骨髓间充质干细胞诱导为神经样细胞后对大鼠脑缺血模型修复的研究

目的:观察大鼠骨髓间充质干细胞(MSCs)诱导为神经样细胞后对大鼠脑损伤模型修复的情况。方法:取大鼠骨髓作骨髓间充质干细胞的分离和传代培养,贴壁法分离MSCs,并诱导其分化,将经诱导的细胞直接注射移植到大鼠脑缺血模型大鼠的脑组织,检测分化的神经细胞样细胞在神经系统微环境中存活及病理变化。结果:细胞移植组病理变化与脑损伤模型组相比,神经细胞变性、坏死数量明显变少,间质水肿较轻。结论:骨髓间充质干细胞诱导为神经样细胞后移植能改善脑损伤大鼠的功能。

Polymorphisms of microsomal triglyceride transfer protein in different hepatitis B virus-infected patients

AIM: To identify the two polymorphisms of microsomal triglyceride transfer protein (MTP) gene in the Chinese population and to explore their correlation with both hepatitis B virus (HBV) self-limited infection and persistent infection. METHODS: A total of 316 subjects with self-limited HBV infection and 316 patients with persistent HBV infection (195 subjects without familial history), matched with age and sex, from the Chinese Han population were enrolled in this study. Polymorphisms of MTP at the promoter region -493 and at H297Q were determined by the allele specific polymerase chain reaction (PCR). RESULTS: The ratio of males to females was 2.13:1 for each group and the average age in the self-limited and chronic infection groups was 38.36 and 38.28 years, respectively. None of the allelic distributions deviated significantly from that predicted by the Hardy-Weinberg equilibrium. There was a linkagedisequilibrium between H297Q and -493G/T (D’ = 0.77). As the χ2 test was used, the genotype distribution of MTP -493G/T demonstrated a significant difference between the self-limited infection group and the entire chronic group or the chronic patients with no family history (χ2 = 8.543, P = 0.015 and χ2 = 7.199, P = 0.019). The allele distribution at the MTP-493 position also demonstrated a significant difference between the study groups without family history (χ2 = 6.212, P = 0.013). The T allele emerged as a possible protective factor which may influence the outcomes of HBV infection (OR: 0.59; 95% CI: 0.389-0.897). CONCLUSION: The polymorphism of the MTP gene, T allele at -493, may be involved in determining the HBV infection outcomes, of which the mechanism needs to be further investigated.

C-peptide and Diabetic Encephalopathy

With the changes of life style, diabetes and its complications have become a major cause of morbidity and mortality. It is reasonable to anticipate a continued rise in the incidence of diabetes and its complications along with the aging of the population, increase in adult obesity rate, and other risk factors. Diabetic en- cephalopathy is one of the severe microvascular complications of diabetes, characterized by impaired cogni- tive functions, and electrophysiological, neurochemical, and structural abnormalities. It may involve direct neuronal damage caused by intracellular glucose. However, the pathogenesis of this disease is complex and its diagnosis is not very clear. Previous researches have suggested that chronic metabolic alterations, vascular changes, and neuronal apoptosis may play important roles in neuronai loss and damaged cognitive functions. Multiple factors are responsible for neuronal apoptosis, such as disturbed insulin growth factor （IGF） system, hyperglycemia, and the aging process. Recent data suggest that insulin/C-peptide deficiency may exert a primary and key effect in diabetic encephalopathy. Administration of C-peptide partially improves the condition of the IGF system in the brain and prevents neuronal apoptosis in the hippocampus of diabetic patients. Those findings provide a basis for application of C-peptide as a potentially effective therapy for diabetes and diabetic encephalopathy.

Noradrenaline as a putative neurotransmitter mediating hypotension-induced Fos-like immunoreactivity inthe supraoptic nucleus of the rat

Hemorrhage or hypotension induces extensive Foslike immunoreactivity in the magnocellular neurosecretory cells in the supraoptic nucleus of the hypothalamus in rat, especially in the vasopressin neurons. The present study was to explore the neurotransmitter mediating this effect. Microinfusion of the alpha-adrenergic blocker into the supraoptic nucleus reduced the hypotension-induced Fos, whereas beta-antagonist did not affect it significantly. Alpha1- and alpha2-antagonist, prazosin and yohimbine,both reduced the Fos-positive cell counts. However, the effective dosage of yohimbine was much larger. Alpha1-agonist, methoxamine, induced abundant Fos-like immnnoreactivity in the vasopressin cells in this nucleus,while beta-and alpha2-agonist did not elicit such effect.Administration of the noradrenergic re-uptake inhibitor,desipramine, to this nucleus to locally accumulate the spontaneously released noradrenaline from the nerve terminals also induced Fos expression, mostly in the vasopressin cells.

Enhancing the efficiency of direct reprogramming of human primary fibroblasts into dopaminergic neuron-like cells through p53 suppression

Dopaminergic(DA)neuron-like cells obtained through direct reprogramming of primary human fibroblasts offer exciting opportunities for treatment of Parkinson’s disease.A significant obstacle is the low efficiency of conversion during the reprogramming process.Here,we demonstrate that the suppression of p53 significantly enhances the efficiency of transcription factor-mediated conversion of human fibroblasts into functional dopaminergic neurons.In particular,blocking p53 activity using a dominant-negative p53(p53-DN)in IMR90 cells increases the conversion efficiency by 5–20 fold.The induced DA neuron-like cells exhibit dopamine neuron-specific gene expression,significant dopamine uptake and production capacities,and enables symptomatic relief in a rat Parkinson’s disease model.Taken together,our findings suggest that p53 is a critical barrier in direct reprogramming of fibroblast into dopaminergic neurons.

Velvet antler polypeptide is able to induce differentiation of neural stem cells towards neurons in vitro

OBJECTIVE: To investigate the neural differentiation capacity of water extraction of velvet antler.METHODS: Velvet antler(Cervus Nippon Temminck) polypeptide(VAP) was used to differentiate neural stem cells(NSCs) towards neurons in the study. Firstly, we obtain the polypeptides of VAP by water extraction. Secondly, we observed the morphology, assayed the factors in the media by enzyme-linked immunosorbent assay, and detected the special neural molecules by immuno fl uorescence staining. NSCs were cultured on the cell climbing film. After neuronal differentiation, differentiated NSCs were mounted for immunocytochemistry with immunofluorescence technique.RESULTS: The differentiating cells look like neuron,some special factors, such as Glial cell line-derived neurotrophic factor, nerve growth factor, in the media can be detected while differentiated neuron-like cells can express the special neural molecules.CONCLUSION: Differentiation of NSCs towards neurons can be induced by velvet antler polypeptide.

Electrophysiological study on differentiation of rat bone marrow stromal stem cells into neuron-like cells in vitro by edaravone

Objective： To explore the electrophysiological proper- ties of differentiation of rat bone marrow-derived stromal stem cells （rBMSCs） to neuron-like cells in vitro by edaravone, a new type of free radical scavenger. Methods： Stromal stem cells were separated from rat bone marrow with Ficoll-Paque reagent and expanded in different culture medium in vitro, rBMSCs were induced by edaravone containing serum-free L-DMEM. Morphologic observation and Western blot analysis including the expression of Nav1.6, Kvl.2, Kv1.3, Cav1.2 were performed, and whole patch-clamp technique was used. Results： Cyton contraction and long processes were shown in differentiated stromal stem cells. Navl.6, Kvl.2, Kv 1.3 and Cav 1.2 were expressed in both differentiated and undifferentiated ceils. However, the expression of channel proteins in differentiated cells was up-regulated. Consistently, their resting potential and outward currents were also enhanced in the differentiated cells, which was especially significant in the outward rectifier potassium current. Conclusion： In vitro, neuron-like cells derived from rBMSCs, induced by edaravone, possess electrophysiological properties of neurons.

Differentiation of adult human bone marrow mesenchymal stem cells into Schwann-like cells in vitro

Objective: To investigate the differentiative capability of adult human bone marrow mesenchymal stem cells (BMSCs) into Schwann-like cells. Methods: Bone marrows were aspirated from healthy donors and mononuclear cells were separated by Percoll lymphocytes separation liquid ( 1.073 g/ml) with centrifugation, cells were cultured in DMEM/F12 (1:1) medium containing 10% fetal bovine serum (FBS), 20 ng/ml epidermal growth factor (EGF) and 20 ng/ml basic fibroblast growth factor (bFGF). Cells of passage 1 were identified with immunocytochemistry. Results: Mononuclear cells separated by Percoll’s were passaged 10 times by trypsin/ethylenediaminetetraacetic acid (EDTA) digestion in 40 days, and BMSCs increased about 6×107 times in this short period. Immunohistochemistry identified that BMSCs were CD34- and CD31-, but they expressed neuron specific enolase; 0.01%- 0.02% of total cells expressed nestin, the marker for neural progenitor cells; 40%-50% cells stained heavily by neurofilament 200; and no glial fibrillary acidic protein(GFAP) positive cells were identified; S100 expression was detected among 0.1%- 0.2% cells. Conclusions: Bone marrow contains the stem cells with the ability of differentiating into Schwann-like cells, which may represent an alternative stem cell sources for neural transplantation.

Xiao-Xu-Ming decoction extract alleviates LPS-induced neuroinflammation associated with down-regulating TLR4/MyD88 signaling pathway in vitro and in vivo

In the present study, we aimed to investigate the effects of Xiao-Xu-Ming decoction extract(XXM) on lipopolysaccaride(LPS)-induced neuroinflammation in vitro and in vivo. In vitro, the microglia BV2 cells were treated with 200 ng/mL LPS for 24 h to induce inflammatory responses. In vivo, mice were treated with 5 mg/kg LPS to induce inflammatory responses. The NO level was determined by Griess Reagents. The levels of IL-1β, IL-6, TNF-α and MCP-1 were determined by ELISA. The expressions of Iba-1, TLR4 and MyD88 at the protein levels were determined by Western blotting analysis. The mRNA levels of TLR4 and MyD88 were determined by real-time PCR. In vitro, XXM significantly reduced the levels of various pro-inflammatory factors, including NO, IL-1β, IL-6 and TNF-α, induced by LPS in the supernatant of BV2 cells and suppressed expressions of inflammatory proteins TLR4 and MyD88 induced by LPS in BV2 cells. In vivo, XXM significantly inhibited microglia activation, attenuated LPS-induced inflammatory factors and chemokine production, such as IL-1β, IL-6, TNF-α and MCP-1, and inhibited the expressions of inflammatory proteins including TLR4 and MyD88, in the cortex of LPS-induced mice. Our findings suggested that XXM could attenuate LPS-induced neuroinflammation via down-regulating TLR4/MyD88 signaling pathway.

Protective effects of β-dihydroagarofuran-type sesquiterpene against Aβ_(25-35)-induced neuronal apoptosis and oxidative damage

Excessive beta-amyloid （Aβ） plays a detrimental role in the pathogenesis of Alzheimer＇s disease （AD）, which is closely associated with apoptosis and oxidative stress in neurons. Therefore, identification of active small molecules with potent effects on neutralizing Aβ-induced neurotoxicity would be a promising strategy for delaying or preventing AD progression. In the present study, we discovered that pretreatment with CF-1 （（1R,2S,4R,5S,7R,9S, IOS）-1,15-diacetoxy-2-benzoyloxy-9-cinnamoyloxy- β-di-hydroagarofuran）, a sesquiterpene isolated from the seeds of Celastrus flagellaris, attenuated Aβ25_35-induced reduction in cell viability in a concentration-dependent manner, as evaluated by the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay. Above neuroprotective effect of CF-1 was associated with a significant decrease of apoptotic cells as measured by 4,6-diamidino-2-phenylindole （DAPI） staining, which concurrently happened with marked inhibition in the level of cleaved Caspase-3, an apoptotic executive protein. CF-1 pretreatment also markedly reduced the intracellular accumulation of reactive oxygen species （ROS） following Aβ exposure in SH-SY5Y neuroblastoma cells, but such pretreatment had no notable influence on 2,2-diphenyl-l-picrylhydrazyl （DPPH） scavenging. In conclusion, we demonstrated that a novel natural product, CF-1, possessed promising effects against Aβ-induced neuronal apoptosis and oxidative stress, which could be a potential drug lead or candidate for the treatment of Aβ-associated neurotoxicity.

Total synthesis of the aminopropyl functionalized ganglioside GM_1

GM1 is a common ganglioside pentasaccharide present on mammalian cell surface.It has been shown to play important roles in cellular communications and initiation of β-amyloid aggregation.In order to synthesize GM1,an efficient synthetic route was developed via a [3+2] strategy.The GM3 trisaccharide acceptor bearing an azido propyl group at the reducing end was prepared using the traditional acetamide protected sialyl thioglycosyl donor,which gave better stereoselectivity than sialyl donors protected with trichloroacetamide or oxazolidinone.The glycosylation of the axial 4-hydroxyl group of GM3 by the disaccharide donor was found to be highly dependent on donor protective groups.Donor bearing the more rigid benzylidene group gave low glycosylation yield.Replacing the benzylidene with acetates led to productive coupling and formation of the fully protected GM1 pentasaccharide.Deprotection of the pentasaccharide produced GM1 functionalized with the aminopropyl side chain,which will be a valuable probe for biological studies.