CN117797278A - Application of IL-17A mRNA and TLSV nanoparticle/IL-17A mRNA complex in preparation of medicines for preventing or treating tumors - Google Patents
Application of IL-17A mRNA and TLSV nanoparticle/IL-17A mRNA complex in preparation of medicines for preventing or treating tumors Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to application of an IL-17A mRNA and TLSV nanoparticle/IL-17 AmRNA complex in preparation of a medicine for preventing or treating tumors. Aiming at the problems of whether the existing IL-17A mRNA has an anti-tumor effect, how to deliver, how to resist tumors and the like, the invention provides the application of the IL-17A mRNA and TLSV nanoparticle/IL-17A mRNA compound in preparing anti-tumor drugs. The invention discovers the effect of IL-17AmRNA in resisting tumor for the first time, and the TLSV nanoparticle is prepared by wrapping tumor cell lysate by DOTAP cationic phospholipid and amphiphilic diblock copolymer mPEG-PCL, and IL-17A mRNA is delivered by the TLSV nanoparticle, so that the TLSV nanoparticle/IL-17A mRNA compound with the anti-tumor effect is obtained. The IL-17A mRNA and TLSV nanoparticle/IL-17A mRNA compound can effectively inhibit the growth of colorectal cancer tumor tissues in vivo, and provides a new strategy for disease treatment.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to application of an IL-17A mRNA and TLSV nanoparticle/IL-17A mRNA compound in preparation of a medicine for preventing or treating tumors.
Background
Malignant tumors are one of the major diseases leading to death, with colorectal cancer being one of the most common malignant tumors. At present, the means for treating cancer mainly comprise operation treatment, radiotherapy, chemotherapy, immunotherapy and the like, and gene therapy is considered as a potential innovative treatment means for further improving the safety and treatment effect of treatment. Among them, mRNA-based gene therapy is an emerging therapeutic strategy. mRNA molecules can express proteins in cells in a short time, and perform the functions of supplementation, replacement, editing and the like so as to inhibit tumor growth. Compared with other forms of nucleic acid molecules, mRNA has the advantages of higher production efficiency, more flexible gene delivery, capability of avoiding genome insertion and the like, and is an ideal form of tumor gene therapy. Tumor immunity gene therapy plays an anti-tumor role by regulating and controlling the immune response of an organism, and mRNA has a certain development potential in tumor immunity gene therapy strategies due to the advantages. However, how to improve the delivery efficiency of mRNA molecules and find suitable anti-tumor immunostimulation strategies is currently an important research direction for mRNA-based tumor immune gene therapy.
Interleukin 17A (IL-17A) is a member of the IL-17 cytokine family, and acts as an extracellular secretion factor to activate immune cells, up-regulate the expression of the immune chemotactic factor CXCL9, tumor killing factor TNF-alpha, recruit anti-tumor immunity of DC cells and natural killer cells (NK cells), so mRNA encoding IL-17A is a potential therapeutic target of immune genes.
However, the current research only shows that IL-17A protein has anti-tumor effect, and no related research on delivery of IL-17A mRNA to cells for tumor treatment exists, so that further research is required as to whether the IL-17A mRNA can be applied to the anti-tumor field.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: the prior art lacks researches on whether IL-17A mRNA has an anti-tumor effect, how to deliver the mRNA, how to resist tumors and the like.
The technical scheme for solving the technical problems is as follows: provides an application of IL-17A mRNA in preparing medicaments for preventing or treating tumors.
Wherein, in the application, the nucleotide sequence of the IL-17A mRNA is shown as SEQ ID NO. 1.
Nucleotide sequence of SEQ ID NO 1IL-17AmRNA
ATGAGTCCAGGGAGAGCTTCATCTGTGTCTCTGATGCTGTTGCTGCTGCTGAGCCTGGCGGCTACAGTGAAGGCAGCAGCGATCATCCCTCAAAGCTCAGCGTGTCCAAACACTG AGGCCAAGGACTTCCTCCAGAATGTGAAGGTCAACCTCAAAGTCTTTAACTCCCTTGGCGCAAAAGTGAGCTCCAGAAGGCCCTCAGACTACCTCAACCGTTCCACGTCACCCTGGACTCTCCACCGCAATGAAGACCCTGATAGATATCCCTCTGTGATCTGGGAAGCTCAGTGCC
GCCACCAG
CGCTGTGTCAATGCGGAGGGAAAGCTGGACCACCACATGAATTCTGTTCTC
ATCCAGCAAGAGATCCTGGTCCTGAAGAGGGAGCCTGAGAGCTGCCCCTTCACTTTCAG
GGTCGAGAAGATGCTGGTGGGTGTGGGCTGCACCTGCGTGGCCTCGATTGTCCGCCAGGCAGCC。
Wherein, in the above application, the IL-17A mRNA is used by combining with TLSV nanoparticles to form TLSV nanoparticle/IL-17A mRNA complex.
Furthermore, the TLSV nanoparticle is prepared by wrapping tumor cell lysates by DOTAP cationic phospholipid and amphiphilic diblock copolymer mPEG-PCL. The average grain diameter of the TLSV nano-particles is 372.93 +/-24.51 nm, and the average potential is +21.40+/-2.77 mV.
Further, the TLSV nanoparticle comprises the following specific components: 1 to 5 parts of DOTAP, 45 to 50 parts of amphiphilic mPEG-PCL diblock copolymer and 12 to 15 parts of tumor cell lysate.
Preferably, the tumor cell lysate is a lysate of CT26 cells or B16 cells. Wherein the CT26 cell lysate is a whole cell lysate, and the main components comprise: organic components such as protein, nucleic acid, lipid saccharide, and other inorganic components, and protein concentration is less than or equal to 1.2mg/mL. The protein concentration in CT26 cell lysates cannot be too high, otherwise the resulting TLSV nanoparticles cannot bind mRNA molecules electrostatically.
Wherein, in the above application, the tumor is colorectal cancer.
The invention also provides a TLSV nanoparticle/IL-17 AmRNA compound, which is obtained by electrostatic adsorption of TLSV nanoparticles prepared by wrapping tumor cell lysate with DOTAP cationic phospholipid and amphiphilic diblock copolymer mPEG-PCL and IL-17AmRNA molecules.
Preferably, the TLSV nanoparticle is 30-40 parts by weight and the IL-17AmRNA is 1-5 parts by weight.
The invention also provides a preparation method of the TLSV nanoparticle/IL-17 AmRNA compound, which comprises the following steps:
a. re-suspending CT26 cell, freeze thawing repeatedly, crushing, centrifuging to collect supernatant to obtain cell lysate solution;
b. weighing 45-50 parts of mPEG-PCL copolymer and 1-5 parts of DOTAP;
c. dissolving mPEG-PCL copolymer and DOTAP in an organic phase solvent together, then adopting a rotary evaporator to operate for 40-45 min under the water bath condition of 55-60 ℃ so as to evaporate the solvent, and then adding 12-15 parts of cell lysate solution for hydration until complete dissolution to obtain TLSV nanoparticle solution;
d. and incubating 30-40 parts of TLSV nanoparticle solution and 1-5 parts of IL-17A mRNA for 15-30 minutes at room temperature to obtain the TLSV nanoparticle/IL-17 AmRNA compound.
In the preparation method of the TLSV nanoparticle/IL-17 AmRNA complex, the solvent in the step c is at least one of dichloromethane, chloroform, acetone, tetrachloromethane, ethanol, methanol, ethyl acetate or cyclohexane; the organic phase solvent is at least one of ethanol, dichloromethane, chloroform, acetone, tetrachloromethane and methanol.
The invention also provides application of the TLSV nanoparticle/IL-17 AmRNA complex in preparing medicaments for preventing or treating tumors.
Further, the tumor is colorectal cancer.
Compared with the prior art, the invention has the beneficial effects that:
the invention discovers for the first time that the IL-17AmRNA can be used for preparing medicines for preventing or treating tumors, and develops a novel mode for applying IL-17A mRNA for the first time, namely, delivering by combining with TLSV nanoparticles to form a TLSV nanoparticle/IL-17A mRNA complex. The invention develops TLSV nano particles with high delivery efficiency aiming at IL-17AmRNA for the first time, and a compound formed by the TLSV nano particles and the TLSV nano particles can effectively introduce target genes into cells, thereby having good application prospects in gene function research, gene therapy research and clinical application based on IL-17 AmRNA. The TLSV nanoparticle/IL-17A mRNA compound can mediate lymphocytes to effectively inhibit the growth of CT26 cells in vitro; the TLSV nanoparticle/IL-17A mRNA compound can also effectively inhibit the growth of colon cancer tumor tissues in vivo, and provides a new choice for preventing and treating colorectal cancer.
Drawings
FIG. 1 shows the band sizes of IL-17AmRNA of the invention in nucleic acid gels.
FIG. 2 shows gel blocking assays for TLSV and IL-17AmRNA of the invention.
FIG. 3 is a graph showing the killing effect of lymphocyte supernatant mediated in test example 1 of the present invention; a represents CT26 cell viability; b represents cell colonies formed by CT26 stained with crystal violet in a colony formation experiment; c represents counting the number of clones and performing statistical analysis.
FIG. 4 is a graph showing the therapeutic effect of the colorectal cancer abdominal cavity in-situ transplantation model in test example 1 of the present invention; a represents the average tumor weight and b represents the average tumor node number.
FIG. 5 is a graph showing the effect of the colorectal cancer lung metastasis model treatment in test example 1 of the present invention; a represents the average tumor weight and b represents the average tumor node number.
FIG. 6 is a survival curve of mice in the lung metastasis model for colorectal cancer prevention in test example 1 of the present invention.
FIG. 7 is a graph showing the effect of the in vitro anti-tumor mechanism of IL-17AmRNA in test example 1 of the present invention; a represents the statistical analysis of the number of spleen lymphocytes in each group in test example 2; b represents the DC cell positive ratio in each group of spleen lymphocytes in test example 2; c representsCD4 in spleen lymphocytes of each group in Experimental example 2 + T cell positive ratio; d represents CD8 in each group of spleen lymphocytes in test example 2 + T cell positive ratio; e represents the NK cell positive ratio in each group of spleen lymphocytes in test example 2;
FIG. 8 is a graph showing the effect of IL-17AmRNA in vivo on the anti-tumor mechanism in test example 1 of the present invention; a represents a therapeutic mechanism of IL-17AmRNA in situ tumor implantation in the abdominal cavity of CT26 colorectal cancer; b represents the therapeutic mechanism of IL-17AmRNA in CT26 colorectal lung metastases.
Detailed Description
The invention provides an application of IL-17A mRNA in preparing a medicament for preventing or treating tumor.
Conventionally, IL-17A protein is secreted by tumor tissues to up-regulate the expression of immune chemotactic factor CXCL9 and tumor killing factor TNF-alpha, recruit DC cells and natural killer cells (NK cells), and inhibit the formation of tumor microvessels to cooperatively achieve the anti-tumor immunity effect.
The current research is only based on IL-17A protein level antitumor, and no IL-17AmRNA level antitumor research is mainly influenced by the following factors: first is a carrier factor, it is difficult to find a carrier that can bind and efficiently deliver mRNA to cells to function. Second, mRNA is more difficult to deliver than other nucleic acids due to the linear structure and low stability of mRNA molecules.
The present invention overcomes the above problems, and specifically screens out a carrier TLSV nanoparticle capable of efficiently delivering IL-17A mRNA for IL-17AmRNA, which can efficiently bind and deliver IL-17AmRNA into cells, and the resulting complex exhibits excellent effects in preventing or treating tumors.
The invention applies IL-17AmRNA to tumor treatment for the first time, because mRNA molecular weight is small, the mRNA can be compressed better, and the influence by space structure is less; the sequence design is simple; the chemical synthesis is bypassed, the self cells are utilized for production, and the method is simple and efficient.
Aiming at the IL-17AmRNA for the first time, the invention designs the TLSV nano-particle capable of efficiently delivering the IL-17A mRNA, and the IL-17AmRNA can be successfully delivered and applied by forming a complex between the IL-17AmRNA and the nano-particle.
The TLSV nanoparticle developed by the invention is prepared by wrapping tumor cell lysate by DOTAP cationic phospholipid and amphiphilic diblock copolymer mPEG-PCL. The average grain diameter of the TLSV nano-particles is 372.93 +/-24.51 nm, and the average potential is +21.40+/-2.77 mV.
Through a large number of screening, the invention discovers that when the weight part of DOTAP is 1-5 parts, the weight part of the amphiphilic mPEG-PCL diblock copolymer is 45-50 parts, and the tumor cell lysate is 12-15 parts, the obtained TLSV nanoparticle has better effect of delivering IL-17 AmRNA.
In particular, the invention also provides a TLSV nanoparticle/IL-17 AmRNA compound, which is prepared by electrostatically adsorbing TLSV nanoparticles prepared by coating tumor cell lysates with DOTAP cationic phospholipid and amphiphilic diblock copolymer mPEG-PCL and mRNA molecules.
Preferably, the TLSV nanoparticle is 30-40 parts by weight and the IL-17AmRNA is 1-5 parts by weight.
The TLSV nanoparticle/IL-17 AmRNA complex is a relatively safe and efficient mRNA molecule non-viral gene delivery complex, and the preparation of the TLSV/IL-17A nanoparticle complex provides a novel strategy for colorectal cancer treatment.
The method for preparing TLSV nano-particles is a special film hydration method obtained by repeated screening, and comprises the following steps:
a. CT26 cells were resuspended in DPBS, oxidized in incubator with hypochlorous acid for 1 hour, and stirred every half hour. Cells were collected, washed with DPBS, resuspended with MilliQ, and attenuated. The cells were repeatedly freeze-thawed 12 times in liquid nitrogen and 37 ℃ water bath. The cell disruptor was sonicated for 30 minutes. Centrifuge and collect the supernatant. Cell lysate was assayed for protein concentration and a cell lysate solution was obtained diluted with MilliQ to a protein concentration of 1.2mg/mL.
b. Weighing 45-50 parts of mPEG-PCL copolymer and 1-5 parts of DOTAP;
c. the mPEG-PCL copolymer and DOTAP are dissolved in a solvent together, then a rotary evaporator is adopted, the operation is carried out for 40 to 45 minutes under the water bath condition of 55 to 60 ℃ so as to evaporate the solvent, and then 12 to 15 parts of cell lysate solution is added for hydration until the solution is completely dissolved, thus obtaining the TLSV nanoparticle solution.
When the TLSV nanoparticle/IL-17 AmRNA compound is prepared, 30-40 parts of TLSV nanoparticle solution and 1-5 parts of IL-17AmRNA are incubated for 15-30 minutes at room temperature, so that the TLSV nanoparticle/IL-17A mRNA compound is obtained.
The invention also provides application of the TLSV nanoparticle/IL-17 AmRNA complex in preparing a medicament for preventing or treating tumors.
Further, the tumor is colorectal cancer.
The following examples are provided to further illustrate embodiments of the present invention and are not intended to limit the scope of the invention to the examples.
The reagents and starting materials used in the examples were all conventional commercial products.
EXAMPLE 1 preparation of TLSV/IL-17A nanogene complexes
1. TLSV nanoparticle prepared by thin film rotary evaporation method
Trypsinization and obtaining CT26 cells, resuspending the cells to 1X 10 with DPBS as solvent 6 Concentration of individual/mL, CO at 37℃ 2 In a 4% incubator, the mixture was oxidized in hypochlorous acid at a final concentration of 0.642. Mu.L/mL for 1 hour with stirring every half hour. The cells were collected, washed 1 time with DPBS, resuspended with MilliQ to a cell concentration of 1X 10 7 The cells were dispensed into seed retention tubes at a volume of each mL. The cells were repeatedly freeze-thawed 12 times in liquid nitrogen and 37 ℃ water bath. The cell disruptor was sonicated for 30 minutes (950 watts, total duration of work 30 minutes, sonication for 9.9 seconds, gap 5 seconds). Centrifugation at 1500rpm for 1 min, and supernatant was collected. The protein concentration of the cell lysate was measured, and the cell lysate solution obtained was diluted with MilliQ to a protein concentration of 1.2mg/mL and stored at-80℃in a refrigerator.
Cationic lipid (2, 3-dioleoyl-propyl) -trimethylamine (1, 2-dioleoyl-3-trimethylcam-propane, DOTAP) was reacted with methyl polyethylene glycol-polycaprolactone (mPEG-PCL) polymer (molecular weight 4000Da, peg-pcl=2000 Da-2000 Da) at a ratio of 1:9, and dissolving the mixture by using dichloromethane, and putting the mixture on a rotary evaporator for rotary evaporation at 60 ℃ for 45min to form a film. The prepared membrane is taken out and then dissolved in CT26 cell lysate solution, and is oscillated for 5min under the water bath condition of 60 ℃ to obtain TLSV cationic polymer nanoparticle solution with specific concentration, and the TLSV cationic polymer nanoparticle solution is placed in a refrigerator with the temperature of 4 ℃ for standby.
2. Preparation of IL-17AmRNA solutions
The method comprises the steps of taking pVAX1-IL-17A plasmid as a template, carrying out PCR amplification to obtain an IL-17A gene (DNA fragment of ORF frame sequence (shown as SEQ ID NO: 1)), cutting off fragment region gel (shown as figure 1) consistent with the size of a target gene after agarose gel electrophoresis, recycling the fragment by using a gel recycling kit to obtain a purified IL-17APCR product, carrying out overnight transcription by using a T7 in vitro transcription kit with 0.5 mu G of IL-17A gene PCR product as the template at a constant temperature of 37 ℃, then carrying out 5 '-end capping and 3' -end tailing modification at a constant temperature of 37 ℃, wherein the 5 '-end of the modified IL-17AmRNA has a 7-methylguanosine (m 7G) cap structure, and the 3' -end has a poly (A) tail.
3. Preparation of TLSV/IL-17A nanogene complexes
Cationic nanoparticles according to TLSV: IL-17AmRNA at 35:1 (w/w) (gel retardation is shown in figure 2), adding the nanoparticle solution (dispersed in deionized water, concentration is 5 mg/mL) into the IL-17A mRNA solution (dispersed in deionized water, concentration is 1 mg/mL), immediately blowing and mixing by a pipette, and standing for 15 minutes at room temperature to obtain the TLSV/IL-17A nano-gene composite.
EXAMPLE 2 in vitro mediated lymphocyte anti-colorectal cancer assay of the TLSV/IL-17A nano-Gene complexes of the invention
To study the anti-tumor effect of the TLSV/IL-17A nanogene complex in vitro, CT26 cells transfected with the TLSV/IL-17A nanogene complex:
a) Blank control group: 0.9% sodium chloride solution;
b) DMP control group: the DMP cationic nanoparticles were placed in MilliQ;
c) TLSV treatment group: TLSV cationic nanoparticles were placed in MilliQ;
d) IL-17A treatment group TLSV/IL-17A nanogene complexes were placed in MilliQ.
The TLSV/IL-17A nano-gene complex was prepared as in example 3, and the ratio thereof was as follows: IL-17A mRNA: TLSV cationic nanoparticle = 1:35 (W/W) containing 2. Mu.g of IL-17A mRNA2. Mu.g, TLSV/IL-17A nanogene complex was diluted in MilliQ.
The DMP cationic nanoparticle is prepared by mixing DOTAP cationic phospholipid and amphiphilic diblock copolymer mPEG-PCL, and no cell lysis compound is added.
Spleen of healthy BalB/c mice (6-8 weeks old, female) was removed, lymphocytes were isolated with lymphocyte isolation solution, and lymphocytes were inoculated into six well plates (6X 10) 5 Each well) followed by 500 μl of CT26 cell supernatant (3 replicates per group) for each transfection group, and after 72 hours, spleen lymphocyte supernatant was collected and co-cultured with CT26 cells: the activity and growth of CT26 cells after treatment were studied by MTT method (FIG. 3 a) and colony formation assay (FIGS. 3b, c).
From FIG. 3a, it can be seen that the viability of CT26 cells in the IL-17A treated group was significantly reduced. The cell viability of the IL-17A treated group was reduced by 42.78% compared to the untreated group, significantly higher than that of the DMP group (17.25%) and TLSV group (35.36%). P <0.05 is considered statistically significant.
As can be seen from FIGS. 3b, C, the number of clones of C26 was significantly reduced in the IL-17A treated group. Furthermore, we counted the number of clones in each group, and compared with the blank (284.+ -. 25), the number of clones detected in the IL-17A treated group (34.+ -. 4) was significantly smaller than that in the DMP group (206.+ -. 32) and the TLSV group (105.+ -. 13), and P <0.01 was considered statistically significant.
The results of the in vitro experiments show that the TLSV/IL-17A nano-gene complex has remarkable colorectal cancer resisting effect.
EXAMPLE 3 TLSV/IL-17A nanogene complexes of the invention anti-celiac in situ colorectal cancer test
To study the antitumor effect of the TLSV/IL-17A nanogene complex in vivo, colorectal cancer cell peritoneal in situ transplantation tumor models were established subcutaneously in BalB/c mice (6-8 weeks old, females). CT26 colorectal cancer cells cultured in vitro were digested with trypsin and fixed in serum-free, antibiotic-free DMEM medium, and 2X 10 was inoculated into the abdominal cavity of each mouse 5 2 days after cell inoculation, randomized group treatment (5 per group) was started as follows:
a) Blank control group: 0.9% sodium chloride solution;
b) DMP control group: the DMP cationic nanoparticles were placed in MilliQ;
c) TLSV treatment group: TLSV cationic nanoparticles were placed in MilliQ;
d) IL-17A treatment group TLSV/IL-17A nanogene complexes were placed in MilliQ.
The treatment was performed by intraperitoneal injection, and the TLSV/IL-17A nano-gene complex was prepared as in example 1, and the ratio was as follows: IL-17AmRNA: TLSV cationic nanoparticle = 1:35 (W/W), TLSV/IL-17A nanogene complexes were diluted in MilliQ.
The DMP cationic nanoparticle is prepared by mixing DOTAP cationic phospholipid and amphiphilic diblock copolymer mPEG-PCL, and no cell lysis compound is added.
Each mouse was injected at a volume of 70. Mu.L containing 10. Mu.g IL-17AmRNA and 350. Mu.g cationic nanoparticle TLSV. The administration was 1 time per day for 16 times. Animals were sacrificed and dissected on day 18, and peritoneal tumor tissue was isolated for tumor nodule counting and weighing. The tumor weights and the number of nodules of the animals in each group are shown in FIG. 4, wherein FIG. 4a shows the tumor weight, FIG. 4b shows the number of nodules, and P <0.05 is considered statistically significant.
As can be seen from FIG. 4, the TLSV/IL-17A nano-gene complex has the slowest tumor growth in the treatment group, and the tumor growth in the control group is faster, and the TLSV/IL-17A nano-gene complex has stronger tumor growth inhibition effect and better effect than the DMP cationic nanoparticles.
The experimental results show that the TLSV/IL-17A nano-gene complex has remarkable colorectal cancer resistance.
EXAMPLE 4 TLSV/IL-17A nanogene complexes of the invention anti-colorectal cancer lung metastasis assay
In order to study the antitumor effect of the TLSV/IL-17A nano-gene complex in colorectal cancer lung metastasis, a colorectal cancer lung model was established in the abdominal cavity of BalB/c mice (6-8 weeks old, females). The in vitro cultured C26 colon cancer cells were digested with trypsin and fixed in serum-free, antibiotic-free DMEM medium, and each mouse was inoculated with 5X 10 cells intravenously 5 3 days after cell inoculation, randomized group treatment (5 per group) was started as follows:
a) Blank control group: 0.9% sodium chloride solution;
b) DMP control group: the DMP cationic nanoparticles were placed in MilliQ;
c) TLSV treatment group: TLSV cationic nanoparticles were placed in MilliQ;
d) IL-17A treatment group TLSV/IL-17A nanogene complexes were placed in MilliQ.
The TLSV cationic nanoparticle/IL-17 AmRNA gene complex was prepared by intravenous injection, and the ratio was as follows, according to the method of example 1: mRNA: cationic nanoparticle = 1:35 (W/W), cationic nanoparticle/mRNA complexes were diluted in MilliQ. The injection volume of each mouse was 100. Mu.L, which contained 10. Mu.g of mRNA and 350. Mu.g of cationic nanoparticles. The administration was 1 time per day for a total of 12 times. Animals were sacrificed and dissected on day 15, lung tissue isolated and weighed and tumor nodule counted. Analysis of variance for tumor growth inhibition, P <0.05 was considered statistically significant. The tumor weights, tumor node numbers, of the animals in each group are shown in fig. 5, wherein fig. 5a represents the average tumor weight and fig. 5b represents the average tumor node number.
As can be seen from FIG. 5, the TLSV/IL-17A nano-gene composite has slow tumor growth in the treatment group, and has faster tumor growth in the control group, and the TLSV cationic nanoparticle/IL-17 AmRNA gene composite has stronger tumor growth inhibition effect, and compared with the blank control group, the tumor inhibition rate reaches 54.71%.
The experimental result shows that the TLSV cation nanoparticle/IL-17 AmRNA gene complex has remarkable colorectal cancer resistance.
EXAMPLE 5 TLSV/IL-17A nanogene complexes of the invention prevention of colorectal cancer lung metastasis assay
To study the tumor metastasis preventing effect of the TLSV/IL-17A nanogene complex in vivo, balB/c mice (6-8 week old, female) were first treated randomly (5 per group):
a) Blank control group: 0.9% sodium chloride solution;
b) DMP control group: the DMP cationic nanoparticles were placed in MilliQ;
c) TLSV treatment group: TLSV cationic nanoparticles were placed in MilliQ;
d) IL-17A treatment group TLSV/IL-17A nanogene complexes were placed in MilliQ.
Each group was treated by intravenous injection, TLSV cationic nanoparticle/IL-17A mRNA gene complexes were prepared as in example 1, with the following ratios: IL-17AmRNA: cationic nanoparticle = 1:35 (W/W), TLSV/IL-17A nanogene complexes were diluted in MilliQ. The injection volume of each mouse was 100. Mu.L, which contained 10. Mu.g of mRNA and 350. Mu.g of cationic nanoparticles. The administration was 1 time.
A lung metastasis model is then established. In vitro cultured C26 colon cancer cells were trypsinized and fixed in serum-free, antibiotic-free DMEM medium, and each mouse was inoculated intravenously with 5X 10 6 Individual cells. The survival curves of the mice (shown in fig. 6) were made and the median survival time of each mouse was calculated. Wherein, the average median survival time of each group is respectively: blank control group, 17 days; DMP control group, 20.5 days; TLSV control group, 22 days; IL-17A treatment group, 49 days.
The experimental result shows that the TLSV/IL-17A nano-gene complex has remarkable effect of preventing colorectal cancer lung metastasis.
EXAMPLE 6 in vitro anti-tumor mechanism study and results of IL-17AmRNA of the invention
Square according to example 2The spleen lymphocytes from each group were collected as a count statistic by the method, as shown in FIG. 7a, and compared with the untreated group (1.35X10 6 Individual) compared to 1.47×10 lymphocytes from the DMP treated group 6 The TLSV treated group lymphocytes were 1.56X10 6 Personal (P)<0.05 The number of lymphocytes in the TLSV/IL-17A complex treated group reached 1.60×10) 6 Personal (P)<0.01 The number of lymphocytes in the TLSV/IL-17A complex treated group was significantly increased.
The lymphocytes of each group were collected by the method of example 2, by the method of the cell line for DC, CD4 + T cells, CD8 + Cell flow analysis was performed after staining for T cells and NK cell surface markers (DC cell surface markers are CD11c molecules, CD 4) + The T cell surface marker is CD3 and CD4 molecule, CD8 + The T cell surface markers are CD3 and CD8a molecules, and the NK cell surface marker is CD49b. As shown in FIG. 7b, the cell positive rate was 26.78% with that of the DMP-treated group (DC cell positive rate, P<0.05 TLSV treated group (DC cell positive rate: 30.00%, P)<0.05 In comparison to the IL-17A treated group (36.42%) DC cells were significantly increased. As shown in FIG. 7c, IL-17A treatment group CD4 + T cell increase was significant (43.53%), whereas DMP treated group CD4 + T cells 22.23% (P)<0.05 TLSV treated group CD 4) + T cells were 32.94% (P)<0.05). As shown in FIG. 7d, the TLSV/IL-17A complex treated group CD8 + T cell increase was significant (66.29%), whereas DMP treated group CD8 + T cells were 46.84% (P)<0.001 TLSV treatment group CD 8) + T cells were 56.17% (P)<0.05). As shown in FIG. 7e, the NK cell ratio of the IL-17A treated group was 11.04%, while the NK cell ratio of the DMP treated group was 1.65% (P)<0.05 TLSV treated group NK cell fraction cells 3.97% (P)<0.05)。
The results show that after transfection of the TLSV/IL-17A nano-gene complex, the C26 cell supernatant containing the IL-17A can induce lymphocyte proliferation, and the IL-17A can promote proliferation of various immune cells such as DC cells, T cells and NK cells, and the induced immune response synergistically inhibits growth of CT26 cells.
EXAMPLE 7 in vivo anti-tumor mechanism study and results of IL-17AmRNA of the invention
The treatment mechanism of IL-17A in tumor tissue was studied by immunohistochemical analysis of paraffin sections of each group of tumor tissue, as in example 3. The immunohistochemical antibodies were diluted with PBS respectively: IL-17A (1:500), CXCL9 (1:500), TNF- α (1:500) and CD31 (1:50) were used to cover the tissues with antibodies, respectively, incubated, washed, blocked, and then differences in positivity were observed for each group.
As shown in FIG. 8a, IL-17A positive signal was significantly enhanced in tumor tissue in the IL-17A treated group compared to the other groups, indicating that TLSV successfully delivered IL-17AmRNA to tumor cells and secreted IL-17A into tumor tissue. And the CXCL9 and TNF-alpha cytokine positive signals of the tumor tissue sections of the IL-17A treatment group are enhanced, and the secretion of the immune chemotactic factor CXCL9 and the inflammatory factor TNF-alpha in the tumor tissue of the IL-17A treatment group is increased. The intensity of CD31 positive signal of tumor tissue of the IL-17A treatment group is obviously reduced, and the micro-angiogenesis in the tumor tissue of the IL-17A treatment group is obviously inhibited.
The results show that in the CT26 colorectal cancer abdominal cavity in-situ transplantation model, IL-17A promotes apoptosis of tumor cells by promoting expression of inflammatory factors, and inhibits formation of tumor tissue microvessels to cooperatively complete the effect of treating tumors.
The treatment mechanism of IL-17A in tumor tissue was studied by immunohistochemical analysis of paraffin sections of each group of tumor tissue, as in example 4. The immunohistochemical antibodies were diluted with PBS respectively: IL-17A (1:500), CXCL9 (1:500), TNF- α (1:500) and CD31 (1:50) were used to cover the tissues with antibodies, respectively, incubated, washed, blocked, and then differences in positivity were observed for each group.
As shown in FIG. 8b, IL-17A positive signal was significantly enhanced in tumor tissue in the IL-17A treated group compared to the other groups, indicating that TLSV successfully delivered IL-17A mRNA to tumor cells and secreted IL-17A into tumor tissue. And the CXCL9 and TNF-alpha cytokine positive signals of the tumor tissue sections of the IL-17A treatment group are enhanced, and the secretion of the immune chemotactic factor CXCL9 and the inflammatory factor TNF-alpha in the tumor tissue of the IL-17A treatment group is increased. The intensity of CD31 positive signal of tumor tissue of the IL-17A treatment group is obviously reduced, and the micro-angiogenesis in the tumor tissue of the IL-17A treatment group is obviously inhibited.
The results show that in the CT26 colorectal cancer lung metastasis model, IL-17A promotes apoptosis of tumor cells by promoting expression of inflammatory factors, and inhibits formation of tumor tissue microvessels to cooperatively complete the effect of treating tumors.
Claims (10)
- Use of il-17A mRNA in the manufacture of a medicament for the prevention or treatment of a tumor.
- 2. Use according to claim 1, characterized in that: the nucleotide sequence of the IL-17A mRNA is shown as SEQ ID 1.
- 3. Use according to claim 1, characterized in that: the IL-17A mRNA is used by combining with TLSV nano-particles to form TLSV nano-particles/IL-17A mRNA complex.
- 4. Use according to claim 1, characterized in that: the TLSV nanoparticle is prepared from DOTAP cationic phospholipid and amphiphilic diblock copolymer mPEG-PCL coated tumor cell lysate.
- 5. Use according to claim 1, characterized in that: the TLSV nanoparticle comprises the following specific components: 1 to 5 parts of DOTAP, 45 to 50 parts of amphiphilic mPEG-PCL diblock copolymer and 12 to 15 parts of tumor cell lysate.
- 6. Use according to claim 1, characterized in that: the tumor is colorectal cancer.
- 7. A TLSV nanoparticle/IL-17A mRNA complex characterized by: TLSV nanoparticles prepared by wrapping tumor cell lysates by DOTAP cationic phospholipid and amphiphilic diblock copolymer mPEG-PCL are obtained by electrostatic adsorption with IL-17A mRNA molecules.
- 8. A method for preparing a TLSV nanoparticle/IL-17A mRNA complex, which is characterized by comprising the following steps:a. re-suspending CT26 cell, freeze thawing repeatedly, crushing, centrifuging to collect supernatant to obtain cell lysate solution;b. weighing 45-50 parts of mPEG-PCL copolymer and 1-5 parts of DOTAP;c. dissolving mPEG-PCL copolymer and DOTAP in an organic phase solvent together, then adopting a rotary evaporator to operate for 40-45 min under the water bath condition of 55-60 ℃ so as to evaporate the solvent, and then adding 12-15 parts of cell lysate solution for hydration until complete dissolution to obtain TLSV nanoparticle solution;d. and incubating 30-40 parts of TLSV nanoparticle solution and 1-5 parts of IL-17A mRNA at room temperature for 15-30 min to obtain the TLSV nanoparticle/IL-17A mRNA compound.
- 9. The method of preparing a TLSV nanoparticle/IL-17A mRNA complex of claim 8, characterized in that: the solvent in the step c is at least one of dichloromethane, chloroform, acetone, tetrachloromethane, ethanol, methanol, ethyl acetate or cyclohexane; the organic phase solvent is at least one of ethanol, dichloromethane, chloroform, acetone, tetrachloromethane and methanol.
- 10. Use of the TLSV nanoparticle/IL-17A mRNA complex of claim 7 in the manufacture of a medicament for the prevention or treatment of tumors.
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