In-vitro cultivation of normal human oral keratinocytes

目的　建立一个稳定的人正常口腔粘膜角化细胞体外培养体系。方法　取正常口腔粘膜 ,经dispase及胰蛋白酶消化获取细胞 ,采用无血清培养液进行原代及传代培养 ,并进行形态学观察和角蛋白免疫组化染色等一系列细胞定性研究。结果　获取的细胞为单一上皮细胞 ;细胞可连续传 4 - 5代 ,成活 30 - 5 0天 ;细胞呈铺路石状 ,为上皮细胞的典型形态 ;电镜下见细胞具有丰富的张力丝和桥粒等特征性超微结构 ;角蛋白免疫组化染色阳性。结论　应用dispase酶及无血清培养液 ,在无 3T3细胞的条件下可成功进行人正常口腔粘膜角化细胞连续传代培养。     

电离辐射诱导人正常角化细胞衰老死亡的作用机制研究

目的 研究电离辐射对人正常角化细胞的影响,探讨放射治疗引起口腔黏膜炎的可能作用机制.方法 原代培养正常人角化细胞在5 Gy辐射后,SA β-Gal染色法检测细胞衰老情况,CM-H2DCF-DH染色法检测细胞活性氧产生情况,实时荧光定量PCR检测氧化酶基因表达情况,γ-H2AX免疫荧光染色检测细胞DNA修复能力.结果 电离辐射可引起人正常角化细胞衰老死亡,细胞内活性氧大量产生,氧化酶基因表达明显增高,细胞DNA双链断裂.结论 电离辐射引起的正常角化细胞衰老死亡与活性氧产生和DNA损伤有关,这可能是放射治疗引起口腔黏膜炎的作用机制之一.     

KGF-transfected cells can stimulate growth and proliferation of human cultured keratinocytes in vitro

Objective: To establish two stably KGF-transfected, immortalized cell lines. Methods: HaCaT-keratinocytes and KMST-6-fibroblasts were transfected by liposome mediated gene transfer. Transfection effectivity, gene integration and configuration of the transgenic protein were investigated by ELISA, DANN-PCR and β-Gal-staining. Results: Most effective GF producing clones were tested by a colorimetric XTT-test. Conclusion: This is a significant acceleration of cell proliferation and mitosis of human keratinocytes in an Air Liquid Interface (ALI) test system.     

Enhancing effect of tazarotene on the HLA-DR expression of cultured human keratinocytes induced by interferon-gamma

Objective: To investigate the effect of tazarotene on the expression of HLA-DR induced by IFN-γ. Methods: (1) Keratinocytes from normal human skin were cultured in vitro;(2) Tazarotene, IFN-γ and the combination of the two compounds were incubated with the keratinocytes in medium, respectively. The expression of HLA-DR in keratinocytes was determined using immunocytochemistry techniques at 24h after incubation. Results: (1) There was rare expression of HLA-DR in normal human keratinocytes; (2) 10-6mol/L tazarotene failed to induce the expression of HLA-DR in keratinocytes at 24h after incubation; (3) 500 U/ml IFN-γ obviously induced the HLA-DR expression in keratinocytes at 24h after treatment; (4) After 24h, 10-7-10-5 mol/L tazarotene had a significantly enhancing effect on the expression of HLA-DR induced by IFN-γ (P<0.005). Conclusion: Tazarotene up-regulates the expression of HLA-DR in keratinocytes cultured in vitro when combined with IFN-γ . Therefore, the reduction of HLA-DR positive keratinocytes in psoriatic lesions may be attributed to not direct interaction of tazarotene in combination with IFN-γ but other pathways.     

甲壳胺对人角朊细胞培养DNA合成速率的影响

０引言甲壳胺是从甲壳类动物如虾、蟹等外壳中经化学方法提取的一种高分子化合物氨基多糖,有关文献报道了用甲壳胺制作的人工皮肤,手术缝合线对创面愈合具有促进作用［１］．我们采用角朊细胞悬液培养法检测了不同浓度的甲壳胺对角朊细胞ＤＮＡ合成速率的影响．１材料和...     

Ultraviolet B light-induced apoptosis in human keratinocytes enriched with epidermal stem cells and normal keratinocytes

Background  The stem-cell compartment is the primary target for the accumulation of oncogenic mutations. Overexposure to solar ultraviolet radiation is responsible for the development and progression of >90% of skin cancers. Ultraviolet B (UVB) light-induced keratinocyte apoptosis is a strong preventive mechanism against carcinogenesis. The aim of this study was to isolate keratinocytes enriched with putative human epidermal stem cells and to investigate their apoptotic induction by UVB.

Methods  Keratinocytes enriched with putative human epidermal stem cells were isolated by adherence to collagen IV and the expressions of β1-integrin and p63 were investigated. Keratinocytes enriched with putative human epidermal stem cells and normal keratinocytes were irradiated with UVB at 0–80 mJ/cm². The apoptotic response was investigated with phase-contrast microscopy, Hoechst 33342 staining, flow cytometry of annexin V/PI, and procaspase-3 Western blotting.

Results  Keratinocyte enriched with stem cells expressed high levels of p63 protein and β1-integrin and low level of pan-keratin (C11). In comparison to non-irradiated cells, significant apoptosis of keratinocyte enriched with stem cells was found with 40 and 80 mJ/cm² UVB. However, significant apoptosis of normal keratinocytes was only found for 80 mJ/cm² UVB.

Conclusions  Human epidermal stem cells can undergo apoptosis in response to UVB radiation and are more susceptible than other keratinocytes. The method could be used in vitro studies of human epidermal stem cells.

     

三氯乙烯诱导人表皮角质形成细胞凋亡的实验研究

目的 探讨有机溶剂三氯乙烯(TCE)诱导正常人表皮角质形成细胞(NHEK)凋亡的可能机制.方法 TCE处理NHEK后,分光光度法测细胞上清中半胱氨酸蛋白水解酶(Caspase)-3、8和9的活性,膜联蛋白V/碘化丙啶(PI)双染后流式细胞仪(FCM)测细胞凋亡,Rh123/PI双染后FCM测线粒体膜电位(△Ψm);用Caspase-3抑制剂或Caspase-9抑制剂预处理NHEK 1 h,验证Caspase的活化.结果 不同浓度TCE处理NHEK 4 h后培养不同时间,Caspase-3和9活性呈剂量和时间依赖的升高,培养12、24 h,各TCE处理组Caspase-3和9活性与溶剂对照比差异均有统计学意义(均P<0.05),而Caspase-8活性变化不明显.0.125、0.25、0.5、1.0和2.0 mmol/L TCE处理NHEK 4 h后培养12 h,膜联蛋白V~+/PI~-为(20.1±4.1)%、(30.0±7.5)%、(42.1±8.2)%、(56.0±6.1)%和(79.1±4.3)%,除0.125 mmol/L组外,其余组与溶剂对照组(9.4±3.0)%比较,差异均有统计学意义(均P<0.05).膜联蛋白V~+/PI~-与Caspase-3和9活性都呈正相关(r=0.786、0.736,均P<0.05),Caspase-3活性与Caspase~活性也呈正相关(r=0.845,P<0.05),膜联蛋白V~+/PI~-和Caspase-3活性均与Caspase-8活性无相关.100 μmol/L Z-DEVD-FMK预处理使2.0 mmol/L TCE处理的NHEK中Caspase-3活性从(0.963±0.043)nmol pNA·min~(-1)·ml~(-1)下降为(0.349±0.045)nmol pNA·min~(-1)·ml~(-1),膜联蛋白V~+/PI~-从(80.0±5.5)%下降为(16.3±3.2)%(P<0.01),Caspase-9活性变化不大(P>0.05).100μmol/L的Z-LEHD-FMK预处理不仅使得2.0 mmol/L TCE处理的NHEK中Caspase-3活性下降为(0.338±0.011)nmol pNA·min~(-1)·ml~(-1),膜联蛋白V~+/PI~-下降为(16.1±1.7)%,而且Caspase-9活性也从(0.821±0.031)nmol pNA·min~(-1)·ml~(-1)下降为(0.240±0.043)nmol pNA·min~(-1)·ml~(-1),差异有统计学意义(P<0.01).2.0 mmol/L的TCE处理NHEK 4 h后,Rh123荧光强度在培养4、8、12、24 h时均明显低于0 h(18.7±0.5,均P<0.01);0.125、0.5和2.0 mmol/L的TCE处理NHEK 4 h后培养8 h,Rh123荧光强度为16.1±0.5、12.1±0.6和8.1±0.6,均低于溶剂对照组(18.1±0.5,均P<0.01).结论 TCE诱导NHEK凋亡中,包括线粒体膜电位下降和Caspse-9依赖的Caspase-3的激活在内的内部凋亡途径可能发挥重要作用.     

正常人角质形成细胞和黑素细胞体外共培养体系的建立

目的体外建立正常人表皮角质形成细胞和黑素细胞直接接触的共培养体系。方法分别培养角质形成细胞和黑素细胞,体外建立角质形成细胞和黑素细胞直接接触的共培养模型,采用四甲基偶氮唑盐(MTT)比色法、紫外分光光度计检测、左旋多巴(L-Dopa)染色、免疫组织化学染色及透射电镜观察对共培养体系中的黑素细胞和角质形成细胞进行生物学鉴定。结果单独培养时,角质形成细胞呈圆形、"铺路石样"生长,黑素细胞呈两极或多级树突状生长;角质形成细胞和黑素细胞按一定比例混合培养后,2种细胞均迅速增殖,黑素细胞树突多为3～5个,与数十个角质形成细胞呈团块状生长,形成类似黑素单元的结构。共培养体系中的黑素细胞和角质形成细胞具备正常的生物学功能。结论角质形成细胞和黑素细胞直接接触的共培养体系为黑素细胞提供了更接近生理状态的体外研究模型。     

Establishment of a novel method for primary culture of normal human cervical keratinocytes

Background Cervical keratinocytes are recovered at a low numbers and frequently associated with contaminating human fibroblasts which rapidly overgrow the epithelial cells in culture with medium supplemented with 10% fetal bovine serum （FBS）. However, it is difficult to initiate keratinocyte cultures with serum-free keratinocyte growth medium alone because cell attachment can be poor. Therefore, the culture of these cells is extremely difficult. In this study, we described a modified culture medium and coated culture plastics for growing normal human cervical epithelial cells in vitro. Methods Normal cervical epithelial tissue pieces were obtained and digested with type I collagenase to dissociate the cells and a single cell suspension produced. The cells were cultured on plastic tissue culture substrate alone or substrate coated with collagen type I from rat tail, with modified keratinocyte serum-free medium （K-SFM） supplemented with 5% FBS. After attachment, the medium were replaced with K-SFM without FBS. The expression of basal keratins of the ectocervical epithelium, K5, K14 and K19 were assayed by immunofluorescence with monoclonal antibodies to identify the cell purity. Results Our results indicate that cells attached to the culture plastic more quickly in K-SFM supplemented with 5% FBS than in K-SFM alone, as well as to tissue culture plastic coated with collagen type I than plastic alone. The modified medium composed of K-SFM and 5% FBS combined with a specific tissue culture plastic coated with collagen type I from rat tail was the best method for culture of normal cervical epithelial cells. K5, K14 and K19 were assayed and keratinocyte purity was nearly 100%. Conclusion A novel, simple and effective method can be used to rapidly obtain highly purified keratinocytes from normal human cervical epithelium.     

Prolactin concentrations during the normal menstrual cycle: rhythms within rhythms

Prolactin concentrations were measured in frequent blood samples collected over a 2 h period randomly distributed during day and night, during the follicular and the luteal phases of the normal menstrual cycle in women. Episodic fluctuations in prolactin concentrations were observed throughout the 24 h period and the pattern of these fluctuations appeared to vary depending on the time, and sleep wake state. Thus a changing pattern of the 'primary pulse' of prolactin may be responsible for the time related and sleep related changes in prolactin during the 24 h period.     

低场单次激发快速序列在危重病人头颅中的应用

目的探讨SSFSE序列在危重病人头颅在的应用价值。方法选35例日常检查中出现躁动的危重病人分别进行常规头颅FSET,WI和SSFSET,WI检查。结果应用SSFSE序列检查出23例一级图像、2例三级图像：FSE无一级图像、21例三级图像;P〈0．05具有显著性差异,35例病人77个脑病灶SSFSE检出70个,漏诊7个,FSE检出36个,漏诊41个。结论应用SSFSE序列能明显消除生理运动伪影,对危重病人的疾病确诊具有临床意义。     

银屑病患者外周血单个核细胞对自身角质形成细胞的促生长作用

目的：了解银屑病患外周血单个核细胞（PBMCs)对自体表皮角质形成细胞（KCs）增生的作用。方法：分离3例银屑病患的PBMCs,经30Gy钴照射后与来自同一患的KCs进行混合培养,观察KCs增生代谢方面的变化。结果：经过混合培养,来自银屑病患皮损和皮损周围未受累皮肤的KCs在自体的PBMCs作用下均发生快速增生反应。结论：表皮KCs与浸润炎性细胞的相互作用可能诱发KCs的过度增生,在银屑病发病机制中发挥重要作用。     

一氧化氮对体外培养的角质形成细胞血管内皮生长因子表达的影响

目的：探讨一氧化氮(NO)对体外培养的角质形成细胞(KC)血管内皮生长因子(VEGF)表达的影响。方法：以NO供者S-亚硝酰-N-乙酰青霉胺(SNAP)作用于体外培养的KC，用免疫组化方法检测培养的KC VEGF、的表达，用双抗体夹心ELISA法定量检测培养的KC上清液中VEGF的含量。结果：免疫组化染色表明NO可促进体外培养的KC产生VEGF，上清液检测结果亦表明NO可明显增加KCVEGF的分泌，并呈剂量依赖性。结论：NO可能通过对KC VEGF表达的影响而在其生理和病理过程中发挥作用。     

重组人白细胞介素10单克隆抗体对角朊细胞增殖及产生细胞因子的影响

目的 研究重组人白细胞介素10单克隆抗体对无血清培养的角朊细胞增殖、产生细胞因子的影响。方法 用四甲基偶氮唑蓝比色法(MTT)测定对细胞增殖的影响;用小鼠胸腺细胞增殖法检测IL-1;ELISA法检测IL-6、IL-8。结果 抗重组人白细胞介素10单克隆抗体促进角朊细胞增殖与IL-1、IL-6及IL-8的分泌,并呈剂量依赖关系。结论 重组人白细胞介素10单克隆抗体促进角朊细胞增殖与细胞因子分泌。     

三氯乙烯诱导人表皮角质形成细胞凋亡中依赖Caspase-9的Caspase-3激活

目的 探讨有机溶剂三氯乙烯(TCE)诱导正常人表皮角质形成细胞(NHEK)凋亡过程中可能的信号通路.方法 用0.125、0.25、015、1.0、2.0 mmol/L的TCE处理NHEK4 h,并培养4、8、12、24 h,分光光度法测上清中Caspase-9和Caspase-3活性,Annexin-V/PI双染后流式细胞仪(FCM)测细胞凋亡;Caspase-3抑制剂Z-DEVD-FMK和Caspase-9抑制剂Z-LEHD-FMK预处理组,用100 μmol/L ZDEVD-FMK或Z-LEHD-FMK预处理NHEK 1 h,再用2.0mmol/L TCE染毒4 h后培养至12 h.结果不同浓度TCE处理NHEK 4 h后培养不同时间,可诱导Caspase-3和Caspase-9活性升高,培养12、24 h时,各TCE处理组Caspase-3和Caspase-9活性与溶剂对照比差异有统计学意义(P<0.05).不同浓度TCE处理NHEK 4 h后培养12 h.凋亡细胞(Annexin-V+/PI-)所占百分比随TCE剂量增加而升高,除0.125 mmol/L TCE组外,其它各处理组AnnexinV+/PI-细胞所占百分比与对照组比差异均有统计学意义(P<0.05).相关分析显示Annexin-V+/PI-细胞所占百分比与Caspase-3和Caspase-9活性都呈正相关(P<0.05).100 μmol/L Z-DEVD-FMK预处理明显抑制2.0 mmol/L TCE诱导的Caspase-3活性上升和Annexin-V+/PI-细胞所占百分比的增加(P<0.05),对Caspase-9活性影响不明显(P>0.05).100 μmol/L Z-LEHD-FMK预处理不仅抑制2.0mmol/L TCE诱导的Caspase-3活性上升和Annexin-V+/PI-细胞所占百分比的增加,还抑制Caspase-9活性上升(P<0.01).结论 TCE诱导NHEK凋亡中,内部凋亡途径中上游关键酶Caspase-9和下游效应分子Caspase-3均激活.且Caspase-3的激活依赖Casapase-9的活化.     

脂多糖对正常人皮肤成纤维细胞胶原合成的影响

目的:研究脂多糖(lipopolysaccharide,LPS)对正常人皮肤成纤维细胞胶原合成的影响,以阐明LPS在皮肤创伤愈合中的可能作用.方法:体外培养正常人皮肤成纤维细胞,采用3H-脯氨酸掺入法观察不同浓度(0、0.005、0.01、0.05、0.1、0.5和1.0 μg·ml-1)的LPS对成纤维细胞胶原合成的影响.结果:LPS刺激浓度在0.005 μg·ml-1时,皮肤成纤维细胞胶原蛋白合成增加;随着刺激浓度增加,刺激作用增强;但当LPS浓度为0.5 μg·ml-1时,促胶原合成作用开始降低;当浓度达到1.0 μg·ml-1时则呈现抑制效应.结论:在一定浓度范围内LPS促进成纤维细胞胶原合成,表明LPS在皮肤创伤愈合中可能具有重要作用.     

Purification and identification of natural antikeratin autoantibody from normal human sera and its effects on the cultured keratinocytes

Objective: To purify the natural antikemtin autoantibody (AK auto-Ab) and observe its effects on the proliferation of the cultured keratinocytes. Methods: Natural AK auto-Ab was purified by using keratin affinity column, and then the titre and specificity of the Abs were studied by ELISA, immunoperoxidase staining and immuno-electronicmi-croscope. The effect of the purified Abs on the cultured keratino-cytes was assayed by ^3H-TdR incorporation. Results:Natural AK auto-Ab was obtained. The binding activity oflgG AK auto-Ab in purified Ab remained similar to that in pooled human sera, and the specificity of the obtained antibody is strong. The purified antibody could decrease the ^3H-TdR incorporation of the cultured keratinocytes in a dose-dependent manner. Conclusion: The method of purifying AK auto-Ab is simple, practicable and reliable. Natural AK auto-Ab, existing in normal human individuals, has inhibitory effect on the proliferation of the cultured keratinocytes.     

恶性疟原虫红内期的体外培养及其影响因素

液氮内保存的FCC—1株恶性疟原虫红内期用蜡烛缸—平皿法体外连续培养40天。疟原虫复苏后培养的第3天原虫密度开始上升.第11天达到最高峰,其中一皿的红细胞含虫率高达20.56%。影响恶性疟原虫体外培养的有关因素如RPMI—1640培养液,血清和红细胞,本文进行了讨论。     

顺产与剖宫产产妇初乳中抗感染因子浓度的比较

比较顺产与剖宫产产妇初乳中抗感染因子含量的差异。方法采用免疫比浊法测定顺产组及剖宫产组产妇分娩后４８、９６ｈ共１１４份初乳中ＳＩｇＡ、ＩｇＭ、补充Ｃ３、Ｃ４浓度。结论产后４８ｈ,剖宫产组初乳中抗感染因子的浓度明显高于顺产组。