

兔骨髓基质细胞体外培养的生物学特性

目的 研究兔骨髓基质细胞(MSC)体外培养状态下的生物学生长特性,为骨/软骨组织工程提供实验理论和技术支持。方法 抽取兔骨髓分离MSC,连续培养5代。描绘1～5代MSC生长曲线、计算倍增时间,测定细胞贴壁率和细胞活性。结果 第1～3代传代细胞生长曲线基本相同,倍增时间20h,贴壁率无明显差异,接种后12h贴壁率为96％,成活率大致相等,为95％;第4、5代细胞倍增时间延长,贴壁能力及细胞活力下降。结论 原代及第1～3代MSC体外培养生长稳定,增殖迅速,贴壁率高,活力良好,是骨／软骨组织工程良好的种子细胞来源。     

胚胎大鼠神经干细胞体外分化的激光共聚焦显微镜观察

目的:采用激光扫描共聚焦显微镜观察体外培养胎鼠大脑皮质神经干细胞(neural stem cells,NSCs)分化情况。方法:利用无血清培养方法,分离培养胚胎大鼠大脑NSCs,进行体外扩增培养、传代;采用溴脱氧尿嘧啶核苷(bromodeoxyuridine,BrdU)掺入、双重免疫荧光细胞化学标记方法和激光扫描共聚焦显微镜,用神经细胞的特异性抗体(神经元β-微管蛋白、胶质纤维酸性蛋白),鉴定NSCs向神经元与星形胶质细胞分化的情况。结果:从胎鼠大脑皮质及皮质下分离的组织,经原代和传代培养均可形成细胞克隆,并表达神经上皮干细胞蛋白(Nestin)抗原。在血清诱导下,分化后的细胞表达神经元、星形胶质细胞2种神经细胞的特异性抗原,NSCs分化为星形胶质细胞神经元的比例分别为(43.70±8.55)%和(23.00±3.69)%。结论:从胎鼠大脑皮质分离出的细胞可获得呈集落样生长的神经干细胞团,并能表达NSCs的特异性抗原;激光扫描共聚焦显微镜可清晰地观察到培养细胞具有分化为神经元和星形胶质细胞的潜能。     

原代培养冷冻兔角膜缘上皮细胞的增殖活性研究

为探讨低温保存的兔角膜缘上皮细胞体外培养方法和增殖活性，采用不同的冷冻条件保存兔角膜缘上皮组织；通过若丹明B染色方法定量检查体外培养的原代细胞增殖活性，并对含血清培养和无血清培养、消化培养和组织块培养进行比较性研究。结果表明，二甲基亚砜浓度梯度和降温速率对冷冻角膜缘上皮原代细胞的增殖能力均无显著性影响(P&;gt;0．05)。在细胞增殖活性上，血清培养组优于无血清培养组(P&;lt;0．05)；组织块培养组优于消化培养组(P&;lt;0．05)；RPMI1640组优于MEM组和DMEM组(P&;lt;0．05)；3T3细胞滋养层组与巨噬细胞滋养层组结果相似(P&;gt;0．05)。结论：兔角膜缘上皮细胞具有血清依赖性和低温耐受性。     

胰酶联合Ⅱ型胶原酶法体外分离培养软骨细胞的分析

目的 通过原代软骨细胞的体外培养和鉴定,探讨胰酶联合Ⅱ型胶原酶法体外分离培养软骨细胞的可行性.方法 分离出兔鼻中隔软骨组织,用胰酶联合Ⅱ型胶原酶的方法进行消化,获取原代软骨细胞.使用倒置显微镜观察软骨细胞的形态及生长情况,并用甲苯胺蓝染色、Ⅱ型胶原免疫组化及免疫荧光染色进行表型鉴定.结果 软骨细胞原代培养形成单层细胞需要7～8d,传代培养时间约2～3 d,细胞以圆形或类上皮细胞形态为主,甲苯胺蓝染色证实细胞可特异性合成糖胺聚糖,Ⅱ型胶原免疫组织化学染色及免疫荧光染色可证实细胞可特异性分泌Ⅱ型胶原.结论 本研究成功建立了简单易行的胰酶联合Ⅱ型胶原酶法体外分离培养大量纯净的软骨细胞,提高了消化速率,加大了细胞释放率,为组织工程气管软骨种子细胞的获取提供重要的技术支撑.     

人皮肤角质形成细胞库的构建

目的探索人皮肤角质形成细胞的分离、培养、传代、冻存、复苏及鉴定技术。方法采用细胞培养技术及免疫组化技术。结果我们采用细胞培养技术体外分离培养了小儿包皮角质形成细胞,传代后的角质形成细胞,经冻存、复苏后,此角质形成细胞仍既能增殖、又能分化,生长状态与新鲜分离的角质形成细胞相类似。结论酶消化法是快速大量培养皮肤角质形成细胞的简便易行的方法。     

Anti-tumour effects of small interfering RNA targeting anion exchanger 1 in experimental gastric cancer

BACKGROUND AND PURPOSE

Anion exchanger 1 (AE1) is an integral membrane protein found in erythrocytes. Our previous studies have demonstrated that AE1 is expressed in human gastric cancer cells and may be involved in the carcinogenesis of cancer. In this study, we further investigated the role of AE1 in gastric carcinogenesis and the anti-tumour effects of AE1-targeted small interfering RNAs (siRNAs) in two experimental models of gastric cancer.

EXPERIMENTAL APPROACH

Molecular and cellular experiments were performed to elucidate the role of AE1 in the malignant transformation of gastric epithelium and the effects of AE1-targeted siRNAs on gastric cancer cells. The anti-tumour effect of the siRNA was evaluated in vivo in two mouse models, nude mice implanted with human gastric cancer xenografts (Model I) and mice with gastric cancer induced by N-methyl-N-nitrosourea (MNU) and Helicobacter pylori (Model II).

KEY RESULTS

AE1 was found to increase gastric carcinogenesis by promoting cell proliferation. AE1-targeted siRNA significantly suppressed AE1 expression and hindered tumour growth. Furthermore, the siRNA markedly decreased the detection rate of gastric cancer, in parallel with an increase in atypical hyperplasia at the end of the experiment in Model II.

CONCLUSIONS AND IMPLICATIONS

Knockdown of AE1 expression in gastric mucosa by administration of synthetic siRNAs significantly inhibits the growth of gastric cancer and decreases the detection rate of this tumour in experimental mice. These results suggest that AE1 is potentially a key therapeutic target and the silencing of AE1 expression in gastric mucosa could provide a new therapeutic approach for treating gastric cancer.     

Quantitative measurement of acute corneal injury in rabbits with surfactants of different type and irritancy.

We have hypothesized that differences in ocular irritancy are related to differences in extent of initial injury and that, regardless of the processes leading to tissue damage, extent of injury is the primary factor that determines the final outcome of ocular irritation. In previous in vivo confocal microscopic (CM) studies we identified quantifiable differences in the extent of corneal injury occurring with four surfactants (three anionic, one cationic) known to cause different levels of ocular irritation and demonstrated that extent of initial corneal injury was related to the magnitude of cell death. The purpose of this study was to assess the applicability of this hypothesis to a broad sampling of surfactants. Specifically, initial corneal changes induced by seven different surfactants (one anionic, three cationic, three nonionic) were measured by in vivo CM and cell death was measured by an ex vivo live/dead assay. The right eye of each rabbit was treated by placing 10 microl of a surfactant directly on the cornea. Eyes were examined macroscopically and scored for irritation at 3 h and 1 day. At 3 h and 1 day, in vivo CM was used to examine the corneas and quantitate epithelial cell size, epithelial thickness, corneal thickness, and depth of stromal injury. At 3 h and/or at 1 day, corneas were removed and excised regions were placed in culture media containing 2 microM calcein AM and 4 microM ethidium homodimer. Using laser scanning CM, the number of dead epithelial and/or stromal cells in a 300 x 300 x 170-microm3 (xyz) volume of the cornea was determined. In vivo CM and live/dead assay findings revealed three surfactants to affect only the epithelium, three surfactants to affect the epithelium and superficial stroma, and one surfactant to affect the epithelium and deep stroma. Extent of initial corneal injury reflected level of ocular irritation, and magnitude of cell death was related to the extent of initial corneal injury. These findings are consistent with those for known slight, mild, and moderate to severe irritants, respectively. They suggest that our hypothesis is broadly applicable to surfactants. Additionally, we believe these surfactants should be included as part of a new "gold standard" for use in developing and validating in vitro tests to replace the use of animals in ocular irritancy testing.     

Effluxing ABC transporters in human corneal epithelium

ATP-binding cassette (ABC) transporters are able to efflux their substrate drugs from the cells. We compared expression of efflux proteins in normal human corneal epithelial tissue, primary human corneal epithelial cells (HCEpiC), and corneal epithelial cell culture model (HCE model) based on human immortal cell line. Expression of multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1–6 (MRP1–6) and breast cancer resistance protein (BCRP) was studied using quantitative RT-PCR, Western blot, and immunohistochemistry. Only MRP1, MRP5, and BCRP were expressed in the freshly excised human corneal epithelial tissue. Expression of MRP1 and MRP5 was localized predominantly in the basal cells of the central cornea and limbus. Functional efflux activity was shown in the cell models, but they showed over-expression of most efflux transporters compared to that of normal corneal epithelium. In conclusion, MRP1, MRP5, and BCRP are expressed in the corneal epithelium, but MDR1, MRP2, MRP3, MRP4, and MRP6 are not significantly expressed. HCE cell model and commercially available primary cells deviate from this expression profile. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:1087–1098, 2010     

Extent of corneal injury as a biomarker for hazard assessment and the development of alternative models to the Draize rabbit eye test

We have characterized 22 ocular irritants differing in type (surfactants, acid, alkali, bleaches, alcohol, aldehyde, acetone) and severity (slight to severe) by using the low-volume rabbit eye test. Ocular irritation was evaluated by 1) light microscopy to assess pathological changes, 2) in vivo confocal microscopy (CM) to quantify 4-dimensionally (x, y, z, and t) initial corneal injury and later responses in the same eye, and 3) laser scanning CM to quantify initial cell death. These studies revealed that regardless of the processes leading to injury, slight irritants injure the corneal epithelium, mild irritants injure the corneal epithelium and the superficial stroma, and moderate/severe irritants injure the epithelium, deep stroma, and at times the corneal endothelium. Furthermore, extent of initial corneal injury was shown to predict subsequent responses and final outcomes. These findings suggest that extent of corneal injury may be used as a basis for the development of alternative ocular irritation tests. To test the validity of this approach, we have used an ex vivo, rabbit cornea culture model to measure extent of corneal injury following exposure to ocular irritants. Data indicate that the extent of ex vivo corneal injury significantly correlate with the extent of initial injury measured previously in live animals. Overall, these findings indicate that extent of initial corneal injury can be used as a new "gold standard" for the continued refinement and ultimate replacement of the Draize rabbit eye Ocular Irritation Test.     

Culture of the primary corneal epithelium as a potential component of test batteries for eye irritancy testing.

One of the main goals for toxicologists working on the development of in vitro tests is to replace the animal-based eye irritation test. Inflammation is one of the mechanisms which have not been covered sufficiently by the existing in vitro ocular irritancy test systems. As there are major species differences between the human and rabbit eye inflammation mechanisms, the most relevant test system is the human eye itself. The current study focused on an evaluation of the practical availability of human corneal epithelial cells for routine eye irritancy testing. Human corneal epithelium cell cultures were used to assess the effects of lipopolysaccharide on IL-1 beta release. The findings indicated that cytokine release can be augmented by the presence of the complement system, which is normally found in tears. However, the corneal cells were found to be highly resistant to the complement system, which can be attributed to the very high expression of CD59, a powerful complement regulatory protein found in the corneal epithelium. It is estimated that discarded corneas from tissue banks could provide enough material for routine testing by this method.     

Mechanistic studies on transcorneal permeation of pilocarpine.

The mechanism of corneal pilocarpine penetration was studied in the albino rabbit using radiochemical techniques. The apparent rate and extent of pilocarpine accumulation in the aqueous humor and the various cell layers of the cornea were determined for both intact and abraded eyes. For the first time, drug levels were monitored in the epithelium and stroma-endothelium of the intact cornea using a tissue-scraping technique. In addition, a new postinstillation rinsing method was devised to evaluate the rate of corneal uptake. The results demonstrate a dual role for the corneal epithelium, both as a barrier to drug penetration and as a reservoir for drug in the intact cornea. The transcorneal pilocarpine flux is slower than the data appear to indicate, and previous overestimates of the apparent absorption rate constant are due to parallel elimination processes occurring at the absorption site. Pharmacokinetic parameters were determined for each tissue to generate an overall mechanism for corneal permeation.     

芦荟大黄素对人永生化角质形成细胞增殖和凋亡作用研究

目的研究芦荟大黄素（AE）对人永生化角质形成细胞（HaCaT）的生长抑制效应和诱导细胞凋亡能力,探讨其治疗银屑病的可能机制。方法四甲基偶氮唑蓝（MTT）法检测芦荟大黄素对HaCaT细胞增殖的抑制作用,倒置显微镜观察细胞形态学变化,流式细胞术检测细胞周期变化及凋亡。结果芦荟大黄素质量浓度在20～80μg／mL范围内可抑制HaCaT细胞增殖且呈剂量依赖性;镜下观察到细胞稀疏,生长减慢,细胞形态拉长,细胞被阻滞于S期而凋亡。结论芦荟大黄素能抑制HaCaT细胞的增殖、诱导HaCaT细胞的凋亡。可用于治疗银屑病。     

Predicted Permeability of the Cornea to Topical Drugs

Purpose. To develop a theoretical model to predict the passive, steady-state permeability of cornea and its component layers (epithelium, stroma, and endothelium) as a function of drug size and distribution coefficient (). The parameters of the model should represent physical properties that can be independently estimated and have physically interpretable meaning. Methods. A model was developed to predict corneal permeability using 1) a newly developed composite porous-medium approach to model transport through the transcellular and paracellular pathways across the epithelium and endothelium and 2) previous work on modeling corneal stroma using a fiber-matrix approach. Results. The model, which predicts corneal permeability for molecules having a broad range of size and lipophilicity, was validated by comparison with over 150 different experimental data points and showed agreement with a mean absolute fractional error of 2.43, which is within the confidence interval of the data. In addition to overall corneal permeability, the model permitted independent analysis of transcellular and paracellular pathways in epithelium, stroma and endothelium. This yielded strategies to enhance corneal permeability by targeting epithelial paracellular pathways for hydrophilic compounds ( < 0.1 – 1), epithelial transcellular pathways for intermediate compounds, and stromal pathways for hydrophobic compounds ( > 10 – 100). The effects of changing corneal physical properties (e.g., to mimic disease states or animals models) were also examined. Conclusions. A model based on physicochemical properties of the cornea and drug molecules can be broadly applied to predict corneal permeability and suggest strategies to enhance that permeability.     

百草枯致兔眼损伤的病理观察

目的探讨百草枯致[损伤的病理]变,为构建相应动物模型奠定基础。方法将20只普通级健康成年白色新西兰家兔按照随机数字表法分为5组,每组4只。每只家兔左眼结膜囊内滴入百草枯原液100μl后立即将其眼睑轻轻闭合,使药液分别保留30 s(30 s组)、1 h(1 h组)、4 h(4 h组)、8 h(8 h组)和24 h(24 h组);右眼结膜囊内滴入生理盐水100μl作为对照。接触百草枯原液达规定时间后,以生理盐水分别冲洗双眼5 min。应用裂隙灯和角膜荧光素染色技术观察眼结膜、虹膜和角膜的损伤情况并进行评分,记录眼损伤累加最高积分和损伤完全恢复时间。实验第21天处死动物,取角膜组织进行组织病理学观察。结果 30 s组家兔出现轻至中度结膜刺激症状,未见角膜、虹膜损伤,实验第2天眼损伤累加最高积分达峰,为(14.0±2.3)分,眼损伤完全恢复时间为(9.5±0.6)d,角膜组织切片未见明显病理改变。1 h组家兔出现中至重度结膜刺激症状,虹膜无损伤,角膜有局灶性损伤,染毒后(5.5±1.9)d眼损伤累加最高积分达峰,为(47.5±8.5)分,高于30 s组(P<0.05),眼损伤完全恢复时间为(13.5±2.4)d,组织切片见角膜受损部位上皮扁平细胞层缺失。4 h组、8 h组和24 h组家兔除了出现重度结膜刺激症状外,均出现角膜和虹膜损伤,损伤程度随着接触百草枯原液时间的延长而加重,眼损伤累加最高积分分别在染毒后(6.2±1.0)、(7.7±1.0)和(7.2±2.1)d达峰,分别为(67.5±10.5)、(79.5±9.7)和(80.0±9.5)分,均高于30 s组和1 h组(均P<0.05),3组之间差异无统计学意义(P>0.05)。4 h组中有2只家兔分别在实验第12、19天眼损伤完全恢复,另2只和8 h组、24 h组的全部家兔至实验第21天眼损伤未完全恢复。组织病理学观察显示4 h组家兔角膜上皮细胞部分脱落,呈虫蚀样改变;8 h组角膜上皮扁平细胞和棘状细胞脱落,仅存柱状基底细胞,部分深达角膜基质层,角膜基质纤维板层结构紊乱,可见肉芽组织和角膜新生血管;24 h组角膜上皮完全剥脱,仅存基质层、内弹力层和内皮细胞层。结论眼接触百草枯原液30 s即可导致结膜损伤,接触1 h可致角膜损伤,接触时间≥4 h可造成角膜混浊、角膜损伤瘢痕愈合等难以恢复的眼损伤。家兔眼接触百草枯原液≥4 h出现的眼损伤可作为百草枯致眼损伤的动物模型。