周期性拉伸应力与人永生化角质形成细胞的黏附和铺展

背景：皮肤组织所处的力学环境以及上皮细胞的铺展状态与伤口愈合和瘢痕形成过程关系密切。 目的：分析胞外力学刺激对细胞铺展的作用,并检测细胞的增殖状态进一步分析铺展形态对细胞增殖等生理活动的影响。 方法：通过FX-4000柔性基底加载系统对人永生化角质形成细胞(HaCaT)进行周期性拉伸应力正弦波加载,加载程式0.2 Hz,10%拉伸幅度。在0,24,48 h对细胞的铺展形态进行对比,利用流式细胞仪检测分析细胞的增殖,并利用免疫荧光染色方法对比分析纽蛋白在细胞内的分布。 结果与结论：HaCaT细胞在加载24 h后分裂期细胞较多,铺展形态无明显变化;加载48 h后HaCaT细胞形态发生较为明显的变化,分裂期细胞较静态对照组略有减少;拉伸应力作用下纽蛋白的分布由细胞核周围的膜区域向细胞边缘集中。结果表明：适度的力学加载能够在保持细胞铺展和黏附状态下,促进细胞增殖;周期性持续拉伸应力的作用时间是HaCaT细胞铺展形态和与基底黏附位点的重要影响因素。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

抗角蛋白16单克隆抗体对角质质形成细胞增殖活性的影响

目的 研究抗角蛋白单克隆抗体（mAb）对体外培养的角质形成细胞增殖活性与细胞周期的影响。方法 以无血清培养基体外原代培养角质形成细胞。应用^3H-TdR掺入DNA合成测定法,观察抗角蛋白16mAb(anti-K16 mAb)对角质形成细胞代谢增殖的影响。用流式细胞仪分析anti-K16 mAb作用后角质形成细胞的细胞周期变化。结果 mAb作用后,角质形成细胞样品的epm降低,并呈抗体剂量依赖方式。流式细胞信分析表明,处于S期的细胞比例由27％上升至42．2％,同时处于G1期与G2期的细胞比例分别相应下降。结论 anti-K16 mAb可抑制体外培养的角质形成细胞的增殖活性,且将细胞阻滞于S期,无法进入G2期与M期分裂增殖。     

表皮细胞生长因子对角质形成细胞体外增殖作用的研究

目的：观察重组人表皮生长因（Recombinant human epidermal growth factor,rhEGF）对人角质形成细胞的细胞周期以及促切口愈合作用。方法：运用包皮环切术后收集培养角质细胞,不同浓度的重组人表皮细胞生长因子处理细胞,MTT法、↑3HtdR掺入法测细胞生长率;流式细胞仪检测细胞周期。结果：体外实验表明5～100ng·ml^-1重组人表皮细胞生长因子可促进人角质形成细胞生长,其中浓度为10ng·ml^-1促进细胞生长作用最为显著。流式细胞仪检测10ng·ml^-1重组人表皮细胞生长因子处理后细胞增殖明显,S和G2／M期细胞数明显增加。结论：重组人表皮细胞生长因子可促进细胞生长。     

抗角蛋白16单克隆抗体对角质形成细胞增殖活性的影响

目的研究抗角蛋白单克隆抗体(mAb)对体外培养的角质形成细胞增殖活性与细胞周期的影响.方法以无血清培养基体外原代培养角质形成细胞.应用3H-TdR掺入DNA合成测定法,观察抗角蛋白16mAb(anti-K16mAb)对角质形成细胞代谢增殖的影响.用流式细胞仪分析anti-K16mAb作用后角质形成细胞的细胞周期变化.结果mAb作用后,角质形成细胞样品的cpm降低,并呈抗体剂量依赖方式.流式细胞仪分析表明,处于S期的细胞比例由27%上升至42.2%,同时处于G1期与G2期的细胞比例分别相应下降.结论anti-K16mAb可抑制体外培养的角质形成细胞的增殖活性,且将细胞阻滞于S期,无法进入G2期与M期分裂增殖.     

核因子E2相关因子2对人永生化角质形成细胞氧化损伤的保护作用

目的 探讨核因子E2相关因子2(Nrf2)对过氧化氢(H2O2)诱导的人永生化角质形成细胞(HaCaT)氧化损伤的保护作用.方法 用慢病毒转染方式使HaCaT过表达Nrf2(Nrf2/HaCaT),并用MTT法和抗Ki67免疫荧光染色法检测其增殖活性.实验分为Nrf2/HaCaT组和HaCaT组,每组5个样本.应用20...     

纳米二氧化硅对人角质形成细胞超微结构的影响

目的:观察纳米二氧化硅(SiO2)颗粒对人角质形成细胞(HaCaT)的影响.方法:将不同浓度的纳米SiO2悬液加入培养的HaCaT细胞中,在37℃,5%CO2的细胞培养箱中共同孵育4h,收集细胞制样,在透射电镜下观察细胞超微结构的改变.结果:纳米SiO2粒径为40～50nm,在不同浓度下均能进入细胞并对细胞器造成影响,影响的严重程度与纳米SiO2颗粒的浓度呈正相关.结论:纳米颗粒可以通过吞噬作用和直接穿膜两种方式进入细胞,通过其表面能和离子效应影响细胞的超微结构.     

人角质形成细胞Notch信号通路与转化生长因子β调控受体蛋白的作用

背景：在皮肤中受体蛋白酪氨酸磷酸酶kappa的调控至关重要,而转化生长因子β似乎是其调控的上游因子,既然Notch信号和转化生长因子β信号通道如此相关,那么Notch是不是也参加了转化生长因子β信号对受体蛋白酪氨酸磷酸酶kappa的调控呢？ 目的：探讨Notch信号通道在人角质形成细胞中对转化生长因子β调控受体蛋白酪氨酸磷酸酶kappa的作用的影响。 方法：在分别用Jagged-1激活和用Γ-分泌酶抑制剂抑制Notch信号通道后,加入转化生长因子β,同时设立对照组,用Real-time PCR测试人角质形成细胞中受体蛋白酪氨酸磷酸酶kappa mRNA表达量。 结果与结论：覆盖率为40%的角质形成细胞在加入了转化生长因子β后,受体蛋白酪氨酸磷酸酶kappa mRNA量在各时间点均高于对照组。在用Jagged-1激活Notch通道的角质形成细胞中,单独加入Jagged-1、转化生长因子β及两者都加入时均高于对照组(P < 0.05,P < 0.01)。在用γ-分泌酶抑制剂抑制Notch通道的角质形成细胞中,只加入转化生长因子β显著高于对照组(P < 0.01),只加入γ-分泌酶抑制剂和两者均加入时与对照组比较,差异无显著性意义(P > 0.05)。说明加入转化生长因子β导致角质形成细胞中受体蛋白酪氨酸磷酸酶kappa表达增加,而分别对Notch信号进行激活和抑制后发现,受体蛋白酪氨酸磷酸酶kappa信号分别显著增加和显著被抑制。所以在转化生长因子β升高受体蛋白酪氨酸磷酸酶kappa表达过程中Notch信号通道是非常重要且不可或缺的。     

microRNA对角质形成细胞功能的影响☆

背景：以角质形成细胞为基础的再上皮化过程是皮肤创面得以顺利修复的关键环节之一。有研究报道在小鼠缺血性创面中microRNA-210负向调控角质形成细胞的增殖,阻碍创面再上皮化的进行,提示microRNA可能通过影响角质形成细胞的生物学活动,进而参与创面修复过程。 目的：全面了解microRNA对角质形成细胞生物学活动的影响,指导创面愈合研究领域研究方向的选择,以及为异常创面修复的预防和治疗提供理论依据。 方法：以“keratinocyte,microRNA”为检索词检索PubMed,Embase数据库(2011-05),语言限定为英文。共收集文献59篇,阅读题目和摘要进行初筛,排除研究方向与本文无关、内容重复性研究,共保留12篇文章。对所保留文章的参考文献进行手工检索后,另添加文献30篇以及microRNA数据库2个。 结果与结论：microRNA对角质形成细胞的增殖、分化和移行能力具有调控作用,特别在缺血性创面中对再上皮化有阻碍作用,有望成为一种潜在的治疗靶点。但目前大部分研究仍为体外实验,需要将现有发现向动物模型以及临床研究转化。     

用于组织工程构建的表皮角质形成细胞的生物学特性

目的　研究体外培养的人表皮角质形成细胞的生物学特性 ,为构建组织工程皮肤提供技术参数。方法　用Dispase/胰蛋白酶法消化分离人表皮角质形成细胞 ,进行原代及传代培养。计算原代细胞获得率 ,观察各代细胞形态学改变 ,描记细胞生长曲线 ,计算群体倍增时间 (PDT) ,免疫组织化学方法检测细胞角蛋白表达。结果　原代表皮角质形成细胞获得率为 ( 0 .72 6± 0 .3 48)× 10 6 /cm2 ,表皮角质形成细胞体外可培养至第 7代 ,第 2代的PDT最短 ,为( 46.5 7± 2 .2 5 )h ,第 4代的细胞角蛋白表达仍为阳性。结论　综合细胞增殖速率、细胞数量及细胞功能的维持各因素 ,考虑第 3、4代的表皮角质形成细胞是构建组织工程皮肤最佳的种子细胞     

二氧化钛纳米颗粒对人角质形成细胞超微结构的影响及毒性效应

目的:观察无机二氧化钛(TiO2,20～35 nm)纳米颗粒进入人角质形成细胞(HaCaT)后的超微结构变化及毒性效应的影响.方法:将TiO2纳米颗粒与HaCaT细胞共孵育4 h后,收集细胞.采用透射电镜观察TiO2纳米颗粒引起细胞超微结构,变化及在细胞内的分布;采用激光共聚焦扫描显微镜观察TiO2纳米颗粒进入细胞后引起的细胞凋亡和坏死;采用噻唑蓝比色法测定TiO2纳米颗粒对细胞存活的影响.结果:TiO2纳米颗粒通过吞噬方式进入细胞.实验组里TiO2纳米颗粒呈簇状存在于HaCaT细胞内,并使细胞有较大损伤;细胞质有溶解现象,内质网肿胀,线粒体嵴结构紊乱,并肿胀崩解;核周间隙扩大、核固缩.结论:TiO2纳米颗粒具有的毒性效应能够损伤HaCaT细胞超微结构并且抑制细胞的生长.     

丁酸钠对人乳头状瘤病毒诱导的永生化食管上皮细胞恶性转化的促进作用

目的 在人乳头状瘤病毒（HPV）18E6E7基因诱导人胚食管上皮细胞永生化的基础上,观察高、低剂量丁酸钠在细胞恶性转化过程中的促癌作用。方法 永生化食管上皮细胞SHEE先用高剂量于酸钠（80mmol/L),后用低剂量丁酸钠（5mmol/L)各处理8周,再经无丁酸钠条件继续培养14周。用相差显微镜、免疫组织化学SABC法和流式细胞仪检查细胞形态、增殖和调亡状况;用Hoechst33342和碘化丙啶检查活细胞和死细胞;细胞软琼脂集落形成及移植裸小鼠和严重联合免疫缺陷小鼠检查成瘤性。结果 当细胞暴露在80mmol/L丁酸钠,细胞死亡,只剩少量活细胞。在含5mmol/L丁酸钠培养基中细胞出现第一增殖期;撤去丁酸钠,细胞进入危象期,细胞倍增时间延长,如老化细胞。度过危象期,细胞进入第二增殖期,细胞继续增生和异型增生。在第二增殖期末细胞出现恶变,软琼脂培养有大集落形成,移植裸小鼠和SCID小鼠成瘤。结论 SHEE永生化上皮由丁酸钠诱导的恶性变通过了两个阶段的死亡威胁：高浓度丁酸钠引起细胞死亡,缺乏丁酸钠引起细胞危象。高剂量丁酸钠引起永生化细胞死亡,低剂量引起细胞增殖,说明丁酸钠对体外培养细胞有促恶变作用。     

Metastatic dissemination of human malignant oral keratinocyte cell lines following orthotopic transplantation reflects response to TGF-beta 1

This study examined the behaviour of nine human malignant oral keratinocyte cell lines following orthotopic transplantation to the floor of the mouth of athymic mice. Tumourigenesis, local spread, and metastatic dissemination were correlated with known cellular responses to transforming growth factor-beta 1 (TGF-beta 1). Six of nine cell lines were tumourigenic; four of these cell lines showed local spread which was characterized by vascular and bone invasion. Metastatic spread was uncommon, with only 9% of animals with primary tumours developing metastases and these were almost exclusively found in the regional lymph nodes; there was one pulmonary metastasis and no liver deposits. Tumour cell behaviour did not reflect the clinical stage of the original tumours. Cell lines that were resistant to TGF-beta 1-induced growth inhibition were more likely to form primary tumours, exhibit local spread, and metastasize than cells that were growth-inhibited by the ligand. The data demonstrate that tumourigenicity and tumour behaviour in this orthotopic mouse model varied between cell lines and that the pattern of local invasion and metastasis was similar to that seen in human oral cancer. Furthermore, cell lines that were refractory to the growth inhibitory effects of TGF-beta 1 behaved more aggressively than cells that underwent ligand-induced cell-cycle arrest.     

分泌人源性单克隆抗体永生化淋巴细胞系的构建

目的;构建分泌人源性单允隆抗体（ＭｃＡｂ）的永生化Ｂ淋巴细胞系。方法：用正丁醇法提取的人卵巢癌细胞系ａｏｌ１０／１７抗原在含免疫活化剂的无血清培基中免疫人淋巴细胞,将制备ａｏ １０／１７细胞ＤＮＡ转染经体外免疫的淋巴细胞。采用ＥＬＩＳＡ筛选和有限稀释法克隆化。     

Viral integration and fragile sites in human papillomavirus-immortalized human keratinocyte cell lines.

Human papillomavirus (HPV) types 16 and 18 integration sites were mapped in six HPV-immortalized human keratinocyte cell lines by fluorescence in situ hybridization (FISH). Mapping of HPV sequences in these cell lines revealed that HPV integration varied in copy number and location but that integration sites were stable over extended passages in culture. Integration occurred at different sites throughout the genome and did not correspond to the location of specific cellular genes. However, integration sites were consistent with integration near or within known fragile sites in five of the six cell lines. Induction of aphidicolin-sensitive fragile sites in one cell line prior to in situ hybridization revealed that integrated HPV DNA was disrupted by fragile-site expression, suggesting that integration occurred within a fragile site.     

Characterization of three human malignant mesothelioma cell lines

Three human malignant mesothelioma cell lines, designated Mero-14, Mero-25, and Mero-41, have been isolated from effusions and from autopsy material of confirmed cases of malignant mesothelioma. Light and electron microscopy, cytogenetics, growth requirements, and intermediate filament expression of these cell lines were studied and, where possible, compared with the original tumor material of the patient. Cytologic and ultrastructural morphology was consistent with the mesothelial nature of the cells. All cell lines displayed a hyperdiploid karyotype similar to that of the tumor cells obtained directly from the patient. All three malignant mesothelioma cell lines had marker chromosomes 1, 3, 9, and 22, as well as other markers that were occasionally present in these cell lines and in other malignant mesotheliomas studied. Growth kinetic studies in medium supplemented with epidermal growth factor (EGF) showed increased proliferation and a decreased proliferation in medium supplemented with hydrocortisone (HC) or EGF plus HC. The three malignant mesothelioma cell lines were positive for the cytokeratins 7, 8, 18, and 19 based on immunofluorescence and immunoblotting tests with chain-specific monoclonal antibodies. The characteristics of these cell lines support the assumption that Mero-14, Mero-25 and Mero-41 are derived from malignant mesotheliomas and have retained their original character.     

建立永生化的中枢神经干细胞系

神经干细胞是中枢神经系统中具有自我更新和多向分化能力的细胞。体外培养的神经干细胞系具有无限增殖及可分化的特点 ,建立的神经干细胞系可用于对神经系统发育及中枢神经系统疾病治疗的研究。本文对目前已建的细胞系、永生化方法及其用于治疗各种神经损伤的动物模型的进展做了回顾。在治疗研究中 ,通过观察其移植后迁移、存活、整和、迁移及分化情况 ,可以认为神经干细胞的确具有应用前景 ,但仍需进行大量基础研究方可用于临床。     

亚硝基吡啶对人乳头状瘤病毒诱导的食管上皮永生化细胞的促癌作用

目的 观察亚硝基吡啶在细胞恶性转化过程中的促癌作用.方法 用HPV18E6E7诱导食管上皮细胞永生化细胞系SHEE,第17代细胞培养在50 ml培养瓶.加入亚硝基吡啶（Nnitrosopiperidine,NPIP）0,2,4,8 mmol/L作用3周.用相差显微镜检查细胞形态,流式细胞仪检测细胞增殖和凋亡;染色体常规制样,检查染色体众数;细胞软琼脂集落形成及接种裸小鼠检查成瘤性;用Western blot检测HPV18表达.结果 当细胞暴露在8 mmol/L NPIP时细胞死亡增加,只剩少量活细胞.换正常培基代替NPIP,经4周后细胞进入增殖状态,细胞出现增生和异型增生.第8周末细胞软琼脂培养有大集落形成,接种裸小鼠成瘤.2,4 mmol/L组细胞倍增时间延长,细胞未能成瘤.8 mmol/L NPIP组染色体众数61～65,对照组56～61.实验组和对照组HPV阳性.结论 NPIP促进人乳头状瘤病毒诱导人胚食管永生化上皮恶性转化,HPV18E6E7和NPIP能协同作用加速食管上皮恶性转化.     

Consistent chromosomal anomalies in keratinocyte cell lines derived from untreated malignant lesions of the oral cavity

Cytogenetic analysis has been carried out on 8 early passage cell lines derived from 8 untreated human oral squamous cell carcinomas. Clonal aberrations were detected in the karyotypes of each cell line. A high frequency of breakpoints were noted on chromosomes 1, 7, 8, 9, 11, and X. An isochromosome 8 was present in 6 out of 8 cell lines; isochromosome 9 (3 cell lines) and isochromosome 11 (1 cell line) were also found. In 4 out of 8 cell lines the X chromosome harboured breakpoints, a novel finding in oral squamous cell carcinomas. Breakpoints were common on chromosome 1, with 1p12–p13 most frequently involved. Tandem duplication of 11q13–q23, which contains a number of growth regulatory genes, was also noted in 2 cases. We correlate the sites of proto-oncogenes and other growth control genes with chromosomal breakpoints and suggest that several of these may play a role in the pathogenesis of oral cancer. © 1993 Wiley-Liss, Inc.     

Two clonal cell lines of immortalized human corneal endothelial cells show either differentiated or precursor cell characteristics

Access to primary human corneal endothelial cells (HCEC) is limited and donor-derived differences between cultures exacerbate the issue of data reproducibility, whereas cell lines can provide sufficient numbers of homogenous cells for multiple experiments. An immortalized HCEC population was adapted to serum-free culture medium and repeated cloning was performed. Clonally grown cells were propagated under serum-free conditions and growth curves were recorded. Cells were characterized immunocytochemically for junctional proteins, collagens, Na,K-ATPase and HCEC-specific 9.3.E-antigen. Ultrastructure was monitored by scanning and transmission electron microscopy. Two clonal cell lines, HCEC-B4G12 and HCEC-H9C1, could be isolated and expanded, which differed morphologically: B4G12 cells were polygonal, strongly adherent and formed a strict monolayer, H9C1 cells were less adherent and formed floating spheres. The generation time of B4G12 cells was 62.26 +/- 14.5 h and that of H9C1 cells 44.05 +/- 5.05 h. Scanning electron microscopy revealed that B4G12 cells had a smooth cell surface, while H9C1 cells had numerous thin filopodia. Both cell lines expressed ZO-1 and occludin adequately, and little but well detectable amounts of connexin-43. Expression of HCEC-specific 9.3.E-antigen was found commensurately in both cell lines, while expression of Na,K-ATPase alpha1 was higher in H9C1 cells than in B4G12 cells. B4G12 cells expressed collagen IV abundantly and almost no collagen III, while H9C1 cells expressed both collagens at reasonable amounts. It is concluded that the clonal cell line B4G12 represents an ideal model of differentiated HCEC, while H9C1 may reflect features of developing or transitional HCEC.