BACKGROUND: Present studies have demonstrated that during neural development, differentiation of neural stem cells (NSCs) was affected by various regulatory factors from surrounding microenvironment. Sonic hedgehog (Shh) is a key induction signal during neural fetal development, and can be an effective inductor to regulate differentiation of neural cells. OBJECTIVE: To investigate the signal transduction pathway of SHH for differentiation of rhesus monkey bone marrow mesenchymal stem cells (BMSCs) into of neuron-like cells by sonic hedgehog factor. METHODS: Rhesus monkey BMSCs were isolated and cultured by conventional density gradient centrifugation. BMSCs in the induction group were treated with L-DMEM containing FGF2, B27 and fetal bovine serum for preinduction of 24 hours, and then with DMEM supplemented with 0.5 μmol/L retinoic acid or 400 μmol/L SHH for 8 days. Non-induced cells served as control group. Following labeling with neuron enolase, positive cells were screened by flow cytometry. RT-PCR and Western-blot were used to detect SHH- and retinoic acid-induced cell membrane receptor and intracellular signal protein changes. RESULTS AND CONCLUSION: SHH specific membrane receptor Ptc, retinoic acid specific receptor RARα, signal protein molecule ptch1 and Smad expressed in normal cells. Ptc expression upregulated in SHH-induced cells. High expression lasted for a long time with induction time, which was significantly stronger compared with the retinoic acid and control groups (P < 0.01). Intracellular ptch1 protein molecule expression showed similar tendency as this, but could not induce upregulation of RARα expression. During induction, retinoic acid-stimulated cells did not activate Ptc pathway. Four days following induction, RARα expression upregulated and lasted till 6 days, but there were no significant differences. No significant change in ptch1 expression was determined. SHH- and retinoic acid-induced cell Smad molecule expression upregulated, but no significant difference was determined. Results verified that SHH-induced scheme participated in cell induction and differentiation by persistently activating its specific receptors. However, there was no significant receptor pathway crossing between retinoic acid-induced and SHH-induced schemes.

0.05), the drug adverse reaction rates were 25.5% vs 51.4%(P

BACKGROUND:Bone marrow mesenchymal stem cells(BMSCs)differentiating into neural cells is an effective way of cell therapy of nervous system disease.However,the methods used nowadays still need to be improved.OBJECTIVE:To induce the differentiation of rhesus monkey BMSCs into neuron-like cells by using sonic hedgehog factor.METHODS:Rhesus monkey BMSCs differentiating into neuron-like cells was induced by typical retinoic acid and sonic hedgehog factor.Rhesus monkey BMSCs were isolated and cultured by density gradient centrifugation method.Cell growth was observed under an inverted phase contrast microscope and cell growth curve was determined using MTT assay.Flow cytometry was performed to characterize the phenotype of BMSCs,and immunohistochemistry was utilized to assess differentiated cells.Ultra-structure of the differentiated cells was observed by transmission and scanning electron microscopes.RESULTS AND CONCLUSION:Rhesus monkey BMSCs cultured in vitro were identified by flow cytometry,with high homogenicity.Following sonic hedgehog factor disposal for 7 days,differentiated cells were mainly positive for neurone specific enolase,neurofilament protein,Tau and glial fibrillary acidic protein(GFAP).Image statistical analysis found that in sonic hedgehog factor scheme,neural stem cells marker Neetin positive rate was significantly higher compared with the rstinoic acid scheme(P＜0.01).GFAP-positive rate was greater in the retinoic acid scheme than in the sonic hedgehog factor scheme(P＜0.05).Results indicated that sonic hedgehog factor scheme is an effective pathway of rhesus monkey BMSC differentiation into neuron-like cells.

OBJECTIVE To study the effect of nano-alumina(nano-Al2 O3 )on mitophagy in primary cortical neuronal cells from Wistar newborn rats. METHODS The purity of neuronal cells was detected by immunohistochemistry,and the lactate dehydrogenase(LDH)assay was performed to determine the viability of the cells treated with 13 nm nano-Al2 O3 0.5 mmol·L-1 for 12,24 and 48 h,respectively. The mitochondrial membrane potential(MMP)was detected by flow cytometry analysis . The ultrastructure of mitochondria and mitophagy vacuoles was observed by transmission electron microscopy(TEM). Auto-phagic vacuoles were observed by dansylpentanediamine(MDC)staining and the expression of autoph-agy related protein Beclin1 and LC3Ⅱ/ Ⅰ was determined by Western blotting. Mitophagy was observed by Lysotracker and Mitotracker staining respectively. RESULTS More than 95% cells were neuronal cells. The activity of LDH in the supernatant liquid exposed to nano-Al2 O3 for 12 and 24 h groups was sig-nificantly increased compared with the control group(P﹤0.05). After exposure to nano-Al2 O3 ,the mito-chondrial membrane potential was significantly decreased compared with the control group( P ﹤0.01). The results of TEM displayed mitochondrial swelling and the formation of vacuoles and mitophagy in nano-Al2 O3 groups. MDC positive fluorescence particles were observed and the expression of autophagy related protein Beclin1 and LC3Ⅱ/ Ⅰ was increased in nano-Al2 O3 groups compared with the control group( P ﹤ 0. 05 ). The result of Lysotracker and Mitotracker colocalization showed the fusion of mitochondria and lysosomals. CONCULSION Nano-Al2 O3 may induce autophagy and mitochondria damage in neuronal cells while the damaged mitochondria may be removed by mitophagy.

ObjectiveTo investigate the role of p38 and ERK1/2 during rhesus monkeys mesenchymal stem cells differentiated into neuron-like cells.MethodsTo induce the neuronal phenotype,rhesus monkeys mesenchymal stem cells were maintained in sub-confluent cultures in serum-contain medium supplement with Sonic hedgehog.Western blot analysis the change of p38 and ERK1/2 during rhesus monkeys mesenchymal stem cells differentiated into neuron-like cells.Under transmission and scanning electron microscope,ultra-structure of the differentiated cell were observed.ResultsDuring BMSCs differentiated into neuron-like cells by SHH,Mitogen-activated protein kinases (MAPK) involved in their signal transduction,p38 was activated and ERK1/2 was inhibited.P38 inhibitor SB203580 increased induced differentiation time compared with normal induced cells,and inhibited neurite outgrowth.ConclusionActivation of p38 and inhibition of ERK was impacted on differentiation into neuron-like cells from rhesus monkeys mesenchymal stem cells induced by Sonic hedgehog,which may has potential application on neuroprotection of stem cells in Nervous system diseases

OBJECTIVE To explore the potential neurotoxicity of nano-alu mina (<50 n m)in vivo, we treated the ICR mouse with the nano-alu mina to investigate the mitochondrial da mage of nerve cells on morphology and function.METHODS Adult male mice were exposed to nano-alu mina (<50 n m)of 0,25,50 and 75 mg·kg -1 by nasal instillation for 1 month.Then we observed the mitochondrial ultra-structure of the nerve cells in CA3 region of hippoca mpus,and measured the mean dia meter in every group.The activities of Na +-K +-ATPase and Ca2 +-Mg2 +-ATPase were tested by the determination of the inorganic phosphorus,which was the deco mposition product of ATPase.Western blot analysis was used to detect the expression of COX-Ⅳ,Beclin1 ,LC3Ιand LC3Ⅱ.RESULTS Co mpared with 0 and 25 mg·kg -1 groups exposed to Al2 O3 nanopartilces (Al2 O3 NPs),the mitochondria of CA3 region in hip-poca mpus in 50 mg·kg -1 group beca me ede matous and swollen with sparse and broken cristae sur-rounding the nuclear,and the mean dia meter was higher(0.49 ±0.02 μm,P <0.05).But co mpared with 50 mg·kg -1 group,the mitochondria in 75 mg·kg -1 group beca me s maller with inner cristae of high density,and the mean dia meter was lower(0.36 ±0.02 μm,P<0.05).The enzy me activity of the mito-chondria in cerebral cortex decreased dose-dependently with exposure,the activities of Na +-K +-ATPase in 50 and 75 mg·kg -1 groups(6.37 ±0.22 kU·g -1 protein,5.48 ±1 .53 kU·g -1 protein)and Ca2 +-Mg2 +-ATPase in 50 and 75 mg·kg -1 groups (3.21 ±0.99 kU·g -1 protein,3.28 ±0.15 kU·g -1 protein)were lower than the 0 mg·kg -1 group(P<0.05).Meanwhile,the Ca2 +-Mg2 +-ATPase in 50 and 75 mg·kg -1 groups showed lower activities in co mparison with the 25 mg·kg -1 group.The 75 mg·kg -1 group expressed higher level of the COX-Ⅳ protein 1 .35 ±0.66(P<0.05)than other groups.Both expression of Beclin1 protein and rate of LC3Ⅱ/LC3Ⅰin 75 mg·kg -1 group were more than the 0 mg·kg -1 group. CONCLUSION The mitochondrial dysfunction may be the potential neurotoxicity of nano-alu mina,and the da maged mitochondria were cleared by autophagy.

Objective To use hemin as a catalyst in the formation of disulfide bonds in the synthesis of linaclotide. Methods The linaclotide peptide was synthesized by the standard 9-fluorenylmethyl(Fmoc)solid-phase synthetic strategy. Wang resin and Trt-protected cysteine were used in the synthesis. Hemin was used in random oxidation of line linaclotide. The result was compared with those of air,dimethyl sulfoxide(DMSO),and I2 oxidation systems. Results and Conclusion Hemin is a highly effective catalyst for disulfide bond formation in linaclotide synthesis. It overcomes some disadvantages in oxidation reactions with conventional oxidative re-agents,and supplies a convenient way for the synthesis of peptide with concentrated disulfide bonds.

Objective To explore the evaluation value of serum Ghrelin and pleural effusion in patients with acute pancreatitis . Methods Eighty patients with acute pancreatitis treated in our hospital from February 2011 to February 2014 were divided into mild case group (31 patients) and severe case group (49 patients) according to the severity of the disease .Venous blood samples were collected at time point including :admission ,48h after admission and after discharge at empty stomach in the morning ,and CRP level ,WBC ,PCT level of patients were checked .The concentration of serum Ghrelin of patients were mensurated by enzyme linked immunosorbent assay and pleural effusion were diagnosed by sternum .Results Compared with the patients in the mild case group , serum Ghrelin ,CRP level ,APACHE score ,CT score and Ranson score were higher in the severe case group and the hospital day of patients in the severe case group was longer(P<0 .01) .The area under the curve of CRP level ,pleural effusion ,Ghrelin ,Ghrelin combined with pleural effusion of ROC were 0 .708 3 ,0 .749 6 ,0 .852 4 and 0 .910 8 .Ghrelin combined with pleural effusion has the best evaluation effect on the patients with acute pancreatitis .The sensibility ,specificity ,accuracy of CRP were 93 .6% ,69 .4% and 73 .7% ;the sensibility ,specificity ,accuracy of pleural effusion were 75 .2% ,88 .7% and 76 .8% ;the sensibility ,specificity ,accuracy of Ghrelin were 86 .9% ,88 .2% and 85 .3% ;the sensibility ,specificity ,accuracy of Ghrelin combined with pleural effusion were 90 .1% ,92 .6% and 91 .4% .Conclusion Serum Ghrelin and pleural effusion have high sensibility ,specificity and accuracy in pa‐tients with acute pancreatitis and has high clinical value .

Objective To analyze CT findings of insulinoma,and to summarize the causes of missed diagnosis of nontypical insuli-noma.Methods Clinical and CT manifestations of 18 patients with 18 insulinomas were analyzed retrospectively which were proved by surgery and pathology,and the causes of the missed diagnosis of nontypical insulinoma were also summarized.Results 10 patients with 10 insulinoma underwent CT plain scan with isodensity in 9 and slightly lower density in 1 with thread-like capsule.Other 18 patients underwent enhanced CT scan,10 of whom showed obvious enhancement in arterial phase with isodentisy in 6 and slightly higher density in 4 in portal phase,and isodensity in 10 in delayed phase.4 lesions showed mild-to-moderate enhancement in arterial phase with slightly higher density than normal pancreas in 2 and isodentisy in 2 in portal phase,and slightly higher density than nor-mal pancreas in 1 and similar density to pancreas in 3 in delayed phase.In portal phase,the enhanced degree in 8 was similar to the pancreas,and that in 6 was slightly higher or higher than that of pancreas.In delayed phase,13 were similar to the pancreas and other 1 was higher than that.3 of 18 lesions were easily missed,and 4 lesions with missed diagnosis showed isodensity on plain CT and en-hanced CT,and were further detected by other imaging methods.Conclusion Multiphase enhancement CT scanning can be used as the first choice for the insulinoma.