Study of thrombopoietin for gene therapy of thrombocytopenia

Thrombopoietin (TPO) is likely to be a potent, specific and reliable medication in the treatment of thrombocytopenia. A TPO-highly-expressed plasmid pcDNA3-TPO was constructed and a primary study was made on the expression of TPO cDNA in vitro and gene transfer study for thrombocytopenia in vivo. rhTPO showed complete and stable bioactivity by a series of indicators. High expression of TPO was detected in plasma from healthy mice or thrombocytopenia mice model receiving direct intramuscular injection of pcDNA3-TPO. And the platelet level of healthy mice peaked to 1.9-fold of baseline. Mice with CTX-induced thrombocytopenia achieved profound nadirs, acceleration of recovery, even 1.8—2.0-fold supranormal levels of peripheral platelet counts. The results offered experimental support for clinical application of gene therapy for thrombocytopenia via direct intramuscular injection of TPO cDNA.     

Production of inbred and hybrid transgenic mice carrying large (> 200 kb) foreign DNA fragments by intracytoplasmic sperm injection

We have developed a mouse transgenesis technique that facilitates the insertion of large (approximately 200 kilo base pairs) DNA fragments into host genomes of both inbred and hybrid mice. Six inbred and three hybrid transgenic mice carrying a single bacterial artificial chromosome (BAC) clone with genes located in the Down syndrome critical region of human chromosome 21 were produced using this technology.     

Gene expression and immune response kinetics using electroporation-mediated DNA delivery to muscle

BACKGROUND: Injection of DNA encoding exogenic proteins into muscle tissue combined with electroporation often results in a transient increase of the encoded protein concentration in the muscle and the blood. The reduction is normally due to an immune response against the exogenic protein but other factors may also be involved. How various electroporation parameters affect the concentration kinetics of syngenic and exogenic proteins is studied in relation to immune response and muscle damage after electroporation-mediated DNA transfer to muscle. METHODS: Electroporation was applied to mouse quadriceps and rat tibialis anterior muscles after injection of DNA encoding either secreted alkaline phosphatase (SEAP), beta-galactosidase (beta-gal), luciferase or a mouse IgG molecule. Protein concentrations in blood or muscle and antibody responses were measured for a period up to 3 months. Tissue inflammation and muscle cell damage were studied on muscle cross-sections and assessed by measuring the concentrations of creatine phosphokinase (CPK) in blood. RESULTS: Mice with the highest SEAP concentration in blood at day 7 also had the highest rate of decrease afterwards, the strongest antibody responses against SEAP and the highest acute levels of CPK in blood. DNA-transfected muscle fibers were significantly reduced in number from days 7 to 14. Mononuclear cells surrounded the reporter gene expressing muscle fibers, thus indicating a cellular immune response. When using DNA encoding a syngenic protein the protein concentration in blood was relatively stabile over a 3-month period, but showed different kinetics for various electroporation parameters. CONCLUSIONS: Our findings suggest that the optimal electroporation parameters for DNA vaccination may be different from the optimal parameters for long-term expression of genes encoding syngenic proteins.     

Rapid identification of vinca alkaloids by direct-injection electrospray ionisation tandem mass spectrometry and confirmation by high-performance liquid chromatography-mass spectrometry

A simple and rapid method for the identification of Vinca alkaloids from a crude extract of Catharanthus roseus G. Don (Apocynaceae) by direct-injection electrospray ionisation (ESI) and tandem mass spectrometry (MS/MS) has been developed. The alkaloids vindoline, vindolidine, vincristine and vinblastine were evaluated in a commercial extract of C. roseus using this method. Catharanthine and its isomers 19S-vindolinine and vindolinine were detected in the commercial product by direct injection ESI/MS/MS and confirmed by preparation and by HPLC-ESI/MS. For the characterisation of different fragment fingerprints, ESI/MS/MS is a sensitive, rapid and convenient technique by which to identify some constituents in complex and mixed plant extracts.     

Determination in serum of some barbiturates using micellar liquid chromatography with direct injection

A procedure was developed for the determination of several barbiturates, amobarbital, barbital, hexobarbital, and secobarbital, using a C18 column (120 x 4.6mm) and micellar liquid chromatography (MLC) mobile phases containing sodium dodecyl sulfate (SDS) and propanol, butanol, or pentanol as a modifier, with UV detection at 230nm. After the application of an interpretative strategy of optimization, the four barbiturates can be resolved and determined in serum samples, allowing the direct injection in 0.10M SDS-4% (v/v) butanol, pH 7, with an analysis time below 8 min. In the proposed MLC procedure, linearities (r >0.999), limits of detection (ngmL(-1)) in the 30-70 range, repeatabilities, and intermediate precision below 1.8% are adequate for the quantification. The proposed method could be applied to the determination of barbiturates in serum samples with recoveries that agreed with the concentration added.     

Seasonal short-term effects of naltrexone on LH secretion in male carp (Cyprinus carpio L.)

In order to evaluate the influence of the season (the stage of gonad maturity) on the modulatory role of endogenous opioid peptides in LH secretion in fish, sexually mature male carp (Cyprinus carpio L.) were intravenously injected with naltrexone-opioid receptor antagonist (5 or 50 microg kg(-1)) in the period of natural spawning (June) or gonad recrudescence (December). Moreover, the possible involvement of the dopaminergic system was studied in fish pre-treated with pimozide (dopamine receptor antagonist) and in intact fish. Blood samples were taken every minute, up to 10 min after naltrexone injection. In June, naltrexone significantly lowered LH levels in comparison to saline injected males. In December, there were no differences between saline and naltrexone-injected carps. In fish pre-treated with pimozide, neither in June nor in December were any significant differences in LH levels between control group and the groups injected with naltrexone found. The results showed that, in male carp, LH secretion under the influence of naltrexone depends on the stage of gonad maturity what suggests that the feedback of gonadal steroids on LH release could be mediated by the endogenous opioids. The role of dopamine in these processes is also discussed.     

当归注射液对脑缺血/再灌注神经元代谢物的影响

目的：研究当归注射液对脑缺血／再灌注时神经元代谢物及血流速度的作用,阐明当归对脑缺血损伤神经修复过程的影响。方法：雄性SD大鼠69只,体重150～170g,随机分成假手术组(n=4)、缺血损伤组(n=30)和当归治疗组(n=35)。制作右大脑中动脉血供阻断(MCA0)模型。缺血2h后,当归治疗组立即腹腔注射当归注射液(5g／kgbw)。在再灌注后3～4h和5～6h,以磁共振成像(MRI)技术研究大脑T2加权成像(T2WI)和局域质子谱(^1H MRS)的变化,观察当归对成像和神经元代谢物N-乙酰天门冬氨酸(NAA)、肌酸／磷酸肌酸(Cr／PCr)和胆碱(Cho)的影响。激光多普勒血流仪观察当归注射液对血流速度的影响,测定脑表面血管密度。结果：与缺血损伤组比较,当归治疗组的高信号强度区信号减弱、体积小,NAA值大,Cr/NAA和Cho/NAA比值小,再灌注时的血流速度显著加快,单位面积内的血管长度增加。结论：当归注射液加快缺血脑组织的血液循环,改善神经元的代谢。     

Acetylcholinesterase induces neuronal cell loss,astrocyte hypertrophy and behavioral deficits in mammalian hippocampus

Previous studies have demonstrated that acetylcholinesterase (AChE) promotes the assembly of amyloid-beta-peptides into neurotoxic amyloid fibrils and is toxic for chick retina neuronal cultures and neuroblastoma cells. Moreover, AChE is present in senile plaques in Alzheimer's disease (AD) brains. Here we have studied the effect of AChE on astrocytes and hippocampal neurons in vivo. Morphological as well as behavioral disturbances were analyzed after intrahippocampal injection of AChE. Rats were trained in the Morris water maze and assayed for behavioral parameters. Neuronal cell loss was found in the upper leaf of the dentate gyrus in rats injected with AChE in comparison with control animals. Glial fibrillary acidic protein immunoreactivity showed astrocytic hypertrophy and the magnitude of the response was associated with neuronal cell loss. Behavioral results show that injection of AChE produces cognitive impairment demonstrated by an altered water maze performance including (i) a higher escape latency score, (ii) a decreased spatial acuity and (iii) a shorter time of swimming in the platform quadrant. These findings indicate that a local increment in neuronal AChE concentration at the mammalian hippocampus, such as those present in amyloid deposits, may play a role in triggering neuropathological and behavioral changes such as those observed in AD brains.     

Intra-tumoral injection of CpG results in the inhibition of tumor growth in murine Colon-26 and B-16 tumors

Direct tumor injections of CpG (ODN #1826) into murine tumors markedly suppressed the tumor growth and increased the survival of the mice. Tumor growth was reduced by 60–67% in Colon Tumor 26 (CT-26) and B-16 melanoma tumors treated with CpG as compared to untreated one. In CT-26 and B-16 tumors treated with CpG, the average survival of the animals were prolonged to 26 and 28 d as compared to 16 and 18 d in control respectively. Long-term surviving animals in CT-26 tumor groups were also protected from a subsequent injection of a lethal dose of tumor cells. In the present study, effect of CpG was mediated through CD8⁺ T cells, as their depletion resulted in the abrogation of the therapeutic effects of the CpG. It suggests that direct tumor injection might be a simple means of achieving a clinical response in cancer patients.