糖皮质激素瘤内注射治疗婴幼儿血管瘤机理的研究

目的:探讨糖皮质激素治疗婴幼儿血管瘤的机理.方法:对50例不同组别血管瘤(治疗显效组18例,无效组17例,未治疗组15例)及16例正常皮肤标本进行:①凋亡研究:光镜及透射电镜下观察组织形态学变化;TUNEL法检测血管内皮细胞(VEC)的凋亡水平;IHC法检测凋亡调控基因bcl-2,c-myc的表达;②增殖水平研究:IHC法检测PCNA.结果:①组织形态学变化:光镜下各组均无明显炎症及坏死征象,透射电镜观察显效组有较多VEC凋亡征象.②VEC增殖及凋亡:显效组VEC的凋亡水平显著高于无效组、未治疗组及正常皮肤组(TUNEL:47.86±3.14,22.05±3.93,19.20±2.82,35.51±3.81,P＜0.01),而增殖水平明显低于上述三组(PCNA:10.25±3.89,44.33±5.01,56.32±4.12,17.91±3.76,P＜0.01).③凋亡调控情况:显效组bcl-2表达显著低于无效组和未治疗组(21.28±4.70,49.96±5.33,52.51±7.69,P＜0.01);c-myc表达也明显低于后两组(25.20±5.01,51.81±5.68,56.28±6.92,P＜0.01).结论:①糖皮质激素可能通过诱导VEC凋亡及抑制其增殖而促进血管瘤消退.②糖皮质激素对VEC凋亡的影响可能与其下调bcl-2和c-myc的表达水平有关.     

PTSD样大鼠杏仁中央核糖皮质激素受体表达的变化

目的研究创伤后应激障碍(PTSD)样大鼠杏仁中央核(CeA)糖皮质激素受体(GR)表达的变化。方法使用SPS方法建立PTSD样大鼠模型,无连续单一应激(SPS)刺激组为对照组,其余3组分别为SPS刺激后静养24h、7d和14d,其中7d组出现PTSD样症状为PTSD样大鼠组。应用免疫组化、Western blotting定位定量检测各组CeA的GR表达,进行图像分析和统计学处理。结果GR分布在CeA神经元的细胞核中,SPS刺激后24h,GR表达急剧下调,而7d、14d呈现逐渐回升趋势,但仍低于对照组(P<0.01)。结论PTSD样大鼠CeA的GR表达变化,可能是下丘脑-垂体-肾上腺(HPA)轴调节紊乱引发PTSD相关症状的重要因素之一。     

高糖通过SGK_1通路促进人肾小球系膜细胞合成结缔组织生长因子

目的研究血清和糖皮质激素诱导的蛋白激酶1(SGK1)在高糖诱导人肾小球系膜细胞(HMC)产生结缔组织生长因子(CTGF)中的作用,探讨SGK1在糖尿病肾病(DN)肾小球硬化中的作用机制。方法HMC分为低糖组(5·5mmol·L-1D-葡萄糖)、高糖组(25mmol·L-1D-葡萄糖)和甘露醇对照组(19·5mmol·L-1甘露醇和5·5mmol·L-1D-葡萄糖),刺激24、72h后,采用RT-PCR方法和West-ernblot方法检测CTGFmRNA及蛋白的表达;将带有SGK1显性激活型突变体质粒(pIRES2-EGFP-S422DhSGK1,SD)和带有SGK1显性失活型突变体质粒(pIRES2-EGFP-K127NhSGK1,KN)分别瞬时转染HMC;同时,设空质粒(PIRES2-EGFP,FP)转染组和未转染组(NT)为对照。分别用低糖(LG,5·5mmol·L-1D-葡萄糖)和高糖(HG,25mmol·L-1D-葡萄糖)刺激24、72h后,采用RT-PCR方法和Western blot方法检测CT-GF的mRNA及蛋白的表达。结果与低糖组和甘露醇组相比较,在高糖刺激下,HMC中CTGF mRNA及蛋白的表达明显上调;低糖环境下,转染SD的HMC与转染FP、NT组比较,其CTGFmRNA和蛋白的表达明显增加。转染KN的HMC与转染FP、NT组比较,其CTGF mRNA和蛋白的表达差异无显著性;高糖环境下转染SD的HMC与转染KN、转染FP、NT组比较,其CTGF mRNA和蛋白的表达明显增加。转染KN的HMC与转染FP、NT组比较,其CTGF mRNA和蛋白的表达明显减少。结论在DN中,高糖能促进HMC的CTGF mRNA及蛋白的表达,并且可以通过SGK1介导的信号通路来诱导人肾小球系膜细胞增加合成CTGF,这种新发现的信号通路,表明SGK1可能通过促进CTGF的合成来参与糖尿病肾病肾小球纤维化的发生。     

成人特发性血小板减少性紫癜与幽门螺杆菌感染的相关性研究

目的:研究重症成人特发性血小板减少性紫癜(ITP)患者的幽门螺杆菌(Hp)感染率以及Hp感染与患者临床特点之间的相关性。方法:应用14C尿素呼气试验(14C-UBT)检测重症ITP患者与对照人群的Hp感染率;比较Hp阳性和Hp阴性ITP患者的年龄、性别、骨髓巨核细胞数及对糖皮质激素治疗的反应。结果:42例ITP患者中28例14C-UBT阳性(66.7%),明显高于正常对照组19%(P<0.01)。Hp阳性和Hp阴性ITP患者糖皮质激素治疗的有效率分别为93%和21%,两组相比差异十分显著(P<0.01)。结论:ITP患者Hp感染率增高,Hp阳性ITP患者对糖皮质激素治疗更敏感。     

Effect of increased glucocorticoid responsiveness in transformed mouse lung cells

Many transformed mouse lung cells, including LM2 cells, contain activating mutations in the Ki-ras gene and show reduced responsiveness to growth inhibition by glucocorticoids. LM2GR cells, which are LM2 cells stably transfected with a rat glucocorticoid receptor (GR) gene, were used to determine whether increasing glucocorticoid responsiveness can influence aspects of the transformed phenotype. LM2GR cells grew slower and had a lower final saturation density than the parental LM2 cells. Expression of growth-related genes was examined by northern blot analysis. The cells were serum-deprived and treated with fetal bovine serum (FBS), steroid-stripped FBS (ssFBS), dexamethasone, or 12-O-tetradecanoylphorbol-13-acetate. The level and pattern of Ki-ras mRNA expression was similar in both LM2 and LM2GR cells, but histone H4 mRNA was expressed in a more regulated fashion in LM2GR cells. The induction of c-jun and c-fos mRNA expression lasted longer in the LM2GR cells treated with ssFBS; however, the maximal induction was greater in the LM2 cells treated with FBS. LM2GR cells demonstrated similar activator protein-1 (AP-1) activity but higher GR activity than LM2 cells as determined by using AP-1-chloramphenicol acetyltransferase (CAT) and mouse mammary tumor virus-CAT transient transfection assays, consistent with the higher level of GR mRNA in LM2GR cells. Both cell lines exhibited the ability to grow in soft agar and to form tumors in nude mice. These results indicate that introduction of a functional GR transgene into LM2 cells can increase glucocorticoid responsiveness and alter the expression of genes involved in growth regulation but cannot overcome anchorage-independent cell growth or tumorigenicity, apparently because of the presence of an activated Ki-ras gene. © 1995 Wiley-Liss, Inc.     

Stress- and aging-associated modulation of macrophage functions

Effects of environmental (cold) stress and aging on cells in monocyte/macrophage lineage were investigated. We demonstrated that immune suppressive states seen in acute cold-stressed mice (8–10 weeks of age) is attributable to FcγRII^bright suppressor macrophages. Serum corticosterone levels were markedly increased in acute cold-stressed mice. In addition, expression of glucocorticoids (GC) receptor mRNA was observed in FcγRII^bright cells from these mice. The increase of FcγRII^bright cells in peritoneal exudate cells caused by acute cold stress was inhibited by adrenalectomy or administration of a saturating amount of the GC antagonist RU 38486 (mifepristone). On the contrary, administration of the GC agonist, dexamethasone, markedly increased the proportion of FcγRII^bright cells in peritoneal exudate cells of control mice. These results suggest that the generation of FcγRII^bright suppressor cells of monocyte/macrophage lineage by acute cold stress was mediated by action of GC through the GC receptor. We likewise found that the proportion of FcγRII^bright suppressor macrophages is increased in aged mice (22–24 months of age). Meanwhile, activated macrophages which function as antigen presenting cells were decreased in aged rats. Both the basal corticosterone concentrations in serum and the expression of mRNA for GC receptor in peritoneal macrophages increased significantly in aged animals, suggesting that these populational and functional changes of macrophages in aged animals were mediated, in part, by the increased basal levels of GC. This is probably being responsible for immunosenescence.     

Serum withdrawal leads to reduced aryl hydrocarbon receptor expression and loss of cytochrome P4501A inducibility in PLHC-1 cells

Changes in the expression of the aryl hydrocarbon receptor (AHR) have been documented in several systems and in response to a variety of treatments. The significance of these findings is unclear, because the effects of such changes on subsequent responses to AHR ligands seldom have been measured. We tested the ability of changes in serum used in cell culture medium to alter expression of the AHR and induction of cytochrome P4501A (CYP1A) in PLHC-1 teleost hepatoma cells. Culture of early-passage cells in serum-free medium for 2 days led to a loss of CYP1A inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, culture in 10% delipidated calf serum increased the TCDD-induced levels of both CYP1A protein and enzymatic activity relative to levels in cells cultured in 10% complete calf serum. These effects were consistent between 8 and 24hr post-treatment, indicating that the kinetics of induction were unaffected. In cells cultured in serum-free medium for 1 and 2 days there was a progressive loss of CYP1A inducibility. This loss of response paralleled a time-dependent decline in AHR protein, as measured by specific binding of [3H]TCDD. Using an operational model for AHR action in PLHC-1 cells, the measured reduction in AHR could be shown to predict the loss of CYP1A induction. Expression of AHR protein was unaffected by culture in 10% delipidated serum. The effects of serum-free medium and delipidated serum were found only in early-passage cells; inducibility of CYP1A and expression of AHR protein in late-passage cells were unaffected by serum withdrawal. Comparison of early- and late-passage cells revealed a 2-fold greater rate of proliferation in the latter, suggesting that a growth advantage is coincident with loss of the serum-dependency of AHR expression. These results provide a quantitative link between changes in receptor expression and a downstream response, establishing a foundation for future studies of receptor expression and sensitivity to toxic responses in vitro and in vivo.     

Effect of prophylactic intracerebroventricular injection of methylprednisolone on nitrogen and catecholamine excretion in the urine after laparotomy in rats

BACKGROUND: This study was carried out to determine whether the prophylactic injection of glucocorticoid into the intracerebroventricular (i.c.v.) space reduced TNF-alpha and IL-1beta mRNA synthesis in the brain after laparotomy, resulting in a reduction of nitrogen excretion in the urine. PATIENTS AND METHODS: Male SD rats (body wt., 225-250 g, n = 114) were catheterized into the i.c.v. space on day 0. On day 4, the rats were assigned to four groups: (1) Control, (2) laparotomy (Trauma), (3) intraperitoneal (i.p.) injection of methylprednisolone (MP) plus laparotomy (IPMP), and (4) i.c.v. injection of MP plus laparotomy (ICVMP). Either 3 or 24 h after surgery, the animals were sacrificed. TNF-alpha and IL-1beta mRNA levels in tissues, including the brain cortex and hypothalamus, were measured by RT-PCR. The amounts of nitrogen and catecholamine excretion in the 24-h urine were determined. RESULTS: The i.p. injection of MP reduced TNF-alpha and IL-1beta mRNA levels in all the tissues 3 h after laparotomy compared with those of the Trauma group. The icv injection of MP prevented elevation of the TNF-alpha and IL-1beta mRNA levels in the brain (cortex, TNF-alpha, ICVMP 0.43 +/- 0.06, P < 0.05, vs Trauma; cortex, IL-1beta, ICVMP 0.25 +/- 0.09, P < 0.05, vs. Trauma; hypothalamus, TNF-alpha, ICVMP 0.31 +/- 0.04, P < 0.05, vs. Trauma; hypothalamus, IL-1beta, ICVMP 0.25 +/- 0.14, P < 0.05, vs. Trauma), but did not inhibit an increase in TNF-alpha and IL-1beta mRNA levels in the liver and skeletal muscle. Both nitrogen and catecholamine excretions in the urine were decreased by ip and by i.c.v. injection of MP compared to those of the Trauma group (nitrogen, ICVMP 559.3 +/- 52.0 mg/day, P < 0.05, vs. Trauma; catecholamine, ICVMP 13.8 +/- 1.8 microg/day, P < 0.05, vs. Trauma). CONCLUSION: A reduction in TNF-alpha and IL-1beta mRNA synthesis in the brain due to prophylactic injection of MP into the icv space reduced the catabolic response after laparotomy.     

Broadened ligand responsiveness of androgen receptor mutants obtained by random amino acid substitution of H874 and mutation hot spot T877 in prostate cancer

In a subset of endocrine therapy-resistant prostate cancers, amino acid substitutions H874Y, T877A and T877S, which broaden ligand specificity of the ligand binding domain (LBD) of the androgen receptor (AR), have been detected. To increase our knowledge of the role of amino acid substitutions at these specific positions in prostate cancer, codons 874 and 877 were subjected to random mutagenesis. AR mutants were screened in a yeast readout system for responsiveness to 5 alpha-dihydrotestosterone, progesterone and dehydroepiandrosterone. At position 874, only the histidine to tyrosine substitution could broaden AR ligand specificity. At position 877, 4 ligand specificity broadening substitutions were found: T877A, T877S, T877C and T877G. The latter 2 were not found in prostate cancer. The AR mutants were tested in mammalian (Hep3B) cells for responsiveness to 13 different ligands. All mutants displayed their own ligand specificity spectrum. Importantly, AR(H874Y) and AR(T877A) could be activated by cortisol. According to the 3-dimensional structure of the AR LBD, T877 interacts directly with the 17 beta-hydroxyl group of androgens. All amino acid substitutions identified at position 877 had smaller side chains than the threonine in the wild-type receptor, indicating that increased space in the ligand binding pocket is important in broadened ligand specificity. Because H874 does not interact directly with the ligand, its substitution by a tyrosine is expected to change the ligand binding pocket conformation indirectly. For T877C and T877G substitutions, 2-point mutations are required, and for H874Y, T877A and T877S substitutions, only a 1-point mutation is sufficient. This most likely explains that the latter 3 have been found in prostate cancer.