干细胞因子在狼疮肾炎患者肾组织中的表达及意义

目的 探讨干细胞因子(SCF)在狼疮肾炎(LN)患者肾组织中的表达及其与狼疮活动、肾损害有关指标的关系.方法 采用原位杂交和免疫组织化学技术,分别在基因和蛋白质水平检测[34例LN,16例慢性肾小球肾炎(CGN),8名健康对照]肾组织中SCF及其受体c-kit(CD117)的表达,同时检测肥大细胞、巨噬细胞及α-平滑肌肌动蛋白(α-SMA)阳性细胞的浸润情况.计算狼疮肾组织活动指数和慢性指数得分及肾间质损害程度.结果 ①LN及CGN肾组织中SCF的表达及肥大细胞较对照组明显增加(t=6.03～14.25,P<0.01).间质损害程度相似的LN与CGN相比较,肾间质SCF、肥大细胞差异无统计学意义.②SCF与肥大细胞、巨噬细胞、α-SMA及临床指标的相关性:相应部位SCF的表达、α-SMA的表达、肥大细胞、巨噬细胞的浸润数量呈显著正相关(r=0.47～0.86,P<0.01).SCF的表达、α-SMA的表达及巨噬细胞数量与肾病理活动性指数、慢性化指数、肾间质损害程度、尿蛋白定量均呈显著正相关(r=0.34～0.93,P<0.05或0.01),与肌酐清除率(Ccr)呈明显负相关(r=-0.39～0.61,P<0.01).SCF及巨噬细胞与抗dsDNA滴度、补体C3、SLE疾病活动指数(SLEDAI)积分也有显著相关性.肥大细胞数与LN慢性指数特别是肾间质损害程度呈明显正相关(r=0.86,r=0.93,P<0.01)与Ccr呈明显负相关(r=-0.56,P<0.01).与LN活动指标无相关性.结论 SCF在LN肾组织呈广泛表达,其表达量与多种炎症细胞、肾脏固有细胞、肾功能、尿蛋白、疾病活动性及严重程度等密切相关,提示SCF可能是LN病理过程中一个关键的全程参与者.     

Brief Report: Homozygous IL37 mutation associated with infantile inflammatory bowel disease

Interleukin (IL)-37, an antiinflammatory IL-1 family cytokine, is a key suppressor of innate immunity. IL-37 signaling requires the heterodimeric IL-18R1 and IL-1R8 receptor, which is abundantly expressed in the gastrointestinal tract. Here we report a 4-mo-old male from a consanguineous family with a homozygous loss-of-function IL37 mutation. The patient presented with persistent diarrhea and was found to have infantile inflammatory bowel disease (I-IBD). Patient cells showed increased intracellular IL-37 expression and increased proinflammatory cytokine production. In cell lines, mutant IL-37 was not stably expressed or properly secreted and was thus unable to functionally suppress proinflammatory cytokine expression. Furthermore, induced pluripotent stem cell–derived macrophages from the patient revealed an activated macrophage phenotype, which is more prone to lipopolysaccharide and IL-1β stimulation, resulting in hyperinflammatory tumor necrosis factor production. Insights from this patient will not only shed light on monogenic contributions of I-IBD but may also reveal the significance of the IL-18 and IL-37 axis in colonic homeostasis.     

大鼠肺动脉和主动脉成纤维细胞的原代培养及生物学行为研究

目的 建立大鼠肺动脉和主动脉外膜成纤维细胞的体外分离、培养及鉴定的方法,并对其细胞形态和成纤维细胞转化成肌成纤维细胞的特征进行观察.方法 通过精分肺动脉和主动脉,剥取外膜,用10％的胎牛血清低糖DMEM培养基诱导细胞爬出生长.倒置显微镜下观察成纤维细胞形态,并对细胞进行免疫荧光鉴定.通过对α-平滑肌肌动蛋白(α-SMA)表达的观察确定传代细胞是否转化为肌成纤维细胞.结果 成纤维细胞能够迅速从剥取的外膜中爬出生长,波形蛋白细胞免疫荧光染色为阳性.主动脉成纤维细胞和肺动脉成纤维细胞形态差别大,前者为铺路石状,后者呈细长梭形.2种成纤维细胞经传代皆能转化为肌成纤维细胞,免疫荧光染色可见α-SMA大量表达,前者包围着细胞核呈条索状非定向分布,后者平行于细胞长轴呈细丝状表达.结论 该方法所获得的成纤维细胞纯度高,可在体外稳定培养.本研究首次发现了肺动脉和主动脉成纤维细胞形态学及生物行为上的差别,为细胞水平研究主动脉和肺动脉血管重塑,胶原沉积等相同或不同的病理机制提供了充足可靠的靶细胞模型.     

胰岛素对血管平滑肌α肌动蛋白基因变异的影响机制

胰岛素不仅能维持血管平滑肌细胞的静止形态,也能促进血管平滑肌细胞的迁移.胰岛素这种不同的效应是通过血管平滑肌细胞表型标志物α-平滑肌肌动蛋白实现的,因为α-平滑肌肌动蛋白在血管平滑肌细胞静止形态中高表达,而在血管平滑肌细胞迁移形态中低表达.胰岛素维持血管平滑肌细胞静止形态是通过磷脂酰肌醇激酶3途径实现的,而其促进血管平滑肌细胞迁移是通过活化蛋白激酶实现的.因此,在模拟胰岛素抵抗状态,即阻断磷脂酰肌醇激酶3信号通路并保留活化蛋白激酶信号通路,胰岛素可能失去维持血管平滑肌细胞的静止状态,取而代之的是促进血管平滑肌细胞的迁移.α-平滑肌肌动蛋白的变异能引发许多血管性疾病,包括冠心病、缺血性脑卒中等.α-平滑肌肌动蛋白的单基因也变异能引发弥漫性血管疾病,包含了动脉的阻塞及扩大,这在临床上对血管性疾病的研究和治疗有直接的指导作用.     

CD133、α-SMA的研究现状及其与脑缺血后血管生成的研究进展

CD133是血管内皮祖细胞的重要标志蛋白,在肿瘤、心肌缺血、颅脑损伤及脑缺血后均有表达.α-SMA是区分血管平滑肌细胞表型的特征性标记物,表达在血管平滑肌细胞、成纤维细胞、肿瘤等细胞中.近年研究发现,两者表达与脑缺血后血管生成密切相关.鉴于此,本文拟就从CD133及α-SMA的结构、表达部位、分布特性及其与脑缺血后血管生成的关系研究进展做一综述,并对存在的问题进行分析探讨.     

太白楤木对肝纤维化模型大鼠的干预作用

[目的]探讨太白楤木对肝纤维化模型大鼠Ⅰ型胶原纤维(Col-Ⅰ)、Ⅲ型胶原纤维(Col-Ⅲ)、α-平滑肌肌动蛋白(α-SMA)表达的干预作用.[方法]雄性SD大鼠60只,随机分为阴性对照组、模型组、太白楤木大、中、小剂量组和秋水仙碱组,采用40％ CCl4橄榄油腹腔注射诱导肝纤维化模型,2次/周,连续8周.采用苏木精-伊红及Masson染色观察肝组织病理学改变;RT-PCR检测Col-Ⅰ、Col-ⅢmRNA表达水平;Western-blot检测α-SMA的表达水平.[结果]与模型组相比,太白楤木大、中、小剂量组及秋水仙碱组肝细胞变性坏死减轻,纤维条索减少,且秋水仙碱组、太白楤木小、中、大剂量组呈逐渐减轻趋势.与模型组相比,太白楤木大、中、小剂量组及秋水仙碱组Col-Ⅰ、Ⅲ mRNA表达水平均减低(均P＜0.05);与秋水仙碱组比较,太白楤木大剂量组Col-Ⅰ、Ⅲ mRNA表达减低(均P＜0.05).太白楤木大、中、小剂量组和秋水仙碱与模型组相比,a-SMA表达不同程度减低(均P＜0.05),与秋水仙碱组相比,太白楤木大、中剂量组a-SMA表达均减低(均P＜0.05).[结论]太白楤木可通过抑制肝组织中胶原纤维的增生和肝星状细胞的活化,从而减轻肝纤维化.     

Ⅳ型胶原酶门脉灌注对兔肝硬化组织中ɑ-平滑肌动蛋白的影响

目的四氯化碳（CC l4）皮下注射建立兔肝硬化动物模型,观察Ⅳ型胶原酶门脉灌注对肝硬化组织中α-平滑肌动蛋白（α-SMA）表达的影响。方法取雄性新西兰大白兔,采用臀部皮下注射50%CC l4橄榄油0.23 mL/kg,每周2次,共12周制作肝硬化动物模型,注射等量橄榄油作为对照。12周后各组动物均建立门静脉给药通路,将已形成肝硬化并门静脉插管成功的33只兔随机分为两组（组1,组2）,组1有16只、组2有17只。组1经门静脉给药通路注入0.1%Ⅳ型胶原酶1.5 mL,组2注入等量0.9%NaC l,5次/周,共4周。将造模对照组中门静脉插管成功的30只兔同样随机分为两组（组3,组4）,每组15只,处理方法同前。4周后,将各组动物处死后留取肝脏组织,固定后α-SMA免疫组织化学染色,行积分光密度、面密度分析。结果肝硬化动物肝脏α-SMA表达显著增强;门脉灌注0.1%Ⅳ型胶原酶肝硬化动物肝脏纤维化程度明显降低,但α-SMA表达强度显著增高。结论采用门脉灌注0.1%Ⅳ型胶原酶可显著增高α-SMA表达,可能与肝脏肝星状细胞激活有关。     

支气管哮喘大鼠细胞周期蛋白D1与气道重塑关系的研究

目的 观察支气管哮喘(简称哮喘)大鼠模型肺组织中细胞周期蛋白D1的表达变化,以及细胞周期蛋白D1与气道重塑的关系.方法 16只SD大鼠随机分成正常组和哮喘组,每组8只.应用卵清蛋白致敏和激发的方法建立哮喘大鼠动物模型.HE染色观察气道炎症情况,采用马松染色观察气道胶原沉积变化,免疫组织化学和蛋白印迹法观察细胞周期蛋白D1的表达变化,免疫组织化学法观察气道平滑肌肌动蛋白-α表达变化.结果 哮喘组大鼠气道胶原沉积、细胞周期蛋白D1和肌动蛋白-α表达较正常组明显增高(P＜0.05),哮喘组肌动蛋白-α与细胞周期蛋白D1表达呈明显正相关(r1=0.20,P＜0.05; r2=0.53,P＜0.05).结论 细胞周期蛋白D1参与哮喘气道重塑的调节.     

Effects of oxymatrine on experimental hepatic fibrosis and its mechanism in vivo

AIM: Hepatic fibrogenesis has close relation with hepatic stellate cells (HSC)and tissue inhibitors of metalloproteinase (TIMP). Oxymatrine (OM) is a kind of Chinese herb that is found to have some effects on liver fibrosis. We aimed to determine the effects of OM on hepatic fibrosis and explore the possible mechanism. METHODS: Thirty-two rats were randomly divided into four groups; 16 were used to develop hepatic fibrosis by carbon tetrachloride (CCI4) and treated with or without OM, and 16 were used as controls. The expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and α-smooth muscle actin (α-SMA) in the livers of rats was detected by immunohisto-chemical assay. Liver pathology was determined by H&E staining and reticulum staining. RESULTS: In CCl4-injured rats, the normal structure of lobules was destroyed, and pseudolobules were formed. Hyperplasia of fibers was observed surrounding the lobules. While the degree of fibrogenesis in liver tissues was significantly decreased in those rats with OM-treatment compared with those without OM treatment. The pseudolobules were surrounded by strong, multi-layer reticular fibers, which netted into pseudolobules in CCl4-injured rats, however, there was a significant decrease in reticular fibers in OM-treated rats. The expression of TIMP-1 in hepatic cells was weak in control groups, but strong in CCl4-injured groups, however, the expression of TIMP-1 was significantly inhibited by OM (F = 52.93, P<0.05). There was no significant change in the expression of α-SMA between CCl4-injured rats with or without OM treatment (F= 8.99, P>0.05). CONCLUSION: OM effectively inhibits CCl4-induced fibrogenesis in rat liver tissues, probably by reducing the expression level of TIMP-1.     

Acute cryptosporidiosis as a cause of sudden recurrence of digestive symptoms in patients with Crohn's disease

Gastrointestinal symptoms occurring in patients with Crohn's disease (CD) can be related to disease activity or to intercurrent infection. Absence of appropriate stool work-up can lead to misdiagnosis and wrong treatment. We report here two cases of acute cryptosporidiosis in patients with CD. This microorganism can trigger IBD flare or cause severe infections in immunocompromised host. Adding specific search for oocysts of Cryptosporidium parvum using the Ziehl–Neelsen technique to the microbiologic work-up from stools in patients with Crohn's disease seeking medical intervention for sudden exacerbation of digestive symptoms seems to be recommended.     

OCTNs： Will the real 1BD5 gene please stand up？

Crohn's disease and ulcerative colitis are inflammatory disorders of the gastrointestinal tract with a substantial heritable component. The IBD5 region on chromosome 5q31 is one of only two loci widely confirmed to be associated with Crohn's disease in multiple independent cohorts. Although many populations have demonstrated association with IBD5, there remains uncertainty as to the causal variant within the region. A recent report identified polymorphisms in SLC22A4 (OCTN1) and SLC22A5 (OCTN2) as being responsible for the IBD5 association, however, these findings have not been replicated. This review discusses the data evaluating the IBD5 locus and the OCTN genes and their relationship to inflammatory bowel disease. Several other genes, including IRF1 and P4HA2 may be equally as likely to contain the IBD5 causal variant as the OCTN genes.     

Recombinant human soluble tumor necrosis factor receptor fusion protein as treatment for steroid refractory graft-versus-host disease following allogeneic hematopoietic stem cell transplantation

Etanercept is a recombinant human soluble tumor necrosis factor (TNF-alpha) receptor fusion protein that inhibits TNF-alpha, a major mediator in the pathogenesis of graft-versus-host disease (GVHD). The purpose of our study was to evaluate the safety and efficacy of etanercept therapy in 21 patients with steroid-refractory acute GVHD (aGVHD) (n = 13) and chronic GVHD (cGVHD) (n = 8). Etanercept 25 mg was given subcutaneously twice weekly for 4 weeks followed by 25 mg weekly for 4 weeks. At the time of initiation of etanercept, 14 patients had skin, 13 had gastro-intestinal, 5 had liver, 5 had pulmonary, and 4 had oral involvement. Twelve patients (57%) completed 12 doses of therapy. Overall, 11 of 21 patients (52%) responded to the treatment with etanercept, including 6 patients (46%) with aGVHD [n = 4 complete response (CR), n = 2 partial response (PR)] and 5 patients (62%) with cGVHD (n = 1 CR, n = 4 PR). Clinical responses were most commonly seen in patients with refractory gut aGVHD with 55% of the patients having a CR and 9% having a PR. CMV reactivation occurred in 48% of patients, bacterial infections in 14% of patients, and fungal infections in 19% of patients. Fourteen patients (67%) were alive after a median follow-up of 429 days (range 71-1007 days) since initiation of etanercept. Seven patients died, 3 of infections, 2 of refractory aGVHD, and 2 of disease progression. In conclusion, our preliminary data indicate that etanercept is well tolerated and can induce a high response rate in patients with steroid-refractory aGVHD and cGVHD, particularly in the setting of GI involvement.     

A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis

BACKGROUND & AIMS: Myofibroblasts of bone marrow origin have recently been found in a number of parenchymal organs such as the gut and kidney. We have analyzed the origin of myofibroblasts within fibrotic liver in 2 scenarios: (1) 7 male patients (hepatitis B; hepatitis B and D; Wilson's disease; hepatitis B, D, and C; and 3 with hepatitis C) who received liver transplants from female donors and subsequently developed liver fibrosis and (2) a female patient who received a bone marrow transplant from a male donor and subsequently developed hepatitis C-induced cirrhosis. METHODS: Through the use of in situ hybridization for the Y chromosome, we have tracked male cells of extrahepatic origin. The phenotype of these male cells was examined by immunohistochemistry using a panel of antibodies against alpha-smooth muscle actin (alpha SMA), vimentin, fibulin-2, and leukocyte common antigen (CD45). Confocal microscopy was performed to confirm the location of the Y chromosome probe within the myofibroblast nuclei. RESULTS: Significant numbers of Y chromosome-positive cells in fibrotic areas were found to be positive for alpha-SMA, vimentin, and fibulin-2 and negative for CD45, thus having a myofibroblast phenotype. In the liver transplant cases, 6.8%-22.2% of alpha-SMA-positive myofibroblasts contained the Y chromosome. In the female recipient of a bone marrow transplant from a male donor, 12.4% of the myofibroblasts were Y chromosome positive, indicating a bone marrow origin. CONCLUSIONS: There is a significant contribution to liver cirrhosis in humans from extrahepatically derived myofibroblasts in liver disease of diverse etiology.     

钙通道阻滞剂维拉帕米抑制肝星状细胞活化和功能

探讨维拉帕米（Ver）对人肝星状细胞系（HSC）-LX2的活化及分泌转化生长因子（TGF-β）的抑制作用。方法体外用内皮素-1（endothelin,ET-1）刺激HSC-LX2活化建立培养体系,设对照、ET-1和ET-1＋Ver 3组,对照组仅加入培养基,ET-1组加入ET-1和培养基,ET-1＋Ver组加入ET-1、Ver和培养基,分别在（0、12、24、48、72 h）观察HSC分沁α-平滑肌肌动蛋白（α-SMA）和细胞因子的能力,采用细胞爬片和免疫组化技术鉴定活化HSC的细胞形态;流式细胞术分析HSC表达α-SMA水平,ELISA测定不同组HSC分泌TGF-β的浓度。结果与对照组比较,ET-1组HSC活化和分泌TGF-β的能力较强（P〈0.05）;与ET-1组比较,ET-1＋Ver组HSC活化和分泌TGF-β能力较弱（P〈0.05）。结论 Ver体外实验中能抑制HSC的增殖和活化,具有潜在的抗纤维化作用。     

Preterm birth in women with inflammatory bowel disease – the association with disease activity and drug treatment

Background: The inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC) have been associated with an increased risk of preterm birth.

Material and methods: We identified all 246 singleton preterm births among women with IBD between July 2006 and December 2010 as cases and an equal number of controls with IBD from the Swedish national health registers, matched by maternal age, parity and IBD diagnosis (CD/UC). From register data and medical charts, we obtained information on reproductive history, comorbidity, disease activity and drug treatment (corticosteroids, 5-aminosalicylates, sulfasalazine, thiopurines and anti-TNF) as risk factors for preterm birth. Associations were estimated using conditional logistic regression and results were presented as odds ratios (OR) with 95% confidence intervals (CI).

Results: Previous preterm birth was more common among cases, OR 6.13 (95%CI: 2.51–15.01). Significant activity at any time during pregnancy (OR: 2.20; 95%CI: 1.37–3.53), and in particular both in early and in late pregnancy, was more common for cases (OR: 4.78 95%; CI: 2.10–10.9). The OR for immunosuppressive treatment with thiopurines or anti-TNF was 1.88 (1.04–3.39) without significant activity and 12.78 (95%CI: 3.68–44.72) with. The risk for women who discontinued thiopurines was 6.56 (1.44–29.82).

Conclusions: Significant activity and immunosuppressive treatment was associated with preterm birth, particularly in women with both. The existing recommendations to aim at maintaining quiescent disease during pregnancy, even if it means continuing immunosuppressive treatment, are rational.     

生长素在人肝星状细胞中的抗纤维化作用

目的观察生长素（Ghrelin）对血小板源生长因子-BB（PDGF-BB）刺激的体外培养人肝星状细胞（HSCLX2）前胶原I合成及α-平滑肌肌动蛋白（α-SMA）生成的影响。方法体外培养HSC-LX2,分别行Ghrelin、PDGF-BB干预和按0.05、0.1、0.15μmol/L Ghrelin＋PDGF干预细胞。采用定量PCR方法检测各组前胶原I mRNA的表达及Western印迹方法检测α-SMA表达情况。结果定量PCR结果显示PDGF处理组前胶原I mRNA的表达（6.91±0.46）较空白对照组（1.00±0.08）明显升高（P〈0.05）,证明PDGF能够促进HSC胶原合成。Ghrelin组前胶原I mRNA的表达（0.60±0.13）与空白对照组相比无明显变化,差异无统计学意义（P〉0.05）。0.05、0.1、0.15μmol/L Ghrelin＋PDGF共同处理组前胶原I mRNA表达分别为：（3.11±0.28）、（2.03±0.23）、（0.70±0.06）,与PDGF组相比显著降低,差异有统计学意义（P〈0.05）,且随着Ghrelin浓度的增加其抑制作用越明显,呈浓度依赖性关系。Western印迹结果显示Ghrelin＋PDGF共同处理组α-SMA较PDGF组表达降低。结论 PDGF刺激HSC活化后,Ghrelin可抑制其前胶原I及α-SMA生成,具有抗肝纤维化作用,可能成为防治肝纤维化的新途径之一。