Method for obtaining sorted and purified autoimmune vaccine

Abstract

The invention discloses a method for obtaining a sorted and purified autoimmune vaccine, which comprises the following steps: S1, extracting 30ml of autologous blood sample of a patient, and sorting to obtain CD34 + cells; s2, carrying out flow analysis on the CD34 + cells to ensure that CD34 + is greater than or equal to 90%, counting by using a counting instrument, and adjusting the concentration of the CD34 + cells to be 1 * 106 / mL; s3, the extracted cells are placed in a 42 DEG C constant-temperature water bath kettle for heat shock treatment, and the heat shock treatment lasts for two hours; s4, collecting the cells obtained in the step S3, transferring the cells into liquid nitrogen at-196 DEG C and a water bath kettle at 37 DEG C, and repeatedly freezing and thawing for multiple times; s5, collecting after detecting that no living cells exist by using a cell counter, and centrifuging for 10 minutes at the rotating speed of 6000 r / min under the centrifugal condition of 4 DEG C; s6, after the centrifugation is finished, discarding the supernatant and leaving the precipitate, and resuspending by using normal saline; and S7, irradiating 200 GY through gamma rays for 60 minutes, collecting the sample, sending the sample, and inspecting the sample for later use after the sample is qualified.

Description

Technical Field

[0001] The present invention belongs to the technical field of vaccine culture, and in particular, relates to a method for obtaining an autoimmune vaccine after sorting and purification. Background Art

[0002] CD34+ can differentiate and develop into LC, which is the only antigen presenting cell in the epidermis. After being stimulated by vaccines made by CD34+, epidermal LCs will migrate to the draining lymph nodes and activate T lymphocytes to exert immune effects. Existing methods for obtaining autologous immune vaccines lack specific antigens for LC to induce them to migrate to lymph nodes and activate T lymphocytes to exert immune effects. Summary of the invention

[0003] In view of the problem that the existing methods for obtaining autoimmune vaccines lack specific antigens for LC to induce them to migrate to lymph nodes and transform into T lymphocytes to exert immune effects, the present invention provides a method for obtaining autoimmune vaccines after sorting and purification.

[0004] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0005] A method for obtaining a sorted and purified autoimmune vaccine comprises the following steps:

[0006] S1. Extract 25-35 ml of the patient's autologous blood sample and obtain CD34+ cells after sorting;

[0007] S2. Perform flow cytometry analysis on CD34+ cells to ensure that CD34+ is ≥90%. Count with a counter and adjust the CD34+ cell concentration to 1*10 ^6 / mL;

[0008] S3, placing the extracted cells in a constant temperature water bath at 40°C-43°C for heat shock treatment, the heat shock treatment lasts for two hours;

[0009] S4, collecting the cells obtained in step S3, transferring them to liquid nitrogen at -190°C to -200°C and a 37°C water bath for repeated freezing and thawing;

[0010] S5. After detecting the absence of viable cells using a cell counter, the cells were collected and centrifuged at 6000 rpm for 10 min at 4°C.

[0011] S6. After centrifugation, discard the supernatant and keep the precipitate, and resuspend it with physiological saline;

[0012] S7. Irradiate with γ-rays for 200 GY. After 50-70 minutes, collect the samples and send them for inspection before use.

[0013] Furthermore, the optimal value of extracting the patient's autologous blood sample in step S1 is 30 ml.

[0014] Furthermore, the optimal temperature for placing the cells extracted in step S3 in a constant temperature water bath is 42°C.

[0015] Furthermore, the optimal liquid nitrogen temperature for transfer to liquid nitrogen in step S4 is -196°C.

[0016] Furthermore, in step S4, the number of freeze-thaw cycles is 3, and the freeze-thaw cycle time is 5 min / time.

[0017] Furthermore, the optimal time for γ-ray irradiation in step S7 is 60 minutes.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] Through the above scheme, after the vaccine is injected into the human epidermis, it can stimulate the migration of Langerhans cells in the epidermis and activate T cells, so that T cells can play functions such as cellular immunity and immune regulation. The recycling of T cells is conducive to extensive contact with antigenic substances entering the body, strengthening the immune response and maintaining immune memory for a longer period of time. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The present invention is an overall flow chart of a method for obtaining an autoimmune vaccine after sorting and purification. DETAILED DESCRIPTION

[0021] In order to facilitate the understanding of those skilled in the art, the present invention is further described below in conjunction with embodiments and drawings. The contents mentioned in the implementation modes are not intended to limit the present invention.

[0022] like Figure 1 As shown, this embodiment provides a method for obtaining a sorted and purified autoimmune vaccine, comprising the steps of:

[0023] S1. Extract 30 ml of the patient's autologous blood sample and obtain CD34+ cells after sorting;

[0024] S2. Perform flow cytometry analysis on CD34+ cells to ensure that CD34+ is ≥90%. Count with a counter and adjust the CD34+ cell concentration to 1*10 ^6 / mL;

[0025] S3, placing the extracted cells in a 42°C constant temperature water bath for heat shock treatment, the heat shock treatment lasts for two hours;

[0026] S4. Collect the cells obtained in step S3, transfer to -196°C liquid nitrogen and 37°C water bath, and freeze and thaw repeatedly for 3 times, with the freezing and thawing time of 5 min / time.

[0027] S5. After detecting the absence of viable cells using a cell counter, the cells were collected and centrifuged at 6000 rpm for 10 min at 4°C.

[0028] S6. After centrifugation, discard the supernatant and keep the precipitate, and resuspend it with physiological saline;

[0029] S7. Irradiate with γ-rays for 200 GY. After 60 minutes, collect the samples and send them for inspection before use.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] Through the above scheme, after the vaccine is injected into the human epidermis, it can stimulate the migration of Langerhans cells in the epidermis and activate T cells, so that T cells can play functions such as cellular immunity and immune regulation. The recycling of T cells is conducive to extensive contact with antigenic substances entering the body, strengthening the immune response and maintaining immune memory for a longer period of time.

[0032] The above is a detailed introduction to a method for obtaining a sorted and purified autoimmune vaccine provided by the present application. The description of the specific embodiment is only used to help understand the method and its core idea of ​​the present application. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. A method for obtaining an autoimmune vaccine after sorting and purification, characterized in that: Includes steps: S1. Extract 25-35 ml of the patient's autologous blood sample and obtain CD34+ cells after sorting; S2. Perform flow cytometry analysis on CD34+ cells to ensure that CD34+ is ≥90%. Count with a counter and adjust the CD34+ cell concentration to 1*10 ^6 / mL; S3, placing the extracted cells in a constant temperature water bath at 40°C-43°C for heat shock treatment, the heat shock treatment lasts for two hours; S4, collecting the cells obtained in step S3, transferring them to liquid nitrogen at -190°C to -200°C and a 37°C water bath for repeated freezing and thawing; S5. After detecting the absence of viable cells using a cell counter, the cells were collected and centrifuged at 6000 rpm for 10 min at 4°C. S6. After centrifugation, discard the supernatant and keep the precipitate, and resuspend it with physiological saline; S7. Irradiate with γ-rays for 200 GY. After 50-70 minutes, collect the samples and send them for inspection before use.

2. The method for obtaining the autoimmune vaccine after sorting and purification according to claim 1, characterized in that: The optimal value for extracting the patient's autologous blood sample in step S1 is 30 ml.

3. The method for obtaining the autoimmune vaccine after sorting and purification according to claim 2, characterized in that: The optimal temperature for placing the cells extracted in step S3 in a constant temperature water bath is 42°C.

4. The method for obtaining the autoimmune vaccine after sorting and purification according to claim 3, characterized in that: The optimal liquid nitrogen temperature for transferring to liquid nitrogen in step S4 is -196°C.

5. The method for obtaining the autoimmune vaccine after sorting and purification according to claim 4, characterized in that: The number of freeze-thaw cycles in step S4 is 3, and the freeze-thaw cycle time is 5 min / cycle.

6. The method for obtaining the autoimmune vaccine after sorting and purification according to claim 5, characterized in that: The optimal time for γ-ray irradiation in step S7 is 60 minutes.

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