Experimental research of adipogenic differentiative ability of adipose tissue-derived stromal cells in patients with type 2 diabetes mellitus / 重庆医学

Objective To investiagte the adipogenic differentiative ability of adipose tissue-derived stromal cells(ADSCs) between the patients with type 2 diabetes mellitus(T2DM) and healthy persons.Methods The adipose tissues were taken from the adipose tissue in T2DM patients and healthy persons for separating and culturing ADSCs.The cells of third generation were taken for inoculation.The difference in cellular phenotype and growth speed were compared between the two groups.Adding adipogenesis inducing fluid,the adipogenic differentiative situation was observed in the two groups.The oil red O was added on 14 d for conducting the cell staining and observation.The oil red O was extracted by isopropanol,and the cellular absorbances were compared between two groups.Meanwhile,the expression of PPAR-γ,C/EBP-α and C/EBP-β on 14 d of adipogenic differentiation were compared between two groups by using qPCR method.Results The cellular phenotype and growth speed of ADSCs had no statisticat difference between T2DM patients and healthy persons.On 14 d of adipogenic differentiation,the oil red O absorbance value of AD-SCs in T2DM patients was significantly higher than that in the healthy persons,and the expression of PPAR-γ,C/EBP-α and C/EBP-β were significantly higher than those in the healthy persons.Conclusion The adipogenic differentiative ability of ADSCs in T2DM patients is obviously higher than that in healthy persons,which may be one of causes easy to be obese in T2DM patients.

Impact of SPK1 Modified ADSC on Osteogenesis of Tissue Engineered Bone / 华中科技大学学报(医学版)

Objective To investigate the impact of sphingosine kinase 1 (SPK1) modified adipose tissue-derived stromal cells(ADSC)on tissue engineered bone osteogenesis.Methods ADSC cells isolated from SD rat fat cells were divided into CON group and SPK1 group,and then the cells were respectively infected with 10 MOI CON and SPK1 lentivirus for 48 h.The infection efficiency was confirmed by using flow cytometry.Alizarin red and oil red O was used to stain the cells 14 days after ADSC infection,and osteogenic and adipogenic ability was evaluated by detecting A595nm and A490 nm.In the meantime,the activity change of alkaline phosphatase(ALP)was detected.The SD rat femoral defect model was created,and then after combining ADSC with β-TCP,the tissue engineered bone was pressed to the defect site.The repairment of bone defect was detected by X-ray in 4,6 weeks.After infection of CON and SPK1 virus,bone morphogenetic protein(BMP7)expression in ADSC of these two groups was detected.Results The infection efficiency of CON and SPK1 lentivirus was 94.4％ vs.94.9％ respectively by flow cytometry.The SPK1 protein expression level in CON group and SPK1 group was (0.73±0.10) vs.(1.29±0.17)(P＜0.05).The A value of CON and SPK1 group at 595 nm was (0.20±0.02) vs.(0.41±0.01) (P＜0.05),respectively.The A value of CON and SPK1 group at 490 nm was (0.72±0.01) vs.(0.51±0.02)(P＜0.05),respectively.The expression level of ALP in CON and SPK1 group was (1.42±-0.09) vs.(2.68±0.09) (P＜0.01),respectively.In the repairment of bone defect,high density tissue at rat bone defect was significantly larger in SPK1 group than in CON group in 4 weeks,and in 6 weeks,bone defect of SPK1 group was healed,but CON group still had defect.The expression level of BMP7 in CON and SPK1 group was (1.13±0.16) vs.(4.46±0.23)(P＜0.05),respectively,48 h after infection.Conclusion SPK1 modified ADSC has an enhanced osteogenesis ability in vitro and in vivo,which was related to the activation of BMP7.

Transfection of adenovirus containing hepatocyte growth factor gene into adipose tissue-derived stromal cells / 国际外科学杂志

Objective To observe the efficiency of infection of adenovirus containing hepatocyte growth factor(Ad-HGF) on adipose derived stem cells and to prove whether the valid HGF can appear after infection and the multiplicity of infection. Methods We use the digestion separation method and the attachingwall characteristic of the adipose-derived stem cells to separate the human adipose-derived stem cells. Adipose-derived stem cells were infected by the vector of adenovirus (Ad-GFP) which carries the GFP gene,and the GFP acts as the indicating gene to determine the infection efficiency of recombinant adenovirus to adipose- derived stem cells. HGF-ELISA was used to detect HGF as expression-secretion. Results The adherent cells displayed themselves as fibroblast in morphology. The primary cultured cells fusion can arrive to 70% - 80% in 7 - 10 days. The infected HGF can be highly expressed in 48hours. Conclusion Adenovirus can meditate the expression of HGF gene in adipose-derived stem cells effectively.

BONE REGENERATION WITH ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELL AND HA/TCP

The effect of growth factors on osteogenic differentiation of adipose tissue-derived stromal cells

Future cell-based therapies such as tissue engineering will benefit from a source of autogenous pluripotent stem cells. There are embryonic stem cells (ESC) and autologous adult stem cells, two general types of stem cells potentilally useful for these applications. But practical use of ESC is limited due to potential problems of cell regulation and ethical considerations. To get bone marrow stem cells is relatively burden to patients because of pain, anesthesia requirement. The ideal stem cells are required of such as the following advantages: easy to obtain, minimal patient discomfort and a capability of yielding enough cell numbers. Adipose autologus tissue taken from intraoral fatty pad or abdomen may represent such a source. Our study designed to demonstrate the ability of human adipose tissue-derived stromal cells (hATSC) from human abdominal adipose tissue diffentiating into osteocyte and adipocyte under culture in vitro conditions. As a result of experiment, we identified stromal cell derived adipose tissue has the multilineage potentiality under appropriate culture conditions. And the adipose stromal cells expressed several mesenchymal stem cell related antigen (CD29, CD44) reactions. Secondary, we compared the culture results of a group of hATSC stimulated with TGF-beta1, bFGF with a hATSC group without growth factors to confirm whether cytokines have a important role of the proliferation in osteogenic differentiation. The role of cytokines such as TGF-beta1, bFGF increased hATSC's osteogenic differentiation especially when TGF-beta1 and bFGF were used together. These results suggest that adipose stromal cells with growth factors could be efficiently available for cell-based bone regeneration.