小干扰RNA对特异性沉寂淋巴细胞白细胞介素-2基因表达和功能的影响

目的 探讨体外转录法合成的小干扰RNA（siRNA）对大鼠淋巴细胞白细胞介素（IL）-2基因的表达和功能的影响。方法 设计并合成3条siRYA（siRNA-1、siRNA-2、siRNA-3）在阳离子脂质体的介导下转染淋巴细胞。于转染后0、24、48h收集细胞，用半定量逆转录-聚合酶链反应（RT-PCR）方法检测IL-2mRNA的变化；酶联免疫吸附试验（ELISA）方法检测IL-2表达的变化。结果 体外合成的3条siRNA通过脂质体转染淋巴细胞后，均能不同程度地抑制I[,-2的表达。转染后24h的ELISA方法检测显示siRNA-1、siRNA-2、siRNA-3组的抑制率分别为（79．20±1．83）％、（66．50±1．42）％、（43．90±1．22）％，以siRNA-1的IL-2抑制作用最强。半定量RT-PCR方法检测显示siRNA-1、6iRNA-2、siRNA-3转染后的IL-2mRNA均受到不同程度的抑制，其中siRNA-2组的抑制作用最明显（82，10±1．85）％。结论 siRNA可特异性的抑制淋巴细胞IL-2基因的转录和表达，为研究siRNA在移植免疫耐受及防移植物抗宿主病（GVHD）方面的应用提供了依据。     

体外转染小鼠白细胞介素12基因抑制膀胱癌EJ细胞的实验研究

目的将含有小鼠白细胞介素12（mIL-12）全长基因的质粒转染到膀胱癌EJ细胞中,观察脾细胞对转染mIL-12膀胱癌细胞的杀伤作用。方法用脂质体转染法将含有mIL-12全长基因质粒转染到EJ细胞中,ELISA法检测IL-12表达水平。在脾细胞作用0～2 d时ELISA法连续检测上清干扰素γ（INF-γ）水平变化。转染后40 h加入制备好的小鼠脾细胞悬液约1×10^6作用24 h后,MTT法检测对膀胱癌细胞的抑制率。结果转染后40、60 h检测到IL-12的高表达（375±15） pg／ml和（400±50）pg／ml,较未转染组9～15 pg／ml明显增高;在EJ细胞表面可以检测到IL-12的表达,而对照组则无;加入脾细胞悬液1 d后,脾细胞加转染后的EJ细胞组的抑制率为58．7％（P〈 0．025）,而脾细胞加未转染EJ细胞组、单纯EJ细胞组以及转染的EJ细胞组差异无统计学意义（P〉 0．05）。脾细胞加入转染后EJ细胞上清24 h的。INF-γ为（155±21）pg／ml,与未转染组（65±8）pg／ml比较差异有统计学意义（P〈0．05）。结论体外转染mIL-12基因到膀胱癌EJ细胞中能够表达IL-12,在小鼠脾细胞存在的条件下,通过一系列的免疫调节作用,诱导T细胞、NK细胞等产生INF-γ等细胞因子从而杀伤膀胱癌细胞。     

热休克因子1基因转染对烧伤血清刺激的巨噬细胞炎性介质表达的影响

目的了解外源性热休克因子1（HSF1）对烧伤血清刺激的巨噬细胞中相关炎性介质基因表达的影响。方法制备严重烧伤和正常大鼠血清；构建pcDNA3．1／HSF1重组载体。将培养的RAW264．7巨噬细胞株转染（具体分为：未转染、转染空载体、转染重组载体）后再分别使用前述2种血清刺激。另取部分未行血清刺激的转染重组载体的巨噬细胞，用蛋白质印迹法检测其HSF1蛋白表达水平；反转录-PCR法检测经血清刺激的细胞中肿瘤坏死因子α（TNF-α）、高迁移率族蛋白B1（HMGB1）和白细胞介素10（IL-10）基因表达水平。结果已转染重组载体的细胞株较稳定，检出有HSF1部分活化。反转录-PCR检测到，未转染的正常血清刺激的巨噬细胞中TNF—α、HMGB1、IL-10的基因仅有少量表达，而烧伤血清刺激能明显上调其基因表达（分别为0．910±0．100、0．860±0．020、0．430±0．010）；与转染空载体＋烧伤血清组（上述3项指标分别为0．800±0．050、0．880±0．030、0．420±0．010）比较，转染重组载体＋烧伤血清组可明显抑制巨噬细胞中TNF—α和HMGB1的基因表达并上调IL-10的基因表达（分别为0．130±0．100、0．450±0．020、0．450±0．020）。结论严重烧伤后给予HSF1可以抑制巨噬细胞产生的某些促炎介质的表达，适当上调某些抗炎介质的表达，对机体发挥保护作用。     

人白细胞介素-10基因的克隆与逆转录病毒载体的构建

目的克隆、测序人白细胞介素(hIL)-10基因片段,构建逆转录病毒载体(MSCV- hIL-10)。方法采用刀豆蛋白A活化的人外周血单个核细胞培养提取总RNA,设计随机引物并逆转录反应合成hIL-10 cDNA第1链,并以hIL-10特异性引物行PCR扩增,获得hIL-10克隆基因片段,将hIL-10克隆基因片段,经双酶切后定向插入到逆转录病毒载体(MSCV)中,用脂质体法转染PT67包装细胞,以G418筛选阳性克隆。结果聚合酶链反应(PCR)扩增出534 bp特异性片段,测序结果表明同源性与GenBank报道完全一致,hIL-10基因片段插入MSCV载体中经转染包装后得到2×10~7 CFU／ml的分泌hIL-10的病毒悬液,hIL-10的分泌量为1220ng／10~6细胞·d~(-1)。结论成功克隆hIL-10开放阅读框架,成功构建MSCV-hIL-10重组质粒,获得高滴度的分泌hIL- 10的病毒悬液,hIL-10的表达可以应用于移植排斥的基因治疗。     

超声微泡介导白细胞介素-1受体拮抗蛋白基因体外转染兔软骨细胞的研究

目的 观察超声介导下携白细胞介素-1受体拮抗蛋白基因(IL-1Ra)的微泡体外转染兔软骨细胞的效率和表达.方法 体外分离培养兔软骨细胞,分为单纯质粒组(P)、微泡+质粒组(M+P)、超声+质粒组(U+P)和超声+微泡+质粒组(U+M+P),照射后48 h,荧光显微镜和流式细胞术检测转染效率,逆转录-聚合酶链反应(RT-PCR)和Western blot法检测IL-1Ra基因的mRNA和蛋白表达,台盼兰染色检测细胞生存率.结果 U+M+P组转染效率较其他三组明显提高,为(11.6±1.0)%;且eGFP-C1-IL-1Ra质粒经超声微泡介导转染后可表达IL-1Ra的mRNA和蛋白.结论 超声微泡可介导IL-1Ra基因在兔软骨细胞内的转染和表达,可望成为软骨损伤基因治疗的新方法.     

hIL-10基因修饰的骨髓间充质干细胞的体外免疫抑制作用

目的研究hlL．10基因修饰的骨髓间充质干细胞（mesenchymalstarecell,MSC）对异种混合T淋巴细胞增殖的影响。方法反转录PCR克隆hlL-10基因并构建慢病毒hlL-10／pLOX—CWgfp,然后将hIL．IO／pLOX—CWgfp转入豚鼠MSC内;ELISA法测定hIL-10基因修饰的MSC（hlL-10-MSC）培养上清液内hIL-10含量;CCK-8法检测hIL-10-MSC和其培养上清液对体外混合淋巴细胞培养的影响。结果hIL-10．MSC培养上清液的hIL-10水平明显高于未转染hIL-10者（P〈0．05）;hIL-10-MSC能抑制异种T淋巴细胞混合培养的增殖反应（P〈0．05）;加入IL-2能逆转MSC的抑制作用（P〈0．05）;hIL-10-MSC培养上清液能抑制异种T淋巴细胞混合培养的增殖反应（P〈0．05）。结论hIL-10-MSC和其培养上清液对混合异种淋巴细胞培养有显著的抑制作用。     

白细胞介素-10基因转染对活化巨噬细胞炎症介质产生的抑制作用

目的 探讨炎症诱导启动子指导人白细胞介素-10(hIL-10)基因的表达及其对炎症介质产生的效应.方法 诱导性真核表达载体pSAA3 hIL-10脂质体转染体外培养巨噬细胞,LPS(10 mg/L)活化后观察巨噬细胞hIL-10的表达(RT-PCR,ELISA法),测定肿瘤坏死因子-α(TNF-α)、IL-6的产生(ELISA法).结果 RT-PCR证明hIL-10可在巨噬细胞表达;巨噬细胞LPS(10 mg/L)活化12 h有hIL-10的表达(2.67 μg/L),活化24 h及48 h的巨噬细胞上清hIL-10显著增加(1.87～5.71倍);转基因预处理显著下调活化细胞TNF-α(25.0%～61.4%,P＜0.05)的产生,显著减少IL-6产生(54.8%～63.9%,P＜0.05).结论 LPS可活化pSAA3 hIL-10表达体系在巨噬细胞诱导表达,白细胞介素-10基因转染显著下调活化MΦ炎症介质的产生.     

人IL-1受体拮抗蛋白重组逆转录病毒载体的构建、鉴定及在骨关节炎软骨细胞中的表达

目的 构建含人IL-1受体拮抗蛋白(IL-1 receptor antagonist, IL-1Ra)逆转录病毒表达载体(PLXRN-IL-1Ra),体外转染人骨关节炎(osteoarthritis, OA)软骨细胞,研究其相关特性.方法 利用细菌内同源重组技术快速构建PLXRN-IL-1Ra逆转录病毒重组质粒,经测序及酶切鉴定正确后转染PT67细胞,包装成为重组PLXRN-IL-1Ra逆转录病毒,并使用小鼠肾成纤维细胞系NIH/3T3对病毒进行滴度测定.实验分为3组未转染组(A组)、PLXRN空质粒转染组(B组)、PLXRN-IL-1Ra转染组(C组),病毒感染人OA软骨细胞后,RT-PCR检测细胞内IL-1Ra基因的转录和表达;ELISA法检测细胞培养上清液中人IL-1Ra蛋白表达.结果 酶切鉴定及基因测序证实重组逆转录病毒质粒中含有人IL-1Ra cDNA,测定包装的病毒滴度为3 × 104 CFU/mL.原代软骨细胞体外培养呈多角形或梭形,甲苯胺蓝染色见细胞内有紫色异染颗粒.RT-PCR结果显示在C组出现311 bp人IL-1Ra mRNA片段,A、B组未见人IL-1Ra mRNA的表达带,GAPDH在各组均有表达.ELISA检测发现C组细胞上清有一定量的人IL-1Ra表达,蛋白浓度为(60.47±15.13)ng/L,A组和B组均无人IL-1Ra表达.结论 构建的IL-1Ra逆转录病毒表达载体成功地感染人OA软骨细胞,并在体外获得稳定表达,为将表达人IL-1Ra基因的人OA软骨细胞用于OA基因治疗提供了实验依据.     

白细胞介素-4和γ-干扰素基因联合治疗胶质瘤

目的 探讨逆转录病毒载体介导的白细胞介素(IL)-4和γ-干扰素(IFN)基因联合治疗胶质瘤的作用。方法构建携带IL-4或IFN-γ基因的逆转录病毒载体，将其导入逆转录病毒包装细胞；将携带IL-4和IFN-γ基因的逆转录病毒包装细胞分别或联合注射到脑内荷瘤大鼠的肿瘤组织中，观察其治疗作用。结果成功获得携带治疗基因的逆转录病毒包装细胞PA317IFN-γ和PA317IL-4，所产病毒滴度分别为2x10^6cfu／ml和2．5x10^6cfu／ml。包装细胞瘤内注射能够诱导大鼠产生抗肿瘤免疫反应，杀伤肿瘤细胞，联合应用具有协同治疗作用。结论携带IL-4或IFN-γ基因的逆转录病毒包装细胞瘤内注射是一种有效的胶质瘤基因治疗方法，两者联合应用具有协同治疗作用。     

人白细胞介素-10/人肝再生增强因子融合基因的构建及在肝细胞中的表达

目的 利用人自细胞介索(IL)-10和肝再生增强因子(ALP)构建融合基因hIL-10／ALR和真核表达载体,验证其在肝细胞中的表达。方法 以富含疏水氨基酸(Gly-Ser)n的相应寡核苷酸接头序列为媒介,通过hIL-10和hALR克隆载体构建真核质粒表达载体pcDNA3hIL-10／ALR,纯化后转染L02肝细胞,并以逆转录-聚合酶链反应(RT-PCR)方法检测其表达。结果 成功构建hIL-10／ALR融合基因,其真核表达载体pcDNA3hIL-10／ALR可在L02肝细胞中有效表达。结论通过融合基因转导方法可能实现IL-10和ALR的各自功能,为肝纤维化、肝硬变的基因治疗探索有效手段。     

Circulating interleukin 6 and interleukin 10 in community acquired pneumonia

BACKGROUND: Inflammatory cytokine concentrations correlate with severity of sepsis. We hypothesised that patients with community acquired pneumonia (CAP) associated with systemic inflammatory response syndrome (SIRS) would have greater interleukin 6 (IL-6) production due to activation of the inflammatory cytokine cascade, matched by a significant anti-inflammatory cytokine response. Interleukin 10 (IL-10) was evaluated as a potential surrogate marker of severity of sepsis in CAP and age related impairment of the cytokine response was studied in elderly patients with CAP. METHODS: Circulating immunoreactive IL-6 and IL-10 levels were measured in 38 patients with CAP subdivided into a group fulfilling the criteria for SIRS (n = 28) and a non-SIRS group (n = 10) in a variety of age groups and correlated with APACHE II scores. RESULTS: 80% had circulating IL-6 levels (median 46.7 pg/ml, range 4.6-27,000) and 60% had circulating IL-10 levels (median 15.5 pg/ml, range 2.5-765). Concentrations of both were significantly increased in patients with SIRS compared with non-SIRS patients. Those with activation of the inflammatory cytokine cascade (IL-6 positive) produced more IL-10 than IL-6 negative patients. Older patients had a similar cytokine response. Both cytokines correlated positively with APACHE II scores. CONCLUSIONS: This is the first demonstration of circulating IL-10 in CAP. A greater counter-inflammatory response in patients with SIRS and in IL-6 positive patients suggests a potential immunomodulatory role for IL-10 in controlling the inflammatory cytokine response in CAP. IL-10 concentrations correlate with severity of illness in CAP and may be of prognostic importance. There is no age related impairment in the cytokine response.     

Bone formation in interleukin‐4 and interleukin‐13 depleted mice

Background and purpose?Cytokines play an important role in the complex process of bone formation. We have previously found an altered skeletal phenotype with reduction of cortical bone mass in mice depleted of the 2 cytokines interleukin‐4 (IL‐4) and interleukin‐13 (IL 13). The present study was performed to investigate a potential role of IL‐4 and IL‐13 in fracture healing and bone induction by demineralized xenogenic bone matrix (DXBM).

Methods?Callus formation in IL‐4-^/-IL‐13-^/- (IL‐4/13 knockout) and wild‐type (WT) male mice was compared using a standardized fracture model. The capacity of IL‐4^-/-IL‐13^-/- and WT male and female mice to form heterotopic bone was compared using intramuscular implants of DXBM. Bone formation and mechanical properties were evaluated by pQCT, ash weight, 3‐point bending, radiology, and immunohistology.

Results?In the fracture investigation substantial amounts of new bone formation by 5 weeks were found, but no differences in radiographical healing, callus volume, BMD, BMC, or mechanical properties were detected between IL‐4^-/-IL‐13^-/- and WT mice. In the DXBM investigation radiographic analysis confirmed mineralization of implants in both groups, but no difference in the amount of mineral deposition (net bone formation) between IL‐4^-/-IL‐13^-/- and WT mice was found. Immunohistology showed inhibition of autonomic nerves in the capsule of the IL‐4^-/-IL‐13^-/- group along with a lack of vascularization within the implants.

Interpretation?Depletion of IL‐4 and IL‐13 does not cause any major alteration in fracture healing or heterotopic bone formation in mice. The pattern of autonomous nerve expression and expression of markers of neovascularization is, however, altered to some extent by the absence of IL‐4 and IL‐13.     

Decreased interleukin generation in patients with cancer of the digestive system. A correlation between interleukin 1 and interleukin 2 production

The generating capacity of interleukin 1 (IL-1) and interleukin 2 (IL-2) by peripheral blood mononuclear cells (PBMNC) was measured in 40 patients with digestive cancer (20 localized and 20 disseminated) and 20 age- and sex-matched control subjects. The localized carcinoma patients showed normal IL-1 production and a significantly depressed IL-2 production (p<0.05) when compared to the healthy individuals. The disseminated carcinoma patients exhibited a significant impairment of both IL-1 and IL-2 production in comparison with the healthy controls (IL-1: p<0.001, IL-2: p<0.001) and the localized carcinoma patients (IL-1: p<0.001, IL-2: p<0.001). A significant correlation was observed between IL-1 and IL-2 generation in all the cancer patients (r=0.458, p<0.01). These results suggest that progressive tumor growth may result in decreased interleukin production by the host PBMNC, and that related mechanisms, which are more susceptable to lymphocytes than monocytes, may be involved in the impairment of both IL-1 and IL-2 production.     

Decreased interleukin generation in patients with cancer of the digestive system. A correlation between interleukin 1 and interleukin 2 production

The generating capacity of interleukin 1 (IL-1) and interleukin 2 (IL-2) by peripheral blood mononuclear cells (PBMNC) was measured in 40 patients with digestive cancer (20 localized and 20 disseminated) and 20 age- and sex-matched control subjects. The localized carcinoma patients showed normal IL-1 production and a significantly depressed IL-2 production (p less than 0.05) when compared to the healthy individuals. The disseminated carcinoma patients exhibited a significant impairment of both IL-1 and IL-2 production in comparison with the healthy controls (IL-1: p less than 0.001, IL-2: p less than 0.001) and the localized carcinoma patients (IL-1: p less than 0.001, IL-2: p less than 0.001). A significant correlation was observed between IL-1 and IL-2 generation in all the cancer patients (r = 0.458, p less than 0.01). These results suggest that progressive tumor growth may result in decreased interleukin production by the host PBMNC, and that related mechanisms, which are more susceptible to lymphocytes than monocytes, may be involved in the impairment of both IL-1 and IL-2 production.     

Genetic predisposition to latex allergy: role of interleukin 13 and interleukin 18

BACKGROUND: Occupational exposure of healthcare workers to natural rubber latex has led to sensitization and potentially life-threatening anaphylaxis. Although environmental exposure to natural rubber latex products is necessary for sensitization, it is not sufficient. A number of genetic factors also seem to contribute to the latex sensitization; however, the multigenic nature of the allergic phenotype has made the identification of susceptibility genes difficult. The current study tests the hypothesis that known functional polymorphisms in genes encoding interleukin 4, interleukin 13, and interleukin 18 occur in a higher frequency in healthcare workers with natural rubber latex allergy. METHODS: Four hundred thirty-two healthcare workers with occupational exposure to natural rubber latex were screened using a clinical history questionnaire and latex-specific immunoglobulin E serology. Genomic DNA was extracted from peripheral blood lymphocytes and analyzed for single-nucleotide polymorphisms in candidate genes of interest. Data from cases and controls were analyzed by nominal logistic regression, with P < 0.05 considered significant. RESULTS: The latex allergy phenotype was significantly associated with promoter polymorphisms in IL13 -1055 (P = 0.02), IL18 -607 (P = 0.02), and IL18 -656 (P = 0.02) compared with nonatopic controls. CONCLUSIONS: The significant association of IL13 and IL18 promoter polymorphisms with latex allergy suggests a potential location for genetic control in the induction of latex allergy in individuals and extends the understanding of the genetic basis for the induction of immediate-type hypersensitivity in healthcare workers occupationally exposed to natural rubber latex.     

Human mesangial cells synthesize interleukin 1 alpha but not interleukin 1 beta, interleukin 1 receptor antagonist, or tumour necrosis factor.

There is controversy over whether mesangial cells synthesize and release IL-1 and TNF, and many of the positive experiments were performed before specific reagents and molecular probes were available. Consequently we have stimulated human mesangial cells using protocols known to stimulate the synthesis of other cytokines. No mRNA for IL-1 beta or TNF could be detected in quiescent or proliferating mesangial cells irrespective of whether they had been exposed to cytokines or not. In contrast mRNA for IL-1 alpha was detected in cells stimulated with IL-1 beta 10 ng/ml or with TNF 500 ng/ml; IL-1 alpha was also detected in cell lysates from stimulated mesangial cells. We could not detect mRNA for IL-1 receptor antagonist in any of the cell preparations. These results suggest that mesangial cells are unlikely to be a major source of IL-1 beta or TNF.     

Precursor frequency and lytic specificity of interleukin 2 and interleukin 4-responsive murine lymphocytes

The response to IL-2 and IL-4 of several functionally defined lymphoid cell types was assessed at the individual responding cell level in limiting dilution experiments in which exogenous IL-2 or IL-4 was used to promote cellular activation and expansion. Splenic precursors that give rise to alloreactive CTL in LDA supplemented with IL-2 were approximately 3-fold more frequent than those detected in IL-4. Of special interest was the observation that cytolytic cultures arising in primary allogeneic LDA supplemented with IL-4 exhibited a greater degree of lytic specificity than those arising in IL-2. This difference may be due to an inherently broader specificity of CTL cultured in IL-2 or to a concomitant activation of CTL and lymphokine-activated killer(s) (LAK) in individual IL-2 supplemented microcultures. In this regard, estimation of LAK precursor(s) (LAK-P) frequencies in LDA cultures established without an overt antigenic stimulus revealed that IL-2 activates a 20-fold higher frequency of LAK-P than does IL-4. Finally, an examination of the proliferative response to IL-2 and IL-4 of several CTL and PTL clones demonstrated that IL-4 responsive cells comprise a subset of cells that respond to IL-2, irrespective of their functional phenotype.     

Sequential interleukin 2 and interleukin 2 receptor levels distinguish rejection from cyclosporine toxicity in liver allograft recipients

Previous studies of renal transplant recipients have demonstrated that allograft rejection is accompanied by an increase in plasma and urinary levels of interleukin 2 and its soluble receptor before the development of clinical symptoms. After measuring interleukin 2 and interleukin 2 receptor levels in the plasma, bile, and urine of liver transplant recipients, we found that rejection is preceded by elevation of plasma and biliary levels of both substances, that cyclosporine toxicity did not affect either of these levels, and that urinary levels of the substances are unaffected in either condition. Levels of interleukin 2 and interleukin 2 receptors increased in bile earlier than in plasma, and interleukin 2 levels did not overlap among stable patients and those experiencing rejection, whereas levels of interleukin 2 receptors did. Serial measurements of interleukin 2 levels, particularly in the product of the transplanted organ, provide a reliable assessment of the immunologic status of the allograft.     

Soluble interleukin 2-receptor alpha secretion is related to altered interleukin 2 production in thermally injured patients

This study examines the relationship between the capacity for interleukin-2 (IL2) production and the magnitude of the in vitro and in vivo secretion of IL2R alpha in 43 patients with major burns (30-90 per cent total body surface area). Throughout the postburn period a significant (P less than 0.001-0.05) proportion of patients studied demonstrated increasingly high levels of serum IL2 ranging from 2 to over 500 U/mL. Serum IL2R alpha also increased, reaching its highest levels at 15-40 days postburn, while serum IL2 gradually declined. In this period in vitro IL2 production and IL2R alpha secretion in patient's cultures were significantly reduced compared to control. However, in parallel cultures supplemented with exogenous IL2, IL2R alpha levels could be significantly increased (2.5 fold). IL2R alpha levels also approached normal in peripheral blood mononuclear cell cultures from recovering patients whose in vitro IL2 production had improved. These observations suggest that in the burn patient altered synthesis and/or secretion of the soluble form of IL2R alpha may be related to IL2 content. Above physiological levels of IL2R alpha and its ligand in postburn serum also indicate that thermal injury induces strong in vivo activation of the lymphoid system.