Preparation method of human body foreign body phagocytosis lysosome immune TH cells

Abstract

The invention provides a preparation method of a human body foreign body phagocytosis lysosome immune TH cell. Specifically, the killing performance of TH cells is activated through a phagocyte immune method, and Th cell separation is realized through a magnetic bead separation process. Through verification and analysis, the separated Th cell has a very effective killing effect on virus infection, can effectively inhibit proliferation of viruses such as H IV and the like in the cell, reduces cytopathy, and provides reference for effective prevention or treatment of virus infection.

Description

Technical Field

[0001] The invention relates to the technical field of TH cell extraction, and in particular to a method for preparing foreign body phagolysosome immune TH cells in human body. Background Art

[0002] Helper T cells (Th cells) are a subset of T lymphocytes with CD4 as a surface marker. They play a key role in the body's immune response, can recognize specific antigens, and initiate a series of immune responses to help other immune cells.

[0003] Viral infection refers to the process in which viruses invade host cells and replicate in them, causing damage or dysfunction of host cells. Viral infection can affect multiple organs and systems, causing everything from mild colds to serious infectious diseases such as AIDS and hepatitis.

[0004] The use of immune cells to treat viral infections is a hot technical topic in this field. How to obtain high-purity, effective and safe Th ​​cells has been a hot topic of research in recent years. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention provides a method for preparing foreign body phagolysosomal immune TH cells in the human body. The present invention activates the killing performance of TH cells by phagocytic cell immunity method, and realizes Th cell separation by magnetic bead sorting process. After verification and analysis, the Th cells obtained by separation have a very effective killing effect on viral infection, can effectively inhibit the proliferation of viruses such as HIV in cells, and reduce the occurrence of cytopathic effects, providing a reference for the effective prevention or treatment of viral infection.

[0006] Specifically, the present invention first provides a method for preparing foreign body phagolysosomal immune TH cells in the human body, wherein the method comprises the following steps:

[0007] 1) Phagolysosome isolation;

[0008] 2) Phagolysosomal immune stimulation of peripheral blood T lymphocytes;

[0009] 3) Isolation of peripheral blood T lymphocytes;

[0010] 4) CD4 + Th cell isolation;

[0011] 5) CD3 + CD4 + Th cell isolation;

[0012] 6) CD28 + CD3 + CD4+ Th cell isolation;

[0013] 7) CD30 - CD28 + CD3 + CD4 + Th cell isolation;

[0014] 8) CD30 obtained in step 7) - CD28 + CD3 + CD4 + Th cells can be cultured in vitro.

[0015] Preferably, in step 1), 5-10 mg of human liver tissue is used as a sample, and phagolysosomes therein are separated using a lysosome separation kit;

[0016] Preferably, in step 2), 2-5 mL of the phagolysosomes separated in step 1) are added to 15 mL of human peripheral blood for immune stimulation treatment at 37° C. for 2-3 h;

[0017] Preferably, in step 3), the peripheral blood of step 2) is centrifuged at 1500-2000 r / min for 15-30 minutes, the upper plasma is drawn, inactivated at 56° C. for 30-45 minutes, and then centrifuged for standby use; the blood cell pellet is resuspended in human lymphocyte separation solution;

[0018] Preferably, in step 4), anti-CD4 antibody-coated immunomagnetic beads are added to step 3), mixed and incubated at 4° C. for 2-3 hours. After the incubation, the beads are placed in a sorting magnetic field, magnetically separated and eluted to obtain CD4+Th cells, which are then resuspended in 1-1.5 mL of 0.9% saline;

[0019] Preferably, in step 5), the anti-CD3 antibody-coated immunomagnetic beads are added to the cell suspension in step 4), mixed and incubated at 4° C. for 2-3 hours. After the incubation, the beads are placed in a sorting magnetic field, and after magnetic separation and elution, CD3 + CD4 + Th cells, resuspended in 1-1.5 mL of 0.9% saline;

[0020] Preferably, in step 6), the anti-CD28 antibody-coated immunomagnetic beads are added to the cell suspension in step 5), mixed and incubated at 4° C. for 2-3 hours. After the incubation, the beads are placed in a sorting magnetic field, and after magnetic separation and elution, CD28 + CD3 + CD4 + Th cells, resuspended in 1-1.5 mL of 0.9% saline;

[0021] Preferably, in step 7), anti-CD30 antibody-coated immunomagnetic beads are added to the cell suspension in step 6), mixed and incubated at 4° C. for 2 hours. After the incubation, the remaining cell suspension is collected after magnetic separation, and the cell precipitate is collected by centrifugation and resuspended in 1 mL of 0.9% saline;

[0022] Preferably, step 8) in vitro culturing comprises: culturing the obtained CD30 - CD28 + CD3 + CD4 + Th cells were resuspended in proliferation cell culture medium and cultured at 5% CO2 and 37°C. Fresh culture medium was replaced every 2 days. After one week of continuous culture, high-purity CD30 - CD28 + CD3 + CD4 + Th cells.

[0023] Preferably, the proliferating cell culture medium comprises: RPMI-1640 culture medium containing 10-15 ng / ml IL-2, 15-20 ng / ml IL-6, 10-15 ng / ml CaCl2, 10-15% FBS, 20-30 ng / ml TPO, and 1-2% P / S.

[0024] Another object of the present invention is to provide a human body foreign body phagolysosomal immune TH cell prepared by the above method.

[0025] Another object of the present invention is to provide a use of the human body foreign body phagolysosomal immune TH cells prepared by the above method in the preparation of drugs for the treatment of viral infections.

[0026] Preferably, the viral infection includes but is not limited to HIV infection.

[0027] The above technical solution of the present invention has the following advantages over the prior art: the present invention activates the killing performance of TH cells by phagocytic cell immunity method, and realizes Th cell separation by magnetic bead sorting process. After verification and analysis, the Th cells obtained by separation have a very effective killing effect on viral infection, can effectively inhibit the proliferation of viruses such as HIV in cells, and reduce the occurrence of cytopathic effects, providing a reference for the effective prevention or treatment of viral infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The killing performance of Th cells isolated by the present invention against HIV virus infection was evaluated. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0030] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0031] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0032] Example 1

[0033] A method for extracting foreign body phagolysosomal immune Th cells in human body, the method comprising the following steps:

[0034] 1) Phagolysosome separation: 5 mg of human liver tissue was used as a sample, and the phagolysosomes were separated using a lysosome separation kit (purchased from Guangzhou Wei Bo Technology Co., Ltd., catalog number 3106671). For detailed steps, please refer to the kit manual;

[0035] 2) Phagolysosome immunostimulation of peripheral blood T lymphocytes: 2 mL of phagolysosomes separated in step 1) was added to 10 mL of human peripheral blood for immunostimulation treatment at 37° C. for 2 h;

[0036] 3) Isolation of peripheral blood T lymphocytes: take the peripheral blood described in step 2), centrifuge at 1500r / min for 15 minutes, aspirate the upper plasma, inactivate at 56°C for 30 minutes, and then centrifuge for later use; resuspend the blood cell pellet in human lymphocyte separation solution (purchased from Hangzhou Lianke Biotechnology Co., Ltd., product number LSM02);

[0037] 4) CD4 + Th cell separation: add immunomagnetic beads coated with anti-CD4 antibody (purchased from Abcam, catalog number ab133616) to step 3), mix well and incubate at 4°C for 2 hours. After the incubation, place it in a sorting magnetic field for magnetic separation, and then elute with 1×PBS (pH=7.0) to obtain CD4 + Th cells, resuspended in 1 mL of 0.9% saline;

[0038] 5) CD3 + CD4 +Th cell separation: add anti-CD3 antibody (purchased from Abcam, catalog number ab16669) coated immunomagnetic beads to the cell suspension in step 4), mix well and incubate at 4°C for 2 hours. After the incubation, place it in a sorting magnetic field for magnetic separation, and then elute with 1×PBS (pH=7.0) to obtain CD3 + CD4 + Th cells, resuspended in 1 mL of 0.9% saline;

[0039] 6) CD28 + CD3 + CD4 + Th cell separation: add anti-CD28 antibody (purchased from Abcam, catalog number ab243228) coated immunomagnetic beads to the cell suspension in step 5), mix well and incubate at 4°C for 2 hours. After the incubation, place it in a sorting magnetic field for magnetic separation, and then elute with 1×PBS (pH=7.0) to obtain CD28 + CD3 + CD4 + Th cells, resuspended in 1 mL of 0.9% saline;

[0040] 7) CD30 - CD28 + CD3 + CD4 + Th cell separation: add immunomagnetic beads coated with anti-CD30 antibody (purchased from Abcam, catalog number ab252561) to the cell suspension in step 6), mix well and incubate at 4°C for 2 hours. After the incubation, collect the remaining cell suspension after magnetic separation, collect the cell pellet by centrifugation, and resuspend in 1 mL of 0.9% saline;

[0041] 8) CD30 obtained in step 7) - CD28 + CD3 + CD4 + Th cells were resuspended in proliferation cell culture medium and cultured at 5% CO2 and 37°C. Fresh culture medium was replaced every 2 days. After one week of continuous culture, high-purity CD30 - CD28 + CD3 + CD4 + Th cells.

[0042] Wherein, the proliferating cell culture medium includes: RPMI-1640 culture medium containing 10ng / ml IL-2, 15ng / ml IL-6, 10ng / ml CaCl2, 10% FBS, 20ng / ml TPO, and 1% P / S.

[0043] Example 2

[0044] A method for extracting foreign body phagolysosomal immune Th cells in human body, the method comprising the following steps:

[0045] 1) Phagolysosome separation: 10 mg of human liver tissue was used as a sample, and the lysosome separation kit (purchased from Guangzhou Wei Bo Technology Co., Ltd., catalog number 3106671) was used to separate the phagolysosome therein. For detailed steps, please refer to the kit manual;

[0046] 2) Phagolysosome immunostimulation of peripheral blood T lymphocytes: 5 mL of phagolysosomes separated in step 1) was added to 15 mL of human peripheral blood for immunostimulation treatment at 37° C. for 3 h;

[0047] 3) Isolation of peripheral blood T lymphocytes: take the peripheral blood described in step 2), centrifuge at 2000r / min for 30 minutes, aspirate the upper plasma, inactivate at 56°C for 45 minutes, and then centrifuge for later use; resuspend the blood cell pellet in human lymphocyte separation solution (purchased from Hangzhou Lianke Biotechnology Co., Ltd., product number LSM02);

[0048] 4) CD4 + Th cell separation: add immunomagnetic beads coated with anti-CD4 antibody (purchased from Abcam, catalog number ab133616) to step 3), mix well and incubate at 4°C for 3 hours. After the incubation, place it in a sorting magnetic field, and after magnetic separation and elution, obtain CD4 + Th cells, resuspended in 1.5 mL of 0.9% saline;

[0049] 5) CD3 + CD4 + Th cell separation: add immunomagnetic beads coated with anti-CD3 antibody (purchased from Abcam, catalog number ab16669) to the cell suspension in step 4), mix well and incubate at 4°C for 3 hours. After the incubation, place it in a sorting magnetic field, and after magnetic separation and elution, obtain CD3 + CD4 + Th cells, resuspended in 1.5 mL of 0.9% saline;

[0050] 6) CD28 + CD3 + CD4 + Th cell separation: add immunomagnetic beads coated with anti-CD28 antibody (purchased from Abcam, catalog number ab243228) to the cell suspension in step 5), mix well and incubate at 4°C for 3 hours. After the incubation, place it in a sorting magnetic field, and after magnetic separation and elution, obtain CD28 + CD3 +CD4 + Th cells, resuspended in 1.5 mL of 0.9% saline;

[0051] 7) CD30 - CD28 + CD3 + CD4 + Th cell separation: add immunomagnetic beads coated with anti-CD30 antibody (purchased from Abcam, catalog number ab252561) to the cell suspension in step 6), mix well and incubate at 4°C for 3 hours. After the incubation, collect the remaining cell suspension after magnetic separation, collect the cell pellet by centrifugation, and resuspend in 1.5 mL of 0.9% saline;

[0052] 8) CD30 obtained in step 7) - CD28 + CD3 + CD4 + Th cells were resuspended in proliferation cell culture medium and cultured at 5% CO2 and 37°C. Fresh culture medium was replaced every 2 days. After one week of continuous culture, high-purity CD30 - CD28 + CD3 + CD4 + Th cells.

[0053] Wherein, the proliferating cell culture medium includes: RPMI-1640 culture medium containing 15ng / ml IL-2, 20ng / ml IL-6, 15ng / ml CaCl2, 15% FBS, 30ng / ml TPO, and 2% P / S.

[0054] Comparative Example 1

[0055] The method steps are the same as those in Example 1, except that the immune stimulation treatment in step 2 is not performed.

[0056] Verification Example

[0057] Take 0.1mL 1×10 5 293T cells / mL were inoculated in a 96-well plate, and 50 μL of HIV virus with a titer of 1.12E+06TU / mL was inoculated in the above 96-well plate and incubated at 37°C for 2 h;

[0058] After the incubation, 0.1 mL of 1×10 6 CD30 cells / mL cultured in Example 1 - CD28 + CD3 + CD4 + Th;

[0059] In treatment group 2, 0.1 mL of 1×10 6 CD30 cells / mL cultured in Example 2 - CD28 + CD3 + CD4 + Th;

[0060] In treatment group 3, 0.1 mL of 1×10 6 CD30 cells / mL cultured in Comparative Example 1 - CD28 + CD3 + CD4 + Th;

[0061] Treatment group 4 was added with an equal amount of cell culture medium;

[0062] After the addition was completed, the 96-well plate was incubated at 37° C. for 12 h, the cytopathic effect (CPE) was observed under a microscope, and the HIV virus content in the cell body and supernatant was detected by qPCR. The results are shown in Table 1.

[0063] Table 1: Cytopathic Effect Assay

[0064]

[0065] Among them, the CPE judgment criteria are: no syncytium formation "-", 2-5 syncytial cells in each well "+", 5-10 syncytial cells in each well "++", and >10 syncytial cells in each well "+++".

[0066] From the results in Table 1, it can be seen that the CD30 isolated by the present invention - CD28 + CD3 + CD4 + Th cells can effectively inhibit the cytopathic effects caused by HIV virus, and the analysis of viral load by qPCR showed that the amount of HIV virus in cells and supernatant was significantly reduced, which has significant anti-HIV virus efficacy. However, the amount of HIV virus in treatment group 3 did not drop sharply. It is speculated that the reason may be that the killing ability of Th cells without phagolysosomal immunity is limited, resulting in a weakened inhibitory effect on HIV virus.

[0067] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing foreign body phagolysosomal immune TH cells in human body, characterized in that: The method comprises the following steps: 1) Phagolysosome isolation; 2) Phagolysosomal immune stimulation of peripheral blood T lymphocytes; 3) Isolation of peripheral blood T lymphocytes; 4) CD4 + Th cell isolation; 5) CD3 + CD4 + Th cell isolation; 6) CD28 + CD3 + CD4 + Th cell isolation; 7) CD30 - CD28 + CD3 + CD4 + Th cell isolation; 8) CD30 obtained in step 7) - CD28 + CD3 + CD4 + Th cells can be cultured in vitro.

2. The method according to claim 1, characterized in that Step 1) Human liver tissue is used as a sample, and phagolysosomes are isolated using a lysosome isolation kit.

3. The method according to claim 1, characterized in that Step 2) 2-5 mL of the phagolysosomes separated in step 1) are added to 15 mL of human peripheral blood for immune stimulation at 37° C. for 2-3 hours.

4. The method according to claim 1, characterized in that Step 3) taking the peripheral blood described in step 2), centrifuging at 1500-2000 r / min for 15-30 minutes, aspirating the upper plasma, inactivating at 56° C. for 30-45 minutes, and then centrifuging for later use; Resuspend the blood cell pellet in human lymphocyte separation medium.

5. The method according to claim 1, characterized in that Step 4) Add the immunomagnetic beads coated with anti-CD4 antibody to step 3), mix well and incubate at 4°C for 2-3 hours. After the incubation, place it in a sorting magnetic field, and after magnetic separation and elution, obtain CD4 + Th cells were resuspended in 1-1.5 mL of 0.9% saline.

6. The method according to claim 1, characterized in that Step 5) Add the anti-CD3 antibody-coated immunomagnetic beads to the cell suspension in step 4), mix well and incubate at 4°C for 2-3 hours. After the incubation, place it in a sorting magnetic field, and after magnetic separation and elution, obtain CD3 + CD4 + Th cells were resuspended in 1-1.5 mL of 0.9% saline.

7. The method according to claim 1, characterized in that Step 6) Add the immunomagnetic beads coated with anti-CD28 antibody to the cell suspension in step 5), mix well and incubate at 4°C for 2-3 hours. After the incubation, place it in a sorting magnetic field, and after magnetic separation and elution, obtain CD28 + CD3 + CD4 + Th cells were resuspended in 1-1.5 mL of 0.9% saline.

8. The method according to claim 1, characterized in that Step 7) Add anti-CD30 antibody-coated immunomagnetic beads to the cell suspension in step 6), mix well and incubate at 4°C for 2 hours. After the incubation, collect the remaining cell suspension after magnetic separation, collect the cell pellet by centrifugation, and resuspend in 1 mL of 0.9% saline.

9. The method according to claim 1, characterized in that Step 8) in vitro culture includes: obtaining CD30 - CD28 + CD3 + CD4 + Th cells were resuspended in proliferation cell culture medium and cultured at 5% CO2 and 37°C. Fresh culture medium was replaced every 2 days. After one week of continuous culture, high-purity CD30 - CD28 + CD3 + CD4 + Th cells.

10. The method according to claim 9, characterized in that The proliferating cell culture fluid comprises: RPMI-1640 culture medium containing 10-15 ng / ml IL-2, 15-20 ng / ml IL-6, 10-15 ng / ml CaCl2, 10-15% FBS, 20-30 ng / ml TPO, and 1-2% P / S.

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