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BACKGROUND:Currently, there is no uniform, standardized approach to isolate, purify and proliferate bone marrow mesenchymal stem cells. Chlormethylbenzamido-1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (CM-Dil) is a stable, reliable, high marking and simple marker.
OBJECTIVE:To develop the methods for isolation, culture and identification of rat bone marrow mesenchymal stem cells in vitro.
METHODS:Two male Sprague-Dawley rats, weighing 50-100 g were taken to col ect the bilateral femur and tibia bone marrow under sterile conditions, and then, primary bone marrow mesenchymal stem cells were isolated and cultured using bone marrow adherent separation and density gradient centrifugation. cells were amplified and purified through timely and repeated passage, and labeled at the third generation with fluorescent dyes CM-Dil in vitro as a source of donor cells.
RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells were cultured successful y in vitro using bone marrow adherent separation and density gradient centrifugation separation methods, but the former was superior to the latter in the number of cultured cells significantly, while the two methods were not different significantly in terms of cellviability and proliferation. Flow cytometry results showed that the positive rates of cultured cells were 17.5%for CD34, 97.9%for CD44, and 91%for CD90. CM-Dil can label bone marrow mesenchymal stem cells successful y, which is a stable, reliable, high marking and simple marker.

Objective To investigate the effects of glutamate receptor signaling on melanoma cell dendrite morphology and cytoskeleton protein. Methods A metastatic human malignant melanoma cell line WM451LU was cultured and transfected by recombinant adenovirus vector carrying a cDNA encoding microtubule-associated protein 2a (MAP2a). MK-801, an antagonist of N-methyl-D-aspartate receptor (NMDAR), and CPCCOEt, an antagonist of metabotropie glutamate receptor 1 (mGluR1), were used to treat transfected or untrausfeeted WM451LU cells. Confocal microscopy and three dimensional atomic force microscopy were used to assess subcellular location of NMDAR2A, mGluR1 and MAP2a as well as the dis-tribution of α-tubulin in and dendrite morphology of WM451LU cells. The proliferation of WM451LU cells was estimated by cell survival growth curve. Results Confocal laser microscopy revealed that NMDAR2A, mGluRl and MAP2a were mainly co-localized in melanoma cell dendrites. Both MK-801 and CPCCOEt increased the density of microtubules in cell dendrites and dendritic branching of WM451LU cells, and both effects of MK-801 and CPCCOEt were enhanced by the expression of MAP2a. Furthermore, the proliferation of WM451LU cells was significantly inhibited by MK-801 of 100 μmol/L and CPCCOEt of 10 μmol/L. Conclusions In melanoma cells, glutamate receptors may participate in the development of dendrites, and anta- gonists of glutamate receptors could inhibit the proliferation of melanoma cells.

Objective To explore the prevalence of parenteral nutrition-associated liver disease (PNALD) and its risk factors in elderly people after gastrointestinal operation. Methods Seventy-five patients received parenteral nutrition (PN) after gastrointestinal operation were retrospectively analyzed. Age, height, body mass index, suftering diseases, history of diseases, time of therapy, total calorie, nonprotein calorie, the kind and amount of fat emulsion and amino acid, the amount of glucose, non-protein energy to nitrogen ratio, ratio of glucose to lipid, liver function, renal function and blood routine were collected. Results The prevalence of PNALD was 25.3% (19/75). The total calorie, nonprotein calorie, the amount of protein, the amount of glucose and ratio of glucose to lipid were obviously higher in PNALD group than in non-PNALD group [(24.0±6.5) vs. (20.7±5.4)kcal·kg-1·d-1, (20.5±5.5)vs. (17.2±4.8)kcal·kg-1·d-1, (1.0±0.3)vs. (0.9±0.2)g ·kg-1·d-1, (2.9±0.9)vs.(2.3±0.9)g·kg-1·d-1, 1.5±0.7 vs. 1.1±0.5; all P<0.05], while the hemoglobin was lower in PNALD group [(97.4±15.1)vs. (110.1±19.1)g/L, P<0.05]. The kind of fat emulsion and amino acid, gender, history of diseases, suftering diseases, body mass index, serum albumin, leukocyte levels and renal function were comparable between the two groups (χ2=0.114,0.843,0.116,0.531,0.344,1.588,0.006,0.063 and 0.549, all P>0.05). Conclusions The prevalence of PNALD is 25.3% in 75 elderly patients after gastrointestinal operation. Total calorie, the amount of glucose and the ratio of glucose to lipid should be reduced in these patients for preventing PNALD.

Objective　To evaluate the effects of lipid emulsion on parenteral nutrition associated liver dis ease (PNALD) in old tumor patients. Methods　A retrospective study was performed with 402 patients in Renji Hospital from January 2003 to December 2008. Patients were retrieved according to the following criteria: (1)age ≥60 years; (2) confirmed diagnosis of tumor, had no evidence of metastasis, and tumor was completely resected before receiving parenteral nutrition; (3) liver and kidney function was in normal range before receiving parenteral nutrition; (4) parenteral nutrition days ≥7; and (5) parenteral nutrition was infused in "all in one" bag via central venous. Patients with history of viral hepatitis or died in parenteral nutrition episode were excluded. These 402 patients aged (71.7 ±6.8) years and the average parenteral nutrition time was (10. 2 ±5.9) (range, 7-61 )days. In 311 patients (77.4％), non-protein calorie was obtained from carbohydrate and lipid and 91 patients (22. 6％ ) just obtained non-protein calorie from carbohydrate.　Results　The total prevalence of PNALD was 15.2％ (61/402). The prevalence of PNALD was 8.8％ (8/91) in patients receiving parenteral regiment without lipid and 17.0％ (53/311) in patients receiving parenteral nutrition with lipid, and there was no significant difference in prevalence of PNALD between two groups (χ2 = 3.72, P = 0.07 ). Lipid type and amount showed no significant effects on PNALD ( P ＞ 0.05 ). The fever days ( P ＜ 0. 001 ), baseline level of alanine transaminase (P ＜0. 001 ) and γ-glutamyltransferase (P ＜0. 001 ) were risk factors for liver injury by logistic regression. Conclusion　Lipid emulsion can be safely used in old tumor patients without affecting the occurrence of PNALD.

Objective:To explore the expression of cyclooxygenase-2(COX-2) and S-100 positive dendritic cell in laryngeal carcinoma tissue and their clinical significance.Method:Sixty-five samples of laryngeal carcinoma and thirty-four biopsies of adjacent noncancerous tissue were obtained. Immunohistochemical technique(SP method) was used to detect the expression of COX-2 and S-100 positive dendritic cell, and the relationship of their expression with clinical pathological parameters and prognosis was analyzed.Result:The rates of COX-2 expression were 63.08%(41/65)and 14.70%(5/34)in laryngeal carcinoma and control group, respectively. The difference was significant(P＜0.05).The positive expression of COX-2 was correlated with T and clinical stage in laryngeal carcinoma(all P＜0.05).The rates of S-100 positive dendritic cell expression were 61.54%(40/65)and 0 in laryngeal carcinoma and control group, respectively. The difference was significant(P＜0.05).S-100 positive dendritic cells showed significant differences between early and late clinical stage and lymph node metastasis (all P＜0.05).Kaplan-Meier survival analysis showed that patients with positive expression of COX-2 and S-100 positive dendritic cell had worse disease-free and overall survival(P＜0.05).Cox regression analysis showed that S-100 positive dendritic cell was indicated as an independent prognostic factor for survival(P＜0.05).Conclusion:COX-2 and S-100 positive dendritic cell are highly expressed in laryngeal carcinoma tissue. It suggests that the expression of COX-2 and S-100 positive dendritic cell is related to the process of carcinogenesis and may be the important indicators in laryngeal carcinoma for prognosis.

Objective:To investigate the expression and clinical significance of Ang-2 and MMP-7 protein in laryngeal carcinoma tissue.Method:Immunohistochemical technique was used to detect the expression of Ang-2 and MMP-7 protein in 65 tissues of laryngeal carcinoma and 34 biopsies of adjacent non-cancerous tissue. The relationship between the expression of Ang-2 and MMP-7 and invasion, metastasis or prognosis in laryngeal carcinoma tissue was analyzed.Result:The positive rates of Ang-2 and MMP-7 were significantly higher in laryngeal carcinoma tissue than those in adjacent non-cancerous tissue(P＜0.05).The level of Ang-2 and MMP-7 expression had no significant correlations with the age and course as well as the smoking, drinking, histological differentiation of carcinoma and clinical classification (P＞0.05).While the expression of Ang-2 significantly differed between patients with different T stage and clinical stage(P＜0.05), and the expression of MMP-7 was notably correlated with the T stage,clinical stage and lymph node metastases (P＜0.05).There was a correlation between the expression of Ang-2 and MMP-7 (P＜0.05).The Kaplan-Meier survival analysis showed that patients with positive expression of Ang-2 had worse overall survival(P＜0.05).However,MMP-7 expression was not related to overall survival or disease-free survival (all P＞0.05).Cox regression analysis indicated that Ang-2 and MMP-7 expression were independent prognostic factors of laryngeal carcinoma.Conclusion:Overexpression of Ang-2 and MMP-7 was observed in laryngeal carcinoma and they might be served as an objective indicator for biological behaviour and prognosis.

OBJECTIVE To investigate the expression of Cyclooxygenase-2(COX-2)andVascular endothelial growth factor(VEGF)in laryngeal carcinoma tissue and its clinical significance. METHODS Sixty-five samples of laryngeal carcinoma and thirty-four biopsies of adjacent noncancerous tissue were immunohistochemically examined for expression of COX-2 and VEGF, whose relationship with clinicopathological parameters was also analysied. RESULTS Percentages of COX-2 and VEGF expression in laryngeal carcinoma tissue were 63.08% and 70.77% respectively, which were higher than those in adjacent noncancerous tissue(P

Objective To obtain Chinese hamster ovary (CHO) cell lines that stably express a targeting complement inhibitor CR2-CD59.Methods The recombinant plasmid PEE14.1-CR2-CD59 was constru-cted by cloning the DNA fragment CR2-CD59 into plasmid PEE14.1,and the obtained plasmid was transfected into CHO cells by FuGENE 6.The clones with stable high expression of target fragment were selected by methionine sulfoximine (MSX),the expression of CR2-CD59 was analyzed by ELISA,SDS-PAGE and Western blotting analysis.Results Several stable expression clones were obtained,and CR2-CD59 was highly expressed in the secret form in CHO cells.SDS-PAGE analysis showed that the molecular weight of the recombined protein CR2-CD59 was consistent with the predicted one.ELISA and Western blotting results revealed that the CR2-CD59 could react with both anti-human CR2 and anti-human CD59 polyclonal antibodies.Compared with serum-containing medium,the protein was highly expressed in serum-free medium (P