非程序性冷冻仪冻存人胚胎干细胞的方法

在人胚胎干细胞（human embryonic stem，hES）的研究中，有效的冻存方法非常重要。目前，冷冻Es细胞主要有两种方法：慢速冷冻一快速复温和超快速玻璃化法。慢冻法需要昂贵的程序性冷冻仪，而玻璃化法步骤繁琐且只能冻存小量细胞．不能满足临床应用。因此，本研究旨在寻找非程序性冷冻仪冻存大量hES细胞的方法。     

Long-term application of diethylstilbestrol upregulates expressions of μ- and m-calpains in pituitary intermediate lobe of female Wistar rats

BACKGROUND: During formation of prolactin neoplasia, how cells and its structure in adenohypophysis affect prolactin cells should be further studied. Intermediate lobe can be regarded as a driving region to release prolactin (PRL) and may promote formation of prolactin neoplasia in pituitary anterior lobe. OBJECTIVE: To observe the effect of diethylstilbestrol (DES) on the expressions of μ and m-calpains in pituitary intermediate lobe of female Wistar rats. DESIGN: Observational contrast animal study. SETTING: Beijing Neurosurgical Institute. MATERIALS: A total of 21 female Wistar rats, 3 weeks old weighing 70–80 g were housed with free access to tap water and standard pellet food. They were kept in a CL-grade condition, at (24±1)℃ and a humidity of (55±5)%, and with a 12 hours day-night cycle. Caprine anti-μ- and m-calpains antibodies were provided by Santa Cruz Biotechnology, CA, USA; rabbit-anti-PRL antibodies by Dako, Denmark; rabbit-anti-ACTH antibody by Boster Company, Wuhan. METHODS: The experiment was carried out in Pathophysiological Department and Animal Laboratory, Beijing Neurosurgical Institute from August 2006 to January 2007. ① Rats were randomly divided into groups with 7 in each group, including vehicle control group, in which rats were injected intraperitoneally with sun-flower seed oil (1 mL/kg, twice a week) for 16 weeks; DES group, where animals were administered with DES (5 mg/kg, twice a week) for 16 weeks; DES + vehicle control group, in which DES was administered for 12 weeks at the same dose with those in DES group, and then was discontinued and replaced by sun-flower seed oil (1 mL/kg, twice a week) for the following 4 weeks. ② At 16 weeks later, pituitary tissue was dealt with HE staining and PRL immunohistochemical examination to observe evoke of tumor; meanwhile, immunohistochemical examination was used to observe expression of PRL of pituitary anterior lobe, expressions of μ- and m-calpains of pituitary intermediate lobe and distribution of adrenocorticotropin. MAIN OUTCOME MEASURES: ① Expression of PRL of pituitary anterior lobe, expressions of μ- and m-calpains of pituitary intermediate lobe and distribution of adrenocorticotropin. ② Morphological observation of pituitary tissue. RESULTS: All 21 rats were involved in the final analysis. ① Results of immunohistochemical examination: Morphological changes of neoplasia in DES group were strongly positive to PRL, and this suggested that formation of prolactin adenoma was observed in pituitary tissue. As compared with vehicle control group, expression of adrenoeorticotropic hormone (ACTH) was increased in both DES group and DES + vehicle control group. In addition, expressions of μ- and m-calpains in pituitary intermediate lobe were higher in DES group than that in vehicle control group. Otherwise, expressions of m-calpains in pituitary intermediate lobe was decreased in DES + vehicle control group, but expression of μ-calpains was still increased. ② Morphological observation of pituitary tissue: Gland tubes were orderly arranged in rats in vehicle control group. Anterior pituitary gland in rats of DES group demonstrated an apparent disappearance of gland tubes and a relatively large-scaled vasculature formation, namely the vascular lake lined by tightly arranged endothelial cells. Local integrated tumor cell arrangements were also detected. In addition, the border between the IL and the anterior lobe was locally blurred. The definite tumor-like changes in pituitary tissues were confirmed in 6 of 7 female Wistar rats in DES group, and one spontaneous occurrence of tumor formation was found in vehicle control group. In DES + vehicle control group, DES withdrawal led to the subtile emergence of gland tube cavity, although tumor-like cells still existed in 4 of 7 rats, suggesting occurrence of the tumor regression due to the withdrawal of DES. CONCLUSION: A long-term application of DES can enhance the expressions of ubiquitours neutral cysteine protease in pituitary intermediate lobe and this suggests that both of them play a key role in release of hormone and formation of prolactin neoplasia through directly promoting PRL expression and release of neighboring pituitary intermediate lobe.     

Autonomic dysregulation as a basis of cardiovascular, endocrine, and inflammatory disturbances associated with obstructive sleep apnea and other conditions of chronic hypoxia, hypercapnia, and acidosis

Obstructive sleep apnea has traditionally been viewed as a structural disease. A multitude of systemic endocrine and cardiovascular abnormalities have been previously attributed to the prevalence of obesity in these patients. A growing body of clinical evidence, however, points to a relationship between sleep apnea and its systemic abnormalities independent of obesity. We hypothesize that this association is based on a maladaptive autonomic response of chemoreceptors, reacting to the hypoxia, hypercapnia, and acidosis of sleep apnea. The elevated sympathetic response triggers an inflammatory cascade that results in a myriad of downstream consequences including insulin resistance, hypertension, diabetes, atherosclerosis and metabolic syndrome. The sympathetic bias and endocrine disturbances may further exacerbate sleep disturbance in a potentially pernicious cycle. Our proposal may extend to any chronic respiratory or metabolic conditions that manifest hypoxia, hypercapnia, and acidosis and elicit a maladaptive autonomic and inflammatory response.     

Molecular characterization of the full-length genome of the Japanese encephalitis viral strain K87P39

We have determined the complete nucleotide and deduced amino acid sequences of the Japanese encephalitis virus (JEV) strain K87P39, isolated from a pool of circulating Culex tritaeniorhynchus mosquitoes in Korea. In comparison with 27 fully sequenced JEV genomes currently available, we found that the 10968-nucleotide RNA genome of K87P39 has a nine-nucleotide deletion in the 3' nontranslated variable region and that its single open reading frame has a total of eight amino acid substitutions. The K87P39 isolate is highly similar to other JEV isolates, and homology ranges from 97.9 to 89.0% at the nucleotide level, and 99.1 to 96.7% at the deduced amino acid level. Phylogenetic analyses using the full-length sequence of the 27 available JEV genomes showed that the K87P39 strain is most closely related to six Chinese SA14 derivatives and that it is distantly related to the Australian FU, Korean K94P05 and Japanese Ishikawa strains. In addition, we also found that phylogenetic relationships based on the full-length genome are highly similar to those based on the E gene, indicating that phylogenetic analysis of the E gene will be useful for studying the genetic relationships among JEV isolates. We therefore performed a more extensive E gene-based phylogenetic analysis on a selection of 70 JEV isolates available from GenBank, which represent a temporally and geographically wide variety of JEV strains.     

The incorporation of iodine in thyroid hormone may stem from its role as a prehistoric signal of ecologic opportunity: an evolutionary perspective and implications for modern diseases

To optimize fitness under conditions of varying Darwinian opportunity, organisms demonstrate tremendous plasticity in their life-history strategies based on their perception of available resources. Higher-energy environments generally promote more aggressive life-history strategies, such as faster growth, larger adult size, greater genetic variation, shorter lifespan, larger brood sizes, and offspring ratio skewed towards the larger-sized gender. While numerous mechanisms regulate life-history plasticity including genetic imprinting, methylation, and growth factors, evidence suggests that thyroid hormone plays a central role. Given the pivotal adaptive role of thyroid hormone, the teleology of its dependence on dietary iodine for production remains unexplained. We hypothesize that iodine may have emerged as a substrate for production of thyroid hormone in prehistoric ecosystems because the former represented a reliable proxy for ecologic potential that enabled the latter to modulate growth, reproduction, metabolic rate, and lifespan. Such a scenario may have existed in early marine ecosystems where ocean-surface vegetation, which concentrates iodine for its antimicrobial and antioxidant properties, formed the basis of the food chain. Teleologic parallels can be drawn to the food-chain accumulation of antimicrobials that also exhibit antioxidant properties and promote adult size, brood size, and offspring quality by modulating central hormonal axes. As each higher species in the food chain tunes its life-history strategy based on iodine intake, the coupling of this functional role of iodine with its value as a resource signal to the next member of the food-chain may promote runaway evolution. Whereas predators in prehistoric ecosystems successfully tuned their life-history strategy using iodine as a major input, the strategy may prove maladaptive in modern humans for whom the pattern of iodine intake is decoupled from resource availability. Iodine acquired through sodium iodide supplementation may independently contribute to some biologic dysfunctions currently attributed to sodium.     

The link between T helper balance and lymphoproliferative disease

Lymphoproliferative disorders comprise a heterogeneous group of neoplasms whose behaviors range from indolent to very aggressive. The increased incidence seen in the context of immunodeficiency provides evidence that the host immune system plays a vital role in their pathogenesis. We believe that T-helper (Th)-2 dominant states favor development of lymphoproliferative disorders, including lymphoma, and conversely T-helper (Th)-1 immunity protects against lymphoproliferative disease. The age distribution of lymphomas favors childhood and post-reproductive senescence, suggesting that exposure to these periods of Th-2 bias constitutes a key risk factor for developing the disease. The tendency of lymphomas to arise in Th-2 biased locations such as mucosal interfaces, immunoprivileged sites, and regions of B-cell differentiation may likewise reflect a corresponding spatial predilection. Various clinical conditions or treatments that shift Th1/Th2 balance, including HIV infection, transplant-related immune suppression, and autoimmune disorders, can also influence the status of lymphoproliferative diseases.     

Identification of human antitumor cytotoxic T lymphocytes epitopes of recoverin, a cancer-associated retinopathy antigen, possibly related with a better prognosis in a paraneoplastic syndrome

Cancer-associated retinopathy (CAR) is a rare paraneoplastic syndrome, and the recoverin-specific autoantibody is suggested to contribute to the pathogenesis of retinopathy, including apoptosis of retinal cells. Because it is known that CAR(+) cancer patients have a preferable prognosis, we hypothesized that aberrantly expressed recoverin in cancer cells can become a target of cytotoxic T lymphocytes (CTL). Here we tested nine recoverin-derived HLA-A24-binding peptides for their capacity to elicit antitumor CTL. We observed recoverin-specific CTL responses in two HLA-A24(+) CAR(+) cancer patients. In addition, the CTL responses were obtained from three of ten CAR(-) cancer patients and two of six healthy individuals. The CTL precursor frequency of CAR(+) cancer patients and that of CAR(-) cancer patients was higher than that of healthy individuals. Of nine recoverin peptides, R49 (QFQSIYAKF), R49.2 (QFQSIYAKFF), and R64 (AYAQHVFRSF) were discovered to induce the peptide-specific CTL. Taken together, our present data suggest that peripheral activation of recoverin-specific antitumor CTL is likely to contribute to the preferable prognosis of CAR(+) cancer patients. Moreover, in cases other than CAR(+) cancer patients, recoverin may offer the opportunity to design epitope-based immunotherapeutic approaches for treating HLA-A24(+) cancer patients with a recoverin-expressing tumor.     

A pilot study of bortezomib in Korean patients with relapsed or refractory myeloma

Recent clinical trials showed that bortezomib, a novel proteasome inhibitor, had therapeutic activity in multiple myeloma. However, there was no data about the feasibility of bortezomib in Korean patients. We performed a pilot study of bortezomib in patients with relapsed or refractory myeloma (1.3 mg/m2 twice weekly for 2 week in a 3-week cycle). Seven patients were enrolled. The median age of patients was 59 yr. All patients previously received VAD (vincristine, doxorubicin and dexamethasone) and thalidomide chemotherapy. Three patients previously received alkylator-containing chemotherapy and 4 patients, autologous stem cell transplantation. Bortezomib monotherapy resulted in 3 partial remissions (43%), 3 no changes (43%) and 1 progressive disease (14%). One patient who had no response to bortezomib monotherapy experienced partial remission after addition of dexamethasone to bortezomib. The most common serious toxicity was thrombocytopenia (grade 3/4, 10 of 20 cycles (50%)) and grade 3 peripheral neuropathy was developed in 2 of 20 cycles (10%). Drug-related adverse event led to discontinuation of bortezomib in 1 patient. There was no treatment related mortality. Overall, bortezomib seems to be effective and feasible. Conduction of larger clinical studies on Korean patients is necessary to characterize clinical efficacy and safety of bortezomib more precisely.     

The effect of beta adrenergic stimulation on QT and QTc interval in syncope children with or without coexisting ventricular arrhythmias

We investigated the effect of beta-adrenergic stimulation on the heart rate and QT interval in syncope children with or without coexisting ventricular arrhythmias (VA). Of the 24 children who presented with syncope or presyncope and showed negative tilt test, 13 were classified into a group with VA and the remaining 11 without VA. The provocative test was performed in bolus infusion and continuous infusion. RR, QT, and QTc intervals on routine 12-lead surface electrocardiogram were obtained during each stage of isoproterenol infusion. In all cases, malignant ventricular arrhythmia and syncope were not induced by isoproterenol provocative test. RR and QT intervals were shortened and QTc intervals were prolonged as the isoproterenol dose was increased in both groups and methods. The QTc interval reached its peak level after the bolus injection of 1.0 microgram and during the continuous infusion of 0.03 microgram/kg/min. The two groups showed no significant difference in the QTc interval change according to the infusion methods. This study indicates that changes in the heart rate and QT interval by beta-adrenergic stimulation were not different according to the coexisting ventricular arrhythmias in syncope children with negative head-up tilt test.