Dissertation > Excellent graduate degree dissertation topics show

Study on the Apoptosis of K562 Cells Induced by Glucosamine Sulfate and Berbamine

Author: GengCaiHong
Tutor: FuChunJing
School: Zhengzhou University
Course: Pathology and Pathophysiology
Keywords: Glucosamine sulfate Berbamine Caspase-3 Survivin Cytochrome C
CLC: R733.7
Type: Master's thesis
Year: 2007
Downloads: 103
Quote: 0
Read: Download Dissertation

Abstract

Leukemia is a common clinical hematopoietic malignancies, the effects of modern conventional treatment has reached a plateau, the majority of patients can not tolerate chemotherapy drugs side effects or because of the leukemia cells resistant to chemotherapy drugs lead to treatment failure . Leukemic cells induced apoptosis is one of the strategies to treat leukemia, the leukemia cell inducer looking for efficiency and low toxicity is particularly urgent. The in-depth study of the mechanisms of tumor apoptosis, will be a good foundation treatment of malignant tumors. Glucosamine sulfate (glucosamine, GS) is an important derivative of glucosamine, an important raw material for the synthesis of amino viscous polysaccharide of the human body, glycoproteins, glycolipids, and other macromolecules. Recent experimental study revealed GS to a new role in the biological research has found that glucosamine hydrochloride K562 cells induced differentiation to macrophages direction, its sulfate can induce apoptosis of K562 cells. GS from natural organisms, safe and reliable, drug side effects; has a strong penetration than the higher bioavailability of similar products; quickly into the interior of the cell; few studies in hematological malignancies. Reported GS activation of Caspase-3 and Bcl-2 protein levels, and induced apoptosis through the mitochondrial pathway down Bcl-2, changing the transmembrane potential, promote membrane permeability pore opening and activation of Caspase- 3 is realized. Berbamine (Berbamine, BBM) is a plant extract from the Chinese herbal Berberidaceae (Berberis) of a two benzyl isoquinoline alkaloids, berbamine its derivatives are calmodulin antagonist (Calmodulin antagonist). Recent studies have found that the berbamine role reversal leukemia resistant, but also have significant anti-tumor effect. BBM chronic myeloid leukemia blastic phase K562 cells Imatini resistant K562 cells and primary leukemia cell growth was significantly inhibited, and induce apoptosis in leukemia cells, but its role in the molecular mechanism is not yet very clear. The research reported BBM obvious K562 cells during apoptosis induced Caspase-3 expression levels. The cross point of these two drugs in K562 cells, so we have chosen these two drugs used in combination, to explore whether inducing apoptosis pathway additivity, which could further enhance the induction of K562 apoptosis. Survivin is a unique member of the recently discovered IAPs family, Survivin gene is located on chromosome 17q25, a slight expression in normal human placenta and thymus tissue, almost undetectable in other normal tissues. Survivin is overexpressed nearly all common malignant tissue and leukemia have high expression often prompt patients with a poor prognosis. Survivin can suppress apoptosis, and promote cell proliferation, and to play an important role in cell mitosis. Survivin inhibits apoptosis pathway downstream of Caspase-3 and Caspase-7 zymogen into the activity of Caspase-3 and Caspase-7, which play an anti-apoptotic role. Therefore, the target gene, can provide a good way for gene therapy of leukemia. Caspases (cystein aspartate-specific proteases) is a group having a high degree of homology, and matured with cytokine and apoptosis-related protease, plays an important role in the apoptosis signal transduction. Caspases family was found since its members have more than 14 kinds, wherein Caspase-3 are expressed in many normal cells or tumor cells is an important effector molecule in the activated reactive Caspases waterfall, can activate the other of Caspases, is called \amplifier \Caspase-3 is the activation of a key enzyme in the apoptotic process, is the major effector molecule of apoptosis, and its activity changes directly affect apoptosis. During execution of apoptosis, Caspase-3 as a key enzyme in the apoptotic cascade pathway, may all or part of recognition and hydrolysis of the corresponding substrate protein. Once apoptotic signaling pathways activated Caspase-3 capable of intracellular the corresponding protein degradation, dying cells irreversible. Cytochrome C (cytochrome, Cyt c) is a water-soluble protein, located between the mitochondrial outer membrane connected with endometrial relaxation. The Cyt c In addition to the participation in cellular respiration activities, significance activate Caspases and induction of apoptosis. , When moved to the cytoplasm from the mitochondria bit activating factor -1 (apoptotic protease-activating factor, Apaf-1) binding and apoptosis, and procaspase-9 interacting, the formation of apoptotic composite body, which activates caspase-3, start the apoptotic cascade triggering apoptosis. In this study, we have chosen of Survivin of Cyt c, Caspase-3 These three have the basis of the molecular mechanism of close contact with the protein as a study GS and BBM combination induced apoptosis of K562 cells. The aim of the present study was designed to study GS the BBM joint and separate medication-induced apoptosis of K562 cells molecular mechanisms: First, to detect different concentrations of GS BBM alone proliferation inhibition rate of K562 cells, derived both alone best effective concentration; then this combination of two optimal concentration, observation of apoptosis induced by the combination of cells, to provide experimental basis for clinical drug combination; detect the two drugs alone and Survivin in combination induced apoptosis Caspase-3 protein expression levels change Cyt c protein content in the cytoplasm to explore the molecular mechanisms in inducing apoptosis of K562 cells and observe whether the two synergistic pro-apoptotic effects. Cell culture methods to different doses of the drug treatment of chronic myeloid leukemia K562 cells blastic phase, inverted microscope to observe changes in cell morphology and photographed; depicts cell growth curve count cell growth and cell counting method; MTT colorimetric assay inhibition of proliferation and calculate the optimal concentration of growth inhibition rate to identify the two drugs alone; and the two combined application of optimal concentration again detect the inhibition of cell proliferation and calculate the rate of proliferation inhibition; take the logarithmic phase of growth period cells were randomly divided into four groups: control group (K562 cells not treated with drugs), experimental group 1 (5.0 mmol / L GS treatment of K562 cells), experimental group 2 (10mg / L BBM treated K562 cells), experimental 3 (5.0mmol/LGS and 10 mg / L the BBM joint processing of K562 cells); flow cytometry (FCM) detection rate and detection of early apoptosis Caspase-3, Survivin protein expression levels change; Western blot detection of drug action before and after the mitochondrial Cyt c protein released into the cytoplasm of the change. The results of MTT assay results is 5.0mmol / L GS and 10mg / L BBM optimal concentration to inhibit cell proliferation, and the experimental group 3 inhibited the proliferation of K562 cells was significantly enhanced. Apoptosis rate in each experimental group apoptosis rate was significantly higher than that in the control group (P <0.01), rate of apoptosis was significantly higher (71.09 ± 1.24)% at 72h experimental combination presents a strong synergistic The enhancement effect. The experimental group 3 Caspase-3 positive expression rate of survivin protein in 72 hours (78.46 ± 0.12)% and (8.24 ± 0.45)%, the former is significantly higher than that in the control group (7.56 ± 1.33)% (P <0.01), while the latter is significantly lower than the control group (38.24 ± 0.14)% (P <0.05). Western blot showed that the the 72h experimental group 3 cytoplasmic Cyt c protein content was significantly higher than that of the control group and the experimental group 1 and group 2 (P <0.05). Conclusion 1. GS, BBM alone and the combination therapy can inhibit the proliferation of K562 cells inhibited the proliferation of K562 cells, and combination therapy is more obvious. 2. Cell apoptosis were detected early apoptosis rate of K562 cells was significantly higher than combination therapy and medication alone group, both used in combination can enhance the induction of apoptosis of K562 cells, a synergistic effect, and there is a certain The time-dependent manner. 3. Associated with elevated cytosolic Cyt c protein content in the experimental process down, Caspase-3 protein expression of Survivin protein upregulation. 4. Both monotherapy induced apoptosis of K562 cells mainly through mitochondria-Caspases ways, and speculated that the combination therapy induced apoptosis enhanced because of the sum of this pathway. 5. Reduction of survivin protein can to some extent alleviated the inhibition of Caspase-3, to further enhance the induction of apoptosis of K562 cells.

Related Dissertations

CLC: > Medicine, health > Oncology > Hematopoietic and lymphoid neoplasms > Leukemia
© 2012 www.DissertationTopic.Net Mobile