CN110438079A - A kind of TangshenosideⅠ improves the purposes of NK cell killing activity in vitro - Google Patents
A kind of TangshenosideⅠ improves the purposes of NK cell killing activity in vitro Download PDFInfo
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- 210000000822 natural killer cell Anatomy 0.000 title claims abstract description 76
- 238000000338 in vitro Methods 0.000 title claims abstract description 26
- 230000000694 effects Effects 0.000 title claims abstract description 24
- 230000022534 cell killing Effects 0.000 title claims abstract description 22
- 241000756943 Codonopsis Species 0.000 claims abstract description 41
- 230000002708 enhancing effect Effects 0.000 claims abstract description 12
- 239000001963 growth medium Substances 0.000 claims abstract description 12
- 239000012930 cell culture fluid Substances 0.000 claims description 10
- 210000004027 cell Anatomy 0.000 abstract description 19
- 230000002147 killing effect Effects 0.000 abstract description 12
- 230000004663 cell proliferation Effects 0.000 abstract description 8
- 239000004615 ingredient Substances 0.000 abstract description 4
- 230000001737 promoting effect Effects 0.000 abstract description 3
- 102000001398 Granzyme Human genes 0.000 description 11
- 108060005986 Granzyme Proteins 0.000 description 11
- 102000004503 Perforin Human genes 0.000 description 10
- 108010056995 Perforin Proteins 0.000 description 10
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 10
- 229930192851 perforin Natural products 0.000 description 10
- 230000035755 proliferation Effects 0.000 description 7
- 238000000034 method Methods 0.000 description 6
- 238000005259 measurement Methods 0.000 description 5
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- 238000005406 washing Methods 0.000 description 3
- 241000208340 Araliaceae Species 0.000 description 2
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 2
- 235000003140 Panax quinquefolius Nutrition 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
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- 229940079593 drug Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000008434 ginseng Nutrition 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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Abstract
The invention discloses the purposes that a kind of TangshenosideⅠ improves NK cell killing activity in vitro.The experimental result explanation of the embodiment of the present invention, two kinds of ingredient Radix Codonopsis alkynols, TangshenosideⅠ in Radix Codonopsis have the function of the in vitro culture of NK cell not exactly the same, Radix Codonopsis alkynol can remarkably promote the in-vitro multiplication of NK cell, it can also be improved NK cell killing activity, although TangshenosideⅠ cannot be obviously promoted the in-vitro multiplication of NK cell, but the killing activity of NK cell can be significantly improved, and Radix Codonopsis alkynol, TangshenosideⅠ will not influence CD3 in NK cellCD56+Cell proportion.Therefore, Radix Codonopsis alkynol can be used for promoting NK cell Proliferation in vitro, enhance its killing activity, it can be used for preparing the culture medium for promoting NK cell proliferation in vitro, enhancing its killing activity, TangshenosideⅠ can be used for enhancing NK cell killing activity in vitro, can be used for preparing the culture medium of enhancing NK cell killing activity.
Description
Technical field
The invention belongs to immunocyte fields, are related to the in vitro culture of immunocyte, and in particular to a kind of TangshenosideⅠ is external
Improve the purposes of NK cell killing activity.
Background technique
Natural kill (Natural killer, NK) cell is the lymphoid cell with panimmunity function, is accounted for about
The 10%~15% of peripheral blood lymphocytes.The NK cell of people refers generally to CD3CD56+Lymphocyte.NK cell is that body is anti-swollen
Tumor, anti-infectious the first line of defence, tumour and viral identification and killing inorganization histocompatibility complex (MHC) are limited,
There is preferable development prospect independent of antibody, therefore in the immunotherapy field of tumour.
NK cell have wider antitumor spectra, an important mechanisms for NK cell killing target cell are as follows: release perforin and
Granzyme B causes target cell necrosis or apoptosis.After NK cell touches tumour cell in vivo, perforation will be contained by exocytosis
The cytotoxic substance of element and granzyme B discharges, and perforin forms aperture, of release in target cell surface by polymerization
Granzyme B enters target cell, can evoke the death of cell by a variety of different approaches, such as evoke the chain reaction of caspases, draw
The activity of target cell DNA degradation is played, then cracking causes apoptosis of tumor cells.
Whether NK cell can play a role in oncotherapy, and maximum key is the quantity of cell and thin to tumour
The killing ability of born of the same parents.However, growth rate is limited in vitro for NK cell, it is difficult to meet needs in quantity, killing activity also needs
Reinforce.
Radix Codonopsis has the function of tonifying middle-Jiao and Qi, enhancing immunity of organisms, and Radix Codonopsis alkynol and TangshenosideⅠ are that content is non-in Radix Codonopsis
Two kinds of often low ingredients, research is few, and there is presently no reported its influence to NK cell Proliferation and killing activity.
Summary of the invention
The present invention is directed to overcome the deficiencies of the prior art and provide a kind of TangshenosideⅠ to improve NK cell killing activity in vitro
Purposes.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of TangshenosideⅠ enhances the purposes of NK cell killing activity in vitro.
A kind of Radix Codonopsis alkynol is used to prepare the purposes of the culture medium of enhancing NK cell killing activity.
A kind of culture medium enhancing NK cell killing activity, which includes NK cell culture fluid and TangshenosideⅠ.
Preferably, TangshenosideⅠ concentration is 40~60 μM.
It is highly preferred that TangshenosideⅠ concentration is 50 μM.
The utility model has the advantages that
The embodiment of the present invention the experiment results show that two kinds of ingredient Radix Codonopsis alkynols, TangshenosideⅠs in Radix Codonopsis to NK cell
In vitro culture has the function of not exactly the same, and Radix Codonopsis alkynol can remarkably promote the in-vitro multiplication of NK cell, can also be improved
NK cell killing activity can significantly improve NK cell although TangshenosideⅠ cannot be obviously promoted the in-vitro multiplication of NK cell
Killing activity, and Radix Codonopsis alkynol, TangshenosideⅠ will not influence CD3 in NK cellCD56+Cell proportion.Therefore, Radix Codonopsis alkynes
Alcohol can be used for promoting NK cell Proliferation in vitro, enhance its killing activity, can be used for preparing promotion NK cell proliferation in vitro, increases
The culture medium of its strong killing activity, TangshenosideⅠ can be used for enhancing NK cell killing activity in vitro, can be used for preparing enhancing NK
The culture medium of cell killing activity.
Detailed description of the invention
Fig. 1 is the 10th day NK cell flow cytometer testing result；
Fig. 2 is the flow cytomery result of NK cell after Radix Codonopsis alkynol, TangshenosideⅠ culture 48h, in which: A is Radix Codonopsis
Alkynol, B are TangshenosideⅠ；
Fig. 3 is the influence of Radix Codonopsis alkynol, TangshenosideⅠ culture 48h to granzyme B, perforin expression level in NK cell,
In: A is control group, and B is Radix Codonopsis alkynol, and C is TangshenosideⅠ.
Specific embodiment
It is specific with reference to the accompanying drawings and examples to introduce essentiality content of the present invention, but guarantor of the invention is not limited with this
Protect range.
One, test material
Lymphocyte separation medium, Shanghai Yuan Ye Biotechnology Co., Ltd；
NK cell culture fluid CellGro SCGM, German CellGenix company；
RhIL-2, Xiamen Amoytop Biotech Co., Ltd.；
Radix Codonopsis alkynol, Shanghai Yuan Ye Biotechnology Co., Ltd, HPLC purity >=95%；
TangshenosideⅠ, Wei Keqi Biotechnology Co., Ltd, Sichuan Province, HPLC purity >=98%；
Perforin and granzyme B primary antibody, Santa Cruz company, the U.S..
Two, test method
1, NK cell culture and identification
100mL healthy adult human peripheral is taken, collects peripheral blood mononuclear cells (PBMC) with lymphocyte separation medium, so
It is afterwards 5 × 10 with the NK cell culture fluid adjustment cell density added with 200U/mL rhIL-256 well culture plates are added in/mL,
Every hole 3mL, in 37 DEG C, 5%CO2Culture, every 2~3d half, which is measured, to be changed liquid 1 time.
Collect the 10th day NK cell, flow cytomery CD3CD56+Cell proportion.
2, proliferation rate, the CD3 of CCK-8 method measurement NK cellCD56+The measurement of ratio
The 10th day NK cell of culture is taken, it is 5 × 10 that density, which is made, with NK cell culture fluid4The cell suspension of/mL, is inoculated in
96 orifice plates, every 100 μ L of hole, are divided into control group, Radix Codonopsis alkynol group, TangshenosideⅠ group.After being inoculated in 96 orifice plate adaptability cultures for 24 hours,
Control group, which is changed to fresh NK cell culture fluid, to be continued to cultivate, and Radix Codonopsis alkynol group or TangshenosideⅠ group are changed to containing 50 μM of parties
The NK cell culture fluid of ginseng alkynol or TangshenosideⅠ continues to cultivate.Every group of 8 multiple holes, wherein 5 multiple holes are used to measure proliferation rate,
Remaining 3 multiple holes are for CD3 after flow cytometer measurement drug culture 48hCD56+Ratio.
Continue after cultivating 48h, the CCK-8 solution of 10 μ L is added in every hole, continues to be incubated for 4h, in 450nm wavelength microplate reader
Each hole optical density (OD) value is measured, drug Radix Codonopsis alkynol, TangshenosideⅠ is calculated to the proliferation rate of NK cell according to following formula: increasing
Grow rate (%)=[(medicine group OD value-control group OD value)/control group OD value] × 100%.
3, granzyme B, perforin expression are horizontal in Western blot method measurement NK cell
The 10th day NK cell of culture is taken, it is 5 × 10 that density, which is made, with NK cell culture fluid4The cell suspension of/mL, is inoculated in
24 orifice plates, every hole 1mL are divided into control group, Radix Codonopsis alkynol group, TangshenosideⅠ group.It is right after being inoculated in 24 orifice plate adaptability cultures for 24 hours
It is changed to fresh NK cell culture fluid according to group to continue to cultivate, Radix Codonopsis alkynol group or TangshenosideⅠ group are changed to containing 50 μM of Radix Codonopsis
The NK cell culture fluid of alkynol or TangshenosideⅠ continues to cultivate, and cell is collected in washing after 48h, thin with Westernblot method measurement NK
Granzyme B, perforin expression are horizontal in born of the same parents, and the specific method is as follows:
With cell pyrolysis liquid lytic cell, supernatant is collected by centrifugation, BCA method measures protein concentration, takes 40 μ g albumen loadings,
Albumen is separated by electrophoresis through 10%SDS-PAGE, then on Protein transfer to pvdf membrane, will be to contain with wet robin (50V, 3h)
Room temperature closes 2h in the confining liquid of 5% skimmed milk power, is separately added into perforin and granzyme B primary antibody, and 4 DEG C overnight, adds after washing film
Enter the secondary antibody of horseradish peroxidase-labeled, 37 DEG C of incubation lh, sufficiently after washing, with Imager photographic analysis.
4, statistical method
It is analyzed using SPSS16.0 software, data are indicated with mean ± standard deviation, and comparison among groups use One-wayANOVA
It examines, it is statistically significant that P < 0.05 represents difference.
Three, test result
1, NK cell culture and identification
NK cell flow cytometer testing result is as shown in Figure 1, CD3 within 10th dayCD56+Cell proportion be (84.4 ±
1.6) % (84.5%, 82.7%, 85.9%), meets the phenotypic features of NK cell.
2, Radix Codonopsis alkynol, TangshenosideⅠ are to NK cell-proliferation activity and CD3CD56+The influence of cell proportion
The results are shown in Table 1 on the influence of the proliferation rate of NK cell by Radix Codonopsis alkynol, TangshenosideⅠ culture 48h, as seen from Table 1, party
Ginseng alkynol can remarkably promote the proliferation of NK cell, and TangshenosideⅠ to NK cell without had significant proliferation facilitation.
1 each group OD value of table and Radix Codonopsis alkynol, TangshenosideⅠ are to the proliferation rate of NK cell
The flow cytomery result of NK cell is as shown in Fig. 2, CD3 after Radix Codonopsis alkynol, TangshenosideⅠ culture 48hCD56+Ratio be respectively (84.2 ± 1.8) % (84.9%, 85.5%, 82.1%), (83.5 ± 1.7) % (84.2%, 81.6%,
84.8%), with no significant difference before Radix Codonopsis alkynol, TangshenosideⅠ culture, illustrate Radix Codonopsis alkynol, TangshenosideⅠ to CD3 in NK cell
CD56+Cell proportion has no significant effect.
3, the influence of Radix Codonopsis alkynol, TangshenosideⅠ to granzyme B, perforin expression level in NK cell
Radix Codonopsis alkynol, TangshenosideⅠ culture 48h are to the influence result of granzyme B in NK cell, perforin expression level as schemed
Shown in 3, it can be seen from figure 3 that Radix Codonopsis alkynol, TangshenosideⅠ can promote the expression of granzyme B, perforin in NK cell, and Radix Codonopsis
The facilitation of glycosides I is clearly more powerful.
It is above-mentioned the experiment results show that two kinds of ingredient Radix Codonopsis alkynols, TangshenosideⅠ in Radix Codonopsis have the in vitro culture of NK cell
Play the role of not exactly the same, Radix Codonopsis alkynol can remarkably promote the in-vitro multiplication of NK cell, can also be improved NK cell killing
Activity, although TangshenosideⅠ cannot be obviously promoted the in-vitro multiplication of NK cell, the killing that can significantly improve NK cell is living
Property, and Radix Codonopsis alkynol, TangshenosideⅠ will not influence CD3 in NK cellCD56+Cell proportion.Therefore, Radix Codonopsis alkynol can be used
In promotion NK cell Proliferation in vitro, enhance its killing activity, can be used for preparing promotion NK cell proliferation in vitro, enhances its killing
Active culture medium, TangshenosideⅠ can be used for enhancing NK cell killing activity in vitro, can be used for preparing enhancing NK cell killing
Active culture medium.
The effect of above-described embodiment is specifically to introduce essentiality content of the invention, but those skilled in the art should know
Protection scope of the present invention should not be confined to the specific embodiment by road.
Claims (5)
1. the purposes that a kind of TangshenosideⅠ enhances NK cell killing activity in vitro.
2. the purposes that a kind of Radix Codonopsis alkynol is used to prepare the culture medium of enhancing NK cell killing activity.
3. a kind of culture medium for enhancing NK cell killing activity, which includes NK cell culture fluid and TangshenosideⅠ.
4. culture medium according to claim 3, it is characterised in that: TangshenosideⅠ concentration is 40~60 μM.
5. culture medium according to claim 4, it is characterised in that: TangshenosideⅠ concentration is 50 μM.
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| CN201910862232.0A CN110438079B (en) | 2019-09-12 | 2019-09-12 | Application of codonopsis pilosula glucoside I in-vitro improvement of killing activity of NK (Natural killer) cells |
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