OBJECTIVETo study the dynamic changes in the protein marker expression in the spermatogonial stem cells (SSCs) of mice at different ages by iTRAQ protein mass spectrometry and to screen new markers using the bioinformatic proteome database.

METHODSBased on the postnatal weeks, we divided 80 healthy male C57BL/6 mice into eight age groups of equal number, harvested their testicular tissues, extracted proteins following purification of the SSCs by compound enzyme digestion and magnetic-activated cell sorting. Then we analyzed and identified proteins using two-dimensional electrophoresis, protein mass spectrometry, and protein database information.

RESULTSTotally, 248,510 mass spectra were obtained from the MS experiment and 1132 proteins were identified. By the criteria of >1.2-fold for protein abundance difference and P value <0.05, we identified 298 differentially expressed proteins and 9 currently known makers of SSCs (PCNA, GFRalpha1, CDH1, Annexin A7, UCHL1, VASA, CD49f, CD29, and PLZf). Compara- tive analysis showed different expressions of the proteins in the SSCs of the mice of different ages, and the differences in the expressions of GFRalpha1, CD49f, and CD29 were consistent with the findings in other published literature. Ten proteins (P63, CD71, CD98, K19, ACE, K18, K15, K17, SH2, and SH3) were selected as SSC markers to be further studied.

CONCLUSIONThe proteins in SSCs are differentially expressed in mice of different ages. The technology of iTRAQ protein mass spectrometry can be used to analyze and compare the proteome information of mouse SSCs, obtain differentially expressed proteins in mice of different ages, and thus offers a new ap- proach to further analysis and study of the function and roles of these differential proteins.

Adult Stem Cells ; cytology ; metabolism ; Age Factors ; Animals ; Biomarkers ; analysis ; metabolism ; Cell Separation ; methods ; Electrophoresis, Gel, Two-Dimensional ; Male ; Mass Spectrometry ; Mice ; Mice, Inbred C57BL ; Proteins ; analysis ; metabolism ; Spermatogonia ; cytology

In order to identify Peucedani Radix, Peucedani Decursivi Radix and their adulterants, the internal transcribed spacer 2 (ITS2) regions of Peucedani Radix, Peucedani Decursivi Radix and their adulterants were amplified and bidirectionally sequenced based on the Principles for Molecular Identification of Traditional Chinese Materia Medica Using DNA Barcoding, which has been promulgated by Chinese Pharmacopoeia Commission. Sequences were analyzed and assembled by Codon Code Aligner V3. 7.1. The relevant data were analyzed by MEGA 5. 0. Species identification analyses were performed by using the nearest distance methods and neighbor-joining (NJ) methods. The result showed that the ITS2 sequence lengths of Peucedani Radix were 229-230 bp and the average intra-specific genetic distances were 0.005. The ITS2 sequence lengths of Peucedani Decursivi Radix were 227 bp and the sequences contained no variation site. The average inter-specific K2P genetic distance of Peucedani Radix, Peucedani Decursivi Radix and their adulterants species were 0.044 and 0.065 respectively. The minimum inter-specific divergence is larger than the maximum intra-specific divergence of Peucedani Decursivi Radix. The nearest distance methods and NJ trees results indicated that Peucedani Radix, Peucedani Decursivi Radix and their adulterants species could be identification clearly. The ITS2 regions can stably and accurately distinguish Peucedani Radix, Peucedani Decursivi Radix and their adulterants.
Apiaceae ; classification ; genetics ; DNA Barcoding, Taxonomic ; methods ; DNA, Ribosomal Spacer ; Drug Contamination

BackgroundEndostatin (ES) is currently the strongest endogenous angiognesis inhibitor,and it can inhibit the occurrence of neovascularization.Various studies demonstrated that the poly RGD sequence can enhance the function of the ES gene.ObjectiveThis study was to evaluate the use of gene therapy of modified ES for alkaline burn-induced corneal neovascularization (CNV).MethodsOne hundred and two clean SD rats were randomly divided into the normal control group,the pCI empty vector group,the pCI-ES group,and the pCI-RGDRGDES group.Corneal neovascularization models were established by placing a piece of 3 mm filter paper with 1 mol/L NaOH at the central cornea for 40 seconds.3 μg of the pCI blank vector,ES-tranfected pCI blank vector,or RGDRGD-ES-transfected pCI vector was injected into the superior bulbar conjunctiva after the alkali burn twice at 1-week intervals.Area of CNV and edema of the cornea in the various groups of rats were examined daily under the slit lamp biomicroscope.1,4,7 and 14 days after operation,the rats were sacrificed by the excessive anesthesia method and corneal tissues were obtained to evaluate pathological changes.The expression of CD34 in vascular endothelial cells was detected by immunochemistry to calculate the corneal neovascular density.The expressions of VEGF mRNA and Flk-1 protein in the corneas were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis.The use and maintenance of animals followed the Statement of ARVO.Results Seven to fourteen days after corneal alkali-burning,the corneal neovascular area was smaller in the pCI-ES group and pCI-RGDRGD-ES group compared with the normal control group and pCI blank vector group (P＜0.05,P＜0.01 ),and nevascular area in the pCI-RGDRGD-ES group was smaller than that in the pCI-ES group (P＜0.05).The expression level of CD34 was significantly lower in the pCI-ES group and pCI-RGDRGD-ES group than that in the normal control group and pCI blank vector group (P＜0.05,P＜0.01 ),and the expression level of CD34 was further declined in the pCI-RGDRGD-ES group compared with the pCI-ES group (P＜0.05 ).Compared with the normal control group and pCI vector group,the expressions of the Flk-1 protein and VEGF mRNA were decreased in the pCI-ES group and pCI-RGDRGD-ES group on the fourth day after corneal alkali-burning (P＜0.01,P＜0.05 ),and those in the pCI-RGDRGD-ES group were less than the pCI-ES group (P＜ 0.05,P＜ 0.05 ).Conclusions Subconjunctival injection of both ES and modified RGDRGD-ES genes result in significant suppression of CNV in vivo,and modified RGDRGD-ES appears to be more effective than native ES.The main mechanism of ES in inhibiting neovascularization is to downregulate the expression of VEGF and Flk-1.

Objective To investigate the diagnosis and treatment of mixed connective tissue disease(MCTD)complicating pulmonary hypertension(PAH) in childhood in order to improve the recognition of this disease.Method According to the symptoms,signs,past history,labratory examinations,the child′s disease was diagnosed and treated,and the relative literature was reviewed.Results The main symptom of the child was interruptable apsychia.Ultrasound showed severe PAH,positive of anti-RNP antibody.After given immunosuppressant and decreased PAH,the patient′s condition was more improved.Conclusions MCTD complicating PAH in childhood onstes delitescently,and it′s difficult to diagnose.Recognition should be elevated to diagnose and treat it earlier.The prognosis can be improved.

OBJECTIVEStudy the effects of β-glucan in highland barley on blood glucose and serum lipid in high fat diet induced C57 mouse.

METHODSUsing table of random number, 40 male C57BL/6 mice were randomly divided into 4 groups (10 mice in each group) by weight: high dosage group (4% β-glucan and high fat diet), low dosage group (2% β-glucan and high fat diet), high fat diet group and normal control group. Food-intake and body weight of C57 mouse were observed. Glucose tolerance tests and examinations of fasting blood glucose were performed at the end of 11 weeks of intervention. Mice were sacrificed after 12 wk of treatment, and serum specimens were obtained to test relevant biochemical indicators.

RESULTSAfter 12 weeks raise, among high dosage group, low dosage group, high fat diet group and normal control group, the weight was (32.8 ± 1.5), (40.4 ± 1.9), (40.7 ± 2.1) and (33.5 ± 1.3) g, respectively (F = 55.26, P < 0.05); average food intake was (3.48 ± 0.56), (3.69 ± 0.76), (3.66 ± 0.81) and (3.54 ± 0.61) g/d respectively (F = 0.26, P > 0.05); fasting blood-glucose was (5.29 ± 1.59), (6.13 ± 1.75), (7.63 ± 1.09) and (4.24 ± 0.98) mmol/L respectively (F = 9.54, P < 0.01); serum insulin level was (1.97 ± 0.10), (2.44 ± 0.24), (3.02 ± 0.36) and (1.48 ± 0.28) ng/ml respectively (F = 47.58, P < 0.01); the area under blood glucose concentration curve was (25.81 ± 1.44), (30.42 ± 2.01), (35.17 ± 1.20) and (21.03 ± 1.24) mmol×L(-1)×h(-1), respectively (F = 64.98, P < 0.05); insulin resistance index was (9.84 ± 3.78), (13.69 ± 4.48), (21.54 ± 3.27) and (5.81 ± 1.59) respectively (F = 30.18, P < 0.01); serum total cholesterol (TC) level was (4.05 ± 0.88), (4.30 ± 0.48), (4.73 ± 0.66) and (3.37 ± 0.40) mmol/L respectively (F = 6.70, P < 0.01); serum triglyceride (TG) level was (0.90 ± 0.09), (0.98 ± 0.09), (1.05 ± 0.06) and (0.76 ± 0.26) mmol/L respectively (F = 6.75, P < 0.01); serum high-density lipoprotein cholesterol (HDL-C) level was (2.91 ± 0.59), (3.34 ± 0.46), (4.89 ± 0.42) and (3.24 ± 0.37) mmol/L respectively (F = 31.73, P < 0.01); serum low-density lipoprotein cholesterol (LDL-C) level was (0.25 ± 0.15), (0.42 ± 0.19), (0.72 ± 0.12) and (0.32 ± 0.11) mmol/L, respectively (F = 17.27, P < 0.01); free fatty acids (FFA) level was (1.06 ± 0.03), (1.05 ± 0.05), (1.18 ± 0.32) and (1.04 ± 0.02) mmol/L, respectively (F = 1.36, P > 0.05); HDL-C/LDL-C was (13.77 ± 5.51), (9.11 ± 3.53), (7.04 ± 1.65) and (11.21 ± 3.31), respectively (F = 5.24, P < 0.01).

CONCLUSIONThe β-glucan in highland barley reduced the serum glucose and serum lipid, as well as insulin resistance and the risk of arterial sclerosis in high-fat induced C57 mouse.

Animals ; Blood Glucose ; Cholesterol ; blood ; Cholesterol, LDL ; blood ; Diet, High-Fat ; adverse effects ; Glucose Tolerance Test ; Hordeum ; Lipids ; blood ; Male ; Mice ; Mice, Inbred C57BL ; Triglycerides ; blood ; beta-Glucans ; pharmacology

Mesenchymal stem cells (MSC) have attracted high attention to their various origins, capability of multi-lineage differentiation, supporting hematopoiesis and regulating immunity. Consequently, MSC show great potential for tissue engineering and cell/gene therapy. The bone marrow microenvironment plays an important role in the pathogenesis of several hematological malignancies. It was confirmed that as key components of the hematopoietic microenvironment, MSC correlated complexly with tumor microenvironment. Recent reports showed that MSC from some patients with AML, MDS, ALL and MM harboured cytogenetic alterations. In addition, the phenotype, ability of differentiation and immunoregulatory function of MSC displayed different degree of abnormalities, suggesting that MSC played a role in the pathophysiological mechanism of malignant hematopoietic diseases. Besides, MSC have been found to participate in drug resistance of antileukemic therapy. Hematopoietic stem cell transplantation (HSCT) has become an important treatment approach for the malignant hematopoietic diseases in recent years. Because of the advantages of supporting hematopoiesis and regulating immunity, MSC are used to promote the engraftment and prophylaxis/treatment of GVHD. This review summarized briefly the abnormalities of mesenchymal stem cells in malignant hematological diseases and MSC research advances on cell therapy.
Bone Marrow Cells ; pathology ; Hematologic Neoplasms ; pathology ; therapy ; Humans ; Mesenchymal Stromal Cells ; cytology ; pathology

0.05),the mean FA on the left was higher than the right(t=1.912,P

Background Corneal epithelial abrasion results in corneal ulcer and stroma cloudy evenb irreversible visual impairment.Previous drugs for corneal epithelial injury can only alleviate the inflammatory irritation.So it is very important to seek a drug which regulate the growth of corneal epithelium.Objective This study was to investigate the effects of recombinant human BIGH3 protein eye drops on corneal epithelial abrasion.Methods Fifty right eyes of 50 clean adult New Zealand white rabbits were collected.Two rabbits were sacrificed right away following establishment of corneal epithelial abrasion models (0 hour group).The other 48 rabbits were randomly divided into recombinant human epidermal growth factor (EGF) derivative group (positive control group),normal saline solution group (negative control group),0.25％ or 0.5％ recombinant human BIGH3 protein eye drops group.Corneal abrasion models were created with alcohol corrosion method with a defect area of 7 mm2.The corresponding eye drops were used separately in 4 groups for four times per day after operation.Experimental eyes were examined by the slit lamp microscope,and fluorescein vital staining were performed 12,24,36,48,72 hours after operation.Planimetry was performed and the corneal photographs were analyzed with computer software.The rabbits were sacrificed 12,24,36,48 and 72 hours after operation,respectively,and the histopathological examination of corneal tissue was carried out.Results No obvious irritation response was seen after administered of eye drops in the recombinant human EGF derivative group,normal saline solution group,0.25％ and 0.5％ recombinant human BIGH3 protein eye drops groups.Histopathological examination revealed a full-thickness defect of corneal epithelium after modeling.The defect area was gradually smaller with time lapse,and corneal epithelium migrated from periphery toward the center zone.Corneal epithelial cells increased with time lapse.Compared with normal saline solution group,the defect area of corneal epithelium lessened 12,24,36,48 hours after operation in the 0.25％,0.5％ recombinant human BIGH3 protein eye drops groups and recombinant human EGF derivative group (all at P =0.000),but at 12and 24,36 hours after operation,no significant differences were found between the recombinant human EGF derivative group and normal saline solution group (P =0.321,0.057,0.126).The defect area was smaller in the 0.5％recombinant human BIGH3 protein eye drops group than that of the recombinant human EGF derivative group at various time points (P=0.042,0.039,0.025,0.008).However,significant smaller defect area was exhibited only at 12 hours and 24 hours after operation in the 0.25％ recombinant human BIGH3 protein eye drops group (P=0.047,0.042).No significant differences were seen in corneal defect area at various time points between 0.25％ and 0.5％recombinant human BIGH3 protein eye drops groups (P =0.358,0.259,0.108,0.062).In addition,the corneal defect area was (0.51 ±0.42)mm2 72 hours after operation in the normal saline group;while that in the recombinant human EGF derivative group and recombinant human BIGH3 protein eye drops groups was disappeared.The repairing curves in the recombinant human BIGH3 protein eye drops groups were superior to those of the recombinant human EGF derivative group and normal saline solution group.Conclusions 0.25％ and 0.5％ recombinant human BIGH3 protein eye drops have facilitation effect on the growth of corneal epithelial cells and the healing of corneal injury.

Objective To explore the changes of structure and function of mitochondria in liver tissue of mice with acute liver injury induced by volatile oils from ArtemisiaeArgyi Folium(VOAAF).Methods VOAAF was obtained by hydrodistillation extraction.specific pathogen free (SPF) Institute of Cancer Research (ICR) mice in half male and half female were randomized into five groups,including control group(n =10),model (carbon tetrachloride,CCl4) group (n =10),low dose (1.9 g · kg-1,n=10),middle dose (2.3 g · kg-1,n =10),and high dose(2.7 g· kg-1,n =12) VOAAF group(test group).The biological samples were obtained in 6 h after gavaging equal volume (20 mL · kg-1) drugs or solvent to mice at single dose.Aspartate transaminase (AST) in serum was detected.Morphological change of mitochondria in hver tissue was observed by transmission electron microscope.Change of mitochondrial membrane potential in liver tissue was examined by JC-1.Change of mitochondrial swelling sensibility in liver tissue was observed by spectrophotometer.Activity change of Na +-K +-ATPase,Ca2+-ATPase and Ca2+-Mg2+-ATPase in liver tissue were detected by phosphorus determination method.Results The levels of AST in control group and low,middle,high dose test group were (154 ± 27),(224 ±56),(251 ±64),(316 ± 101) U · L-1 (P ＜0.05).The mitochondria got swollen and hydropic in some degree,mitochondrial membrane potemial were 31.35 ±9.14,22.06-6.18,21.34 ±5.99,16.48 ±4.88(P ＜0.05).The mitochondrial swelhng sensibility were (1.43 ±0.69) +10-2,(0.68 ±0.28) ×10-2,(0.40 × ±0.18) + 10-2,(0.26 ±0.10) × 10-2 (P ＜ 0.05).The activity change of Na+-K+-ATPase were 4.40 ± 0.90,2.60 ± 0.60,2.20±0.80,2.10 ±0.80(P ＜0.05).The Ca2 +-ATPase were 4.20 ±0.80,2.70 ±0.50,2.50 ±0.80,2.20 ± 0.60 (P ＜ 0.05).The Ca2 +-Mg2 +-ATPase were 3.90 ± 0.70,2.80 ± 0.60,2.60 ± 0.90,2.50 ± 0.70 (P ＜ 0.05).All changes were related to dose variation trend of volatile oils.Conclusion The disorder of the structure and function of mitochondria could be the mechanism of acute liver injury induced by VOAAF in mice.

Objective To analyze the spatio-temporal process on 2009 influenza A (HlNl) pandemic in Changsha and the influencing factors during the diffusion process. Methods Data were from the following 5 sources, influenza A (HlNl) pandemic gathered in 2009, Geographic Information System (GIS) of Changsha, the broad range of theorems and techniques of hot spot analysis, spatio-temporal process analysis and Spearman correlation analysis. Results Hot spot areas appeared to be more in the economically developed areas, such as cities and townships. The cluster of spatial-temporal distribution of influenza A (HlNl) pandemic was most likely appearing in Liuyang city (RR=22.70,P<0.01). The secondary cluster would include districts as Yuelu (RR=6A9,P< 0.01) , Yuhua (RR=81.63, P<0.01). Xingsha township appeared as the center in the Changsha county (RR=2.90, P<0.01) while townships as Yutangping (RR=19.31, P<0.01) , Chengjiao (RR=73.14,P<0.01) and Longtian appeared as the center in the west of Ningxiang county (RR= 14.43,P<0.01) and Wushan as the center in the Wangcheng county (RR= 13.84,P<0.01). As time went on, the epidemic moved towards the eastern and more developed regions. Regarding factor analysis, population, the amount of students, geographic relationship and business activities etc. appeared to be the key elements influencing the transmission of influenza A (H1N1) pandemic. At the beginning of the epidemic, population density served as the main factor (r=0.477, P<0.05) but during the initial and fast growing stages, it was replaced by the size of students to serve as the important indicator (r=0.831, P<0.01; r=0.518, P<0.01). However, during the peak of the epidemics, the business activities played an important role (r=-0.676, P<0.01). Conclusion Groups under high risk and districts with high incidence rates were shifting, along with the temporal process of influenza A(H1N1) pandemic, suggesting that the protection measures need to be adjusted, according to the significance of influencing factors at different stages.