Effects Of Rehmannia Glutinosa Oligosaccharide On The Proliferation, Anti-oxidization And Paracrine Of Adipose Tissue-derived Mesenchymal Stromal Cells

Background: Adipose tissue-derived mesenchymal stromal cells (ADMSCs) are a kind of adult stem cells sourced from fat. ADMSCs have the potency to differentiate into many kinds of tissue such as cardiac muscle, endothelium and nerve. Human adipose tissue is resourceful and can be obtained from abandoned adipose tissues of patients receiving abdominal surgery. It is a safe and acceptable operation to patients to get a great quantity of ADMSCs. ADMSCs can be used as seed cells to regenerate the tissues such as cardiac muscle. In January 2007, Spainish researcher witnessed the first experimental treatment of angina pectoris and heart disease by the way to transplant adult stem cells which obtained from adipose tissues into patients' hearts. The clinical use of ADMSCs in China is hampered by the poor condition of foundational study. Rehmannia glutinosa oligosaccharide (RGO) is effective ingredient isolated from rehmannia, a kind of Chinese herbs. Its molecular weight is 1000-3000. It consists of 60% tetrasaccharide and 20% trisaccharide. The aim of the study is to investigate the effect of RGO on ADMSCs' proliferation, its anti-oxidization and paracrine and provide experimental data for the effective application of RGO.Aim: To investigate the effect of RGO on ADMSCs' proliferation. To investigate the effect of RGO on ADMSCs anti-oxidization induced by H₂O₂. To investigate the effect of RGO on the secretion of VEGF and HGF secreted by ADMSCs.Contents and methods: The study divided into three parts for the aforementioned objectives.Part one: The human ADMSCs (hADMSCs) from abandoned adipose tissues of patients receiving abdominal surgery were isolated and cultured. The cells identification of hADMSCs was done by immunohistochemical method for surface molecules CD44,CD105,CD45,CD34. After the demonstration of hADMSCs, cells were cultured with different concentration of RGO (0,1mg/L,10mg/L,100mg/L,400mg/L) in vitro. The effects of different culture medium on hADMSCs proliferation were detected by MTT (methyl thiazolyl tetrazolium) colorimetry.Part two: Cultured hADMSCs were divided into three groups as the normal group (without any treatment, N group), H₂O₂ group (treated with 0.1 mmol/ L H₂O₂, H group), and 0.2g/L RGO + H₂O₂ group (treated with 0.2g/L RGO +0.1 mmol/ L H₂O₂, R group). After 1,6,24h , the apoptotic rate was determined by flow cytometry. The influence of RGO on apoptosis of ADMSCs induced by hydrogen peroxide (H₂O₂) was investigated.Part three: ADMSCs were cultured with different concentration of RGO (0,1,10,100,400mg/L). At 72 hours after culture, ELISA was used to detect the level of VEGF and HGF in different culture mediums.Results: ADMSCs were identified by positive expression of CD44 and CD105 but negative expression of CD34 and CD45. According to MTT colorimetry's results, there was little and non-significant difference of proliferation between the groups of 1mg/L, ]0mg/L and contrast group (P>0.05) .Both group of 100mg/L and group of 400mg/L show significant pushing effect on hADMSCs's proliferation. The comparison between those two groups showed significant difference and the pushing effect of group of 100mg/L was much better than effect of group of 400mg/L (P<0.05).After 1,6 and 24 h, the apoptotic rate of H group was obviously higher than that of N group and R group (P<0.01), and apoptotic rate of H group was evidently higher than that of R group (P<0.01).It proved that RGO can attenuate apoptosis of hADMSCs induced by H₂O₂.At 72 hours after culture, ADMSCs of groups of 100 mg/L and 400 mg/L had significant proliferation on ADMSCs (P<0.01), and the result of the former was obviously higher than the latter.Conclusion:1 .RGO have positive effects on the proliferation of hADMSCs.2.RGO can attenuate apoptosis of hADMSCs induced by H₂O₂.3.RGO (100mg/l and 400mg/l) has positive effects on ADMSCs' proliferation and can promote secretion of VEGF and HGF indirectly.