原代培养的细胞在生理上接近体内的状态,在细胞生物学研究中具有重要价值.气道(气管和支气管)上皮细胞较难培养,对生长条件要求高,目前国内这方面的报道很少.我们通过多年实践摸索,已能稳定地培养猪气道上皮细胞,现介绍其具体方法及细胞微管的检测和绿色荧光蛋白(GFP)的表达。

0 引言 神通营养液是以中药苦苣、当归、薏苡仁、枸杞子等为主要成分由河南鹤壁市高科技攻关组研制提供,临床用于解烟毒10+a,效果良好,现已被中国吸烟与健康协会推荐为保健品.我们通过体外实验对其抗肿瘤作用进行探讨,为神通营养液在防治肿瘤方面的应用,提供可靠的实验依据.

To investigate the effect of lithium on cell cycle progression of airway epithelial cells, primary pig tracheobronchial epithelial cells were incubated with lithium chloride (LiCl) at different concentrations (0, 5 mmol/L, and 10 mmol/L) and time (12 h, 16 h and 24 h). After the treatment, cells were counted, cell cycle profile was measured by BrdU labeling and flow cytometry, and expression of cyclin D1 and cyclin B1 were detected by Western blotting. The results showed that after 24h of 10mmol/L but not 5mmol/L LiCl treatment, proliferation of cells was slowed down as manifested by delayed confluence and cell number accumulation (P<0.05). Lithium did not change the percentage of cells in S phase (P>0.05), but 24 h incubation with 10 mmol/L LiCl induced a G2/M cell cycle arrest. Furthermore, 10mmol/L LiCl elevated cyclin D1 expression after 12h treatment, while expression of cyclin B1 increased more significantly after 24h incubation. These data demonstrate that lithium inhibits proliferation of pig airway epithelial cells by inhibiting cell cycle progression, and suggest that lithium-sensitive molecule(s) such as glycogen synthase kinase 3 may have a role in the regulation of growth of airway epithelial cells.

AIM: To detect the role of cyclic nucleotides in the alleviation of hypoxic pulmonary vasoconstriction (HPV) in chronic hypoxic animals. METHODS: The intracellular cAMP and cGMP of the cultured porcine pulmonary arterial smooth muscle cells (PASMC) and endothelial cells (PAEC) were assayed by RIA. The length of single PASMC during acute hypoxia was measured by imaging analysis system. RESULTS: The basal levels of cAMP and cGMP in PASMC and cGMP in PAEC of Chronic hypoxic groups decreased remarkably compared with normoxic groups ( P 0.01). Under acute hypoxia, the contents of cAMP and cGMP in PASMC of chronic hypoxic groups increased significantly ( P 0.01). Meanwhile, the percentage of PASMC with weak constrictive response in chronic hypoxic group was higher than that of control group. CONCLUSION: It's suggested that the changes of cAMP and cGMP in chronic hypoxic PASMC and PAEC might contribute to the increase in the basic tension of pulmonary artery and the alleviation of HPV in chronic hypoxic animals. [

应用原位杂交、免疫细胞化学及免疫斑点法观测香烟烟雾提取物(CSE)和酪氨酸激酶抑制剂tyrphostin25作用后,培养的猪气道上皮细胞上皮钙粘附素(E-cd)mRNA和蛋白、增殖细胞核抗原(PCNA)的表达及培养上清液中可溶型E-cd(sE-cd)含量的变化.结果发现,加入CSE作用4 h后,C4组细胞内E-cd表达比对照N4组降低了36%(P<0.01),而24 h后,C24组E-cd表达为对照N24组的1.72倍(P<0.01).PCNA表达量C24组细胞为N24组的1.38倍(P<0.01),而加入tyrphostin 25和CSE作用24 h后,CT24组细胞比N24组降低了40%(P<0.01).培养上清液中sE-cd的含量C4组和CT24组分别为相应N组的1.34倍和2.02倍,C24组比N24组降低了14%.结果表明:酪氨酸激酶活化时,E-cd滞留在胞浆中,细胞增殖能力增强;酪氨酸激酶活性抑制时,E-cd稳定表达于膜上,细胞增殖能力减弱.培养上清液中sE-cd的含量可间接反映气道上皮细胞损伤修复的状态.

采用3H-TdR掺入、结晶紫染色以及PCNA免疫细胞化学方法观察异搏定对缺氧和4种缩血管物质促肺动脉平滑肌细胞增生的影响.结果发现:①4种缩血管物质(ET-1、ATⅡ、A23178、KCl)均有不同程度的促肺动脉平滑肌细胞(PASMC)DNA合成的作用,其作用可被Ca2+通道阻滞剂异搏定阻断;②缺氧所致PASMC增殖的3项指标变化同样可被异搏定阻断.表明[Ca2+]i升高可能为缺氧和缩血管物质引起肺血管SMC收缩和增生的信息传递的共同途径.

体外进行器官培养 ,通过免疫组织化学和原位杂交方法检测香烟烟雾提取物 （CSE）作用 48、 72 h,小鼠气管上皮细胞上皮钙粘附素 （E- cd）表达的变化。结果表明 ,正常对照组 E- cd分布于气管假复层纤毛柱状上皮细胞连接处的胞膜上 ,5 0 % CSE作用后 ,膜上 E- cd表达减少 ,随着作用时间延长 ,胞浆内 E- cd表达明显增加 （P＜0 .0 5 ） ,而细胞内 E- cd m RNA含量无明显变化。提示 ,CSE对小鼠气管上皮细胞 E- cd表达的调控在翻译后水平 ,胞膜上 E- cd表达的下调与吸烟所致气道上皮细胞的损伤修复过程相关。

目的:Alzheimer病(Alzheimer disease, AD)的主要脑病理改变是神经原纤维缠结(neurofibrillary thangles, NFT)和老年斑(senile plaques, SP),且前者与AD临床痴呆症状呈相关关系.

The effects of erythropoietin (EPO)3 -enhancer on endothelium-dependent and endothelium-independent proliferation caused by hypoxia in cultured porcine pulmonary artery smooth muscle cells (PASMCs) were investigated with MTT test, H(3)-TdR incorporation and flow-cytometry. The results showed that (1) PASMCs exposed to hypoxia for 24 h proliferated significantly, which was suppressed by pretransfecting wild type EPO3 -enhancer fragments into PASMCs, but not by protransfecting mutant fragments; and (2) the conditioned medium of pulmonary artery endothelial cells (PAECs) exposed to hypoxia for 24 h promoted the proliferation of PASMCs. This effect was abolished when wild type EPO3 -enhancer fragments were transfected, but it persisted when mutant fragments were transfected. The results suggest that (1) the conditioned medium of hypoxic PAECs induces proliferation of PASMCs. This may be because that not only PAECs are sensitive to hypoxia, but also PASMCs respond to hypoxia directly; and the hypoxic responses of both endothelial cells (ECs) and smooth muscle cells (SMCs) can be inhibited by exogenous EPO3 -enhancer fragments. (2) Since there is a HIF-1 binding site in EPO3 -enhancer, there may be a common pathway for HIF-1 hypoxia signal transduction in hypoxic responses of ECs and SMCs.

应用酶消化法体外培养猪气道上皮细胞(AEC) ,加入一定浓度梯度的白细胞介素-1β(IL-1β)和肿瘤坏死因子α(TNF-α),应用免疫细胞化学染色和原位杂交方法观测AEC中E钙粘着蛋白(ECD)表达的变化.结果发现: 正常AEC中ECD表达于相邻上皮细胞间的胞膜上,其mRNA也有一定含量.一定浓度梯度的IL- 1β和TNF-α作用24 h后,胞膜上ECD表达减弱,而胞浆内ECD表达在低浓度时升高,高浓度时降低(P<0.01),同时ECD mRNA含量在低浓度时增多,而在高浓度时维持正常水平 (P>0.05).结果提示,IL-1β和TNF-α在翻译或翻译后水平使膜上ECD表达下调,参与了AEC的损伤修复反应.

To investigate whether the change of E-cadherin (ECD) expression plays a role in the injury and repair of airway epithelial cells (AEC) caused by smoking, porcine AECs were cultured by using an enzyme-dispersed method. After exposure of the AECs to cigarette smoke extract (CSE), the ECD expression in the cells was detected by using immunocytochemistry and in situ hybridization. The results showed that ECD was distributed on the plasma membrane at the cell junctions of AECs. After exposure to 20 % CSE, the membranous ECD expression was decreased, the cytoplasmic ECD expression was increased (P < 0.01) as the exposure time went on. But the content of ECD mRNA in the AECs did not chang. It suggests that the change of ECD expression is regulated at the posttranslational level and plays a role in the injury and repair of AEC caused by smoking.

目的观察香烟烟雾提取物（ＣＳＥ）对气道上皮细胞（ＡＥＣ）、上皮钙粘附素（ＥＣＤ）表达的影响，探讨其表达变化在吸烟致ＡＥＣ损伤中的作用。方法体外培养小鼠气管环及猪ＡＥＣ，加入一定浓度的ＣＳＥ，应用免疫组化染色和图像分析法检测ＡＥＣ中ＥＣＤ表达的变化。结果正常ＡＥＣ中ＥＣＤ表达于相邻上皮细胞连接处的胞膜上，２０％ＣＳＥ作用１２和２４小时后，膜上ＥＣＤ表达减少，胞浆内表达量分别为正常对照组的１６１和１３２倍（Ｐ均＜００１），且２４小时表达量为１２小时的１２１倍（Ｐ＜００１）。结论ＣＳＥ使ＡＥＣ中ＥＣＤ膜上表达下调，胞浆内表达增高，该表达变化可能介导了吸烟对ＡＥＣ的损伤作用。

At high concentration (50 μg/ml), diallyl trisulfide (DATS) had an inhibitory effect on T cell activation (compared with control group,P<0.05). But at appropriate concentrations (3.125–12. 5 μg/ml), DATS augmented the activation of T lymphocytes by Con A (compared with control group,P<0. 01). The augmentation of T cell activation by DATS was related to its inhibitory effect on the production of nitric oxide (NO) by macrophages. In a wide range of concentrations (1–100 μg/ml), DATS can inhibit the production of NO by macrophages (P<0.05,P<0.01). In addition, DATS can antagonize the inhibition of tumor-derived immunosuppressive factors produced by S180 cells and Ehrlich ascitic cancer cells on the activation of T cells, and reduce the inhibitory rate significantly (P < 0.01). DATS, despite its inhibition of the production of NO by macrophages, can significantly enhance the production of hydrogen peroxide (H2O2) by macrophages. When macrophages were pretreated with DATS for 24 h, the cytotoxicity % of macrophages to three tumor cell lines was significantly higher than that in corresponding control group (P<0.05,P<0.01). In the presence of both DATS and LPS, the cytotoxicity of macrophages was further enhanced so that the cytotoxicity % of macrophages to tumor cells was significantly higher than either that in the presence of DATS alone or that in the presence of LPS alone (P<0. 05,P<0. 01). These results indicate that DATS can augment the activation of T cells and enhance the anti-tumor function of macrophage, suggesting that DATS may be potentially useful in tumor therapy.

Effects of Chinese Medicinal Preparation "Re Du Qing" (RDQ) on the activation, proliferation and membrane fluidity of T lymphocytes from human peripheral blood were studied by means of 3H-TdR incorporation and DPH fluorescence polarization. The results showed that "RDQ" can: 1) significantly inhibit the activation of T lymphocytes; 2) restrain the proliferation of activated T lymphoblasts in the presence of exogenous interleukin-2 (IL-2); and 3) increase the membrane fluidity of T lymphocytes and antagonize the decreased fluidity of lymphocyte membrane mediated by Con A or PHA. The functional abnormalities of T lymphocytes in some autoimmune diseases such as arthritis and the usefulness of RDQ in the treatment of these diseases were also discussed.

A549, a human lung cancer cell line, spontaneously produces a tumor-derived immunosuppressive factor (TDSF) which inhibits the production and action of interleukin 1 (IL-1). After exposure of macrophages to TDSF for 5 h, the production of IL-1 by macrophages was significantly inhibited. The inhibition was much stronger if TDSF existed continuously in macrophage culture. The response of thymocytes treated with nylon wool to exogenous IL-1 was significantly suppressed in the presence of TDSF, suggesting that TDSF can inhibit the action of IL-1. The thymocytes untreated with nylon wool could proliferate after being stimulated with Con A. The proliferation was significantly suppressed by TDSF in a dose-dependent manner. These results suggest that the inhibitory action of TDSF on T cell activation is associated with IL-1, and that TDSF might exert an inhibitory action on other reactions mediated by IL-1. Furthermore, TDSF can reduce the supplementation of new T cells by inhibiting the proliferation of thymocytes.