BACKGROUND: The increasing prevalence of diabetes mellitus has been become one of the diseases which threaten the heath of human being in the 21st century. Islet transplantation is considered to be the most effective approach to cure type Ⅰ diabetes mellitus. However, lack of donor tissue limits the application of this therapy. However, recent progress of stem cell research shows that stem cell therapy may be a potential means to solve this problem.OBJECTIVE: To take activin A and all-trans retinoic acid (AR) in inducing the differentiation of bone marrow mesenchymal stem cells (MSCs) and explore its possibility DESIGN: A randomized controlled experiment.SETTING: Institute of Biotechnology, Academy of Military Medical SciencesMATERIALS: This experiment was conducted at the Institute of Biotechnology, Academy of Military Medical Sciences from November 2004to June 2005. Six male Sprague-Dawley rats, with body mass of 150-160g, were provided by the Experimental Animal Center of Academy of Military Medical Sciences.METHODS: Femoral bone marrow of the rats was extracted under aseptic condition. Bone marrow mesenchymal stem cells (MSCs) were isolated with density gradient centrifugation. Passaged MSCs were randomly divided into 4 groups: high concentration of glucose (HG), AR, beta-mercaptoethanol (ME) and negative control groups. MSCs were induced to differentiate into IPCs with conditional medium containing high concentration glucose, activin A, RA and ME etc. After induction, phenotypes of differentiated cells were examined by immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR).MAIN OUTCOME MEASURES: Expression of insulin and glucagon of differentiated cells were examined by immunocytochemistry. Insulin-1 mR-NA expression of differentiated cells was detected by RT-PCR.RESULTS: After bone marrow mesenchymal stem cells were induced,there were scattered insulin-and glucagon-positive cells in the HG group,many insulin-and glucagon-positive cells in the AR and ME groups, and these cells formed insulin-like structure. The expression of insulin-1mRNA could be observed in the HG, AR and ME groups. Insulin-and glucagonpositive cells and the expression of insulin-1mRNA were not observed in the negative control group.CONCLUSION: We adopt an induction scheme based on AR and other matured factors, and successfully make bone marrow mesench.ymal stem cells induce and differentiate into insulin positive reaction cells and form insulin-like structure, but its induction efficiency needs further improvement.

With suspension adapted recombinant Chinese hamster ovary (CHO) cell lines 11G-S expressing human pro-urokinase (pro-UK) as the object of study, a serum-free medium for the cultivation of recombinant CHO cells in suspension was formulated by using Plackett-Burman design and response surface methodology. The two-level Plackett-Burman design was used to evaluate the effect of 10 medium supplements on the growth of the 11G-S cells in suspension culture. Among the 10 medium supplements, insulin, transferrin, and putrescine were identified as the most significant factors (P < 0.05). The response surface methodology with three factors and three levels was used to determine the optimal levels of these factors. And a serum-free medium, SFM-CHO-S for recombinant CHO cells suspension culture was formulated. The maximum cell density of 11G-S cells in SFM-CHO-S in suspension batch culture reached 4.12 x 10(6) cells/mL with a maximum pro-UK activity at 5614 IU/mL, which was superior to the commercial serum-free medium for recombinant CHO cells.
Animals ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Culture Media, Serum-Free ; Genetic Engineering ; Insulin ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; Transferrin ; pharmacology ; Urokinase-Type Plasminogen Activator ; biosynthesis ; genetics

In the light of Chinese hamster ovary (CHO) cell line 11G-S expressing human recombinant pro-urokinase, the differences of gene expression levels of the cells in different growth phases in both batch and fed-batch cultures were revealed by using gene chip technology. Then, based on the known cell cycle regulatory networks, the transcriptional profiling of the cell cycle regulatory networks of the cells in batch and fed-batch cultures was analyzed by using Genmapp software. Among the approximate 19 191 target genes in gene chip, the number of down-regulated genes was more than those of up-regulated genes of the cells in both batch and fed-batch cultures. The number of down-regulated genes of the cells in the recession phase in fed-batch culture was much more than that of the cells in batch culture. Comparative transcriptional analysis of the key cell cycle regulatory genes of the cells in both culture modes indicated that the cell proliferation and cell viability of the cells in both batch and fed-batch cultures were mainly regulated through down-regulating Cdk6, Cdk2, Cdc2a, Ccne1, Ccne2 genes of CDKs, Cyclin and CKI family and up-regulating Smad4 gene.
Animals ; Batch Cell Culture Techniques ; CHO Cells ; Cell Cycle Proteins ; genetics ; Cell Line ; Cricetinae ; Cyclin-Dependent Kinase 2 ; genetics ; Cyclin-Dependent Kinase 6 ; genetics ; Gene Expression Profiling ; Gene Expression Regulation ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; Smad4 Protein ; genetics ; Urokinase-Type Plasminogen Activator ; biosynthesis ; genetics

By using the cell density, cell viability, Pro-UK activity, specific consumption rate of glucose (q(glc)), specific production rate of lactate (q(lac)), yield of lactate to glucose (Y(lac/glc)) and as the evaluation indexes, the growth and metabolism characteristics of pro-urokinase (Pro-UK) expressing CHO cells in serum-free suspension batch culture were examined and compared to those in serum-containing suspension batch culture. We observed hardly differences in growth and metabolism characteristics between the CHO cell populations grown in serum-free suspension batch culture and serum-containing suspension batch culture. The optimal mathematical model parameters for the CHO cells grown in suspension batch culture were obtained by non-linear programming of data representing the growth, substrate consumption and product formation of the CHO cells during logarithmic growth phase using MATLAB software, and the kinetic model of the cell growth and metabolism in serum-free culture were established.
Animals ; Bioreactors ; CHO Cells ; Cricetinae ; Cricetulus ; Culture Media, Serum-Free ; Culture Techniques ; methods ; Kinetics ; Models, Theoretical ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Urokinase-Type Plasminogen Activator ; biosynthesis ; genetics ; metabolism

Taking a suspension adapted recombinant CHO cell line, 11G-S expressing human Pro-urokinase (Pro-UK) as the object of study, the impacts of different feeding nutrients, the start time of feeding and cell inoculation density on the growth and Pro-UK production of 11G-S cells in serum-free fed-batch culture were evaluated in 100 mL shacking flasks. The results indicated that amino acids, serum-free supplements and inorganic salts played important role in cell growth, cell viability and protein expression. And the effects of cells fed-batch culture was much better with the initial cell inoculation density at 3 x 10(5)-4 x 10(5) cells/mL and the start time of feeding set at 72 h, a maximum viable cells density of 7.8 x 10(6) cells/mL with a peak Pro-UK activity at 8570 IU/mL was achieved during 12 d fed-batch culture. Further, the mu of the 11G-S cells at the middle phase of the fed-batch culture, and both the q(glu) and q(gln) of the 11G-S cells at the middle and later phases of the fed-batch culture was higher than that of the 11G-S cells at the same phase of the batch culture, respectively.
Animals ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Culture Media, Serum-Free ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Urokinase-Type Plasminogen Activator ; biosynthesis ; genetics ; metabolism