| Part I: establishment of osteoporosis model and isolation and identification of mouse adipose derived stem cellsObjective: The pathogenesis of osteoporosis is osteogenic activity decreased and osteoclast activity increased during the bone remodeling process, resulting in the balance of bone metabolism was destructed, causing the bone tissue dissolution was greater than the bone formation. Eventually it leading to the cortical bone became thinning, the bone trabecular density decreased, so the osteoporosis occured, and the incidence of fracture increased as well as the bone healing ability reduced[1]. The source of adipose derived stem cell(ASCs) was rich, and easy to get, it was confirmed that ASCs has the potential to differentiate into bone tissue by the recent researches[2],but weather osteoporosis adipose stem cells have the same ability of bone formation has not been reported. In this experiment, the mice osteoporosis model was established, while isolation and culture of osteoporosis mice adipose stem cells. Methods:(1) Female C57BL/6 mice were randomized into experimental group( ovariectomized group) and control group(sham operation group).The ovariectomized group suffered bilateral ovarian resection during 10-week old( weigh 18~20 grams). 6 mice were picked from each group since 4 weeks and 8 weeks after the surgery individually, then the BMD of the middle femoral shaft,bone volume fraction,trabecular number,trabecular thickness and trabecular spacing were measured by micro-CT under anesthesia to prove the osteoporosis mice model was successful from the iconography perspective. Besides, another 6 mice were randomly picked from the ovariectomized group and the sham group, and their knee joints were sectioned then stained with MASSON and HE, so we can judge whether the osteoporosis mice model was successful or not by observing the trabecular bone density and cortical bone thickness.(2)After execute mice into death by overdose pentobarbital sodium intraperitoneal injection, then disinfect the whole body by 75% alcohol, get the inguinal adipose tissue from the osteoporosis mice, and cut it by using tissue culture method, then culture the primary ASCs in the culture medium which containing 10% FBS, passage ASCs by enzyme digestion,finally the OP-ASCs can be obtained by the second generation.(3)Use the flow cytometry to demonstrated the characteristics of stem cells of osteoporosis mice adipose derived stem cells. Results: 1.Castration method to establish female C57 BL mice osteoporosis model, tissue block method get osteoporosis mice fat stem cells of mouse femoral shaft micro CT flat sweep and results of three-dimensional reconstruction and mice metaphyseal section HE, Masson staining showed that operation group compared with the control group mice osteoporosis. From the image learning organization and learning that castration method to construct osteoporosis mice model successfully, by flow cytometry osteoporosis mice adipose stem cells showed that the stem cell surface antibody positive, in line with the characteristics of stem cells that obtained by tissue block method since osteoporotic mice inguinal fat cells indeed osteoporosis mice fat stem cells. Conclusion: Through the method of castration, we successfully established the animal model of osteoporosis, and obtained OP-ASCs from indirect inguinal fat in osteoporotic micethe by tissue block method..Part II Changes of Wnt signaling pathway in adipose derived stem cells in osteoporotic miceObjective:(OP-ASCs) have been prepared for find out the differential expression of Wnt signaling pathway related genes in osteoporosis adipose stem cells[3-5]. Previous studies demonstrated that Wnt signaling pathway played a crucial role in bone differentiation in bone marrow mesenchymal stem cells(BMSCs), embryonic stem cells, and normal ASCs.But in the OP-ASCs, did the Wnt signaling pathway played the same crucial role like other stem cells ? If yes, and what's the molecular mechanism of osteogenic differentiation regulation ? The aim of our study was to detect the expression changes of crucial genes in Wnt signaling pathway by up-regulating /down-regulating Wnt / beta catenin signaling pathway, then compare it with the crucial genes expression changes of Wnt signaling pathway in other stem cells, help to explore the specific molecular mechanism of how the Wnt signaling pathway regulate the OP-ASCs differentiate into bone tissue, it can help lay the molecular foundation for the clinical application of OP-ASCs into bone defects restorement. Methods: LiCl group and DKK-1 group were respectively defined that whether 50mg/ml LiCl or 0.1?g/ml recombinant mouse DKK-1 protein were added into the culture medium which containing 10% FBS for OP-ASCs, and the OP-ASCs in the culture medium which containing 10% FBS was used for control. The total RNA will be extracted from 3 groups at day 1 and day 3, then the different genes related to Wnt signal pathway were screened by Wnt signal pathway gene chip combined with Real-time PCR. Result: We find that at day1, the OP-ASCs LiCl group compared with control group, Cond 1 genes expression was up-regulated whereas Porcn?Sfrp 1?Sfrp 2?Sfrp 4?Wnt 5a genes expression were down-regulated, while Axin 1 ? Fzd 6 ? Wnt 5a genes expression were up-regulated in OP-ASCs DKK-1 group compared with OP-ASCs control group at day 1. At day 3 we find that Wnt 2 gene expression were up-regulated while Nlk?Porcn?Wnt 5a genes expression were down-regulated in the OP-ASCs LiCl group, however Wif 1 ? Wnt 5a genes expression were up-regulated in OP-ASCs DKK-1 group compared with OP-ASCs control group. Conclusion: 1.In OP-ASCs, the canonical Wnt signaling pathway also works in the regulation of these genes, we hypothesized that added LiCl raised the OP-ASCs activation of canonical Wnt signaling pathway by up regulated Wnt 2 gene expression, reduced the expression of inhibitions of the Wnt signaling pathway Porcn. SFRP1, SFRP 2, SFRP4 gene, so that the downstream genes Ccnd1 expression; and DKK-1 join up the canonical Wnt signaling pathway inhibitors Axin 1, Wif1 gene, thus inhibiting the canonical Wnt signaling pathway. 2. LiCl adding to downregulated the key factor of noncanonical Wnt signaling pathway Wnt 5a, Nlk gene expression and thus inhibition the non canonical Wnt signaling pathway, and DKK-1 upregulated the key factor of noncanonical Wnt signaling pathway Wnt 5a, Fzd6 gene expression to activate the noncanonical Wnt signal pathway. |
PDF Full Text Request