Study On The Mechanism Of Angelica Polysaccharide Regulating Bone Marrow Mesenchymal Stem Cell Of CML Senescence And Inhibiting The Proliferation Of Leukemia Stem Cells

Background : Stem cells have the ability of self-renewal and multi differentiation, which is one of the key factors to maintain the structure and function of tissues and organs. The occurrence and development of human aging and senile diseases are closely related to stem cell aging. Previous research proof that hematopoietic stem cells(HSCs) aging and various senile diseases are closely related, mainly for hematopoietic system recession caused by severe anemia, a weakened immune system defenses reducing, cell proliferation and differentiation of runaway which may cause leukemia. Studies have shown that with the gradual increase in the age, hematopoietic inductive micro environment ecological niches are reducing the number and recessing the function, resulting in HSC number decreasing and function reduced. In the end, elderly related diseases eventually occurrence. Stem cells is an important model to study cell aging. In-depth study of stem cell aging and delaying stem cell senescence of the modern biological mechanism, not only has important scientific significance in promoting the study of aging but also have immeasurable social value in the prevention of age-related diseases and treatment of degenerative diseases.Hematopoietic inductive microenvironment(HIM) is the place of HSCs surviving, proliferating and differentiating. It is not only the support of HSC survival, but also creates a lot of hematopoiesis regulation factors, affecting the cellular signal transduction on the function plays a role in regulation. The modern theory of hematopoietic is that HIM’s core component is bone marrow stromal cells, it constitutes a specific ecological niches regulating HSCs function. Bone marrow stromal cells(BMSCs) is composed of a variety of cells in mixed cell populations. Most of them originated from bone marrow mesenchymal stem cells of bone mesenchymal stem cells(BMSCs). Therefore, it is necessary to study the effects of bone marrow mesenchymal stem cells on the senescence of hematopoietic stem cells.Chronic myeloid leukemia(chronic granulocytic leukemia(CML) is a hematopoietic stem cell clonally proliferating disease. CML has bone marrow hyperplasia of myeloid cell, peripheral blood leukocyte increased and splenomegaly as the main feature, and a high prevalence of the elderly. Studies have shown that the development of leukemia is closely related to the development of leukemia stem cells(stem cell leukemia, LSC). At present, it is believed that LSC evolved from normal HSC, especially HSC aging or HIM changes are the key factors in the formation of LSC, while LSC is also the difficulty of clinical treatment of leukemia. So far there is no ideal treatment for leukemia except HSC transplantation. But the HSC transplantation is complex, especially difficult to obtain ideal donor cells. And because of it costly, most patients have no therapeutic opportunities. How to cure leukemia is still difficult to overcome the bottleneck of the medical profession.Angelica is a traditional Chinese medicine clinical drug for enriching the blood. And angelica polysaccharide(ASP) is one of the main active ingredients of the drug. Our research has shown that ASP has an important regulatory effect on the proliferation and differentiation of HSCs. ASP can delay the HSCs aging, promote the hematopoietic function of bone marrow in anemia animals, and has a good protective effect on immune organs. Recently, we have found that ASP can promote the aging of LSC, and have a better therapeutic effect on transplanted leukemia. The role of ASP in the "two-way regulation" is not clear. If we can find an effective components of natural drugs which can not only inhibit the proliferation and differentiation of leukemia cells, but also can have a good protective effect on the normal hematopoietic cells of bone marrow. That will bring new therapeutic approaches to the treatment of leukemia and other tumors.Objective:In this paper, the new modern theory and technology of stem cells, traditional Chinese medicine "Qi-Xue" theory and aging theory combined in order to research the following contents: 1. With the age increasing, how are the aging biological characteristics of bone marrow HSCs and BMSCs in mice and human; 2. Bone marrow mesenchymal stem cells’ aging biology and its influence to the LSC; 3. The mechanism of ASP regulating BMSC aging and inhibiting LSC. The research has important theoretical value to explain the theory of stem cell aging and the theory of ‘Qi-Xue’ in Chinese medicine, and also has important application value for clinical treatment of leukemia.Method:1. Male C57BL/6 mice were divided into A group(3~4 week old), B group(2 month old), C group(6 month old), D group(12 month old), E(18 month old). Extract bone marrow mononuclear cells from each group, using the modified MACS method to separate and purify the Sca-1+HSC/HPCs. The percentage of positive cells in each age group was observed by aging related SA-β-gal staining, and the colony forming ability of Sca-1+HSC/HPCs in different age groups were compared with the mixed colony of hematopoietic progenitor cells(CFU-Mix).2. Take bone marrow from the normal human, then divide them into young group(3 cases, 26~31 years), middle-aged group(3 cases, 52~59 years) and older age group(3 cases, 62 years old), using the modified MACS method to separate and purify the CD34+CD38-cells. Flow cytometry was used to detect the purity, trypan blue staining was used to detect cell survival rate and SA-β-gal staining was used to detect cellular senescence.3. The human bone marrow mononuclear cells were isolated and cultured in vitro for 14-21 days. The number of fibroblast colony forming units(CFU-F) was counted under the inverted microscope. CD34, CD45, CD73, CD90, and CD105 expression in bone marrow mesenchymal stem cells were analyzed by flow cytometry. Flow cytometry was used to detect the cell phenotype of the third generation of BMSCs. SA-beta-gal staining was observed in the percentage of positive cells in each age group. CCK-8 test compared the ability of CFU-F forming in each age group’s BMSCs.4. Take bone marrow from the normal human in 3 cases. Five cases of chronic myeloid leukemia patients were divided into control group and ASP group. Control group, conventional culture while ASP group cultured with 10 g/ml Angelica polysaccharide. CFU-F number of each group were counted, CCK-8 was detected in each group BMSC proliferation, SA- beta-gal staining to detect the positive rate of aging cells. The osteogenic differentiation ability of BMSC was detected by alkaline phosphatase staining, and oil red O staining was used to detect the ability of BMSC to differentiate into fat.5. To detect the contents of SOD, total glutathione, MDA and superoxide in every groups, and observe the oxidative damage and antioxidant capacity of the cells in the groups. Elisa was used to detect the content of AngiopoietinⅠ(Ang Ⅰ)to detect the supporting role of BMSC in each group in hematopoietic system.6. RT-PCR and Western blotting were used to detect the gene p19 Arf, p21Cip1/Waf1 m RNA, p16INK4 a, p53 expressing of BMSC, and the expression of senescence associated protein P16INK4 a, P21Cip1/Waf1, P19, P53 in all groups.7. The third generations of the groups of BMSCs were added to the lower zone of the transwell plate, the LSCs were added to upper zone, each hole has added 106 LSCs. Culture 24 h, take out the upper layer of CD34+CD38-cells for CFU-Mix culture and CCK-8 proliferation ability test.Result: 1. The percentage of Sca-1+cells before MACS cells was(0.19 1.02). The purity of Sca-1+ cells was up to 0.83 93.66% after purification. There was no significant difference in the percentage of Sca-1+ cells in each age group. The CD34+CD38- cell population ratio per 106 BMNCs was 1.76 0.34% before MACS separation and purification. The proportion of CD34+CD38-cells in 106 BMNCs was 91.15 2.41% after MACS purification. The CD73, CD90, CD105 antigen are high expressed in BMSCs, while CD34, CD45 antigen are low expressed.2. With age increasing the ability of generating CFU-F of Youth group, middle age group and elderly group declined progressively. The number of CFU-Mix of human CD34+CD38- cells with aging gradually decreased, and also the number of cells in CFU-Mix decreased gradually. SA-β-gal staining of BMSCs positive rate increased with aging. The quantity of CFU-Mix formation of mice Sca-1+HSC/HPCs in A group, B group, C group, D group and E group reduced with aging gradually. The number of CFU-Mix of mice Sca-1+HSC/HPCs decreased gradually. The number of SA-β-gal staining positive cells of murine BMSC increased gradually with aging. The proliferation of mouse or human BMSC showed a gradual decreasing trend with aging.3. LSC and BMSC co-culture results showed that the number of LSC colony in the blank control group and the CML group was significantly higher than that in the other groups, and the number of cells in the colony was also higher than that in the other groups. CML-ASP group set fall significantly less than the number of CML group, which support that the prompt intervention of ASP BMSC have inhibitory effect on the proliferation of LSC and ASP can delay senescence of BMSCs. What is the mechanism of inhibiting LSC proliferation remains to be investigated.4. CML patients with BMSC compared with healthy people’s BMSC, the ability to form CFU-F decreased, while the role of CML in patients with BMSC ASP, the ability to form CFU-F significantly improved. The number of BMSC stained positive for SA-β-gal was significantly increased in the leukemia group, while the number of SA-β-gal positive cells was significantly decreased in the ASP group. The ability of BMSC to differentiate into bone and fat in the leukemia group was significantly lower than that in the ASP group, and the ability of BMSC to differentiate into bone and fat was significantly higher than that in the leukemia group., the activity of SOD, total glutathione and angiogenesis in CML group content significantly decreased, but MDA and superoxide were significantly increased, and superoxide dismutase(SOD) of BMSCs in the CML-ASP group, total glutathione and Angiopoietin I content was increased and the content of MDA and superoxide were significantly reduced in CML- ASP group.5. PCR showed that p16INK4 a, p19 ARF, p53 and p21cip1 / WAF1 gene was expressed in each group of BMSC, and the p16INK4 a p19ARF, p53 and p21Cip1/Waf1 m RNA expression levels of CML group were significantly higher than other groups, CML-ASP group’s gene expressing level is lower than that in CML group. The result of Western blot suggests that the BMSC in four groups all have expressed the p16INK4 a, P21Cip1/Waf1, p53 and P19 protein, CML group was significantly higher, CML-ASP group’s corresponding protein expression was lower than that of leukemia group, which suggest that ASP can delay the senescence of human BMSCs. The mechanism may be related to the p16INK4a-Rb and p19Arf-Mdm2-p53-p21Cip1/Waf1 signaling pathway controlled by ASP.Conclusion: 1. HSCs and BMSCs of mice and human appear senescence with age, which showed that the proliferation and differentiation ability decreased, and the senescence of HSCs and BMSCs may have relationship.2. With the increasing of age, human bone marrow mesenchymal stem cells appear senescence on hematopoietic system, especially the supporting function to hematopoietic stem cell decreased, which may lead to hematopoietic stem cells into leukemia stem cells, and suffering from leukemia. And because of the existence of leukemia stem cells, the effect of leukemia treatment is not good and easy to relapse. This part of the experiment support that angelica polysaccharide may inhibit leukemic stem cell proliferation and differentiation through delaying mesenchymal stem cell senescence and reducing the prevalence rate of chronic myeloid leukemia(CML).3. Angelica polysaccharide can regulate CML bone marrow mesenchymal stem cell aging, so as to inhibit the proliferation of leukemia stem cells. And the possible mechanism may be improving the cell’s antioxidant capacity and reducing cell oxidative damage. It may also be related to the regulation of p16INK4a-Rb and p19Arf-Mdm2-p53-p21Cip1/Waf1 signaling pathways. This discovery could be a new way for us to explore new methods for the treatment of CML in the future.