人白细胞介素12双亚基共表达真核载体的构建

目的 :为满足基因治疗的需要构建人白细胞介素 12 (hIL 12 )双亚基共表达质粒。方法 :先从人胚肾组织的逆转录产物中用PCR方法扩增出它的两个亚基P4 0 和P3 5的cDNA全长 ,分别克隆入真核表达载体pcDNA3.1(+/ )中构建单亚基质粒———P(+) /P4 0 和P( ) /P3 5,然后再将二者串联克隆入真核表达载体 pcDNA3.1(+)中得到P(+) /IL 12质粒。脂质体转染HepG2后 2 4,48hELISA检测细胞培养上清内hIL 12蛋白质表达。结果 :P(+) /IL 12质粒经酶切和测序证明两亚基连接方向正确 ,序列无突变 ;ELISA检测细胞上清结果证实可表达hIL 12蛋白质。结论 :成功构建P4 0 和P3 5双亚基真核共表达质粒———P(+) /IL 12 ,为模拟hIL 12生理表达方式 ,简化hIL 12基因治疗操作奠定了基础。     

当归多糖组分促进淋巴细胞增殖及对IL-2和IFN-γ的诱生作用

目的:观察当归多糖及其3个组分对淋巴细胞增殖和细胞因子IL-2、IFN-γ的影响.方法:机械分散法制备小鼠单个脾细胞悬液;MTT法检测细胞增殖;酶联免疫法测定培养上清液中IL-2和IFN-γ的浓度.结果:当归多糖及其3个组分在30～100 μg/ml剂量范围内,能显著促进脾细胞的增殖;其中组分AP-3能够显著促进巨噬细胞、混合淋巴细胞的增殖反应,剂量依赖性地增加细胞上清液中IL-2和IFN-γ的浓度.结论:当归多糖能够活化巨噬细胞和T细胞,还可以通过细胞因子IL-2、IFN￣γ发挥免疫调节作用.     

饲料中添加钴、维生素B12对羔羊生长的影响

选用产期接近的3月龄小尾寒羊30只,随机分为二组(试验Ⅰ、试验Ⅱ),每组15只。试验Ⅰ饲喂本场加工的羔羊精饲料,试验Ⅱ饲喂本场加工的羔羊精饲料基础上,每公斤饲料中分别添加钴0.4mg和维生素B120.30mg,试验结果表明:试验Ⅰ与试验Ⅱ日增重相比差异极显著(P<0.01)。     

人巨细胞病毒感染对肾移植受者血清白细胞介素－6水平的影响

应用ＰＣＲ检测ＨＣＭＶ－ＤＮＡ，ＥＬＩＳＡ检测ＨＣＭＶ－ＩｇＭ、ＩｇＧ，诊断肾移植受者ＨＣＭＶ感染，６５例受者中ＨＣＭＶ感染者３９例，非感染者２６例。应用ＭＴＴ法检测受者血清ＩＬ－６生物活性，阐明了ＨＣＭＶ感染对肾移植受者血清ＩＬ－６水平的影响。结果表明：感染与非感染组间血清ＩＬ－６水平差异无显著性（Ｐ＞０．０５）；６例原发性感染者血清ＩＬ－６水平随感染时间延长呈增高及降低双相改变，表明慢性迁延性感染者血清ＩＬ－６水平降低。临床工作中监测ＨＣＭＶ感染的肾移植受者血清ＩＬ－６水平变化具有重要意义。     

Serum and Cerebrospinal Fluid Vitamin B12 Levels in Demented Patients with CH3—B12 Treatment—Preliminary Study—

Abstract: The vitamin B12 (VB12) parameter was studied in the serum and cerebrospinal fluid (CSF) of 14 demented patients. Eleven of these patients were in a state of dementia of the degenerative type such as Alzheimer's disease, senile dementia and Pick's disease. The serum VB12 concentration in all the patients was within normal limits, I.e. 500–1,300 pg/ml. There was no significant difference between the CSF-VBl2 levels and the severity of dementia. The serum and CSF-VB12 levels of the demented patients did not show any significant elevation after the oral administration of CH3–Bl2, 2 mg per day. On the other hand, there was a marked elevation of both the serum and CSF-VB12 after an oral medication (2 mg per day) plus intramuscular administrations (500 μg per day). These results confirm that the intramuscular administration of CH3–B12 is an effective way to get a higher value of the serum and CSF-VB12 levels.     

Intranasal treatment with ovalbumin but not the major T cell epitope ovalbumin 323–339 generates interleukin-10 secreting T cells and results in the induction of allergen systemic tolerance

BACKGROUND: Nasal administration of major peptide T cell epitopes gives contradictory data on the induction of peripheral tolerance. OBJECTIVE: To compare the prophylactic effect of intranasal treatment (INT) on the development of an allergic response, using either ovalbumin (OVA) or its major T cell epitope OVA 323-339 (OVAp). METHODS: BALB/c mice were treated intranasally with OVA or OVAp and subsequently immunized s.c. with OVA. Anti-OVA-specific antibody, T cell proliferation and cytokine responses were analysed. In an adoptive transfer model using OVAp specific TCR transgenic (Tg) T cells from D011.10 mice, in vivo tracking and characterization of transferred T cells in the cervical, inguinal and bronchial lymph nodes (BLN) and in the spleen were determined by FACS analysis. RESULTS: Prophylactic INT with OVA induced T cell tolerance towards subsequent OVA s.c. immunizations, inhibiting OVA specific T cell proliferation, IgE and IgG1 production, in contrast to INT with OVAp, which was unable to induce tolerance. In vivo analysis of transferred OVA-specific TCR Tg T cells showed that INT with OVA induced a preferential activation of T cells in BLN, as opposed to a broad, systemic activation with OVAp. In vivo, OVAp INT led to faster and more sustained cell division cycles than OVA INT. Ex vivo, tolerance to OVA was associated with the generation of IL-10 secreting CD4(+) T cells in BLN of OVA-treated mice only. CONCLUSION: INT with OVA but not with OVAp led to regional (as opposed to systemic) T cell activation and the induction of IL-10 secreting CD4(+) T cells in BLN, potentially critical steps in the induction of T cell-specific tolerance via the nasal route.     

Allergen activates peripheral blood eosinophil nuclear factor-κB to generate granulocyte macrophage-colony stimulating factor, tumour necrosis factor-α and interleukin-8

BACKGROUND: Allergic inflammation is characterized by the influx and activation of eosinophils. Cytokines generated by both resident and infiltrating cells are responsible for the initiation and maintenance of this pathogenesis. This study focuses on allergen-induced activation of eosinophil NF-kappaB and generation of granulocyte macrophage-colony stimulating factor (GM-CSF), TNF-alpha, and IL-8. METHODS: Peripheral blood eosinophils were enriched to >99.9% by Percoll gradient sedimentation and negative magnetic affinity chromatography. NF-kappaB activation by 10 microg/mL house dust mite (HDM) extract was demonstrated immunocytochemically using a monoclonal antibody against the active form of NF-kappaB (NF-kappaBa). The authenticity of NF-kappaB was confirmed by Western blot. Cytokine production was assessed both by immuno-staining of eosinophils and by assay of cytokines in the cell supernatant. RESULTS: Activation of peripheral blood eosinophils from atopic, but not non-atopic, donors induced activation of NF-kappaB, which peaked at 4 h and was accompanied by a decline in IkappaB-alpha. The activation of authentic NF-kappaB was confirmed in gel shift assays. Supershift assays showed p65 to be the major subunit of eosinophil NF-kappaB. Immunofluorescent confocal microscopy demonstrated localization of NF-kappaBa to the nucleus. Following activation, cytokine immunoreactivity was seen in a fraction of the eosinophils and cytokines were released into the supernatant. The NF-kappaB inhibitors, calpain inhibitor 1 (10 microm), pentoxifylline (0.5 mm), pyrrolidine dithiocarbamate (PDTC, 10 microm) or gliotoxin (1 pg/mL) reduced the generation of GM-CSF, TNF-alpha and IL-8 in parallel with their inhibition of NF-kappaB. CONCLUSIONS: HDM allergen activates human eosinophil NF-kappaB leading to the production of the cytokines GM-CSF, TNF-alpha and IL-8. We speculate that a role for eosinophil NF-kappaB-dependent cytokines is to act as an autocrine loop augmenting the survival of eosinophils in vivo.     

爆炸伤创面IL-1、IL-6、TNF-α、CRP的测定

[目的]检测兔肢体爆炸伤创面组织IL-1、IL-6、TNF-α和CRP含量。[方法]新西兰大白兔24只,随机分为炸伤后即刻取材组（A组,n=12）和炸后1h取材组（B组,n=12）;0.9g铜壳单质猛黑索金炸药（RDX）以海绵间隔5cm,绑于左下肢股部中段前外侧,电引爆,分别取爆炸中心区（Ⅰ区）、爆炸边缘区（Ⅱ区）、爆震区（Ⅲ区）的肌肉组织,测IL-1、IL-6、TNF-α、CRP的含量并取对侧肢体肌肉组织做对照研究。[结果]炸伤后两组各区标本IL-1、IL-6、TNF-α、CRP含量均比正常组织高,差异显著（P〈0.05）,且从Ⅰ区、Ⅱ区到Ⅲ区,含量依次降低,差异显著（P〈0.05）：A组：Ⅱ区与Ⅲ区的IL-1含量差别有显著意义（P〈0.05）。Ⅰ区与Ⅲ区、Ⅱ区与Ⅲ区的IL-6含量差别有极显著意义（P〈0.01）。Ⅰ区与Ⅲ区的TNF-α、CRP含量差别有极显著意义（P〈0.01）,Ⅰ区与Ⅱ区、Ⅱ区与Ⅲ区的含量差别有显著意义（P〈0.05）。其余各区的含量无统计学差异（P〉0.05）;B组：Ⅱ区与Ⅲ区的Ⅱ-1、IL-6、TNF-α含量差别有显著意义（P〈0.05）。Ⅰ区与Ⅲ区、Ⅰ区与Ⅱ区、Ⅱ区与Ⅲ区的CRP含量差别有显著意义（P〈0.05）。其余各区的含量无统计学差异（P〉0.05）。A、B两组比较,炸后组织中上述因子表达虽有增强,但差异无显著意义（P〉0.05）。[结论]肢体爆炸伤1h内,创面组织中IL-1、IL-6、TNF-α、CRP表达增强,不同区域间表达有差异。     

内皮细胞生长因子诱导大鼠血管内皮细胞增殖过程中FKBP12的表达

目的探讨内皮细胞生长因子(Endothelial cell growth factor，ECGF)诱导的血管内皮细胞(Vascular endothelial cell，VEC)增殖过程中FK506结合蛋白12(FK506 binding protein 12，FKBP12)表达的变化，为VEC的鉴定提供新的方法。方法培养大鼠VEC并传代。倒置显微镜下观察其在ECGF作用下的增殖情况，检测Ⅷ因子相关抗原、FKBP12和不同时期FKBP12 mRNA水平的表达。结果VEC表达FKBP12；VEC传代后第4天FKBP12 mRNA表达水平达到最高，6—8d下降。结论FKBP12既可作为VEC的标志物，又可反映VEC的增殖情况。     

表达白细胞介素18的条件增殖腺病毒在肾癌Ketr-3细胞中的生物活性及抗肿瘤作用

目的 观察表达白细胞介素18(IL-18)基因的条件增殖腺病毒在肾癌Ketr-3细胞中的生物活性及其对Ketr-3细胞的杀伤作用.方法 通过荧光显微镜观察表达绿色荧光蛋白的条件增殖腺病毒(ZD55-EGFP)在肾癌Ketr-3细胞中的感染和增殖情况.分别将表达IL-18的条件增殖腺病毒(ZD55-IL-18)及表达IL-18的普通腺病毒(Ad-IL-18)感染人肾癌Ketr-3细胞系,通过Western blot法检测病毒E1A和IL-18蛋白的表达;免疫细胞化学染色检测IL-18抗原表达;TUNEL法检测Ketr-3细胞的凋亡情况;噻唑蓝(MTT)比色法检测Ketr-3细胞存活情况.结果 ZD55-EGFP能有效感染肾癌Ketr-3细胞并在其中大量增殖.Westem blot检测结果 发现ZD55-IL-18能在肿瘤细胞内表达E1 A并有效介导IL-18表达,在病毒感染Ketr-3细胞48 h后,ZD55-IL-18、Ad-IL-18处理组的IL-18蛋白表达量分别为255.6±3.1、118.7±2.90免疫组织化学检测显示ZD55-IL-18、Ad-IL-18处理组的IL-18抗原阳性率分别为(82.4±3.2)%和(23.4±1.9)%.TNUEL检测结果 显示ZD55-IL-18、Ad-IL-18处理组的细胞凋亡率分别为(52.2±3.5)%和(25.5±1.9)%.病毒感染4 d后,MTT检测结果 显示ZD55-IL-18、Ad-IL-18处理组细胞存活率分别为(32.6±2.3)%和(73.3±2.5)%,表明ZD55-IL-18对Ketr-3细胞有显著的杀伤作用.结论 ZD55-IL-18能在Kerr-3细胞中高效特异性表达IL-18基因并显示出良好的抗肿瘤作用.