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.     

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

目的 观察脾动脉缩窄对肝硬化门静脉高压大鼠脾脏iNOS、Th1/Th2型细胞因子表达的影响,并探讨机制.方法 肝硬化门静脉高压大鼠随机分3组(n=10):假手术组(SOG)、脾动脉缩窄术组(SAC)和脾动脉结扎术组(SAL);正常大鼠10只行假手术作为对照组(NCG).免疫组化法测脾脏iNOS表达,RT-PCR法测脾脏IFN-γ、IL-4mRNA表达,对iNOS与IFN-γ、IL-4表达量作相关分析.结果 SOG脾脏iNOS明显高于NCG(P＜0.01),SAC和SAL明显低于SOG(P＜0.01).SOG脾脏IFN-γmRNA和IFN-γ/IL-4明显低于NCG(P＜0.01),IL-4mRNA明显高于NCG(P＜0.01);SAC脾脏IFN-γmRNA高于SOG(P＜0.05),SAC和SAL脾脏IL-4mRNA低于SOG(P＜0.05),而IFN-γ/IL-4高于SOG(P＜0.05).iNOS与IFN-γ负相关(r=-0.672,P＜0.01),与IL-4正相关(r=0.634,P＜0.01).结论 脾动脉缩窄术后门静脉高压大鼠脾脏iNOS表达降低,IFN-γ/IL-4升高,脾脏Th1/Th2失衡改善可能与术后iNOS表达降低有关.     

CD40在肝硬化门静脉高压症脾脏中的表达与临床意义

Objective To investigate the expression and clinical significance of CD40 in the spleen of cirrhotic portal hypertension. Methods Expression of CD40 was determined with S-P immunohistochemistry in spleen specimens from 50 cases of cirrhotic portal hypertension and 15 healthy individuals. Results The CD40 positive rates in normal spleen and cirrhotic spleen were 86.7% and 36.0%, respectively. There was a significant difference between the 2 groups (P＜0.05). There was a negative correlation between the expression of CD40 and Child grades of liver function and cirrhotic splenic types(P＜0. 05). Conclusion CD40 might reflect the changes of splenic immune function,which might be one of more exact clinical examination indexes of splenic immune function.     

CD40在肝硬化门静脉高压症脾脏中的表达与临床意义

目的 探讨CD40在肝硬化门静脉高压症患者脾脏中的表达及临床意义.方法 采用免疫组织化学S-P法测定50例肝硬化门静脉高压症患者和15例正常脾脏中CD40的表达情况.结果 CD40在正常脾脏与巨脾组织中的表达率分别为86.7%和36.0%,存在显著性差异(P＜0.05).CD40表达与肝功能Child分级及巨脾病理分级均呈负相关(P＜0.05).结论 CD40可能为临床评估肝硬化门静脉高压症患者脾脏免疫功能状态的重要指标.

Abstract：

Objective To investigate the expression and clinical significance of CD40 in the spleen of cirrhotic portal hypertension. Methods Expression of CD40 was determined with S-P immunohistochemistry in spleen specimens from 50 cases of cirrhotic portal hypertension and 15 healthy individuals. Results The CD40 positive rates in normal spleen and cirrhotic spleen were 86.7% and 36.0%, respectively. There was a significant difference between the 2 groups (P＜0.05). There was a negative correlation between the expression of CD40 and Child grades of liver function and cirrhotic splenic types(P＜0. 05). Conclusion CD40 might reflect the changes of splenic immune function,which might be one of more exact clinical examination indexes of splenic immune function.     

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.     

Relations of transcription expression of IL—2 with nuclear factor of activated T cells as well as changes of C—Fos and C—Jun after trauma

Objective:To observe the relations among expression of interleukin-2(IL-2)in spleen lymphocytes,DNA binding activity of nuclear factor of activated T cells(NFAT)and expression of the partly family members C-Fos,C-Jun after trauma.Methods:A murine closed trauma model was used,animals were sacrificed6,12hours and 1,4,7,10,14days,respectively after injury,Spleen lymplocytes were isolated from injured mice and stimulated with concanavalin-A,The culture supernatants were harvested and assayed for IL-2activity,Total RNA was extracted from spleen lymphocytes and assayed for IL-2mRNA.Nuclear protein was extracted,and the DNA binding activity of NFAT was measured using an electrophoretic mobility shift assay(EMSA),the expressions of C-Fos,C-Jun protein determined by Western blot analysis.Results:The expressions of IL-2 activity and IL-2mRNA in spleen lymphocytes were decreased in injured mice compared with those in control mice,and the most obvious decrease appeared on the 4th day after injury,The DNA binding activity of NFAT decreased gradually and reached the minimum that was only41%of the control on the 4th day after injury,which was cloely associated with the decline of IL-2activity and IL-2mRNA.An decrease in the expression ofC-Fos on the lst and 4th day after injury,trauma had no significant effect on the C-Jun expression.Conclusions:These results suggest that the inhibition of IL-2 expression is partly due to the impairment in the activation of NFAT in injured mice;and the decline in the DNA binding activity of NFAT is partly due to trauma block in the C-Fos expression.     

Effect of isoflurane on expression of IL-1β mRNA,IL-6 mRNA and TNF-α mRNA In the hippocamapus of immature rats

Obiective To investigate the effect ofisoflurane on expression of IL-1β mRNA,IL-6 mRNA and TNF-α mRNA in the hippocampus of immature rats.Methods sixty-four 7-clay-old SD rats were randomly assigned into 2 groups(n=32 each):control group(group C)and isoflurane group(group S).group S was exposed to 1.5% isoflurane for 6 h while group C to air.Fore animals were killed before anesthesia(T0,baseline),at 2,4,6 h(T1-3)of isoflurane anesthesia and 4,6,12 and 24 h after anesthesia(T4-7).The hippocampi were immediately removed for determimation of the expression of IL-1β mRNA,IL-6 mRNA and TNF-α mRNA by RT-PCR.Results Compared with group C,the expression of IL-1β mRNA at T1-5,IL-6 mRNA at T2.3 and TNF-α mRNA at T1-6 in the hippocampus was upregulated in group S.Conclusion The expression of IL-1β mRNA,IL-6 mRNA and TNF-β mRNA was elevated in the hippocampus of immature rats after being exposed to isoflurane.     

Effect of isoflurane on expression of IL-1β mRNA,IL-6 mRNA and TNF-α mRNA In the hippocamapus of immature rats

Obiective To investigate the effect ofisoflurane on expression of IL-1β mRNA,IL-6 mRNA and TNF-α mRNA in the hippocampus of immature rats.Methods sixty-four 7-clay-old SD rats were randomly assigned into 2 groups(n=32 each):control group(group C)and isoflurane group(group S).group S was exposed to 1.5% isoflurane for 6 h while group C to air.Fore animals were killed before anesthesia(T0,baseline),at 2,4,6 h(T1-3)of isoflurane anesthesia and 4,6,12 and 24 h after anesthesia(T4-7).The hippocampi were immediately removed for determimation of the expression of IL-1β mRNA,IL-6 mRNA and TNF-α mRNA by RT-PCR.Results Compared with group C,the expression of IL-1β mRNA at T1-5,IL-6 mRNA at T2.3 and TNF-α mRNA at T1-6 in the hippocampus was upregulated in group S.Conclusion The expression of IL-1β mRNA,IL-6 mRNA and TNF-β mRNA was elevated in the hippocampus of immature rats after being exposed to isoflurane.     

血液净化治疗多器官功能障碍综合征引起Th1/Th2漂移的研究

目的研究连续性血液净化治疗（CBP）对多器官功能障碍综合征（multiple organ dysfunction syndrome，MODS）患者循环Th1／Th2的影响及其引起Th1／Th2漂移的机制。方法18例多器官功能障碍综合征患者行CBP治疗。采用中心静脉留置单针双腔导管建立血管通路，采用连续性血液净化系统进行前稀释连续性静脉-静脉血液透析滤过（CVVHDF）。治疗前及治疗后24、48、72h采血检测Th1／Th2，同时检测血液中IL4和IFN-1浓度。结果MODS患者经CBP治疗后，Thl表达逐渐增加，而Th2表达逐渐下降，IFN-γ和IL-4的浓度均下降；Th1／Th2和IFN-γ／IL-4比值随治疗时间的增加升高；Th1／Th2和IFN-γ/IL-4比值呈正相关。结论CBP治疗可能可改善MODS患者的Th1／Th2平衡漂移，可能与改善机体内IFN-γ/IL-4比值有很大的相关性。     

Th1/Th2细胞因子mRNA的表达与心脏移植免疫耐受的关系

目的探讨Th1/Th2细胞因子信使核糖核酸(mRNA)表达改变与心脏移植免疫耐受的关系. 方法采用大鼠腹部心脏移植模型,将30只大鼠随机分成对照组、排斥反应组、免疫耐受组,每组10只.观察移植心脏存活时间,供心病理学改变,受者脾和心脏中Th1/Th2细胞因子白细胞介素2(IL-2)、γ干扰素(IFN-γ)、白细胞介素4(IL-4)、白细胞介素10(IL-10) mRNA表达水平. 结果免疫耐受组供心存活时间为85.28±7.48天,较排斥反应组的7.33±1.03天显著延长(P＜0.01);排斥反应组供心见大量炎性细胞浸润,免疫耐受组供心仅见少量炎性细胞浸润;排斥反应组Th1细胞因子IL-2、IFN-γ mRNA表达较对照组增强,免疫耐受组减弱;排斥反应组Th2细胞因子IL-4、IL-10 mRNA表达较对照组减弱,免疫耐受组增强. 结论 Th1/Th2细胞因子的动态平衡在移植耐受中起重要作用,Th1向Th2偏离是移植耐受的机制之一.     

慢性非细菌性前列腺炎/慢性骨盆疼痛综合征患者外周血Th1/Th2细胞的检测

目的:探讨慢性非细菌性前列腺炎/慢性骨盆疼痛综合征(CAP/CPPS)患者外周血Th1/Th2细胞分布的变化情况及其在CAP/CPPS临床分型中的意义。方法:采用流式细胞术检测35例CAP/CPPS患者和12例健康体检者外周血CD3+CD8-T细胞胞内细胞因子干扰素γ(IFN-γ)和白细胞介素4(IL-4)的表达。结果:与正常对照组相比,ⅢA型、ⅢB型CAP/CPPS患者的Th1细胞数均升高,Th1/Th2比值升高,差异有显著性(P<0.05),Th2细胞数差异无统计学意义(P>0.05);ⅢA型与ⅢB型患者比较,Th1、Th2细胞数与Th1/Th2比值差异均无统计学意义(P>0.05)。结论:CAP/CPPS患者Th1型反应模式占优势状态,Th1/Th2平衡失调,Th1/Th2平衡向Th1方向变化,提示Th1细胞在CAP/CPPS的病理发生中可能起重要作用。     

T-cell activation follows Th1 rather than Th2 pattern in haemodialysis patients.

BACKGROUND: Patients on chronic intermittent haemodialysis (HD) show an impaired cellular and humoral immune response that clinically appears with frequent infectious complications and low vaccination responses. This immune defect strongly correlates with reduced in vitro proliferative responses of T cells. The defect is localized in antigen presenting cells, which show a decreased co-stimulatory activity. Furthermore, the impaired immune response correlates with an increased production of pro-inflammatory cytokines. In response to primary activation, CD4 positive T helper (Th) cells mainly differentiate into either Th1 or Th2 cells. Th1 cells support cell mediated immunity whereas Th2 cells enhance humoral immune responses. Since both types of responses mutually inhibit each other, the impaired humoral immune response seen in HD patients could either be due to a reduced number of Th2 cells or to a predominant Th1 response. METHODS: We analysed the Th cell profile in HD patients using flow cytometry. Monocytic cytokine expression was analysed using both flow cytometry and enzyme linked immunoadsorbant assays. RESULTS: Our data demonstrate that the cytokine differentiation profile in circulating T cells from HD patients is dysregulated and characterized by an increase in Th1 cells, but a normal amount of Th2 cells. Moreover, the skewed helper cell responses correlate with a higher percentage of monocytes capable of secreting the Th1 promoting cytokine interleukin 12 (IL-12). CONCLUSIONS: Our findings contribute to a better understanding of the pathogenesis of impaired cellular immune functions in dialysis patients and, in particular, the decreased antibody production after vaccination. They provide a link between overproduction of pro-inflammatory cytokines (IL-12) and imbalanced T-cell activation.     

Th1/Th2类细胞因子转换对小鼠心脏移植物存活的影响

目的 探讨Th1/Th2类细胞因子转换对小鼠心脏移植物存活时间的影响。方法 采用小鼠腹部心脏移植模型，分为同种异体移植组（A组）、同种异体移植+免疫抑制处理组（B组）和同系移植组（C组），每组20对。观察移植物存活时间、供心病理改变、受鼠脾和供心内IFN-γ，IL-2，IL-4及IL-10 mRNA的表达水平。结果 A组及B组移植物平均存活时间分别为（7.8±0.77）d和（14.80±1.01）d，C组移植物存活均超过28d；3组间差异均有显著性（P<0.05）。A组与B组、C组比较，移植物的心肌细胞变性坏死严重，并有大量炎性细胞浸润。A组受鼠脾脏及供心内IFN-γ和IL-10 mRNA表达比其余两组明显增强；3组移植心组织IL-2及IL-4 mRNA均无表达；A组脾脏IL-2 mRNA表达最强；B组脾脏IL-4 mRNA表达明显强于其余两组。结论 Th1/Th2转换在延长移植物存活过程中起重要作用；IL-10也参与移植物排斥反应过程。     

成骨肉瘤患者TH1/TH2亚群漂移研究

观察成骨肉瘤患者辅助性Ｔ淋巴亚群中Ｔｈ１／Ｔｈ２亚群的变化,为细胞因子免疫治疗提供依据。方法应用放射免疫法以及酶联免疫吸附法检测成骨肉瘤患者血清中ＩＬ－２、ＩＬ－４、ＩＬ－６、ＴＮＦ－α的含量,检测外周血单个核细胞培养上清中ＩＬ－２、ＩＬ－４含量。结果成骨肉瘤血清中及培养上清中ＩＬ－２减少,而ＩＬ－４、ＩＬ－６、ＴＮＦ－α含量增加。结论成骨肉瘤患者中存在有Ｔｈ１／Ｔｈ２平衡失调,其中Ｔｈ１亚群功能抑制,Ｔｈ２亚群功能亢进,它们与肿瘤在宿主体内生长密切相关。     

凝血酶对狼疮肾炎末梢血单个核细胞分泌Th1/Th2细胞因子的影响

目的探讨狼疮肾炎(LN)患者凝血活性对其Th1/Th2平衡的影响。方法LN患者20例,按红斑狼疮病情活动指数(SLEDAI)分为稳定组11例(SLEDAI<9分),活动组9例(SLEDAI≥9分)。健康对照组8例。采集末梢血单个核细胞(PBMC),进行高、低浓度凝血酶刺激下体外培养。ELISA法检测培养液上清IL-10、IFN-γ水平。RT-PCR检测IL-10和IFN-γmRNA表达。结果凝血酶呈浓度依赖性上调LN患者PBMCIL-10表达,但不影响IFN-γ表达,故增加IL-10/IFN-γ比值。结论凝血酶加重狼疮肾炎患者PBMC分泌Th1/Th2细胞因子的平衡紊乱。     

The role of Th1/Th2 cell chemokine expression in hypertrophic scar

The aim of this study was to study the role of Th1/Th2 cell‐associated chemokines in the formation of hypertrophic scars in rabbit ears. Twenty‐six New Zealand white rabbits were used to establish the hypertrophic scar model of rabbit ear and the normal scar model of rabbit's back. Two rabbits were sacrificed on days 0 and 21, 28, 35, 42, 49, 56, and 63 after operation. The specimens were stained with haematoxylin‐eosin (HE). Scar elevation index (SEI) was used to detect the expression of 10 chemokines related to Th1/Th2 cells in both scar formation expressions. Real‐time polymerase chain reaction (PCR) results showed that two chemokines (CXCL10, CXCL12) were highly expressed during the formation of normal scar, and there was almost no expression during the formation of hypertrophic scar (*P < 0.05). The chemokines (CCL2, CCL3, CCL4, CCL5, CCL7, CCL13, CX3CL1) were almost non‐expressed in the formation of normal scars but were expressed for a long time in the formation of hypertrophic scars. The four chemokines, CCL2, CCL4, CCL5, and CX3CL1, maintained a long‐term high expression level during the formation of hypertrophic scars (P < 0.01). There were also three chemokines (CCL14, CCL19, CCL21) that were almost undetectable in normal scarring, but there was transiently low‐level expression (P < 0.05) only during the peak proliferative phase in proliferative scarring. Th1/Th2 cell‐associated chemokines are different in the type, quantity and expression, and maintenance time of rabbit ear hypertrophic scars.