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NK cell in-vitro induced amplification method

A technology of NK cells and cells, applied in the field of biomedicine, can solve the problems of cumbersome operation process of immunomagnetic bead sorting method, unsuitable for clinical large-scale application, and safety is not guaranteed. The effect of increasing the number

Inactive Publication Date: 2015-05-27
浙江卫未生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, in vitro expansion of NK cells mainly adopts the method of co-culture of feeder cells and immunomagnetic bead sorting, but the co-culture of feeder cells is to add NK cells to the modified tumor cell line, so that the modification is clinically applicable. The safety is not guaranteed, and the immunomagnetic bead sorting method is cumbersome and costly, and is not suitable for large-scale clinical application

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Extraction and separation experiment of Clover medicinal material

[0042] Grouping of extracts: According to the preparations selected during extraction, the extracts are divided into 9 groups, named respectively: TH-t, TH-p, TH-a, TH-b, TH-w, TH-w1, TH-w2 , TH-w3 and TH-w4;

[0043] The extraction method of each group of cloverleaf extract: refer to the literature [Yang Yang. Study on the chemical constituents of Sage Ganxi and Polygonum cephalum. Master's degree thesis of the Second Military Medical University. Shanghai: Second Military Medical University, 2009] separation and purification, Checked by high performance liquid chromatography (HPLC). Can refer to figure 1 About the roadmap of Clover Extraction Technology.

[0044] TH-t part: take 200g of dried medicinal material of Clover, add 5 times the volume of 80% ethanol aqueous solution to soak for 24 hours, heat and reflux for extraction, a total of 3 times, each 40min, combine the extracts, filter...

Embodiment 2

[0053] Example 2: Effect experiment of different concentrations of Clover extracts on NK cell proliferation

[0054] NK cell culture and identification:

[0055] Take 10ml of peripheral anticoagulant blood from healthy blood donors, separate mononuclear cells with lymphocyte separation medium, and use NK medium containing 500U / ml rhIL-2 to adjust the cell density to 3.5×10 5 / mL, seeded in 6-well cell culture plate, 5ml per well, at 37°C, 5% CO 2 Culture in an incubator, change the medium once every 2 days, and adjust the cell density to 5×10 5 NK cells cultured for 7 days were collected, and PE-labeled CD3 and FITC-labeled CD56 were added to detect cell surface markers.

[0056] The CCK8 method was used to detect the effects of different clover extracts on the growth of human NK cells:

[0057] NK cells cultured for 7 days were taken to make 5×10 4 / mL cell suspension, add to 96-well cell culture plate, 200μl / well. Experimental group: A total of 11 concentration groups w...

Embodiment 3

[0060] Example 3: NK cell amplification multiple detection on the amplification product

[0061] Effects of four kinds of clover extracts on NK cell proliferation:

[0062] In the experiment, it was found that the extracts of Clover clover had a concentration window phenomenon on the growth of NK cells, and some of the extracts (Th-t, Th-p, Th-w2 and Th-w3) promoted the proliferation of NK cells more obviously. Therefore, in this experiment, these four extracts were selected for long-term proliferation experiments on NK cells. The number of NK cells induced by these four extracts was higher than that of the control group at each time period; when cultured to the 16th day, the NK cell proliferation induced by the extracts Th-t, Th-p, Th-w2 and Th-w3 The multiples were 2468 times, 1646 times, 6334 times and 5373 times, which were significantly higher than those of the control group (263 times), and there were statistical differences (p Figure 10-13 .

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Abstract

The invention provides an NK cell in-vitro induced amplification method which comprises the following step: adding a radix tetrastigme extract into an NK cell culture solution, wherein the concentration of the radix tetrastigme extract is 0.00625-9.76 mu g / ml, and the radix tetrastigme extract is radix tetrastigme total extract Th-t, boiled desugarized part Th-w3, petroleum ether part Th-p or water part desugarized part Th-w2. According to the method, abundant NK cells with higher application safety, higher purity and higher killing activity can be obtained under in-vitro culture conditions, so that the cells achieve the quality indexes for safe and effective clinical application. The method is simple to operate, low in cost and suitable for large-scale application. The NK cells obtained by the method have obviously higher cell number, purity, killing activity and cell factor secretion level. When the method is used for culture, the cell number is enhanced by thousands of times.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a method for inducing and expanding NK cells in vitro using a trefoil extract. Background technique [0002] In recent years, tumor biotherapy has become a research hotspot in the field of tumor treatment. NK cells are an independent group of lymphocytes. NK cells show biological effects mainly through cytotoxic activity, and can directly kill tumor cells, virus-infected cells, and intracellular parasites. NK cells can recognize their own normal cells and abnormal tissue cells, and only kill abnormal cells without cytotoxicity to host normal tissue cells. In addition, NK cells play an important role in immune regulation and differentiation of certain immune cells by secreting a variety of cytokines. NK cells mainly recognize tumors in a non-restricted manner by the major histocompatibility complex (MHC), and kill tumor cells through granzyme B (granzyme B) and perforin (p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 刘军权陈锦阳杨阳
Owner 浙江卫未生物医药科技有限公司
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