CN108753817A - The enhanced cell for enhancing the method for the anti-cancer ability of cell and being obtained using this method - Google Patents
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Abstract
The present invention provides a kind of method of the anti-cancer ability of enhancing CIK cell, and the method includes the PD-1 genes of CIK cell are knocked out based on CRISPR/Cas9 technologies.The present invention also provides using enhanced CIK cell made from this method.It is demonstrated experimentally that CIK cell anti-liver cancer and anti-cell ability can effectively be enhanced by knocking out the PD-1 genes of CIK cell, a kind of new effector cell is provided for tumour cell immunization therapy.
Description
Technical field
The present invention relates to biotechnologies, more particularly it relates to which a kind of struck based on CRISPR/Cas9 technologies
Except the PD-1 genes of CIK cell to enhance the method for the anti-tumor capacity of CIK cell.
Background technology
Hepatocellular carcinoma (HCC) is one of the most common reason of cancer related mortality in the world, and shelter has mortality of malignant tumors
The second of rate, prognosis are poor.Liver cancer treatment traditional at present relates generally to partially hepatectomized, liver transfer operation, RF ablation
(RFA) and Arterial chemotherapy and embolization.The state of an illness has been enter into the mid and late liver cancer stage when being clarified a diagnosis due to most patients, passes at present
The therapy of system is difficult for patients with terminal and provides clinical effective benefit.This 5 years survival rate for also resulting in liver cancer is protected always
It holds in 10-20% or so.
In order to improve the survival rate of liver cancer, scholars have investigated various new therapeutic scheme, wherein CIK cell of adopting is controlled
Treatment is one of wherein promising treatment.CIK cell is produced through amplification in vitro culture by human peripheral blood mononuclear cell in blood
Raw heterogeneous cell mass, cell composition have CD4+T cell, CD8+T cell, CD3+CD56+Cell, NK cells etc., wherein main
That play antitumor action is CD3+CD56+Cell, can, NK like cell restricted by non-MHC to kill mechanism thin to tumour
Born of the same parents play lethal effect.Research shows that CIK cell has lethal effect to kinds of tumor cells, and normal tissue cell is without killing
Hinder function, this makes CIK cell clinically have broad application prospects.Have multiple CIK cell clinical tests at present opening
Zhan Zhong.For example, Lee team report CIK cell can significantly improve the median survival interval of patient as the auxiliary treatment of liver cancer patient,
Reduce tumor recurrence rate.
But since CIK cell killing ability is still weak, clinically often needs repeatedly to input a large amount of CIK cells or combine it
He treats, this causes patient to spend increase.In addition some patients stop treatment because being difficult to be resistant to repeatedly infusion, and treatment is caused to be imitated
Fruit is limited.Therefore in recent years, many researchs concentrate on how improving in the ability of CIK cell.For example, Schlimper is thin by CIK
Dysuria with lower abdominal colic contaminates the specific chimeric receptor for CEA antigens to enhance anti-CEA+Colon cancer cell function.Paola Iudicone reports
Road interleukin-15 can enhance cytotoxicity of the CIK cell to epithelial cancer cell line by congenital approach.And it is compiled by gene
The means of collecting give CIK cell and carry out related gene knockout to enhance the anti-tumor capacity of CIK cell also gradually by scholar's weight
Depending on.
PD-1 also known as the programmed cell death factor 1 are one of T cell surface Inhibitory receptors, and the T for being mainly expressed in activation is thin
Born of the same parents, while it is also one of the molecular marker that T cell is exhausted, in cancer and chronic infection, up-regulated expression.More and more
Evidence shows PD-1 in vivo and in vitro to CD8+The effector function of T cell shows to negatively affect.Previous research has been reported
Road increases the effector T cell function in tumour with antibody blocking PD-1 and improves antitumous effect.
The short palindrome repetitive sequence (CRISPR) of regular intervals of time focused recently and CRISPR GAP-associated protein GAPs (Cas) (CRISPR-
Cas9) system becomes the powerful for gene editing.It is previous studies have shown that using Cas9 RNP (Cas9：sgRNA
Ribonucleoprotein) targeting individual gene significantly improves the genome targeting efficiency of Cas9 mediations and reduces site
Dependence undershooting-effect.However for heterogeneous CIK cell, the CIK having not yet to see after literature research knockout PD-1 is thin
The function situation of born of the same parents.
Invention content
The problem to be solved in the present invention is, passes through the Cas9 RNP knockout Cytotoxic T lymphocytes mediated or CIK cell
Can PD-1 genes enhance the antitumor, particularly anti-cancer ability of cell.The present inventor confirms, passes through electroporation Cas9
The PD-1 gene knockouts that RNP is carried out are technically feasible and efficient；In addition, in Cytotoxic T lymphocytes or CIK cell
PD-1 knockouts enhance cellullar immunologic response and cytotoxicity of the cell to liver cancer cell lines.
Therefore, it is an object of the present invention to provide the PD-1 genes of cell are knocked out based on CRISPR/Cas9 technologies to increase
The method of the anti-cancer ability of strong cell, the sgRNA of the targeting PD-1 genes used in this method, and obtain enhanced thin
Born of the same parents.
Other objects of the present invention are to provide the use of sgRNA or enhanced cells in the preparation of medicament for cancer treatment
On the way, and include the present invention sgRNA kit.
Technical scheme is as follows.
On the one hand, the present invention provides a kind of method of the anti-cancer ability of enhancing cell, and the method includes being based on
CRISPR/Cas9 technologies knock out the PD-1 genes of the cell.
Specifically, the method for the present invention includes the following steps：By Cas9 albumen and target PD-1 gene extrons -1
SgRNA electricity is gone in cell, wherein the cell is the cytotoxic T lymph for tumour specific antigen or tumor associated antigen
Cell or CIK cell.
The nucleotide sequence of the wherein described sgRNA is as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3：
SEQ ID NO.1(PD-1 sgRNA1)：GTCTGGGCGGTGCTACAACT
SEQ ID NO.2(PD-1 sgRNA2)：TGTAGCACCGCCCAGACGAC
SEQ ID NO.3(PD-1 sgRNA3)：ACCGCCCAGACGACTGGCCA
Preferably, the nucleotide sequence of the sgRNA is as shown in SEQ ID NO.1.
Preferably, the Cas9 albumen and sgRNA carry out electricity turn with 1: 0.5-2 mass ratio；
Preferably, the Cas9 albumen, sgRNA and cell are with 9.44 (μ g)：9-12(μg)：1-5×106A cell carries out
Electricity turns.
Specific implementation mode according to the present invention, Cas9 albumen use the EnGen Cas9NLS (20uM) of NEB companies, use
Amount can be 3-5 μ l (9.44 μ g-16.1 μ g)；SgRNA is the sgRNA that in-vitro transcription generates, and dosage can be 3-5 μ l (9 μ g-
15μg)。
Specific implementation mode according to the present invention, the Cas9 albumen, sgRNA and cell are with 9.44 (μ g)：9-12(μg)：
5×106A cell carries out electricity and turns.
Specific implementation mode according to the present invention uses Lonza 4D-Nucleofector X Unit electroporations with EO-
115 electric carryover sequences carry out electricity and turn.
Preferably, the cell is CIK cell, and wherein CD3+CD56+Cell proportion >=20%, CD3+CD8+Cell
Ratio >=50%.
Specific implementation mode according to the present invention, the described method comprises the following steps：
1) CIK cell is obtained, wherein the CD3 of the CIK cell+CD56+Cell proportion >=20%, CD3+CD8+Cell ratio
Example >=50%；
Specific implementation mode according to the present invention can prepare CIK cell by the mononuclearcell in peripheral blood；
2) the sgRNA electricity of Cas9 albumen and targeting PD-1 gene extrons -1 is gone in CIK cell；Wherein, described
Cas9 albumen, sgRNA and CIK cell are with 9.44 (μ g)：9-12(μg)：5×106A cell and use Lonza 4D-
Nucleofector X Unit electroporations carry out electricity with EO-115 electricity carryover sequences and turn；
Specific implementation mode according to the present invention, before electricity turns, electricity consumption turns liquid and is resuspended CIK cell, at the same by Cas9 albumen and
SgRNA is added other electricity and turns to be incubated at room temperature in liquid 10 minutes, and cell suspension then is added in Cas9 albumen and sgRNA mixed liquors
In, it carries out electricity and turns.
Preferably, the method is further comprising the steps of：
3) by the CIK cell turned through electricity in 37 DEG C, 5%CO2It is lower to stand 24 hours.
Specific implementation mode according to the present invention, after standing, the T that the CIK cell turned through electricity can be suspended in preheating is thin
In born of the same parents' culture medium, in 37 DEG C, 5%CO2Every 2 days of lower culture, wherein cell culture medium with it is fresh containing 5% people's AB types blood plasma and
100U/mL recombinant human il-2s complete medium half, which is measured, changes liquid.
Anti-cancer ability of the present invention be preferably anti-liver cancer and anti-, colorectal cancer, breast cancer, lymthoma, leukaemia, lung cancer,
Gastric cancer, cancer of pancreas, posterior peritoneum tumour, the cancer of the esophagus, cholangiocarcinoma, gallbladder cancer, duodenal cancer, thyroid cancer, nasopharyngeal carcinoma, laryngocarcinoma,
Malignant mela noma, carcinoma of endometrium, cervical carcinoma, oophoroma, kidney, carcinoma of urinary bladder, prostate cancer, osteosarcoma, metastatic carcinoma of bone or
The ability of the cancers such as glioblastoma, more preferably anti-liver cancer and anti-ability.
On the other hand, the present invention is provided using enhanced cell made from the above method.Wherein it is preferred in the present invention
Enhanced cell in, knockout rate >=40% of PD-1 genes.Preferably, the present invention is provided using above method enhancing obtained
Type CIK cell.
Another aspect, it is described the present invention also provides the sgRNA for knocking out PD-1 genes based on CRISPR/Cas9 technologies
The nucleotide sequence of sgRNA is as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.Preferably, the sgRNA
Nucleotide sequence as shown in SEQ ID NO.1.
Further aspect, the present invention provide a kind of pharmaceutical composition, and described pharmaceutical composition includes above-mentioned sgRNA or enhanced
Cell.Preferably, described pharmaceutical composition includes above-mentioned sgRNA or enhanced CIK cells.
Correspondingly, in the preparation of medicament for cancer treatment the present invention also provides above-mentioned sgRNA or enhanced cells
Purposes；
Preferably, the cancer be liver cancer, colorectal cancer, breast cancer, lymthoma, leukaemia, lung cancer, gastric cancer, cancer of pancreas,
Posterior peritoneum tumour, the cancer of the esophagus, cholangiocarcinoma, gallbladder cancer, duodenal cancer, thyroid cancer, nasopharyngeal carcinoma, laryngocarcinoma, malignant mela noma,
Carcinoma of endometrium, cervical carcinoma, oophoroma, kidney, carcinoma of urinary bladder, prostate cancer, osteosarcoma, metastatic carcinoma of bone or glioblastoma etc.,
Preferably liver cancer.
On the other hand, the present invention provides a kind of for knocking out cell, preferably CIK cell based on CRISPR/Cas9 technologies
The kit of PD-1 genes, the kit include：Target the sgRNA of PD-1 gene extrons -1；
Preferably, the nucleotide sequence of the sgRNA such as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3 institutes
Show；It is highly preferred that the nucleotide sequence of the sgRNA is as shown in SEQ ID NO.1.
Preferably, the kit further includes Cas9 albumen；
Preferably, the kit further includes turning the reagent of sgRNA and/or Cas9 albumen for electricity.
The present invention provides what is carried out based on electroporation Cas9 RNP to be directed to tumour specific antigen or tumor associated antigen
Cytotoxic T lymphocytes or CIK cell PD-1 gene knockout methods, wherein use filter out for outside PD-1 the 1st
3 sgRNA of aobvious subsequence and specific electric carryover sequence.It is demonstrated experimentally that the method for the present invention can make the PD-1 of CIK cell
Gene knockout rate reaches 41.23 ± 0.52%, before knocking out and after knocking out the PD-1 expression rates of CIK cell be respectively 4.54 ±
0.28% and 1.81 ± 0.31%；ELISPOT and cellulotoxic experiment are shown simultaneously, the CIK provided by the invention knocked out after PD-1
The IFN-γ secretion function of cell and the lethal effect of liver cancer cell lines is enhanced.Therefore, the PD-1 of CIK cell is knocked out
Gene can effectively enhance CIK cell anti-liver cancer and anti-cell ability, and obtained enhanced CIK cell is tumour cell immunization therapy
A kind of new effector cell.
Description of the drawings
Hereinafter, carry out the embodiment that the present invention will be described in detail in conjunction with attached drawing, wherein：
Fig. 1 shows the flow cytometry of the immunophenotype of in vitro culture CIK cell.Wherein, Figure 1A to Fig. 1 D is aobvious
Show the analysis result of CIK cell marker CD3, CD4, CD8, CD56；Fig. 1 E are shown further gates CD45RA by CD3+ cells
And CD27, to show the memory cell of CIK cell and the ratio of effector cell；Fig. 1 F show the expression feelings of PD-1 in CIK cell
Condition.
Fig. 2 shows that the PD-1 of CIK cell knocks out the testing result of situation.Wherein, Fig. 2A shows on PD-1 genes 3
SgRNA target sites, wherein bold Italic indicate that sgRNA targeting sequencings, runic indicate PAM sequences；Fig. 2 B are shown using TIDE points
The PD-1 for analysing different sgRNA knocks out efficiency (average value ± SEM, n=3), and experiment carries out in three biology repeat；Fig. 2 C are aobvious
Show that the PD-1 that different sgRNA are detected using T7E1 mispairing restriction analysis methods knocks out efficiency, M, marker；WT, wild type；Ctrl is
Control group transfects unrelated sgRNA+Cas9 albumen；Fig. 2 D show that compared with wild-type sequence (WT), PD-1 knocks out site
Indel situations, wherein sgRNA target sites show that PAM sequences are shown in bold with bold Italic, and deletion mutation is aobvious with middle horizontal line
Show, insertion mutation is shown with underscore, and the PCR product of each sample is subcloned, and is carried out to the allele of each clone
Sequencing.16/40 indicates the number of the clone containing mutation allele in total clone of sequencing.
Fig. 3 shows that the immunophenotype for the CIK cell and wild type CIK cell that PD-1 is knocked out changes.Wherein Fig. 3 A-1 and
Fig. 3 A-2 show the result by gating CD3+ cell analysis PD-1+ cells；Figure Fig. 3 B-1 and Fig. 3 B-2 are shown by gating CD3
The result of+cell analysis CD4+ and CD8+ cells；Fig. 3 C-1 and Fig. 3 C-2 show by gate CD3+ cell analysis CD45RA+/
CD27+, CD45RA-/CD27+, CD45RA-/CD27- and CD45RA+/CD27- cell results.
Fig. 4 shows that the IFN-γ of the CIK cell that PD-1 is knocked out and wild type CIK cell generates measurement result.
Fig. 5 shows the cell of the CIK cell that PD-1 is knocked out and wild type CIK cell to SMMC-7721 liver cancer cells system
Toxicity test result.
Specific implementation mode
The present invention is described below with reference to specific embodiments.It will be appreciated by those skilled in the art that these embodiments are only
For illustrating to invent, do not limit the scope of the invention in any way.
Experimental method in following embodiments is unless otherwise specified conventional method.Medicine as used in the following examples
Material raw material, reagent material etc. are commercially available products unless otherwise specified.Wherein, Bel7402 SMMC-7721 is thin
Born of the same parents are to buy from ATCC, in the DMEM high glucose mediums containing 10%FBS and 100U/ml penicillin, 100mg/ml streptomysins
Culture.All cells are incubated at 37 DEG C, 5%CO2Cell incubator.Cas9 albumen is the EnGen Cas9 purchased from NEB companies
NLS (20uM) can use 3-5 μ l (9.44 μ g-16.1 μ g).Use Lonza4D-Nucleofector X Unit electroporations.
About statistical analysis：All experiments are independent in triplicate.As a result it is indicated with average value ± standard error.It is only using double tails
Vertical T examines to determine statistical significance.The significant property of group difference is assessed using 6 softwares of GraphPad prism.P value <
0.05 is considered to have statistical significance.
Embodiment 1CIK cell culture
Human peripheral is derived from the patients with hepatocellular carcinoma of Beijing Shijitan Hospital, has obtained the written consent of liver cancer patient,
And obtain Hospital Ethical Committee's approval.
Scheme (Schmidt-wolf IG et al., Use of a SCID of the cultural method of CIK cell according to forefathers' research
mouse/human lymphoma model to evaluate cytokine-induced killer cells with
potent antitumor cell activity.J.Exp.Med.1991；174(1)：139-149).In brief, the 0th day
40ml peripheral bloods are obtained from patients with hepatocellular carcinoma, PBMC cells are detached with Ficoll-Hypaque gradient centrifugations, by PBMC
Cell is suspended in GT-T551 serum free mediums (TAKARA companies, Japan), and 5% people's AB types blood plasma, 1000U/mL is added
IFN-γ(PeproTech).1st day, 50ng/mL anti-cd 3 antibodies (eBiosciences), 100U/mL recombinant human il-2s is added
In (eBioscience) to cell culture fluid.Hereafter contained 5% people's AB types blood plasma and 100U/mL recombinant human il-2s every 2 days
(e-Bioscience) fresh GT-T551 serum free mediums (TAKARA companies, Japan) half amount changes liquid, keeps cell concentration
It is 2 × 106/ ml collects CIK cell on the 15th day, analyzes the phenotype and cytotoxicity of CIK cell, all products are without bacterium, branch
Substance or fungi, endotoxin < 5Eu.
Flow cytometry is used to monitor the immunophenotype of in vitro culture CIK cell.The results are shown in Figure 1, CD3+Cell accounts for
98.53 ± 1.08%, CD4 of CIK cell+Cell accounts for CD3+23 ± 5.541%, CD8 of CIK cell+Cell accounts for CD3+CIK is thin
58.8 ± 4.834% and CD56 of born of the same parents+Account for CD3+The ratio of CIK cell reaches 23.73 ± 2.04%.In addition, PD-1+Account for CD3+
The ratio of CIK cell reaches 4.54 ± 0.2845%, this and substantially similar (the Olioso P of pertinent literature CIK cell immunophenotype
Et al., Immunotherapy with cytokine induced killer cells in solid and
hematopoietic tumours：a pilot clinical trial.Hematol.Oncol.2009；27(3)：130-
139), show successful activation amplification cultivation CIK cell.
Embodiment 2SgRNA is designed and the external T7 of sgRNA is transcribed
The 1st exon sequences of PD-1 are obtained from NCBI, use two CRISPR design tools (http：//
Crispr.mit.edu and https：//portals.broadinstitute.org/gpp/public/) design sgRNA, it is comprehensive
3 gRNA are picked out in conjunction：
SEQ ID NO.1(PD-1 sgRNA1)：GTCTGGGCGGTGCTACAACT
SEQ ID NO.2(PD-1 sgRNA2)：TGTAGCACCGCCCAGACGAC
SEQ ID NO.3(PD-1 sgRNA3)：ACCGCCCAGACGACTGGCCA
Using pX330 plasmids (Addgene plasmid #4223) as template, the few core of sequence is targeted with T7 promoters+20bp
Thuja acid+20bp sgRNA scaffer are as forward primer (TAATACGACTCACTATAGNNNNNNNNNNNNNNNNNNN
(20bp target sequences) GTTTTAGAGCTAGAAATAGC).Specifically, three forward primers are respectively：
SEQ ID NO.4 (forward primer of PD-1 sgRNA1)：
SEQ ID NO.5 (forward primer of PD-1 sgRNA2)：
SEQ ID NO.6 (forward primer of PD-1 sgRNA3)：
Reverse primer is；
SEQ ID NO.7 (reverse primer)：AGCACCGACTCGGTGCCACT.
By PCR amplification T7-sgRNA in-vitro transcription templates, adsorption column purified pcr product, and use HiScribe T7
Quick High Yield RNA Synthesis Kit (NEB, USA) in-vitro transcription goes out sgRNA.Use RNA clean&
ConcentratorTM-25 (ZYMO RESEARCH, USA) purifying RNAs simultaneously elute in the water of no RNA enzyme, make immediately after elution
With or -80 DEG C preservation, a concentration of 3 μ g/ μ l.
Embodiment 3It prepares the CIK cell of PD-1 knockouts and detects knockout situation
Pass through 4D- using P3 Primary Cell 4D-Nucleofector X Kit (Lonza, Germany)
Nucleofector System X (Lonza, Germany), with the implementation of 9.44 μ g Cas9 albumen (NEB, USA) and 12 μ g
Any, electroporation 5 × 10 in the sgRNA of three kinds of targeting PD-1 exons 1s prepared by example 26The CIK prepared in a embodiment 1
Cell.Wherein electricity preheats 50ml serum-free GT-T551 culture mediums before turning, and collects CIK cell, centrifuges and 70 μ l electricity are added and turn liquid weight
Outstanding cell, while 30 μ l electricity are added in Cas9 albumen and sgRNA and are turned in liquid, incubation at room temperature after ten minutes, by Cas9 albumen and
SgRNA mixed liquors are added in cell suspension, are turned with EO-115 electricity using Lonza 4D-Nucleofector X Unit electroporations
Program carries out electricity and turns.After electroporation, cell stand 24 hours, then by cell be resuspended in 2ml pre-temperatures contain 5% people's AB type blood plasma
It in the GT-T551 culture mediums of 100U/mL recombinant human il-2s, and is transferred in 12 porocyte plates, and in 37 DEG C, 5%CO2Culture
It is cultivated in case, cell culture medium contains 5% people's AB types blood plasma and 100U/mL recombinant human il-2's complete mediums in every 2 days with fresh
Half amount changes liquid.
Analysis and cloned sequence analysis (are inserted into) by decomposing tracking by T7El mispairing repairing analysis method, TIDE to check
Mutation.7th day harvest cell after electrotransfection, extracts genomic DNA with genome DNA extracting reagent kit, uses high-fidelity Q5
PCR kit expands PD-1 gene knockout location proximate DNA fragmentations：
SEQ ID NO.8 (forward primer PD-F)：CCAGCACTGCCTCTGTCACTCTCG；
SEQ ID NO.9 (reverse primer PD-R)：ACGTCGTAAAGCCAAGGTTAGTCCC.
T7E1 mispairing digestions point are carried out to PCR product after purification using EnGen mutation detection kits (NEB, the U.S.)
Analysis method analyzes DNA mutation situation.Sanger sequencings, sequencing result are carried out to PCR product using primer PD-F as sequencing primer
Use network tool (http：//tide.nki.nl) carry out TIDE analysis sequencings.Use pGEM-T EASY Vector
The PCR fragment of purifying is cloned into pGEM-T EASY carriers to detect mutant by systems kits (Promega, the U.S.)
Allele.40 bacterium colonies are sequenced the PCR connection products sample each converted using universal primer M13F in total.Used
All PCR methods follow the specification or standard molecule cloning approach of manufacturer's offer.
As a result referring to Fig. 2.The result shows that the knockout efficiency highest of sgRNA1, reaching 41.23 ± 0.5239%, (TIDE divides
Analysis method).It further expands and has been subcloned the target areas sgRNA1 and identify mutation allele.It was found that 40 sequencing equipotentials
16 in gene are mutant, it was confirmed that PD-1 knocks out the catastrophe in site, and as shown in Figure 2 D, all mutation are accurately
It is happened at sgRNA1 target areas.This illustrates the PD-1 genes in successful knockout CIK cell.
CIK cells of the PD-1 obtained through knockout be referred to as " PD-1 KO/CIK cells ", " PD-1KO-CIK cells " or
" enhanced CIK cell ".
Embodiment 4The performance detection of enhanced CIK cell
(1) immunophenotyping
By 5 × 106The CIK cell that a wild type CIK cell and electricity turn latter 7th day is resuspended in 100 μ l PBS, is then added
Enter monoclonal antibody CD3-PerCP-Cy5.5, CD4-FITC, CD8-APC, CD56-PE, CD45RO-PE, CD45RA-FITC,
CD27-Brilliant Violet421, CD279 (PD-1)-PE use FACSAriar streamings after 4 DEG C are protected from light incubation 30 minutes
Cell instrument detects sample.All antibody are purchased from BD Bioscience (San Diego, CA, USA).Use FlowJo
V.10.2 (TreeStar, Ashland, OR, USA) analyzes flow cytometry data.
The CIK cell after PD-1 is knocked out is assessed using flow cytomery CD4, CD8, CD45RA, CD27, CD279
Immunophenotype, although PD-1 baseline express relatively low, percentage of the PD-1+CD3+ cells in wild type CIK cell
It is 4.54 ± 0.2845%, but is 1.81 ± 0.3121% (Fig. 3 A-1 and Fig. 3 A- in the CIK cell of PD-1 gene knockouts
2) it, is compared with wild type CIK cell, there is notable difference, there is statistical significance.Expression by CD4 and CD8 and primary tape
T cell (CD45RA+/CD27+, TN), center memory T cells (CD45RA-/CD27+, TCM), effect memory T cells
The feature of (CD45RA-/CD27-, TEM) and effector T cell (CD45RA+/CD27-, Teff) come assess CIK knock out PD-1 after
Immunophenotype (Fig. 3 B-1 and Fig. 3 B-2 and Fig. 3 C-1 and Fig. 3 C-2).Compared with compareing CIK cell, T cell is knocked out in PD-1
Middle immunophenotype is roughly the same, no significant change, this illustrates that the PD-1 for knocking out CIK cell does not influence the normal phenotype of CIK cell
Change.
(2) cytokine secretion
With E: T=20: 1 ratio by 5 × 103A SMMC-7721 cells (target cell) and 1 × 105A wild type CIK cell
Or PD-1 KO-CIK cells (effector cell) are added to after being co-cultured 24 hours in complete GT-T551 culture mediums, abandon supernatant,
Using IFN-γ ELISPOT kits (Dakewei, China) by calculating spot number come the ability of thinner intracrine IFN-γ
Strong and weak situation.Tablet is scanned with ELISPOTCTL readers (Cell Technology Inc, Columbia, MD), is used in combination
ELISPOT softwares (AID, Strassberg, Germany) analysis result.The results are shown in Figure 4, the spot of PD-1 KO-CIK cells
Points are apparently higher than wild type CIK cell (90.00 ± 1.528 VS 24.00 ± 2.517), have statistical significance.This also table
The knockout of bright PD-1 enhances cellullar immunologic response of the CIK cell to liver cancer cell lines.
(3) to the cytotoxicity of liver cancer cells
The cell toxicant of CIK cell and PD-1 KO-CIK cells is detected by the cytotoxicity analysis of luciferase mediation
Property.By slow-virus infection structure with luciferase and GFP albumen SMMC-7721 liver cancer cells (referring to Wang Xiaomin et al.,
Application of the bis- mark technologies of GFP and Luc in mouse tumor model foundation, experimental animal and comparative medicine, 2010,30 (1)：2-
7), i.e. fLuc-EGFP/SMMC772 liver cancer cells, then by the cell and effector cell (wild type CIK cell or PD-1 KO/
CIK cell) according to different effect targets than combined inoculation in saturating 96 orifice plate of fluoroscopic examination of white bottom, wherein fLuc-EGFP/
SMMC7721 liver cancer cells quantity is fixed as 5000 cells, and effector cell is separately added into according to effect target than 10: 1,5: 1,2.5: 1
5×104、2.5×104、1.25×104Cell, in 37 DEG C co-culture 16-18h after, be added 100 μ l substrate mixtures after immediately in
Multi-function microplate reader detects relative intensity of fluorescence.
Cell killing rate is calculated according to following equation：((target imitates the fluorescence number that cell co-cultures hole to % killings rate=100-
Value)/(Target cell wells fluorescence values) × 100)
The results are shown in Figure 5.When it is 10: 1,5: 1,2.5: 1 to imitate target ratio, PD-1 KO-CIK cells and wild type CIK are thin
Born of the same parents are respectively 90.77 ± 0.72%VS50.23 ± 2.66%, 67.03 ± 3.22%VS to the killing rate comparison of liver cancer cells
32.43 ± 1.33%, 37.67 ± 4.14%VS 22.07 ± 2.63%, P < 0.05, difference have statistical significance.This shows
It is compared with wild type CIK cell, the killing rate higher of the PD-1 KO-CIK cells in liver cancer patient source to liver cancer SMMC-7721.
This also illustrates that PD-1 knockouts can enhance CIK cell anti-liver cancer and anti-cell ability.
Specific description of embodiments of the present invention above is not intended to limit the present invention, and those skilled in the art can be according to this
Invention is variously modified or deforms, and without departing from the spirit of the present invention, should all belong to the model of appended claims of the present invention
It encloses.
Claims (10)
1. a kind of method of the anti-cancer ability of enhancing cell, the method includes by Cas9 albumen and targeting PD-1 gene extrons
The sgRNA electricity of son -1 is gone in cell, wherein the cell is the cell for tumour specific antigen or tumor associated antigen
Malicious T lymphocytes or CIK cell.
2. according to the method described in claim 1, it is characterized in that, the nucleotide sequence of the sgRNA such as SEQ ID NO.1,
Shown in SEQ ID NO.2 or SEQ ID NO.3；
Preferably, the nucleotide sequence of the sgRNA is as shown in SEQ ID NO.1.
3. method according to claim 1 or 2, which is characterized in that the Cas9 albumen and sgRNA are with 1: 0.5-2 matter
Amount turns than carrying out electricity；
Preferably, the Cas9 albumen, sgRNA and cell are with 9.44 (μ g): 9-12 (μ g): 1-5 × 106A cell carries out electricity and turns,
More preferably with 9.44 (μ g): 9-12 (μ g): 5 × 106A cell carries out electricity and turns；
Preferably, the cell is CIK cell, and wherein CD3+CD56+Cell proportion >=20%, CD3+CD8+Cell proportion >=
50%.
4. according to the method in any one of claims 1 to 3, which is characterized in that the described method comprises the following steps：
1) CIK cell is obtained, wherein the CD3 of the CIK cell+CD56+Cell proportion >=20%, CD3+CD8+Cell proportion >=
50%；
2) the sgRNA electricity of Cas9 albumen and targeting PD-1 gene extrons -1 is gone in CIK cell；Wherein, the Cas9 eggs
In vain, sgRNA and CIK cell are with 1 (μ g): 1-1.5 (μ g): 5 × 106A cell and use Lonza 4D-Nucleofector
X Unit electroporations carry out electricity with EO-115 electricity carryover sequences and turn；
Preferably, the method is further comprising the steps of：
3) by the CIK cell turned through electricity in 37 DEG C, 5%CO2It is lower to stand 24 hours.
5. using enhanced cell made from method any one of Claims 1-4；
Preferably, knockout rate >=40% of the PD-1 genes of the enhanced cell；
Preferably, the enhanced cell is enhanced CIK cell.
6. sgRNA, the nucleotide sequence such as SEQ of the sgRNA for knocking out PD-1 genes based on CRISPR/Cas9 technologies
Shown in ID NO.1, SEQ ID NO.2 or SEQ ID NO.3；
Preferably, the nucleotide sequence of the sgRNA is as shown in SEQ ID NO.1.
7. a kind of pharmaceutical composition, described pharmaceutical composition includes the enhanced CIK cell or claim described in claim 5
SgRNA described in 6.
8. the sgRNA described in enhanced cell or claim 6 described in claim 5 is preparing the drug for treating cancer
In purposes；
Preferably, the cancer is liver cancer, colorectal cancer, breast cancer, lymthoma, leukaemia, lung cancer, gastric cancer, cancer of pancreas, rear abdomen
Film tumour, the cancer of the esophagus, cholangiocarcinoma, gallbladder cancer, duodenal cancer, thyroid cancer, nasopharyngeal carcinoma, laryngocarcinoma, malignant mela noma, uterus
Endometrial carcinomas, cervical carcinoma, oophoroma, kidney, carcinoma of urinary bladder, prostate cancer, osteosarcoma, metastatic carcinoma of bone or glioblastoma, preferably
Liver cancer.
9. a kind of kit for knocking out cell, preferred PD-1 genes of CIK cell based on CRISPR/Cas9 technologies, described
Kit includes：Target the sgRNA of PD-1 gene extrons -1；
Preferably, the nucleotide sequence of the sgRNA is as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3；More
Preferably, the nucleotide sequence of the sgRNA is as shown in SEQ ID NO.1.
10. kit according to claim 9, which is characterized in that the kit further includes：Cas9 albumen；And/or
Turn the reagent of sgRNA and/or Cas9 albumen for electricity.
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