OBJECTIVESTo assess the culture and differentiation of neural stem cells in embryonic mice and set up a basis for further research in to neural stem cells.

METHODSEmbryonic cortices of mice were dissociated and single cell suspensions were achieved by mechanical methods in sterile conditions, and cells were seeded in uncoated plate in N2 medium. The cells were passaged by mechanical methods, frozen and thawed by general procedure. They were identified by immunocytochemical techniques.

RESULTSNeural stem cells from embryonic mice were successfully cultured forming typical neurospheres in suspension. Neurons, astrocytes and oligodendrocytes were differentiated from neural stem cells, with a ratio of 7%, 85% - 90% and 2% - 4% respectively.

CONCLUSIONSNeural stem cells, which can be cultured and passaged steadily in vitro and they are the ideal cell sources for cell transplantation and gene therapy.

Animals ; Cell Differentiation ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Immunohistochemistry ; Mice ; Mice, Inbred BALB C ; Neurons ; cytology ; Stem Cells ; cytology

OBJECTIVETo research the genetic diversity of different Rhodiola rosea geographical populations in Tianshan Mountain, China;

METHODThe genetic diversity of eighteen R. rosea geological populations from six niches was estimated using amplified fragment length polymorphism (AFLP) markers. The data of amplified bands were analyzed by the software POPGENE v1.31 (32-bit) and SPSS.

RESULTThe nine primers employed produced a total of 238 discernable and reproducible amplified fragments. There were 228 polymorphic bands. The percentage of polymorphic bands with in different populations was 95.6%. Genetic diversity analysis showed that average number of alleles per loci was Na = 1.4883, effective number of alleles per loci Ne = 1.3907, Neis gene diversity index H = 0.2170, Shannon's information index I = 0.3108, the percentage of polymorphic loci P = 52.71, genetic differentiation among populations Gst = 0.364; UPGMA cluster analysis based on genetic distance data divided eighteen populations into two clusters: Cluster I composed of twelve populations and Cluster II 6 populations which distributed in attitude upper 3 175 m;

CONCLUSIONOur researches suggest that the best niche of R. rosea was at attitude between 3 150-3 250 m; this region is important for the conservation of R. rosea germplasm resource.

Amplified Fragment Length Polymorphism Analysis ; China ; Genetic Variation ; Phylogeny ; Polymorphism, Genetic ; Rhodiola ; classification ; genetics

OBJECTIVE: To explore the molecular pathological mechanism of liver metabolic disorder in severe spinal muscular atrophy (SMA). METHODS: The transgenic mice with type Ⅰ SMA (Smn^-/- SMN2^0tg/2tg) and littermate control mice (Smn^+/- SMN2^0tg/2tg) were observed for milk suckling behavior and body weight changes after birth. The mice with type Ⅰ SMA mice were given an intraperitoneal injection of 20% glucose solution or saline (15 μL/12 h), and their survival time was recorded. GO enrichment analysis was performed using the RNA-Seq data of the liver of type Ⅰ SMA and littermate control mice, and the results were verified using quantitative real-time PCR. Bisulfite sequencing was performed to examine CpG island methylation level in Fasn gene promoter region in the liver of the neonatal mice. RESULTS: The neonatal mice with type Ⅰ SMA showed normal milk suckling behavior but had lower body weight than the littermate control mice on the second day after birth. Intraperitoneal injection of glucose solution every 12 h significantly improved the median survival time of type Ⅰ SMA mice from 9±1.3 to 11± 1.5 days (P < 0.05). Analysis of the RNA-Seq data of the liver showed that the expression of the target genes of PPARα related to lipid metabolism and mitochondrial β oxidation were down-regulated in the liver of type Ⅰ SMA mice. Type Ⅰ SMA mice had higher methylation level of the Fasn promoter region in the liver than the littermate control mice (76.44% vs 58.67%). In primary cultures of hepatocytes from type Ⅰ SMA mice, treatment with 5-AzaC significantly up-regulated the expressions of the genes related to lipid metabolism by over 1 fold (P < 0.01). CONCLUSION Type Ⅰ SMA mice have liver metabolic disorder, and the down-regulation of the target genes of PPARα related to lipid and glucose metabolism due to persistent DNA methylation contributes to the progression of SMA.
Mice ; Animals ; PPAR alpha ; Liver Diseases ; Muscular Atrophy, Spinal/genetics* ; Mice, Transgenic ; Body Weight ; Glucose

OBJECTIVE To explore the molecular mechanism of urolithin A inhibition of human hepatoma cells growth. METHODS Hepatoma Huh-7 cells were treated with different concentrations of urolithin A(Uro-A). The inhibition rate of Huh-7 cells survival was detected by CCK-8 assay and the IC50 was calculated. Cell proliferation was detected by colony formation assay and cell migration ability was assessed by cell wound healing experiment. Glucose uptake and lactate level in culture medium through colorimetry and the ATP production in cell through chemiluminescence method was analyzed. Western blotting was applied to detect protein expression levels of glucose transporter(GLUT1), key enzymes of glycolysis(HK2, PFKM, LDHA), p53, p-p38 and Bcl-2 after treatment with different concentrations of Uro-A. Flow cytometry and TUNEL method were used to detect apoptosis rate. RESULTS The results of CCK-8 showed that Uro-A significantly inhibited the proliferation of Huh-7 cells, and the IC50 was(48.54±1.21) μmol·L−1. The ability of clone formation and migration decreased after Uro-A treatment. Cellular glucose uptake and level of lactic acid and ATP production were down regulated in Huh-7 cells treated with Uro-A. The results showed that expression of glycolytic key proteins GLUT1, PKM2, LDHA and HK2 decreased. Western Blotting further research indicated that the p53 and p-p38 were activated, while the Bcl-2 was down-regulated. Flow cytometry data and TUNEL method revealed that the induction of apoptosis by Uro-A was remarkably increased. CONCLUSION These findings suggest that Uro-A can suppress Huh-7 cell proliferation and migration. The possible mechanism is the inhibition of glycolysis by p53, p-p38 and Bcl-2, which prevent cell growth and finally induce apoptosis.

OBJECTIVETo identify the feasibility and safety of fossa infratemporalis approach for blind-needle at sphenopalatine ganglion so as to provide anatomical evidence for the operation and the prevention of non-immediate adverse reaction.

METHODSThe variations of pterygopalatine fossae in sixty dry skulls were observed by selecting measuring points for facial skull width. The brains of six wet skulls were taken out,then acupuncture of fossa infratemporalis approach was applied. Sphenopalatine ganglion was separated accurately with the pterygopalatine segment of maxillary arteria retained in the pterygopalatine fossa after its paries posterior was opened. We detected whether the needle was inserted into pterygopalatine fossa. Measurements showed needle inserted depth, facial skull width,the distance between the needle and sphenopalatine ganglion,the distance between the needle and the pterygopalatine segment of maxillary arteria,the distance between the pterygopalatine segment of maxillary arteria and the crotaphitic nerve in pterygopalatine fossa.

RESULTSThe distance between the slight hollow under bilateral arcus superciliaris was selected as skull width, and 3 dry skulls showed the variation of pterygopalatine fossa. Needles were inserted into the pterygopalatine fossae of the wet skulls (12 times). The proportion of the inserting depth to the distance between the slight hollow under bilateral arcus superciliaris was 44%-54%. Only twice did the needle contact sphenopalatine ganglion. The average distances between the sphenopalatine ganglion and the needle were (5.88±3.70) mm in the left side and (6.43±5.54) mm in the right side. The average distances between the needle and the pterygopalatine segment of maxillary arteria were (2.77±3.99) mm left and (2.53±3.10) mm right. The average distances between the pterygopalatine segment of maxillary arteria and the crotaphitic nerve in pterygopalatine fossa were (2.83±4.05) mm left and (2.67±4.95) mm right. The mean data between the two sides had no statistic significance about all the above indices (all>0.05).

CONCLUSIONSFossa infratemporalis approach is feasible for blind-needle at sphenopalatine ganglion with less possibility to contact it. The effect of treating nasitis may achieved by little distance to nerve. Pricking at the pterygopalatine segment of maxillary arteria may induce non-immediate adverse reaction. The safety and efficacy should be comprehensively considered. There is a proportional relationship between the width of the skull and the insertion depth of the needle. The inserting depth of 44 percent may appropriate accounted for skull width.