VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------------***------------- UNDERGRADUATE THESIS TITLE: EFFECT OF VIBLASTINE ON TRANSCRIPT EXPRESSION OF IMMUNE RELATED GENES IN DENDRITIC CELL OF CHRONIC LYMPHOCYTIC LEUKEMIA Student name : TRAN THI THUY DUNG Class : K63CNSHE Student code : 637407 Faculty : BIOTECHNOLOGY Supervisor : Assoc.NGUYEN THI XUAN MSc.NGUYEN THANH HUYEN HANOI – 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------------***------------- UNDERGRADUATE THESIS TITLE: EFFECT OF VIBLASTINE ON TRANSCRIPT EXPRESSION OF IMMUNE RELATED GENES IN DENDRITIC CELL OF CHRONIC LYMPHOCYTIC LEUKEMIA Student name : TRAN THI THUY DUNG Class : K63CNSHE Student code : 637407 Faculty : BIOTECHNOLOGY Supervisor : Assoc.NGUYEN THI XUAN MSc.NGUYEN THANH HUYEN HANOI – 2022 DECLARATION I hereby declare that the graduate thesis work is mine. All research results have been results during the implementation of the topic. The results, the data are completely true, never appeared in any scientific report. I also guarantee that the references and useful information for the topic are clearly cited and all helps are appreciated.
Hanoi, December 5th, 2022 Student Tran Thi Thuy Dung ACKNOWLEDGEMENTS During the process of implementing my graduation project, I have received a lot of attention and help from individuals and groups. First of all, I would like to express my respect and deep gratitude to Associate Professor Ph. D Nguyen Thi Xuan and MSc. Nguyen Thanh Huyen for giving me the opportunities to carry out this work, and their huge efforts, enthusiasm, and supports throughout the duration of the undergraduate thesis.
Secondly, I would like to thank the teachers in the Faculty of Biotechnology who helped and taught me during my training at the University. Especially, the teachers of the Microbial Biotechnology department who gave me advice during carrying out. Finally, I would like to sincerely thank my family members and friends who always trust, support and encourage me to complete this report. Sincerely thank! Hanoi, December 5th, 2022 Student Tran Thi Thuy Dung ii CONTENTS DECLARATION .iii LIST OF ABBREVIATIONS.
v LIST OF FIGURES .vii LIST OF TABLES. Objectives and Requirements. Acute Lymphocytic Leukemia (ALL). Acute myeloid leukemia (AML).
Chronic myeloid leukemia (CML). Chronic Lymphocytic Leukemia (CLL). Leukemia in the world and in Vietnam. In the world.
The immune related genes. Pro-apoptotic group. MATERIALS AND METHODS. Subjects for research.
Tools and Devices. Collecting the samples. Culturing peripheral mononuclear blood cells (PBMCs). Synthesis of cDNA from total RNA.
RESULTS AND DISCUSSION. Expression levels of A20, CYLD, OTUB1, OTUB2 and CEZANNE genes in patients with Chronic lymphocytic leukemia. Expression level of A20 gene in patients with Chronic lymphocytic leukemia. Expression level of CYLD gene in patients with Chronic Lymphocytic Leukemia.
Expression level of OTUB1 gene in patients with Chronic lymphocytic leukemia. Expression level of OTUB2 gene in patients with Chronic lymphocytic leukemia. Expression level of CEZANNE gene in patients with Chronic lymphocytic leukemia. Expression level of BAX gene in patients with Chronic lymphocytic leukemia.
CONCLUSION AND SUGGESTION. 46 iv LIST OF ABBREVIATIONS CLL Chronic lymphocytic leukemia ALL Acute lymphocytic leukemia AML Acute myeloid leukemia CML Chronic myeloid leukemia MOM Mitochondria outer membrane PTP Permeability transition pore cDCs conventional dendritic cells DUB Deubiqitinase NK-κB Nuclear factor kappa-light-chain-enhancer of activated B CCR7 C-C chemokine receptor 7 HLA Human Leucocyte Antigen GM-CSF Granulocyte-macrophage colony- stimulating factor PBMC Peripheral blood mononuclear cell DC Dendritic cell VinB Vinblastine ABL Abelson murine leukemia BCR Breakpoint TKI Tyrosine kinase inhibitor FLT3 FM’s Related Receptor Tyrosine Kinase 3 PCR Polymerase-Chain-Reaction Ag Antigen OTU Ovarian tumor domain (OTU) HCC Hepatocellular carcinoma v RNA Ribonucleic acid DNA Deoxyribonuclec acid vi LIST OF FIGURES Figure 2.1: Acute Lymphocytic Leukemia (ALL) .2: Acute myeloid leukemia (AML) .3: Chronic myeloid leukemia (CML) .4: Chronic Lymphocytic Leukemia (CLL) and normal blood cell .5: Comparison between normal blood and Leukemia .6: The structure of A20 .7: The structure of OTUB1 .8: The structure of CYLD .9: Chemical structure of Vinblastine .1: Phase cleavage after centrifugation to isolate PBMCs .1: DCs are differentiated from PBMCs: A. DCs at 40X microscopy, B. PBMCs at 20X microscopy .2 Electrophoresis image after RNA extraction .3: The graph of transcript expression of A20 .4: The graph of transcript expression of CYLD .5: The graph of transcript expression of OTUB1 .6: The graph of transcript expression of OTUB2 .7: The graph of transcript expression of OTUD7 .8: The graph of transcript expression of BAX.
43 vii LIST OF TABLES Table 3.1: Sequence of immune related genes .1: List of RNA extraction from the cultured cell sample. Preface Leukemia is a cancer of the blood-forming tissues including the bone marrow and the lymphatic system, caused by a sudden increase in the number of white blood cells in the body. White blood cells are an important part of the immune system. They protect the body from invasion by bacteria, viruses and fungi as well as from abnormal cells and other foreign substances.
Clinically, leukemia is defined as a hematopoietic stem cell malignancy, correlating with an elevated white blood cell count in the peripheral blood and bone marrow (pokharel 2012). Leukemia consists of four main types: Acute Lymphocytic Leukemia – ALL, Chronic Lymphocytic Leukemia – CLL, Acute Myeloid Leukemia – AML, Chronic Myeloid Leukemia – CML. Leukemia can occur in either lymphocytes or myeloid leukocytes. Cancer that develops in the white blood cells is called lymphocytic leukemia.
The deubiquitin gene group A20, CYLD, OTUB1, OTUB2 and Cezanne encode a number of proteins belonging to the deubiquitinase (DUB) group. DUB plays several roles in the ubiquitin pathway. One of the most well-known functions of DUB is to remove monoubiquitin and polyubiquitin chains from proteins. Ubiquitin is bound to proteins to regulate protein degradation via proteasomes and lysosomes, coordinate cell localization of proteins, activate and inactivate proteins, and regulate protein–protein interactions (Schnell 2003, Michael H.
DUB is able to reverse these effects by cleaving the peptide or isopeptide bond between ubiquitin and its substrate protein. The role of DUB in diseases remains unclear. However, their involvement in disease is anticipated due to known roles in physiological processes that are implicated in disease states including cancer and neurological disorders (Singhal 2008). Although there are many studies on mutations and expression of A20, OTUB1, OTUB2, CYLD and Cezanne genes in cancer, the expression levels of these genes in cancer patients in Vietnam have not been extensively studied…In addition, the BCL-2 family relate to the regulation of apoptoic cell death, consisting of anti- apoptotic and pro- apoptoic members.
BAX is one of genes belong to the BCL-2 which trigger the release of caspases from death antagonists via heterodimerization and also by inducing the release of mitochondrial apoptogenic factors into the cytoplasm via acting on mitochondrial permeabiity transition pore, thereby leading to caspase activation (Tsujimoto 2001). Vinblastine is a component of a number of chemotherapy regimens. It is also used to treat histiocytosis according to the established protocols of the Histiocytosis Association. Adverse effects of vinblastine include hair loss, loss of white blood cells and blood platelets, gastrointestinal problems, high blood pressure, excessive sweating, depression, muscle cramps, vertigo and headaches.
As a vesicant, vinblastine can cause extensive tissue damage and blistering if it escapes from the vein from improper administration. At very low concentrations they suppress microtubule dynamics and at higher concentrations they reduce microtubule polymer mass (Jordan MA 2004). Recent findings indicate that they also produce microtubule fragments by stimulating microtubule minus-end detachment from their organizing centers. Dose-response studies further indicate that enhanced microtubule detachment from spindle poles correlate best with cytotoxicity (Salerni BL 2010).
But research into the mechanism is still ongoing as recent studies also show vinblastine inducing apoptosis that is phase- independent in certain leukemia. Vinblastine is reported to be an effective component of certain chemotherapy regimens, particularly when used with bleomycin, and methotrexate in chemotherapy for Stage IA or IIA Hodgkin lymphomas. The inclusion of vinblastine allows for lower doses of bleomycin and reduced overall toxicity with larger resting periods between chemotherapy cycles (Salerni BL 2010). Addtionally, the study focus on immune related genes in dendritic cells of Chronic Lymphocytic Leukemia.
Dendritic cells (DCs) have 2 been used in therapeutic cancer vaccines with some success. To be effective, DCs from cancer patients must be able to take up tumor-specific antigens and present them in an immune-stimulatory manner to immunologically competent T cells. These cells must then recognize and react to these antigens before maturing into cytotoxic cells that destroy the tumor. Studies on the function of DC and T cells in CLL have revealed some information about these cells' ability to perform the functions required for a DC-based therapeutic approach to this disease.
Therefore, under the guidance of Assoc. Nguyen Thi Xuan and Ms. Nguyen Thi Thanh Huyen, I conduct the project: “Effect of vinblastine on transcript expression of immune related genes in dendritic cell of chronic lymphocytic leukemia patients”. This study will make a contribution in providing a new insight about effect of vinblastine toward dendritic cells of chronic lymphocytic leukemia (CLL).
Objectives and Requirements 1.1 Objectives Evaluating the quality of peripheral mononuclear blood cells (PBMCs) and the differentiation of dendritic cells in the isolation and culture. Evaluating the transcript expression levels of immune related genes: A20, CYLD, OTUB1, OTUB2, CEZANNE and BAX in dendritic cell of CLL patients when is treated with different concentrations of VinB 1. Requirements Collecting the blood samples of CLL Isolating Peripheral mononuclear blood cells (PBMCs) Differentiating PBMCs into dendritic cells (DCs) and treating them with VinB at different concentrations. Using realtime-PCR technique to evaluate the expression of immune- related genes.
Leukemia There are two main types of leukemia: acute and chronic leukemia, depending on how quickly the disease progresses. In acute leukemia, infected white blood cells do not work or function like normal white blood cells; while in chronic leukemia it can act like normal white blood cell. Therefore, chronic leukemia can be serious because it is indistinguishable from normal white blood cells. Furthermore, there are two subtypes of each type of leukemia depending on the size and shape of the white blood cells: lymphoid and myeloid.
In general, leukemia is divided into four main subtypes including: ALL - 10%, CLL - 35%, Leukemia Acute myeloid leukemia (AML – 33%) and Chronic myeloid leukemia (CML – 14%) (Dong, Shi et al. The types of leukemia differ in their pathogenesis, origin, incidence, and prognosis. Acute Lymphocytic Leukemia (ALL) Acute lymphoblastic leukemia is the most common blood cancer in children and young adults, especially those between the ages of 2 and 5 (Inaba 2013, Facts 2017). ALL is a hematopoietic stem cell malignancy derived from a B- or T-lymphocyte progenitor cell lineage and driven by a variety of genetic aberrations including mutations, chromosomal translocations, and heterochromia.
multiples in genes involved in lymphocyte development and cell cycle regulation (Hunger 2015). In patients with ALL, the bone marrow makes too many immature lymphocytes. These lymphocytes do not function like normal lymphocytes and are not able to fight infections. The most common signs are swollen lymph nodes, hepatosplenomegaly, fever, anemia, signs of hemorrhage, and bone pain.
Genetic factors that can cause the disease include Down syndrome, Fanconi anemia, Bloom syndrome, ataxia-telangiectasia and Nijmegen fracture syndrome (Bielorai , German 1997, Chessells 2001, Shah 2013). Other predisposing factors include 4 exposure to ionizing radiation, pesticides, certain solvents, or viruses such as Epstein-Barr virus and human immunodeficiency virus (Sehgal 2010). ALL affected approximately 876,000 people globally in 2015 and resulted in approximately 111,000 deaths. In the United States, ALL is the most common cancer and causes the highest mortality from cancer in children.
The incidence of ALL in the United States is estimated at 1. In 2016 alone, an estimated 6590 new cases were diagnosed, with more than 1400 deaths from ALL (according to the American Cancer Society) (Disease 2016).1: Acute Lymphocytic Leukemia (ALL) (Ahmed, Yigit et al.