中枢和外周神经损伤后胞体区看家基因表达的变化

目的观察并比较几种常用的看家基因,包括β-actin,GAPDH,18s RNA,cyclophilin A等在中枢和外周神经损伤后胞体分布区神经组织中表达的变化.方法Wistar雄性大鼠随机分为右侧坐骨神经夹伤,右侧坐骨神经结扎和胸段脊髓半横断三组.手术后7d,用相对定量RT-PCR的方法检测几种看家基因在受损神经元胞体分布区神经组织中的表达水平.结果GAPDH和Cyclophilin A的表达在不同模型中的变化幅度较小,损伤与对照侧比较差异均无显著性.β-actin和18sRNA的表达在不同模型中变化不同,在脊髓损伤和坐骨神经结扎后表达的变化有统计学意义.结论本研究结果为研究神经系统损伤后相关基因表达变化时内参照基因的选择提供参考,同时也为进一步认识看家基因更加广泛的功能提供依据.     

总钙水平测定不适用于肝移植术中高钙血症和低钙血症的诊断

目的观察肝移植术中离子钙和总钙浓度的变化,探讨监测总钙是否适用于钙紊乱的诊断。方法20例肝移植患者,全身麻醉下行改良背驮式肝移植,分别于术前、无肝前期、无肝期30 min以及下腔静脉开放后5、30、120 min时采集动脉血,测定离子钙和总钙水平,并按公式(离子钙=总钙×0.48)依据总钙计算出离子钙水平。结果所有患者均完成手术,术中输液总量为3500～10 000 ml,包括晶体、白蛋白、新鲜冰冻血浆、冷沉淀和红细胞,补氯化钙3.5～8.0 g。检测所得离子钙和计算得出的离子钙,除术前外,差异有统计学意义(P<0.01)。若依据总钙浓度判断,术前12例血钙正常,而以测定的离子钙来判断,则其中4例是低钙血症(假阴性);在无肝前期,依据总钙浓度判断,4例血钙正常,其余16例均为高钙血症,但是若以离子钙作为诊断标准,血钙正常的4例均为低钙血症,而诊断为高钙血症的16例,仅有3例是高钙血症,余下的13例中有7例是低钙血症;无肝30 min时及移植肝恢复血流30 min时的情况和无肝前期类似;移植肝恢复血流120 min时,依据总钙水平,所有患者均为高钙血症,但离子钙测定提示7例血钙正常。无肝前期之后,依据总钙水平诊断术中各期高钙血症、低钙血症的特异性或敏感性较低。结论肝移植术中总钙及离子钙浓度变化明显,相对于总钙水平,肝移植术中离子钙水平的变化能更真实反映机体的钙紊乱。     

High osteoprotegerin serum levels in primary biliary cirrhosis are associated with disease severity but not with the mRNA gene expression in liver tissue

The influence of osteoprotegerin and RANKL as regulators of osteoclastogenesis and bone remodeling in liver disease and in the development of osteoporosis in primary biliary cirrhosis (PBC) is uncertain. Therefore, 68 women with PBC and 20 healthy females were studied by assessing circulating osteoprotegerin and RANKL. Bone mineral density and markers of bone turnover were measured as well. Osteoprotegerin-mRNA expression was also assessed in liver tissue from 16 patients and 5 controls. Osteoprotegerin was higher in PBC than in controls (5.4 ± 0.2 vs. 2.9 ± 0.2 pM/l, P < 0.0001), whilst RANKL was lower in patients than in controls (0.39 ± 0.06 vs. 1.40 ± 0.16 pM/l, P < 0.0001). Osteoprotegerin was more elevated in patients with more advanced disease, as defined by bilirubin above 1.2 mg/dl (6.6 ± 0.6 vs. 5.2 ± 0.2 pM/l, P = 0.02) or by Mayo over 4 (5.9 ± 0.3 vs. 4.8 ± 0.2 pM/l, P = 0.02). Osteoprotegerin and RANKL were unrelated with osteoporosis, and no associations were found with markers of bone remodeling, except for RANKL, which was particularly decreased in patients with low osteocalcin. This marker of bone formation was also higher in patients with elevated circulating osteoprotegerin. Liver osteoprotegerin gene expression was similar in patients and controls, and no correlation was found between liver osteoprotegerin-mRNA and patients’ respective circulating levels. In conclusion, osteoprotegerin and RANKL are abnormal in patients with PBC, regardless of osteoporosis. The elevated circulating osteoprotegerin is associated with the severity of disease, but not with gene expression in the liver.     

Vascular adventitia is a suitable compartment to transplant transduced vascular smooth muscle cells for ex vivo gene expression

Vascular smooth muscle cells (VSMC) are ideal for systemic gene therapy because of their proximity to blood vessels and they have demonstrated long-term exogenous gene expression in vivo. However, the procedure generally followed to seed the transduced VSMC onto arteries denuded of endothelial cells usually induces stenosis and thrombosis, with a consequent high risk for use in humans. We demonstrate here that the vascular adventitia is a suitable place to introduce transduced VSMC and to secrete therapeutic proteins into the blood stream by a simple procedure, avoiding postoperative vascular complications. Transduced VSMC, with the retroviral vectors carrying the human growth hormone gene (hGH), were seeded into the adventitia of the rat abdominal aorta by single injection of a cell suspension. Based on the hGH and anti-hGH production in serum and on histological analysis of the removed aortas, we demonstrated hGH production over the 2-month experimental period. None of the animals used in the experiment showed stenosis, thrombosis, or other vascular or visible physiological abnormalities.     

Transforming growth factor beta 1 gene expression in human airways.

BACKGROUND--Asthmatic airways have a characteristic deposition of connective tissue under the epithelial basement membrane, but the mediators involved in this alteration are unknown. Several authors have postulated that transforming growth factor beta 1 (TGF-beta 1) could be overexpressed in asthmatic airways. METHODS--Lung samples from 16 asthmatic patients, six patients with chronic obstructive pulmonary disease (COPD), and six non-obstructed smokers were analysed. RNA was extracted from these tissues to measure expression of TGF-beta 1 by Northern blot analysis using a cDNA probe for TGF-beta 1. The level of expression was quantitated by densitometry using glyceraldehyde 3-phosphate dehydrogenase mRNA as a control. TGF-beta 1 was localised to specific cell types in these lungs by immunohistochemical analysis using polyclonal antibodies specific for intracellular and extracellular TGF-beta 1. RESULTS--The 2.5 kb TGF-beta 1 mRNA was seen in all 18 samples analysed by Northern blotting and densitometric analysis showed no difference between the asthmatic group (mean (SD) 108% (43%)), the group with COPD (122% (33%)), and the non-obstructed group (100% (49%)). The TGF-beta 1 precursor was immunolocalised throughout the airway wall including the epithelium and in alveolar macrophages. The mature TGF-beta 1 was localised primarily within the connective tissue of the airway wall. These patterns of expression of both forms of TGF-beta 1 were similar in lungs from asthmatic patients, those with COPD, and controls. CONCLUSIONS--While TGF-beta 1 mRNA and protein are abundantly expressed in human lungs, there is no clear difference in expression between the airways of asthmatic subjects and those of smokers with and without COPD.     

Transition protein 1 mRNA expression is not related to pregnancy rate in azoospermic men undergoing TESE-ICSI

Our laboratory has previously reported significantly low levels of protamine (PRM) gene expression in infertile men. This drop was correlated with a low pregnancy rate, suggesting that PRMs may be useful as predictive factors for the outcome of testicular sperm extraction and intracytoplasmic sperm injection (TESE-ICSI) in azoospermic men. Transition nuclear proteins (TPs) are expressed earlier in spermatogenesis than protamines and are required for normal sperm development. In the present study, we examined the expression of the transition nuclear protein 1 gene (TNP1) in azoospermia and its relationship with TESE-ICSI outcomes. The cellular expression of TNP1 mRNA in spermatids was quantified by in situ hybridisation on paraffin sections of testis biopsies from 21 men with obstructive azoospermia and 23 men with non-obstructive azoospermia. Cases of non-obstructive and obstructive azoospermia did not differ significantly in terms of TNP1 expression. Furthermore, TNP1 mRNA expression was similar in non-pregnant and pregnant couples. Hence, the pregnancy rate was not related to TNP1 mRNA expression levels in azoospermia. Our results emphasise the value of TNP1 as a reliable predictive marker for the presence of spermatids/spermatozoa in the testis biopsies used for TESE but also indicate that expression of the TNP1 gene (believed to be a major player in spermiogenesis and required for production of normal sperm) may not be a predictive factor for successful post-ICSI embryonic development.     

Separation of sperm cells by sedimentation technique is not suitable for in vitro fertilization purposes

Summary. Two methods of sperm preparation for in vitro fertilization were compared: the swim-up technique vs. the migration-sedimentation technique. The study comprised fresh semen samples obtained from 25 couples treated in the In Vitro Fertilization Unit. Oocytes aspirated in a single cycle were divided into two groups, each inseminated by sperm prepared by one of these techniques. Motility, degree of motility, and normal morphology were improved by both methods. The improvement was greater when the migration-sedimentation technique was applied. However, fertilization rate was significantly higher after the swim-up technique. In order to clarify this contradiction, an additional group of 26 semen samples was divided and then prepared by the swim-up or migration-sedimentation techniques. Sperm quality was examined up to 72 h after separation. Compared with the swim-up technique, sperm characteristics were better after separation by the migration-sedimentation technique. However, this difference abated after 24 h. The better results of the swim-up technique in the 'survival experiment' may explain its improved performance in in vitro fertilization, despite lower separation capacity. Thus, the migration-sedimentation technique is not recommended for sperm preparation in in vitro fertilization.
Migration-sedimentation technique (MST)—     

Enhanced expression of cyclo-oxygenase isoenzyme 2 (COX-2) in asthmatic airways and its cellular distribution in aspirin-sensitive asthma

BACKGROUND: There are two isoforms of cyclo-oxygenase (COX), namely COX- 1 and COX-2. COX-1 is constitutively expressed in most tissues and in blood platelets. The metabolites derived from COX-1 are probably involved in cellular housekeeping functions. COX-2 is expressed only following cellular activation by inflammatory stimuli and is thought to be involved in inflammation. METHODS: The expression of COX-1 and COX-2 isoenzymes has been studied in the bronchial mucosa of 10 normal and 18 asthmatic subjects, 11 of whom had aspirin-sensitive asthma (ASA) and seven had non-aspirin-sensitive asthma (NASA) RESULTS: There was a significant fourfold and 14-fold increase, respectively, in the epithelial and submucosal cellular expression of COX-2, but not of COX- 1, in asthmatic patients. There was no significant difference in the total number of cells staining for either COX-1 or COX-2 between subjects with ASA and NASA, but the number and percentage of mast cells that expressed COX-2 was significantly increased sixfold and twofold, respectively, in individuals with ASA. There was a mean fourfold increase in the percentage of COX-2 expressing cells that were mast cells in subjects with ASA and the number of eosinophils expressing COX- 2 was increased 2.5-fold in these subjects. CONCLUSION: COX-2-derived metabolites may play an essential part in the inflammatory processes present in asthmatic airways and development of drugs targeted at this isoenzyme may have therapeutic potential in the treatment of asthma. Mast cells and eosinophils may also have a central role in the pathology of aspirin-sensitive asthma.


     

Pax3 gene expression is not altered during diaphragmatic development in nitrofen-induced congenital diaphragmatic hernia

Background/PurposeMalformations of the pleuroperitoneal folds (PPFs) have been identified as the origin of the diaphragmatic defect in congenital diaphragmatic hernia (CDH). Pax3, expressed in muscle precursor cells (MPCs), plays a key role in regulating myogenesis and muscularization in the fetal diaphragm. Pax3 mutant mice display absence of muscular diaphragm. However, the distribution of muscle precursor cells is reported to be normal in the PPF of the nitrofen-CDH model. We designed this study to investigate the hypothesis that Pax3 gene expression is unaltered in the PPF and developing diaphragm in the nitrofen-induced CDH model.MethodsPregnant rats were treated with nitrofen or vehicle on gestational day (D) 9 and sacrificed on D13, D18, and D21. Pleuroperitoneal folds (D13) and developing diaphragms (D18 and D21) were dissected, total RNA was extracted, and real-time quantitative polymerase chain reaction was performed to determine Pax3 messenger RNA levels. Confocal immunofluorescence microscopy was performed to evaluate protein expression/distribution of Pax3.ResultsRelative messenger RNA expression levels of Pax3 in PPFs and developing diaphragms were not significantly different in the nitrofen group compared with controls. Intensity of Pax3 immunofluorescence was also not altered in PPFs and developing diaphragms of the nitrofen group compared with controls.ConclusionPax3 gene expression is not altered in the PPFs and developing diaphragm of nitrofen-CDH model, suggesting that the diaphragmatic defect is not caused by disturbance of myogenesis and muscularization.     

肺癌组织中p16基因mRNA表达及其临床意义

目的　检测肺癌组织中p16基因mRNA的表达情况 ,探讨p16基因在肺癌发生发展过程中的作用。方法　应用逆转录 聚合酶链反应 (RT PCR)方法检测肺癌组织中p16基因mRNA的表达。结果　肺良性疾病组织、癌周正常肺组织p16基因mRNA表达高于肺癌组织 ,差异有显著性 (P分别 <0 .0 5 ) ;小细胞肺癌p16基因mRNA阳性率 (10 0 %)高于非小细胞肺癌 (4 6.0 %)P <0 .0 5 ;临床Ⅰ、Ⅱ期p16基因mRNA阳性率 (75 .0 %)高于临床Ⅲ、Ⅳ期 (3 4.1%) ,P <0 .0 5 ;无淋巴结转移的病例p16基因mRNA阳性率 (73 .3 %)高于有淋巴结转移的病例 (3 3 .3 %) ,P <0 .0 5 ;高、中分化病例组p16基因mRNA表达阳性率 (5 4.5 %)与低分化病例组 (4 4 .0 %)之间差异无显著性 (P >0 .0 5 )。结论　p16基因的异常改变可能参与了非小细胞肺癌发生发展过程 ,p16基因mRNA的表达检测作为一种辅助指标。     

Determination of tobramycin in saliva is not suitable for therapeutic drug monitoring of patients with cystic fibrosis

BACKGROUND: Tobramycin, used in the treatment of infections caused by Gram-negative bacteria, requires therapeutic drug monitoring (TDM) due to its narrow therapeutic index. The collection of blood for these assays may cause pain and trauma to the child and/or be difficult because of limited access to appropriate blood vessels. We undertook an evaluation of the role of saliva concentrations in the TDM of once-daily tobramycin therapy in patients with cystic fibrosis. METHODS: Fourteen patients (mean age 15 years) with cystic fibrosis were enrolled at Women's and Children's Hospital, Adelaide (WCH). All patients received once-daily dose of intravenous tobramycin for 2-3 weeks and had plasma levels measured once a week. At the same time of blood sampling at 1 and 6 h after initiation of tobramycin infusion, the patients also provided saliva samples. For collection of saliva, the Salivette (Sarstedt Laboratories) system was used which was developed specifically for saliva sampling. Concentrations in blood and saliva were measured by the Beckman Synchron CX system, which is utilized for routine assays of plasma tobramycin. RESULTS AND CONCLUSION: Tobramycin could not be detected in saliva within the first 6 h after a once-daily dosing. Therefore, plasma cannot be substituted with saliva for the TDM of tobramycin using the clinical routines at WCH.     

Interleukin-6 and interleukin-10 plasma levels and mRNA expression in polytrauma patients

Purpose: Host response to polytrauma occasionally has unpredictable outcomes. Immune response is a major factor influencing patient''s outcome. This study evaluated the interaction of two main cytokines in immune response after major trauma, specifically interleukin-6 (IL-6) and interleukin-10 (IL-10). Plasma level of these cytokines is determined by mRNA expression of these cytokines genes which may decide the outcome of polytrauma patients. Methods: This prospective multicenter trial held at four trauma centers enrolled 54 polytrauma patients [Injury Severity Score (ISS) 16]. Plasma levels and mRNA expression of IL-6 and IL-10 were measured for 5 days after trauma. Clinical evaluation was conducted to observe whether patients endured multiple organ dysfunction syndrome (MODS) and death. MODS evaluation was performed using sequential organ failure assessment (SOFA). Trauma load which in this study is represented with ISS, plasma level, expression of cytokine genes and patient''s outcome were examined with correlation test and statistical analysis. Results: The elevated IL-6/IL-10 ratio indicated increased activity of systemic inflammation response, especially pro-inflammation response which bears higher probability of progressing to MODS and death. The decline of IL-6/IL-10 ratio with heavy trauma load (ISS > 30) showed that compensatory antiinflammation response syndrome (CARS) state was more dominant than systemic inflammatory response syndrome (SIRS), indicating that malfunction and failure of immune system eventually lead to MODS and deaths. The statistical significance in plasma level of cytokines was found in the outcome group which was defined as bearing a low trauma load but mortality. Conclusion: The pattern of cytokine levels in inflammation response has great impact on the outcome of polytrauma patients. Further study at the genetic level is needed to investigate inflammation process which may influence patient''s outcome.