From The Nourishing Marrow And Blood Particles Regulation Of ¦Â-globin And Related Genes Expression To Explore The Molecular Mechanisms Of Renal Marrow Theory

This research was directed by the academic ideas in modern research and clinical practice for the theory of "kidney producing marrow". Supported by NSFC,we had studied the whole clinical effect and molecular mechanism of the therapy for β -thalassemia treated by Yi Sui Sheng Xue particle through combined theoretical thinking of Traditional Chinese Medicine with advanced technique. Techniques such as enzyme linked immunosorbent assay, reverse transcription-polymerase chain reaction, laser cnfocal microscopy scanning technique, real-time polymerase chain reaction and differential display reverse transcriptase polymerase chain reaction was used for study. We have studied expression of hematogenesis stimulating factor, interaction of DNA sequence of DNase I hypersensitive site 2 in β - globin gene cluster locus control region and protein in nucleus, expression of the transcriptional factors that play important roles in globin gene expression and erythroid differentiation and expression of ferritin gene coherent to haematolysis. The objective was to investigate the molecular mechanism of regulation the β - globin gene expression of β-thalassemia patients treated by this drug. We have obtained high academic value findings. Theoretical StudiesThe kidney producing marrow theory includes four parts. Those are the internal relationship of kidney producing marrow and marrow storing pneuma, kidney producing marrow and marrow nourishing bone, brain communicating kidney producing marrow, kidney producing marrow and marrow promoting hemogenesis. Apprehension in the kidney producing marrow theory was deepen through analyzing and summing up the past dynasties literature. Four parts including the dependability of kidney producing marrow and neurological function, studies in the material foundation of kidney producing marrow and marrow nourishing bone, objectivity of kidney producing marrow and marrow promoting hemogenesis and molecular mechanisms of kidney producing marrow was studied by analyzed pertinent literature of modern research in this theory. The objective was to summarize the achievement in the theoretical studies and trying to find outnew study way for classical theory of TCM.We investigated regulation and switching mechanisms of globin gene expression in five parts that were globin gene organization and temporal and spatial expression order, globin gene structure and expression, the transcription factors that play important roles in globin gene expression and erythroid differentiation, globin gene cis acting elements and functions of the J3 -globin gene cluster locus control region. Penetrating apprehended thalassemia pathogenesy can provide theoretical foundation. Summaring the advisor's research achievement in J3 -thalassemia treated by TCM invigorating the kidney and promotating hemogenesis would be study deeply in advisor's academic thoughts and scientific research ideas.Empirical studies in regulation mechanisms of globin gene and related genes expressionThe whole clinical effect of 3 -thalassemia treated by this drug was studied at two parts. Results were as follow: clinical observation was used random mono-blind placebo parallel comparison. The result demonstrated that hematologic parameter such as hemoglobin, red blood cell count, reticulocyte count and fetal hemoglobin of treated group increased obviously. No marked change was found in the placebo group. B-mode ultrasonography showed that spleen diameter of sufferer post-treatment was obviously less than that of prior treatment. At post-treatment sufferers' TCM symptom was improved notably. Hematologic parameter heighten was concord to the amendment of clinical symptom. Laser confocal microscopy scanning technique showed that the fluorescence intensity of deoxyribonucleic acid and ribonucleic acid in marrow erythropoiesis reinforced obviously at post-treatment. This result hinted that this drug can maintain normal structure and physiological morphous of earlier period hematopoietic cell.Techniques of enzyme linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR) were used to detect the genetic expression of haemopoiesis stimulating factor in peripheral blood and marrow. The result showed that this agent can stimulate genetic expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in marrow, interfere in the procedure of hemopoietic progenitor cell differentiation, promote activeerythropoiesis and relief the symptom of anaemia. This drug can increase the genetic expression of c-kit, which is a proto-oncogene in peripheral blood; regulate the signal conduction pathway of stem cell factor/ c-kit system. Thus the pathobiology changes in cell differentiation of erythropoiesis and multi-hematopoietic cell was reablement. This drug also can raise the level of erythropoietin in peripheral blood , promote the active erythrocytopoiesis, increase fetal hemoglobin synthesis and depress poison function result in free a -hemoglobin deposition. Erythropoietin receptor gene expression was cut down. So invalid erythropoiesis decreased and normal haematopoiesis would be recovered. From those results we supposed that this agent could regulate genetic expression of haemopoiesis stimulating factor, so interfere in the physiological functions of SCF-GM-CSF-EPO system. Those results hint that physiological functions of SCF-GM-CSF-EPO system in hemopoietic progenitor cell differentiation were one material foundation of kidney producing marrow and marrow promoting hemogenesis.The experimental result of gel lag test displayed that the interaction of the hypersensitive site 2(HS2) in the P-globin gene cluster locus control region and neucleoprotein in karyocyte had been changed after treatment. The sequence of HS2 includes two motifs specific binding GATA-1 and GATA-2 (trans-acting factor in globin gene expression and erythroid differentiation). Because of this change, the synthesis of fetal hemoglobin was increased. As a result, poisonous function generated through liberated a -globin chain deposited on competent cell in liver and hemocyte. This effect can regulate erythro- ancestral cell differentiation and erythropoiesis, so promote valid erythropoiesis.We studied the genetic expression of the transcriptional factors that play important roles in globin gene expression and erythroid differentiation at prior treatment and post-treatment by real-time PCR. The results showed that this drug could elevate the genetic expression of the trans-acting factors GATA-1 and GATA-2 notably. GATA-1 may promote the Y -globin gene expression and increase the production of fetal hemoglobin. It also can induce a protein stabilizing free alpha-haemoglobin generating. This protein, named Alpha Haemoglobin Stabilizing Protein (AHSP), is an abundant, erythroid-specific protein that forms astable complex with free alpha-haemoglobin but not with beta-haemoglobin or haemoglobin A. AHSP specifically protects free alpha-haemoglobin from precipitation in solution and live cells. GATA-2 in coordination with GATA-1 could promote the hemopoietic progenitor cell differentiation and increase the active erythrocytopoiesis. The gene expression of nucleic factor-erythroid 2 (NF-E2) had risen obviously in marrow's nucleated cell after treatment. NF-E2 is transcriptional factor that play important roles in globin gene expression and erythroid differentiation. Increasing its gene expression would advance globin genetic transcription and active erythropoiesis. It also can regulate ferric absorption and metabolism, relieve ferrugination in vivo. Hence, this effect could depress ferri burden in sufferer's organism. However, this drug has no regulative obvious, function on erythroid kruppel-like factor (EKLF)and embryonic/fetal -like globin gene-activating Kriippel-like factor (FKLF).The result of different display RT-PCR showed ferroprotein genetic expression decreased obviously in marrow karyocyte of sufferers after treated by this drug three month latter. It could regulate NF-E2 gene expression, which concerned with ferroprotein metabolism. This result hints that this agent can promote absorption and metabolism of ferri and cut down ferri burden in vivo efficiently, so as to control endosomatic ferrum accumulation, because it can interfere in metabolic pathway of ferritin. Empirical study demonstrated hypothetical rationality that the therapy of Traditional Chinese Medicine could change the expression of correlated functional gene.New ideas brought by this studyIt was first time that studied the molecular mechanism of P -thalassemia treated by TCM from changes in regulation of the expression of globin gene and important significant transcriptional factors in globin gene expression and erythroid differentiation and function's alteration of DNA-protein interaction at HS2 in ￡ -LCR. There was not found that analogic investigative reports which concerning with study in the mechanism of curative effect for thalassemia therapy.The physiological function of SCF-GM-CSF-EPO system promote erythroid differentiation and mature in several parts, which were regulating the gene expression stimulating factor in haematopoiesis, intervention in the physiological function of SCF-GM-CSF-EPO system, enhancement in erythrocytic...