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.     

Analysis of anti-zona pellucida antibody and tumor necrosis factor-α,γ-interferon and interleukin-2 in sera from patients with premature ovarian failure

Objective:To investigate the role and the clinical significance of anti-zona pellucidaantibody (AzpAb) and tumor necrosis factor-α(TNF-α),γ-interferon(IFN-γ) and inter-leukin-2 (IL-2) in sera from patients with premature ovarian failure (POF).Methods: The AzpAb in the serum of POF patient was analyzed by means ofELISA. The levels of TNF-α, IL-2 and IFN-γ in the serum were determined by meansof radioimmunoassay (RIA).Results:The level of serum AzpAb in the POF patients was significantly higher thanthat of the normal controls(P＜0.001). The levels of TNF-α and IL-2 were significantlyreduced (P＜0. 001), and the level of IFN-γ was significantly elevated (P＜0.01). Thelevels of above three cytokines in AzpAb positive group were significantly higher thanthose of the negative group in POF patients.Conclusion: This study suggested that AzpAb, TNF-α, IFN-γ and IL-2 might playimportant roles in the pathogenesis of autoimmune POF.     

FK506对干扰素-γ和肿瘤坏死因子-α诱导间充质干细胞免疫抑制的影响

目的 观察用免疫抑制剂FK506对干扰素(IFN)-γ和肿瘤坏死因子(TNF)-α诱导的间充质干细胞(MSCs)免疫抑制功能的影响及其作用机制.方法 将经过不同炎症因子预处理的小鼠MSCs与脾细胞共培养,并在FK506作用72 h后,以噻唑蓝(MTT)比色法检测脾细胞增殖;以流式细胞术检测脾细胞凋亡;以实时定量聚合酶链反应(Real-time PCR)法检测MSCs发挥免疫抑制作用的主要分子,观察FK506对其影响.结果 IFN-γ和TNF-α联合预处理后的MSCs可以显著抑制脾细胞增殖,促进脾细胞凋亡,凋亡率可达(53.5±2.5)%,明显高于IFN-γ或TNF-α预处理的MSCs组以及对照组(P＜0.05),并且IFN-γ和TNF-α仅可以显著上调MSCs中诱导型一氧化氮合酶(iNOS)的表达,MSCs的免疫抑制作用可以被iNOS特异性抑制剂1400W所拮抗;但FK506则可以抑制MSCs中iNOS的表达,使MSCs对脾细胞增殖的抑制作用显著降低,脾细胞凋亡减少,凋亡率降至(44.3±3.2)%(P＜0.05).结论 FK506通过抑制IFN-γ和TNF-α联合预处理的MSCs中iNOS表达从而抑制其免疫抑制功能.

Abstract：

Objective To investigate the mechanism of immunosuppressant FK506 on the immunesuppression of mesenchymal stem cells ( MSCs) induced by interferon ( IFN) -γ and tumor necrosis factor (TNF)-α. Methods MSCs pretreated with inflammatory cytokines were co-cultured with mice spleen cells. After incubation with FK506 for 72 h, proliferation of spleen cells was measured by methyl thiazol tetrazolium (MTT) assay and the apoptosis was assessed by flow cytometry. Real-time polymerase chain reaction (PCR) was used to detect the major molecule that mediated the immunosuppression of MSCs and the effect of FK506 on the molecule. Results MSCs pretreated with inflammatory cytokines could inhibit the proliferation of spleen cells and increase the apoptosis of spleen cells, and the apoptosis rate was (53. 5 ±2. 5) % , which was higher than that of MSCs pretreated with IFN-γ or TNF-α and control group ( P ＜0. 05), and IFN-γ and TNF-α upregulated inducible nitric oxide synthase (iNOS) expression in MSCs significantly, and this immunosuppression could also be antagonized by a specific inhibitor 1400W. However,FK506 could inhibit iNOS expression in MSCs pretreated with IFN-γ and TNF-α, the inhibition of spleen cells proliferation by MSCs was reduced significantly, and the apoptosis rate of spleen cells was reduced to (44. 3 ±3. 2)% (P＜0. 05). Conclusion FK506 inhibits the immunosuppression of MSCs through downregulation of iNOS expression in MSCs pretreated with IFN-γ and TNF-α.     

早期应用冬眠合剂对严重烫伤大鼠小肠的保护作用

Objective To study the protective effect of early application of lytic cocktail on small intestine of severely scalded rats. Methods Sixty-six male SD rats were divided into sham injury group (SI, n =6) , scald group (S, n = 30) and scald + lytic cocktail group (SL, n =30) according to the random number table. After anesthesia, rats in the latter 2 groups were inflicted with 30% full-thickness scald, while rats in S group were sham scalded with 37 ℃ water. Resuscitation was carried out by intraperitoneal injection with 2 mL · kg-1 · %TBSA-1 lactated Ringer's solution in all rats; meanwhile 12 mL/kg lytic cocktail [ 1 mL pethidine (50 mg/mL) + 1 mL chlorpromazine (25 mg/mL) + 1 mL promethazine (25 mg/mL) + 125 mL saline] was hypodermically injected to rats in SL group, while 12 mL/kg saline was injected into rats in the other 2 groups. Samples of blood and small intestine were harvested from S and SL groups at post scald hour (PSH) 3, 6, 12, 24, 48 and from SI group at PSH 3, with 6 rats in each group at each time point. Pathological changes in intestine were observed, and the expression of intercellular adhesion molecule 1 (ICAM-1) and CD68 were determined with immunohistochemistry at PSH 24 for S and SL groups and at PSH 3 for SI group. Plasma levels of D-lactate, diamine oxidase (DAO) , IL-1β, TNF-α, IL-10 were determined with ELISA. Data were processed with one-way analysis of variance. Results (1) At PSH 24, mild hemorrhage, inflammatory cell infiltration and epithelial cell shedding were observed in small intestinal mucosa of rats in S group.Compared with S group, the intestinal villi of SL group were arranged regularly without obvious hyperemia and edema. (2) Expression levels of ICAM-1 and CD68 [(1. 69 ± 0.27)%, (0.80 ±0.09)%] in S group were significantly higher than those in SI group [(0.77 ± 0. 10) % , (0. 30 ± 0. 05) % , with F value respectively 77. 303 and 66. 933 , P < 0.05 or P < 0.01] and SL group [(0. 53 ± 0.09) % , (0. 32 ± 0. 06) % , with F value respectively 77. 303 and 66. 933 , P values all below 0.01]. (3) D-lactate levels of rats in SL group were significantly lower than those of rats in S group at PSH 12, 24 (with F value respectively 20. 936 and 19. 854, P values all below 0.01), while DAO levels of rats in SL group were significantly lower than those of rats in S group at PSH 3, 12 (with F value respectively 21. 930 and 11. 342 , P values all below 0. 05).(4) The levels of IL-1β and TNF-α in S group were significantly higher than those of SI group at each time point (P values all below 0.01). The levels of IL-1β and TNF-α in SL group were significantly higher than those of S group at PSH 6, 12 and 24 (with F value respectively 96. 517 , 17.365, 79.715 and 21. 328, 17. 682, 28.424, P <0.05 or P <0.01). IL-10 level in SL group was higher than that in S group at each time point, and the differences were statistically significant at PSH 6 and 24 (with F value respectively 8. 668, 19. 634, P < 0.05 or P < 0.01). Conclusions Early administration of lytic cocktail can attenuate edema and injury of intestinal mucosa in severely scalded rats. The mechanism may lie in that it can reduce the expression of ICAM-1 in intestinal mucosa, decrease the number of intestinal inflammatory cells and regulate the levels of inflammatory cytokines.     

早期应用冬眠合剂对严重烫伤大鼠小肠的保护作用

Objective To study the protective effect of early application of lytic cocktail on small intestine of severely scalded rats. Methods Sixty-six male SD rats were divided into sham injury group (SI, n =6) , scald group (S, n = 30) and scald + lytic cocktail group (SL, n =30) according to the random number table. After anesthesia, rats in the latter 2 groups were inflicted with 30% full-thickness scald, while rats in S group were sham scalded with 37 ℃ water. Resuscitation was carried out by intraperitoneal injection with 2 mL · kg-1 · %TBSA-1 lactated Ringer's solution in all rats; meanwhile 12 mL/kg lytic cocktail [ 1 mL pethidine (50 mg/mL) + 1 mL chlorpromazine (25 mg/mL) + 1 mL promethazine (25 mg/mL) + 125 mL saline] was hypodermically injected to rats in SL group, while 12 mL/kg saline was injected into rats in the other 2 groups. Samples of blood and small intestine were harvested from S and SL groups at post scald hour (PSH) 3, 6, 12, 24, 48 and from SI group at PSH 3, with 6 rats in each group at each time point. Pathological changes in intestine were observed, and the expression of intercellular adhesion molecule 1 (ICAM-1) and CD68 were determined with immunohistochemistry at PSH 24 for S and SL groups and at PSH 3 for SI group. Plasma levels of D-lactate, diamine oxidase (DAO) , IL-1β, TNF-α, IL-10 were determined with ELISA. Data were processed with one-way analysis of variance. Results (1) At PSH 24, mild hemorrhage, inflammatory cell infiltration and epithelial cell shedding were observed in small intestinal mucosa of rats in S group.Compared with S group, the intestinal villi of SL group were arranged regularly without obvious hyperemia and edema. (2) Expression levels of ICAM-1 and CD68 [(1. 69 ± 0.27)%, (0.80 ±0.09)%] in S group were significantly higher than those in SI group [(0.77 ± 0. 10) % , (0. 30 ± 0. 05) % , with F value respectively 77. 303 and 66. 933 , P < 0.05 or P < 0.01] and SL group [(0. 53 ± 0.09) % , (0. 32 ± 0. 06) % , with F value respectively 77. 303 and 66. 933 , P values all below 0.01]. (3) D-lactate levels of rats in SL group were significantly lower than those of rats in S group at PSH 12, 24 (with F value respectively 20. 936 and 19. 854, P values all below 0.01), while DAO levels of rats in SL group were significantly lower than those of rats in S group at PSH 3, 12 (with F value respectively 21. 930 and 11. 342 , P values all below 0. 05).(4) The levels of IL-1β and TNF-α in S group were significantly higher than those of SI group at each time point (P values all below 0.01). The levels of IL-1β and TNF-α in SL group were significantly higher than those of S group at PSH 6, 12 and 24 (with F value respectively 96. 517 , 17.365, 79.715 and 21. 328, 17. 682, 28.424, P <0.05 or P <0.01). IL-10 level in SL group was higher than that in S group at each time point, and the differences were statistically significant at PSH 6 and 24 (with F value respectively 8. 668, 19. 634, P < 0.05 or P < 0.01). Conclusions Early administration of lytic cocktail can attenuate edema and injury of intestinal mucosa in severely scalded rats. The mechanism may lie in that it can reduce the expression of ICAM-1 in intestinal mucosa, decrease the number of intestinal inflammatory cells and regulate the levels of inflammatory cytokines.     

脾动脉缩窄对门静脉高压大鼠脾脏iNOS表达及Th1/Th2平衡的影响

Objective To investigate the effect and the potential mechanism of splenic artery coarctation on the expression of iNOS and Th1/Th2 cytokines in spleen of cirrhotic rats with portal hypertension (PHT). Methods Cirrhotic rats were randomized into 3 groups (n= 10):sham operation group (SOG), splenic artery coarctation group (SAC) and splenic artery ligation group (SAL). Ten normal rats treated with sham operation were employed to serve as normal control group (NCG). Immunohistochemial staining was used to observe iNOS. RT-PCR was used to detect IFN-γ and IL-4mRNA. The Pearson's correlation analysis was used to investigate the relationship between iNOS and IFN-γ or IL-4. Results The expression of iNOS was increased significantly in spleen of cirrhotic rats as compared with NCG(P＜0. 01). It was decreased after SAC and SAL compared with SOG (P＜0. 01). The expression of IFN-γmRNA and IFN-γ/IL-4 of SOG were decreased but IL-4mRNA increased significantly than that of NCG(P＜0.01). IFN-γmRNA was increased after SAC compared with SOG (P＜0.05). IL-4mRNA was decreased and IFN-γ/IL-4 increased after SAC and SAL compared with SOG (P＜0. 05). The expression of iNOS was negatively correlated with the expression of IFN-γmRNA(r=-0.672, P＜ 0.01 ) and positively correlated with the expression of IL-4 mRNA (r=0.634,P＜0. 01). Conclusion The expression of iNOS is decreased and IFN-γ/IL-4 increased after SAC in spleen of cirrhotic rats with PHT and it may improve Th1/Th2 polarization by reducing the expression of iNOS.     

选择性肺叶隔离对胸科手术患者氧合及细胞因子的影响

Objective To determine the effect on concentrations of interleukin (IL-6) and tumor necrosis factor-α (TNF-α) in bronchoalveolar lavage fluid (BALF) and serum and oxygenation during selective lobar blockade. Methods Thirty patients undergoing esophagectomy or lobectomy were randomly assigned to the total lung collapse (TLC) group (n=15) and the selective lobar blockade (SLB) group( n=15). Anesthesia was induced and maintained with target-controlled infusion of propofol and remifentanil. After intubating with a 8.0 mm internal diameter single -lumen endotraeheal tube, by the guidance of fiberoptic bronchoscope, a 9F coopdech endobronchial blocker was placed into the target lobe in the SLB group, whereas the blocker was placed into the mainstem bronchus in the TLC group. Intermittent arterial blood gas analysis was performed at the following times: 15 min after two lung ventilation in the lateral decubitus position(T1)); 30 min (T2)and 60 min (T3) after TLC or SLB respectively; 15 min after recovering to two lung ventilation (T4), peak inspiratory airway pressure (Ppeak) was also recorded. BALF and blood samples were collected at T, and T4, the concentrations of IL-6 and TNF -α were measured using enzyme -linked immunosorbent assay. Results airway pressure increased significantly after the beginning of one-lung ventilation (F=215.746,P<0.05)in both groups, with more increasing extent in group T (F=53.798, P<0.01).Significant trends were found toward a better improvement in oxygention index with the group S compared with the group T after the beginning of one lung ventilation (F=1 3.747, P<0.05). IL-6 and TNF-αconcentrations of the serum and BALF collected at T4 increased significantly in both groups, but the concentrations of IL-6 and TNF-α in serum and BALF in the group S was lower than those in the TLC group (IL-6:F=1503.734,P<0.01;TNF-α:F=1423.486,P<0.05). The incidence of postoperative complications was comparable between both groups. Conclusion The SLB strategy improves oxygenation and decreases the proinflammatory cytokines during thoracic surgery.     

选择性肺叶隔离对胸科手术患者氧合及细胞因子的影响

Objective To determine the effect on concentrations of interleukin (IL-6) and tumor necrosis factor-α (TNF-α) in bronchoalveolar lavage fluid (BALF) and serum and oxygenation during selective lobar blockade. Methods Thirty patients undergoing esophagectomy or lobectomy were randomly assigned to the total lung collapse (TLC) group (n=15) and the selective lobar blockade (SLB) group( n=15). Anesthesia was induced and maintained with target-controlled infusion of propofol and remifentanil. After intubating with a 8.0 mm internal diameter single -lumen endotraeheal tube, by the guidance of fiberoptic bronchoscope, a 9F coopdech endobronchial blocker was placed into the target lobe in the SLB group, whereas the blocker was placed into the mainstem bronchus in the TLC group. Intermittent arterial blood gas analysis was performed at the following times: 15 min after two lung ventilation in the lateral decubitus position(T1)); 30 min (T2)and 60 min (T3) after TLC or SLB respectively; 15 min after recovering to two lung ventilation (T4), peak inspiratory airway pressure (Ppeak) was also recorded. BALF and blood samples were collected at T, and T4, the concentrations of IL-6 and TNF -α were measured using enzyme -linked immunosorbent assay. Results airway pressure increased significantly after the beginning of one-lung ventilation (F=215.746,P<0.05)in both groups, with more increasing extent in group T (F=53.798, P<0.01).Significant trends were found toward a better improvement in oxygention index with the group S compared with the group T after the beginning of one lung ventilation (F=1 3.747, P<0.05). IL-6 and TNF-αconcentrations of the serum and BALF collected at T4 increased significantly in both groups, but the concentrations of IL-6 and TNF-α in serum and BALF in the group S was lower than those in the TLC group (IL-6:F=1503.734,P<0.01;TNF-α:F=1423.486,P<0.05). The incidence of postoperative complications was comparable between both groups. Conclusion The SLB strategy improves oxygenation and decreases the proinflammatory cytokines during thoracic surgery.     

IFN-γ和TNF-α诱导的骨髓间充质干细胞的免疫抑制作用及环孢素A对其的影响

目的 观察γ干扰素(IFN-γ)和肿瘤坏死因子α(TNF-α)诱导的骨髓问充质干细胞(MSC)的免疫抑制作用及环孢素A(CsA)对其的影响.方法 取Balb/c小鼠骨髓,分离、培养、纯化及扩增MSC,分别以IFN-γ、TNF-α和IFN-γ+TNF-α(终浓度均为20 ng/ml)预处理MSC 12 h,然后与Balb/c小鼠脾细胞共培养,脾细胞以刀豆蛋白A激活72 h.混合细胞培养实验分8组进行:(1)脾细胞组;(2)MSC组,MSC+脾细胞;(3)IFN-γ处理组,IFN-γ预处理的MSC+脾细胞;(4)TNF-α处理组,TNF-α预处理的MSC+脾细胞;(5)联合处理组,IFN-γ与TNF-α联合预处理的MSC+脾细胞;(6)CsA组,CsA+脾细胞;(7)CsA联合预处理组:IFN-γ与TNF-α联合预处理的MSC+CsA+脾细胞;(8)1400W组,IFN-γ与TNF-α联合预处理的MSC+脾细胞+1400W[诱导型-氧化氮合酶(iNOS)抑制剂].各组脾细胞数量均为5000个,MSC与脾细胞按1:10混合,CsA浓度为10μg/ml.培养72 h后,以四甲基偶氮唑盐法检测脾细胞增殖情况,同时以碘化丙啶和Annexin-V凋亡双染试剂盒检测脾细胞的凋亡情况;以实时聚合酶链反应法检测经炎症因子处理的MSC的iNOS mRNA表达情况,观察CsA对MSC的iNOS mRNA表达的影响.结果 MSC组、IFN-γ处理组和TNF-α处理组的MSC对脾细胞的增殖无明显抑制作用(P>0.05),而联合处理组的脾细胞增殖明显受到抑制,CsA组的脾细胞增殖同样受到抑制,但IFN-γ与TNF-α联合预处理MSC产生的这种免疫抑制作用却可以被CsA所抑制(CsA联合预处理组,P<0.05).联合处理组、CsA组和CsA联合预处理组的脾细胞凋亡率明显高于脾细胞组、MSC组、IFN-γ处理组和TNF-α处理组(P<0.05),而联合处理组和CsA组的脾细胞凋亡率又明显高于csA联合预处理组(P<0.05).野生型MSC、分别以IFN-γ或TNF-α单独刺激的MSC均不表达iNOS mRNA,而以IFN-γ和TNF-a联合处理的MSC则高表达iNOS mRNA,但这种高表达可被CsA所抑制(P<0.05).1400W组的脾细胞增殖受到明显抑制(P<0.05).结论 IFN-γ和TNF-α联合预处理的MSC在体外可抑制脾细胞的增殖,促进脾细胞凋亡,CsA可抑制该种MSC的这一作用,其机制可能与CsA下调lFN-7和TNF-α联合预处理的MSC的iNOS表达有关.     

冬眠合剂对严重烫伤大鼠肺损伤的保护作用

目的 观察早期应用冬眠合剂对严重烫伤大鼠肺损伤是否具有保护作用. 方法建立Ⅲ度烫伤大鼠模型,随机分为冬眠合剂组和对照组(未用冬眠合剂),每组36只.分别在伤后3、5、7、10 d测定大鼠动脉血氧分压,检测肺组织中过氧化物酶(MPO)活性、丙二醛(MDA)含量、肿瘤坏死因子α(TNF-α)及γ干扰素(IFN-γ)蛋白表达,动态观察伤后肺组织病理学变化. 结果与对照组大鼠伤后3 d动脉血氧分压(8.86±0.23)kPa(1 kPa=7.5 mm Hg)比较,冬眠合剂组该时相点明显升高[(12.58±0.41)kPa,P<0.01].冬眠合剂组大鼠伤后各时相点肺组织MPO活性和MDA含量下降(P<0.05或P<0.01);伤后3、5、7 d肺组织TNF-α表达显著下调(P<0.05或P<0.01),伤后5、7、10 d肺组织IFN-γ的表达亦显著下调(P<0.01).冬眠合剂组大鼠肺泡间质水肿减轻,炎性细胞浸润减少. 结论大鼠烫伤后及时复苏并给予冬眠合剂,能适度抑制机体应激反应并下调促炎因子表达,改善早期肺功能.     

氩氦刀冷冻消融治疗兔VX2肾癌外周血细胞因子的变化

目的研究冷冻消融治疗兔VX2肿瘤后外周血细胞因子的变化。方法建立兔VX2肾癌模型,将38只荷瘤兔随机分为：冷冻消融组（A组）,根治性肾切除组（B组）,肿瘤对照组（C组）,应用酶联免疫吸附试验法测定治疗前、治疗后21d外周血Th1型细胞因子（IFN-γ、TNF-α和Th2型细胞因子（IL-4、IL-6）浓度的变化。结果治疗后21d组间组内比较显示：冷冻消融组外周血IFN-γ、TNF-α浓度和IFN-γ／／IL-4比值明显升高,IL-6明显降低,与治疗前差异存在统计学意义（P〈0．05）,IL-4与治疗前相比差异无统计学意义（P〉0．05）;冷冻消融组外周血IFN-γ／、IL-4、IL-6浓度和IFN-γ／IL-4比值与肿瘤对照组相比,差异均有统计学意义（P〈0．05）;与根治肾切除组相比,冷冻消融组TNF—α浓度明显升高,IL-6明显降低,差异均有统计学意义（P〈0．05）,两组IFN-γ／、IL-4浓度差异无统计学意义（P〉0．05）。结论冷冻消融冶疗肾癌可使外周血IFN-γ／、TNF-α、IFN-γ／IL-4比值升高,IL-6浓度降低,增强机体的抗肿瘤免疫功能。     

肌球蛋白轻链激酶介导缺氧后肠上皮屏障功能紊乱的研究

目的 了解肌球蛋白轻链激酶(MLCK)在缺氧后肠上皮屏障功能损害中的作用.方法 建立单层肠上皮细胞缺氧模型,根据缺氧时间不同将细胞分为正常对照组(缺氧0 h),缺氧2、6、8、12、24 h组,(5-氯萘-1-磺酰)-1-六氢-1,4-双苯妥英钠(ML-9)处理组(缺氧6 h并用终浓度为100μmol/L的MLCK特异性抑制剂ML-9处理).用电阻仪测定单层肠上皮细胞跨细胞电阻(TER);免疫荧光法检测单层肠上皮细胞紧密连接蛋白ZO-1的变化;蛋白质印迹法检测肠上皮细胞磷酸化肌球蛋白轻链(p-MLC)及MLCK蛋白表达.结果 缺氧6、8、12、24 h组单层肠上皮细胞TER值分别为(422±17)、(427±27)、(403±40)、(426±22)Ω,均低于正常对照组[(451±27)Ω,P<0.05],ML-9处理组为(558±110)Ω,显著高于各单纯缺氧组(P<0.01).缺氧6 h组单层肠上皮细胞紧密连接蛋白ZO-1发生明显改变,主要表现为不规则,出现明显皱褶、圆滑排列程度减弱,甚至有断裂及锯齿等不连续现象.ML-9组肠上皮细胞紧密连接蛋白ZO-1近似于正常对照组,排列较规则,皱褶及锯齿状改变较单纯缺氧组减少或消失.各单纯缺氧组肠上皮细胞MLCK、p-MLC蛋白表达水平均较正常对照组增加.结论 MLCK可介导缺氧引起的肠上皮屏障功能紊乱.     

Regulation of Nitric Oxide Synthesis in Wounds by IFN-γ Depends on TNF-α

Macrophage-derived nitric oxide is a critical mediator in wound healing. Its regulation in vivo, however, remains unclear. We hypothesized that interferon (IFN)-γ plays an important role in the regulation of nitric oxide in wounds. Groups of 12 male IFN-γ -knockout mice and wild-type controls underwent dorsal skin incision and polyvinyl alcohol sponges were inserted subcutaneously. Mice were sacrificed 10 days later to determine wound breaking strength and reparative collagen deposition. Synthesis of nitric oxide (NO), tumor necrosis factor (TNF)-α, and IFN-γ was measured in the wound. Wound-derived macrophages were tested for NO synthesis in the presence or absence of IFN-γ, TNF-α, and anti-TNF-α antibody. In a separate experiment, IFN-γ -knockout mice and wild-type controls were treated with molsidomine, a nitric oxide donor. It was found that wound collagen deposition and wound breaking strength were impaired in IFN-γ-knockout mice (p <. 05). Impaired healing was reflected in diminished synthesis of TNF-α and NO in wounds (p <. 05). In vivo treatment with molsidomine reversed impaired healing in IFN-γ-deficient mice. Ex vivo, addition of IFN-γ stimulated the synthesis of TNF-α and NO in wound-derived macrophages. IFN-γ -induced NO synthesis by wound-derived macrophages was abolished by anti-TNF-α-antibody-treatment, which could be fully reversed by exogenous TNF-α. Thus we conclude that IFN-γ-deficiency impairs wound healing and diminishes NO synthesis in wound-derived macrophages. The stimulatory effect of IFN-γ on macrophage NO production depends on endogenous TNF-α synthesis.     

辅助性T细胞亚群分化在Ⅲ型前列腺炎免疫发病机制中的作用

目的 探讨前列腺液(EPS)中CD+4T细胞亚群辅助性T细胞(Th细胞)分化在Ⅲ型前列腺炎免疫发病机制中的作用. 方法门诊诊断前列腺炎患者76例,年龄18～47岁,平均32岁.患者均有慢性前列腺炎典型临床症状,病程均＞3个月.按美国国立卫生院分类方法分为ⅢA型组(47例)、ⅢB型组(29例).其中ⅢA型组根据炎症程度又分为ⅢAl组(轻度炎症26例)和ⅢA2组(重度炎症21例).另设健康对照组(16例),年龄19～45岁,平均31岁.采用双抗体夹心酶联免疫法检测EPS中Th1类细胞因子(IFN-γ)、Th2类细胞因子(IL-4)水平及Th1/Th2比值(IFN-γ/IL-4),比较各组问差异.结果 ⅢA、ⅢB组IFN-γ水平[(134.78±43.67),(109.82±30.09)pg/m1]与对照组[(60.63±15.16)pg/m1]比较,差异有统计学意义(P＜0.05),ⅢA组较ⅢB组升高更明显(P＜0.05);ⅢA组IL-4水平[(51.99±20.59)pg/m1]与对照组[(53.88±17.92)pg/m1]比较差异无统计学意义P＞0.05),ⅢB组IL-4水平[(76.40±17.99)pg/m1]明显上调(P＜0.05);ⅢA组IFN-γ/IL-4水平(2.94±1.12)明显高于对照组(1.20±0.48,P＜0.05),Ⅲ B组(1.49±0.48)无明显改变(P＞0.05).与对照组比较,Ⅲ A1组IL-4水平[(63.03±18.86)pg/m1]无明显变化(P＞0.05),而ⅢA2组水平[(30.20±13.16)pg/m1]显著下调(P＜0.05);ⅢA1组和m A2组IFN-γ水平均明显上调[(127.65±36.57),(143.49±50.76)pg/m1],P＜0.05),但2组间差异无统计学意义(P＞0.05);ⅢA2组IFN-γ/IL-4明显高于ⅢA1组(3.67±0.82 vs 2.34±0.97,P＜0.05).结论 ⅢA型前列腺炎Th1细胞分化占优势,Th1/Th2平衡向Th1漂移,以细胞免疫反应为主;Th细胞分化也参与了ⅢB型前列腺炎的发病,但Th1/Th2处于相对平衡状态.Th1的优势分化可能是导致前列腺局部炎症发展的原因之一.     

环孢素A对不明原因复发性流产患者外周血IFN-γ、TNF-α的调控

目的探讨环孢素A(CsA)对不明原因复发性流产(uRM)患者外周血干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)表达的影响。方法收集79例正常育龄期妇女的外周血标本,采用流式细胞仪检测其外周血CD3~+CD4~+T细胞中IFN-γ、TNF-α的表达水平,制定正常参考范围。入选36例IFN-γ、TNF-α表达量异常升高的uRM患者,于月经第1天口服CsA50mg,2次/d,直至黄体中期;收集uRM患者治疗前后的外周血,采用流式细胞仪检测IFN-γ、TNF-α的表达水平并进行比较。结果正常育龄期妇女外周血CD3~+CD4~+T细胞中IFN-γ、TNF-α的表达水平呈正态分布,以百分位数P10~P90(分别为6.7%~30.5%,12.6%~41.0%)作为正常参考范围。IFN-γ和/或TNF-α异常升高的uRM患者服用CsA后,其表达量均显著降低[IFN-γ:(36.7±9.3)%vs.(28.5±11.0)%,TNF-α:(52.8±7.2)%vs.(40.9±13.8)%,P<0.01]。结论CsA可以下调uRM患者外周血中IFN-γ、TNF-α的表达量,但其具体机制尚需进一步研究。     

γ干扰素对瘢痕疙瘩成纤维细胞转化生长因子β/Smad信号通路的作用

目的 了解γ干扰素(IFN-γ)对瘢痕疙瘩Fh(KFb)中TGF-β/Smad信号通路的作用,探讨IFN一γ治疗病理性搬痕的可能机制. 方法 切取3例患者的瘢痕组织,体外分离培养KFb,实验选用第3～5代细胞.(1)将KFb分为:对照组,加无血清DMEM培养;TGF-β_1组,用10 ng/mL的TGF-β_1单独作用;IFN-γ组,用100 ng/mL的IFN-γ单独作用;TGF-β_1+IFN-γ组,10 ng/mL的TGF-β_1与100 nS/mL的IFN-γ联合作用.采用实时荧光定量RT-PCR、蛋白质印迹法和免疫荧光细胞化学染色法,分别检测结缔组织生长因子(CTGF)的mRNA、蛋白表达,以及α平滑肌肌动蛋白(α-SMA)的蛋白表达与阳性细胞表达情况.(2)另取KFb,用10 ng/mL的IFN-γ作用,于作用前及作用后30 min和1、2、4、6、8 h通过实时荧光定量RT-PCR检测Smad 3和Smad 7的mRNA表达,于作用前及作用后1、2、4、6、8 h用蛋白质印迹法检测Smad 3和Smad 7的蛋白表达.(3)另取KFb,根据添加的IFN-γ终浓度不同分为1、10、100 ns/mL IFN-γ组,均作用4 h;设立未添加IFN-γ的KFb为对照组.同前检测各组Smad 3和Smad 7的mRNA及蛋白表达. 结果 (1)IFN-γ组KFb CTGF的mRNA和蛋白表达量为0.017±0.009与1.198±0.004,较对照组(0.024±0.013与1.229±0.011)显著减少(P<0.05);TGF-β1+IFN-γ组CTGF的mRNA和蛋白表达量为0.634±0.138与1.204±0.010,较TGF-β_1组(1.331±0.298与1.727±0.004)显著减少(P<0.01).IFN-γ组KFb中,α-SMA阳性细胞荧光强度(0.922±0.059)和α-SMA蛋白表达量(0.3051±0.0031)较对照组(1.055±0.005与0.4513±0.0094)显著减少(P<0.01);TGF-β1+IFN-γ组SMA阳性KFb荧光强度(1.129±0.004)和SMA蛋白表达量(0.6734±0.0098)较TGF-β_1组(1.270±0.005与1.38420.0024)显著减少(P<0.01).(2)10 ng/mL IFN-γ作用后第1个时相点,Smad 3的mRNA和蛋白表达量均出现一过性增高,随后降低,mRNA表达量于作用后4 h降至最低点,随后缓慢上升,至作用后8 h仍低于作用前(P<0.01);其蛋白表达量于作用后2~8 h显著低于作用前(P<0.01).而Smad 7的mRNA和蛋白表达量在INF-γ作用后逐渐增高,分别于作用后2、4 h达峰值随后降低,至作用后8 h仍高于作用前(P<0.05).(3)与对照组比较,1、10、100 ng/mL IFN-γ组Smad 3的mRNA及蛋白表达量显著减少(P<0.05或P<0.01),Smad 7的mRNA及蛋白表达量显著增加(P<0.05或P<0.01),且随IFN-γ浓度升高,减少或增高幅度愈为显著. 结论 IFN-γ呈时间和剂量依赖方式下调Smad 3、上调Smad 7,降低基础状态下或经TGF-β_1诱导后KFb的CTGF和α-SMA表达量,表现出对TGF-β/Smad 信号通路的显著拮抗作用,这可能是IFN-γ治疗病理性瘢痕的重要机制.     

干扰素-γ和肿瘤坏死因子-α对精子受精能力的影响及机制初探

目的:观察IFN-γ和TNF-α对正常精子顶体酶活性和顶体反应率的影响,并对其变化机制进行初步探讨。方法:36例精液常规分析基本正常标本,经75%Percoll分离后,分别或联合使用IFN-γ和TNF-α处理(终浓度均为30ng/ml)。采用BAEE/ADH联合法测定精子顶体酶活力的变化。用三色染色法观察精子顶体反应率的变化。用高效液相色谱法(HPLC)检测精子NO含量。用试剂盒法测定Na+-K+-ATPase、Ca2+-ATPase和SOD活性。结果:IFN-γ和TNF-α单独及联合使用时,均可显著降低精子顶体酶活性和顶体反应率(P<0.05或P<0.01),且TNF-α的抑制作用更强些;IFN-γ可使精子Na+-K+-ATPase、Ca2+-ATPase和SOD活性显著降低(P<0.01),且两者协同作用更低,但TNF-α对Na+-K+-ATPase和Ca2+-ATPase活性几乎无作用;IFN-γ、TNF-α及两者合用时均使精子NO含量显著增加(P<0.01)。结论:IFN-γ和TNF-α对精子顶体酶活性及顶体反应率有一定的抑制作用,并且可能通过对精子Na+-K+-ATPase、Ca2+-ATPase、SOD活性以及NO含量等多方面影响来实现的。