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1.
Zhongguo Wei Zhong Bing Ji Jiu Yi Xue ; 22(12): 719-22, 2010 Dec.
Article in Chinese | MEDLINE | ID: mdl-21190596

ABSTRACT

OBJECTIVE: To design a novel bioreactor of bioartificial liver system by using expanded and differentiated human bone marrow mesenchymal stem cells (hBMMSCs) as active cells. METHODS: hBMMSCs were isolated from bone marrow of volunteers and grown to 10(7) population, and then replanted into hollow fiber cartridge to expand continuously for 10 days. They were incubated in differentiation medium containing recombinant human hepatocyte growth factor (rhHGF), recombinant human fibroblast growth factor 4 (rhFGF-4), recombinant human oncostatin M (rhOSM), and the cells were induced to differentiate into hepatocyte-like cells for 21 days. The functions of the differentiated cells, such as synthesis of albumin (Alb), alpha fetoprotein (AFP) were determined. Eighteen days later, the functions of metabolism of ammonia and benzodiazepines, and synthesis of urea were monitored. The cellular synthesis rate of Alb was measured with flow cytometer. The glucose levels in the medium were measured during entire culture process. RESULTS: (1)Glucose-uptake in the cartridge was increased during the culture period, and at the end of culture, the number of cells in the cartridge increased to 10(9). (2)After induction, AFP was detected on day 6, reaching the peak level on day 12. Alb was detected on day 9. Eighteen days after being induced, the clearance rate of ammonia and benzodiazepines in the cartridge was 2.0-2.7 mmol/24 hours and 3.2-3.8 mg/24 hours, respectively, and urea production rate was 1.8-2.2 mmol/24 hours. (3)At the end of the culture, 66.18%-76.91% of the cells showed positive Alb expression. CONCLUSION: hBMMSCs can be multiply to construct a novel bioreactor of bioartificial liver system in a hollow fiber cartridge.


Subject(s)
Bioreactors , Bone Marrow Cells/cytology , Liver, Artificial , Mesenchymal Stem Cells/cytology , Adolescent , Cell Differentiation , Cells, Cultured , Humans , Young Adult
2.
Cell Biol Int ; 32(8): 959-65, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18486499

ABSTRACT

We have used a uniform design to explore the most effective directed differentiation medium (MEDDM) for differentiating mouse bone marrow mesenchymal stem cells (mMSCs) into hepatocytes. Our methods involved arranging eight differentiation medium groups following uniform design. Flow cytometry was used to evaluate the percentage of ALB+ and CK18+ cells in each group. Factors and their concentrations in the MEDDMs were then identified. The MEDDMs were evaluated by their ability to differentiate mMSCs into hepatocytes by RNA and protein expressions and synthesis functions. FGF at 35 ng/ml and OSM at 30 ng/ml in the medium yielded the highest percentage of ALB+ and CK18+ cells. During directed differentiation using MEDDMs, ALB, CK18, TTR, AFP mRNAs were expressed. ALB and CK18 proteins were detected in the cells. The differentiated cells produced albumin and urea in a time dependent manner. Uniform design was adequate for choosing the MEDDM of mMSCs. MEDDM containing 35 ng/ml FGF and 30 ng/ml OSM was effective in differentiating mMSCs into hepatocytes.


Subject(s)
Cell Differentiation , Culture Techniques , Hepatocytes/cytology , Mesenchymal Stem Cells/cytology , Animals , Biomarkers/analysis , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Culture Media , Epidermal Growth Factor/metabolism , Fibroblast Growth Factor 4/metabolism , Gene Expression , Hepatocyte Growth Factor/metabolism , Hepatocytes/metabolism , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Oncostatin M/metabolism , Phenotype
3.
Acta Pharmacol Sin ; 27(12): 1567-74, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17112410

ABSTRACT

AIM: The role of caveolin-1 (Cav-1) in angiogenesis remains poorly understood. The endothelial nitric oxide (NO) synthase (eNOS), a caveolin-interacting protein, was demonstrated to play a predominant role in vascular endothelial growth factor (VEGF) -induced angiogenesis. The purpose of our study was to examine the role of Cav-1 and the eNOS complex in NO-mediated angiogenesis. METHODS: Human umbilical vein endothelial cells (HUVEC) were isolated and cultured in 3-D fibrin gels to form capillary-like tubules by VEGF stimulation. The expression of Cav-1 and eNOS was detected by semiquantitative RT-PCR. The HUVEC were treated with antisense oligonucleotides to downregulate Cav-1 expression. Both transduced and non-infected HUVEC were cultured in fibrin gels in the presence or absence of VEGF (20 ng/mL) and NG-nitro-L-arginine methyl ester (L-NAME; 5 mmol/L). NO was measured using a NO assay kit and capillary-like tubules were quantified by tubule formation index using the Image J program. RESULTS: RT-PCR analysis revealed that Cav-1 levels steadily increased in a time-dependent manner and reached their maximum after 5 d of incubation, but there were no obvious changes in eNOS mRNA expression in response to VEGF in the fibrin gel model. VEGF (20 ng/mL) can promote NO production and the formation of capillary-like tubules, and this promoting effect of VEGF was blocked by the addition of L-NAME (5 mmol/L). When transduced HUVEC with the antisense Cav-1 oligonucleotides were plated in the fibrin gels, the capillary-like tubules were significantly fewer than those of the non-infected cells. The capillary-like tubules formation and NO production of transduced HUVEC with the antisense Cav-1 oligonucleotides cultured in fibrin gels showed no responses to the addition of VEGF (20 ng/mL) and L-NAME (5.0 mmol/L). CONCLUSION: NO was a critical angiogenic mediator in this model. Cav-1 was essential for NO-mediated angiogenesis and may be an important target of anti-angiogenesis therapy.


Subject(s)
Caveolin 1/metabolism , Endothelial Cells/metabolism , Neovascularization, Physiologic/physiology , Nitric Oxide Synthase Type III/biosynthesis , Caveolin 1/genetics , Cells, Cultured , Endothelial Cells/cytology , Humans , NG-Nitroarginine Methyl Ester/pharmacology , Neovascularization, Physiologic/drug effects , Nitric Oxide Synthase Type III/genetics , Oligonucleotides, Antisense/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Umbilical Veins/cytology , Vascular Endothelial Growth Factor A/pharmacology
4.
Hepatol Res ; 31(4): 223-31, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15862781

ABSTRACT

AIM: : It is imperative to explore some ways to gain the functional hepatocytes for hepatocyte transplantation. Bone marrow stem cells can differentiate into hepatocytes in vivo and in vitro. We select fibroblast growth factor-4 (FGF-4), oncostatin M (OSM), hepatocyte growth factor (HGF) and epithermal growth factor (EGF) as differentiation factors, and design the appropriate directed differentiation medium in order to gain hepatocytes through directed differentiation of bone marrow stem cells. METHODS: : Bone marrow mononuclear cells (BMMCs) were cultured in the directed differentiation media including FGF-4, OSM, HGF and EGF. In the course of cell differentiation, cell morphology was observed, and the expression patterns of some genes of the hepatocyte were validated and confirmed by RT-PCR. The ALB-, and CK18-expressed cells were gone further step to be confirmed by Western blot analysis, immunofluorescence and flow cytometric analysis. Hepatocyte functional activity, including glycogen synthesis and urea production, were confirmed by periodic acid-Shiff (PAS) staining and urea assay. RESULTS: : Some epithelial-like cells or polygonal cells appeared in the directed differentiation medium within 12 days, and the number and sizes of colonies of epithelial-like cells or polygonal cells increased in the course of the cell directed differentiation. AFP, HNF-3ss, ALB and CK18 mRNA expressions first appeared within day 7, and lasted throughout the later directed differentiation. TTR, G-6-P and TAT mRNA expressions could be detected within day 14, and their expressions lasted in the course of the later directed differentiation. ALB and CK18 were confirmed to exist in the differentiated BMMCs by Western blot analysis. ALB was found in the cytoplasm and cell membrane, while CK18 scattered in the cytoplasm by immunofluorescent staining. On day 21,the ratio of ALB-positive cells was 69.45%, and the ratio of CK18-positive cells was 67.36%. The accumulation of glucogen was detected in the cytoplasm of the differentiated cells. The directed differentiated BMMCs produced urea 3 days later, and they produced urea in a time-dependent manner. CONCLUSIONS: : BMMCs could differentiate into hepatocytes or hepatocyte-like cells in the differentiation media including HGF, FGF-4, EGF, and OSM. These hepatocyte-like cells were identified at the gene level and protein level. Furthermore, these hepatocyte-like cells had some hepatocellular synthesis and metabolism functions.

5.
Acta Pharmacol Sin ; 26(4): 469-76, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15780197

ABSTRACT

AIM: To design the effective directed differentiation medium to differentiate bone marrow cells into hepatocyte-like cells. METHODS: Bone marrow cells were cultured in the directed differentiation media including fibroblast growth factor-4 (FGF-4) and oncostatin M (OSM). Hepatocyte-like cells from directed differentiation of bone marrow cells were identified through cell morphology, RNA expressions by reverse transcriptase-polymerase chain reaction (RT-PCR), protein expressions by Western blot, and hepatocellular synthesis and metabolism functions by albumin ELISA, Periodic acid-Shiff staining and urea assay. RESULTS: Some epithelial-like cells or polygonal cells appeared and increased in the course of the cell directed differentiation. Hepatocyte nucleur factor-3beta (HNF-3beta, albumin (ALB), cytokeratin 18 (CK18), transthyretin (TTR), glucose-6-phosphate (G-6-Pase), and tyrosine aminotransferase (TAT) mRNA were expressed in the course of the directed differentiation. The directed differentiated cells on d 21 expressed HNF-3? ALB, and CK18 proteins. The directed differentiated cells produced albumin and synthesized urea in a time-dependent manner. They could also synthesize glycogen. CONCLUSION: Our differentiation media, including FGF-4 and OSM, are effective to differentiate bone marrow cells into hepatocyte-like cells, which could be used for hepatocyte resources for bioartificial liver or hepatocyte transplantation.


Subject(s)
Albumins/biosynthesis , Bone Marrow Cells/cytology , DNA-Binding Proteins/biosynthesis , Hepatocytes/cytology , Keratins/biosynthesis , Nuclear Proteins/biosynthesis , Transcription Factors/biosynthesis , Albumins/genetics , Animals , Cell Culture Techniques/methods , Cell Differentiation/drug effects , DNA-Binding Proteins/genetics , Fibroblast Growth Factor 4 , Fibroblast Growth Factors/pharmacology , Glucose-6-Phosphate/biosynthesis , Glucose-6-Phosphate/genetics , Hepatocyte Nuclear Factor 3-beta , Hepatocytes/metabolism , Keratins/genetics , Mice , Mice, Inbred C57BL , Nuclear Proteins/genetics , Oncostatin M , Peptides/pharmacology , Prealbumin/biosynthesis , Prealbumin/genetics , Proto-Oncogene Proteins/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Transcription Factors/genetics
6.
Zhongguo Wei Zhong Bing Ji Jiu Yi Xue ; 15(2): 114-6, 2003 Feb.
Article in Chinese | MEDLINE | ID: mdl-12857476

ABSTRACT

OBJECTIVE: To investigate the pharmacokinetics of vancomycin in continuous veno-venous hemofiltration(CVVH) in order to determine appropriate vancomycin dosing strategies for patients receiving CVVH. METHODS: The serum concentrations of vancomycin were measured by TDx and the pharmacokinetics parameters were calculated. RESULTS: The pharmacokinetics of vancomycin during CVVH was fitted with open two-compartment model. At the beginning of CVVH, the pharmacokinetic parameters were: maximum plasma concentration (Cmax)=22.18 mg/L, minimum plasma concentration attained (Cmin)=5.82 mg/L, half-life of drug (T1/2)=5.75 h, apparent volume of distribution (Vd)=21.92 L, total body clearance of drug(CL)=3.49 L/h. On the 16 d of CVVH, the pharmacokinetic parameters were Cmax=38.70 mg/L, Cmin=16.50 mg/L, T1/2=33.32 h, Vd=12.92 L, CL=0.38 L/h. CONCLUSION: CVVH can significantly augment the clearance of vancomycin in acute renal failure patients. Dosing strategies for individualization of vancomycin therapy in patients receiving CVVH are proposed. Monitoring the serum concentration during CVVH is necessary.


Subject(s)
Hemofiltration/methods , Vancomycin/pharmacokinetics , Vancomycin/therapeutic use , Half-Life , Humans , Male , Middle Aged , Renal Dialysis/methods
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