CN109529050A - The application that 9 δ 2T cell of V γ, the drug ZOL and hMSH2 for treating lung cancer act synergistically - Google Patents
The application that 9 δ 2T cell of V γ, the drug ZOL and hMSH2 for treating lung cancer act synergistically Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, specifically, providing the application of a kind of 9 δ 2T cell of V γ, the drug for treating lung cancer and ZOL and hMSH2 synergistic effect.The present invention provides application of the synergistic effect in the cytotoxic activity related keyword factor for regulating and controlling 9 δ 2T cell of V γ of ZOL and hMSH2.Present invention discover that secretion of the ZOL and hMSH2 synergistic effect by RANTES in inhibition 9 δ 2T cell of V γ, while promoting the secretion of IFN-γ, fibroblast growth factor, colony stimulating factor, IP-10, MIP-1 α, MIP-1 β and IL family few members.Therefore, the synergistic effect of ZOL and hMSH2 can be used as a kind of novel drug, quickly activate 9 δ 2T cell of V γ, have potential therapeutic value to tumour cell.
Description
Technical field
The present invention relates to field of biotechnology, in particular to a kind of 9 δ 2T cell of V γ, treat the drug of lung cancer with
The application of ZOL and hMSH2 synergistic effect.
Background technique
Mankind's gamma delta T cells are a specific groups in T lymphocyte, are obtained because the T cell receptor of expression is TCR γ δ
Name.Gamma delta T cells have both the double characteristic of natural killer cells and T cell, can be with MHC (Major histocompatibility
Complex, MHC) non-limiting way identifies and killing tumor cell, have very importantly in antitumor immunity of organism
Position.
9 δ 2T cell of V γ is the major subpopulations of peripheral blood gamma delta T cells, accounts for about the 50~95% of its sum, to identify phosphoric acid
Changing antigen and secretion Th1 cytokines is main feature.In recent years, work of the 9 δ 2T cell of V γ in antitumor immunity of organism
With more and more attention has been paid to and have become tumor vaccine cells and adopt the up-and-coming youngster for the treatment of.It is currently known 9 δ 2T cell of V γ
A variety of solid tumors and the blood tumor cell including lung cancer can be killed in vivo and in vitro, however, 9 δ 2T cell recognition of V γ kills
The molecular mechanism of lung cancer it is not immediately clear.How to utilize 9 δ 2T cell of V γ and improves the killing ability of 9 δ 2T cell of V γ not
There is enough theoretical basis and using means.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide the synergistic effect of ZOL and hMSH2 in activation 9 δ 2T cell killing tumour of V γ
Application in key factor, to alleviate the effective means for lacking activation 9 δ 2T cell of V γ in the prior art, and to 9 δ 2T of V γ
The unclear technical problem of cell killing tumour mechanism.
The second object of the present invention is to provide a kind of 9 δ 2T cell of V γ, with alleviate lack one kind in the prior art can be with
The immunocyte of Efficient killing effect tumour cell.
The third object of the present invention is to provide a kind of drug for the treatment of cancer, with alleviate lack one kind in the prior art can
With effective treating cancer, especially entity tumor, and it is free from side effects, highly-safe drug.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
The synergistic effect of ZOL and hMSH2 is in following A)-E) at least one of in application:
A) inhibit RANTES in 9 δ 2T cell of V γ (activated regulators of normal T-cell expression and secretion, Regulated
Upon activation normal T cell expressed and secreted factor) secretion；
B) promote the secretion of IFN-γ (interferon-γ, Interferon- γ) in 9 δ 2T cell of V γ；
C) promote 9 δ 2T cell of V γ in FGF (fibroblast growth factor, Fibroblast growth family),
G-CSF (granulocyte colony stimulating factor, Granucyte-colony stimulating factor) or GM-CSF (granulocyte
At least one of macrophage stimulation factor, Granucyte-macrophage-colony stimulating factor)
Secretion；
D) promote IP-10 (interferon-γ inducible protein 10, Interferon gamma-induced in 9 δ 2T cell of V γ
Protein 10), MIP-1 α (Macrophage inflammatory protein-α, Macrophage inflammatory protein-1 α) or
Point of at least one of MIP-1 β (macrophage inflammatory protein -1 β, Macrophage inflammatory protein-1 β)
It secretes；
E) promote 9 δ 2T cell of V γ in IL-1 β, IL-1 γ a, IL-2, IL-4, IL-6, IL-7, IL-8, IL-9, IL-12,
The secretion of at least one of IL-13 or IL-18.
Further, the concentration of the ZOL is 5-30 μM.
Further, the hMSH2 is located at lung carcinoma cell surface.
Further, the lung carcinoma cell includes that GLC-82, NCI-H446 or hMSH2 are overexpressed in NCI-H520 at least
One kind, preferably hMSH2 are overexpressed NCI-H520.
Further, the hMSH2 is overexpressed NCI-H520 by that will contain the Transfected Recombinant Plasmid of coding hMSH2 gene
NCI-H520 is obtained.
Further, the expression vector in the recombinant plasmid is cFugw plasmid.
A kind of 9 δ 2T cell of V γ, the 9 δ 2T cell of V γ are activated by ZOL and hMSH2.
A kind of drug for the treatment of cancer, the effective component of the drug include ZOL and hMSH2.
Further, the effective component of the drug includes the cell of ZOL and cell surface expression hMSH2.
Further, the cancer includes lung cancer (Lung cancer, LC), cutaneum carcinoma, kidney, breast cancer or prostate
Cancer.
Compared with prior art, the invention has the benefit that
The present invention provide ZOL and hMSH2 synergistic effect in following A)-E) and at least one of in application: A) inhibit 9 δ of V γ
The secretion of RANTES in 2T cell；B) promote the secretion of IFN-γ in 9 δ 2T cell of V γ；C) promote 9 δ 2T cell of V γ in FGF,
The secretion of at least one of G-CSF or GM-CSF；D) promote in 9 δ 2T cell of V γ at least one in IP-10, MIP-1 α or MIP-1 β
The secretion of kind；E) promote IL-1 β, IL-1 γ a, IL-2, IL-4, IL-6, IL-7, IL-8, IL-9, IL- in 9 δ 2T cell of V γ
12, the secretion of at least one of IL-13 or IL-18.ZOL and hMSH2 synergistic effect can promote 9 δ 2T cell recognition of V γ and killing
Tumour cell, present invention discover that ZOL and hMSH2 synergistic effect is promoted simultaneously by the secretion of RANTES in inhibition 9 δ 2T cell of V γ
Into IFN-γ, the secretion of FGF, CSF, IP-10, MIP-1 α, MIP-1 β and IL family few members.This is thin to improve 9 δ 2T of V γ
Born of the same parents' identification and killing tumor cell provide theoretical explanation, and the synergistic effect of ZOL and hMSH2 can be thin with 9 δ 2T of fast activating V γ
Born of the same parents improve the ability of V γ 9 δ 2T cell recognition and killing tumor cell.Therefore, the synergistic effect of ZOL and hMSH2 can be used as
A kind of newtype drug quickly activates 9 δ 2T cell of V γ, has potential therapeutic value to tumour cell.
Detailed description of the invention
Fig. 1 is that hMSH2 and various concentration ZOL collaboration activation 9 δ 2T cell of V γ is thin to NCI-H520 in the embodiment of the present invention 3
The result figure of born of the same parents' lethal effect；
Fig. 2 is that ZOL activates 9 δ 2T cell of V γ to the result figure of NCI-H446 lethal effect in the embodiment of the present invention 3；
Fig. 3 A is ImmunohistochemistryResults Results of the hMSH2 in cancerous lung tissue LC-1 in the embodiment of the present invention 3；
Fig. 3 B is immunohistochemical staining result of the hMSH2 in cancerous lung tissue LC-2 in the embodiment of the present invention 3；
Fig. 3 C is immunohistochemical staining result of the hMSH2 in cancerous lung tissue LC-3 in the embodiment of the present invention 3；
Fig. 3 D is the representative result of cancerous lung tissue HE dyeing in the embodiment of the present invention 3；
The immunohistochemical staining result that Fig. 3 E is hMSH2 in cancer side control tissue (relatively normal) in the embodiment of the present invention 3；
Fig. 3 F is the negative control of hMSH2 immunohistochemical staining in the embodiment of the present invention 3；
Fig. 4 A is that phosphorylation antigen cooperates with the result figure for promoting IL-1b secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 4 B is that phosphorylation antigen cooperates with the result figure for promoting IL-1ra secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 4 C is that phosphorylation antigen cooperates with the result figure for promoting IL-2 secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 4 D is that phosphorylation antigen cooperates with the result figure for promoting IL-4 secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 4 E is that phosphorylation antigen cooperates with the result figure for promoting IL-5 secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 4 F is that phosphorylation antigen cooperates with the result figure for promoting IL-6 secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 4 G is that phosphorylation antigen cooperates with the result figure for promoting IL-7 secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 4 H is that phosphorylation antigen cooperates with the result figure for promoting IL-8 secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 4 I is that phosphorylation antigen cooperates with the result figure for promoting IL-9 secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 4 J is that phosphorylation antigen cooperates with the result figure for promoting IL-10 secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 4 K is that phosphorylation antigen cooperates with the result figure for promoting IL-12 secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 4 L is that phosphorylation antigen cooperates with the result figure for promoting IL-13 secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 4 M is that phosphorylation antigen cooperates with the result figure for promoting IL-15 secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 4 N is that phosphorylation antigen cooperates with the result figure for promoting IL-17 secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 4 O is that phosphorylation antigen cooperates with the result figure for promoting IL-18 secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 5 A is that phosphorylation antigen cooperates with the result figure for promoting IFN-γ secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 5 B is that phosphorylation antigen cooperates with promotion eosinophil chemotactic factor activation to become with hMSH2 in the embodiment of the present invention 4
Change the result figure of the factor (Eosinophil chemotactic proteins, Eotaxin) secretion；
Fig. 5 C is that phosphorylation antigen cooperates with the result figure for promoting FGF-basic secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 5 D is that phosphorylation antigen cooperates with the result figure for promoting G-CSF secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 5 E is that phosphorylation antigen cooperates with the result figure for promoting GM-CSF secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 5 F is that phosphorylation antigen cooperates with the result figure for promoting IP-10 secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 5 G is that phosphorylation antigen cooperates with promotion monocyte chemoattractant protein-1 with hMSH2 in the embodiment of the present invention 4
The result figure of (Monocyte Chemoattractant Protein-1, MCP-1) secretion；
Fig. 5 H is that phosphorylation antigen cooperates with the result figure for promoting MIP-1a secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 5 I is that phosphorylation antigen cooperates with the result figure for promoting MIP-1b secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 5 J is that phosphorylation antigen cooperates with promotion PDGF-bb (platelet derived growth with hMSH2 in the embodiment of the present invention 4
The factor, Platelet-derived growth factor-bb) secretion result figure；
Fig. 5 K be the embodiment of the present invention 4 in phosphorylation antigen cooperateed with hMSH2 promotion VEGF (vascular endothelial growth factor,
Vascular endothelial growth factor) secretion result figure；
Fig. 5 L is that phosphorylation antigen cooperates with the result figure for promoting RANTES secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 5 M is that phosphorylation antigen cooperates with promotion TNF (tumor necrosis factor, Tumor with hMSH2 in the embodiment of the present invention 4
Necrosis factor)-a secretion result figure；
Fig. 5 N is that phosphorylation antigen cooperates with the result figure for promoting TNF-beta secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 5 O is that phosphorylation antigen cooperates with the result figure for promoting IL-21 secretion with hMSH2 in the embodiment of the present invention 4；
Fig. 5 P is that phosphorylation antigen cooperates with the result figure for promoting IL-22 secretion with hMSH2 in the embodiment of the present invention 4.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.
The synergistic effect of ZOL and hMSH2 is in following A)-E) at least one of in application:
A) inhibit the secretion of RANTES in 9 δ 2T cell of V γ；
B) promote the secretion of IFN-γ in 9 δ 2T cell of V γ；
C) promote fibroblast growth factor, granulocyte colony stimulating factor or granulocyte macrophage in 9 δ 2T cell of V γ
The secretion of at least one of cell stimulation factor；
D) promote the secretion of at least one of IP-10, MIP-1 α or MIP-1 β in 9 δ 2T cell of V γ；
E) promote 9 δ 2T cell of V γ in IL-1 β, IL-1 γ a, IL-2, IL-4, IL-6, IL-7, IL-8, IL-9, IL-12,
The secretion of at least one of IL-13 or IL-18.
ZOL and hMSH2 synergistic effect can promote 9 δ 2T cell recognition of V γ and killing tumor cell, present invention discover that ZOL
With hMSH2 synergistic effect by inhibiting the secretion of RANTES in 9 δ 2T cell of V γ, while promoting IFN-γ, FGF, CSF, IP-
10, the secretion of MIP-1 α, MIP-1 β and IL family Some cytokines.This is raising 9 δ 2T cell recognition of V γ and killing tumour
Cell provides theoretical explanation, and ZOL and hMSH2 synergistic effect can improve 9 δ 2T cell of V γ with 9 δ 2T cell of fast activating V γ
The ability of identification and killing tumor cell, and the expression by regulating and controlling the related keyword factor reaches body and coordinates Immune discrimination
The purpose of killing tumor cell.Therefore, the synergistic effect of ZOL and hMSH2 can be used as a kind of newtype drug, quickly activate V γ 9
δ 2T cell has potential therapeutic value to tumour cell.
RANTES is the chemotactic factor (CF) being found earliest in CC family.RANTES to lymphocyte, Monocytes/Macrophages and
NK cell etc. has apparent chemotactic activity, being capable of the specifically panimmunities cell such as chemotactic memory T cell and monocyte, and energy
The function of mediating effect+6 cell participates in regulation immune response.
IFN-γ is as a kind of important inflammatory factor, mainly by the T cell and the secretion generation of NK cell in immune system.
IFN-γ the autoimmunity adjusting of body, acquired immunity adjusting, inflammation, tumour and aging be immune and body hematopoiesis and
Apparent inhibiting effect is all had in the proliferation of cell, apoptosis.
CSF is the type cytokines that find in carrying out hematopoietic cell in vitro study, can stimulate multipotential stem cell and not
Proliferation of hematopoietic progenitors with the Development And Differentiation stage breaks up, and corresponding cell colony is formed in semisolid culturemedium.
GM-CSF plays a key effect in the development and mature and T cell proliferation and activation of Dendritic Cells, even
Connect congenital and Acquired immune response.
G-CSF is mainly generated by the monocyte and macrophage of the activation of endotoxin, TNF-α and IFN-γ.It can be used for
The recovery of caused leukopenia and neutrophilic granulocytopenia accelerates after prevention and treatment tumor radiotherapy, chemotherapy.
FGF is a kind of polypeptides matter that can promote fibroblastic growth.Can promote fibroblast mitosis, in
The growth of endoderm cell can also stimulate vascularization, play a role in wound healing and limb regeneration.
IP-10 receptor is CXCR3.IP-10 is mainly derived from Monocytes/Macrophages and T lymphocyte, and variant can increase
Add receptor active.
MIP includes MIP-1 α and MIP-1 β.Belong to Chemokines CC C subfamily, receptor is a variety of CC-chemokine receptors
(CCR).Granulocyte can be activated, CD8 is adjusted+T cell is sticked with vascular endothelial cell, participates in hematopoietic regulation, and induced NK cell increases
It grows and activates.
IL is a type cytokines, transmitting information, activation with adjust immunocyte, mediate T, B cell activation, proliferation with
Break up and plays an important role in inflammatory reaction.
The present invention passes through the collaboration stimulation of two kinds of target antigens of ZOL and hMSH2, finds in 9 δ 2T cell culture of V γ
The secretion of RANTES is significantly lowered in clear, IFN-γ, FGF, CSF, IP-10, MIP-1 α, MIP-1 β and IL family few members
Secretion dramatically increases, disclose two kinds of target antigens of ZOL and hMSH2 to 9 δ 2T cell of V γ collaboration stimulation effect and
While relevant molecule mechanism, important theoretical foundation is provided to establish and optimizing tumour immunity therapeutic strategy of adopting, is had
Important realistic meaning.
In the present invention, one is preferably carried out in mode, and the concentration of ZOL is 5-30 μM.Small molecule phosphate antigen can be used as
The activator of 9 δ 2T cell of V γ enhances the cytotoxic activity of 9 δ 2T cells against tumor cells of V γ.ZOL (nitrogenous diphosphonate azoles
Come phosphonic acids, Zoledronic acid) cytotoxic activity of 9 δ 2T cell of V γ can be obviously improved with the synergistic effect of hMSH2, it mentions
The content of high specific key factor, to promote the antineoplastic immune of body.It is 2 μM that the concentration of ZOL is typical but non-limiting,
5 μM, 8 μM, 10 μM, 13 μM, 15 μM, 17 μM, 20 μM, 24 μM, 25 μM or 30 μM.
In the present invention, one is preferably carried out in mode, and hMSH2 is located at lung carcinoma cell surface.
In the present invention, one is preferably carried out in mode, and lung carcinoma cell includes that GLC-82, NCI-H446 or hMSH2 are overexpressed
At least one of NCI-H520, preferably hMSH2 are overexpressed NCI-H520.GLC-82, NCI-H446 and NCI-H520 are
Human lung cancer cell line, wherein the hMSH2 expression on the surface GLC-82 and NCI-H446 is higher, passes through the mistake in NCI-H520
The hMSH2 height ectopic expression of cell surface also may be implemented in expression hMSH2 gene, and then realizes and more rapidly activate 9 δ 2T of V γ
Cell.
In the present invention, one is preferably carried out in mode, and hMSH2 is overexpressed NCI-H520 by that will contain coding hMSH2 base
The Transfected Recombinant Plasmid NCI-H520 of cause is obtained.Cell surface can be improved by being overexpressed in NCI-H520 cell in hMSH2
The film ectopic expression of hMSH2 is horizontal, so that the lung carcinoma cell for promoting film hMSH2 to be overexpressed is by the antigen recognizing of 9 δ 2T cell of V γ
The ability of Receptor recognition and combination eventually leads to the immune clearance of tumour cell.
In the present invention, one is preferably carried out in mode, and carrier is cFugw plasmid.
A kind of 9 δ 2T cell of V γ, 9 δ 2T cell of V γ are activated by ZOL and hMSH2.The V γ activated by ZOL and hMSH2
There is 9 δ 2T cells stronger tumor cytotoxic activity, especially hMSH2 to derive from the NCI-H520 cell that hMSH2 is overexpressed
When, the identification lethal effect of 9 δ 2T Cells on Lung Cancer cell of V γ significantly increases.
A kind of drug for the treatment of cancer, the effective component of drug include ZOL and hMSH2.It is used since the present invention tests discovery
When ZOL and hMSH2 is overexpressed 9 δ 2T cell of NCI-H520 Co stituation V γ, the cell toxicant of 9 δ 2T cell of V γ can be significantly increased
Activity.HMSH2 is present in most of tumor cell surfaces, can quickly activate 9 δ of V γ by the synergistic effect of ZOL and hMSH2
2T cell recognition kills tumour.The use in conjunction of ZOL and hMSH2 can be used for the treatment of tumour.
In the present invention, one is preferably carried out in mode, and the effective component of drug includes ZOL and cell surface expression hMSH2
Cell.The drug can quickly activate 9 δ 2T cell of V γ, improve body to the Scavenging activity of tumour cell.
In one embodiment of the present invention, cancer includes LC, cutaneum carcinoma, kidney, breast cancer or prostate cancer.
Present invention will be further explained by specific examples below, it should be understood, however, that, these embodiments are only
It is used, is but should not be understood as present invention is limited in any form for being described in more detail.
The culture of 1 tumour cell of embodiment and the amplification of effector cell
NSCLC cell line NCI-H520 and NCI-H446 are purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's cell culture
Center, with the RPMI1640 culture medium secondary culture containing 10%FCS.The sun of flow cytometry tumor cell surface hMSH2
Property rate.
It separates and is amplified from the 9 δ 2T cell of effect V γ of young healthy adult human volunteers' peripheral blood PBMCs with containing IL-2's
1640 complete medium secondary culture of RPMI.
The foundation of 2 hMSH2 of embodiment overexpression NCI-H520 lung carcinoma cell model
The coded sequence of hMSH2 gene is cloned and is connected into Fugw-EGFP expression vector.Construct successful Fugw-
EGFP-hMSH2 recombinant plasmid with Lipo2000 be transfected into NCI-H520 cell establish Fugw-EGFP-hMSH2 be overexpressed lung cancer it is thin
Born of the same parents' model.With overexpression of the WB and FCM detection hMSH2 in Fugw-EGFP-hMSH2-NCI-H520 lung cancer mode cell.
FCM analysis shows, Fugw-EGFP-hMSH2 be overexpressed NCI-H520 mode cell surface hMSH2 ectopic expression
It is significantly increased compared with NCI-H520-Fugw (empty carrier transfection group), NCI-H520 (wild group) and homotype IgG control group.It transfected
We see that transfection reagent Lipo2000 induction, ectopic expression film hMSH2 is significantly increased simultaneously in journey.
Under conditions of imitating target than 10: 1, it is amplified from the 9 δ 2T cell of effect V γ of two healthy volunteers respectively to film
The killing-efficiency that hMSH2 is overexpressed NCI-H520 mode cell is up to 60-70%.
Collaboration stimulation of 3 ZOL of embodiment to hMSH2 activation 9 δ 2T cell of V γ
Fugw-EGFP-hMSH2 is handled respectively with 30 μM of initial concentration of ZOL is overexpressed NCI-H520 lung cancer mode cell
With wild type NCI-H520 cell (final concentration is respectively 0 μM, 5 μM, 20 μM), kit is killed with CytoTox96 on-radiation
Lethal effect of the analyzing activated 9 δ 2T cell of people V γ to group of cells.The 9 δ 2T cell of V γ that observation ZOL activates hMSH2 is to lung
The lethal effect of cancer mode cell.
9 δ 2T cell of V γ can be Antigen-activated by some natural or synthetic phosphorylations.In killing experiments it was found that
Lipo2000 transfection can cause the up-regulation of NCI-H520 lung cancer mode cell surface hMSH2 ectopic expression and 9 δ 2T cell of V γ swollen to target
The lethal effect of oncocyte increases, and illustrates a kind of hMSH2 film dystopy mechanism driven by the non-peptides antigen of phosphoric acid salt.In order into
One step proves the lethal effect of 9 δ 2T cell of V γ that non-peptides phosphorylation antigen is mediated in hMSH2 to Lung Squamous Carcinoma Cells, Wo Menyong
The Diphosphonate ZOL pretreatment Fugw-EGFP-hMSH2- of various dose is overexpressed NCI-H520 lung cancer mode cell, observes ZOL
The synergistic effect of the lung cancer cell-mediated to 9 δ 2T of V γ identification killing with hMSH2.As a result as shown in Figure 1, ZOL pretreatment can be shown
It writes enhancing Fugw-EGFP-hMSH2 and is overexpressed NCI-H520 lung squamous cancer mode cell to the killing sensibility of 9 δ 2T cell of V γ, and
This killing sensibility is dose-dependent.ZOL (20 μM) can equally activate V to activate cell in born of the same parents and kill to NCI-H446
There is (such as Fig. 2) in effect.Immunohistochemistry detection is carried out to cancerous lung tissue, analyzes the expression of hMSH2 in cancerous lung tissue, as a result
As shown in Fig. 3 A- Fig. 3 F, the Showed by immune group result of Lung Cancer Tissue Microarray, 3 squamous cell lung carcinoma after births and endochylema are compared with cancer
There is the ectopic expression of different degrees of hMSH2 albumen in other control tissue, the expression of hMSH2 is then significantly dropped in neoplastic cell nuclei
Low, preceding 2 cancerous lung tissues become apparent.LC-1 (Fig. 3 A), LC-2 (Fig. 3 B) and LC-3 (Fig. 3 C) are respectively 3 cancerous lung tissues；
HE (Fig. 3 D), Normal (Fig. 3 E) and NC (Fig. 3 F) respectively refer to the HE dyeing of cancerous lung tissue, compare by relatively normal lung cancer carcinoma,
The negative control of immunohistochemical staining.
4 ZOL of embodiment cooperates with the key factor of activation 9 δ 2T cell killing lung cancer of V γ with hMSH2
It will be amplified from the 9 δ 2T cell of V γ (9 δ 2T1, V γ of V γ, 9 δ 2T2) of different adult healthy volunteers peripheral bloods respectively
It is incubated altogether with wild type NCI-H520 (9 δ 2T+520 of V γ), Fugw-EGFP-hMSH2-NCI-H520 (9 δ 2T+520+M of V γ) respectively
It educates, V is jointly processed by with ZOL processing 9 δ 2T cell of V γ (9 δ 2T+Z of V γ) and Fugw-EGFP-hMSH2-NCI-H520 and ZOL
9 δ 2T cell of γ (9 δ 2T+520+M+Z of V γ), collects the total incubated cell culture supernatant of each experimental group, is compared point with Bioplex
The variation of the cytokine secretions such as each group effect V γ 9 δ 2T cell ILs, CSFs, MIPs spectrum is analysed, and observes ZOL and hMSH2 in lung
Cancer cell activates the synergistic effect in 9 δ 2T cell cytokine secretion of V γ.
Compare the 9 δ 2T of V γ of 9 δ 2T cell of V γ, wild type NCI-H520 stimulation through Bioplex cell multiplex factorial analysis
Cell, Fugw-EGFP-hMSH2 are overexpressed the 9 δ 2T cell of V γ of NCI-H520 stimulation, wild type NCI-H520 cell adds ZOL to pierce
Sharp 9 δ 2T cell of V γ, Fugw-EGFP-hMSH2 are overexpressed in the 9 δ 2T cells and supernatant of V γ that NCI-H520 adds ZOL to stimulate
The express spectra of 31 kinds of cell factors.Phosphorylation antigen cooperates with the secretion result such as Fig. 4 A- figure for promoting 31 kinds of cell factors with hMSH2
Shown in 4O and Fig. 5 A- Fig. 5 P: in 31 kinds of cell factors, most of members of IL family, including IL-1 β, IL-1 γ a, IL-2,
IL-4, IL-6, IL-7, IL-8, IL-9, IL-12, IL-13 and IL-18 are incubated altogether in the 9 δ 2T cell of V γ of NCI-H520 activation
It educates in culture supernatant and significantly increases, and IL-5, IL-10, IL-15, IL-17, IL-21 and IL-22 secretion are then without significant changes.With
9 δ 2T cell of control group V γ is compared, and NCI-H520 is incubated for FGF, G-CSF, GM-CSF etc. in 9 δ 2T cells and supernatant of V γ altogether
Secretion dramatically increase；Meanwhile the concentration of IP-10, MIP-1a, MIP-1 β etc. also significantly increase, the secretion of RANTES was then once shown
Downward trend is write, declines after being incubated for altogether with the NCI-H520 Lung Squamous Carcinoma Cells handled through ZOL and becomes apparent, prompts ZOL and hMSH2
It is negative sense to the synergistic effect that it is secreted.Especially it is noted that in NCI-H520 activation 9 δ 2T cells and supernatant of V γ
IFN-γ secretion also obviously increases, and ZOL and hMSH2 have significant synergistic effect (figure to promotion 9 δ 2T cell secretion of gamma-IFN of V γ
5A)。
In Fig. 4 A- Fig. 4 O and Fig. 5 A- Fig. 5 P: 9 δ 2T1 of 1-V γ；9 δ 2T1+ wild type NCI-H520 of 2-V γ；3-Vγ9δ
2T1+ZOL；9 δ 2T1+Fugw-EGFP-hMSH2 of 4-V γ is overexpressed NCI-H520+ZOL；5-Vγ9δ2T2；9 δ 2T2+ of 6-V γ is wild
Raw type NCI-H520；7-Vγ9δ2T2+ZOL；9 δ 2T2+Fugw-EGFP-hMSH2 of 8-V γ is overexpressed NCI-H520+ZOL.
The present invention mediates the cell of 9 δ 2T Cells on Lung Cancer of V γ identification killing, molecule to imitate to illustrating hMSH2 with ZOL and cooperate with
It should be with dependent interaction mechanism, and the immune therapeutic strategy tool of adopting of the lung cancer of foundation and optimization based on 9 δ 2T cell of hMSH2 and V γ
There are important theory and realistic meaning.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
It can make and other reasonably be altered or modified in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
- The synergistic effect of 1.ZOL and hMSH2 is in following A)-E) at least one of in application:A) inhibit the secretion of RANTES in 9 δ 2T cell of V γ；B) promote the secretion of IFN-γ in 9 δ 2T cell of V γ；C) promote fibroblast growth factor, granulocyte colony stimulating factor or granular leukocyte macrophage in 9 δ 2T cell of V γ The secretion of at least one of stimulating factor；D) promote the secretion of at least one of IP-10, MIP-1 α or MIP-1 β in 9 δ 2T cell of V γ；E) promote IL-1 β, IL-1 γ a, IL-2, IL-4, IL-6, IL-7, IL-8, IL-9, IL-12, IL- in 9 δ 2T cell of V γ The secretion of at least one of 13 or IL-18.
- 2. application according to claim 1, which is characterized in that the concentration of the ZOL is 5-30 μM.
- 3. application according to claim 1, which is characterized in that the hMSH2 is located at lung carcinoma cell surface.
- 4. application according to claim 3, which is characterized in that the lung carcinoma cell include GLC-82, NCI-H446 or HMSH2 is overexpressed at least one of NCI-H520, and preferably hMSH2 is overexpressed NCI-H520.
- 5. application according to claim 4, which is characterized in that the hMSH2 is overexpressed NCI-H520 and is compiled by that will contain The Transfected Recombinant Plasmid NCI-H520 of code hMSH2 gene is obtained.
- 6. application according to claim 5, which is characterized in that the expression vector in the recombinant plasmid is cFugw plasmid.
- 7. a kind of 9 δ 2T cell of V γ, which is characterized in that the 9 δ 2T cell of V γ is activated by ZOL and hMSH2.
- 8. a kind of drug for the treatment of cancer, which is characterized in that the effective component of the drug includes ZOL and hMSH2.
- 9. drug according to claim 8, which is characterized in that the effective component of the drug includes ZOL and cell surface Express the cell of hMSH2.
- 10. drug according to claim 9, which is characterized in that the cancer includes lung cancer, cutaneum carcinoma, kidney, breast cancer Or prostate cancer.
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