Establishment and characterization of EBV positive B-lymphoblastoid cell lines derived from cancers

다국어 초록 (Multilingual Abstract)

The international agency for research on cancer estimates that one in five cancer cases worldwide are caused by infection, with most caused by viruses. Viruses have been central to modern cancer research and provide profound insights into both infectious and non-infectious cancer causes. Many studies have been conducted on this, and the link between virus infection and cancer development is increasing (1, 2).
Epstein-Barr virus (EBV), one of the typical virus that causes cancer, is a γ-herpes viruses. EBV infection of resting B lymphocytes in vitro give rise to spontaneous outgrowth of EBV-transformed cell lines, referred to as lymphoblastoid cell lines (LCLs) in previous studies (2, 3). But, in this study, the cell lines established by the proliferation of EBV-infected lymphocytes in tissue during cell culture for establishment of tumor cell lines are referred LCLs.
These LCLs are easy to handle and to maintain so that they are suitable material for biological research (4). Although there are many studies relating LCLs with cancer therapy (5, 6, 7), there has not yet been a study to confirm the presence of anticancer
effect in the same patient by using LCLs itself.
Therefore, we performed type analysis and characterization of LCLs which were established by in vitro proliferation of EBV-infected lymphocytes in tissues during cell culture for establishment of tumor cell lines and we tried to show anticancer effect by approach of new immunotherapy for cancer by these LCLs having EBV specificity.
All LCLs were found to have different origins, EBV was predominantly infected with lymphocytes, and EBNA 1, EBNA 3A, EBNA 3C, BARF 1 and LMP 1 genes were expressed by EBV infection. Using a specific cell surface marker, we could prove that most of the LCLs correspond to B cells. The proliferation assay of LCL having the characteristics of EBV showed that it was sensitive to ganciclovir, and in order to confirm whether it had an anticancer effect in fact, co-culture with cancer cell was performed in vitro and identified that cancer growth tended to be suppressed.
In this study revealed that characterization of EBV positive B-lymphoblastoid cell lines naturally derived from cancer patients by wright staining, PCR, RT-PCR, western blot, Flow cytometry and cell proliferation assays, and identified that LCLs induce apoptosis of cancer cell line. I think that this steady study of LCLs could act as a biomaterial that can be used to treat cancer and disease related to EBV in the future.

목차 (Table of Contents)

• Introduction 1
• Materials and Methods 2
• Establishment and maintenance of Epstein-Barr virus (EBV) cell lines 2
• Detection of mycoplasma contamination 3
• Wright staining 4
• Growth properties and morphology in vitro 4
• DNA profiles 5
• Western blotting analysis 5
• PCR assays and Reverse Transcription-PCR assays 7
• Apoptosis assay 9
• Results 11
• Morphological character of established EBV positive B-lymphoblastoid cell lines in vitro 12
• Mycoplasma test 12
• Detection of mycoplasma contamination 13
• Identification of lymphocytes stained by wright stain 15
• Detection of latent genes from EBV positive B-lymphoblastoid cell lines by PCR analysis 17
• Detection of latent genes from EBV positive B-lymphoblastoid cell lines by RTPCR analysis 17
• EBV positive B-lymphoblastoid cell lines express the EBV latent protein EBNA 1, lymphocyte and B cell marker 21
• Analysis of cell marker expression in Epstein-Barr virus (EBV) positive Blymphoblastoid cell lines by Flow cytometry (FACS) 24
• Drug sensitivity analysis 28
• Co-culture analysis of Epstein-Barr virus (EBV) positive B-lymphoblastoid cell line with cancer cell line induces apoptosis in cancer cell 29
• Discussion 33
• References 38