This study was aimed to explore the methodology regarding culture, proliferation and purification of adipose-derived stromal cells (ADSCs), and to study their biological characteristics. ADSCs were obtained using type I collagenase digestion method. Cell growth was observed, and cell viability were detected under different digestion period by MTT. The ADSCs were then identified and induced. The results showed that adherent cells digested by type I collagenase for 60 min had a strong proliferation capability. After the induction of different inducers these adherent cells could differentiate into nerve cells and fat cells. The best digestion period was proved to be of 60 minutes in the experiment. The results indicate that stem cells with multilineage differentiation ability could be separated from adipose tissue, namely ADSCs.
Adipocytes ; cytology ; Adipose Tissue ; cytology ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Neurons ; cytology ; Primary Cell Culture ; Stem Cells ; cytology ; Stromal Cells ; cytology

0.05). Conclusion There are MA and mutation of p53 gene in T-cell lymphoma though no significant correlation between them. But, MA positive cases might experience high mutation of p53 gene in T-cell lymphoma.

Objective To establish a method to determine six kinds of PAEs in human serum simultaneously,and on this basis to understand the level of PAEs in human body.Methods Ultrasonic extraction,gas chromatography-mass spectrometry-selected ion monitoring(GC-MS-SIM) detection and quantitative analysis based on internal standard were used to detect six kinds of PAEs in human serum simultaneously.Finally this method was used in fifty human serum samples.Results The results showed that this method had a good linear relation in the range from 10 to 1 000 ng/ml,and the correlation coefficients of standard curve were higher than 0.999.The average recovery rate and the relative standard deviation(RSD,n=6) of the target compounds in standard-addition blank was 94.5% and 4.4% respectively,the lower limits of detection for DEP,DMP,DBP,BBP,DEHP,DNOP were 0.31,0.85,0.90,0.63,0.88,0.41 ng/ml respectively,and the recovery rates of all samples ranged from 67.98% to 110.8%.Conclusion This method has good recovery rate,reproducibility,lower limit of detection and can be used for the determination of many kinds of PAEs substances in human serum.

Objective To investigate the effects of cyclooxygenase-2 (COX-2) selective inhibitor combined with 5-lipoxygenase (5-LOX) inhibitor docebenone (AA861) on cell proliferation and migration of human colon cancer cell line HT-29. Methods Cultured in vitro of HT-29 cells in high metastatic colon cancer, A A861 and celecoxib on colon cancer cell drugs for 48 h, the cell proliferation was assayed by MTT; the migration of cell was detected by Transwell; immunofluorescence staining of intracellular changes in ICAM-1 protein, RT-PCR detect the gene expression of ICAM-1 and VEGF. Results MTT and Transwell experiments showed that the inhibition on celecoxib and AA861 on colon cancer was dose-dependent and time-dependent. And the inhibition of AA861 and celecoxib 100 μmol/L alone on colon cancer cells proliferation were 43.2 % and 42.8%, and when the two drugs 100 μmol/L combined on colon cancer cells the inhibition was 53.8%, and the difference between alone group and combined group was statistical (P ＜0.001). The inhibition of AA861 and celecoxib 100 μmol/L alone on colon cancer cells migration were 32.0 % and 29.3%, and when the two drugs 100 μ,mol/L combined on colon cancer cells the inhibition is 57.8 %, and the difference of between alone group and combined group was statistical (P ＜0.001). Immunefluorescence staining and RT-PCR results suggested that the two drugs can inhibit ICAM-1 and VEGF protein and gene expression, and when the two drugs combined, a stronger inhibition effect appeared than used alone (P＜0.001). Conclusion Low-dose celecoxib and AA861 combined has a synergistic inhibited effect on colon cancer cells invasion and metastasis, and the mechanism relates with the VEGF and ICAM-1 expression.

Objective To study the effect of 5-lipoxygenase(5-LOX) inhibitor nordihyroguaiaretic acid (NDGA) combined the selective cyclooxygenase-2 (COX-2) inhibitor Celecoxib on the apoptosis of human colon carcinoma cell line HT-29. Methods Different concentration of NDGA and Celecoxib combinations were used to process cancer cell, and thiazolyl blue tetrazlium bromide (MTT) and phase contrast microscope and Annexin V/PI fluorescence staining and reverse transcription polymerase chain reaction (RT-PCR) were used to study the proliferation inhibited effect and apoptosis induced effect caused by combination of NDGA combined Celecoxib. Results MTT results showed that the viability of NDGA group, Celecoxib group and the group of NDGA combined Celecoxib (0.432±0.024,0.425±0.013,0.303±0.014 vs 0.693±0.018,t＝18.79,25.75,37.64,P＜0.01) was obviously lower than control group. The group of NDGA combined Celecoxib was significantly lower than NDGA group or Celecoxib group (t＝10.21, 14.14,P＜0.01). Under inverted phase contrast microscope, cell morphology significantly changed, and the group of NDGA combined Celecoxib changed most obviously. Apoptosis was observed by laser scanning confocal microscope (LSM) after NDGA and Celecoxib were used to process the HT-29. RT-PCR showed that up-regulation of Caspase-3 after treatment, and the combination of two drugs increased the most. Conclusions NDGA combined Celecoxib inhibited proliferation and induced apoptosis in human colon carcinoma cell line HT-29, and combined therapy had better effect than that of any drug used separate-ly. The mechanism may be associated with up-regulation of Caspase-3.

Objective To observe the expression of microtubule-associated protein 1 light chain 3 II (LC3II) and microtubule-associated protein 2 (MAP-2) in CA1 area of hippocampus in vascular dementia rats. Methods 48 Sprague-Dawley rats were randomly divided into sham group and model group. Meanwhile, each group was further divided into 1, 2, 4 and 8 weeks subgroups (n=6). Vascular dementia model was established by blocking four vessels. The expressions of LC3II and MAP-2 protein were detected with immunohistochemistry in the CA1 area of hippocampus. Results The expression of LC3II significantly increased, and the expression of MAP-2 decreased in the model group compared with the sham group at every time point (P<0.001). The expression of LC3II was negatively correlated with MAP-2 at every time point in the model group (r=-0.723, P<0.05). Conclusion It may play an important role for the occurrence and development of vascular dementia that the expression of LC3II increased and MAP-2 descreased in CA1 area of hippocampus in rats.

To investigate the association of HLA-A0205 and HLA-A30 with latent autoimmune diabetes mellius in adults (LADA) in Chengdu Hans, 121 subjects (41 cases of LADA, 40 cases of T2DM, and 40 normal controls) were enrolled in the study. The frequencies of HLA-A0205 and HLA-A30 were determined by nested PCR-SSP and direct sequencing, respectively. The allele frequencies of patient groups and of normal controls were compared by chi-square test using SPSS 11.0 (alpha = 0.05). Hardy-Weinberg equilibrium was tested with use of software HWE (alpha = 0.05). Data from the subjects showed: HLA-A0205 was present in 1 patient with LADA and in 1 normal control (2.44% and 2.5%, respectively), HLA-A30 was present in 2 patients with LADA, in 2 patients with T2DM and in 1 normal control (4.87%, 5.0% and 2.5%, respectively). There was no statistically significant difference between the allele frequencies of the three groups. These results suggest that HLA-A0205, HLA-A30 may not be related to LADA in Chengdu Hans. Yet, further studies with larger sample size may be needed to warrant this conclusion.
Adult ; Age of Onset ; Autoimmune Diseases ; genetics ; Case-Control Studies ; China ; Diabetes Mellitus ; genetics ; Female ; Genotype ; HLA-A Antigens ; genetics ; Humans ; Male ; Polymerase Chain Reaction ; methods ; Polymorphism, Single-Stranded Conformational

[Abstract ] Objective The purpose of this study was to observe the effects of dl-3n-butylphthalide (NBP) sodium chloride injection post-processing on the expressions of X-inhibitor of apoptosis (XIAP) and Bcl-2/adenovirus E1B19kDa interacting protein 3 (BNIP3) in the hippocampus CA1 neurons of focal cerebral ischemia reperfusion (IR) rats, and to investigate the brain-protection mechanisms of NBP. Methods A total of65 adult male Sprague-Dawley rats were divided into five groups of equal number, sham op-eration, IR, and low-,medium -and high-dose NBP, according to the random number table. The IR models were established by modified ligation of the middle cerebral artery.The animals in the NBP groups received intra-abdominal injection of NBP at 2, 4, and 6 mg/kg, re-spectively.All the rats were sacrificed at 24 hours after modeling,neurological scores obtained by Zea Longa, the volume of infarction measured by TTC staining, the number of apoptotic cells counted by TUNEL, and the expressions of XIAP and BNIP3 detected by immunohistochemistry and real-time PCR. Results The neural function defect scores were markedly lower in low-, medium-and high-dose NBP groups than in IR model rats (P<0.05), with statis-tically significant differences among the three dose groups (P<0.05).The volume of infarction was remarkably higher in the low-dose than in the medium-and high-dose NBP groups (P<0.05).The number of apoptotic cells in the hippocampus CA1 neurons was de-creased in the NBP groups as compared with the IR models (P<0.05).The XIAP-and BNIP3-positive cells were significantly in-creased in the IR model rats as compared with the sham operation group ([22.31 ±0.94] and [60.13 ±2.59]/HP vs [3.07 ±1.43] and [5.78 ±0.44]/HP, P<0.05).In comparison with the IR models, the NBP-treated rats showed a progressively increased number of XIAP-positive cells in low-, medium-, and high-dose groups ([28.70 ±1.18], [32.79 ±0.88], and [37.01 ±1.24]/HP) (P<0.05) but a decreased number of BNIP3-positive cells in the three dose groups ([52.07 ±1.02], [40.30 ±2.00], and [31.04 ± 0.43]/HP) (P<0.05).Similarly, the expression of XIAP mRNA was up-regulated while that of BNIP3 mRNA down-regulated in the NBP treatment groups as compared with the IR model rats, both in a dose-dependent manner (P<0.05). Conclusion NBP post-processing has a neuroprotective effect on IR rats, which is associated with its impact on the expressions of XIAP and BNIP3.

Objective To observe the effects of autophagy on the expression of synaptic plasticity related protein, growth-associated pro-tein-43 (GAP-43) and microtubule associated protein-2 (MAP-2), in CA1 area of hippocampus of vascular dementia rats. Methods Nine-ty-six healthy male Sprague-Dawley rats were randomly divided into sham group, vascular dementia model group (VD group), autophagy in-hibitor 3-methyl adenine preconditioning group (3-MA group) and autophagy agonist rapamycin preconditioning group (Rap group). Each group was divided randomly into subgroups of one week, two weeks, four weeks and eight weeks after modeling, six rats in each group. The vascular dementia rat model was established with modified Pulsineli's four-vessel occlusion. The expression of GAP-43 and MAP-2 in CA1 area of hippocampus were detected with immunohistochemistry. Results Compared with the sham group, the expression of GAP-43 protein increased, and the expression of MAP-2 protein decreased at every time point in VD group (P<0.01). Compared with VD group, the expres-sion of both GAP-43 and MAP-2 increased in 3-MA group (P<0.05), and decreased in Rap group (P<0.05). Conclusion Autophagy may in-hibit the expression of synaptic plasticity related protein, GAP-43 and MAP-2, in CA1 area of hippocampus in vascular dementia rats, indi-cating inhibition of autophagy may promote synaptic remodeling.

Objective The metabolism and excretion of isochlorogenic acid B in rats were investigated by UHPLC-MS.Methods Feces,urine and plasma were individually collected before and at different time points after administration of 20 mg·kg-1.Post-prepared samples were analyzed by UHPLC-MS.Results According to the retention times,m/z,characteristic fragment ions and related literature,a total of 22 metabolites were detected,of which 18 metabolites were present in rat feces,3 metabolites in urine and one metabolite in plasma.The main metabolic pathways contain hydrolysis,hydrogenation,methylation,sulfation and so on.The cumulative excretion of isochlorogenic acid B and its main metabolite chlorogenic acid in feces and urine was further performed.Conclusion The metabolism and excretion of isochlorogenic acid B in rats were explored to provide experimental basis for its further research and development.