Objective To explore a kind of simple and high efficient approach to differentiate human embryonic stem cells(hESCs)into neural stem cells(NSCs).Methods hESCs were cultured in bacterial culture dish filled with serum free medium to gain embryoid bodies.Then the mature embryoid bodies grew in the special medium including B27 and noggin by adherent culture to differentiate into NSCs. Results The hESCs kept floating in the bacterial culture dish and growing all the time and then formed mature embryoid bodies 7 to 10 days later.The embryoid bodies could be differentiated into highly pure (96.4%)nestin positive cells.And these cells were differentiated into all kinds of neural cells if cultured further.Conclusions This kind of method is less time-consuming,cheaper,and more efficient than those of the results in literatures reported.It affords very good source of seed cells for cell transplantation therapy in the future.

Though mesenchymal stem cells (MSC) have been clinically used to repair a variety of damaged tissues, the underlying mechanisms remain elusively as the majority of the ex vivo expanded MSC die shortly after transplantation. To explore the mechanism in which the death cells play tissue repair effect, apoptosis of rat bone marrow MSC was induced by culturing cells in the conditions of hypoxia or/and serum-free medium, and the subcellular structures in the supernatants were analyzed. The results showed that apoptosis occurred in the presence of either hypoxia or serum-free condition as well, and the apoptotic proportion reached up to (17.44 ± 2.15) after the cells were treated by hypoxia plus serum free culture for 72 hours. The flow cytometric analysis of the sub-cellular substances harvested by ultracentrifugation of the supernatants found that the MSC released substantial amount of membrane microparticles into the supernatants, which expressed CD29, CD44A and Annexin-V-binding phosphatidylserine. It is concluded that the MSC can release membrane microparticles after induction, the amount of these membrane microparticles was around 15-fold of the parent cell numbers. The membrane microparticles is the mediators in the cross-talk between the transplanted cells and their surrounding tissues. This study provides some novel information for the mechanisms of MSC therapy.
Animals ; Apoptosis ; Cell Count ; Cell Hypoxia ; Cell-Derived Microparticles ; metabolism ; Cells, Cultured ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Rats ; Rats, Wistar

The biological properties of cultured mesenchymal stem cells (MSC) have been intensively investigated, while there is still a paucity of information about the definite in vivo sites that harbor these stem cells due to the lack of specific surface markers. Previous data have demonstrated that human and murine MSC can be isolated from the compact bones. To investigate if it is the case for other species, the femurs from Wistar rats, Beagles, C57 mice and New Zealand rabbits were collected, minced and digested with collagenase type I. The digested bone fragments were seeded into the medium for human bone marrow culture after removal of the suspended cells in the digestion. The results showed that the fibroblast-like cells were observed to migrate from the bone fragments after several days of culture, and they gradually formed an adherent confluent layer. The adherent cells could be passaged and expressed homogenously the mesenchymal cell marker vimentin. Differentiation assays showed that these cells had the capacity to differentiate into osteoblasts and adipocytes. In conclusion, the results here provide new information for the further investigations on the in vivo biological features of MSC in the context of the simplicity of the compact bone structure.
Animals ; Bone and Bones ; cytology ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Dogs ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred C57BL ; Rabbits ; Rats ; Rats, Wistar

Mesenchymal stem cell (MSC)-based cell therapy has shifted into clinical trials to repair the damage of various tissues. In this setting, the survival of the transplanted cells contributes critically to the therapeutic effectiveness. To investigate the in vivo tracing of MSCs, a recombinant retroviral vector carrying firefly-luciferase reporter gene [pL (FLUC) SN] was constructed and several GPE+86 cell clones that stably expressed fluc were selected. The retroviral supernatants were collected and used to transfect MSC derived from C57 mice. The cells were then screened with G418 and the expression of the exogenous gene was identified by luciferase enzyme activity analysis. Labeled mouse MSCs (2x10(6)) were injected into skeletal muscles, and the in situ expression was noninvasively tracked by in vivo bioluminescence imaging for 1, 3 and 6 days after transplantation. The results showed that the survival rates of the grafted cells dropped sharply with time, they were 57.2+/-11.7%, 8.6+/-2.5% and 5.4+/-3.1% on day 1, 3 and 6 after transplantation, and no fluorescent signals above background were detected on day 10. It is concluded that the method described above could be used for in vivo tracing of grafted cells. Furthermore, MSCs could not survive even transplanted into the none-ischemic skeletal muscles.
Animals ; Bone Marrow Cells ; cytology ; Bone Marrow Transplantation ; methods ; Cell Survival ; Female ; Genetic Vectors ; Green Fluorescent Proteins ; Luminescent Measurements ; methods ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred C57BL

Objective To understand the epidemic situation of soil-transmitted nematodiasis in the national surveillance site in Henan Province. Methods Over 1000 fecal samples from inhabitants in Huaiyang County of Henan Province,which was a national surveillance site,were collected each year from 2006 to 2015,the eggs of soil-transmitted nematodes and other intesti-nal helminths were examined by Kato-Kats technique. The cellophane swab method was used to detect Enterobius vermicularis eggs in children aged 3 to 12 years. In addition,the soil samples were collected from vegetable fields,lavatories,courtyards and kitchens of 10 families randomly selected in each year to examine Ascaris eggs by a modified saturated sodium nitrate floatation method. Results From 2006 to 2015,10419 persons were investigated,and the eggs of five species of intestinal helminths, Ascaris lumbricoides,Trichuris trichiura,hookworm,E. vermicularis,and Trichostrongylus orientalis,were detected,The aver-age infection rate of soil-transmitted nematodes in residents in Huaiyang County was 3.69%. The intensity of infection was mild and a family clustering was obvious. Both the infection rates of E. vermicularis in children and soil-transmitted nematodes in vil-lagers had no significant differences between different genders (both P>0.05). The infection rates of soil-transmitted nema-todes,A. lumbricoides and E. vermicularis all reached the highest in the age group of 1-10 years. For different education back-ground,the people with primary school education had the highest infection rate,and the infection rate showed a decreasing trend with the increase of the educational level. The infection rate of soil-transmitted nematodes in the national surveillance site in Henan Province showed a decreasing trend from 2006 to 2015. Unfertilized and fertilized A. lumbricoides eggs were detected in the soil samples,but the positive rate was very low. Conclusions In the recent 10 years,the infection rate of soil-transmit-ted nematodes in the national surveillance site in Henan Province shows a decreasing trend and maintains at a low level. The in-fection shows a family clustering. The children,especially those aged 3-9 years are the main infected population,and E. vermic-ularis infection is the key point of prevention and control.

Objective To explore the relationship between sleep status and the risk of diabetes in adults.Methods The baseline data of 53 260 subjects who were aged 30-79 years and had been enrolled into China Kadoorie Biobank (CKB) study from Suzhou,Jiangsu province were analyzed.Multiple logistic regression models were used to investigate the association between sleep status and diabetes after adjusting for potential confounders.Results Among 53 260 subjects,5.3％ had diabetes.The proportions of difficultly falling asleep,early morning arousal and snoring frequently was 7.2％,10.0％ and 29.5％,respectively.There were 22.6％ of subjects reporting sleep duration ≤6 hours.After controlling for possible confounders,the subjects with difficulty falling sleep (OR=1.63 for male,95％ CI:1.30-2.05;OR=1.48 for female,95％ CI:1.27-1.73),early morning arousal (OR=1.37 for male,95％CI:1.12-1.68;OR=1.31 for female,95％CI:1.14-1.51) or snoring frequently (OR=1.16 for male,95％CI:1.00-1.34;OR=1.39 for female,95％CI:1.23-1.57) had a higher risk of diabetes.Using hypnotics regularly was associated with the risk of diabetes in females (OR=1.42,95％CI:1.06-1.92).Compared with 8 hours sleep duration daily,shorter sleep duration (≤ 6 hours) was associated with risk of diabetes in both males (OR=1.37,95％CI:1.17-1.60) and females (OR=1.24,95％ CI:1.08-1.41).No statistical significant association was found between longer sleep duration (≥9 hours) and the risk of diabetes.Conclusion Sleep problems,including difficulty falling asleep,early morning arousal,snoring frequently and shorter sleep duration,were associated with the risk of diabetes,but no statistical significant association was observed between longer sleep duration and the risk of diabetes.

Objective To report a pedigree with tyrosinemia type Ⅱ,and to analyze its causative mutations.Methods Clinical data were obtained from a 10-year-old male proband with tyrosinemia type Ⅱ,and analyzed retrospectively.Blood and urine samples were collected from 19 persons in 3 generations of the pedigree,and the amino acid level was detected in these samples.Genomic DNA was extracted from all of the 19 family members,and mutations in the tyrosine aminotransferase (TAT) gene were detected.Results The patient developed photophobia at 2 months after birth,and the symptom was gradually aggravated after that.At the age of 6 years,ocular pain and photophobia occurred.At the age of 8 years,linear keratotic plaques occurred on his fingertips and soles of both feet,with obvious tenderness.Ophthalmic examination showed no obvious abnormalities in corneal staining or ocular fundus.Skin examination showed multiple linear keratotic plaques on the fingers and soles of both feet.The serum tyrosine level was 825.64 μmol/L,and the level of p-hydroxyphenyllactic acid in urine was 161.4 μmol/L.Genetic testing showed 2 novel mutations,including c.236G ＞ A at position 236 in exon 2 of the TAT gene causing the substitution of glycine by glutamic acid (p.Gly79Glu),and c.1141G ＞ T at position 1141 in exon 10 of the TAT gene leading to the formation of a premature termination codon instead of glutamic acid (p.Glu381*).The proband was the only patient in the family.Some members in the patrilineal family carried the mutation c.1141G ＞ T (p.Glu381*),and some in the maternal family carried the mutation c.236G ＞ A (p.Gly79Glu).Conclusion This is the first case of tyrosinemia type Ⅱ reported in the domestic population,and 2 novel heterozygous mutations were identified in the TAT gene,which may lead to the occurrence of tyrosinemia type Ⅱ in the patient.

OBJECTIVETo explore the proangiogenic activity of exsomes released by human umbilical cord mesenchymal stem cells (MSCs) stimulated by erythropoietin and platelet-derived growth factor BB (PDGF-BB).

METHODSHuman umbilical cord-derived MSCs were seeded and maintained in culture overnight. The media were then replaced by alpha-MEM containing EPO (1 U/ml) and/or PDGF-BB (50 ng/ml), and the culture was maintained for 72 hours. The exosomes from the culture supernatants were isolated with a routine ultra-catrifagation method. Flow cytometric analysis was performed to identify the origin of the exosomes, and their morphological features were observed by using a transmission electron microscopy. The exosomes were added at a concentration of 10 µg/ml into the culture system of human umbilical cord vein endothelial cells. MTT assay was used to evaluate the proliferative status. The Matrigel assay was used to observe the formation of net-work structures which were calculated after culture for 12 hours.

RESULTSFlow cytometric analysis showed that microparticles released by human umbilical cord MSCs expressed CD9, CD63 and CD81, which was in accordance with the surface molecular features of exosomes. Under an electron microscope, the exosomes took the featured cystic shape. The protein contents of exosomes released by untreated, EPO-stimulated, PDGF-BB-stimulated and EPO plus PDGF-BB stimulated MSCs (10 cells) were 256±124 µg, 1021±392 µg, 830±265 µg and 2207±733 µg, respectively. The results revealed that MSCs treated by EPO and PDGF-BB released significantly higher amounts of exosomes (P<0.01). MTT assay proved that the exosomes from EPO and PDGF-BB treated MSCs had more potent proliferation-promoting activity on human umbilical cord vein endothelial cells than those from untreated MSCs. The Matrigel assay showed that the numbers of capillary-like structures in untreated, EPO-, PDGF-BB and EPO plus PDGF-BB-treated groups were 2.6±0.84, 4.6±1.57, 4.2±0.78 and 6.3±1.34 per high power objective. Treatment with EPO or PDGF-BB dramatically enhanced the numbers of capillary liue structure, compared with that of untreated group (P<0.01) and those in EPO and PDGF-BB combination group was significantly greater than those of EPO or PDGF-BB group (P<0.01).

CONCLUSIONEPO and PDGF-BB can stimulate MSCs to release exosomes with more potent proangiogenic activity.

Pathological specimen sampling is not only the prerequisite of a good pathological diagnosis, but also the primary clinical skill that must be mastered by the standardized residency training trainees (resident trainees) in clinical pathology department. In view of the problems and difficulties encountered in the teaching of specimen sampling, through five years of exploration and attempt, this paper has gradually established a new model with five basic elements, including theory teaching, practice teaching, promoting teaching effect by examination, learning from senior students, and review teaching. The results of evaluation analysis and questionnaire survey show that the teaching mode can make the trainees master the methods of specimen sampling quickly and efficiently, learn and improve clinical skills in practice, and lay a solid foundation for the subsequent standardized training of histopathological diagnosis.

Objective To construct recombinant eukaryotic expression plasmid encoding Swedish and Flemish mutations of amyloid precursor protein (APP) fused with fluorescent protein and to investigate the APP cleavage progress. Methods The last 300 bases of APP (named as C99 containing Flemish mutation), together with cyan and yellow fluorescence sequence (named as CFP and YFP,respectively) were obtained by polymerase chain reaction (PCR). The 54 bases in the middle of APP sequence were synthesized (named as 54 bp containing Swedish mutation). The 4 fragments mentioned above (CFP, YFP, C99 as well as 54 bp) were inserted into the vector pcDNA3.0. By genetic engineering, the recombinant plasmid pcDNA3.0-CFP-54bp-YFP-C99 was constructed and identified by enzyme digestion, PCR and sequencing. Then the plasmid was transfected into SH-SY5Y cells. Its expression was examined by fluorescence confocal microscopy and the fluorescence resonance energy transfer (FRET) signal was collected. The amyloid beta (A) deposition was detected by immunocytochemistry. Results (1) DNA sequencing showed the sequence of the constructed recombinant plasmid was correct. (2) FRET and two types of fluorescence could be seen by the spectrum confocal fluorescence microscopy. (3) The expression product of fusion gene was correct and cleaved by and secretases. (4) The A deposition was detected in the cell membrane, cytoplasma and intercellular space. Conclusion (1) The fusion protein can generate A by and γproteolytic processing. (2) It is for the first time to observe the APP cleavage by FRET. (3) It is also the first time to find that APP may be cleaved during its transportation from cell plasma to cell membrane. (4) C99 is very important for the correct cleavage of APP. Our test data strongly suggest that C99 may function as the signal peptide. It might guide and direct the APP to the right location for the cleavage.