The Effect of Abnormal Expression of HIF-1α in Acute Leukemia BMSCs on Bone Marrow Angiogenesis and the Intervention Mechanism of Ginsenoside Rg3
|School||Third Military Medical University|
|Keywords||Acute leukemia Hypoxia-inducible factor -1α Vascular endothelial growth factor Microvessel density Angiogenesis Ginsenoside Rg3 MAPK PI3K/Akt Bone marrow stromal cells|
Objective: bone marrow microenvironment neovascularization plays an important role in the onset and progression of acute leukemia, but its mechanism is not yet fully elucidated. The bone marrow microenvironment and leukemia cells can be through the secretion of pro-angiogenic factors or media to promote angiogenesis, both equally important role. The study found that the bone marrow angiogenesis occurs not only occur in acute lymphoblastic leukemia and acute myeloid leukemia. Angiogenesis is a very complex pathophysiological process, the abnormal expression of oncogenes and tumor suppressor genes, local tissue response to hypoxia, cytokines, etc. play an important role. As an important transcription factor hypoxia-inducible factor-1α (hypoxia inducible factor-1α, HIF-1α), regulation of a variety of promoting the expression of angiogenic genes and factors, may be involved in acute leukemia angiogenesis. Previous studies have found, in the genesis and development of a variety of malignant solid tumors, HIF-1α expression have obvious abnormalities, and with tumor neovascularization and prognosis; increased expression of HIF-1α and tumor suppressor genes inactivation and tissue hypoxia related. There are also high expression of HIF-1α in acute leukemia bone marrow stromal cells, but there is no direct evidence of the relationship between vascular with leukemia bone marrow microenvironment newborn. The purpose of this experiment is to research on the basis of HIF-1α and regulation of the downstream vascular generate relevant factor VEGF expression in acute leukemia bone marrow stromal cells in the characteristics and laws of analysis of HIF-1α and acute leukemia bone marrow microvessel density between relations; explore the mechanism of HIF-1α signal transduction pathways involved in acute leukemia bone marrow angiogenesis; and further study of anti-angiogenic drugs Rg3 on the inhibition produced by vascular bone marrow of acute leukemia and its related signaling pathways for HIF-1α in leukemia bone marrow The hematopoietic microenvironment exception to provide reliable experimental evidence. Methods: 1. Acute leukemia bone marrow stromal cells of HIF-1α abnormal expression generated with the bone marrow vascular relationship study collected 15 cases of acute myeloid leukemia patients, 10 patients with acute lymphoblastic leukemia patients and 10 normal controls bone marrow biopsy specimens and bone marrow stromal cells: (1) The application of immunohistochemistry SP method in the detection of bone marrow biopsy specimens HIF-1α, VEGF expression and microvessel density value (MVD); (2) isolated and cultured in patients with acute leukemia and normal bone marrow stromal cells; (3) Western blot assay HIF-1α, VEGF expression in acute leukemia bone marrow stromal cells. Role in its mechanism to collect the initial issuance of the 31 cases of patients with leukemia bone marrow samples. Rg3 against acute leukemia bone marrow angiogenesis, divided into of acute lymphoblastic groups and ANLL group. 28 cases of normal bone marrow samples as a control group: (1) isolated and cultured in patients with acute leukemia and normal bone marrow stromal cells; (2) Drug grouping: the ginsenoside Rg3 role of bone marrow stromal cells concentration were 20,40,60,80,100 mg / L, 6, 12, 24 and 48 h of culture; (3) MTT assay ginsenoside Rg3 on bone marrow stromal cells best time to the role and function of the concentration; (4) RT-PCR detection ginsenoside Rg3 treated acute leukemia bone marrow stromal cell HIF-1α and VEGF mRNA expression changes; (5) Western blot method, immunofluorescence assay ginsenoside Rg3 treated acute leukemia bone marrow stromal cell expression of HIF-1α, VEGF, p-Akt, p-ERK protein expression changes. Group and acute lymphoblastic leukemia (ALL) group of HIF-1α expression was significantly higher than the normal control group. Abnormal acute leukemia bone marrow stromal cells of HIF-1α expression generated with the bone marrow vascular relationship (1) acute myeloid leukemia (AML) (P lt; 0.05), acute myeloid leukemia group with acute lymphoblastic leukemia group difference was not statistically significant (P gt; 0.05); (2) acute myeloid leukemia (AML) group and acute lymphoblastic leukemia (ALL) group VEG expression was also significantly higher than the control group (P lt; 0.05), acute myeloid leukemia group with acute lymphoblastic leukemia group showed no statistical significance (P gt; 0.05); (3) acute myeloid leukemia group and acute lymphoid cell leukemia MVD was higher than that in the normal control group (P lt; 0.05), acute myeloid leukemia group with acute lymphoblastic leukemia group difference was not statistically significant (P gt; 0.05); (4) bone marrow biopsy specimens, acute leukemia group HIF-1α and VEGF expression, MVD was being related (r = 0.848, P lt; 0.05; r = 0.211, P lt; 0.05), of VEGF expression and MVD count was being related (r = 0.249, P lt; 0.05); (5 ) bone marrow stromal cells, acute leukemia group expression of HIF-1α and VEGF expression, MVD was being related (r = 0.683, P lt; 0.05; r = 0.374, P lt; 0.05), of VEGF expression and MVD count was positively correlated (r = 0.491, P lt; 0.05). Best action time of the study (1) 2. Rg3 against acute leukemia bone marrow angiogenesis and its mechanisms MTT assay ginsenoside Rg3 on bone marrow stromal cells for 24h, the optimal concentration 40ug/mL; ( 2) warp the ginseng saponins Rg340ug/mL role after 24 h, acute leukemia bone marrow stromal cell expression of HIF-1α, VEGF mRNA expression and protein expression were reduced compared with the control group, there is a significant difference (P lt; 0.05); (3) ginseng - Rg3 can inhibit a key protein signaling pathway of Akt, ERK1 / 2 phosphorylation of proteins, which were blocking the PI3K/Akt and MAPK pathways, compared with the control group were significantly different (P lt; 0.05). Conclusion 1. Acute leukemia bone marrow microvessel density was significantly higher than the control group, suggesting that angiogenesis may play an important role in the occurrence and development of acute leukemia. 2.HIF-1α highly expressed in acute leukemia and bone marrow biopsy specimens and bone marrow stromal cells and high expression of HIF-1α may promote acute leukemia angiogenesis by upregulating the expression of VEGF, HIF-1α expression in bone marrow stromal cells may acute leukemia play an important role in the occurrence and development. Ginsenoside Rg3 by HIF-1α inhibit VEGF mRNA and protein expression; MAPK, PI3K/Akt two ways to have an important role in regulation inhibit the expression of HIF-1α protein; ginsenoside Rg3 might inhibit angiogenesis acute leukemia play an important role.