肿瘤坏死因子对人精子顶体酶和顶体反应的影响及机制探讨

目的 研究肿瘤坏死因子(TNF-α)对正常精子顶体酶活性和顶体反应的影响及其机制。方法采用BAEE/ADH联合法测定顶体酶和三色染色法技术测定顶体反应。结果 TNF-α可显著降低精子顶体酶的活性和顶体反应(P<0.01;P<0.01),并且它可使精子中的Ca2+-ATPase和SOD的活性显著降低(P<0.05;P<0.001);但TNF-α可使精子中NOS活性增强及NO含量增加(P<0.001;P<0.001);对Na+-K+-ATPase活性影响不明显(P>0.05)。结论 TNF-α对精子顶体酶及顶体反应有一定的抑制作用,并且可能通过降低Ca+-ATPase活性,自由基和NO及增加NOS等多种途径来实现的。     

重组人肿瘤坏死因子-α对人正常精子运动影响的实验研究

目的 观察重组人肿瘤坏死因子-α(rhTNF-α)在体外对人正常精子运动的影响.方法 40例健康自愿者正常精液,Peroll梯度离心将精子密度调整为10×106/ml,分别与对照组以及终浓度为30 pg/ml、60 pg/ml、90 pg/ml、270 pg/ml的rhTNF-α孵育.在0.5h、1h、2h、3h、4h时间段取样,用CASA检测精子运动参数变化.结果 rhTNF-α终浓度为60、90、270 pg/ml各组精子的活力、直线速度、曲线速度、平均速度和前向运动明显低于对照组,差异均有统计学意义(P＜0.01),且270 pg/ml组的rhTNF-α对精子作用最为显著.结论 一定浓度的rhTNF-α可明显抑制人正常精子运动.     

卵巢早衰患者血清抗透明带抗体和肿瘤坏死因子-α、γ-干扰素及白细胞介素-2的分析

目的 :探讨抗透明带抗体 ( Azp Ab)和肿瘤坏死因子 -α( TNF-α) ,γ-干扰素( IFN-γ)及白细胞介素 -2 ( IL-2 )在卵巢早衰 ( POF)发病中的作用及其临床意义。方法 :以定量酶联免疫吸附试验 ( EL ISA)测定 POF患者血清中 Azp Ab水平 ,放射免疫测定 IFN-γ、IL-2和 TNF-α的水平。结果 :POF组 Azp Ab明显高于正常对照组 ( P<0 .0 0 1 ) ,TNF-α和 IL -2显著降低 ( P<0 .0 0 1 ) ,IFN-γ明显升高 ( P<0 .0 1 )。 POF患者中 ,Azp Ab阳性组 ,以上三种细胞因子水平均明显高于 Azp Ab阴性组。结论 :Azp Ab,TNF-α,IFN-γ和IL-2在自身免疫性 POF的发病中起着重要作用。     

肿瘤坏死因子α和干扰素α对膀胱癌患者LAK细胞增殖和细…

为探讨联合应用白细胞介素２（ＩＬ－２）和干扰素（ＩＦＮ）或肿瘤坏死因子（ＴＮＦ）对膀胱癌患者ＬＡＫ细胞的增殖和细胞毒作用的影响，采用淋巴细胞分离液梯度离心法从２１例膀胱癌患者外周血分离ＬＡＫ细胞并用ＩＬ－２激活培养，细胞于９６孔细胞培养板用细胞计数方法观察了不同浓度ＴＮＦα和ＩＦＮα对细胞增殖的影响，用膀胱癌细胞系ＢＩＵ－８７和ＥＪ细胞作为靶细胞，用ＭＴＴ法测定ＬＡＫ细胞的细胞毒。结果：ＴＮＦα呈     

橙皮苷对急性肝损伤模型小鼠肿瘤坏死因子 α和干扰素 γ表达的影响

目的 研究橙皮苷对刀豆蛋白A（Con A）致小鼠急性肝损伤的保护作用及其对肿瘤坏死因子-α（TNF-α）和干扰素-γ（IFN-γ）表达的影响。方法72只SPF级C57BL/6雄性小鼠,随机均分为正常对照组、模型对照组和橙皮苷组。橙皮苷组给小鼠连续灌胃橙皮苷悬浊液1 000 mg.kg-1,灌胃10 d,模型对照组灌胃等量0. 5%羧甲基纤维素钠,模型对照组和橙皮苷组均用Con A诱导小鼠急性肝损伤模型,测定血清丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）的含量;苏木精 伊红（HE）染色检查肝组织病理变化,反转录 聚合酶链反应（RT-PCR）法检测肝组织TNF-α和IFN-γ mRNA水平,酶联免疫吸附测定（ELISA）法检测血清TNF-α和IFN-γ水平。结果橙皮苷预处理后,与模型对照组比较,橙皮苷组ALT和AST水平显著降低（P<0. 01）,肝细胞坏死及炎性细胞浸润明显减轻,肝组织TNF-α和IFN-γ mRNA表达明显下调（P<0. 01）。橙皮苷组血清TNF-α和IFN-γ水平2 h分别为（717. 05±205. 22）,（611. 06±92. 82）pg.mL-1,6 h分别为（811. 56±167. 47）,（786. 19±215. 44）pg.mL-1。橙皮苷组TNF-α和IFN-γ水平较模型对照组明显降低（P<0. 01）,但Con A注射6 h,橙皮苷组TNF-α水平与模型对照组比较,差异无统计学意义（P＞0. 05）。结论橙皮苷预处理对Con A致小鼠急性肝损伤具有保护作用,其作用机制与其抑制TNF-α和IFN-γ的表达有关。     

肿瘤坏死因子α和干扰素α对膀胱癌患者LAK细胞增殖和细胞毒作用的影响

为探讨联合应用白细胞介素２（ＩＬ２）和干扰素（ＩＦＮ）或肿瘤坏死因子（ＴＮＦ）对膀胱癌患者ＬＡＫ细胞的增殖和细胞毒作用的影响，采用淋巴细胞分离液梯度离心法从２１例膀胱癌患者外周血分离ＬＡＫ细胞并用ＩＬ２激活培养，细胞于９６孔细胞培养板用细胞计数方法观察不同浓度ＴＮＦα和ＩＦＮα对细胞增殖的影响，用膀胱癌细胞系ＢＩＵ８７和ＥＪ细胞作为靶细胞，用ＭＴＴ法测定ＬＡＫ细胞的细胞毒。结果：ＴＮＦα呈剂量依赖性地加强由ＩＬ２诱导的ＬＡＫ细胞增殖，１０００Ｕ／ｍｌ的ＩＦＮα于４８小时也对ＬＡＫ细胞产生刺激作用，用５０００Ｕ／ｍｌ的ＴＮＦα可增强ＬＡＫ细胞对ＥＪ和ＢＩＵ８７细胞的细胞毒杀伤，ＩＦＮα对此则无明显影响。结果提示ＴＮＦα和ＩＦＮα增强膀胱癌患者ＬＡＫ细胞的增殖并在细胞毒杀伤中起重要作用，对临床膀胱癌的免疫治疗提供一定依据。     

干扰素α、丝裂霉素和顺铂对肿瘤坏死因子α抗膀胱肿瘤作用的影响

为探讨细胞因子和抗癌药物对肿瘤坏死因子抗膀胱癌作用的影响,将膀胱癌细胞系BIU-87作体外培养,并将肿瘤坏死因子α和不同浓度的丝裂霉素(MC)、顺铂(CDDP)以及干扰素(IFN-α)作用于细胞,用MTT法测定细胞毒效果。TNF-α对膀胱癌细胞BIU-87有明显的浓度依赖性抑制作用,MC(2.5μg-5.0μg/ml)与TNF-α(10000U/ml)作用于细胞后的细胞活力及增殖明显低于单用TNF-α或MC实验组,说明MC可增强TNF-α对BIU-87细胞的抑制,两者发挥协同作用。虽然CDDP与TNF-α作用细胞后细胞活性及增殖也低于单用TNF-α组,然而与CDDP对照组之间却无显著性差异。IFN-α(2500U-5000U/ml)与TNF-α(10000U/ml)作用于细胞后的细胞活性及增殖明显被抑制。TNF-α与IFN-α或MC对膀胱癌细胞的抑制有协同作用。该研究将对临床应作TNF-α治疗膀胱肿瘤提供一定的理论依据。     

孕酮对人精子受精能力的影响

用不同浓度孕酮处理人精子，研究孕酮对人精子穿透去透明带金黄地鼠卵能力的影响。结果发现低浓度孕酮使人精子的穿卵率和穿卵指数均有极显著的提高，而高浓度孕酮则对人精子穿卵率和穿卵指数有降低作用。     

肿瘤坏死因子α-308多态性与弱精子症关系的研究

目的:探讨肿瘤坏死因子α(TNFα)基因启动子区-308位点基因型多态性与弱精子症的关系。方法:采用等位基因特异多聚酶链式反应(ASPCR)和聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)法分别对187例男性不育患者(分3组:A组,60例,为弱精子症组;B组,65例,为少弱精子症组;C组,62例,为正常精液不育组)的TNFα基因-308位点基因型进行分析。采用放射免疫分析法测定患者精浆中的TNF-α水平,并对各组数据进行统计学分析。结果:观察各组TNFα-308位点基因型GA/AA频率,与C组(8.06%)相比,A组(21.67%)和B组(26.15%)显著增高,差异均有统计学意义(P<0.05)。采用Spearman方法分析,各组间TNFα-308位点等位基因GA+AA类型与a+b级精子百分率呈显著负相关关系(r=-0.690,P<0.05)。观察各组精浆TNF-α水平,与C组[(4.03±0.66)ng/ml]相比,A组[(4.23±0.45)ng/ml]和B组[(4.29±0.47)ng/ml]显著升高,差异均有统计学意义(P<0.05)。而A组与B组相比,差异无统计学意义(P>0.05)。观察TNFα-308位点不同等位基因(GA+AA、GG)间的精浆TNFα水平,与GG类型组[(4.06±0.45)ng/ml]相比,GA+AA类型组[(4.61±0.29)ng/ml]显著升高,差异有统计学意义(P<0.05)。结论:不论精子密度如何,TNFα-308GA/AA的频率与a+b级精子百分率呈显著负相关关系,其机制之一可能与精浆TNF-α水平有关。据此推测,治疗弱精子症,抗TNFα方案可能有效;另外,精浆TNFα水平的检测可作为男性不育症的辅助诊断指标。     

γ干扰素与肿瘤坏死因子α对肠上皮屏障功能影响的实验研究

目的 观察炎症介质γ干扰素及TNF-α联合作用对肠上皮屏障功能的影响并探讨其分子机制.方法建立人肠上皮细胞株Caco-2单层细胞培养模型,分别在预处理的24孔板与6孔板中采用DMEM培养基培养.将2种培养板中细胞均按随机数字表法分为对照组(常规培养)、γ干扰素组(加入终浓度为10 ng/mL γ干扰素培养)、TNF-α组(加入终浓度为10 ng/mL TNF-α培养)、γ干扰素+TNF-α组(加入终浓度均为10 ng/mL的γ干扰素与TNF-α培养).24孔板细胞处理后0 h(即刻)及6、12、24、36、48 h,用电阻测定仪检测肠上皮细胞跨上皮电阻(TER);于48 h分别采用异硫氰酸荧光素-葡聚糖荧光示踪法与免疫荧光法,检测肠上皮细胞通透性及紧密连接咬合蛋白的分布与形态变化.6孔板细胞处理24 h时,用蛋白质印迹法检测咬合蛋白、磷酸化肌球蛋白轻链(pMLC)、肌球蛋白轻链激酶(MLCK)蛋白表达.对数据进行单因素方差分析与t检验.结果 (1)对照组各时相点肠上皮细胞TER无明显变化(F=0.86,P＞0.05);γ干扰素组与TNF-α组TER虽逐渐降低,但与处理后0 h比较差异均无统计学意义(F值分别为1.69、2.47,P值均大于0.05);γ干扰素+TNF-α组TER从24 h起显著低于处理后0 h(t=4.97,P＜0.05),并明显低于其余3组(F=11.54,P＜0.05).(2)γ干扰素+TNF-α组的肠上皮细胞通透性[(1197±215)pmo1]显著高于对照组、γ干扰素组与TNF-α组[(303±93)、(328±76)、(797±177)pmol,t值分别为4.8、5.0、6.9,P值均小于0.01].(3)24 h时各组咬合蛋白表达量无明显变化(F=0.26,P＞0.05).48 h时对照组咬合蛋白排列规则;γ干扰素组及TNF-α组咬合蛋白排列不规则;而γ干扰素+TNF-α组咬合蛋白排列不连续,发生明显重分布,胞质内分布增加.(4)γ干扰素+TNF-α组pMLC蛋白表达量(0.95±0.05)显著高于对照组、γ干扰素组与TNF-α组(0.57±0.12、0.56±0.07、0.59±0.10,F=17.97,P＜0.01),MLCK蛋白表达量(1.57±0.36)也显著高于其余3组(0.85±0.18、1.04±0.23、1.00±0.07,F=9.05,P＜0.05).结论γ干扰素与TNF-α联合作用通过增加MLCK及pMLC蛋白表达,引起肠上皮屏障功能损害.

Abstract：

Objective To investigate the effect of combination of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha ( TNF-α ) on intestinal epithelial barrier function. Methods The Caco-2 monolayers were cultured in DMEM nutrient solution, and then they were inoculated in 24-well or 6-well plate with Transwell inserts. They were divided into control group ( ordinary treatment), IFN-γ group ( with addition of 10 ng/mL IFN-γ), TNF-α group (with addition of 10 ng/mL TNF-α), and IFN-γ plus TNF-α group (with addition of 10 ng/mL TNF-α and 10 ng/mL IFN-γ ). Monolayers inoculated in 24-well plate were collected for determination of transepithelial electrical resistance (TER) with an ohmmeter at post treatment hour (PTH) 0, 6, 12, 24, 36, and 48, the permeability of monolayers with fluorescein isothiocyahate-labeled dextran (FITC-dextran) tracer method at PTH 48, the distribution and morphological change of tight junction occludin with immunofluorescence assay at PTH 48. Monolayers inoculated in 6-well plate were collected for determination of protein expression of occludin, myosin light chain kinase (MLCK), and phosphorylated MLC (pMLC) with Western blot at PTH 24. Data were processed with one-way analysis of vaiiance and t test. Results ( 1 ) There was no obvious difference in TER in control group at each time point ( F = 0. 86, P ＞ 0.05 ). TER in IFN-γgroup and TNF-α group were gradually decreased during PTH 6-48,but showed no statistical difference as compared with that at PTH 0 ( with F value respectively 1.69, 2.47,P values all above 0.05 ). TER in IFN-γplus TNF-α group was significantly decreased from PTH 24 as compared with that at PTH0 ( t =4.97, P ＜0.05) and that in each of the other three groups ( F =11.54,P ＜ 0.05 ). (2) The permeability of monolayers in IFN-γplus TNF-α group [( 1197 ± 215 ) pmol] was significantly higher than that in control group, IFN-γ group, and TNF-α group [( 303 ± 93 ), ( 328 ± 76),(797 ± 177) pmol, with t value respectively 4.8, 5.0, 6.9, P values all below 0.01]. (3) There was no statistical difference in occludin expression at PTH 24 among four groups ( F = 0.26, P ＞ 0.05). The occludin in control group at PTH 48 was regular in arrangement, while that in IFN-γand TNF-α groups was irregular in arrangement. The arrangement of occludin in IFN-γ plus TNF-c group at PTH 48 was interrupted,with obvious redistribution in cytoplasm. (4) The protein expression of pMLC in IFN-γ plus TNF-α group (0.95 ±0.05) was significantly higher than that in control group, IFN-γ group, or TNF-α group (0.57 ±0.12, 0.56 ±0.07, 0.59 ±0. 10, respectively, F = 17.97, P ＜0.01). The protein expression of MLCK in IFN-γplus TNF-α group (1.57 ±0.36) was also significantly higher than that in control, IFN-γ, TNF-α groups (0.85 ± 0.18, 1.04 ± 0. 23, 1.00 ± 0.07, respectively, F = 9.05, P ＜ 0.05 ). Conclusions Combination of IFN-γand TNF-α can induce intestinal epithelial barrier dysfunction by up-regulating MLCK protein expression and promoting MLC phosphorylation.     

肿瘤坏死因子联合干扰素γ治疗原发性肝癌的实验研究

肿瘤坏死因子 (ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒ,ＴＮＦ)具有显著抗肿瘤作用 ,但在临床上单独应用ＴＮＦ治疗肿瘤 ,效果并不理想。主要问题是低剂量ＴＮＦ不足以有效地杀灭肿瘤 ,若增加剂量又可能会出现恶液质、休克等严重毒副作用。本研究以已建立的人肝癌裸小鼠原位移植模型为实验对象 ,采用体外细胞毒性试验和瘤体内注射的直接给药途径 ,观察干扰素γ(Ｉｎｔｅｒｆｅｒｏｎγ ,ＩＦＮγ)增强ＴＮＦα治疗原发性肝癌 (ｈｅｐａｔｏｃｅｌｌｕｌａｒｃａｒｃｉｎｏｍａ ,ＨＣＣ)的效果。1.材料与方法 :ＢＡＬＢ/ＣＡ裸小鼠 ,建立人…     

肿瘤坏死因子-α和神经病理性疼痛

背景 神经病理性疼痛(Neuropatlic pain,NP)是神经系统损伤引起的一种慢性疼痛,其发病机制复杂,至今尚缺乏有效的治疗药物.目的 肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)是神经损伤和神经炎症过程早期释放的重要致炎细胞因子.近年来,许多研究显示外周及脊髓免疫细胞激活后表...     

γ-干扰素加强肿瘤坏死因子相关凋亡诱导配体抑制胆管癌细胞的作用

目的　探讨肿瘤坏死因子 (TNF)相关凋亡诱导配体 (TRAIL)对人胆管癌的作用及γ 干扰素 (IFN γ)对TRAIL抗瘤活性的影响。方法　应用透射电镜、琼脂糖凝胶电泳和流式细胞仪 (FCM ) ,研究TRAIL对人胆管癌细胞的抑制作用及IFN γ对TRAIL抗瘤活性的影响。结果　透射电镜和DNA琼脂糖凝胶电泳可见到典型细胞凋亡特征。FCM分析显示 :浓度为 0、1、10、10 0、10 0 0 μg/L的TRAIL 2 4h引起QBC93 9细胞的凋亡率分别为 (1.66± 0 .73 ) %、(8.83± 0 .5 4) %、(2 2 .3 0± 0 .64 ) %、(4 2 .5 0± 0 .47) %、(4 9.0 6± 0 .72 ) % ,与对照组比较差异有显著性 (P <0 .0 1)。IFN γ与TRAIL 10 0 μg/L联合应用 ,当IFN γ浓度大于 5 0U /ml或 10 0U /ml时 ,IFN γ作用时间超过 2 4h分别与单用TRAIL组比较 ,明显加强QBC93 9细胞凋亡 (P <0 .0 1)。结论　TRAIL通过诱导胆管癌细胞凋亡而起到抑癌作用 ,IFN γ能加强TRAIL诱导胆管癌细胞的凋亡。     

孕酮对人精子受精能力的影响

用不同浓度孕酮（Ｐ）处理人精子，研究孕酮对人精子穿透去透明带金黄地鼠卵能力的影响。结果发现低浓度孕酮（２０μｍｏｌ／Ｌ）使人精子的穿卵率和穿卵指数均有极显著的提高，而高浓度孕酮（１０００μｍｏｌ／Ｌ）则对人精子穿卵率和穿卵指数有降低作用。提示人工授精时可用低浓度孕酮处理穿卵率低下的病人精子，以提高人工授精的成功率     

肿瘤坏死因子α对膀胱肿瘤细胞作用的影响因素探讨

目的 探讨肿瘤坏死因子抗膀胱癌细胞作用的影响因素。方法 将肿瘤坏死因子α（ＴＮＦ－α）和不同浓度的丝裂霉素（ＭＣ），顺铂（ＣＤＤＰ）以及干扰素α（ＩＦＮ－α）作用于体外培养的膀胱癌细胞系ＢＩＵ－８７，用噻唑蓝（ＭＴＴ）法测定细胞毒效果。结果 ＴＮＦ－α对ＢＩＵ－８７细胞有明显的浓度依赖性抑制作用，ＭＣ（２．５～５．０ｍｇ／Ｌ）或ＩＦＮ－α（２５００Ｕ－５０００Ｕ／ｍｌ）与ＴＮＦ－α（１００００Ｕ／     

肿瘤坏死因子受体在人精子的表达及其意义

目的　证实肿瘤坏死因子受体 (TNF R2 )在人精子有表达 ,为TNF α对精子的直接作用提供实验证据。　方法　用Western印迹法检测TNF R2 在 15例生育者精子的表达。　结果　 15例生育者的精子均有TNF R2 表达。　结论　TNF R2 在人精子有表达 ,该受体可能参与了TNF α在人精子的信号转导及对精子功能的调控     

γ干扰素与肿瘤坏死因子α对肠上皮屏障功能影响的实验研究

Objective To investigate the effect of combination of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha ( TNF-α ) on intestinal epithelial barrier function. Methods The Caco-2 monolayers were cultured in DMEM nutrient solution, and then they were inoculated in 24-well or 6-well plate with Transwell inserts. They were divided into control group ( ordinary treatment), IFN-γ group ( with addition of 10 ng/mL IFN-γ), TNF-α group (with addition of 10 ng/mL TNF-α), and IFN-γ plus TNF-α group (with addition of 10 ng/mL TNF-α and 10 ng/mL IFN-γ ). Monolayers inoculated in 24-well plate were collected for determination of transepithelial electrical resistance (TER) with an ohmmeter at post treatment hour (PTH) 0, 6, 12, 24, 36, and 48, the permeability of monolayers with fluorescein isothiocyahate-labeled dextran (FITC-dextran) tracer method at PTH 48, the distribution and morphological change of tight junction occludin with immunofluorescence assay at PTH 48. Monolayers inoculated in 6-well plate were collected for determination of protein expression of occludin, myosin light chain kinase (MLCK), and phosphorylated MLC (pMLC) with Western blot at PTH 24. Data were processed with one-way analysis of vaiiance and t test. Results ( 1 ) There was no obvious difference in TER in control group at each time point ( F = 0. 86, P ＞ 0.05 ). TER in IFN-γgroup and TNF-α group were gradually decreased during PTH 6-48,but showed no statistical difference as compared with that at PTH 0 ( with F value respectively 1.69, 2.47,P values all above 0.05 ). TER in IFN-γplus TNF-α group was significantly decreased from PTH 24 as compared with that at PTH0 ( t =4.97, P ＜0.05) and that in each of the other three groups ( F =11.54,P ＜ 0.05 ). (2) The permeability of monolayers in IFN-γplus TNF-α group [( 1197 ± 215 ) pmol] was significantly higher than that in control group, IFN-γ group, and TNF-α group [( 303 ± 93 ), ( 328 ± 76),(797 ± 177) pmol, with t value respectively 4.8, 5.0, 6.9, P values all below 0.01]. (3) There was no statistical difference in occludin expression at PTH 24 among four groups ( F = 0.26, P ＞ 0.05). The occludin in control group at PTH 48 was regular in arrangement, while that in IFN-γand TNF-α groups was irregular in arrangement. The arrangement of occludin in IFN-γ plus TNF-c group at PTH 48 was interrupted,with obvious redistribution in cytoplasm. (4) The protein expression of pMLC in IFN-γ plus TNF-α group (0.95 ±0.05) was significantly higher than that in control group, IFN-γ group, or TNF-α group (0.57 ±0.12, 0.56 ±0.07, 0.59 ±0. 10, respectively, F = 17.97, P ＜0.01). The protein expression of MLCK in IFN-γplus TNF-α group (1.57 ±0.36) was also significantly higher than that in control, IFN-γ, TNF-α groups (0.85 ± 0.18, 1.04 ± 0. 23, 1.00 ± 0.07, respectively, F = 9.05, P ＜ 0.05 ). Conclusions Combination of IFN-γand TNF-α can induce intestinal epithelial barrier dysfunction by up-regulating MLCK protein expression and promoting MLC phosphorylation.     

γ干扰素与肿瘤坏死因子α对肠上皮屏障功能影响的实验研究

Objective To investigate the effect of combination of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha ( TNF-α ) on intestinal epithelial barrier function. Methods The Caco-2 monolayers were cultured in DMEM nutrient solution, and then they were inoculated in 24-well or 6-well plate with Transwell inserts. They were divided into control group ( ordinary treatment), IFN-γ group ( with addition of 10 ng/mL IFN-γ), TNF-α group (with addition of 10 ng/mL TNF-α), and IFN-γ plus TNF-α group (with addition of 10 ng/mL TNF-α and 10 ng/mL IFN-γ ). Monolayers inoculated in 24-well plate were collected for determination of transepithelial electrical resistance (TER) with an ohmmeter at post treatment hour (PTH) 0, 6, 12, 24, 36, and 48, the permeability of monolayers with fluorescein isothiocyahate-labeled dextran (FITC-dextran) tracer method at PTH 48, the distribution and morphological change of tight junction occludin with immunofluorescence assay at PTH 48. Monolayers inoculated in 6-well plate were collected for determination of protein expression of occludin, myosin light chain kinase (MLCK), and phosphorylated MLC (pMLC) with Western blot at PTH 24. Data were processed with one-way analysis of vaiiance and t test. Results ( 1 ) There was no obvious difference in TER in control group at each time point ( F = 0. 86, P ＞ 0.05 ). TER in IFN-γgroup and TNF-α group were gradually decreased during PTH 6-48,but showed no statistical difference as compared with that at PTH 0 ( with F value respectively 1.69, 2.47,P values all above 0.05 ). TER in IFN-γplus TNF-α group was significantly decreased from PTH 24 as compared with that at PTH0 ( t =4.97, P ＜0.05) and that in each of the other three groups ( F =11.54,P ＜ 0.05 ). (2) The permeability of monolayers in IFN-γplus TNF-α group [( 1197 ± 215 ) pmol] was significantly higher than that in control group, IFN-γ group, and TNF-α group [( 303 ± 93 ), ( 328 ± 76),(797 ± 177) pmol, with t value respectively 4.8, 5.0, 6.9, P values all below 0.01]. (3) There was no statistical difference in occludin expression at PTH 24 among four groups ( F = 0.26, P ＞ 0.05). The occludin in control group at PTH 48 was regular in arrangement, while that in IFN-γand TNF-α groups was irregular in arrangement. The arrangement of occludin in IFN-γ plus TNF-c group at PTH 48 was interrupted,with obvious redistribution in cytoplasm. (4) The protein expression of pMLC in IFN-γ plus TNF-α group (0.95 ±0.05) was significantly higher than that in control group, IFN-γ group, or TNF-α group (0.57 ±0.12, 0.56 ±0.07, 0.59 ±0. 10, respectively, F = 17.97, P ＜0.01). The protein expression of MLCK in IFN-γplus TNF-α group (1.57 ±0.36) was also significantly higher than that in control, IFN-γ, TNF-α groups (0.85 ± 0.18, 1.04 ± 0. 23, 1.00 ± 0.07, respectively, F = 9.05, P ＜ 0.05 ). Conclusions Combination of IFN-γand TNF-α can induce intestinal epithelial barrier dysfunction by up-regulating MLCK protein expression and promoting MLC phosphorylation.     

肿瘤坏死因子-α、白细胞介素-6和脓毒症

复习肿瘤坏死因子-α、白细胞介素-6和脓毒症发生、发展、变化及治疗的关系。