CN109908326A - People's beta-alexin 1 is promoting the application in NK cell killing activity - Google Patents
People's beta-alexin 1 is promoting the application in NK cell killing activity Download PDFInfo
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- CN109908326A CN109908326A CN201811072521.2A CN201811072521A CN109908326A CN 109908326 A CN109908326 A CN 109908326A CN 201811072521 A CN201811072521 A CN 201811072521A CN 109908326 A CN109908326 A CN 109908326A
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Abstract
The invention discloses a kind of human alpha-defensin albumen to adjust the new application in NK cell killing activity.The human alpha-defensin albumen behaviour-alexin 1 (HBD1) is one of human body natural immune system peptide material, and amino acid sequence is as shown in SEQ ID NO.1.HBD1 of the present invention can effectively facilitate the killing activity of NK cells on cancer cells, have potential pharmaceutical applications.
Description
Technical field
The present invention relates to people's beta-alexins 1 to promote the application in NK cell killing activity, belongs to biomedicine field.
Background technique
It is anti-by a series of inherent immunities and adaptive immunity after stimulation of the normal body by extraneous pathogenic microorganism etc.
Infection should be resisted, exercise immune defense function.Wherein, inherent immunity is the first line of defence of the congenital acquisition of body, occur it is early and
It is swift in response, plays a significant role during body removes pathogen.Natural killer cells (Natural Killer
Cells, NK cells) it is a kind of important inherent immunity cell of body, since lethal effect occurs after it acts on target cell
Early, killing activity is limited without MHC, does not depend on antibody, because of referred to herein as natural killer cells.NK cell not only with it is antitumor, disease-resistant
Poison infection is related with immunological regulation, and participates in the generation of hypersensitivity and autoimmune disease in some cases, can
Identify target cell, killing medium.
Studies have shown that many bioactive substances, such as vitamine D3, vitamin A, lactoferrin etc. can be thin to NK
Born of the same parents have an impact, and promote its killing activity.Alexin is that one kind can kill the microorganisms such as bacterium, fungi and virus and have antitumor
Active polypeptide can pass through genetic engineering mass production.Its molecular weight is 4-5KD, containing 6 conservative cysteine residues,
Form 3 typical intramolecular disulfide bonds.Spatial position according to its cysteine connects order difference, Ren Leifang with disulfide bond
Imperial element can be divided into α alexin, beta-defensin etc., be resistant to first of natural defence line of pathogenic microorganism invasion.So far,
Have found 6 kinds of α alexins (HNP-1~4, HD5, HD6) and 6 kinds of beta-defensins (HBD-1~6) altogether in the mankind.Alexin also by
Report participates in the antineoplastic immune activity of body, has antitumor cell characteristic.Its antitumor action mainly by with peroxidating
Hydrogen enzyme synergistic effect, inhibits the activity of oxidizing ferment, and partly depend on the lipid components of target cell membrane to realize.However it is right at present
The immunoregulation effect of alexin is known little about it.
Summary of the invention
The present invention proposes a kind of new opplication of human alpha-defensin albumen, i.e. its purposes in adjusting NK cell killing activity.
The present inventor's Buchner's bodies are people's beta-alexin 1 (HBD1), and the HBD1 is a kind of peptide material, amino acid sequence such as SEQ
Shown in ID NO.1:
GNFLTGLGHRSDHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK。
In the present invention, the HBD1 can be by extraction, chemical synthesis, recombinant expression and purifying from cell or body fluid.Its
In, genetic engineering preparation is the prefered method of mass production alexin.The present invention relates to the applications of HBD1 a kind of, and HBD1 is used for
Preparation adjusts the regulator of NK cell killing function.
HBD1 of the present invention can promote NK cell for the lethal effect of cancer cell.The cancer cell includes leukaemia
Cell, breast cancer cell, liver cancer cells etc..Preferably, the leukaemia cell is chronic marrow original leukaemia cell, further excellent
It is selected as K562 cell；The breast cancer cell is human breast cancer cell, further preferably human breast cancer cell MCF7；The liver
Cancer cell is human liver cancer cell, and further preferably human liver cancer cell is HepG2.
In the present invention, the application further includes effective concentration of the HBD1 in the regulator.Described is effective
Concentration is 10ng/mL-1000ng/mL, preferably 100ng/mL.
In the present invention, the adjusting NK cell killing function, comprising: promote source of people peripheral blood mononuclear cells (PBMC)
Lethal effect of the NK cell in source to K562 cell.
The present invention also provides one kind to be used for antitumor medicine composition comprising people-alexin 1 (HBD1) and pharmacy
Upper acceptable carrier, wherein the HBD1 is a kind of peptide material, and amino acid sequence is as shown in SEQ ID NO.1:
GNFLTGLGHRSDHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK。
Wherein, the tumour include but is not limited to be leukaemia, breast cancer, liver cancer, the cancer of the esophagus, oophoroma, incidence squama
Shape cell cancer, colon cancer, prostate cancer etc..It preferably, is leukaemia, breast cancer and liver cancer etc..It is further preferred that described white
Blood disease cell is the chronic marrow original leukaemia cell of people, further preferably K562 cell；The breast cancer cell is human breast carcinoma
Cell, further preferably human breast cancer cell MCF7；The liver cancer cells are human liver cancer cell, further preferably human liver cancer
Cell is HepG2.
Wherein, described pharmaceutical composition be formulated into injectable fluid, aerosol, emulsifiable paste, gelling agent, pill, capsule,
Syrup, transdermal patch or excipient.
In the present invention, the people-alexin 1 (HBD1) can effectively adjust the killing activity of NK cell, for inhibiting swollen
The proliferation of tumor, growth, infiltration, transfer, recurrence.
The beneficial effects of the present invention are, HBD1 provided in the present invention can effectively adjust the killing activity of NK cell,
Promote NK cell for the lethal effect of cancer cell, such as lethal effect for K562 cell.The HBD1 albumen, which has to be used as, to be controlled
Treat the potential drug of cancer (such as leukaemia).In addition, HBD1 is as a kind of novel biologically active peptides, antimicrobial spectrum is extensive, can
Broad-spectrum microorganism is quickly killed, and it is less to its bacterium with repellence, and pathogen is also not easy to generate drug resistance to it；
And HBD1 is one kind of alexin in human body natural immune system, it is opposite not have immunogenicity, therefore human body will not be caused
It is highly-safe to the immune response of HBD1；HBD1 can also largely be synthesized by genetic engineering, and cost is relatively low.
Detailed description of the invention
Fig. 1 is the ability of 1 control group of embodiment and HBD1 processing group NK cell killing K562 cell.A. streaming schematic diagram；
B. analysis of statistical results (n=10).
Fig. 2 is the ability of 1 control group of embodiment and HBD1 processing group NK cell killing MCF7 cell.A. streaming schematic diagram；
B. analysis of statistical results (n=10).
Fig. 3 is the ability of 1 control group of embodiment and HBD1 processing group NK cell killing HepG2 cell.A. streaming schematic diagram；
B. analysis of statistical results (n=10).
Specific embodiment
By the following examples and the mode of attached drawing further illustrates the present invention, but does not therefore limit the present invention to institute
Among the scope of embodiments stated.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or
It is selected according to product manual.
Embodiment 1
1. Human peripheral blood NK cells isolate and purify:
1) people peripheral vein heparin anti-coagulating 1ml is taken, 1ml PBS is added and is diluted to half；
2) plus lymphocyte separation medium 1ml is in 15ml centrifuge tube；
3) blood diluted is added in separating liquid test tube using capillary syring slowly along tube wall, makes to be formed with separating liquid
One interface, the two cannot mix；
4) under room temperature, 2200rpm, raising speed 5, reduction of speed 0 are centrifuged 23 minutes；
5) carefully draw mononuclearcell (PBMC) layer with capillary syring, be placed in new 15ml centrifuge tube, be added 5 times with
Upper PBS, 1500rpm × 10 minute wash 2 times, obtain human peripheral PBMC；
6) above-mentioned gained PBMC is resuspended in RoboSepTMIn buffer, adjustment cell concentration is 5 × 107/ml；
7) EasySep is added according to 50ul/ml PBMCTMHuman NK Cell Enrichment Cocktail is mixed
It is even, it is stored at room temperature 10 minutes；
8) concussion mixes EasySepTMD Magnetic Particles.It is added according to 100ul/ml PBMC after mixing
EasySepTMD Magnetic Particles is mixed, is stored at room temperature 5 minutes；
9) RoboSep is addedTMBuffer is into above-mentioned cell suspension, until total volume is that (cell quantity is less than 10 by 5ml8)
Or (cell quantity is in 1-4.25 × 10 by 10ml8Between).Cell is mixed, centrifuge tube is placed on magnet, is placed 5 minutes；
10) supernatant is poured in a new centrifuge tube, gained cell is CD3-CD56+NK cell；
2. the NK cell of processing after purification
HBD1 processing group:
1) above-mentioned NK cell after purification is resuspended using 1640 complete mediums (PRIM 1640+10%FBS+1%PS)；
2) it is added and melts the HBD1 solution in distilled water, until HBD1 final concentration of 100ng/ml, 37 DEG C, 5%CO2Culture
24 hours；
Set control group (distilled water processing) simultaneously:
1) above-mentioned NK cell after purification is resuspended using 1640 complete mediums (PRIM 1640+10%FBS+1%PS)；
2) distilled water of addition and above-mentioned HBD1 solution same volume, 37 DEG C, 5%CO2Culture 24 hours；
3. fluorescence probe marks target cell
1) take K562 cell, using culture medium (PRIM 1640+10%FBS) be resuspended cell, adjustment cell concentration be 1 ×
106/ml；
Or, taking MCF7 cell, cell is resuspended using culture medium (PRIM 1640+10%FBS+1%PS), adjustment cell is dense
Degree is 1 × 106/ml；
Or, taking HepG2 cell, cell is resuspended using culture medium (PRIM 1640+10%FBS+1%PS), adjustment cell is dense
Degree is 1 × 106/ml；
2) Dil cell membrane fluorescence probe solution is added to cell suspension
(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate,
DiIC18 (3)), until final concentration of 5uM；
3) it is protected from light incubation 10 minutes for 37 DEG C；
4) supernatant is abandoned in centrifugation, and PBS is washed 2 times, cell is resuspended using complete medium, adjustment cell concentration is 105/ml。
4. mixed culture and flow cytometer detection
1) HBD1 processing group: by the K562 cell of above-mentioned HBD1 treated NK cell and Dil label, MCF7 cell or
HepG2 cell is according to the effect target of 10:1 than mixing, and 37 DEG C, 5%CO2 is cultivated 4 hours；
Control group: K562 cell, MCF7 cell or the HepG2 that NK cell and Dil that above-mentioned distilled water is handled are marked are thin
Born of the same parents are according to the effect target of 10:1 than mixing, and 37 DEG C, 5%CO2 is cultivated 4 hours；
2) the cell 100ul of above-mentioned mixed culture is taken, 5ul PI is added, room temperature is protected from light dyeing 15 minutes；
3) apoptosis rate of the K562 cell of flow cytometry detection Dil label, MCF7 cell or HepG2 cell.
Killing activity experimental result to K562 cell is as shown in Figure 1, by Flow cytometry result shown in figure 1A
It is found that the apoptosis rate of the chronic marrow original K562 Leukaemia cell of people is respectively 23.5% He in control group and HBD1 processing group
Value < 0.05 34.1%, P illustrates that NK cell is significantly higher than control group to the killing rate of K562 cell in HBD1 processing group；By Figure 1B
Statistical result it is found that 10 repetition test displays, the apoptosis rate of K562 cell is respectively in control group and HBD1 processing group
NK cell is significantly higher than control group to the killing rate of K562 cell in 21.4% and 32.5%, P value < 0.05, HBD1 processing group, says
Bright HBD1 can significantly promote NK cell to K562 killing activity.
Killing activity experimental result to MCF7 cell is as shown in Fig. 2, the Flow cytometry result as shown in Fig. 2A
It is found that the apoptosis rate of human breast cancer cell MCF7 cell is respectively 8.58% and 16.9%, P in control group and HBD1 processing group
Value < 0.05, illustrates that NK cell is significantly higher than control group to the killing rate of MCF7 cell in HBD1 processing group；By the statistics knot of Fig. 2 B
Fruit is it is found that the apoptosis rate of MCF7 cell is respectively 9.1% He in 10 repetition test display control groups and HBD1 processing group
NK cell is significantly higher than control group to the killing rate of MCF7 cell in 15.5%, P value < 0.05, HBD1 processing group, illustrates that HBD1 can
Promote NK cell to the killing activity of MCF7 cell.
Killing activity experimental result to HepG2 cell is as shown in figure 3, the Flow cytometry result as shown in Fig. 3 A
It is found that the apoptosis rate of human liver cancer cell HepG2 cell is respectively 16.9% and 22.4%, P value in control group and HBD1 processing group
< 0.05, illustrate that NK cell is significantly higher than control group to the killing rate of HepG2 cell in HBD1 processing group；By the statistics knot of Figure 1B
Fruit is it is found that the apoptosis rate of HepG2 cell is respectively 17.21% He in 10 repetition test display control groups and HBD1 processing group
NK cell is significantly higher than control group to the killing rate of HepG2 cell in 21.95%, P value < 0.05, HBD1 processing group, illustrates HBD1
NK cell be can promote to HepG2 cell killing activity.
In conclusion people-alexin 1HBD1 has the function of effectively facilitating the killing activity of NK cells against tumor cells.
It will be evident for a person skilled in the art that not departing from disclosed by appended claims of the present invention
Scope and spirit under the premise of, can be with numerous modifications and variations may be made, and these modifications and variations each fall within power of the present invention
In the protection scope that benefit requires.
Sequence table
<110>Shanghai Zhong Ze Biotechnology Co., Ltd
<120>people's beta-alexin 1 is promoting the application in NK cell killing activity
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 47
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 1
Gly Asn Phe Leu Thr Gly Leu Gly His Arg Ser Asp His Thr Asn Cys
1 5 10 15
Val Ser Ser Gly Gly Gln Cys Leu Thr Ser Ala Cys Pro Ile Phe Thr
20 25 30
Lys Ile Gln Gly Thr Cys Thr Arg Gly Lys Ala Lys Cys Cys Lys
35 40 45
Claims (10)
1. a kind of application of human alpha-defensin albumen in the regulator that preparation adjusts NK cell killing activity.
2. application as described in claim 1, which is characterized in that the human alpha-defensin albumen behaviour-alexin 1HBD1, it is described
HBD1 is a kind of peptide material, and amino acid sequence is as shown in SEQ ID NO.1: GNFLTGLGHRSDHYNCVSSGGQCLYSA
CPIFTKIQGTCYRGKAKCCK。
3. application as claimed in claim 1 or 2, which is characterized in that the human alpha-defensin albumen is for promoting NK cell to cancer
The killing activity of cell.
4. application as claimed in claim 3, which is characterized in that the cancer cell is leukaemia cell, breast cancer cell, liver cancer
Cell.
5. application as claimed in claim 4, which is characterized in that the leukaemia cell is the chronic marrow original leukaemia cell of people,
The breast cancer cell is human breast cancer cell, and the liver cancer cells are human liver cancer cell.
6. application as claimed in claim 5, which is characterized in that the chronic marrow original leukaemia cell of people is K562 cell, institute
Stating human breast cancer cell is MCF7, and the human liver cancer cell is HepG2.
7. such as claim 1~2,4~6 described in any item applications, which is characterized in that the NK cell is source of people peripheral blood
The source mononuclearcell PBMC.
8. application as claimed in claim 3, which is characterized in that the NK cell comes for source of people peripheral blood mononuclear cells PBMC
Source.
9. one kind is used for antitumor medicine composition, which is characterized in that it includes people-alexin 1 (HBD1) and pharmaceutically may be used
The carrier of receiving, wherein the HBD1 is a kind of peptide material, and amino acid sequence is as shown in SEQ ID NO.1:
GNFLTGLGHRSDHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK。
10. pharmaceutical composition as claimed in claim 9, which is characterized in that the tumour is leukaemia, breast cancer and liver cancer.
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| CN201811072521.2A CN109908326A (en) | 2018-09-14 | 2018-09-14 | People's beta-alexin 1 is promoting the application in NK cell killing activity |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998007833A1 (en) * | 1996-08-22 | 1998-02-26 | The Trustees Of The University Of Pennsylvania | Compositions and methods for use of defensin |
| WO2007047512A2 (en) * | 2005-10-14 | 2007-04-26 | Musc Foundation For Research Development | Inhibition of pax2 by defb1 induction as a therapy for cancer |
| CN102481379A (en) * | 2009-08-24 | 2012-05-30 | 菲吉尼克斯公司 | Targeting pax2 for the treatment of breast cancer |
| WO2013172932A1 (en) * | 2012-05-15 | 2013-11-21 | University Of Southern California | Colon cancer tumor suppressor gene, b-defensin 1, predicts recurrence in patients with stage ii and iii colon cancer |
| CN105031613A (en) * | 2015-07-08 | 2015-11-11 | 武汉胜达康生物科技有限公司 | Use of human beta-defensin-1 in preparation of drugs for treating or preventing hepatitis B virus infection |
-
2018
- 2018-09-14 CN CN201811072521.2A patent/CN109908326A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998007833A1 (en) * | 1996-08-22 | 1998-02-26 | The Trustees Of The University Of Pennsylvania | Compositions and methods for use of defensin |
| WO2007047512A2 (en) * | 2005-10-14 | 2007-04-26 | Musc Foundation For Research Development | Inhibition of pax2 by defb1 induction as a therapy for cancer |
| CN102481379A (en) * | 2009-08-24 | 2012-05-30 | 菲吉尼克斯公司 | Targeting pax2 for the treatment of breast cancer |
| WO2013172932A1 (en) * | 2012-05-15 | 2013-11-21 | University Of Southern California | Colon cancer tumor suppressor gene, b-defensin 1, predicts recurrence in patients with stage ii and iii colon cancer |
| CN105031613A (en) * | 2015-07-08 | 2015-11-11 | 武汉胜达康生物科技有限公司 | Use of human beta-defensin-1 in preparation of drugs for treating or preventing hepatitis B virus infection |
Non-Patent Citations (1)
| Title |
|---|
| CHELSEY J.JUDGE 等: "HBD-3 induces NK cell activation, IFN-γ secretion and mDC dependent cytolytic function", 《CELL IMMUNOL》 * |
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