CN108295078B - A kind of application of STAT3 inhibitor in terms of enhancing NK cells against tumor cells lethality - Google Patents
A kind of application of STAT3 inhibitor in terms of enhancing NK cells against tumor cells lethality Download PDFInfo
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- CN108295078B CN108295078B CN201810310476.3A CN201810310476A CN108295078B CN 108295078 B CN108295078 B CN 108295078B CN 201810310476 A CN201810310476 A CN 201810310476A CN 108295078 B CN108295078 B CN 108295078B
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Abstract
The invention discloses a kind of application of STAT3 inhibitor in terms of enhancing NK cells against tumor cells lethality.Those skilled in the art will know that, activation STAT3 can promote the proliferation of tumour cell and weaken its sensibility to immunocytes such as NK cells, the activation of STAT3 can also inhibit the immunocytes such as NK cell to the lethal effect of tumour, adjusting between disorder immunocyte simultaneously, inhibits migration of the immunocyte to tumour.It is a discovery of the invention that Lobocrassin C, Lobocrassin D are STAT3 inhibitor, Lobocrassin C, Lobocrassin D are by inhibiting STAT3 expression to activate the lethality of NK cells against tumor cells；Lobocrassin E does 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 application of STAT3 inhibitor in terms of enhancing NK cells against tumor cells lethality.
Background technique
In recent years, the immunotherapy of tumour in terms of achieve huge progress, but main research is all
It is for expansion such as T cells in specific immune system.However, with to the natural kill for 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 cell unlike B cell does not need specificity can kill target cell, have immune clearance and be immunized
The functions such as monitoring.Therefore NK cell is resisting tumour, especially plays in the removing of transfer oncocyte and microscopic tumor cells form important
Effect.
STAT3 and STAT5 is the important component of JAK-STAT access.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 cell
To the lethal effect of tumour, while the adjusting between disorder immunocyte, inhibit migration of the immunocyte to tumour.Therefore inhibit
STAT3 signal path in tumor microenvironment is perhaps one of the approach for treating certain tumours.STAT5 both can promote tumour cell
Growth and development can promote the development and cytotoxicity of NK cell again.The STAT5 level of NK cell is inhibited not only to damage NK cell
Normal function, and promote tumour growth.Expression of the STAT5 in NK cell is thus promoted to be conducive to the treatment to tumour
(bibliography: the Advances in antitumor activity of STAT3, STAT5 in NK cell, translational medicine e-magazine 2017 the 4th
Phase volume 5).
Lobocrassin C, Lobocrassin D, Lobocrassin E are diterpene isolated from a kind of coral
Class compound, the following (bibliography: Terpenoids from the octocorals Menella sp. of structural formula
(Plexauridae)and Lobophytum crassum(Alcyonacea).MarDrugs,2012,10:427-438)。
The prior art did not report that Lobocrassin C, Lobocrassin D were used as the purposes of STAT3 inhibitor,
Do not reported that the lethality of NK cells against tumor cells can be improved in it.
Summary of the invention
It is an object of that present invention to provide a kind of STAT3 inhibitor answering in terms of enhancing NK cells against tumor cells lethality
With.
Above-mentioned purpose of the invention is achieved by following technical solution:
Lobocrassin C or Lobocrassin D are used as the purposes of STAT3 inhibitor.
Lobocrassin C or Lobocrassin D are used to enhance the purposes of NK cells against tumor cells lethality.
Further, the tumour is gastric cancer.
A kind of STAT3 inhibitor, containing Lobocrassin C or Lobocrassin D, also containing can pharmaceutically receive
Carrier or excipient, pharmaceutically acceptable dosage form is made.
Further, the pharmaceutically acceptable carrier or excipient include one or more solids, semisolid or liquid
Body auxiliary material.
Further, the pharmaceutically acceptable dosage form include 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 Lobocrassin C or
Pharmaceutically acceptable dosage form is made also containing pharmaceutically acceptable carrier or excipient in Lobocrassin D.
Further, the pharmaceutically acceptable carrier or excipient include one or more solids, semisolid or liquid
Body auxiliary material.
Further, the pharmaceutically acceptable dosage form include tablet, capsule, granule, injection, pill,
Syrup, powder, paste.
Further, the tumour is gastric cancer.
Technical effect of the invention:
It is a discovery of the invention that Lobocrassin C or Lobocrassin D be STAT3 inhibitor, Lobocrassin C or
Lobocrassin D is by inhibiting the expression of STAT3 to improve the lethality of NK cells against tumor cells.
Detailed description of the invention
Fig. 1 is each group NK cell STAT3 expression quantity and the lethality to stomach cancer cell SGC-7901, BCG-823.
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.
Embodiment 1:
One, experimental material
RPMI-1640, fetal calf serum (FBS) are purchased from GIBCO company；NK cell culture medium is 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 centrifugation are acquired, separation obtains peripheral blood mononuclear cells
(PBMC).PBMC is adjusted to initiator cell with the NK cell culture fluid containing 5% autologous plasma, 80U/ml gentamicin by Day 0
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 are changed liquid 1 time, are 1.0 × 10 by cell number control6/
L.Administration group (Lobocrassin C group, Lobocrassin D group, Lobocrassin E group) is basic with control group cultural method
Unanimously, 10 μM of Lobocrassin C, Lobocrassin D or Lobocrassin E are only additionally added in day 0.
2, Western blot measures the expression quantity of STAT3 in each group NK cell
10 each group NK cell of Day is collected, extracts total protein with cell pyrolysis liquid lytic cell.Prepare 12%SDS- propylene
Constant pressure 60V in glue is concentrated in acrylamide gel, loading after albumen is mixed with 5 × loading buffer, and separation gel adds to 120V,
It is completed to electrophoresis, albumen is gone on pvdf membrane (constant current 150mA, 3h).It will be washed after film closing 2h with TBST with 5% skim milk
Film 3 times, each 10min, the secondary antibody of horseradish peroxidase label is added with working concentration 1:500,4 DEG C of overnight incubations in primary antibody
(STAT3 is rabbit-anti 1:10000；β-actin is the anti-1:12000 of mouse).It is incubated for 2h, is shone with ECL luminous agent moulding piece, development,
Using the gray level ratio of STAT3 and internal reference β-actin as STAT3 expression quantity.Control group STAT3 expression quantity is normalized, is calculated
The normalized value of each administration group STAT3 expression quantity.
3, NK cell measures the lethality of stomach cancer cell SGC-7901
The culture of stomach cancer cell SGC-7901: being removed from liquid nitrogen SGC-7901 cell strain, is placed in rapid fluid resuscitation in warm water,
It is inoculated in the 50mL culture bottle containing 10% fetal calf serum RPMI-1640 culture medium, 37 DEG C, 5%CO2Environment culture, every 2d half
Amount changes liquid, observes cell growth status and changes liquid and passage in time.
Each group NK cell measures the lethality of stomach cancer cell SGC-7901: Day 10, thin as target using SGC-7901 cell
Born of the same parents (2 × 104/ hole, 100 holes μ l/), 10:1 imitates target than 96 orifice plates are added, and separately sets target cell group and effector cell's group.Every group sets 3
Multiple holes are placed in CO2Incubator.After 18h, 20 μ l CCK-8 reagents are added in every 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 value-effector cell organizes OD value)/target cell group OD value × 100%.
4, NK cell measures the lethality of stomach cancer cell BCG-823
The culture of stomach cancer cell BCG-823: being removed from liquid nitrogen BCG-823 cell strain, is placed in rapid fluid resuscitation in warm water, connects
Kind is in the 50mL culture bottle containing 10% fetal calf serum RPMI-1640 culture medium, and 37 DEG C, 5%CO2Environment culture, every 2d half are measured
Liquid is changed, cell growth status is observed and changes liquid and passage in time.
Each group NK cell measures the lethality of stomach cancer cell BCG-823: Day 10, thin with logarithmic growth phase BCG-823
Born of the same parents are target cell (2 × 104/ hole, 100 holes μ l/), 10:1 imitates target than 96 orifice plates are added, and separately sets target cell group and effector cell's group.
Every group sets 3 multiple holes, is placed in CO2Incubator.After 18h, 20 μ l CCK-8 reagents are added in every 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 value-effector cell
Group OD value)/target cell group OD value × 100%.
5, statistical method
One-way analysis of variance is carried out using SPSS19.0 software, P < 0.05 is that difference is statistically significant.
Three, experimental result
1, the expression quantity of STAT3 compares in each group NK cell
STAT3 expression quantity is substantially less than control group in Lobocrassin C group, Lobocrassin D group NK cell,
STAT3 expression quantity and control group are without significant difference in Lobocrassin E group NK cell.
The results are shown in Table 1.
2, lethality measurement result of each group NK cell to stomach cancer cell SGC-7901
Lobocrassin C group, Lobocrassin D group NK cell are significantly high to the lethality of stomach cancer cell SGC-7901
In control group, Lobocrassin E group NK cell is to the lethality and control group of stomach cancer cell SGC-7901 without significant difference.
As a result as shown in table 1 and Fig. 1.
3, lethality measurement result of each group NK cell to stomach cancer cell BCG-823
Lobocrassin C group, Lobocrassin D group NK cell are significantly high to the lethality of stomach cancer cell BCG-823
In control group, Lobocrassin E group NK cell is to the lethality and control group of stomach cancer cell BCG-823 without significant difference.
As a result as shown in table 1 and Fig. 1.
1 each group NK cell STAT3 expression quantity of table and the lethality to stomach cancer cell SGC-7901, BCG-823
The result shows that Lobocrassin C, Lobocrassin D be STAT3 inhibitor, Lobocrassin C,
Lobocrassin D is by inhibiting STAT3 expression to activate the lethality of NK cells against tumor cells；Lobocrassin E does not have
Standby this effect.
Embodiment 2:
A kind of STAT3 inhibitor, containing Lobocrassin C or Lobocrassin D, also containing can pharmaceutically receive
Carrier or excipient, pharmaceutically acceptable dosage form is made；Pharmaceutically acceptable carrier or excipient include it is a kind of or
Many kinds of solids, semisolid or Auxiliary Liquid Material, pharmaceutically acceptable dosage form include tablet, capsule, granule, injection,
Pill, syrup, powder, paste.
Embodiment 3:
It is a kind of for enhancing the pharmaceutical preparation of NK cells against tumor cells lethality, containing Lobocrassin C or
Pharmaceutically acceptable dosage form is made also containing pharmaceutically acceptable carrier or excipient in Lobocrassin D；Medicine
Acceptable carrier or excipient include one or more solids, semisolid or Auxiliary Liquid Material on, pharmaceutically acceptable
Dosage form 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 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 (1)
1.Lobocrassin C or Lobocrassin D are used to prepare the drug of enhancing NK cells against tumor cells lethality
Purposes, the tumour are gastric cancer.
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