Method for cultivating lymph-node autologous CIK (cytokine-induced killer) cells and application of lymph-node autologous CIK cells

Abstract

The invention belongs to the field of biotechnology and microbial animal cell lines and particularly relates to a method for cultivating lymph-node autologous CIK (cytokine-induced killer) cells and application of the lymph-node autologous CIK cells. The CIK cells are obtained by in-vitro cultivation of isolated lymph node tissue obtained in surgery in patients with lung cancer and are compared with the CIK cells obtained from cultivation of isolated peripheral blood, biological characteristics of the CIK cells and cytotoxicity effect on pulmonary granule cancer cells are observed, the result shows that the isolated lymph node issue of the surgical patients can be completely used for cultivation of CIK, in-vitro tumor-cytotoxicity cells are powerful in activity, and the activity and cancer inhibition function are prior to the CIK cells of the peripheral blood. The CIK cells can be used for preparing pulmonary granule and cancer inhibition products and made into compounds with chemotherapy drugs as well as used for adoptive immunity and intervention for postoperative patients with lung cancers, and occurrence rate for recurrence and metastasis of the postoperative patients with the lung cancers is beneficially decreased.

Description

technical field

[0001] The invention belongs to the fields of biotechnology and microbial animal cell lines, and in particular relates to a method for culturing autologous CIK cells in lymph nodes and an application thereof. Background technique

[0002] According to literature reports, the morbidity and mortality of lung cancer rank first among all malignant tumors, of which non-small cell lung cancer (NSCLC) accounts for 80%, which is characterized by high incidence, high mortality and low survival rate. Although the current intervention measures such as surgery, radiotherapy, and chemotherapy are constantly improving, the overall survival rate of lung cancer patients has not been significantly improved, and the 5-year survival rate is less than 15%. However, its severe toxicity can easily cause the persistence of low immunity in tumor patients; the poor immunogenicity of lung cancer cells further increases the immune escape of tumors, which seriously hinders the further t...

Examples

Embodiment 1

[0058]1) Induction and expansion of CIK cells in lymph nodes and peripheral blood CIK cells

[0059] (1) 50 mL of isolated venous blood from lung cancer patients was stored at 16-20°C, and the peripheral blood was diluted by Ficoll density gradient centrifugation (density 1,077 g / mL): 50 mL of peripheral blood + 40 mL of normal saline Blood samples were separated and centrifuged by Ficoll (2000 rpm, 670 (×g) for 20 minutes), and the misty peripheral blood mononuclear cell layer in the middle layer was taken and left in the incubator for 2 hours, and then the suspension cells were used to prepare CIK;

[0060] (2) About 10 g of isolated mediastinal lymph node tissue from lung cancer patients (paraffin section staining and pathology confirmed no metastasis), stored at 16-20°C, shredded, removed adipose tissue and various fiber components, and then ground, After washing and resuspending in 0.9% sodium chloride solution, the cells were collected by filtering with a cell strainer. ...

Embodiment 2

[0073] Example 2 Antiproliferation and Cytotoxicity Tests on A549 Cells

[0074] Peripheral blood obtained before operation and lymph nodes obtained during operation were cultured for 14 days to obtain CIK cells from each patient according to the method 1.3.2, and reinfused continuously for 4 days starting from the 15th day, (lymph node group d1+peripheral blood group d2) alternately 2 reinfusions; peripheral blood was drawn on the 14th day and 7 days after the reinfusion to detect carcinoembryonic antigen (CEA), blood routine (to assess the incidence of bone marrow suppression), and to observe the incidence of rash, fever, and allergic events;

[0075] The SPSS11.0 software was used, and the data were represented by x±s; the comparison between the inhibition rates detected by the CCK8 method was performed by the t test, and the difference was statistically significant when P<0.05;

[0076] The results of the anti-proliferation and cytotoxic effects of lymph node CIK cells and...

Embodiment 3

[0079] Embodiment 3 clinical intervention experiment

[0080] For the unbalanced immune system of lung cancer patients, the intervention strategies of activated killer T cells combined with dendritic cells (CIK-DC) cultured and activated from autologous lymph nodes of patients with lung cancer surgery, combined with chemotherapy can inhibit the recurrence and metastasis of lung cancer patients and improve the patient's resistance. Cancer immunity, thereby improving the disease-free survival and overall survival of lung cancer patients.

[0081] According to the point of view of immune staging of lung cancer, the number of CD8+T lymphocytes and FOX-P3+T lymphocytes corresponds to the traditional pathological stage, which represents tumor size T; CD45RO+ memory T cells corresponds to the traditional pathological stage, which represents distant metastasis; M; Thus, the traditional pathological TNM staging of lung cancer is leapfrogged to the TNM staging representing the body's an...

technical field

[0001] The invention belongs to the fields of biotechnology and microbial animal cell lines, and in particular relates to a method for culturing autologous CIK cells in lymph nodes and an application thereof. Background technique

[0002] According to literature reports, the morbidity and mortality of lung cancer rank first among all malignant tumors, of which non-small cell lung cancer (NSCLC) accounts for 80%, which is characterized by high incidence, high mortality and low survival rate. Although the current intervention measures such as surgery, radiotherapy, and chemotherapy are constantly improving, the overall survival rate of lung cancer patients has not been significantly improved, and the 5-year survival rate is less than 15%. However, its severe toxicity can easily cause the persistence of low immunity in tumor patients; the poor immunogenicity of lung cancer cells further increases the immune escape of tumors, which seriously hinders the further t...

Examples

Embodiment 1

[0058]1) Induction and expansion of CIK cells in lymph nodes and peripheral blood CIK cells

[0059] (1) 50 mL of isolated venous blood from lung cancer patients was stored at 16-20°C, and the peripheral blood was diluted by Ficoll density gradient centrifugation (density 1,077 g / mL): 50 mL of peripheral blood + 40 mL of normal saline Blood samples were separated and centrifuged by Ficoll (2000 rpm, 670 (×g) for 20 minutes), and the misty peripheral blood mononuclear cell layer in the middle layer was taken and left in the incubator for 2 hours, and then the suspension cells were used to prepare CIK;

[0060] (2) About 10 g of isolated mediastinal lymph node tissue from lung cancer patients (paraffin section staining and pathology confirmed no metastasis), stored at 16-20°C, shredded, removed adipose tissue and various fiber components, and then ground, After washing and resuspending in 0.9% sodium chloride solution, the cells were collected by filtering with a cell strainer. ...

Embodiment 2

[0073] Example 2 Antiproliferation and Cytotoxicity Tests on A549 Cells

[0074] Peripheral blood obtained before operation and lymph nodes obtained during operation were cultured for 14 days to obtain CIK cells from each patient according to the method 1.3.2, and reinfused continuously for 4 days starting from the 15th day, (lymph node group d1+peripheral blood group d2) alternately 2 reinfusions; peripheral blood was drawn on the 14th day and 7 days after the reinfusion to detect carcinoembryonic antigen (CEA), blood routine (to assess the incidence of bone marrow suppression), and to observe the incidence of rash, fever, and allergic events;

[0075] The SPSS11.0 software was used, and the data were represented by x±s; the comparison between the inhibition rates detected by the CCK8 method was performed by the t test, and the difference was statistically significant when P<0.05;

[0076] The results of the anti-proliferation and cytotoxic effects of lymph node CIK cells and...

Embodiment 3

[0079] Embodiment 3 clinical intervention experiment

[0080] For the unbalanced immune system of lung cancer patients, the intervention strategies of activated killer T cells combined with dendritic cells (CIK-DC) cultured and activated from autologous lymph nodes of patients with lung cancer surgery, combined with chemotherapy can inhibit the recurrence and metastasis of lung cancer patients and improve the patient's resistance. Cancer immunity, thereby improving the disease-free survival and overall survival of lung cancer patients.

[0081] According to the point of view of immune staging of lung cancer, the number of CD8+T lymphocytes and FOX-P3+T lymphocytes corresponds to the traditional pathological stage, which represents tumor size T; CD45RO+ memory T cells corresponds to the traditional pathological stage, which represents distant metastasis; M; Thus, the traditional pathological TNM staging of lung cancer is leapfrogged to the TNM staging representing the body's an...

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