China ; Occupational Health Services

To develop an analytic method for qualitative and quantitative analysis of dodecatetraenamides A, B in 42 samples of two official species of Asari Radix et Rhizoma( ARR) (37 samples of Asarum heterotropoides var. mandshuricum with different collection time and 5 samples of Asarum sieboldiivar. seoulense). The HPLC-IT-TOF-MS/MS methods for the qualitative and UPLC-PDA methods for the quantitative analysis were established. Dodecatetraenamides A, B were identified by comparing the retention time, UV absorption spectrum and quasi-molecular ion peak [ M + H]+ with the reference compound using HPLC-IT-TOF-MS/MS. The content of dodecatetraenamides A and B in ARR were determined by UPLC-PDA. The separation was successfully carried out on a ACQUITY UPLC BEH C18 (2.1 mm x 100 mm, 1.7 µm) column eluted with mobile phases of water (A) and acetonitrile (B) in gradient program (0-3 min, 35% B; 3-5 min, 35%-36% B; 5-6 min, 36%-43% B; 6 min-11 min 43% B; 11-12 min, 43%-100% B). The column temperature was 45 °C, and the detection wavelength was set at 254 nm. The flow rate was 0.6 mL · min(-1). On one level mass spectrometry scanning, the results showed that the quasi-molecular ion [M + H] + of both dodecatetraenamides A and B were m/z 248.20. The quantitative method with UPLC-PDA has made the baseline separation of the constituents, which were reported as mixtures in the most literatures. The average recovery of dodecatetraenamides A and B were 97.90% and 99.86%, the relative standard deviation were 0.4% and 1.1%, respectively. The contents of dodecatetraenamides A, B in all ARR samples was in the range of 0.11-3.89 and 0.24-6.65 mg · g(-1). Their contents reduced with the extension of storage time. Compared with the samples of 2013, the average content of the two constituents in the samples collected in year 2002-2003 reduced 34% and 36%, respectively (P < 0.05). Compared the A. sieboldii var. seoulense and A. heterotropoides var. mandshuricum with the same collective time and production area, the average contents of the two constituents in latter were up to (1.59 ± 0.75) mg · g(-1) and (2.90 ± 1.17) mg · g(-1), respectively, significantly higher than that in A. sieboldii var. seoulense (dodecatetraenamide A were (0.78 ± 0.52) mg · g(-1), dodecatetraenamide B were (1.69 ± 0.83) mg · g(-1)) (P < 0.05). The content of the dodecatetraenamide A in overground part was in the range of 0.11-0.33 mg · g(-1), dodecatetraenamide B was 0. 24-0.60 mg · g(-1), which were much lower than that of the underground part of ARR (dodecatetraenamide A was in the range of 0.73-3.89 mg · g(-1), dodecatetraenamide B was 2.11-6.24 mg · g(-1)). The method was certified to be simple, accurate and reliable and could be used for qualitative and quantitative analysis of dodecatetraenamide A and B in different species of ARR, also can be used for the comprehensive quality control of traditional Chinese medicine, Asari Radix et Rhizoma.
Amides ; chemistry ; Asarum ; chemistry ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Mass Spectrometry ; Molecular Structure ; Rhizome ; chemistry

AIM: To investigate the ocular complication after radiation therapy for nasopharyngeal carcinoma(NPC).METHODS: The authors performed a previous study on keratopathy in 213 NPC patients who received first stage radiation and had at least 10 months of follow-up. These patients were categorized into three groups depending on NPC clinic stages. Rates and proportions of keratopathy occurring in these groups were compared and analyzed with Chi-square Test and Spearman rank correlation coefficient.RESULTS: Radiation keratopathy developed in 19 patients, about 8.9% (19/213). The latency value was 3 to 30days. The effect of NPC clinic stages and radiation did on the development of keratopathy was not statistically significant (P＞0.05).CONCLUSION: The NPC clinic stages and radiation doses plays few effects on the development of keratopathy. It may play a key role that corneal nerves damage induced ocular surface diseases. It can not be excluded that individuals have different sensitivities to radiation.

Objective To investigate the possibility of inducing mesenchymal stem cells(MSCs)from human umbilical cord Wharton's Jelly to differentiate into spermatogonia.Methods To isolate,culture and purify MSCs with adherent method,the growth and proliferation of human umbilical cord-derived MSCs were observed,and their immunophenotypes were determined by flow cytometry;MSCs of the third generation were divided into 2 groups to be induced and cultured,MSCs of the control group were cultured in basal medium,while those of the experimental group with conditional medium.The morphologic and ultrastructure changes of control group and experimental group cells were compared with phase contrast microscopy,electron microscopy(EM)and transmission electron microscope(TEM)respectively ;the spermatogonial cells differentiated were then evaluated by immunohistochemistry stained for CD117and CD49f ;the method of Western-blot was used to test if the cells induced could express CD49f.Results A population of MSCs were isolated from human umbilical Wharton's Jelly;they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling;After induction,the shape of MSCs changed greatly from the fibroblast to the round,even familiar to the tadpole;expressed the known molecular markers of spermatogonial cells,such as CD49f,CD117.Conclusion The induced MSCs not only undergo spfermatogonial-cell like morphologic changes,ultramicrostructure mature with increasing cell organs,but also express the spermatogonial cell markers,which show that human umbilical cord derived MSCs are capable of differentiating into spermatogonial cell.

Objective To observe clone ability of human umbilical cord mesenchymal stem cells (MSCs) into infertile mouse seminife-rous tubules and the effects of MSCs on reproductive function.Methods Busulfan was used to destroy endogenous spermatogenesis of the recipient mice.To isolate,culture and purify MSCs with adherent method before marked with Brdu and Hoechst 33258 respectively,and then transplanted into the seminiferous tubules by microinjection.The survival of MSCs in recipient testes were evaluated by immunohistochemistry stained for Brdu and Fluorescent microscopy for Hoechst 33258 observation at different times.The diameter of seminiferous tubules was detected with HMIAS-2000 high-definition colored analyzing system for medical pictures.SPSS 13.0 software was used to analyze the data.Results The dosage of Busulfan resulted in 15% death in the mice,the testis of survived mice showed only basilar membrane in seminiferous tubules after 4 weeks.A lot of purified MSCs were obtained at the third generation and transplantation them into mouse seminiferous tubules survive for at least 4 months and appear to migration.The average diameter in experimental groups were higher than those in controls not only on 26 days but also on 120 days(P

OBJECTIVETo investigate the correlation of the onset time of sudden deafness and blood rheology using a large-scale retrospective study.

METHODSOne hundred and ninety-eight patients of sudden deafness were examined with blood rheology and compared with 60 healthy people. Thirty patients were as subjects to study the circadian of blood rheology in order to search whether the blood rheology of morning was the highest. By divided 24 hours into twelve groups, each group contained two hours. Then the 198 patients were separated into twelve groups according to the time of onset. The blood rheology of each group was analyzed and compared with each other to observe which group was the highest.

RESULTSMost blood rheology index of the patients were higher than that of the healthy people. The onset time in most of the patients was during 4 am-8 am, but 45 patients during 4 am-6 am and 40 patients during 6 am-8 am. By analyzing and comparing the blood rheology of each group, the results showed that the blood rheology index in the group of 4 am-8 am was highest. And the blood rheology index of sudden deafness was highest in the morning.

CONCLUSIONSThere was a close relation between blood rheology and sudden deafness. The peak time of onset was 4 am-8 am, and the patients whose blood rheology were the highest should be more predisposed to sudden deafness than the other patients.

Adolescent ; Adult ; Aged ; Case-Control Studies ; Child ; Circadian Rhythm ; Female ; Hearing Loss, Sudden ; blood ; physiopathology ; Hemorheology ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Young Adult

OBJECTIVE: To perform the proteome analysis of conditioned medium prepared from mouse embryonic fibroblast feeder layers by 2-dimensional (2D) electrophoresis and mass spectrometry and to find out the possible differentiation-inhibitory factor in conditioned medium. METHODS: Feeder layers were prepared by 60Co gamma-irradiation on mouse embryonic fibroblast. Insulin-transferrin-sodium selenite supplemented medium was used to culture the feeder layers for 24 hours. The condioned medium prepared from mouse embryonic fibroblast feeder layers were made into powder by lyophilization, the redissolved solution was applied to Sephadex G-50 gel filtration chromatography, and then cold acetone was used to precipitate the proteins in the eluted solution. The protein samples were applied to 2D electrophoresis. The 2D images were analyzed by 2D image analysis software. Selected protein spots were digested by trypsin, analyzed by mass spectrometry, and then searched against the NCBInr batabase using Mascot MS/MS Ions Search. RESULTS: The protein samples extracted from mouse embryonic fibroblast feeder layers conditioned medium could be used for 2D electrophoresis. On 2D images, there were (221+/-67) spots. Most of the proteins were located in the region of MW 20 approximately 70 kD, pI 4 approximately 8. Using mass spectrometry, we preliminarily identified 13 spots: 3 keratins, 3 transferrins, 1 trypsin precursor, 2 unknown proteins (3 spots), 1 connexin 46, 1 beta-galactoside binding protein, and 1 secreted protein, acidic and rich in cysteine. CONCLUSION Conditioned medium prepared from mouse embryonic fibroblast feeder layers contain beta-galactoside binding protein and secreted protein, acidic and rich in cysteine.
Animals ; Cells, Cultured ; Culture Media, Conditioned ; chemistry ; Cysteine ; chemistry ; Electrophoresis, Gel, Two-Dimensional ; Embryo, Mammalian ; Fibroblasts ; cytology ; Galactosides ; chemistry ; Mass Spectrometry ; Mice ; Proteome

OBJECTIVETo investigate the effect of Tauroursodeoxycholic acid (TUDCA) on Taurodeoxycholic acid (TDCA)-induced HepG2 cell apoptosis and to clarify the molecular mechanism of its anti-apoptosis effect of TUDCA.

METHODSMorphologic evaluation of apoptotic cells was performed by Hoechst 33258 staining and electron microscope. DNA fragment was detected by electrophoresis on 1.5% agarose gels. Apoptosis rate was measured by flow cytometry using PI dye. Following incubation of HepG2 cells either with TDCA alone, or coincubation with TUDCA and TDCA, the releasing level of cytochrome c from mitochondria into cytosol was determined by western blot, also the activity of caspase-3, 8, 9.

RESULTSIncubating the cells with 400 micromol/L TDCA for 12 h induced the cells apoptosis significantly. The apoptotic rate decreased from 50.35% +/- 2.20% to 13.78% +/- 0.84% after coincubation with TUDCA, and this anti-apoptotic effect of TUDCA was confirmed by morphological and DNA ladder detection. TUDCA significantly inhibited the release of cytochrome C from mitochondria into cytosol, and the activity of caspase-9, 3 (t > or = 13.00, P < 0.01), especially at 12 h, caspase-3 activity decreased by 54.9% (t = 16.88, P < 0.01) and 52.5%, however it had no obvious effect on the activity of caspase-8 (t = 1.94, P > 0.05).

CONCLUSIONSTUDCA prevents HepG2 cells apoptosis induced by TDCA through modulating mitochondrial membrane stability, inhibiting the release of cytochrome c and the activation of procaspase-9 and 3. Anti-apoptotic mechanism of TUDCA may be considered to be one of the most important reasons that TUDCA exerts significant efficacy in the treatment of cholestatic liver diseases.

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; pathology ; Caspase 3 ; Caspase 9 ; Caspases ; metabolism ; Cytochromes c ; pharmacology ; Humans ; Liver Neoplasms ; pathology ; Taurochenodeoxycholic Acid ; pharmacology ; Taurodeoxycholic Acid ; analogs & derivatives ; pharmacology ; Tumor Cells, Cultured

OBJECTIVETo investigate the effect of chemically synthetic small interfering RNA (siRNA) targeting connective tissue growth factor (CTGF) on the synthesis and secretion of extracellular matrix (ECM) in hepatic stellate cells (HSC).

METHODSChemically synthetic siRNA targeting CTGF was transfected into HSC T6 (an active HSC line in rats) by oligofectamine package, and untreated HSC T6 were used as control. Total RNA and protein of the cells, after their incubation with siRNA for 24, 48 and 72 hours, were extracted, and the supernatants were collected. The expressions of CTGF and type I and III collagen genes were detected by means of reverse transcription-polymerase chain reaction (RT-PCR) and/or Western blot. Contents of hyaluronic acid and type III pro-collagen in the supernatants were determined by radioimmunoassay.

RESULTSThe expression of CTGF at mRNA and protein level and type I and III collagen at mRNA levels were markedly down-regulated in siRNA-transfected HSCs. The contents of hyaluronic acid and type III pro-collagen in the supernatants decreased by 46%+/-7%, 52%+/-7%, 53%+/-7% and 29%+/-18%, 29%+/-7%, 27%+/-5%, compared with those of the blank control at 24, 48 and 72 hours.

CONCLUSIONSChemically synthetic anti-CTGF siRNA can significantly inhibit CTGF gene expression in HSC, and markedly reduce the synthesis and secretion of ECM including type I and III collagen and hyaluronic acid. The siRNA-directed suppression of CTGF gene in HSC was maintained for 72 hours. This suggests that chemically synthetic siRNA may be a potential in preventing and treating liver fibrosis and may have a promising future for development

Cell Line ; Connective Tissue Growth Factor ; Extracellular Matrix ; metabolism ; Gene Targeting ; Humans ; Immediate-Early Proteins ; genetics ; Intercellular Signaling Peptides and Proteins ; genetics ; Liver ; cytology ; metabolism ; RNA, Small Interfering ; genetics

OBJECTIVETo investigate the anti-fibrogenesis property of intraportal vein small interfering RNA (siRNA) injection targeting connective tissue growth factor (CTGF) in a rat model of liver fibrosis induced by carbon tetrachloride (CCl4) and its effect on hepatic stellate cell (HSC) activation.

METHODS24 male rats were randomly divided into four group. rats received CCl4 by subcutaneous injections every three days for 6 consecutive weeks, and meantime they also obtained either siRNA targeting CTGF (as CTGF siRNA group), saline (as model group) or a control siRNA (as control siRNA group) by intraportal vein injection to rats liver at the same approach. Other rats received saline intraportal vein injection for 6 weeks (as normal control group). The expressions of CTGF and a-SMA protein were detected by Western blot. Hepatic histology was evaluated by HE staining and Sirius red staining. The collagen staining areas were measured quantitatively using a computer-aided manipulator with slight modifications. The number of active HSC were evaluated by immunohistochemistry.

RESULTSSix weeks after CCl4 injection, prominent upregulations were observed in the expressions of CTGF and a-SMA protein in saline or control siRNA-treated rats livers. In rats with CTGF siRNA treatment, the protein expressions of CTGF and a-SMA in liver decreased by 95%+/-2% and 86%+/-11% (F=21.234 and 12.473, P<0.01) respectively, the number of active HSC in liver decreased by 76%+/-9% (F=9.179, P<0.01) as compared to the model group. The attenuation of liver fibrosis was also observed in rats with CTGF siRNA treatment.

CONCLUSIONIntraportal vein siRNA injection targeting CTGF could significantly inhibit CTGF gene expression in rats, thereby attenuate liver fibrosis by decreasing the number of active HSCs.

Animals ; Connective Tissue Growth Factor ; genetics ; Gene Silencing ; Hepatic Stellate Cells ; metabolism ; Liver Cirrhosis ; genetics ; metabolism ; pathology ; therapy ; Male ; RNA, Small Interfering ; genetics ; Rats ; Rats, Sprague-Dawley