Extremities ; Humans ; Joint Prosthesis

To establish a method for quantitative measurement of phagocytosis, the phagocytic process of apoptotic granulosa cells by monocytes was imitated in vitro. Monocytes and granulosa cells were isolated from Kunming mice and cultured. Granulosa cells were induced to apoptosis by garlic, and then co-cultured with monocytes. At different time points (1 h, 2 h, 3 h, 4 h, 5 h), co-cultured cells were observed by microscope after Wright's staining. The results showed that at the beginning of morphological changes in apoptotic granulosa cells, monocytes captured the apoptotic cells. Meanwhile, the apoptosis of granulosa cells were progressing. Debris was found in phagocytic vacuole. At the point of 3 h after co-culture, the ratio of monocytes which attached to apoptotic granulosa cells to those which engulfed the apoptotic cells was close to one. Namely, half of monocytes were in the state of recognition and half were in the state of engulfment, and this time point was named as 'half phagocytic period'. Regression analysis showed that the equation of linear regression was y = -0.247x +1.644 (y represents Attachment/Engulfment ratio, x represents co-culture time), R(2)=0.912, F=31.095, P=0.011 (<0.05), T= -5.576, P=0.011 (<0.05). In conclusion, the present mode of phagocytosis in vitro can be used as a method to quantitatively assay some effective factors such as medicines which could enhance or restrain phagocytosis.
Animals ; Apoptosis ; Coculture Techniques ; Female ; Granulosa Cells ; cytology ; Mice ; Monocytes ; cytology ; Phagocytosis

Purpose To explore the immunohistochemical features of neuroendocrine markers, CKpan and TTF-1 and their relationship to TNM stage and prognosis in small cell lung cancer (SCLC). Methods The expression of NSE, CgA, Syn, CD56, TTF-1 and CK-pan in SCLC tissue specimens were detected using immunohistochemical EnVision indirect method. Clinical data and TNM stage of 90 patients were collected and the overall survival ( OS) was followed up by telephone. Results Of 90 cases of SCLC, the vast majority were occured in the elderly men. The ratio of man to woman was 5 to 1. The median age was 64 years old. The stageⅠ+Ⅱ was 21 cases, 30 in stageⅢand 39 cases in stage IV. The positive rate of immunohistochemical staining of neuroendocrine markers for NSE, CgA, Syn and CD56 were 83. 3%, 70%, 65. 5% and 86%, respectively. The positive rate of CKpan, TTF-1 were 92. 2% and 81. 1%. Kaplan-Meier analysis indicated that the expression of TTF-1 and NSE were significantly correlated with the TNM stage and over-all survival of patients with SCLC (P<0. 05). The median OS was 8 months in positive expression of TTF-1, which was higher than those in negative expression of TTF1 (5. 5 months)(P=0. 000). The median OS was 7 months in NSE positive expression which was lower than those in negative expression of NSE (11 months)(P=0. 009). The median OS of stageⅠ+Ⅱ,ⅢandⅣwere 16 months, 9 months and 4 months with significant difference ( P=0. 000 ) . Cox multivariate analysis indicated the TTF-1 expression and TNM were independent prognostic factors for the OS of the SCLC patients. Conclusion Most of SCLC has neuroendocrine differentiation, expression CKpan and TTF-1. The expression of TTF-1 may be negative correlation but NSE positive correlation with the prognosis of SCLC patients. And the TTF-1 expression and TNM may be independent prognostic factors for the OS of the SCLC patients.

Objective To explore the feasibility of culturing dermal papillae cells (DPC) of hu- man hair in a serum-flee medium,and to observe the growth characteristics of these cells.Methods Cell culture flasks (plates) were pretreated with fibronectin,and DPC (2nd passage) were incubated with Williams E serum-flee medium supplemented with insulin-transferrin-selenite (ITS).Cells were observed by an inverted phase-contrast microscope.Proliferation of DPC was evaluated with 3-(4,5-dimethylthia- zol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and by their growth curve.Results In a serum-free medium,2nd passage DPC adhered to the flask surface within two to four hours of incubation; two to three days later,confluence,of the cells was observed,without noticeable proliferation.Four days later,cell connection was interrupted,isolated cells or cell clusters were seen,and detachment of some cells from the flask surface was observed.One to two weeks later,most cells had died.After incubation with 4% bovine serum for ten hours,cell proliferation was observed surrounding the remaining viable cell colonies. DPC growth curve showed stagnant phase and slow growth phase;however,log growth phase was not ob- served.Conclusion DPC could be successfully cultured in serum-free medium.However,the culture con- dition needs to be further optimized.

OBJECTIVETo evaluate the effects of Chlamydia pneumoniae infection on oxidative stress and the development of atherosclerosis in C57BL/6J mice.

METHODSForty-eight C57BL/6J mice were divided into 4 groups including infection of CP and cholesterol diet, cholesterol diet, infection of CP and control. Atherosclerotic lesions were measured in the aortic root at 40 weeks after the primary infection. Production of superoxide was measured by lucigenin-enhanced chemiluminescence response and evaluated in situ with laser scanning confocal microscope.

RESULTSInfected mice fed with an atherogenic diet developed significantly larger lesion areas compared with the single atherogenic diet mice (135 249 +/- 43 748 microm2 vs. 96 378 +/- 30 945 microm2, P < 0.05). Superoxide generation was higher in aortic arches of the infected mice or atherogenic diet mice compared with the control mice (1974.25 +/- 650.49, 701.00 +/- 105.16, 455.62 +/- 77.54 counts.mg(-1).min(-1) vs. 142.25 +/- 31.82 counts.mg(-1).min(-1), respectively, P < 0.001).

CONCLUSIONChlamydia pneumoniae infection accelerates atherosclerotic lesion development in diet-induced hypercholesterolemic mice. Generation of reactive oxygen species may contribute to atherosclerotic development by Chlamydia pneumoniae infection.

Animals ; Antibodies, Bacterial ; blood ; Atherosclerosis ; etiology ; Chlamydia Infections ; complications ; metabolism ; Chlamydophila pneumoniae ; immunology ; Male ; Mice ; Mice, Inbred C57BL ; Oxidative Stress ; Superoxides ; metabolism

Curcumin has been reported to possess antitumor activity with low toxicity. However, the clinical application of curcumin has been significantly limited by its instability and poor metabolic property. In order to overcome these limitations and discover novel small molecules with potential antitumor activity, 29 curcumin mimics were synthesized, which were confirmed by 1H NMR and HR-MS, and their cytotoxic property was evaluated against five human cancer cell lines in vitro. Compounds 16, 18 and 19 exhibited good cytotoxic property, their IC50 value were even below 5 micromol x L(-1) to some cancer cell lines, 5-9 times better than curcumin.
Antineoplastic Agents ; chemical synthesis ; pharmacology ; Cell Line, Tumor ; Curcumin ; analogs & derivatives ; chemical synthesis ; pharmacology ; Drug Screening Assays, Antitumor ; Humans ; Inhibitory Concentration 50

OBJECTIVETo investigate the quantity and function of circulating dendritic cells (DC) in patients with chronic idiopathic thrombocytopenic purpura (ITP).

METHODSHigh dose dexamethasone (HD-DXM) at a dose of 40 mg orally per day for four consecutive days was the initial treatment for chronic ITP patients. Flow cytometry was used to analyze the number of myeloid DC (mDC), plasma cytoid DC (pDC) and CD4+FOXP3+ T cells in patients before and after the treatment, meanwhile the co-stimulatory molecules on circulating DCs were assayed as well. Monocyte-derived DCs and CD4+ T cells were co-cultured with autologous or allogeneic normal fresh platelets and after 6 days of incubation H-TdR was used to assay the proliferation of CD4+ T cells.

RESULTSThe absolute numbers of circulating mDC and pDC were not significantly different between pre-treatment patients and healthy controls (P > 0.05 and P >0.05). However, percentage of CD4+ FOXP3+ T cells was decreased (P < 0.01), and their percentage was inversely correlated with the number of pDC and mDC (r = -0.396, P =0.045 and r = -0.410, P =0.037). The initial response rate to HD-DXM was 92.3%. After 4-days treatment, CD4 FOXP3+ Treg cells increased (P <0.01) while pDCs decreased (P <0.01). Although mDCs increased after HD-DXM (P <0.05), their CD11c expression level was decreased (P < 0.01), the mean fluorescence intensity (MFI) decreased from 340 +/- 30 before treatment to 199 +/- 21 after treatment. The inverse correlation between pDCs and CD4+ FOXP3+ Treg cells remained (r= -0.524, P =0.006) while that between mDCs and Treg cells disappeared (r = - 0.360, P =0.071). The MFI of CD86 on DCs was higher in ITP patients than in healthy controls (P <0.05), while the proportions of CD86, CD40, CD80 and the MFI of CD40, CD80 in ITP patients were normal (P > 0.05). DCs from chronic ITP patients co-cultured with autologous or allogeneic platelets were highly efficient in stimulating autologous CD4+ T cells proliferaton as compared to those derived from healthy donors (P < 0.05 and P <0.05).

CONCLUSIONDCs may play a role in the pathogenesis of chronic ITP in relation with CD4+CD25+ Treg cells.

Adult ; CD4-Positive T-Lymphocytes ; immunology ; Dendritic Cells ; immunology ; metabolism ; pathology ; Female ; Humans ; Male ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic ; blood ; immunology

OBJECTIVETo determine the effects of PCMV-FGEV transfection on the profile of SMMC-7721 hepatocellular in vitro in vivo.

METHODSSMMC-7721 hepatocellular was transfected with PCMV-FGEV antisense, PCMV-VEGF sense and empty vector plasmid encapsulated by lipofectamine. The positive cell clones were selected with G418. The stable transfection and expression of VEGF in the SMMC-7721 hepatocellular were determined by in situ hybridization and immunochemical analysis. The effect of PCMV-FGEV transfection on SMMC-7721 hepatocellular proliferation was observed by MTT colorimetric assay. Flow cytometry was used to determine the effects of PCMV-FGEV transfection on cell apoptosis of SMMC-7721. The growth of transfected cells was also observed in nude mice.

RESULTSThere was reduced VEGF expression in SMMC-7721 transfected with PCMV-FGEV confirmed by in situ hybridization and immunohistochemical analysis. There was no effect of PCMV-FGEV transfection on cell proliferation and cell apoptosis of SMMC-7721 in vitro. The growth of cell with PCMV-FGEV transfected was slow in nude mice (vivo) and accompanied with obvious apoptosis. The latent time of tumor in the antisense mice group was 25.0+/-1.8 days, which was longer than that in sense and control group significantly (F=19.455, P<0.01). On the other hand, the average tumor weight in antisense group (0.96 g+/-0.28 g) was the smallest among the three groups (F=21.501, P<0.01).

CONCLUSIONSThe expression of VEGF was inhibited by PCMV-FGEV. There was no effect on cell proliferation and cell apoptosis of SMMC-7721 by transferring PCMV-FGEV gene into SMMC-7721 cells in vitro. But in vivo it can inhibit tumor growth and induce cell apoptosis.

Apoptosis ; Cell Division ; Cell Line, Tumor ; Humans ; Immunohistochemistry ; Liver Neoplasms ; pathology ; therapy ; RNA, Antisense ; therapeutic use ; Transfection ; Vascular Endothelial Growth Factor A ; analysis ; antagonists & inhibitors ; genetics

OBJECTIVETo investigate the significance of PKC and p44/42 mitogen-activated protein kinase (MAPK) signal transduction in ischemic preconditioning (IP).

METHODSThrough liver cell IP models, PKC inhibitor and MEK inhibitor were utilized to analyze the phosphorylation level of p44/42 MAPK and cell viability was also observed. Rat liver IP models were established which were treated with various drugs. Then the phosphorylation level of p44/42 MAPK in vivo and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) concentrations were detected. And cellular structures were observed under light microscopy.

RESULTSSimilar results were obtained in vivo and in vitro IP models. Compared with the ischemia reperfusion (IR) group in vivo, the phosphorylation level of p44/42 MAPK was obviously increased in IP treated rats (q = 27.217, P < 0.01), and the cellular structure injured slightly. The concentrations of serum ALT and AST in IP group were significantly lower than those in IR group (281.0 U/L +/-35.6 U/L vs 762.8 U/L +/-130.5 U/L and 407.7 U/L +/-73.7 U/L vs 820.9 U/L +/-111.3 U/L, P < 0.01). However, opposite changes were found in PKC and MEK inhibited groups, when compared to IP group. The phosphorylation level of p44/42 MAPK was obviously decreased, the liver tissues injured evidently, and the concentrations of serum ALT and AST (645.61 U/L +/-90.4 U/L, 678.6 U/L +/-136.5U/L and 466.2 U/L +/-82.8 U/L, 732.9 U/L +/-91.1 U/L, respectively) were significantly greater than those in IP group.

CONCLUSIONThese results suggest that p44/42 MAPK pathway plays a vital role in the protection of hepatocytes in ischemic preconditioning.

Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Hepatocytes ; cytology ; physiology ; Humans ; Ischemic Preconditioning ; Liver ; blood supply ; cytology ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Phosphorylation ; Protein Kinase C ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction

OBJECTIVETo explore the effects of mitochondrial pathways on apoptosis in colon carcinoma cells induced by Tumor necrosis factor related apoptosis inducing ligand and offer evidences for TRAIL application in clinic.

METHODSApoptosis, integration of mitochondria (including DeltaPsim, cardiolipin), activity of Caspase-9 and release of cytochrome c in colon carcinoma cells SW1116 treated with TRAIL, were detected by means of flowcytometry, fluorometer method and western-blot at the different time point.

RESULTSAfter treated with TRAIL for 4 hours, the apoptosis index was 32.98%, and the damage of mitochondria occurred with DeltaPsim, cardiolipin decreased, and the activity of Caspase-9 and cytochrome c increased. The Caspase-9 activity at 24 hour was (48.12+/-2.21)micromol.L(-1).h(-1).mg(-1) protein. Mitochondrial damage induced by TRAIL could be inhibited by Caspase inhibitor Z-VAD. fmk.

CONCLUSIONMitochondrial pathways involved in the apoptosis of colon carcinoma cell induced by TRAIL. Cytochrome c was released and Caspase-9 was activated in the Caspase-dependent manner after the damage of mitochondrial.

Apoptosis ; Cardiolipins ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Cytochromes c ; metabolism ; Humans ; Mitochondria ; metabolism ; Mitochondrial Membranes ; metabolism ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; metabolism