CN111996166B

CN111996166B - Loaded DC cell, DC-CIK cell and application in tumor cell treatment

Info

Publication number: CN111996166B
Application number: CN202010927781.4A
Authority: CN
Inventors: 单海华
Original assignee: Yousai Medical Technology Tianjin Co ltd
Current assignee: Yousai Medical Technology Tianjin Co ltd
Priority date: 2020-09-07
Filing date: 2020-09-07
Publication date: 2022-10-25
Anticipated expiration: 2040-09-07
Also published as: CN111996166A

Abstract

The invention discloses a loaded DC cell, a DC-CIK cell and application in tumor cell treatment. The research result of the invention shows that the 2'-FL load can improve the activity of the DC cell, and the DC cell loaded by the 2' -FL can not only promote the in-vitro proliferation of the CIK cell, but also improve the killing power of the CIK cell to the tumor cell. DC-CIK cells prepared by replacing conventional DC cells which are not loaded with DC cells with 2' -FL loaded DC cells can be used for preparing antitumor drugs and are input into a tumor patient body for cell therapy after in vitro co-culture; the DC cell loaded by the 2' -FL can also be directly used for preparing an anti-tumor medicament, and CIK cells in a patient body are activated for cell therapy after the DC cell is input into the patient body of a tumor patient.

Description

Loaded DC cell, DC-CIK cell and application in tumor cell treatment

Technical Field

The invention belongs to the field of biology, relates to cell therapy of tumors, and particularly relates to a loaded DC cell, a DC-CIK cell and application of the loaded DC cell and the DC-CIK cell in tumor cell therapy.

Background

The DC-CIK is the CIK cells and the DC cells which are cultured together in vitro, and the anti-tumor activity of the ClK cells is enhanced while the maturation of the DC cells is promoted. The DC cells can activate the immune system of a human body, can monitor and kill tumor cells for a long time, and show a strong antigen presenting function; the CIK cells can directly and accurately 'click' tumor cells and remove micro lesions without affecting normal tissues, so that the DC and the CIK cells are cultured in a combined mode (namely the DC-CIK cells), specific immune killing can be highlighted, and strong non-specific anti-tumor effects can be displayed, and the strategy deployment is similar to the strategic deployment of mutual cooperation between 'scouts' and 'artillery' in a battlefield. Besides the characteristics of high tumor killing activity and wide tumor killing range, the compound also has the characteristic of reducing immune tolerance, and can effectively prevent certain autoimmune diseases. The combination of the two has obvious effect in the biological immunotherapy, fully mobilizes autoimmunity, inhibits and kills tumor cells, and more importantly, does not damage normal tissues, effectively improves the life quality of tumor patients, prolongs the life cycle.

Research shows that the killing power of the CIK cells to the tumor cells can be further improved by selecting a proper loading substance to load the DC cells and then co-culturing the DC cells and the CIK cells.

2' -fucosyllactose (2 ' -fucosyllactose,2' -FL) is one of the major oligosaccharides found in breast milk, and is high in content and most widely studied at present. 2' -FL has a variety of health effects including: is degraded and metabolized by intestinal probiotics, and provides energy to promote self growth; the specific binding of pathogenic bacteria in the intestinal tract prevents the adhesion of the pathogenic bacteria to the epithelial cells of the intestinal tract; has the function of immunoregulation in the intestinal lymphatic system and the whole body.

At present, no research report that 2' -FL is used for CIK cell in-vitro culture after loading DC cells to promote CIK cell proliferation and improve lethality is available.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a loaded DC cell, a DC-CIK cell and application thereof in tumor cell treatment, so that a large number of DC-CIK cells with high lethality can be rapidly obtained in vitro, and further, the cells can be 'massively' and 'efficiently' used for being infused into a tumor patient body for tumor treatment.

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

a DC-CIK cell, wherein the DC-CIK cell is prepared by co-culturing a DC cell and a CIK cell; the DC cells are 2' -fucosyllactose-loaded DC cells.

The DC-CIK cell is used for preparing antitumor drugs.

A DC cell, which is a 2' -fucosyllactose-loaded DC cell.

The DC cell is used for promoting the proliferation of CIK cells by in vitro culture.

The DC cell is used for improving the killing power of CIK cells in vitro culture.

The DC cell is used for preparing antitumor drugs.

The application of the 2' -fucosyllactose in DC cell in-vitro culture for improving the activity of the DC cell, wherein the activity refers to that the killing power of the DC cell on tumor cells is improved by co-culture with CIK cells.

Has the advantages that:

the experimental result shows that the 2'-FL loaded DC cell can improve the activity of the DC cell, and the 2' -FL loaded DC cell can not only promote the in-vitro proliferation of the CIK cell, but also improve the killing power of the CIK cell on the tumor cell. The DC-CIK cell prepared by replacing the conventional DC cell which is not loaded with the DC cell loaded with the 2' -FL can be used for preparing an anti-tumor medicament, and is input into a tumor patient body for cell therapy after in vitro co-culture; the DC cell loaded by the 2' -FL can also be directly used for preparing an anti-tumor medicament, and CIK cells in a patient body are activated for cell therapy after the DC cell is input into the patient body of a tumor patient. The technical scheme of the invention can achieve the purpose of rapidly obtaining a large amount of DC-CIK cells with high lethality in vitro and further can be used for inputting a large amount of DC-CIK cells and high efficiency DC-CIK cells into a tumor patient body for tumor treatment.

Drawings

FIG. 1 shows the expansion ratio (A) of DC-CIK cells and CD3 cells in control and load groups ⁺ CD56 ⁺ Cell proportion (B);

FIG. 2 is a photograph of a stripped tumor in the blank, control and load groups;

FIG. 3 shows the tumor suppression rate in the control and load groups of nude mice.

Detailed Description

1. Experimental materials

PBMC human peripheral blood mononuclear cells were purchased from Shenzhen luxury Tuo Biotech, inc. and shipped by frozen storage.

Human peripheral blood dendritic primary cells (DC primary cells) were purchased from Yaji Biotechnology, inc. of Shanghai, frozen for shipment.

HepG2 cells were purchased from ATCC, used after resuscitative subculture, and grown in nude mice as log-phase cells.

RPMI-1640 culture medium, fetal bovine serum, AIM-V serum-free culture medium, and human AB serum were purchased from Gibco, USA.

2'-fucosyllactose (2' -FL, hereinafter abbreviated) has a purity of 98% or more.

Male healthy SPF-grade BALB/c-nu nude mice are purchased from Nanjing university model animal institute, are 7-8 weeks old, and have a body weight of 24-26 g.

2. Experimental methods

1. Resuscitation and passage of PBMC human peripheral blood mononuclear cells

(1) Preparing sterile water at 37 ℃ 5 minutes earlier (preparing sterile water 1L for later use one day earlier, pouring out a 300mL microwave oven, slightly heating, and adjusting to 37 ℃);

(2) putting the PBMC freezing tube into water at 37 ℃, holding the upper part of the PBMC freezing tube by hand without immersing the cover seam into the water, shaking the PBMC freezing tube to melt the cells within 1 minute as far as possible (not more than 2 minutes, taking out and seeing down the PBMC freezing tube at intervals, stopping water bath when the cells are melted to leave small granular ice-like substances);

(3) centrifuging the freezing tube (900 rpm, 1 min), spraying 75% ethanol on the tube wall for sterilization, after ethanol volatilization, aseptically sucking supernatant freezing liquid, discarding, adding 1mL culture medium, mixing, adding into culture bottle (T25), inoculating, adding culture medium, and adjusting the content of CO at 37 deg.C and 5% ₂ And (5) performing conditioned culture. The culture medium is RPMI-1640 medium containing 1% double antibody and 10% fetal bovine serum.

(4) And culturing the cells until the density reaches more than 80 percent, and then carrying out passage.

2. Resuscitation and passage of DC cells

(2) putting the DC cell freezing tube into water at 37 ℃, holding the upper part of the DC cell freezing tube by hand without immersing the cover seam into the water, shaking the freezing tube to melt the cells within 1 minute as much as possible (not exceeding 2 minutes, taking out the DC cell freezing tube at intervals, and stopping water bath when the cells are melted to leave small granular ice materials);

(3) centrifuging the frozen tube (900 rpm, 1 min), spraying 75% ethanol on the tube wall for sterilization, sterilizing, collecting supernatant, discarding, adding 1mL culture medium, mixing, adding into culture flask (T25), inoculating, adding culture medium at 37 deg.C and 5% CO ₂ And (5) performing conditioned culture. The culture medium isRPMI-1640 medium containing 1% double antibody and 10% fetal bovine serum.

(4) Cells were passaged when they reached 80% confluence.

3. CIK cell culture

Subcultured PBMC were aspirated off the medium, digested with 0.25% trypsin, and resuspended in AIM-V medium containing 5% human AB serum to a concentration of 1.5X 10 ⁶ The final concentration of IFN-. Gamma.added to the cell suspension was 1000IU/mL, the CO content was 5% at 37 ℃% ₂ After the conditioned culture for 24h, adding anti-CD 3McAb with the final concentration of 80 mug/L and rhIL-2 with the final concentration of 1000IU/mL for continuous culture, supplementing AIM-V culture solution containing 5% human AB serum, 80 mug/L anti-CD 3McAb and 1000IU/mLrhIL-2 every 2-3 d, and maintaining the cell concentration at 1.5 x 10 ⁶ The volume is/mL. CIK cells were collected for further 7 d.

4. DC cell culture and Loading

Subcultured DC cells were removed, the medium was aspirated, digested with 0.25% trypsin, and resuspended in AIM-V medium containing 5% human AB serum to a concentration of 1.0X 10 ⁶ The final concentration of IL-4 and GM-CSF was 500IU/mL in the cell suspension/mL, and the final concentration was 5% CO at 37 ℃ ₂ Culturing under conditions, adding AIM-V culture solution containing 5% human AB serum, 500IU/mLIL-4 and 1000IU/mLGM-CSF every 2-3 days, and maintaining cell concentration at 1.0 × 10 ⁶ and/mL. Addition of 50ng/mL TNF-. Alpha.on day 6 stimulated DC maturation. The load of 200ng/mL2'-FL was added on day 7, and incubated with DC cells for 24h, and the DC cells loaded with 2' -FL were collected on day 8. Meanwhile, DC cells without 2' -FL load are set as a control group.

5. Grouping and DC-CIK cocultivation

The control group and the load group are divided, the DC cells without 2'-FL load are used for co-culturing with CIK cells in the control group, and the DC cells with 2' -FL load are used for co-culturing with CIK cells in the load group. The co-culture method is as follows: and respectively collecting CIK cells and DC cells, washing with PBS, respectively resuspending with AIM-V culture solution containing 5% human AB serum, and mixing the DC cells and the CIK cells according to the quantitative ratio of 1 ⁵ The volume is/mL. And collecting DC-CIK cells which are co-cultured for 7 d.

6. Cell proliferationFold and CD3 ⁺ CD56 ⁺ Cell ratio determination

And respectively collecting DC-CIK cells co-cultured for 7d in the control group and the load group, counting the total number of the cells subjected to culture and amplification by using a cell counting method, and calculating the ratio of the total number of the cells to the number of the DC-CIK cells at 0d, wherein the ratio is the cell amplification multiple.

CD3 ⁺ CD56 ⁺ The cell proportion is measured by a flow method, and the specific method comprises the following steps: respectively collecting DC-CIK cells co-cultured for 0d and 7d in the control group and the load group, washing for 2-3 times by PBS, resuspending, and adjusting the cell concentration to 5 multiplied by 10 ⁶ PermL, 100. Mu.L of cell suspension was placed in a special flow cytometer tube, cells were labeled with FITC-CD3 and PE-CD56 flow antibodies, incubated at 4 ℃ for 30min, and then analyzed for cell phenotype by flow cytometry.

7. Determination of in vivo tumoricidal Activity of cells in nude mice transplantation tumor assay

Taking HepG2 cells in logarithmic growth phase, digesting, washing with PBS, and adjusting the cell concentration to 4 multiplied by 10 ⁷ Each nude mouse was inoculated with 0.2mL of the vaccine subcutaneously on one side of the back. The tumor mass is about 80mm ³ Then, the treatment was carried out by randomly dividing the test sample into a blank group, a control group and a load group, each group containing 5 animals, according to the following method:

blank group: injecting normal saline around the tumor body;

control group: the control group of "5, subgroup and DC-CIK Co-culture" above was injected around the tumor for 7 days of Co-culture of DC-CIK cells (5X 10) ⁶ Individual cells, 0.2 mL);

and (3) load group: injecting the DC-CIK cells of the load group co-culture 7d (5X 10) in the above "5, group and DC-CIK co-culture" around tumor body ⁶ Cell, 0.2 mL).

1 injection was given every 3d as described above for a total of 5 injections. After 24h of the last injection for 1 time, the nude mice were sacrificed, tumor tissues were stripped, photographed, tumor weight was weighed, and the tumor inhibition rate was calculated according to the following formula:

tumor inhibition (%) = (mean tumor weight of blank-DC-mean tumor weight of CIK) divided by mean tumor weight of blank × 100%.

8. Statistical method

Statistical analysis was performed using SPSS software. Data are presented as mean ± s, and comparisons between groups were performed using the t-test.

3. Results of the experiment

1. Cell proliferation fold and CD3 ⁺ CD56 ⁺ Cell ratio determination

Control group and load group DC-CIK cell amplification fold and CD3 ⁺ CD56 ⁺ The cell ratios are shown in table 1 and fig. 1, for example. The result shows that the DC-CIK cells prepared by co-culturing the DC cells loaded with 2' -FL and the CIK cells have stronger proliferation activity and higher CD3 ⁺ CD56 ⁺ Cell ratio. As is known to those skilled in the art, CD3+ CD56+ cells are the main effector cells of CIK cells, and are the cells of DC-CIK cells which play the most important role in killing tumor cells. Therefore, the 2' -FL-loaded DC cells can promote the proliferation of CIK cells in vitro.

TABLE 1 fold expansion of DC-CIK cells and CD3 in control and load groups ⁺ CD56 ⁺ Proportion of cells

2. Killing activity of DC-CIK cells on tumor cells in vivo

Fig. 2 is a photograph of the peeled tumors of the blank group, the control group and the load group, and tables 2 and 3 show the in vivo tumor inhibition rates of the control group and the load group. The result shows that the DC-CIK cells of the load group with the same quantity have stronger in-vivo tumor killing and inhibiting activity than the DC-CIK cells of the control group. Therefore, the 2' -FL loaded DC cells can improve the killing power of CIK cells on tumor cells.

TABLE 2 in vivo tumor inhibition rates in control and load groups of nude mice

The experimental result shows that the 2'-FL load can improve the activity of the DC cell, and the DC cell loaded by the 2' -FL can promote the in-vitro proliferation of the CIK cell and improve the killing power of the CIK cell on the tumor cell. The DC-CIK cell prepared by replacing the conventional DC cell which is not loaded with the DC cell loaded with the 2' -FL can be used for preparing an anti-tumor medicament, and is input into a tumor patient body for cell therapy after in vitro co-culture; the DC cell loaded by the 2' -FL can also be directly used for preparing an anti-tumor medicament, and CIK cells in a patient body are activated for cell therapy after the DC cell is input into the patient body of a tumor patient.

Claims

1. A DC-CIK cell, comprising: the DC-CIK cell is obtained by co-culturing a DC cell and a CIK cell; wherein the DC cells are 2' -fucosyllactose-loaded DC cells.

2. The application of the DC cells in CIK cell in-vitro culture for promoting CIK cell proliferation and improving the killing power of the CIK cells is disclosed, and the DC cells are DC cells loaded with 2' -fucosyllactose.