Leukocyte extract and preparation method and application thereof

An extract and white blood cell technology, applied in the field of white blood cell extract and its preparation, can solve the problems of decreased ability to secrete active cytokines, unclear active substances, and difficult preservation of activity, so as to promote cell regeneration, improve the function of activated cells, The effect of low production cost

Inactive Publication Date: 2018-09-25
上海蕙禾生物科技事务所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] (2) The leukocytes in human peripheral blood have been aging, and the ability to secrete active cytokines has begun to decline, and the use of bovine serum as a culture additive when expanding lymphocytes also brings various unknown risks
[0016] (3) Cosmetics using biologically active substances as raw materials have problems such as unclear active substances and difficult preservation of activity
[0017] In addition, when the existing leukocyte extracts are applied to cosmetics, there are also the following technical defects: the cosmetic formulations that add leukocyte extracts are only for the purpose of preserving or exerting the function of cytokines in the leukocyte extracts
How to rationally select the appropriate cosmetic components, which can fully exert synergistic effects with cytokines, so as to enhance the effect of activating cells has not been reported yet.

Method used

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  • Leukocyte extract and preparation method and application thereof
  • Leukocyte extract and preparation method and application thereof
  • Leukocyte extract and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, the preparation of leukocyte extract

[0064] The preparation of leukocyte extract comprises the steps:

[0065] (1) Collect the umbilical cord blood of the newborn of the healthy puerpera, and separate the upper umbilical cord plasma and the lower layer of the buffy coat by density gradient centrifugation of human lymphocyte separation fluid. The umbilical cord plasma is inactivated at 56°C to obtain the inactivated umbilical cord Plasma, spare.

[0066] (2), expansion of umbilical cord blood T lymphocytes: separate mononuclear cells (PBMC) from the buffy coat of step (1), get PBMC cells and inoculate into GT551 serum-free medium (purchased from Japanese TAKARA company), adjust The concentration of PBMC cells was 1×10 6 / ml, add 500U / ml IL-2 and PBMC cells with a volume fraction of 5% inactivated umbilical cord plasma, at 37°C, 5% CO 2 After one week, the cells were centrifuged and the supernatant was discarded to obtain induced amplified lymphocytes. ...

Embodiment 2

[0071] Embodiment 2, ELISA kit detects fibroblast growth factor (bFGF) content in the supernatant

[0072] Natural state human lymphocytes can secrete various cytokines, the preparation method of Example 1 can obtain various cytokines secreted by natural state human lymphocytes, including growth factors, interleukins, colony stimulating factors and interferons .

[0073] We take one of various cytokines as a representative (such as: bFGF, IL-2, GM-CSF and IFN-γ), respectively detect the content of these four cytokines in the leukocyte extract prepared in Example 1, And bFGF was used as the quantitative index of cytokines in leukocyte extracts, the specific process is as follows:

[0074] ELISA kit (Shanghai Enzyme Biotechnology Co., Ltd.) was used to quantitatively detect the contents of cytokines in the supernatant before ultrafiltration and the retentate after ultrafiltration using 1KD ultrafiltration membranes in three batches, and the results are as follows: Table 1 show...

Embodiment 3

[0081] Example 3. Detection of proliferative activity of leukocyte extracts on human fibroblasts (HFF-1)

[0082] HFF-1 cells in logarithmic growth phase (purchased from Shanghai Institute of Biological Sciences, Chinese Academy of Sciences) were cultured with serum-free medium (medium composition: DMEM+sodium pyruvate+double antibody), the specific steps are as follows:

[0083] (1) Adjust the concentration of HFF-1 cells to 4000 cells / 200ul, add them into a 96-well plate, and inoculate the cells to 4000 cells / 200ul / well.

[0084] (2), the leukocyte extract prepared in Example 1 was added to each well according to the volume percentage of HFF-1 cell inoculum amount, 2%, 4%, 6%, 8%, 10%, without adding Example 1. The HFF-1 cells in the logarithmic growth phase of the prepared leukocyte extract were used as a control group, and triplicate wells were set up respectively.

[0085] (3), in an incubator at 37 degrees, 5% CO 2Incubate under conditions for 72h observation, then add...

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Abstract

The invention discloses a preparation method of leukocyte extract. Neonate cord blood is used directly as a raw material subjected to human lymphocyte separation density-gradient centrifugation to obtain PBMCs (peripheral blood mononuclear cells); amplification culture is performed via cord serum separated from the cord blood; centrifugal separating is performed to obtain supernate; leukocyte extract is acquired by ultra-filtration. The preparation method has no need for bovine serum, no heterogeneous animal proteins or stimulating factors are involved, and safety of application in human bodies is good. The leukocyte extract prepared via the method has rich natural human cellular factors, wherein bFGF (basic fibroblast growth factor) reaches 60 pg/ml and above, the yield is high, and the production cost is low. The invention also provides application of the leukocyte extract in the activation and restoration of cells. The leukocyte extract may be added to cosmetics to provide synergy with other components in cosmetic formulations; the leukocyte extract can cooperate to improve cell mitochondrial functionality, complete nutrients for cell metabolism are provided, and the cell activating function of the leukocyte extract is significantly improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a white blood cell extract and its preparation method and application. Background technique [0002] Leukocytes are the general term for blood cells other than red blood cells and platelets in the blood, and include five types: neutrophils, eosinophils, basophils, lymphocytes, and monocytes. They are mainly involved in the defense and immune function of the body. Among them, neutrophils account for 50%-70% of white blood cells, have active deformation movement and phagocytosis ability, and can engulf bacteria. Eosinophils dampen allergic reactions and kill parasites. Basophils may cause allergic reactions. After leaving blood vessels, monocytes enter the connective tissue around blood vessels to become macrophages, which can eliminate invading pathogenic microorganisms, phagocytose foreign particles and aging cells, and also participate in the regulation of immune resp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K8/98A61K8/64A61K8/67A61K8/73A61K8/92A61Q19/08A61Q19/00
CPCA61K8/64A61K8/675A61K8/735A61K8/922A61K8/97A61K8/983A61K8/99A61K2800/592A61Q19/00A61Q19/08
Inventor 徐荻
Owner 上海蕙禾生物科技事务所
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