The Isolation And Culture Of Mesenchymal Stem Cells From Human Adipose Tissue & The Effect Of Phosphatidylcholine On Cultured Human Adipocytes

Background:The cellular component of tissue engineering play a key role in bringing these tissue engineered constructs from the laboratory bench to the clinical bedside. In recent years, there were evidence that some stem cells cells existed in adipose tissue, which was called AMSCs (adipose tissue derived marrow mesenchymal stem cells) . AMSCs were proved in the following years that they could be induced to different into osteoblast cartilage lipocytes cardiac muscle cells neuronal lineages, etc. However,the ideal source of stem cells from adipose tissue still remains unclear,whether adipose-derived stem cells are the origin of differentiation cells ,which are all worth discussing deeply.OBJECTIVE: To find a method which can be used for isolation and cultivation of mesenchymal stem cells from human adipose tissue in vitro,providing a experimental evidence for AMSCs'application extensively. METHODS: The adipose tissue was obtained from subcutaneous adipose tissue of abdominal surgery patients under aseptic condition, cultured with collagenase digestion, The cells were observed under inverted microscope each day and cell growth was studied with cell counting, Calculate the population doubling time of the cells; Immunocytochemistry was used to determine the surface molecule CD44; the cells was in DMEM medium supplemented with 10% fetal boven serum,1%ABAM,1μmol/L dexamethasone,10μmol/L insulin,0.5mmol/LIBMX for 1 week as adipogenic-inducing culture, observing the cells'change in shape and using red oil "O"staining to determine the fat grain in cytoplasm.RESULTS: There was a large amount ofmesenchymal stem cells in human adipose tissue, the most cells were shuttle-like in shape, and growed as fibroblats-like. There were also some muti-shape cells among the shuttle-like cells. The population doubling time of the cells was about 55 hours, the cells'proliferation rate were not slow obviously after being cultivated for 10 generations; Immunocytochemical staining showed that 70 percent of cells were CD44 positive; and they could differentiate into mature adipocytes: after being adipogenic-inducing cultured for 2-4 days, there were vacuoles in cytoplasm, which reached the peak after 1 week, the small vacuoles fused to form bigger vacuoles, and the cells began to excrete fat grain. Red oil "O" staining demonstrated that the vacuoles in cytoplasm were really fat grain.CONCLUSION: 1,In this experiment, AMSCs had been cultured successfully from human adipose tissue using collagenase digestion way. the way of culture is convenient and easier to carry out. 2,Immunocytochemical staining showed that 70 percent of cells were CD44 positive, which showed that the cultured cells was marrow mesenchymal stem cells, but the component of cells was multiplicate.3,The AMSCs was induced to be mature adipocytes successfully using the medium including dexamethasone,insulin and IBMX, the effect of induce in this way was obviously. PartⅡ:The effect of phosphatidylcholine on cultured human adipocytesBackground.In rencent years, in Europe and the United States a number of scientists are exploring a reduing fat technology without pain or hurt, which is known as "the dissolving fat injection", that is, subcutaneous injecting a drug called "Phosphatidylcholine choline mixture" to achieve dissolved fat results. As we know from above that Phosphatidylcholine for partial dissolving fat is still in exploration stage. Whether Phosphatidylcholine can dissolve fat tissues, or whether the drug has side effects on the human body are worth exploring. There has been little research in this field.OBJECTIVE: To explore the effect of phosphatidylcholine on fat gather in cells during the human AMSCs were differentiated into mature adipocytes, and the effect of phosphatidylcholine on the mature adipocytes' vitality, discussing the feasibility of phosphatidylcholine which is as dissolving fat drug.METHODS: Adipose tissue-derived stem cells were cultivated in vitro. Several thickness (0,0.05%,0.5%,5%) of phosphatidylcholine were added to the culture media of human adipocytes, calculating fat content in cells using red oil "O" staining, determining the OD score on enzyme-mark intrument, the data obtained were statistically analyzed by computer with the software package SPSS10.0 for windows. The mature adipocytes'growth were disturbed by using Several thickness (0,0.05%,0.5%,5%) of phosphatidylcholine, observing the cells vitality using AO/EB staining after 3 days,5 days and 7 days since disturbence. RESULTS: Phosphatidylcholine inhibited obviously fat gather during differentiation. The statistics results was: the OD value among several thickness had significant (P＜0.05) comparing to the control group at the same time.The OD value among several time group had significant (P＜0.05) at the same thickness. AO/EB staining results:the nucleolus of control group displayed bright green fluorescence, which showed that the cells'vitality of control group were good. At the same time, comparing to the control group, 0.05% Phosphatidylcholine group to induce mature fat cells to apoptosis after three days'effect, the nuclear displayed yellow green fluorescence. Concentration of 0.5% Phosphatidylcholine group in 3 days, 5 days, 7 days cells were all characterized necrosis state with a bright red fluorescence nuclear. 5% Phosphatidylcholine group did the most serious damage to cells, the cells began to necrosis completely after 5 days, mostly cells began to be dissolved after 7 days.CONCLUSION: 1,Phosphatidylcholine could inhibit fat gather during AMSCs differentiation:the higher was thickness, the more obviously was the inhibition function at the same time; the longer was time, the results showed that the 5% group had the most obviously destroy function. 2,The author observed that phosphatidylcholine had the destroy function to the cells' membrane, so its destroy function to the cells wound have some relation with this function.