| ObjectiveStem cells, in terms of embryonic stem cells, are multipotent stem cells that can differentiate into a variety of cell types, including:bone, cartilage, fat, muscle, nerve,tendons, and so on. This has been shown in vitro or in vivo. MSCs have turnned into hot cells of tissue engineering seed cells recently with these characters:easily obtained, forceful proliferation ability, large-scale expansion in vitro without losing pluripotency, the weak immunogenicity, strong immune tolerance, low immune rejection.Currently bone marrow mesenchymal stem cells (BMSCs) mainly obtained from animals and experimental results based on them canot applied to human body, because of the biological differences between animals and human.How to selecte proper donor's age and location of human bone marrow mesenchymal stem cells (human bone mesenchymal stem cells, hBMSCs) through clinical research and choose the optimum culturing method to make cells rapidly proliferation and maintain directional differentiation, is a problem to be solved at present.According to Chinese medicine theory, kidney is regarded as the "birth of the country. " Spleen as the "acquired this. " Spleen of the health movement, subtle chemical and biological weapons, to be driven by means of kidney, kidney essence is also dependent on the cultivation of water and nourishing food essence in order to keep filling and maturity. Mutual assistance between the spleen and kidney, promote each other. As the "Lord of renal development, the main bone marrow", treatment, bone disease often start from the kidney, and spleen Qi based therapy is the treatment by acting in the role of the spleen into the sky.Strong muscle healthy strength drink is the Product of Buzhong Yiqi decoction which Old Traditional Chinese Medicine Te Tao Deng studied. Small molecules can control stem cell proliferation, differentiation, desting. includes several bioligical processes, apoptosis in tumor, degenerative diseases and so on. have broad application prospects. Experimental study of classical prescription for the active ingredient in the screening and found a new compound K9(three-ring monoterpenes)and the test results by MTT K9 (three-ring monoterpenes)that as low toxicity compounds, may launched MSCs differentiation. This experiment is determing hBMSCs to cartilage induced by K9 differentiation.Method1. We collect the bone marrow of the patients undergone surgical operation in Guangzhou University of Traditional Chinese Medicine, First Affiliated Hospital during from February 2010 to March 2011. We adopted adherent whole blood cell culture propagation hBMSCs, passed to two generations, by flow cytometer technology to detect cell surface CD45, CD44 antigen; exploring clinical conserving method of hBMSCs.2. By comparing the hBMSCs first passage time (80% of cell fusion time) among young, middle-aged and elderly patients and bone marrow fluid fat rate (%) among the iliac, femoral, vertebral bone, combined with MTT activity detecting, to determing the age and the best parts of specimens from hBMSCs.3. hBMSCs were induced to cartilage differentiation with different concentrations of K9, after that 3d,7d,14d, using toluidine blue staining glycosaminoglycans, immune staining collagenâ…¡and aggrecan, to further explore the effect of K9 promoting hBMSCs cartilage differentiationResult1. the method of adherent culturing is easy to be operated and suitable for clinical use for workers. Flow cytometry revealed: the cultured cells expressied CD44, but not the expression of antigen CD45, proved adherent whole blood culture specimens from hBMSC not only simple, but reliable. CD44 positive rate was 42.8%, the cells continue to passage for the hBMSCs further purification, 2. hBMSCs first passage time were divided into old age group> middle-aged group> youth Group. And comparied by youth group, middle group passage time has been extended, but no significant difference, time extended in the elderly group and significantly different (P<0.05).3,The samples from different sources of bone marrow, fat percentage (%) were divided into femoral> iliac) vertebrae. Compared with the vertebral bone, iliac bone group fat rates (%) no significant difference (P> 0.05), femoral fat group significantly different rates (P<0.05)4. MTT was involed to detecte the proliferative activity of hBMSCs among the different ages, different parts of the bone marrow.Results showed that: within 72h, hBMSCs cell proliferation is very clear in the youth group. OD values of the middle age group, elderly group were significantly (P<0.05 or <0.01) compared with the young group. In 72h, hBMSCs cell proliferation is very clear in the vertebral bone group.OD values were significantly (P<0.05 or<0.01). between the femoral group and iliac bone group compared with the vertebral bone group5. Toluidine blue staining showed that:the extracellular matrix were dyed with purple, metachromasia, indicating that there are acidic glycosaminoglycans production; immunohistochemistry showed that:hBMSCs can secrete cartilage cells differentiated phenotype of secretory collagen II and aggrecan by K9conclusionWe can culture bone marrow mesenchymal stem cells (BMSCs) from the human femur, iliac bone, vertebral bone and other tissues, all adherent blood method cultured specimens from human bone marrow mesenchymal stem cells should bepassaged to three generations to purify cell lines and can be used in othre research field.
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