CN108210485A - 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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- CN108210485A CN108210485A CN201810306177.2A CN201810306177A CN108210485A CN 108210485 A CN108210485 A CN 108210485A CN 201810306177 A CN201810306177 A CN 201810306177A CN 108210485 A CN108210485 A CN 108210485A
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- Prior art keywords
- pavidolide
- 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
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
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.Research shows that TLR7 agonists can enhance lethality of people's CIK cell to tumour cell.Pavidolide D and Pavidolide E are diterpene-kind compound isolated from a kind of coral, present invention discover that, Pavidolide D and Pavidolide E are effective agonist of TLR7, and Pavidolide D and Pavidolide E activate lethality of the CIK cell to tumour cell by improving the expression of 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.Research shows 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).
Pavidolide D and Pavidolide E are diterpene-kind compound isolated from a kind of coral, and chemistry is tied
Following (the bibliography of structure formula：Pavidolides A-E,new cembranoids from the soft coral
Sinulariapavida.Tetrahedron Lett,2012,53:5759-5762)。
The prior art did not report that Pavidolide D and Pavidolide E were used as the purposes of TLR7 agonists, did not had yet
Had been reported that it can activate lethality of the 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：
Pavidolide D or Pavidolide E are used as the purposes of TLR7 agonists.
Pavidolide D or Pavidolide E are used to enhance purposes of the CIK cell to tumor cell killing potential.
Further, the tumour is gastric cancer.
A kind of TLR7 agonists, containing Pavidolide D or Pavidolide E, also containing pharmaceutically acceptable
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 Pavidolide D or
Also containing pharmaceutically acceptable carrier or excipient, pharmaceutically acceptable dosage form is made in Pavidolide E.
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 Pavidolide D and Pavidolide E are effective agonist of TLR7, by improving TLR7
Expression so that activate CIK cell to the lethality of tumour cell.
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 embodiment
The specific guarantor for introducing essentiality content of the present invention, but the present invention not being limited with this with reference to the accompanying drawings and examples
Protect range.
Embodiment 1：
First, 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 Chinese Academy of Sciences's Shanghai cell bank.
2nd, experimental method
1st, 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 adds in 1000U/ml's
IFN-γ is placed in 37 DEG C, 5%CO2Incubator culture for 24 hours, then adds in the AntiCD3 McAb McAb that mass concentration is 50ng/ml,
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/ml rhIL-26/L.Administration group (Pavidolide D
Group, Decaryiol C groups, Pavidolide E groups) it is basically identical with control group cultural method, only 10 μ are additionally added in day 0
M Pavidolide D, Decaryiol C or Pavidolide E.
2nd, 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 treats that electrophoresis is completed, 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, primary antibody is with working concentration 1:500,4 DEG C of overnight incubations add in the secondary antibody of horseradish peroxidase label
(TLR7 is rabbit-anti 1:10000；β-actin resist 1 for mouse:12000).2h is incubated, is shone, developed with ECL luminous agent moulding pieces, with
The gray level ratio of TLR7 and internal reference β-actin is as TLR7 expression quantity.Control group TLR7 expression quantity is normalized, calculates each administration
The normalized value of group TLR7 expression quantity.
3rd, 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 μ l/ holes), 10:1 effect target separately sets target cell group and effector cell's group than adding in 96 orifice plates.Every group sets 3
Multiple holes are placed in CO2Incubator.After 18h, 20 μ l CCK-8 reagents are added in per hole, continue to cultivate 4h.Microplate reader is in 450nm wavelength
Lower detection absorbance, killing rate are calculated with the following formula：
Killing rate (%)=(experimental group OD values-effector cell organizes OD values)/target cell group OD value × 100%.
4th, 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, rapid fluid resuscitation in warm water is placed in, connects
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, observe cell growth status 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 μ l/ holes), 10:1 effect target separately sets target cell group and effector cell's group than adding in 96 orifice plates.
Every group sets 3 multiple holes, is placed in CO2Incubator.After 18h, 20 μ l CCK-8 reagents are added in per hole, continue to cultivate 4h.Microplate reader exists
Absorbance is detected under 450nm wavelength, killing rate is calculated with the following formula：Killing rate (%)=(experimental group OD values-effector cell
Group OD values)/target cell group OD value × 100%.
5th, statistical method
One-way analysis of variance is carried out using SPSS19.0 softwares, P ＜ 0.05 are statistically significant for difference.
3rd, experimental result
1st, the expression quantity of TLR7 compares in each group CIK cell
TLR7 expression quantity is significantly higher than control group in Pavidolide D groups, Pavidolide E 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.
2nd, each group CIK cell is to the lethality measurement result of stomach cancer cell SGC-7901
Pavidolide D groups, Pavidolide E groups CIK cells are significantly high to the lethality of stomach cancer cell SGC-7901
In control group, the Decaryiol C groups CIK cells of diterpene are to the lethality and control group of stomach cancer cell SGC-7901 as a comparison
Without significant difference.As a result as shown in table 1 and Fig. 1.
3rd, each group CIK cell is to the lethality measurement result of stomach cancer cell BCG-823
Pavidolide D groups, Pavidolide E 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 Pavidolide D, Pavidolide E are effective agonist of TLR7, Pavidolide D,
Pavidolide E activate lethality of the CIK cell to tumour cell by improving the expression of TLR7；As a comparison two
Terpenoid Decaryiol C do not have this effect.
Embodiment 2：
A kind of TLR7 agonists, containing Pavidolide D or Pavidolide E, also containing pharmaceutically acceptable
Pharmaceutically acceptable dosage form is made in carrier or excipient；Pharmaceutically acceptable carrier or excipient include a kind of or more
Solid, semisolid or Auxiliary Liquid Material are planted, 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 Pavidolide D or
Also containing pharmaceutically acceptable carrier or excipient, pharmaceutically acceptable dosage form is made in Pavidolide E；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.Pavidolide D or Pavidolide E are used as the purposes of TLR7 agonists.
2.Pavidolide D or Pavidolide E are used to enhance 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 Pavidolide D or Pavidolide E, also contain 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 be used to enhance pharmaceutical preparation of the CIK cell to tumor cell killing potential, it is characterised in that：Contain Pavidolide
Also containing pharmaceutically acceptable carrier or excipient, pharmaceutically acceptable dosage form is made in D or Pavidolide E.
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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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1771931A (en) * | 2004-11-09 | 2006-05-17 | 和记黄埔医药企业有限公司 | Use of diterpene compound in preparingmedicines with synergistic effect to chemical therepy medicines |
| CN101829108A (en) * | 2009-03-10 | 2010-09-15 | 湘北威尔曼制药有限公司 | Application of diterpene ginkgolide |
| CN102675252A (en) * | 2012-05-28 | 2012-09-19 | 南京中医药大学 | Cembrane type diterpenoids with anti-tumor activities and application thereof |
-
2018
- 2018-04-08 CN CN201810306177.2A patent/CN108210485A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1771931A (en) * | 2004-11-09 | 2006-05-17 | 和记黄埔医药企业有限公司 | Use of diterpene compound in preparingmedicines with synergistic effect to chemical therepy medicines |
| CN101829108A (en) * | 2009-03-10 | 2010-09-15 | 湘北威尔曼制药有限公司 | Application of diterpene ginkgolide |
| CN102675252A (en) * | 2012-05-28 | 2012-09-19 | 南京中医药大学 | Cembrane type diterpenoids with anti-tumor activities and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| SHI SHEN: "Pavidolides A-E, new cembranoids from the soft coral sinularia pavida", 《TETRAHEDRON LETTERS》 * |
| 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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