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Functional Characterization of Interleukin-15 Gene Transduction into the Human Natural Killer Cell Line NKL

Author: JiangZuo
Tutor: ZhangJian
School: Shandong University
Course: Microbial and Biochemical Pharmacy
Keywords: Gene transfection IL-15 NK cells NKL cell lines
CLC: R392
Type: Master's thesis
Year: 2008
Downloads: 80
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Abstract

Purpose of NK cells as the body's innate immune important part of the body's anti-tumor, anti-infective first natural line of defense. The emergence of the killing function does not require pre-irritation or sensitization, MHC (histocompatibility complex) restrictive. NK cells are not only capable of mediating the natural killing effect on tumor cells, but also can be adjusted to the specific and non-specific immune response. Adoptive transfer the activation or allogeneic NK cells have been used in the treatment of leukemia and certain solid tumors. Interleukin -15 (IL-15) produced by a variety of tissue and cell subsets of CD34 cells to differentiate into NK cells can be induced with IL-2 has many similarities in function, and can promote the proliferation of NK cells , enhance the cytotoxic activity of NK cells. We have built the IL-15 gene-modified NK-92 cell line, confirmed that the IL-15 gene-modified NK-92 cells can enhance a variety of tumor-killing ability. Compared with the NK-92 cells, NKL cells have different destruction spectrum, such as human gastric cancer cell line SGC7901, NKL killing ability of NK-92 cells, NK-92 can be used as an important supplement. The NKL cell by Robertson et establish derived from CD3-CD16 CD56 large granular lymphocytes in peripheral blood of patients with leukemia. NKL depends on the IL-2 growth, with natural killer activity and ADCC effect, the proliferative response is also similar to the normal CD16 CD56dim NK cells. NKL NK cell lines is most similar to the original NK cells, and therefore another important choice to become adoptive immunotherapy. Established NKL cell lines stably transfected with hIL-15, and that IL-15 gene-modified NKL cells with enhanced proliferation, anti-apoptosis and liver cancer cell killing effect, and to investigate its molecular mechanism. 1. Electric switch to the IL-15 gene stably transfected into NKL cell lines. 2.CTLL-2 proliferation assay measured cell culture supernatant of IL-15 biological activity. 3 ELISA Analysis of the cells was measured in the culture supernatant of IL-15 concentration. 4.RT-PCR to detect the the three NKL cell apoptosis-related genes and destruction-related gene expression levels. 5.CCK method is used to analyze the three NKL cells in different concentrations of IL-2 and IL-15 under the conditions of proliferation levels. Flow cytometry the three NKL cell apoptosis, cycle, and the expression of membrane surface NKG2D and NKG2A molecules. 7.MTT method to detect the three NKL cell cytotoxicity. Results 1. Establish stably expressing human IL-15 gene-modified NKL-IL15 cell lines. 2.IL-15 gene modified NKL cells have stronger proliferative activity in the low-dose IL-2 and IL-15 under the conditions. 3.IL-15 gene to improve the anti-apoptotic effects NKL cell lines. 4.IL-15 upregulated the anti-apoptotic gene expression in the NKL cell lines. 5.IL-15 no significant effect on cell cycle gene NKL cell lines. 6.IL-15 gene-modified NKL cells in vitro liver cancer cells with enhanced cytotoxic activity. 7.IL-15 genes were upregulated the NKL cell killing molecules expression. 8.IL-15 gene-modified NKL cells in the body can effectively inhibit the proliferation of liver cancer cells. Conclusion as an important part of the innate immune NK cells are the first line of defense against viral infections and tumor cells without antigen pre-role, can directly kill tumor and virus-infected target cells in tumor immune surveillance and early play an important role in the process of anti-infection immunity. Application of NK cells is an important means of tumor biological treatment, the NK-92 cells has entered clinical trials. In order to overcome some of the deficiencies in the application of NK cell lines, gene modification has become the primary means to enhance the activity of NK cells. To date, IL-2, IL-15, SCF, CD20 and other genes modified NK-92 cells, important progress. NK cell lines in order to obtain clinical applications, we have successfully established the IL-15 gene-modified NKL cell lines under the conditions of the IL-15 gene-modified NKL cell lines in the low-dose IL-2 and IL-15 showed stronger proliferation ability, and showed strong anti-apoptotic role in serum starvation state. Further studies showed that the IL-15 gene-modified NKL cells, anti-apoptotic gene Bcl-2, Bcl-xl, Mcl-1 expression levels were significantly increased apoptotic gene Bim, Noxa, Fas expression level decreased. More important is the NKL-IL-15 cells not only in vitro can effectively kill liver cancer cells HepG2, H7402, PLC/PRF-5, also significantly enhanced in vivo growth of human liver tumor inhibition. This anti-tumor effect and NKL-IL15 cells of IFN-γ, FasL and Perforin gene expression levels related. Thus, the biological characteristics of the human IL-15 gene-modified NKL cells more conducive to clinical adoptive immunotherapy Application.

Functional Characterization of Interleukin-15 Gene Transduction into the Human Natural Killer Cell Line NKL

Abstract

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