CN108295060A - A kind of TLR7 agonists are in enhancing CIK cell to the application in terms of tumor cell killing potential - Google Patents
A kind of TLR7 agonists are in enhancing CIK cell to the application in terms of tumor cell killing potential Download PDFInfo
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- CN108295060A CN108295060A CN201810306179.1A CN201810306179A CN108295060A CN 108295060 A CN108295060 A CN 108295060A CN 201810306179 A CN201810306179 A CN 201810306179A CN 108295060 A CN108295060 A CN 108295060A
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- Prior art keywords
- flexilarin
- cell
- pharmaceutically acceptable
- tlr7
- cik
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
Abstract
The invention discloses a kind of TLR7 agonists in enhancing CIK cell to the application in terms of tumor cell killing potential.Improving CIK cell function by different approaches enhances its hot spot to tumor cell killing potential as Recent study.Studies have shown that TLR7 agonists can enhance lethality of people's CIK cell to tumour cell.Flexilarin B and Flexilarin C are diterpene-kind compound isolated from a kind of coral, present invention discover that, Flexilarin B and Flexilarin C are effective agonist of TLR7, and Flexilarin B and Flexilarin C activate lethality of the CIK cell to tumour cell by the expression for improving TLR7；Comparison diterpene compound Decaryiol C 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 TLR7 agonists are in enhancing CIK cell to the application in terms of tumor cell killing potential.
Background technology
In recent years, the killing cell (CIK cell) of application cell factor induction carries out the clinical test for the treatment of malignant tumor
At home and abroad carry out successively, CIK cell immunization therapy shows to be substantially better than other adoptive immunotherapies in clinical treatment
Powerful advantages, thus be applied in clinical cancer therapy more and more widely.CIK cell can be used as individual treatment side
Method can also be combined with surgical operation, radiotherapy, chemotherapy and carry out complex treatment.
Improving CIK cell function by different approaches enhances its heat to tumor cell killing potential as Recent study
Point.Studies have shown that TLR7 agonists can enhance lethality (bibliography of people's CIK cell to tumour cell：TLR7 agonists
Enhance the research of people's CIK cell killing ability, Journal of Immunology the 7th phase of volume 32 in July, 2016).
Flexilarin B and Flexilarin C 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 B and Flexilarin C were used as the purposes of TLR7 agonists, did not had yet
Had been reported that its lethality that can activate CIK cell to tumour cell.
Invention content
The purpose of the present invention is to provide a kind of TLR7 agonists in terms of enhancing CIK cell is to tumor cell killing potential
Using.
The above-mentioned purpose of the present invention is achieved by following technical solution：
Flexilarin B or Flexilarin C are used as the purposes of TLR7 agonists.
Flexilarin B or Flexilarin C are for enhancing purposes of the CIK cell to tumor cell killing potential.
Further, the tumour is gastric cancer.
A kind of TLR7 agonists also contain pharmaceutically acceptable containing Flexilarin B or Flexilarin C
Pharmaceutically acceptable dosage form is made in carrier or excipient.
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 pharmaceutical preparation of the CIK cell to tumor cell killing potential, containing Flexilarin B or
Flexilarin C also contain pharmaceutically acceptable carrier or excipient, pharmaceutically acceptable dosage form are 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.
Further, the tumour is gastric cancer.
The technique effect of the present invention：
It is a discovery of the invention that Flexilarin B and Flexilarin C are effective agonist of TLR7, Flexilarin B
Lethality of the CIK cell to tumour cell is activated by the expression for improving TLR7 with Flexilarin C.
Description of the drawings
Fig. 1 is each group CIK cell TLR7 expression quantity and the lethality to stomach cancer cell SGC-7901, BCG-823.
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, AIM-V culture solution, fetal calf serum (FBS) are purchased from GIBCO companies.
Recombinated interleukin-2 (rhIL-2), recombinant interferon-γ (IFN-γ), AntiCD3 McAb McAb, recombinant human leucocyte
1 α of interleukin (rhIL-1 α) is purchased from double aigret medicine companies；Lymphocyte separation medium is purchased from Nycomed Pharma companies.
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 CIK 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 AIM-V culture solutions 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 1000U/ml's
IFN-γ is placed in 37 DEG C, 5%CO2For 24 hours, the AntiCD3 McAb McAb that mass concentration is 50ng/ml is then added in incubator culture,
The rhIL-1 α of the rhIL-2 and 100U/ml of 300U/ml；Addition in every 3 days later is big mould containing 5% autologous plasma, 80U/ml celebratings
Cell number control is 1.0 × 10 by the AIM-V culture solutions of element, 1000U/mlrhIL-26/L.Administration group (Flexilarin B
Group, Decaryiol C groups, Flexilarin C groups) it is almost the same with control group cultural method, only 10 μ are additionally added in day 0
M Flexilarin B, Decaryiol C or Flexilarin C.
2, Western blot measure the expression quantity of TLR7 in each group CIK cell
15 each group CIK 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
(TLR7 is rabbit-anti 1:10000；β-actin are mouse anti-1:12000).It is incubated 2h, is shone with ECL luminous agent moulding pieces, development, with
The gray level ratio of TLR7 and internal reference β-actin is as TLR7 expression quantity.Control group TLR7 expression quantity is normalized, each administration is calculated
The normalized value of group TLR7 expression quantity.
3, CIK cell measures 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 CIK cell measures the lethality of stomach cancer cell SGC-7901：Day 15 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, CIK cell measures 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 CIK cell measures the lethality of stomach cancer cell BCG-823：Day 15 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 TLR7 compares in each group CIK cell
TLR7 expression quantity is significantly higher than control group in Flexilarin B groups, Flexilarin C group CIK cells, as right
Than TLR7 expression quantity in the Decaryiol C group CIK cells of diterpene and control group without significant difference.
The results are shown in Table 1.
2, lethality measurement result of each group CIK cell to stomach cancer cell SGC-7901
Flexilarin B groups, Flexilarin C groups CIK cells are significantly high to the lethality of stomach cancer cell SGC-7901
In control group, lethality and control group of the Decaryiol C groups CIK cells of diterpene to stomach cancer cell SGC-7901 as a comparison
Without significant difference.
As a result as shown in table 1 and Fig. 1.
3, lethality measurement result of each group CIK cell to stomach cancer cell BCG-823
Flexilarin B groups, Flexilarin C groups CIK cells are significantly higher than the lethality of stomach cancer cell BCG-823
Control group, as a comparison the Decaryiol C groups CIK cells of diterpene to the lethality of stomach cancer cell BCG-823 and control group without
Significant difference.
As a result as shown in table 1 and Fig. 1.
1 each group CIK cell TLR7 expression quantity of table and the lethality to stomach cancer cell SGC-7901, BCG-823
The result shows that Flexilarin B, Flexilarin C are effective agonist of TLR7, Flexilarin B,
Flexilarin C activate lethality of the CIK cell to tumour cell by the expression for improving TLR7；As a comparison two
Terpenoid Decaryiol C do not have this effect.
Embodiment 2：
A kind of TLR7 agonists also contain pharmaceutically acceptable containing Flexilarin B or Flexilarin C
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：
It is a kind of for enhancing pharmaceutical preparation of the CIK cell to tumor cell killing potential, containing Flexilarin B or
Flexilarin C 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 (10)
1.Flexilarin B or Flexilarin C are used as the purposes of TLR7 agonists.
2.Flexilarin B or Flexilarin C are for enhancing purposes of the CIK cell to tumor cell killing potential.
3. purposes according to claim 2, it is characterised in that：The tumour is gastric cancer.
4. a kind of TLR7 agonists, it is characterised in that：Containing Flexilarin B or Flexilarin C, also contains and pharmaceutically may be used
With the carrier or excipient of receiving, pharmaceutically acceptable dosage form is made.
5. pharmaceutical preparation according to claim 4, it is characterised in that：The pharmaceutically acceptable carrier or excipient
Including one or more solids, semisolid or Auxiliary Liquid Material.
6. pharmaceutical preparation according to claim 4, it is characterised in that：The pharmaceutically acceptable dosage form includes piece
Agent, capsule, granule, injection, pill, syrup, powder, paste.
7. a kind of for enhancing pharmaceutical preparation of the CIK cell to tumor cell killing potential, it is characterised in that：Contain Flexilarin
B or Flexilarin C 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.
9. pharmaceutical preparation according to claim 7, it is characterised in that：The pharmaceutically acceptable dosage form includes piece
Agent, capsule, granule, injection, pill, syrup, powder, paste.
10. according to any pharmaceutical preparations of claim 7-9, it is characterised in that：The tumour is gastric cancer.
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| CN201810306179.1A CN108295060A (en) | 2018-04-08 | 2018-04-08 | A kind of TLR7 agonists are in enhancing CIK cell to the application in terms of tumor cell killing potential |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101018567A (en) * | 2004-07-20 | 2007-08-15 | 先灵公司 | Induction of apoptosis in Toll-like receptor expressing tumor cells |
| CN102666541A (en) * | 2009-10-22 | 2012-09-12 | 吉里德科学公司 | Derivatives of purine or deazapurine useful for the treatment of (inter alia) viral infections |
-
2018
- 2018-04-08 CN CN201810306179.1A patent/CN108295060A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101018567A (en) * | 2004-07-20 | 2007-08-15 | 先灵公司 | Induction of apoptosis in Toll-like receptor expressing tumor cells |
| CN102666541A (en) * | 2009-10-22 | 2012-09-12 | 吉里德科学公司 | Derivatives of purine or deazapurine useful for the treatment of (inter alia) viral infections |
Non-Patent Citations (2)
| Title |
|---|
| YUN-SHENG LIN ET.AL: "cembrane diterpenoids from the taiwanese soft coral sinularia flexibilis", 《TETRAHEDRON》 * |
| 陈艳媛: "TLR7激动剂增强人CIK细胞杀伤功能的研究", 《免疫学杂志》 * |
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