DC-CIK cell loaded with liver cancer tumor antigen as well as preparation method and application of DC-CIK cell

Abstract

The invention relates to the technical field of cell culture. The invention provides a liver cancer tumor antigen-loaded DC-CIK cell as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) incubating a liver cancer cell under an acidic condition, and centrifuging to obtain supernate; (2) centrifuging the supernate at a high speed, collecting precipitate, and drying to obtain the liver cancer tumor antigen peptide; (3) mixing the liver cancer tumor antigen peptide with Hsp70 protein, and reacting to obtain compound protein; (4) mixing the compound protein and the DC cells, and co-culturing to obtain modified DC cells; and (5) mixing the modified DC cell and the CIK cell, and co-culturing to obtain the DC-CIK cell loaded with the liver cancer tumor antigen. The Hsp70 protein is combined with the liver cancer tumor antigen peptide, so that the presentation efficiency of the DC cells is improved. By dynamically regulating and controlling parameters such as proportion, time and cell factors, the specific killing ability of the CIK cells is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a DC-CIK cell loaded with liver cancer tumor antigens, and a preparation method and application thereof. Background Art

[0002] Dendritic cells (DCs), an important type of antigen-presenting cell in the body, can effectively take up and process tumor antigens, then present them to T and B cells via MHC class I and MHC class II molecules, activating them and generating specific anti-tumor immune responses. DCs can be produced by inducing and culturing bone marrow and peripheral blood cells in vitro, but the effectiveness of DCs alone in activating T cells after antigen loading is limited, and large-scale production is difficult.

[0003] CIK cells are derived from human peripheral blood mononuclear cells (PBMCs) cultured in vitro with various cytokines for a period of time. They possess non-major histocompatibility antigen (MHC)-restricted tumoricidal activity. However, traditional CIK therapy lacks tumor specificity and is not sufficiently targeted for liver cancer. Summary of the Invention

[0004] The purpose of the present invention is to provide a DC-CIK cell loaded with liver cancer tumor antigens and its preparation method and application, so as to realize the production of efficient and specific anti-liver cancer immune cell preparations, and have significant clinical transformation potential.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing DC-CIK cells loaded with liver cancer tumor antigens, comprising the following steps:

[0007] (1) Incubate liver cancer cells under acidic conditions and obtain the supernatant after centrifugation;

[0008] (2) centrifuging the supernatant at high speed, collecting the precipitate, and drying it to obtain the liver cancer tumor antigen peptide;

[0009] (3) mixing the liver cancer tumor antigen peptide and the Hsp70 protein to obtain a composite protein after reaction;

[0010] (4) mixing the complex protein and DC cells, and co-culturing to obtain modified DC cells;

[0011] (5) The modified DC cells and CIK cells were mixed and co-cultured to obtain DC-CIK cells loaded with liver cancer tumor antigens.

[0012] Preferably, the incubation in step (1) is carried out in a citric acid and sodium hydrogen phosphate buffer solution with a pH value of 3.0 to 3.5, the incubation time is 6 to 12 hours, the centrifugal speed is 650 to 700 r / min, and the centrifugation time is 8 to 10 minutes.

[0013] Preferably, the rotation speed of the high-speed centrifugation in step (2) is 20,000 to 22,000 r / min, the centrifugation time is 22 to 26 min, and freeze drying is used during the drying.

[0014] Preferably, the reaction system in step (3) is as follows: 60-65 mg / L of liver cancer tumor antigen peptide, 40-150 mg / L of Hsp701, 0.8-1.2 mmol / L of MgCl2, and 0.6-0.8 mmol / L of ADP obtained in step (2) are dissolved in PBS buffer;

[0015] The reaction temperature is 35-37° C., and the reaction time is 150-180 min.

[0016] Preferably, the mixing method in step (4) is to adjust the concentration of DC cells to 2-3×10 6 / mL, and then add the complex protein to make the final concentration of the complex protein 80-100mg / L.

[0017] Preferably, the co-cultivation temperature in step (4) is 35-37° C., and the co-cultivation time is 24-36 h.

[0018] Preferably, the mixing method in step (5) is: inoculating the modified DC cells and CIK cells into a culture medium, wherein the ratio of the modified DC cells to the CIK cells during inoculation is 1:6-8;

[0019] The culture medium is based on 1640 culture medium and further contains 12-15 μg / mL of 4-hydroxyethylpiperazineethanesulfonic acid, 400-450 IU / mL of IL-2, and 50-60 μg / mL of IFN-γ.

[0020] Preferably, the co-cultivation temperature in step (5) is 35-37° C., and the co-cultivation time is 8-10 h.

[0021] The present invention also provides DC-CIK cells loaded with liver cancer tumor antigens prepared by the preparation method.

[0022] The present invention also provides the use of the DC-CIK cells loaded with liver cancer tumor antigens in the preparation of medicines for treating liver cancer.

[0023] The present invention provides a DC-CIK cell loaded with a liver cancer tumor antigen, and a preparation method and application thereof, comprising the following steps: (1) incubating liver cancer cells under acidic conditions and obtaining a supernatant after centrifugation; (2) centrifuging the supernatant at high speed, collecting the precipitate, and drying to obtain a liver cancer tumor antigen peptide; (3) mixing the liver cancer tumor antigen peptide with Hsp70 protein to obtain a composite protein after reaction; (4) mixing the composite protein with DC cells and co-culturing to obtain modified DC cells; (5) mixing the modified DC cells with CIK cells and co-culturing to obtain DC-CIK cells loaded with a liver cancer tumor antigen. The present invention improves the presentation efficiency of DC cells by combining Hsp70 protein with the liver cancer tumor antigen peptide. By dynamically regulating parameters such as ratio, time, and cytokines, the specific killing ability of CIK cells is enhanced. DETAILED DESCRIPTION

[0024] The present invention provides a method for preparing DC-CIK cells loaded with liver cancer tumor antigens, comprising the following steps:

[0025] (1) Incubate liver cancer cells under acidic conditions and obtain the supernatant after centrifugation;

[0026] (2) centrifuging the supernatant at high speed, collecting the precipitate, and drying it to obtain the liver cancer tumor antigen peptide;

[0027] (3) mixing the liver cancer tumor antigen peptide and the Hsp70 protein to obtain a composite protein after reaction;

[0028] (4) mixing the complex protein and DC cells, and co-culturing to obtain modified DC cells;

[0029] (5) The modified DC cells and CIK cells were mixed and co-cultured to obtain DC-CIK cells loaded with liver cancer tumor antigens.

[0030] In the present invention, the incubation in step (1) is preferably carried out in a citric acid and sodium hydrogen phosphate buffer solution with a pH value of 3.0 to 3.5, more preferably in a citric acid and sodium hydrogen phosphate buffer solution with a pH value of 3.2 to 3.3, the incubation time is preferably 6 to 12 hours, more preferably 9 hours, the centrifugal speed is preferably 650 to 700 r / min, more preferably 675 r / min, and the centrifugal time is preferably 8 to 10 minutes, more preferably 9 minutes.

[0031] In the present invention, the rotation speed of the high-speed centrifugation in step (2) is preferably 20,000 to 22,000 r / min, more preferably 21,000 r / min, the centrifugation time is preferably 22 to 26 min, more preferably 24 min, and freeze drying is preferably used for drying.

[0032] In the present invention, the reaction system in step (3) is preferably: 60-65 mg / L of liver cancer tumor antigen peptide, 40-150 mg / L of Hsp701, 0.8-1.2 mmol / L of MgCl2, and 0.6-0.8 mmol / L of ADP are obtained in step (2), and dissolved in PBS buffer. More preferably, 63 mg / L of liver cancer tumor antigen peptide, 45 mg / L of Hsp701, 1 mmol / L of MgCl2, and 0.7 mmol / L of ADP are obtained in step (2);

[0033] The reaction temperature is preferably 35-37° C., more preferably 36° C., and the reaction time is preferably 150-180 min, more preferably 165 min.

[0034] In the present invention, the mixing method in step (4) is preferably: adjusting the concentration of DC cells to 2-3×10 6 / mL, and then add the complex protein to make the final concentration of the complex protein 80-100 mg / L. More preferably, the concentration of DC cells is adjusted to 2.5×10 6 / mL, and then the complex protein was added to make the final concentration of the complex protein 90mg / L.

[0035] In the present invention, the co-culture temperature in step (4) is preferably 35-37°C, more preferably 36°C, and the co-culture time is preferably 24-36 hours, more preferably 30 hours.

[0036] In the present invention, the mixing method in step (5) is preferably: inoculating the modified DC cells and CIK cells into the culture medium, wherein the ratio of the modified DC cells to the CIK cells during inoculation is 1:6 to 8, more preferably 1:7;

[0037] The culture medium is preferably based on 1640 culture medium, and further contains 12-15 μg / mL of 4-hydroxyethylpiperazineethanesulfonic acid, 400-450 IU / mL of IL-2, and 50-60 μg / mL of IFN-γ. It is further preferably based on 1640 culture medium, and further contains 13-14 μg / mL of 4-hydroxyethylpiperazineethanesulfonic acid, 25 IU / mL of IL-2, and 55 μg / mL of IFN-γ.

[0038] In the present invention, the co-culture temperature in step (5) is preferably 35-37° C., more preferably 36° C., and the co-culture time is preferably 8-10 h, more preferably 9 h.

[0039] The present invention also provides DC-CIK cells loaded with liver cancer tumor antigens prepared by the preparation method.

[0040] The present invention also provides the use of the DC-CIK cells loaded with liver cancer tumor antigens in the preparation of medicines for treating liver cancer.

[0041] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1

[0043] A method for preparing DC-CIK cells loaded with liver cancer tumor antigens comprises the following steps:

[0044] (1) HepG2 liver cancer cells were incubated in a citric acid and sodium hydrogen phosphate buffer solution at pH 3.5 for 6 h and centrifuged at 650 rpm for 10 min to obtain the supernatant;

[0045] (2) The supernatant was centrifuged at 20,000 rpm for 26 min, the precipitate was collected, and freeze-dried to obtain the liver cancer tumor antigen peptide;

[0046] (3) Mixing the liver cancer tumor antigen peptide, Hsp70 protein, MgCl2, and ADP to obtain a reaction system of 60 mg / L liver cancer tumor antigen peptide, 140 mg / L Hsp70, 1.2 mmol / L MgCl2, and 0.6 mmol / L ADP, and reacting at 37°C for 150 min to obtain a composite protein;

[0047] (4) Use culture medium to adjust the concentration of DC cells to 3×10 6 / mL, then add the complex protein to make the final concentration of the complex protein 80mg / L, and culture at 37℃ for 24h to obtain modified DC cells;

[0048] (5) The modified DC cells and CIK cells were inoculated into the culture medium at a ratio of 1:8, and co-cultured at 35°C for 10 h to obtain DC-CIK cells loaded with liver cancer tumor antigens.

[0049] Note: The culture medium in step (5) is based on 1640 medium and also contains 12 μg / mL of 4-hydroxyethylpiperazineethanesulfonic acid, 400 IU / mL of IL-2, and 60 μg / mL of IFN-γ.

[0050] Example 2

[0051] A method for preparing DC-CIK cells loaded with liver cancer tumor antigens comprises the following steps:

[0052] (1) HepG2 liver cancer cells were incubated in a pH 3.0 citric acid and sodium hydrogen phosphate buffer for 12 h and centrifuged at 700 rpm for 8 min to obtain the supernatant;

[0053] (2) The supernatant was centrifuged at 22000 rpm for 22 min, the precipitate was collected, and freeze-dried to obtain the liver cancer tumor antigen peptide;

[0054] (3) Mixing the liver cancer tumor antigen peptide, Hsp70 protein, MgCl2, and ADP to obtain a reaction system of 65 mg / L liver cancer tumor antigen peptide, 150 mg / L Hsp70, 0.8 mmol / L MgCl2, and 0.8 mmol / L ADP, and reacting at 35°C for 180 min to obtain a composite protein;

[0055] (4) Use culture medium to adjust the concentration of DC cells to 2×10 6 / mL, then add the complex protein to make the final concentration of the complex protein 100mg / L, and culture at 35℃ for 36h to obtain modified DC cells;

[0056] (5) The modified DC cells and CIK cells were inoculated into the culture medium at a ratio of 1:6, and co-cultured at 37°C for 8 h to obtain DC-CIK cells loaded with liver cancer tumor antigens.

[0057] Note: The culture medium in step (5) is based on 1640 medium and also contains 4-hydroxyethylpiperazineethanesulfonic acid 15 μg / mL, IL-24 50 IU / mL, and IFN-γ 50 μg / mL.

[0058] Example 3

[0059] A method for preparing DC-CIK cells loaded with liver cancer tumor antigens comprises the following steps:

[0060] (1) HepG2 liver cancer cells were incubated in a pH 3.3 citric acid and sodium hydrogen phosphate buffer for 8 h and centrifuged at 675 rpm for 9 min to obtain the supernatant;

[0061] (2) The supernatant was centrifuged at 21000 rpm for 24 min, the precipitate was collected, and freeze-dried to obtain the liver cancer tumor antigen peptide;

[0062] (3) The liver cancer tumor antigen peptide, Hsp70 protein, MgCl2, and ADP were mixed to obtain a reaction system of 63 mg / L liver cancer tumor antigen peptide, 145 mg / L Hsp70, 1 mmol / L MgCl2, and 0.7 mmol / L ADP. The reaction was carried out at 36°C for 165 min to obtain a composite protein.

[0063] (4) Use culture medium to adjust the concentration of DC cells to 2.5×10 6 / mL, then add the complex protein to make the final concentration of the complex protein 90mg / L, and culture at 36℃ for 30h to obtain modified DC cells;

[0064] (5) The modified DC cells and CIK cells were inoculated into the culture medium at a ratio of 1:7, and co-cultured at 36°C for 9 hours to obtain DC-CIK cells loaded with liver cancer tumor antigens.

[0065] Note: The culture medium in step (5) is based on 1640 medium and also contains 4-hydroxyethylpiperazineethanesulfonic acid 13 μg / mL, IL-24 25 IU / mL, and IFN-γ 55 μg / mL.

[0066] Test example: cell killing test

[0067] The DC-CIK cells loaded with liver cancer tumor antigens prepared in Examples 1 to 3 were collected as effector cells. The DC-CIK cells loaded with liver cancer tumor antigens prepared in Example 1 were dispersed in RMPI-1640 medium containing 10% fetal bovine serum to a final concentration of 2×10 5 The same method was used to prepare sample 2 containing DC-CIK cells prepared in Example 2 and sample 3 containing DC-CIK cells prepared in Example 3. Untreated conventional CIK cells were treated in the same way to obtain control samples.

[0068] HepG2 liver cancer cells were used as target cells and inoculated into samples 1 to 3 and the control sample (the concentration after inoculation was 1×10 4 Cells were incubated at 37°C, 5% CO₂ for 24 h, and then CCK-8 reagent was added for a further 3 h. The killing activity was calculated.

[0069] The results showed that the killing activity of sample 1 against HepG2 liver cancer cells was 78.11%; the killing activity of sample 2 against HepG2 liver cancer cells was 75.79%; the killing activity of sample 3 against HepG2 liver cancer cells was 79.03%, and the killing activity of the control sample against HepG2 liver cancer cells was 61.29%.

[0070] As can be seen from the above examples, the present invention provides a DC-CIK cell loaded with a liver cancer tumor antigen, and a preparation method and application thereof, comprising the following steps: (1) incubating liver cancer cells under acidic conditions and obtaining a supernatant after centrifugation; (2) centrifuging the supernatant at high speed, collecting the precipitate, and drying to obtain a liver cancer tumor antigen peptide; (3) mixing the liver cancer tumor antigen peptide with Hsp70 protein to obtain a composite protein after reaction; (4) mixing the composite protein with DC cells and co-culturing to obtain modified DC cells; (5) mixing the modified DC cells with CIK cells and co-culturing to obtain DC-CIK cells loaded with a liver cancer tumor antigen. The present invention improves the presentation efficiency of DC cells by combining Hsp70 protein with liver cancer tumor antigen peptides. By dynamically controlling parameters such as ratio, time, and cytokines, the specific killing ability of CIK cells is enhanced.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing DC-CIK cells loaded with liver cancer tumor antigens, characterized in that: The steps include: (1) Incubate liver cancer cells under acidic conditions and obtain the supernatant after centrifugation; (2) centrifuging the supernatant at high speed, collecting the precipitate, and drying it to obtain the liver cancer tumor antigen peptide; (3) mixing the liver cancer tumor antigen peptide and the Hsp70 protein to obtain a composite protein after reaction; (4) mixing the complex protein and DC cells, and co-culturing to obtain modified DC cells; (5) The modified DC cells and CIK cells were mixed and co-cultured to obtain DC-CIK cells loaded with liver cancer tumor antigens.

2. The preparation method according to claim 1, characterized in that The incubation in step (1) is carried out in a citric acid and sodium hydrogen phosphate buffer solution with a pH value of 3.0 to 3.5, the incubation time is 6 to 12 hours, the centrifugal speed is 650 to 700 r / min, and the centrifugal time is 8 to 10 minutes.

3. The preparation method according to claim 2, characterized in that The speed of the high-speed centrifugation in step (2) is 20,000 to 22,000 r / min, the centrifugation time is 22 to 26 minutes, and freeze drying is used during the drying.

4. The preparation method according to claim 3, characterized in that The reaction system in step (3) is as follows: 60-65 mg / L of liver cancer tumor antigen peptide, 40-150 mg / L of Hsp701, 0.8-1.2 mmol / L of MgCl2, and 0.6-0.8 mmol / L of ADP obtained in step (2) are dissolved in PBS buffer; The reaction temperature is 35-37° C., and the reaction time is 150-180 min.

5. The preparation method according to claim 4, characterized in that The mixing method in step (4) is as follows: the concentration of DC cells is adjusted to 2-3×10 6 / mL, and then add the complex protein to make the final concentration of the complex protein 80-100mg / L.

6. The preparation method according to claim 5, characterized in that The co-cultivation temperature in step (4) is 35-37° C., and the co-cultivation time is 24-36 h.

7. The preparation method according to claim 6, characterized in that The mixing method in step (5) is as follows: inoculating the modified DC cells and CIK cells into a culture medium, wherein the ratio of the modified DC cells to the CIK cells during inoculation is 1:6-8; The culture medium is based on 1640 culture medium and further contains 12-15 μg / mL of 4-hydroxyethylpiperazineethanesulfonic acid, 400-450 IU / mL of IL-2, and 50-60 μg / mL of IFN-γ.

8. The preparation method according to any one of claims 1 to 7, characterized in that The co-cultivation temperature in step (5) is 35-37° C., and the co-cultivation time is 8-10 h.

9. DC-CIK cells loaded with liver cancer tumor antigens prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the DC-CIK cells loaded with liver cancer tumor antigens according to claim 9 in the preparation of a drug for treating liver cancer.