BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are in advantages of easy collection and amplification in vitro, but the culture and induction in vitro still need to be modified. OBJECTIVE: To investigate the differentiated potency of BMSCs into ostcoblasts by modified in vitro culture methods and inductor matching. DESIGN: Observational study. SETTING: Department of Medical Laboratory, General Hospital of Guangzhou Military Area Command of Chinese PLA. MATERIALS: This study was performed in the Stem Cell Tissue Engineering Laboratory, Department of Medical Laboratory, Genera Hospital of Guangzhou Military Area Command of Chinese PLA from July 2004 to June 2006. Twenty adult SD rats of clean grade, irrespective of gender, weighing 140-180 g were provided by Animal Experimental Center, General Hospital of Guangzhou Military Area Command of Chinese PLA. The experimental animals were disposed according to ethical criteria. Β-sodium glycerophosphate, dexamethasone, and vitamin C were provided by Sigma Company, USA; goat-anti-rat osteocalcin antibody by DSL Company, USA; S-P immunohistochemical kit by Maixin Biotechnology Developing Co., Ltd., Fujian. METHODS: Cells were cultured and induced into osteoblasts by modified methods. Separation and culture of BMSCs: By anesthesia, bone marrow of bilateral femur and tibia was separated to prepare single cell suspension; subsequently, the suspension was inoculated in culture bottle, and the culture media was semi-quantitatively changed after 48 and 96 hours in order to get rid of non-adherent hematopoietic cells. The liquid was changed every three days to further get rid of non-adherent cells. At 80% cell confluence, the medium was digested with 0.25% trypsin and cells were subcultured in the ratio of 1:2. MSCs in the second generation were inoculated in 6-well culture plate and glass fiat plate; after 48 hours, basic culture fluid was removed. Differentiation of BMSCs into osteoblasts: Subcultured BMSCs differentiated into osteoblasts induced by dexamethasone (10 mmol/L), β-sodium glycerophosphate (10-7 mol/L), and vitamin C (50 mg/L). MAIN OUTCOME MEASURES: Ten days after differentiation by modified culture methods and inductor matching, alkaline phosphatase activity was measured with Ca-Co technique. Twelve days after culture, osteocalcin secretion was detected with immunohistochemical method. Two weeks after culture, cell mineralization was detected with Von kossa staining. RESULTS: Alkaline phosphatase activity: Alkaline phosphatase staining of cells was apparent; gray-black particles or massive precipitations were observed in cytoplasm after positive reaction; regions expressing alkaline phosphatase activity were brown-black. Osteocalcin secretion: Osteocalcin was apparently positive; nucleus was blue; cytoplasm was brown. Cell mineralization: Induced cells grew layer by layer, and adiaphanous nodus was observed at the same time. Black mineralized nodus granules in various sizes were observed after Von kossa staining, and this suggested that mineralized apposition occurred. CONCLUSION: BMSCs may be successfully cultured and induced into osteoblasts by modified culture method.

BACKGROUND:With the potential of multi-directional differentiation and high proliferation, bone marrow mesenchymal stem cells (BMSCs) have wide application prospect in tissue engineering. OBJECTIVE:To investigate changes in cells and bioactivity in the differentiation from BMSCs into osteoblasts. DESIGN, TIME AND SETTING:A randomized control experiment was performed at the Laboratory of Stem Cells and Tissue Engineering, Department of Medical Experiment, General Hospital of Guangzhou Military Area Command of Chinese PLA from July 2004 to June 2006. MATERIALS:Twenty adult SD rats of both genders were used for bone marrow collection. METHODS:Rats were equally and randomly divided into an experimental group and a control group. BMSCs were isolated from adult rats by modified bone marrow culture method. BMSCs were inoculated in basic medium containing 10% fetal bovine serum, high glucose DMEM, 100 U/L penicillin, 100 U/L streptomycin and 2 mmol/L glutamine in the control group. BMSCs were inoculated in conditioned medium (basic medium, 10 mmol/L β-glycerophosphoric sodium, 10-7 mol/L dexamethasone, 50 mg/L vitamin C). Cell slide was prepared. MAIN OUTCOME MEASURES:Inverted microscope and transmission electron microscope were used to observe cell appearance. Gomori modified calcium-cobalt method was applied to do alkaline phosphatase staining. Immunocytochemistry was employed to measure osteocalcin expression. RESULTS:Forty-eight hours after inoculation, a mass of BMSCs adhered to a wall. Seventy-two hours later, BMSCs proliferated and well grew. Seven to nine days later, cells were grown to 80% confluency. Transmission electron microscope showed BMSCs with big cell nucleus and immature appearance. After in vitro osteoblast induction, BMSCs proliferated, aggregated, had node and mineralized. One week later, alkaline phosphatase activities were expressed in BMSCs. Two weeks later, osteocalcin expression was detected. CONCLUSION:After one week of in vitro osteogenic induction, BMSCs enter the process of osteoblast transformation, remain proliferative activities, and can be used as seed cells in bone tissue engineering

Objective To study the levels of IL 6 production in peripheral blood mononuclear cells (PBMC) in 25 patients with inactive and active (include mild、 moderate and severe) ulcerative colitis(UC) and age, sex matched 20 healthy subjects.Methods Peripheral blood monocytes (PBMC) were stimulated with PHA for 48 hours to induce IL 6 production. IL 6 content in the culture medium was assayed by using B9 cells. Results IL 6 production was significantly increased in PBMC from active UC. There was an increasing tendency with severity of disease. But no significant difference was found between the extent of the lesious. Conclusions The levels of IL 6 production in PBMC can be an indicator of the activity of UC.

Adult ; Female ; Humans ; Intestinal Neoplasms ; Male ; Middle Aged ; Sarcoma, Myeloid

Objective To investigate an association between Toll-like receptor(TLR)4 Asp299Gly or CD14 C-260T polymorphisms and colorectal cancer in Chinese patients.Methods By a method of polymerase chain reaction-based restriction fragment length polymorphism(PCR-RFLP), TLR4 Asp299Gly and CD14 C-260T polymorphisms in unrelated 110 patients with colorectal cancer and 160 healthy controls in Chinese Han race population were genotyped.Results Significant differences of CD14 genotypes were found between healthy controls and the patients with colorectal cancer.The fre- quency of the CC genotype in healthy controls was extremely lower than that in the group of colorectal cancer(15.6% vs.31.8%,P=0.0027,OR=0.3968,95%CI:0.2209-0.7129)while the frequency of the CT genotype in healthy controls was significantly higher than that in the group of colorectal cancer (48.1% vs.30.9%,P=0.0056,OR=2.074,95%CI:1.246-3.452).No TLR4 Asp299Gly mutant genotype was detected in any patients and healthy controls in Chinese Han population.Conclusion The polymorphism of CD14 C-260T but not TLR4 Asp299Gly is associated with colorectal cancer in Chinese patients.

Objective To investigate the regulation effect of promoter methylation of deathassociated protein kinase (DAPK) on mRNA and protein expression of DAPK in tissue of primary gastric cancer (GC). Methods The cancerous and noncancerous samples from 62 patients with GC were determined by RT-PCR for mRNA expression of DAPK. The DAPK promoter methylation was detected by methylation-specific PCR. The protein expression of DAPK in 34 patients with methylation was determined by Western blot. Results mRNA and protein expre.ssions of DAPK in cancerous tissues were reduced significantly compared to noncancerous tissues (0. 2863d±0. 2027 vs 0. 57364±0. 1968,0. 2616±0. 0913 vs 0. 65294±0. 1808, P＜0.01). Methylation frequency of DAPK in cancerous tissues was higher than that in noncancerous tissues (54.8% vs 17.7%, P＜0.01). Furthermore, DAPK mRNA expression was decreased in methylation group compared to unmethylation group (0.1399±0. 0835 vs 0. 46404±0. 1569, P＜0. 01). Moreover, a significant correlation was demonstrated between the TNM stage and DAPK promoter methylation (P = 0. 04). Conclusion Expression of DAPK is down-regulated in cancerous tissues at mRNA and protein levels. Low expression of DAPK is associated with hypermethylation of the promoter of DAPK gene.

Objective To estimate reliability of magnetic resonance elastography (MRE) in measuring liver stiffness of volunteers and patients with chronic liver disease and to assess inter-rater consistency.Methods MRE was performed on a 3.0 T scanner in all subjects,including 24 volunteers (control group) and 64 patients with liver disease (chronic liver disease group).Liver stiffness was measured blindly by two raters.The pathological fibrosis score was applied as a standard reference for liver fibrosis in 22 patients.The intraclass correlation coefficient (ICC) was used to evaluate inter-rater reliability.The differences of liver stiffness between two groups were evaluated using non-parametric MannWhitney U test.Spearman analysis was used to analyze the correlation between fibrosis stages and liver stiffness.Results The intraclass correlation coefficient of liver stiffness was perfect (ICC =0.99,P ＜ 0.01)between two raters.There was significant difference of mean stiffness between control group and patient group (U =90.5,P ＜0.01) with(2.35 ±0.34) kPa and(4.17 ± 0.47) kPa,respectively.The correlation between fibrosis stage (3,3,5,5 and 6 patients in fibrosis stage S0,S1,S2,S3 and S4) and stiffness (2.13,3.25,3.82,5.45 and 7.35 kPa) was very strong (r =0.96,P ＜0.01).Conclusion MRE is a reliable and promising tool to measure liver stiffness and to assess liver fibrosis.

Adult ; Agriculture ; Antibody Formation ; Female ; Humans ; Immunity, Cellular ; Male ; Middle Aged ; Occupational Exposure

Through retrieval of all medical journals of CNKI and VIP from Jan. 1996 to Oct. 2012, ninety-nine articles were selected and analyzed. The result shows that reliable effect can be found in pain relieving, especially in pain of muscles and soft tissues. In mechanism research, action mechanism of Fu's subcutaneous needling (FSN) on pain relieving is expounded through theory of traditional medicine and modern medicine. Although the effect of FSN on pain relieving has been confirmed by numerous clinical trials, it is still lack of explanation on mechanism revealing. The further studies should focus on mechanism expounding, improving research methods and selecting more objective and reasonable evaluation system. Therefore, the effectiveness and scientificalness of FSN can be further enhanced.
Acupuncture Analgesia ; instrumentation ; methods ; Acupuncture Therapy ; instrumentation ; methods ; Humans ; Pain Management ; instrumentation ; methods ; Randomized Controlled Trials as Topic

Objective To determine the frequency of mutations in PINK1 in Chinese Han people with sporadic early-onset Parkinsonism (EOP). Methods DNA sequencing was used to detect point mutations and small deletions/insertions, and quantitative real-time PCR was carried out to detect deletions/insertions and rearrangements in 149 patients and 150 healthy controls. Results Four heterozygous mutations in PINK1 were identified, including 3 missense mutations (c.832C>G, c. 938C>T, c.1 220G>A) and ex 3-8 del. A novel single nucleotide polymorphism (SNP) c.899+18G>A and 14 reported SNPs were identified. Chi-square test showed that c.189C> T and c.960-5G﹥A had significant difference in the genotype frequencies and allele frequencies between the patients and the controls (for c.189C>T genotype χ2=21.244，P<0.0001; T allele χ2=24.353，P<0.0001, and for c.960-5G﹥A genotype's χ2=6.524，P =0.038; A allele χ2=6.725，P=0.0095). Conclusion About 3.35% Chinese Han patients with EOP carry mutations in PINK1. Two SNPs c.189C>T and c.960-5G>A may contribute to the risk of EOP in Chinese Han people.