CN114561361A

CN114561361A - DC cell with high proliferation and migration activity and application thereof in cellular immunotherapy

Info

Publication number: CN114561361A
Application number: CN202210221287.5A
Authority: CN
Inventors: 不公告发明人
Original assignee: Huaian Taikairui Pharmaceutical Technology Co ltd
Current assignee: Huaian Taikairui Pharmaceutical Technology Co ltd
Priority date: 2022-03-09
Filing date: 2022-03-09
Publication date: 2022-05-31

Abstract

The invention discloses a DC cell with high proliferation and migration activity and application thereof in cellular immunotherapy. The invention discovers that the DC cell with high GBE1 expression has high proliferation activity and high migration activity, and the DC cell can certainly improve the cellular immunotherapy effect when being applied to cellular immunotherapy, and has a promising application prospect.

Description

DC cell with high proliferation and migration activity and application thereof in cellular immunotherapy

Technical Field

The invention belongs to the field of immune cell therapy, relates to dendritic cells, and particularly relates to a DC cell with high proliferation and high migration activity and application thereof in cellular immunotherapy.

Background

Glycogen branching enzyme (GBE1) protein molecules belong to members of the alpha-amylase family, are mainly involved in the formation of glycogen branches in human cells, and are one of the major enzymes in the process of glycogen formation. The formation of glycogen branches increases the water solubility of glycogen on the one hand, and facilitates the storage of the glycogen; on the other hand, glycogen-binding proteins can be provided with docking sites, including glycogen metabolism-related enzymes, regulatory proteins and the like.

Early researches show that the high expression of GBE1 can effectively improve the migration activity of NK cells, and meanwhile, the proliferation activity and the killing activity of the NK cells can be enhanced.

Dendritic Cells (Dendriti Cells, DCs) are the only antigen presenting Cells with stimulation of naive T lymphocytes. It can capture tumor antigen and present it to T cell, generate tumor specific T cell immune response, and has high IL-2 secreting ability. The dendritic Cells are the most powerful professional antigen presenting Cells (antigen presenting Cells APC) in the human body and are an indispensable component of the immune system. DC cells are also of great interest in cellular immunotherapy.

The effect of high GBE1 expression on DC cell proliferation and/or migration activity is currently unknown.

Disclosure of Invention

The invention aims to provide a DC cell with high proliferation and high migration activity and application thereof in cellular immunotherapy.

The purpose of the invention is realized by the following technical scheme:

a DC cell with high proliferation and migration activity, wherein the DC cell highly expresses GBE 1.

The application of the DC cells in cellular immunotherapy.

Use of high expression of GBE1 for increasing DC cell proliferation activity and/or migration activity.

The technical effects are as follows:

the invention discovers that the DC cell with high GBE1 expression has high proliferation activity and high migration activity, and the DC cell can certainly improve the cellular immunotherapy effect when being applied to cellular immunotherapy, and has a promising application prospect.

Drawings

FIG. 1 shows the growth state of DC cells observed under an inverted microscope.

FIG. 2 shows the results of detecting the expression level of GBE1 protein in each group.

FIG. 3 is a photograph of 0, 24h scratch for each group of DC cells.

FIG. 4 shows the proliferation activity of DC cells in each group.

Detailed Description

The following presents a more detailed description of the invention in connection with the examples, but it will be appreciated by those skilled in the art that the scope of the invention is not limited to the examples.

First, experimental material

Human peripheral blood lymphocyte separation medium and GM-CSF were obtained from Beijing Solebao scientific Co. Recombinant human IL-4 was purchased from Wuhan Feien Biotech, Inc. RPMI-1640 medium, fetal bovine serum was purchased from Gibco, and double antibody was purchased from Biyunnan. Ad-GFP and Ad-GBE1-GFP were purchased from the Kjeldahl gene. GBE1 antibody was purchased from Abcam and other antibodies were purchased from petunia.

Second, Experimental methods

1. Culture of DC cells

1.1 isolation of mononuclear cells

Taking peripheral anticoagulation blood of a healthy volunteer, uniformly mixing the anticoagulation blood with physiological saline in equal volume, carefully adding the anticoagulation blood onto the liquid surface of the human peripheral blood lymphocyte separation liquid, and centrifuging for 20 minutes by 400g (about 1500 rpm, a horizontal rotor with the radius of 15 cm), wherein cells in a centrifuge tube are divided into four layers from top to bottom: the first layer is a plasma layer, the second layer is an annular milky white lymphocyte layer, the third layer is a transparent separation liquid layer, and the fourth layer is a red blood cell layer. Collecting the second layer of cells, mixing with normal saline, centrifuging at 400g (same as above) for 20 min, collecting the precipitate, and washing the precipitate for 2 times to obtain peripheral blood mononuclear cells.

1.2 Induction of DC cells by mononuclear cells

Culturing peripheral blood mononuclear cells in RPMI-1640 medium (complete medium) containing 10% fetal bovine serum and 1% double antibody for 3 hr at 25cm²Placing 1X 10 culture flask⁶Cells, in principle, are as much as possible provided a DC adherent surface. The flask was shaken with a slight force to elute the non-adherent T cells and aspirate the liquid and non-adherent cells. Care was taken to wash out T cells as much as possible, leaving adherent DC cells. Replace with new medium and repeat the previous procedure 1 time. Adding RPMI-1640 medium containing 10% fetal bovine serum, 1% double antibody, 1000U/mL GM-CSF and 500U/mL IL-4 at 37 deg.C and 5% CO₂And continuously culturing under the saturated humidity condition, changing the liquid once for half 2d, and supplementing the cell factors in full quantity. And continuously culturing for 3d and 7d, observing the growth morphology of the cells under an inverted microscope, and collecting the cells at 7d to complete DC cell induction culture.

2. Construction and detection of DC cells highly expressing GBE1

The best MOI of 80 was determined according to transfection efficiency and cell status with different MOI values in the preliminary experiments, so the formal transfection experiment was performed with MOI of 80. The experiments were divided into a control group (Blank group), an empty adenovirus-transfected group (Ad-GFP group), and a GBE1 adenovirus-transfected group (Ad-GBE1-GFP group). The Blank group was not transfected, the Ad-GFP group was transfected with null adenovirus, the Ad-GBE1-GFP group was transfected with GBE1 adenovirusDC cells. Each group is provided with 3 multiple holes simultaneously. The specific transfection method is as follows: the 7d cultured DC cells were digested, resuspended in RPMI-1640 medium, and plated at 2X 10⁵One/well was inoculated in 24-well culture plates at 37 ℃ with 5% CO₂After culturing for 12h under saturated humidity condition, the virus stock solution is diluted to 1.0X 10 titer⁸And (3) removing the culture medium in the hole by PFU/mL, sequentially adding a virus diluent and an RPMI-1640 culture medium into the transfection hole, fully and uniformly mixing, after culturing for 24h, removing the original culture medium by suction, washing by PBS, adding the RPMI-1640 complete culture medium, after continuously culturing for 24h, and observing the transfection result under a fluorescence microscope. The cells were collected, washed with PBS, and total protein was extracted, and the expression level of GBE1 protein was measured in each group of cells by western blot method with internal reference GAPDH.

3. DC cell migration Activity assay

Grouping is the same as "construction and detection of DC cells with high GBE1 expression and 2". The 7-day-old DC cells were digested, resuspended in RPMI-1640 medium, and plated at 5X 10⁵One cell/well was inoculated in 6-well plates at 37 ℃ with 5% CO₂After culturing for 12h under saturated humidity condition, the virus stock solution is diluted to 1.0X 10 titer⁸Removing culture medium in the hole, sequentially adding virus diluent and RPMI-1640 culture medium into the transfection hole, mixing well, culturing for 24h, removing original culture medium, washing with PBS, adding RPMI-1640 complete culture medium, culturing for 24h, scratching the center of the culture hole with 1mL gun head, taking pictures at fixed point under an inverted microscope, and taking pictures at 37 deg.C and 5% CO₂After further incubation for 24h under saturated humidity conditions, photographs were taken of the same positions as before. The Image J software measures the width of the scratch and calculates according to the formula:

mobility (%) (1-24 h scratch width ÷ 0h scratch width) × 100%.

4. DC cell proliferation Activity assay

Grouping is the same as "construction and detection of DC cells with high GBE1 expression and 2". The 7d cultured DC cells were digested, resuspended in RPMI-1640 medium, and plated at 5X 10⁵One cell/well was inoculated in 6-well plates at 37 ℃ with 5% CO₂After culturing for 12h under saturated humidity condition, the virus stock solution is diluted to 1.0X 10 titer⁸PFU/mL, discarding medium in wellAdding virus diluent and RPMI-1640 culture medium into the transfection hole in sequence, mixing thoroughly, culturing for 24h, removing the original culture medium, washing with PBS, adding RPMI-1640 complete culture medium, culturing for 24h, collecting cells, washing with PBS, digesting, re-suspending with RPMI-1640 complete culture medium, and mixing at a ratio of 1 × 10⁴And inoculating each cell/well into a 96-well culture plate, after culturing for 48 hours, adding 20 mu L of MTT solution with the concentration of 5mg/mL into each well, culturing for 4 hours, removing supernatant, adding 150 mu L of DMSO, gently shaking, detecting the absorbance (OD value) at 490nm by using a microplate reader, and calculating the proliferation activity of the transfection group according to the following formula by using the OD value, wherein the higher the OD value is, the stronger the cell proliferation activity is. The proliferation activity of cells in Blank group was 100%.

The cell proliferation activity in the transfected group was defined as OD value in the transfected group/OD value in Blank group × 100%.

5. Statistical analysis

Statistical analysis of the data was performed using GraphPad Prism 5 software, and significance analysis of the data was performed using paired t-tests, with P <0.05 considered significant differences.

Third, experimental results

1. DC cell culture

As shown in FIG. 1, the growth state of the cells was good as seen in FIG. 1, and more projections and burrs were observed up to 7 days after the induction culture. Induction of 7d is the normal cycle in which monocytes induce the formation of mature DC cells, and hyperprocesses and increased eruptions are also consistent with the characteristics of mature DC cells.

2. Transfection of DC cells

The detection results of the GBE1 protein expression levels of all groups are shown in FIG. 2, and the GBE1 protein expression level of the Ad-GBE1-GFP group is obviously higher than that of the Blank group and the Ad-GFP group, which indicates that GBE1 is successfully transfected, and GBE1 high-expression DC cells are constructed.

3. DC cell migration Activity

The migration rate of DC cells in each group is shown in Table 1 and FIG. 3, and the migration rate of Ad-GBE1-GFP group is obviously higher than that of Blank group and Ad-GFP group, which indicates that the DC cells with GBE1 high expression have stronger migration activity.

TABLE 1 migration rates of groups of DC cells

	Mobility (%)
		Blank group	31.52±1.87
Ad-GFP panel	34.95±2.03
		Ad-GBE1-GFP group	60.81±2.14

4. DC cell proliferative Activity

The proliferation activity of DC cells in each group is shown in Table 2 and FIG. 4, and the proliferation activity of the Ad-GBE1-GFP group is obviously higher than that of the Blank group and the Ad-GFP group, which indicates that the DC cells with high GBE1 expression have stronger proliferation activity.

TABLE 2 proliferative Activity of DC cells

	Proliferation Activity (%)
		Blank group	100.00±3.75
Ad-GFP panel	102.58±3.23
		Ad-GBE1-GFP panel	131.74±3.51

The experiments show that the DC cell with high GBE1 expression has high proliferation activity and high migration activity, and the application of the DC cell in cellular immunotherapy can certainly improve the cellular immunotherapy effect, so that the application prospect is promising.

Claims

1. A DC cell with high proliferation and migration activity, which is characterized in that: the DC cells highly express GBE 1.

2. Use of the DC cell of claim 1 in cellular immunotherapy.

Use of high expression of GBE1 for increasing DC cell proliferative and/or migratory activity.