In vitro effect of GFP-labeled total RNA of HepG2 cells transfected into dendritic cells

Objective: to explore the feasibility of observing dendritic cells transfected with total RNA of tumor cells with green fluorescent protein as a marker and the feasibility of transfected dendritic cells as a potential immunotherapy vaccine. Methods: plasmid pGFPC3 was stably transfected into HepG2 cells. Total RNA was extracted from HepG2GFP with Trizol. Peripheral blood mononuclear cells (PBMCs) of patients with liver cancer induced dendritic cells and transfected with total RNA. The transfection effect was observed by fluorescence microscope, the phenotypic changes were detected by flow cytometry, and the secretion of IL 12 in the supernatant of dendritic cells was detected by ELISA. Cytotoxic effect of CTLs was detected by MTT assay. Results: GFP was stably expressed in HepG2GFP cells, which showed green fluorescence under fluorescence microscope, and dendritic cells transfected with total RNA of HepG2GFP cells were also stably expressed. After transfection, CD80 (13.2% to 86.7%), HLADR (38.9% to 97.9%), CD83 (0.9% to 97. 1%) and CD86 (3 1.2% to. The secretion of IL 12 in the supernatant was significantly increased [61.3 8.10000001CTLs activated by dendritic cells transfected with total HepG2GFP RNA showed effective specific lysis on HepG2 cells. Conclusion: GFP can be used as a marker to observe the effect of transfection of dendritic cells with total RNA of tumor cells. Dendritic cells transfected with total RNA of tumor cells can be used as a promising immunotherapy vaccine.

Dendritic cells; Green fluorescent protein; RNA transfection

Objective: To explore the feasibility of observing the effect of transfection of hepatocellular carcinoma (HCC) RNA into dendritic cells (DCs) with green fluorescent protein (GFP) as a marker, and the feasibility of preparing vaccine by transfection of tumor cell RNA into DCs. Methods: the green fluorescent protein particle vector PGPFPC 3 was stably transfected into HCC HepG2, and the total RNA of HepG2GFP was extracted by Trizol method. Dendritic cells were induced by peripheral blood mononuclear cells from patients with liver cancer in vitro, and total RNA was transfected into dendritic cells. The transfection effect was observed under fluorescence microscope, and the phenotypic changes of dendritic cells before and after transfection were detected by flow cytometry. The changes of IL 12 in supernatant before and after transfection were detected by ELISA. MTT assay was used to detect the killing rate of effector cells to target cells. Results: HepG2 cells stably expressing GFP were obtained after transfection of PGPFPC 3, and showed green fluorescence under fluorescence microscope. Under the DCs fluorescence microscope, the total RNA transfected cells showed green fluorescence, with CD80( 13.2% rising to 86.7%), HLADR(38.9% rising to 97.9%), CD83(0.9% rising to 97. 1%) and CD86 (3/kloc). The induced CTL can specifically kill HepG2 cells. Conclusion: GFP can be used as an observation marker of dendritic cells transfected with tumor cells RNA, and dendritic cells transfected with tumor cells RNA can be used as an effective tumor vaccine.

Dendritic cells; Green fluorescent protein; RNA transfection

Chinese drawing number R739.4 1

Introduction to 0

Hepatocellular carcinoma (HCC) is a common malignant tumor, only a few patients are suitable for surgical treatment, with high recurrence rate and poor prognosis, which seriously threatens human health [1]. DCS (Dendritic Cells) is a powerful professional antigen presenting cell in the body. It can effectively ingest and process antigens, and present the processed peptides to static T cells, causing specific immune response against antigens [2]. In similar studies at home and abroad, there is no clear index to observe the effect of tumor RNA transfection on dendritic cells. We stably transfected hepatoma cell HepG2 with green fluorescent protein particle vector PGPFPC 3, and extracted and screened HepG2GFP total RNA to transfect dendritic cells. To observe the expression of green fluorescent protein, specific surface markers and function-related molecules in transfected dendritic cells, and detect the secretion of cytokines in their supernatant, so as to explore the feasibility of using them as tumor vaccines.

1 materials and methods

1. 1 Materials Peripheral blood mononuclear cells of patients with liver cancer are derived from patients undergoing hematopoietic stem cell transplantation in our department. HepG2 hepatocellular carcinoma cell line was routinely subcultured by our department. Flow cytometer (BD), DG3022A enzyme-linked immunosorbent assay (East China Electron Tube Factory) and XIF optical fluorescence microscope (Nicon) are all available in our hospital. Antibodies against FITCCD80, FITCHLADR, PECD83 and PECD86 were purchased from BD company. RhGMCSF, rhIL4 was purchased from Peprorotec company; TNFα was purchased from the Biotechnology Center of our school; The green fluorescent protein carrier PGPFPC 3 was donated by Dr. Jia Jun from Shaanxi Cancer Hospital. Liposome transfection 2000, Trizol reagent from Invitrogen; ; RPMI 1640 medium, fetal bovine serum, G4 18 were purchased from Gibco company, and serum-free Xvivo medium was purchased from BioWhittaker company. . IL 12 ELISA kit was purchased from Bioscience Company. MTT was purchased from Sigma Company.

1.2 methods HepG2 was transfected with PGPFPC 3 and G4 18 were screened for 2 months according to transfection instructions 2000. The screened cells were named HepG2 FP and observed under fluorescence microscope. Total RNA of HepG2 FP was extracted with Trizol reagent and observed under ultraviolet lamp. Store at -80℃ for later use. Total RNA of HepG2 was extracted by the same method. Hematopoietic stem cells from patients with liver cancer were isolated by adherent method, and the adherent cells were induced into DCs under the action of cytokines rhGMCSF and rhIL4. Cell morphology was observed by inverted microscope and electron microscope, and cell phenotype was detected by flow cytometry.

1.2. 1 Transfect dendritic cells, collect dendritic cells cultured for 4 days, transfer them into 6-well plates, transfect HepG2GFP total RNA, and set up a blank control group. Culture for 48 hours. Observe under a fluorescent microscope.

1.2.2 determination of cytokine secretion 48 hours later, the supernatant of DCs in each group was collected, and the secretion of IL 12 in the supernatant of each group before and after transfection was detected by ELISA.

65438 0.2.3 CTL effect dendritic cells were collected in each group. After 30 Gy 60Co irradiation, it was mixed with the recovered non-adherent cells at the ratio of 20∶ 1 and inoculated into a 24-well plate. After 5 days of culture, the collected cells were effector cells. HepG2 cells in logarithmic growth phase were collected as target cells. The effector cells and target cells were inoculated in a 96-well plate at the ratio of 20∶ 1. Set a single target cell group and a single effector cell group at the same time. After 24 h of culture, it was detected by MTT method. The killing efficiency of CTL was calculated according to the following formula: [1- (effective target A490- effective cell A490)/ target cell A490 ]× 65438.

Statistical methods: SPSS 10.0 statistical software was used to analyze the results.

Two results

2. The total RNA of1HepG2 FP was transfected into dendritic cells, and HepG2 FP cells which could stably express GFP were stably transfected and screened. All cells showed green fluorescence under fluorescence microscope (figure 1), and remained unchanged after more than 20 passages. Compared with the control cells, the cells modified by GFP gene have no obvious changes in morphology and growth. GFP expression is not toxic to target cells. After extracting the total RNA from HepG2GFP cells with Trizol, 2 μL of total RNA was taken for gel electrophoresis, and bands of 5 s, 18 s and 28 s were found (Figure 2). After monocytes adhered to the wall, under the action of cytokines GMCSF and IL-4, cell colonies appeared on the second day of culture, but after 5 days, the colonies obviously increased and became larger, and cells with burr-like processes appeared. On the 7th day, the colony began to decrease, resulting in a large number of cells with dendrites (Figure 3A, B). Under the fluorescence microscope, the cells in the DCs group transfected with HepG2 total RNA showed green fluorescence, while the cells in the HepG2 total RNA transfection group and the blank control group showed no obvious fluorescence expression (Figure 4A, B, C).

Fig. 1 fluorescence microscope after stable transfection of PGPFPC 3, the cytoplasm fluorescence of HepG2 was positive by 250 (omitted).

Fig. 22 Electrophoresis of total RNA of HepG2 GFP cells (omitted)

Fig. 3 Dendritic cell morphology (day 7) (omitted)

2.2 Flow cytometry showed that cell phenotype induced the expression of CD80 13.2% in d 7 and DC, while that of Hladr was 38.9%, CD86310.2%, and the low expression of CD83 was only 0.9%. After transfection, the expression of the above molecules increased significantly, which were CD80 86.7%, HLADR 97.9%, CD86 92.5% and CD83 97. 1% respectively, suggesting that the transfected cells have the characteristics of mature dendritic cells.

Fig. 4 Total RNA transfected into DCs×250 (omitted)

2.3ELISA was used to detect the total RNA of HepG2GFP secreted by IL 12.

The secretion of IL 12 in the supernatant of DCs transfection group was significantly higher than that before transfection [(287.4 29.3) ng/l vs (61.3 8.1) ng/l; n=6，P < 0.05。

2.4CTL mediates cytotoxicity of HepG2GFP total RNA. The cytotoxic effect of CTL stimulated by DCs on HepG2 cells was (62.6 2.9)%, that of T cells stimulated by non-transfection DCs was (20.8 65438 0.5)%, and that of T cells not stimulated by DCs was (20.8 65438 0.5)%. The cytotoxic effect of total RNA transfected DC-stimulated CTL on HepG2 cells was significantly higher than that of the two control groups (n=6, P < 0.05), and the cytotoxic effects of total RNA transfected DC-stimulated CTL on SMMC772 1 and K562 cells were (37.7 65438 0.8)% and (26.8 65438) respectively.

3 discussion

DCs is the most powerful full-time antigen presenting cell in the body and plays an extremely important role in T cell immunity [2]. The preparation of vaccines from dendritic cells sensitized with various tumor antigens is a hot research topic in recent years [3-8]. Compared with other methods, the total RNA of tumor cells transfected with DCs has the following advantages: first, it is not limited by known tumor antigens and can induce polyclonal CTL；; Secondly, the RNA needed for transfection can be obtained by amplification, and a small amount of tumor tissue samples can be obtained by amplification [8]. However, there is no clear method to observe the efficiency of total RNA transfection into dendritic cells at home and abroad. We transfected DCs with HepG2GFP total RNA, observed the efficiency of transfection of DCs with GFP, and discussed its feasibility as a tumor vaccine to provide experimental basis for clinical treatment of liver cancer.

In this experiment, the DC cytoplasm transfected with HepG2GFP RNA showed green fluorescence under fluorescence microscope, which confirmed that GFP mRNA could be expressed normally in DC cytoplasm, and the total RNA of tumor cells could also be translated and expressed in DC cytoplasm. After transfection, the expression of CD83, CD80, CD86 and HLADR increased significantly, and the secretion of IL 12 increased significantly. The results showed that RNA transfection promoted the maturation of dendritic cells in vitro. Mature dendritic cells translate the total RNA of tumor cells and present the expressed tumor antigen to T lymphocytes. T lymphocytes induced by this pathway are mainly CD8+ cytotoxic T lymphocytes (CTL). In this experiment, the cytotoxic effect of induced CTL on HepG2 was significantly higher than that of control T lymphocytes. It is proved that the induced T lymphocytes have tumor specificity. Therefore, under the condition that the specific antigen of liver cancer has not been found yet, it can be a development direction of DCs vaccine for liver cancer to transfect the total RNA of liver cancer cells into DCs to prepare tumor vaccine and induce polyclonal CTL against liver cancer cells.

refer to

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