BACKGROUND:The traditional two-dimensional culture system has been widely used in the in vitro culture of human tissue stem cells,but it cannot really simulate the three-dimensional physiological microenvironment in the body, which is not conducive to the study of the biological behavior of human stem cells. OBJECTIVE: To detect the effect of the bioactivity of Col-Tgel in human hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs)in vitro and in vivo,by constructing a three-dimensional culture system stimulating the physiological microenvironment of the body. METHODS:(1)In vitro co-culture:Green fluorescent protein labeled MSCs(MSCs-GFP)and human umbilical cord blood CD34+cells were co-cultured in Col-Tgel for 3 days (three-dimensional culture group). Human umbilical cord blood CD34+cells were cultured in Col-Tgel for 3 days as single culture group. MSCs-GFP and human umbilical cord blood CD34+cells were co-cultured in Transwell chamber for 3 days as two-dimensional culture group. Human umbilical cord blood CD34+cells were cultured routinely as control group. The percentage of CD34+CD38-CD45RA-CD90+cells in each group was measured by flow cytometry. In situ immunofluorescence staining was used to detect the activity of cells that were co-cultured in Col-Tgel.(2)In vivo transplantation:NOD/SCID mice subjected to 24-hour X-ray irradiation were divided into two groups: in experimental group, MSC-GFP cells were resuspended in Col-Tgel and transplanted into the tibia of NOD/SCID mice; in control group, MSCs-GFP were resuspended in PBS and transplanted into the tibia of NOD/SCID mice. The MSC-GFP growth in the bone marrow was detected by two-photon/confocal microscopy at 3 days post transplantation. RESULTS AND CONCLUSION: (1) After co-culture in Col-Tgel for 3 days, the percentage of CD34+CD38-CD45RA-CD90+cells in the three-dimensional culture group was 2.8 times that of the two-dimensional culture group, indicating that the MSCs significantly promoted the expansion of CD34+CD38-CD45RA-CD90+cells in the Col-Tgel. The percentage of CD34+CD38-CD45RA-CD90+cells in the three-dimensional culture group was increased by 4.5 times compared with the single culture group and increased by 1.5 times compared with the control group. Immunofluorescence staining showed that the cell viability of human MSCs and human umbilical cord blood CD34+cells was not affected after co-cultured in Col-Tgel for 3 days.In the in vivo transplantation experiment,MSC-GFP cells could survive in the medullary cavity.In summary, Col-Tgel provides a new strategy for stem cell culture and in vivo growth by forming a three-dimensional system similar to the physiological environment in vivo.

AIMTo investigate the effects of human urotensin II (hUII) on in vivo pia mater microcirculation in rats.

METHODSAdult SD rats were randomly assigned to the following groups: control, sodium chloride injection (NS), UII(10(-6) mol/L), noradrenaline (NA, 10(-6) mol/L), and UII (10(-6) mol/L) + NA (10(-6) mol/L) groups. For recording of microcirculation images in pia mater, skull windows were performed and mounted on the stage of an intravital microscope equipped with a TV camera. Video images of microcirculation were stored by a video cassette recorder. Temporal changes in internal diameter and microcirculatory velocity of microvessels were measured by computer using the Image Pro software. The blood flow in cerebral tissues were measured with PIMII laser Doppler perfusion Imager (Lisca, Sweden).

RESULTSThe internal diameters of arterioles and venules in control group were (35.4 +/- 3.6) microm and (40.6 +/- 8.5) microm, respectively. In UII group, the arterioles and venules contracted immediately after treated with UII and up to the peak at 1 min, the internal diameters of arterioles and venules were (25.6 +/- 3.4) microm and (23.4 +/- 3.3) microm, respectively (P < 0.05). Both microcirculatory velocity in arterioles and venules had no significant changes in UII group (P > 0.05). The blood flow in meninges increased 1 min after treated with UII and up to high peak at 5 min (3.5 +/- 0.4 perfusion unit vs. control 2.3 +/- 0.6, P < 0.05).

CONCLUSIONhUII can contract microvessels in pia mater of rats and increase microcirculatory blood perfusion to cerebral tissue involved.

Animals ; Cerebrovascular Circulation ; drug effects ; Humans ; Male ; Microcirculation ; drug effects ; Rats ; Rats, Sprague-Dawley ; Urotensins ; pharmacology

OBJECTIVETo investigate the association of p15 and pl6 genes deletion and STKI5 gene overexpression in primary hepatocellular carcinoma (PHC).

METHODSThe carcinoma tissue and the adjacent normal tissue were taken from 30 PHC patients during operations who had had neither chemotherapy nor radiotherapy preoperatively. DNA was extracted from the tissues and PCR was used to determine the homozygous deletion of p15 exon2 (pl5E2) and pl6 exon 2 (pl6E2). RNA was extracted, cDNA was synthesized by RT-PCR, and the expression of STKI5 gene was tested by PCR. Beta-actin was used as an internal control. Average density value (ADV) of STK15 gene and that of beta-actin gene were determined in both carcinoma tissue and the adjacent normal tissue.

RESULTSThe rate of p15E2 deletion was 13.3% (4/30) and the rate of p16E2 deletion was 16.7% (5/30) in the carcinoma tissue. The p15E2 and pl6E2 co-deletion rate was 6.7% (2/30). In 19 of the 30 cases (63.3%) the expression of STK15 gene in carcinoma tissue was higher than that in the adjacent normal tissue. The ratio of ADV of STK15 gene to ADV of beta-actin gene (1.53+/-0.31) in the carcinoma tissue was significantly higher than that (0.91+/-0.25) in the paired adjacent normal tissue (t = 2.86).

CONCLUSIONThe homozygous deletion of p15E2 and p16E2 and overexpression of STKI5 gene may play a role in the oncogenesis and malignant progression of PHC.

Aurora Kinase A ; Aurora Kinases ; Carcinoma, Hepatocellular ; genetics ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; Female ; Gene Deletion ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms ; genetics ; Male ; Protein-Serine-Threonine Kinases ; biosynthesis ; genetics

OBJECTIVETo seek a safe, efficient, and cost-effective technique for local thermo-ablation of hepatic cancer.

METHODSThe livers from 16 healthy rabbits were thermocoagulated by diode-laser with scanner fiber tip, 6 w for 10 mins. At the same time, the temperatures were measured at 0, 5 and 10 mm from laser tip. The pre-thermocoagulative liver function was compared with that of 7 days post-thermocoagulation. The pathologic changes were also observed 1 month after laser thermocoagulation.

RESULTSAll the rabbits survived and hepatic tissue temperatures at 0, 5, 10 mm from laser tip reached 96.39 degrees C +/- 3.97 degrees C, 60.79 degrees C +/- 6.21 degrees C, 46.10 degrees C +/- 4.58 degrees C respectively after 10 minutes of thermocoagulation. There were no significant differences in liver function parameters between rabbits of pre-laser thermocoagulation and of post-laser thermocoagulation. Thermocoagulated necrosis of liver tissue with surrounding fibrosis in a diameter of 26.0 mm was formed. Light microscopy revealed coagulative necrosis in the center of the coagulated area without surviving hepatic cells.

CONCLUSIONThe hepatic tissue can be coagulated safely and effectively by diode-laser with scanner fibertip, and such a technique may provide a new method for the treatment of hepatic carcinoma.

Animals ; Female ; Laser Coagulation ; methods ; Liver Neoplasms ; pathology ; surgery ; Male ; Rabbits

Calreticulin (CRT) is an essential Ca(2+)-binding chaperone existing in endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR), and is involved in intracellular Ca(2+) homeostasis and protein folding. Ischemic postconditioning (I-postC), a newly discovered endogenous protective phenomenon, induces CRT up-regulation. The present study aimed to investigate the cardioprotective mechanism of CRT up-regulation induced by hypoxic postconditioning (H-postC). Primary cultured neonatal rat cardiomyocytes were exposed to 2 h of hypoxia followed by 24 h of reoxygenation. Postconditioning was carried out by two cycles of 10 min of reoxygenation and 20 min of rehypoxia after 2 h of hypoxia. Antisense oligodeoxynucleotides (AS-ODNs) were used to inhibit CRT expression 36 h before hypoxia. Cardiomyocytes were randomly divided into 6 groups as follows (n=4): control, hypoxia/reoxygenation (H/R), H-postC, AS, AS + H/R, and AS + H-postC. Morphological studies, lactate dehydrogenase (LDH) activity assay in culture medium, and flow cytometry were used to detect cardiomyocyte necrosis and apoptosis. Intracellular Ca(2+) concentration was detected by fluorescent Fluo-3/AM staining through laser confocal microscope, and p-nitrophenyl phosphate (PNPP) was used as substrate to measure calcineurin (CaN) activity. The expression of CRT, CaN, nuclear factor kappa B (NFκB) and apoptosis-related proteins, such as Bcl-2, Bax and C/EBP homologous protein (CHOP) were detected by Western blot. The results were as follows. (1) H-postC protected neonatal cardiomyocytes from H/R injury. Compared with H/R group, cell survival rate increased by 17.1%, apoptotic rate and LDH leakage decreased by 6.67% and 27.9% in H-postC group, respectively (P<0.05). (2) H-postC induced mild up-regulation of CRT expression. Inhibition of CRT by AS-ODNs attenuated the cardioprotection of H-postC partly. Compared with H-postC group, cell survival rate decreased by 8.98%, and apoptotic rate and LDH leakage increased by 1.74% and 13.6% in AS + H-postC group, respectively (P<0.05), but intracellular Ca(2+) concentration, CaN activity, and expression of CaN and NFκB did not change significantly (P>0.05), suggesting that CRT participates in endogenous protection, not through Ca(2+)-CaN pathway. (3) H-postC inhibited the expression of pro-apoptosis proteins such as Bax and CHOP, but induced up-regulation of anti-apoptosis protein Bcl-2. Inhibition of CRT by AS-ODNs partly inhibited the changes in apoptosis-related proteins expression induced by H-postC, suggesting that CRT participates in the anti-apoptosis effect of H-postC through regulating expression of apoptosis-related proteins. These results indicate that CRT up-regulation induced by H-postC is involved in the cardioprotection through regulating expression of apoptosis-related proteins, not through Ca(2+)-CaN pathway in neonatal cardiomyocytes.
Animals ; Apoptosis ; Calcineurin ; metabolism ; Calreticulin ; metabolism ; Cell Hypoxia ; Cell Survival ; Cells, Cultured ; Ischemic Postconditioning ; Myocytes, Cardiac ; metabolism ; Oxygen ; metabolism ; Rats ; Up-Regulation

Serum pharmacochemistry of traditional Chinese medicine (TCM) is designed to screen the efficacy material base of TCMs from the constituents absorbed into the blood after oral administration. The theory and method is in accordance with the effect characteristics of TCMs, and reflects the interaction between the body and the drugs, has become an effective pathway for researching the efficacy material base of TCMs which has been recognized and used widely. In the paper, the previous research contents and methods of the serum pharmacochemistry of TCM were reviewed, and on the basis of the further validity of the special administration form of the TCM formula and the corresponding property to TCM syndrome, the new strategy of serum pharmacochemistry of TCM integrating the metabonomics technologies was put forward. According to the strategy, we take the biological characters of TCM syndrome as a research starting point, taking TCM formula as object, using the metabolic biomarkers of syndromes or disease to evaluate the therapeutic effect of formula and screen the compounds of TCMs in serum which are highly correlated with the metabolic biomarkers through the correlation analysis, and by further biological validation to finally confirm the efficacy material basis of TCMs. Integrating with the systems biology technologies, the theory and method of serum pharmacochemistry of TCM will further develop, and open a new chapter in the interpretation of the theory of TCM.
Animals ; Drug Therapy ; trends ; Drugs, Chinese Herbal ; chemistry ; pharmacokinetics ; Humans ; Metabolomics ; Serum ; chemistry

OBJECTIVETo study the metabolic chemistry and pharmaco-dynamics characters of ligan from seeds of Arctium lappa.

METHODHPLC method was used in the study. The analysis was carried out on C18 column. The mobile phase was CH3CN-0.05% H3PO4 (36:64) with flow-rate at 0.6 mL x min(-1) and wave-length of 210 nm. The column temperature was kept at 25 degrees C.

RESULTThe results indicated that the ligan was detected in plasma and the main organs 5 min after po. The main metabolic production in plasma was arctigenin. In addition, arctigenin and an unknown product were found in metabolic production in the organs.

CONCLUSIONThe method was stable,simple and reproducible. It can be used to determine the metabolic product of the ligan. The metabolic chemistry of ligan in plasma was obviously different from that in the main organs.

Animals ; Arctium ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Furans ; blood ; metabolism ; Glucosides ; blood ; metabolism ; Lignans ; blood ; isolation & purification ; metabolism ; pharmacokinetics ; Liver ; metabolism ; Male ; Mice ; Plants, Medicinal ; chemistry ; Reproducibility of Results ; Seeds ; chemistry ; Tissue Distribution

AIMTo establish an HPLC-fluorescent spectrometric method for the determination of mexiletine hydrochloride in plasma after derivatization with fluram.

METHODSFluram acetone solution was added to the deproteinized plasma with acetone to obtain the derivative of mexiletine. The HPLC method was performed on a column of Allitima C18 (150 mm x 4.6 mm, 5 microns) with the mobile phase of methanol-water-diethylamine-phosphoric acid buffer (2.4 mol.L-1, pH 4.0) (70:28:2), and the detective wavelength were set at Ex 392 nm and Em 480 nm.

RESULTSMexiletine has a liner range over the concentration range from 0.100-6.400 mg.L-1. The lowest detectable concentration of this method was 5 micrograms.L-1 (S/N > or = 4). The intra-day and inter-day RSDs were 1.34%-5.31%, respectively.

CONCLUSIONThis method is simple, selective and can be used for therapeutic drug monitoring (TDM) and pharmacokinetic studies of mexiletine.

Anti-Arrhythmia Agents ; blood ; pharmacokinetics ; Chromatography, High Pressure Liquid ; methods ; Fluorescamine ; chemistry ; Humans ; Mexiletine ; blood ; pharmacokinetics

OBJECTIVETo investigate the possibility of adipose-derived stromal cells (ADSCs) transfeced by adenovirus containing human bone morphogenetic protein-2 (Ad-hBMP-2) gene and their osteogenic potential.

METHODSADSCs were obtained from inguinal fat tissue of 4 weeks old SD rats. After exposure to adenovirus containing green fluorescent protein(Ad-GFP), fluorescent microscope was used to observe gene transfection effect once 12 hours. After transfected with Ad-hBMP-2, cytochemistry, immmucytochemistry and Western blot were used to examine the expression of alkaline phosphatase (ALP), osteocalcin (OC) and hBMP-2.

RESULTSAfter exposed to Ad-GFP 12 hours, 52% ADSCs were observed being transfected and 48 hours later reached 95%. The double number time belonged after transfecting with Ad-hBMP-2, and cytochemistry, immucytochemistry and Western blot examines indicated positive results of ALP, OC, hBMP-2 after 48 hours.

CONCLUSIONAdipose tissue contains abundant ADSCs which could be transfected as gene vectors by adenovirus, ADSCs transfected with Ad-hBMP-2 can convert to ostoeblasts, and can act as a kind of seed cells for osteo-tissue engineering.

Adenoviridae ; Adipocytes ; Adipose Tissue ; Animals ; Bone Morphogenetic Protein 2 ; Cells, Cultured ; Genetic Vectors ; Humans ; Rats ; Rats, Sprague-Dawley ; Stromal Cells ; Tissue Engineering ; Transfection

OBJECTIVETo analyze and identify fatty acids in the seeds of Sterculia lychnophora.

METHODThe compositions was isolated and determined by GC-MS technique, and area normalization method was used to make quantitative analyze of the content of compositions.

RESULTS21 Fatty acids and 5 other compositions were isolated and determined.

CONCLUSIONThe major fatty acids are 9,12(Z,Z)-octadecadienoic acid(37.96%), hexadecanoic acid(24.77%), 9-(Z)-octadecenoic acid(19.77%) and octadecanoic acid(5.01%).

Fatty Acids, Nonesterified ; chemistry ; isolation & purification ; Fatty Acids, Unsaturated ; analysis ; Gas Chromatography-Mass Spectrometry ; Palmitic Acid ; analysis ; Plants, Medicinal ; chemistry ; Seeds ; chemistry ; Sterculia ; chemistry