CN108464980A - A kind of STAT3 inhibitor, application of the STAT5 activator in terms of enhancing NK cells against tumor cells lethality - Google Patents
A kind of STAT3 inhibitor, application of the STAT5 activator in terms of enhancing NK cells against tumor cells lethality Download PDFInfo
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- CN108464980A CN108464980A CN201810310610.XA CN201810310610A CN108464980A CN 108464980 A CN108464980 A CN 108464980A CN 201810310610 A CN201810310610 A CN 201810310610A CN 108464980 A CN108464980 A CN 108464980A
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Abstract
The invention discloses a kind of application of STAT3 inhibitor, STAT5 activator in terms of enhancing NK cells against tumor cells lethality.One skilled in the art will appreciate that inhibiting the STAT3 in tumor microenvironment that can promote lethal effect of the immunocytes such as NK cells to tumour；STAT5 can not only promote the growth and development of tumour cell but also can promote development and the cytotoxicity of NK cells, the STAT5 levels of NK cells are inhibited not only to damage the normal function of NK cells, and promote the growth of tumour, thus expression of the STAT5 in NK cells is promoted to be conducive to the treatment to tumour.It is a discovery of the invention that Flexilarin F or Flexilarin G are STAT3 inhibitor, STAT5 activator, Flexilarin F or Flexilarin G are by inhibiting the expression of STAT3, the expression of activation STAT5 to improve NK cells against tumor cells lethality；Comparison diterpene Lobocrassin E do not have this effect.
Description
Technical field
The invention belongs to which field is immunized, it is related to diterpene-kind compound and its derivative answering in terms of cellular immunotherapy
With, and in particular to a kind of STAT3 inhibitor, application of the STAT5 activator in terms of enhancing NK cells against tumor cells lethality.
Background technology
In recent years, the immunotherapy of tumour achieves huge progress in clinical research etc., but main research is all
It is for expansion such as T cells in specific immune system.However, with the natural kill to belonging to inherent immunity system
(natural killer, NK) cell research is goed deep into, and the effect in tumour immunotherapy is increasingly taken seriously.NK is thin
Born of the same parents are a kind of independent lymphocyte subgroups, are found to be the initial member of congenital lymphoid cell family recently.With T cell and
The antigenic stimulus that NK cells unlike B cell do not need specificity can kill target cell, have immune clearance and be immunized
The functions such as monitoring.Therefore NK cells are resisting tumour, especially shift played in the removing of oncocyte and microscopic tumor cells form it is important
Effect.
STAT3 and STAT5 is the important component of JAK-STAT accesses.Activation STAT3 can promote the increasing of tumour cell
It grows and weakens its sensibility to immunocytes such as NK cells.In addition the activation of STAT3 can also inhibit the immunocytes such as NK cells
To the lethal effect of tumour, while the adjusting between disorderly immunocyte, inhibit migration of the immunocyte to tumour.Therefore inhibit
STAT3 signal paths in tumor microenvironment are perhaps one of the approach for treating certain tumours.STAT5 can both promote tumour cell
Growth and development can promote development and the cytotoxicity of NK cells again.The STAT5 levels of NK cells are inhibited not only to damage NK cells
Normal function, and promote tumour growth.Expression of the STAT5 in NK cells is thus promoted to be conducive to the treatment to tumour
(bibliography：The Advances in antitumor activity of STAT3, STAT5 in NK cells, translational medicine e-magazine 2017 the 4th
Phase volume 5).
Flexilarin F, Flexilarin G are diterpene-kind compound isolated from a kind of coral, chemistry knot
Following (the bibliography of structure formula：Cembrane diterpenoids from the Taiwanese soft coral
Sinularia flexibillis.Tetrahedron,2009,65:9157-9164)。
The prior art did not report that Flexilarin F, Flexilarin G were used as STAT3 inhibitor, STAT5 is activated
The purposes of agent did not report that it can improve the lethality of NK cells against tumor cells yet.
Invention content
Present invention aims at a kind of STAT3 inhibitor of offer, STAT5 activator to be killed in enhancing NK cells against tumor cells
Application in terms of overstrain.
The above-mentioned purpose of the present invention is achieved by following technical solution：
Flexilarin F or Flexilarin G are used as the purposes of STAT3 inhibitor.
Flexilarin F or Flexilarin G are used as the purposes of STAT5 activator.
Flexilarin F or Flexilarin G are used to enhance the purposes of NK cells against tumor cells lethality.
Further, the tumour is gastric cancer.
A kind of STAT3 inhibitor or STAT5 activator also contain medicine containing Flexilarin F or Flexilarin G
Acceptable carrier or excipient on, are made pharmaceutically acceptable dosage form.
Further, pharmaceutically acceptable carrier or excipient include one or more solids, semisolid or liquid
Auxiliary material；Pharmaceutically acceptable dosage form includes tablet, capsule, granule, injection, pill, syrup, powder, paste.
It is a kind of for enhancing the pharmaceutical preparation of NK cells against tumor cells lethality, containing Flexilarin F or
Flexilarin G also contain pharmaceutically acceptable carrier or excipient, pharmaceutically acceptable dosage form are made.
Further, pharmaceutically acceptable carrier or excipient include one or more solids, semisolid or liquid
Auxiliary material；Pharmaceutically acceptable dosage form includes tablet, capsule, granule, injection, pill, syrup, powder, paste.
Further, the tumour is gastric cancer.
The technique effect of the present invention：
It is a discovery of the invention that Flexilarin F or Flexilarin G are STAT3 inhibitor, STAT5 activator,
Flexilarin F or Flexilarin G are by inhibiting the expression of STAT3, the expression of activation STAT5 to improve NK cells against tumor
Execution.
Description of the drawings
Fig. 1 is for each group NK cell STAT3, STAT5 expression quantity and to stomach cancer cell SGC-7901, BCG-823 lethality.
Specific implementation mode
It is specific with reference to the accompanying drawings and examples to introduce essentiality content of the present invention, but the guarantor of the present invention is not limited with this
Protect range.
Embodiment 1：
One, experiment material
RPMI-1640, fetal calf serum (FBS) are purchased from GIBCO companies；NK cell culture mediums are purchased from Germany CellGro.
Recombinated interleukin-2 (rhIL-2) is purchased from double aigret medicine companies；Lymphocyte separation medium is purchased from Nycomed
Pharma。
SGC-7901 cells, BCG-823 are purchased from Shanghai Cell Bank of the Chinese Academy of Sciences.
Two, experimental method
1, mononuclearcell separation, grouping and NK cell culture
Healthy volunteer's peripheral blood, Ficoll density gradient centrifugations are acquired, separation obtains peripheral blood mononuclear cells
(PBMC).PBMC is adjusted to initiator cell by Day 0 with the NK cell culture fluids containing 5% autologous plasma, 80U/ml gentamicins
Number is 1.0 × 106Cell is divided into control group and administration group, control group routine culture by/L：Day 0 is added 500U/ml's
RhIL-2 is placed in 37 DEG C, 5%CO2Incubator culture, later every 2 days half amounts change liquid 1 time, are 1.0 × 10 by cell number control6/
L.Administration group (Flexilarin F groups, Flexilarin G groups, Lobocrassin E groups) and control group cultural method basic one
It causes, 10 μM of Flexilarin F, Flexilarin G or Lobocrassin E is only additionally added in day 0.
2, Western blot measure the expression quantity of STAT3, STAT5 in each group NK cells
10 each group NK cells of Day are collected, total protein is extracted with cell pyrolysis liquid lytic cell.Prepare 12%SDS- propylene
Acrylamide gel, loading after albumen is mixed with 5 × loading buffer concentrate constant pressure 60V in glue, and separation gel adds to 120V,
It waits for that electrophoresis is completed, albumen is gone on pvdf membrane (constant current 150mA, 3h).It will be washed with TBST after film closing 2h with 5% skim milk
Film 3 times, each 10min, primary antibody is with working concentration 1:500,4 DEG C of overnight incubations, are added the secondary antibody of horseradish peroxidase label
(STAT3, STAT5 are rabbit-anti 1:10000；β-actin are mouse anti-1:12000).It is incubated 2h, shone with ECL luminous agent moulding pieces,
Development, using the gray level ratio of STAT3, STAT5 and internal reference β-actin as STAT5 expression quantity.By control group STAT3, STAT5
Expression quantity normalizes, and calculates the normalized value of each administration group STAT3, STAT5 expression quantity.
3, NK cells measure the lethality of stomach cancer cell SGC-7901
The culture of stomach cancer cell SGC-7901：SGC-7901 cell strains are taken out from liquid nitrogen, are placed in rapid fluid resuscitation in warm water,
It is inoculated in the 50mL culture bottles containing 10% fetal calf serum RPMI-1640 culture mediums, 37 DEG C, 5%CO2Environment culture, half per 2d
Amount changes liquid, observes cell growth status and changes liquid and passage in time.
Each group NK cells measure the lethality of stomach cancer cell SGC-7901：Day 10 is thin as target using SGC-7901 cells
Born of the same parents (2 × 104/ hole, 100 holes μ l/), 10:1 effect target separately sets target cell group and effector cell's group than 96 orifice plates are added.Every group sets 3
Multiple holes are placed in CO2Incubator.After 18h, 20 μ l CCK-8 reagents are added per hole, continue to cultivate 4h.Microplate reader is in 450nm wavelength
Lower detection absorbance, killing rate are calculated with following formula：
Killing rate (%)=(experimental group OD values-effector cell organizes OD values)/target cell group OD value × 100%.
4, NK cells measure the lethality of stomach cancer cell BCG-823
The culture of stomach cancer cell BCG-823：BCG-823 cell strains are taken out from liquid nitrogen, are placed in rapid fluid resuscitation in warm water, are connect
Kind is in the 50mL culture bottles containing 10% fetal calf serum RPMI-1640 culture mediums, 37 DEG C, 5%CO2Environment culture, per 2d, half measures
Liquid is changed, cell growth status is observed and changes liquid and passage in time.
Each group NK cells measure the lethality of stomach cancer cell BCG-823：Day 10 is thin with exponential phase BCG-823
Born of the same parents are target cell (2 × 104/ hole, 100 holes μ l/), 10:1 effect target separately sets target cell group and effector cell's group than 96 orifice plates are added.
Every group sets 3 multiple holes, is placed in CO2Incubator.After 18h, 20 μ l CCK-8 reagents are added per hole, continue to cultivate 4h.Microplate reader exists
Absorbance is detected under 450nm wavelength, killing rate is calculated with following formula：Killing rate (%)=(experimental group OD values-effector cell
Group OD values)/target cell group OD value × 100%.
5, statistical method
One-way analysis of variance is carried out using SPSS19.0 softwares, P ＜ 0.05 are that difference is statistically significant.
Three, experimental result
1, the expression quantity of STAT3, STAT5 compare in each group NK cells
STAT3 expression quantity is substantially less than control group, STAT5 in Flexilarin F groups, Flexilarin G group NK cells
Expression quantity is significantly higher than control group, STAT3, STAT5 table in diterpene compound Lobocrassin E group NK cells as a comparison
Up to amount with control group without significant difference.The results are shown in Table 1.
2, lethality measurement result of each group NK cells to stomach cancer cell SGC-7901
Flexilarin F groups, Flexilarin G group NK cells are significantly higher than the lethality of stomach cancer cell SGC-7901
Control group, diterpene compound Lobocrassin E group NK cells as a comparison to the lethality of stomach cancer cell SGC-7901 with
Control group is without significant difference.As a result as shown in table 1 and Fig. 1.
3, lethality measurement result of each group NK cells to stomach cancer cell BCG-823
Flexilarin F groups, Flexilarin G group NK cells are significantly higher than the lethality of stomach cancer cell BCG-823
Control group, diterpene compound Lobocrassin E group NK cells as a comparison to the lethality of stomach cancer cell BCG-823 with it is right
According to group without significant difference.As a result as shown in table 1 and Fig. 1.
1 each group NK cell STAT3, STAT5 expression quantity of table and the lethality to stomach cancer cell SGC-7901, BCG-823
The result shows that Flexilarin F, Flexilarin G are STAT3 inhibitor, STAT5 activator,
Flexilarin F, Flexilarin G are by inhibiting STAT3, activation STAT5 expression to activate killing for NK cells against tumor cells
Overstrain；Comparison diterpene compound Lobocrassin E do not have this effect.
Embodiment 2：
A kind of STAT3 inhibitor also contains pharmaceutically acceptable containing Flexilarin F or Flexilarin G
Pharmaceutically acceptable dosage form is made in carrier or excipient；Pharmaceutically acceptable carrier or excipient include a kind of or more
Kind solid, semisolid or Auxiliary Liquid Material, pharmaceutically acceptable dosage form includes tablet, capsule, granule, injection, ball
Agent, syrup, powder, paste.
Embodiment 3：
A kind of STAT5 activator also contains pharmaceutically acceptable containing Flexilarin F or Flexilarin G
Pharmaceutically acceptable dosage form is made in carrier or excipient；Pharmaceutically acceptable carrier or excipient include a kind of or more
Kind solid, semisolid or Auxiliary Liquid Material, pharmaceutically acceptable dosage form includes tablet, capsule, granule, injection, ball
Agent, syrup, powder, paste.
Embodiment 4：
It is a kind of for enhancing the pharmaceutical preparation of NK cells against tumor cells lethality, containing Flexilarin F or
Flexilarin G also contain pharmaceutically acceptable carrier or excipient, pharmaceutically acceptable dosage form are made；Pharmacy
Upper acceptable carrier or excipient include one or more solids, semisolid or Auxiliary Liquid Material, pharmaceutically acceptable agent
Type includes tablet, capsule, granule, injection, pill, syrup, powder, paste.The preferred gastric cancer of tumour cell.
The effect of above-described embodiment is specifically to introduce the essentiality content of the present 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 (9)
1.Flexilarin F or Flexilarin G are used as the purposes of STAT3 inhibitor.
2.Flexilarin F or Flexilarin G are used as the purposes of STAT5 activator.
3.Flexilarin F or Flexilarin G are used to enhance the purposes of NK cells against tumor cells lethality.
4. purposes according to claim 3, it is characterised in that：The tumour is gastric cancer.
5. a kind of STAT3 inhibitor or STAT5 activator, it is characterised in that：Containing Flexilarin F or Flexilarin G,
Also contain pharmaceutically acceptable carrier or excipient, pharmaceutically acceptable dosage form is made.
6. pharmaceutical preparation according to claim 5, it is characterised in that：The pharmaceutically acceptable carrier or excipient
Including one or more solids, semisolid or Auxiliary Liquid Material；The pharmaceutically acceptable dosage form include tablet, capsule,
Granule, injection, pill, syrup, powder, paste.
7. a kind of pharmaceutical preparation for enhancing NK cells against tumor cells lethality, it is characterised in that：Contain Flexilarin
F or Flexilarin G also contain pharmaceutically acceptable carrier or excipient, pharmaceutically acceptable dosage form are made.
8. pharmaceutical preparation according to claim 7, it is characterised in that：The pharmaceutically acceptable carrier or excipient
Including one or more solids, semisolid or Auxiliary Liquid Material；The pharmaceutically acceptable dosage form include tablet, capsule,
Granule, injection, pill, syrup, powder, paste.
9. pharmaceutical preparation according to claim 7 or 8, it is characterised in that：The tumour is gastric cancer.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810310610.XA CN108464980A (en) | 2018-04-09 | 2018-04-09 | A kind of STAT3 inhibitor, application of the STAT5 activator in terms of enhancing NK cells against tumor cells lethality |
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| CN201810310610.XA CN108464980A (en) | 2018-04-09 | 2018-04-09 | A kind of STAT3 inhibitor, application of the STAT5 activator in terms of enhancing NK cells against tumor cells lethality |
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2018
- 2018-04-09 CN CN201810310610.XA patent/CN108464980A/en not_active Withdrawn
Non-Patent Citations (2)
| Title |
|---|
| YUN-SHENG LIN ET AL.: "Cembrane diterpenoids from the Taiwanese soft coral Sinularia flexibilis", 《TETRAHEDRON》 * |
| 高程海等: "中国柳珊瑚萜类化合物研究新进展", 《广西科学院学报》 * |
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