Objective To induce adult adult adipose-derived stem cells(ADSCs) in vitro to differentiate into neuronal-like cells,and to analyze the features of their cell morphology and ultrastructure. Methods Adipose stromal cells were obtained and amplified in vitro. Then make use of chemical induction to induce them. Observed ADSC and differentiation of cells in morphology and ultrastructure under inverted microscope and transmission electron microscopy. Immunocytochemistry to detection of Nestin, Neuron Specific Endolase( NSE) ,and glial fibrillary acidic protein(GFAP) expression in cells. Used Real-time PCR to detection of Nestin,GFAP gene mRNA expression before and after induction in ADSC. To observe the morphology and ultrastructure of the cells prior to and after induction under microscope and electron microscope. Results The morphology of ADSC was similar to fibroblasts ,and could be amplified stability within 10 passages in vitro. Some of the cells induced display a typical astrocyte-like cells in ultrastructure. Followed neuronal induction,astrocyte-like cells began to stain brightly for CFAP, Nestin. GFAP stained in both the cytoplasm and nuclei of astrocyte-like cells, but Nestin only stained in the cytoplasm. The peak positive expression rate within 14d following neuronal induction. The rate of positive expression cells was( 14.4 ± 3. 6) % for Nestin, (87. 3 ± 5. 3 ) % for GFAP. Then two kinds of protein expression remained the similar rate. The average relative concentration of GFAP and Nestin gene mRNA have significant statistical difference between ADSC and differented cells analyzed by Real-time PCR (P<0.05).The peak concentration of GFAP was within 20 d after induction,and GFAP was within 14 d after induction. Conclusion In the cytoplasm of adult adipose-derived cells possess Nestin genetic material,which is the marker of neural stem cell. The differential astrocyte-like cells have the typical morphology, ultrastructure and GFAP phenotype of mature astrocytes.

Objective To reseach the time point of the highest percentage of neural precursor cells derived from adipose stromal cells (ADSCs) in vitro, and to observe the ultrastructure features of neural precursor cells. Methods Used the β-mercaptoethanol to induce ADSCs to differentiate into neural precursor cells and neuron-like cells. The morphology of the uninductedcells and inducted cells were observed with inverted phase contrast microscope. The expression of nestin which was the marker of neural precursor cell in each group was detected using immunofluorescence staining method. The ultrastructural feature of cells which was induced for 3 hours were observed. Results The highest ratio of positive expression of nestin was 3 hours following induction,with the ratio ( 86.25 ± 4.82) %. There were many protuberance on the cell membrane under transmission electron microscopy.There were plenty of organelles in the neural precursor cells. The neural precursor cells had a large size nucleus,large nucleoplasmic index, much extended chromatin,and less condensed chromatin. The nucleus had double-layer nuclear envelope, more nuclear pore on the nuclear envelope. Conclusion The time point of the highest percentage of neural precursor cells derived from ADSCs is 3 hours,and the ultrastructral feature of induced neural precursor cells confirm that cells at this time point are in a state of split active period.

OBJECTIVETo observe the effect of Guilingji Capsule (GC) on the fertility, liver functions, and serum lactate dehydrogenase (LDH) of adult male SD rats exposed by 900 MHz cell phone.

METHODSTotally 18 adult male SD rats and 36 adult female rats in child-bearing period were selected and randomly divided into three groups according to weight equilibrium principle, i.e., the normal group, the radiated group, and the GC group, 6 males and 12 females in each group. Male rats in the normal group and all female rats were not radiated. Male rats in the radiated group and the GC group received radiation for 4 h per day, lasting for 18 successive days. Rats in the GC group received GC suspension at the daily dose of 0. 15 g/kg by gastrogavage at the same time. Equal volume of normal saline was administrated to other male rats. Then male rats were mated with corresponding female rats from the 14th radiation night to the 18th radiation night in the ratio of 1:2. Male rats were killed following on the next morning of ending the radiation. Female rats were normally fed and then killed before delivery. The pregnant outcomes of female rats in responding groups (the rates of pregnancy and the number of death fetus, birth weight, body length, and tail length) were observed and compared. Serum alanine aminotransferase (ALT), aspartate transferase (AST), AST/ALT, and LDH levels of the male rats were detected by colorimetry. Histological and morphological changes of liver were observed by HE staining.

RESULTSCompared with the normal group, the pregnancy rates of female rats decreased and the number of death fetus increased, the serum LDH level obviously increased in the radiated group (P < 0.05). Serum levels of ALT, AST, and AST/ALT were no significantly changed in the radiated group. The hepatocyte nuclear atrophy and cytoplasm vacuolar degeneration appeared. Compared with the radiated group, the pregnancy rates increased, the number of death fetus dropped, and the serum level of LDH decreased in the GC group (P < 0.05). There was no obvious change in serum levels of ALT, AST, or AST/ALT. The hepatocyte nuclear atrophy and cytoplasm vacuolar degeneration were significantly attenuated. The histomorphological structures recovered to normal basically in the GC group.

CONCLUSIONSThe pregnancy rates could be decreased, the number of death fetus increased, histomorphological structures abnormal, and serum LDH level increased by exposure toy GSM 900 MHz cell phone. GC could prevent and treat the aforesaid lesion. But there was no statistical difference in serum ALT or AST levels.

Animals ; Cell Phone ; Drugs, Chinese Herbal ; pharmacology ; Female ; Fertility ; Lactate Dehydrogenases ; blood ; Liver ; drug effects ; Male ; Pregnancy ; Radiation ; Rats ; Rats, Sprague-Dawley

Agmatine ; administration & dosage ; pharmacology ; Analgesia ; methods ; Animals ; Injections, Spinal ; Male ; Morphine ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; drug effects

Objective To determine the difference of macrophage RAW264.7 apoptosis induced by Brucella melitensis virulent strain 16M and attenuated strain M5-90 and elucidate the regulatory role of caspases 3,8 and 9.Methods The best multiplicity of infection (MOI) was determined through kinetic analysis of Brucella melitensis strain 16M and M5-90 induced mouse macrophages apoptosis(bacterium ∶ cell =100 ∶ 1,50 ∶ 1,10 ∶1).The infection model was established using the best MOI =50 ∶ 1.The numbers of in vivo bacteria by colony formation units were calculated after macrophages were infected for different times,including 2,4,8,12,24 and 48 h,and the infected cells were collected.The ratios of apoptosis were detected and the regulation of caspases 3,8 and 9 in apoptosis pathway was elucidated by flow cytometry.Results The numbers of 16M in vivo bacteria were 105.4,104.8,105.8,106.5,108.0 and 109.0,respectively and of M5-90 were 106.1,106.2,106.4,106.3,106.1 and 105.0,respectively.The number of in vivo bacteria of 16M was significantly increased than that of M5-90 after infected for 24 h to 48 h.The ratios of apoptosis induced by 16M after infected for 2,4,8,12,24 and 48 h was (2.67 ± 0.09)％,(13.13 ± 0.30)％,(6.56 ± 0.42)％,(6.49 ± 0.28)％,(16.07 ± 0.86)％ and (24.23 ± 1.67)％,respectively,and by M5-90 was (3.62 ± 0.02)％,(32.01 ± 2.59)％,(17.58 ± 0.44)％,(16.09 ± 0.10)％,(62.53 ± 2.70)％ and (85.53 ± 0.15)％,respectively,and by control group was [(1.90 ± 0.20)％,(1.92 ±0.16)％,(1.99 ± 0.03)％,(2.48 ± 0.11)％,(3.56 ± 0.07)％,(5.26 ± 0.33)％].The differences were statistically between groups in same time.The Brucella melitensis vaccine strain M5-90 was more powerful than virulent strain 16M in respect of inducing macrophage apoptosis after infected for 24 to 48 h.Twenty-four hours after infection,the expression of caspases 3,8 and 9 was (1.47 ± 0.05)％,(1.52 ± 0.02)％ and (2.47 ± 0.12)％,respectively,in control group and the expression was (9.70 ± 0.46)％,(6.08 ± 0.56)％ and (35.08 ± 1.64)％,respectively,after infected for 24 h induced by M5-90.The expression of caspases 3,8 and 9 was significantly higher than that control group (P ＜ 0.01).Twenty-four hours after given caspases 3,8 and 9 inhibitor,apoptosis rate in control group was (66.72 ± 1.28)％,in M5-90 group was (22.58 ± 0.55)％,(53.15 ± 1.85)％ and (29.18 ± 0.23)％,respectively,and compared with control group,apoptosis rate of caspases 3,8 and 9 was significantly lower(P ＜ 0.01).Conclusions Apoptosis of macrophage can be induced by Brucella melitensis virulent vaccine strain 16M and attenuated strain M5-90.M5-90 is stronger than that of strain 16M.Caspases 3,8 and 9 can regulate macrophage apoptosis after M5-90 infection.

Objective To observe the changes of cardiac function and carotid artery structure in elderly hypertensive patients with different left ventricular geometric patterns. Methods Seventy-eight elderly patients with essential hypertension were divided into 4 groups according to left ventricular geometric patterns by ultrasonography: normal ventricular geometry group(n=34),concentric remodeling group(n=18),concentric hypertrophy group(n=11)and eccentric hypertrophy group(n=15).The 24-h ambulatory blood pressure,left ventricular function,carotid artery intima-media thickness(IMT),hemodynamic parameters and incidence of plaque were measured and compared among groups.Results Patients in concentric hypertrophy group had higher 24-h average systolic blood pressure in comparison with those in normal ventricular geometry group and concentric remodeling group(P

Understanding the research methods for drug protein targets is crucial for the development of new drugs, clinical applications of drugs, drug mechanisms, and the pathogenesis of diseases. Cellular thermal shift assay (CETSA), a target research method without modification, has been widely used since its development. Now, there are various CETSA-based technology combinations, such as mass spectrometry-based cellular thermal shift assay (MS-CETSA), isothermal dose response-cellular thermal shift assay (ITDR-CETSA), amplified luminescent proximity homogeneous assay-cellular thermal shift assay (Alpha-CETSA), etc., which combine their respective advantages and further expand the application scope of CETSA. These technologies are suitable for the entire drug development chain, from drug screening to monitoring the target binding and off-target toxicity of drugs in patients. Based on the author's research experience, this paper reviews the principles of CETSA and related binding technologies, their application in target discovery, and the progress of data processing and analysis in recent years, aiming to provide reference and reference for the further application of CETSA.

BACKGROUNDAdipose-derived stromal cell (ADSC) differentiation into neural cells in vitro is becoming widely studied. However, there are few reports on astrocytes following differentiation, and particularly on maturation and electrophysiology. In this study, we used various methods to determine ADSC-derived astrocyte maturity.

METHODSChemical induction with isobutylmethylxanthine (IBMX) was used to differentiate adult ADSCs into astrocytes followed by hematoxylin-eosin (HE) staining to observe morphology and transmission electron microscopy for cellular ultrastructure assessment. Immunofluorescence was used to detect expression of neural stem cell marker nestin as well as glial markers glial fibrillary acidic protein (GFAP) and S-100. In addition, we measured membrane potentials in bis-(1,3-dibarbituric acid) trimethine oxanol-labeled ADSCs and astrocytes by stimulation with a high potassium solution under an inverted fluorescence microscope. Finally, cell cycle distribution was detected by flow cytometry.

RESULTSTypical astrocyte morphology was shown by HE staining after 48-hour differentiation. Glial fibril was observed with transmission electron microscopy. GFAP and S-100 were not expressed in the control group, but were expressed within 24-hour differentiation and reached a maximum at day 14 with no change up to day 28. Nestin was weakly expressed in control cells and also reached a maximum at day 14 with the percentage of positive cells constant until day 21 followed by a decrease. Differentiated cell membrane potentials after stimulation with potassium were slightly increased, and then gradually declined over time. There was no significant membrane potential change in the control group. Flow cytometry showed that the percentage of cells in G0/G1 phase was 93% and only 5% in S phase.

CONCLUSIONADSCs were differentiated into mature astrocytes with typical characteristics including morphology, ultrastructure, marker protein expression, mature potassium channels and mitotic capacity.

1-Methyl-3-isobutylxanthine ; pharmacology ; Adipose Tissue ; cytology ; Adult ; Astrocytes ; cytology ; Barbiturates ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Electrophysiology ; methods ; Female ; Flow Cytometry ; Glial Fibrillary Acidic Protein ; metabolism ; Humans ; Male ; Membrane Potentials ; drug effects ; Microscopy, Fluorescence ; S100 Proteins ; metabolism ; Stromal Cells ; cytology ; Young Adult

OBJECTIVETo investigate the effects of Tangzhiping Granule (TZPG) on blood lipids and free fatty acids (FFA) in rats with insulin resistant diabetes (IRD).

METHODSA blank control group consisted of randomly selected normal rats was set up. The remaining rats were established to IRD model by high-fat high-sugar diet feeding and streptozotocin injection. Then the 32 successfully modeled rats were randomized into the model group (treated by saline), the Tangmaikang group (treated with Tangmaikang Granule 1.35 g/kg), and the two TZPG groups treated with high dose (2.70 g/kg) and low dose TZPG (1.35 g/kg) respectively through intragastric infusion for 4 weeks. The body weight (BW), fasting blood glucose (FBG), insulin (INS), blood lipids including triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and FFA were detected, and the insulin sensitivity index (ISI) calculated.

RESULTSCompared with the blank control group, BW, FBG and INS increased while ISI decreased in the model group (P < 0.05 or P < 0.01). All the above-mentioned abnormal indices were improved in the three treated groups (Tangmaikang, high and low dose TZPG group), but the improvements in the high dose TZPG group were more significant than those in the other two groups (P < 0.05 or P < 0.01). Similar outcomes were also seen in blood lipids detection, in which TG, TC, LDL-C and FFA were higher and HDL-C were lower in model rats than those in blank controls, they were improved in the three treated groups (P < 0.05), and the best improvements were seen in the high dose TZPG group (P < 0.05).

CONCLUSIONTZPG could reduce levels of BW, FBG, INS, TC, TG, LDL-C and FFA, and increase levels of ISI and HDL-C in rat model of insulin resistant type 2 diabetes, so as to improve the insulin resistance in them.

Animals ; Diabetes Mellitus, Experimental ; drug therapy ; Diabetes Mellitus, Type 2 ; drug therapy ; Drugs, Chinese Herbal ; therapeutic use ; Fatty Acids, Nonesterified ; blood ; Female ; Insulin Resistance ; Lipids ; blood ; Male ; Phytotherapy ; Random Allocation ; Rats ; Rats, Wistar

Three compounds were isolated from the extract of Taxus cuspidta Sibe et Zucc with the column chromatography on silica gel and preparative HPLC methods. Their structures were identified according to the physicochemical properties and spectral analysis, and they were identified as (E)-1-methoxy-2-O-(p-coumaroyl)-myo-inositol (1), 2-deacetoxy-7beta, 9a, 10beta-trideacetyltaxinine J (2) and (3aS, 4aR, 6S, 8S, 8aS, 9R, 10R, 10aS)-benz[f]azulene-6, 8, 9, 10 (3H)-terol, 3a, 4, 4a, 5, 6, 7, 8, 8a, 9, 10-decahydro-10a-(1-hydroxyl-1-methylethyl)-1, 8a-dimethyl-5-methylene (3). Among them, compound 1 was a new compound, and compounds 2, 3 were two novel natural products.
Azulenes ; chemistry ; isolation & purification ; Coumarins ; chemistry ; isolation & purification ; Inositol ; analogs & derivatives ; chemistry ; isolation & purification ; Molecular Structure ; Plants, Medicinal ; chemistry ; Taxoids ; chemistry ; isolation & purification ; Taxus ; chemistry