Role Of 17-β Estradiol In Osteogenic And Adipogenic Differentiation Of Rat Adipose-derived Stem Cells

Estrogen is a kind of substance which is pertinent to bone metabolism, decomposition and formation of fat as well as its sedimentation. Due to diminution of estrogen and osteoporosis which are triggered by their ongoing ages, mid-aged women will be inevitably bothered by syndromes such as central obesity, augmentation of fat tissues, and etc. Equal heeds should be paid to both the degression of gonads and the physical functions. Osteoblast and lipocyte are probably all derivates of precursor cells or stem cells, and the differentiation balance between them may be subject to same factors. It is still not clear about the catalysis of estrogen and age-related changes to ADSCs. Trough observation on ADSCs of SD rats aging from 3 to 18 months, this study will try to find out the influences exerted by ages and estrogen over exterior proliferation and ontogenesis of ADSCs, and adipogenic differentiation, and discuss the probable functions of age-related changes and estrogen in the regulations ADSCs differentiation. The experiment consists these following contents:1. Adipose-derived stem cells, isolation, purification identification:Depart, purify and culture ADSCs from the SD rat's groin fat by using density gradient centrifugation and adherent of natural law; invert microscope and observe cell morphology; detect the surface markers by flow cytometry. It will exclude the possibilityof being hematopoietic stem cells with mesenchymal stem cell surface antigen CD29 and CD105 positive, CD34 negative. At the same time, if alizarin red and oil red O staining are positive by the induction and differentiation of the bone and the adipogenic, it will prove that the cells cultured and isolated are ADSCs.2. The effects of estrogen on cell growth and proliferation of ADSCs:Through the MTT curves of estrogen's effects on ADSCs of different months, we find that estrogen can promote cells'growth and proliferation, and the capacity of growth and proliferation of ADSCs of 18-month-old is lower than that of the 3-month-old.3. The influence of estrogen on the capacity of ADSCs of different months inducing osteogenic to differentiate in vitro:Objective: To observe the physiological concentration of estrogen (E2) and increasing the age factors on the vitro-induced osteogenic differentiation of ADSCs capacity. Methods: take 3-month-old and 18-month-old third-generation ADSCs prepared (1) ordinary steogenesis induced by fluid-induced group, (2) Add the estrogen-induced fluid induced group, containing a final concentration of estrogen 10-7M (3) Normal routine culture of non-induced group. The alizarin red staining and the biochemical estrogen detection which have some influences on ADSCs that includes the proliferation and osteogenic differentiation. Results: The estrogen induction microscopic observation for 21 days with estrogen-month-old group with alizarin red staining area of more than conventional induction group, the non-induced group were negative. Semi-quantitative RT-PCR tests showed estrogen group compared with conventional induction group expressed strong OCN. Under the same conditions, induced by alizarin red staining area of 3-month-old group are more than 18-month-old group, RT-PCR semi-quantitative detection of OCN expression of 3-month-old group than in the strong 18-month-old group. Conclusion: Estrogen can enhance the in vitro ability to induce osteogenic differentiation. Rat ADSCs in vitro proliferation and induced osteogenic differentiation capacity decline with age-related changes.4. The influence of estrogen on on the capacity of ADSCs of different months inducing adipogenic to differentiate in vitro:Objective: To observe the physiological concentration of estrogen (E2) and increasing the age factors on the ADSCs induced in vitro adipogenic differentiation capacity. Methods: take 3-month-old and 18-month-old third-generation ADSCs setting as follows (1) Conventional adipogenic inducing medium induced group, (2) Add adipogenic inducing medium of estrogen-induced group, containing a final concentration of estrogen 10-7M (3) normal routine cultivation of non-induced group. The oil red O staining and the biochemical estrogen detection which have some influences on ADSCs that includes the proliferation and osteogenic differentiation. Results: The first five days adipogenic microscopic observation group of the same-month-old estrogen-containing oil red O staining area is larger than the conventional induction group, the non-induced group were negative. Semi-quantitative RT-PCR tests showed estrogen group compared with conventional induction group expressed strong LPL. Under the same conditions, induced by oil red O staining area of 3-month-old group are more than 18-month-old group, RT-PCR semi-quantitative detection of LPL expression of 3-month-old group is strong than 18-month-old group.Conclusion: Estrogen can enhance the in vitro ability to induce adipogenic differentiation. Rat ADSCs in vitro proliferation and induced adipogenic differentiation capacity decline with age-related changes.