CN102600454A - Application of CD137L in preparation of medicine for treating non-small cell lung cancer - Google Patents
Application of CD137L in preparation of medicine for treating non-small cell lung cancer Download PDFInfo
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- CN102600454A CN102600454A CN2012101020696A CN201210102069A CN102600454A CN 102600454 A CN102600454 A CN 102600454A CN 2012101020696 A CN2012101020696 A CN 2012101020696A CN 201210102069 A CN201210102069 A CN 201210102069A CN 102600454 A CN102600454 A CN 102600454A
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Abstract
The invention discloses an application of CD137L in preparation of a medicine for treating non-small cell lung cancer. According to the invention, CD137L capable of treating lung cancer, especially non-small cell lung cancer is proposed for the first time, provides possibility and application for development of related medicines as a medicine-screening target, and has great application prospect and market value.
Description
Technical field:
The present invention relates to
The application of CD137L in preparation treatment non-small cell lung cancer drug
Background technology:
Costimulatory molecules (costimulatory molecule) has played extremely important regulating action in immunne response.Costimulatory signal is that Brestcher in 1970 and Cohn at first propose on the basis of t cell activation dual signal theory and confirm; After sensitized T cell does not obtain antigen signals (first signal) through " antigenic peptides-MHC-TCR " triplet; Needing costimulatory molecules provides the activation signal (secondary signal) can activation, and secondary signal is called costimulatory signal.Can costimulatory molecules be divided into two types according to structure: (1) tumor necrosis factor (TNF)-Tumor Necrosis Factor Receptors (TNFR) superfamily, comprise CD27 part (Ligand) (CD27L)-CD27, CD30L-CD30, CD40L-CD40, OX40L-OX40, CD137L-CD137, FasL-Fas etc.; (2) immunoglobulin superfamily is like B7-1/B7-2-CD28, PDL1/PDL2-PD-1 etc.
CD137L (CD137 part) is important costimulatory molecules, is II type transmembrane glycoprotein.The gene of coding human CD137L is positioned at 19p13.3, and its coded product is 254 aminoacid, and wherein 28 aminoacid of cytoplasmic domain are striden 21 aminoacid in film district, 205 aminoacid of extracellular region.Its aminoacid sequence is meyasdasld peapwppapr aracrvlpwa lvagllllll laaacavfla cpwavsgara spgsaasprl regpelspdd paglldlrqg mfaqlvaqnv llidgplswy sdpglagvsl tgglsykedt kelvvakagv yyvffqlelr rvvagegsgs vslalhlqpl rsaagaaala
Ltvdlppass earnsafgfq grllhlsagq rlgvhlhtea rarhawqltq gatvlglfrv tpeipaglps prse, CD137L be expressed in various antigen presenting cells (APC) as: activatory B cell, mononuclear cell, BMDC (DC) etc. the surface.The receptor CD137 (4-1BB) of CD137L mainly is expressed in activated T cell, cell surfaces such as mononuclear cell.CD137L can pass through signal in the approach transfer sell such as TRAF2, TRAF1 and MAPK after acting on CD137, promotes the activation of T cell, propagation, the increase of cytokine secretion, and the inhibition of the inductive apoptosis of activation (AICD).CD137L combines the back to produce with CD137 costimulatory signal can be worked in coordination with the CD28 signal, also can not rely on CD28-B7 and brings into play the common stimulation to the T cell.CD137 also is expressed in the surface of NK cell and has participated in the function adjusting of NK cell, excites the CD137 molecule on the NK cell, can promote NK cell proliferation and IFN
-The secretion of γ, and this activated NK cell passes through and the killing activity of the interaction enhanced CT L of CTL.Salih has reported that at some T and B be on the cell membrane of leukemia tumor cell line, and the composition coexpression of CD137L and CD137 molecule is arranged, and expresses though the both is low, and CD137L and CD137 signal all can promote the propagation of leukemia cell line.
But do not see the report of CD137L in the effect of treatment nonsmall-cell lung cancer.
Summary of the invention:
Technical scheme of the present invention is:
The application of
CD137L in preparation treatment non-small cell lung cancer drug.
Beneficial effect
The present invention proposes CD137L first can treat pulmonary carcinoma, particularly lung cancer in non-cellule type, for the exploitation related drugs, as the drug screening target spot, provides and maybe and use, and has huge applications prospect and market value.
The present invention further invents the expression of CD137L and the relation between lung cancer in non-cellule type (NSCLC) clinical parameter at clinicing aspect, and judging and treat for the prognosis of lung cancer in non-cellule type (NSCLC) provides new theoretical foundation.
The specific embodiment
Below in conjunction with embodiment the present invention is described in further detail.
Embodiment 1
1, the clone of CD137L gene and evaluation:
The take the logarithm total RNA of human lung adenocarcinoma cell A549 cell extraction of trophophase, the mRNA reverse transcription is cDNA.The segmental primer upstream and downstream of clone hCD137L (Genbank accession number GI:209954675) adds the enzyme action site respectively: Not I and Xho I, fragment length are 794 bp.Primer sequence is following: Forward 5 one ATT CGC GGCCGC A TGG AAT ACG CCT CTG ACG CTT C 1, and Reverse 5 one GGC CCT CGA GCG CCG CC TTA TTC CGA CCT CGG TGA AGG GA 1 carry out the PCR reaction; The PCR operating process: 94 ℃ of preparatory degeneration 3 min, 94 ℃ of degeneration 30 S, 68 ℃ of annealing/extension 2 min, 30 circulations, last 70 ℃ are extended 3 min.Cut glue and reclaim the product fragment of corresponding size, and carry out purification with test kit.
2, connect, transform and identify:
Digest clone's product and plasmid pcDNA3 (available from Takara company) behind the purification respectively with restriction endonuclease (restricted enzyme HindIII and Xba I); Reclaim the purpose fragment; Connect back transformed competence colibacillus E.coli DI-I5a (available from Takara company), extracting plasmid with the T4 ligase.Recombiant plasmid is identified called after pcDNA3 one hCD137L through double digestion and gene sequencing.Get pBTdel 1 (available from Omega Bio-Tek company) and pcDNA3 one hCD137L and use Hind m and Xba I double digestion respectively; Obtaining two ends is the CD137L segment in Hind III and Xba I site; And be connected on pBTdel one 279 plasmids of same restriction enzyme site; Recombiant plasmid called after pBTde1.279 one hCD137L that obtains, double digestion is identified.
3, gene transfection lung cancer cell line A549 and normal HELF's strain HELF cell strain:
The take the logarithm A549 cell of trophophase is got proper density and is carried out bed board, gets recombiant plasmid pcDNA3 one hCD137L and pBTdel one 279 one hCD137L respectively, carries out transfection by transfection reagent box description, continues to cultivate.
3, the detection of cell CD137L after the transfection: with the method for RT-PCR and flow cytometry, the expression of CD137L after the detection transfection.
Embodiment 2
Application SABC streptomycete avidin-peroxidase method (Streptavidin-perosidase, SP) detect the expression of CD137L in nonsmall-cell lung cancer (NSCLC):
61 routine nonsmall-cell lung cancer tissue specimens (No.1 Attached Hospital, Nanjing Medical Univ 2006-01---2008-12 underwent operative lung cancer specimen 61 examples; Identify by No.1 Attached Hospital, Nanjing Medical Univ Pathology Deparment) all through 10% formaldehyde fixed; FFPE, the thick serial section of 4 μ m.The anti-people CD137L of rabbit monoclonal antibody is available from Epitomics company, and monoclonal antibody working solution concentration is 1ug/ml, and SP immunohistochemistry test kit steps the neoplasm technology development co. available from Foochow, the DBA colour developing, and haematoxylin is redyed, and strict in steps by specification carries out.Do positive control with the male NSCLC tissue specimen of known CD137L, substitute an anti-negative control of doing with PBS.
Distribution and the expression of 1 CD137L in the non-small cell lung cancerous tissue
2 CD137L express the relation with clinical and pathological data
By sex, age, pathology, wait and analyze by stages, the result shows: the positive expression rate that is expressed in the early stage of lung cancer of CD137L is higher than progressive stage pulmonary carcinoma (P=0.027) with 61 patients with lung cancer, and does not have obviously relevant (P>0.05) with other factors
The relation of the expression of CD137L and patient's clinicopathologic features
The relation of the expression of 3 CD137L and nonsmall-cell lung cancer patient life span
61NSCLC patient is followed up a case by regular visits to, follow up a case by regular visits to 1 ~ 56 month time, meta is followed up a case by regular visits to 35 months time, loses and visits 3 people, and negative group median survival interval is about 15 months; About 21 months of positive group median survival interval, life span is pointed out with the analysis of Log-rank method: CD137L expression positive patient life cycle maybe longer (P=0.023).
The present invention shows; Find that on NSCLC patient's BIAO and BEN visible CD137L builds up in a large number in cytoplasm, CD137/CD137L at first is found in immunocyte, begins to think only to express and the surface of immunocyte; And do not match with the in-vivo tissue experimental result; Explain that CD137 and CD137L maybe be different in the expression position of immunocyte and non-immunocyte, its mechanism it be unclear that, and thinking at present maybe be different with the function of its execution relevant.In 61 routine NSCLC patients; The patient CD137L expression of morning is higher by stages; Possibly point out patient's in early days immunologic function strong slightly; The CD137L of high expressed is activated T cell effectively, and along with the progress of tumor, immunologic function adds that some immunosuppressive factors of autocrine have influenced t cell activation even its propagation a little less than more and more.The single factor analysis prompting of CD137L and existence; The patient of CD137L positive expression is longer than the negative patient who expresses of CD137L total life cycle; Prompting high expressed CD137L possibly be able to prolong patient's existence; Set up the Cox proportional hazards regression models simultaneously and carry out the multiplicity discovery, the expression of pathological staging and CD137L is the prognosis factor that influences NSCLC, shows that CD137L has certain influence to patient's prognosis; Can predict patient's prognosis to a certain extent, for the NSCLC immunization therapy theoretical foundation is provided simultaneously.
Claims (1)
1.CD137L the application in preparation treatment non-small cell lung cancer drug.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2012101020696A CN102600454A (en) | 2011-11-19 | 2012-04-10 | Application of CD137L in preparation of medicine for treating non-small cell lung cancer |
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| CN201110316829 | 2011-11-19 | ||
| CN2012101020696A CN102600454A (en) | 2011-11-19 | 2012-04-10 | Application of CD137L in preparation of medicine for treating non-small cell lung cancer |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101386840A (en) * | 2008-10-31 | 2009-03-18 | 江苏省人民医院 | Construction method of CD3<->CD56<+>NK cell high-efficient multiplication culture system |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101386840A (en) * | 2008-10-31 | 2009-03-18 | 江苏省人民医院 | Construction method of CD3<->CD56<+>NK cell high-efficient multiplication culture system |
Non-Patent Citations (2)
| Title |
|---|
| QUN WANG等: "Analysis of CD137 and CD137L Expression in Human Primary Tumor Tissues", 《CROAT MED J.》 * |
| 张利宁等: "人CD137L在肿瘤细胞上的表达及其功能研究", 《上海免疫学杂志》 * |
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Application publication date: 20120725 |