CN111303265A - One kind contains131I-labeled Caerin1.1 polypeptide and application thereof - Google Patents
One kind contains131I-labeled Caerin1.1 polypeptide and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a biological enzyme inhibitor131The polypeptide of Caerin1.1 marked by I and the application thereof. The invention provides131The Caerin1.1 polypeptide marked by the I is marked by adopting a chloramine-T iodine direct marking method, and the method has high marking rate and strong stability. The invention provides131The Caerin1.1 polypeptide marked by I is characterized in that the Caerin1.1 polypeptide has specificity and can enter tumor cells through the combination of membrane receptors and inhibit the growth and proliferation of the tumor cells,131after the I-Caerin1.1 specificity enters the tumor cells, the I-Caerin1.1 specificity can be utilized131I emission of gamma rays and use for tumor imaging, while also being usable131I launch β ray executionThe tumor internal irradiation treatment realizes the integration of tumor diagnosis and treatment, the visualization of the tumor treatment process improves the tumor treatment effect, and reduces the diagnosis and treatment cost of the tumor.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a biological enzyme inhibitor131The polypeptide of Caerin1.1 marked by I and the application thereof.
Background
The Caerin polypeptide is extracted from glandular secretion of back skin of Rana catesbiana (Litoria) in coastal rainforest of Australia and identifiedA series of polypeptides with biological activity, which contains 9 small molecule biological active peptides with different sequences (now collectively named as: Caerin 1.1-Caerin 1.9). the Caerin1.1 polypeptide (F1 polypeptide) used for the first time in the present study is a biologically active targeting polypeptide which can specifically bind to tumor cell membrane receptors and enter tumor cells, and can inhibit the growth and proliferation of various tumor cells (such as thyroid cancer, lung cancer, esophageal cancer, etc.) in vitro, and Caerin1.1 has more significant advantages than Caerin1.9 in the in vitro inhibition test of various tumor cells (see FIG. 1). in FIG. 1, A represents thyroid papilloma, B represents thyroid anaplastic cancer, C represents lung cancer, D represents esophageal cancer, and has no inhibition effect on normal mammalian cells under the same dosage. furthermore, the Caerin1.1 polypeptide can change several immune related proteins and immune related pathways, such as kinase and K signal pathway, and K signal pathway activation, and can also promote the inflammatory cell response of tumor cells, such as tumor cell migration promotion factor, such as Caerin 1.6, and can be adapted to the early stage of tumor cell migration promotion of immune response of tumor cell (see FIG. 369)125The mark I is marked with a mark I,125compared with the unlabeled Caerin polypeptide (Caerin 1.9, the polypeptide sequence: GLFGVLGSIAKHLLPHVVPVIAEKL-NH2, the invention patent number: ZL 201711167995.0), the I-Caerin polypeptide has better effect of inhibiting the growth of breast cancer cells and has tumor targeting property.
Radionuclide-labeled small-molecule polypeptide molecular probes are the hot spot of current nuclear medicine research, and recently, the radionuclide-labeled small-molecule polypeptides approved by FDA such as:68Ga-PSMA、177Lu-PSMA、177lu-dotatae and the like show good prospects in diagnosis and treatment of tumors. The radioactive iodine-131 is a cheap and easily available radionuclide which can be widely used for diagnosis and treatment in clinic at present, and can be used for integrated research of tumor diagnosis and treatment. Thyroid cancer is the most common malignant tumor in the endocrine system, among which differentiated thyroid cancer (differentiated thyroid cancer)DTC) accounts for about 90% or more, and while the 5-year survival rate of differentiated thyroid cancer is 90% or more, the 5-year survival rate of undifferentiated thyroid cancer is less than 10%, and the degree of malignancy is relatively high. And partially metastatic DTC patients are in early stage or131I, the iodine-uptake capacity is lost in the treatment process, so that the thyroid cancer (RAIR-DTC) which is difficult to treat by iodine develops, and the problem is especially troublesome in clinic at present. Patients with RAIR-DTC have fast disease progression and high mortality, and the search for effective treatment methods is always a research difficulty and a hotspot of thyroid cancer. ATA guidelines strongly recommend that once thyroid cancer patients are diagnosed as iodine refractory, they are no longer eligible to be given further iodine therapy. In recent years, the molecular mechanism of RAIR-DTC is researched, and many molecular targeted therapeutic drugs have wide application prospects. However, the existing benefit of the targeted drugs only improves the Progression Free Survival (PFS) of the patients, and can reduce the tumor to a certain extent, but no research of any targeted drug proves that the Overall Survival (OS) of the patients can be prolonged, and the methods all have adverse reactions with different degrees, once the patients are grouped into clinical experiments, the balance of risk and benefit is realized, which brings difficulty to the selection of the patients and the grasp of the indications.
Therefore, the continued search for new treatments has become a hot spot and difficulty in thyroid cancer, and the present study has thyroid cancer (papillary and undifferentiated carcinoma) as the main treatment131I-Caerin1.1 tumor model study paradigm, labeling of Caerin1.1 including iodine-131, and,131Tumor cell inhibition experiment of I-Caerin1.1 and131I-Caerin1.1 thyroid cancer tumor-bearing mice, showed that131The I-Caerin1.1 has better application prospect.
Disclosure of Invention
The purpose of the present application is to provide a container with the general disadvantages of the prior art131The peptide I marked Caerin1.1 has high purity, simple preparation process, easy storage and high stability after marking, and is expected to become a novel molecular probe for integrating diagnosis and treatment of tumors such as thyroid cancer and the like.
In order to achieve the purpose, the invention adopts the technical scheme that:
a kind of131The preparation process of the I-labeled Caerin1.1 polypeptide comprises the following steps:
s1, 40. mu.L of Caerin1.1 polypeptide solution was put in a 0.5mL EP tube, and 100. mu.L of Na was added131Adding 100 mu L of chloramine-T solution into the solution I to prepare a 240 mu L system to obtain a reaction solution;
s2, placing the reaction solution prepared in the step S1 in a vortex mixer to shake and react for 30min at the temperature of 25 ℃ to obtain a reaction mixture;
s3, separating the reaction mixture obtained in the step S2 by using a paper chromatography131And (3) the Caerin1.1 polypeptide of the I.
Preferably, the amino acid sequence information of the Carein1.1polypeptide is shown in SEQ ID NO. 1.
GLLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH2(SEQ ID NO.1)
Preferably, the131The preparation process of the I-labeled Caerin1.1 polypeptide, wherein the mass concentration of the Caerin1.1 polypeptide solution in the step S1 is 1 mg/mL; the Na is131When the ionizing radiation activity of the solution I is 1mci, the decay number of the nucleus is 3.7X 104kBq; the chloramine-T solution needs to be prepared just before use, and the mass concentration is 1 mg/mL.
The invention also provides the131The application of the I-labeled Caerin1.1 polypeptide in tumor diagnosis and treatment integration.
Tumor immunotherapy refers to killing tumors by the specificity of the patient's immune system and its effects, while the thyroid cancer microenvironment has abundant immune cells, which makes it the best choice for immunotherapy. Therefore, the Caerin1.1 polypeptide is selected for research, the primary structure of the polypeptide is shown as SEQ ID NO.1, and the polypeptide is simple to prepare, high in purity and easy to store. The structure of the compound contains histidine, and whether the compound can be carried out or not can be researched by a chloroammonia-T method131I marking, if marking is successful, then131The I-Caerin1.1 is expected to become a novel molecular probe for diagnosing and treating tumors such as thyroid cancer.
Compared with the prior art, the invention provides131The advantages of the I-labeled cairin 1.1polypeptide are:
(1) the invention provides a catalyst containing131The Caerin1.1 polypeptide marked by the I has high marking rate, high purity and low toxicity;
(2) the invention provides a catalyst containing131The Caerin1.1 polypeptide marked by the I contains histidine in the primary structure of the selected Caerin1.1 polypeptide, can be directly marked by a chloramine-T method, and has simple marking process and low cost;
(3) the invention provides a catalyst containing131The peptide of the Caerin1.1 marked by the I can inhibit the growth and proliferation of various tumor cells such as thyroid cancer, lung cancer, esophageal cancer and the like, and is expected to become a novel molecular probe for diagnosis and treatment of malignant tumors such as thyroid cancer and the like.
Drawings
FIG. 1 MTT inhibition assay of Caerin1.1, Caerin1.9 and P3 control peptides on different tumor cells;
FIG. 2 is a graph showing the survival of B-CPAP and CAL-62 cells at various concentrations of Caerin1.1 polypeptide;
FIG. 3 shows the inhibition of B-CPAP and CAL-62 cells at various gradient concentrations of Caerin1.1;
FIG. 4 shows confocal scan results of Caerin1.1 polypeptide and P3 control polypeptide;
FIG. 5 is a drawing showing131I, measuring the marking rate;
FIG. 6 is a drawing showing131I-the result of the stability determination of the Carein1.1 polypeptide;
FIG. 7 shows different radioactivity131I-Caerin1.1 and Na131I, survival of B-CPAP and CAL-62 cells;
FIG. 8 is a drawing showing131I-the results of the weight change of the mice bearing tumor of the Caerin1.1 polypeptide at different time;
FIG. 9 is a schematic view of131I-the results of tumor volume changes of the mice bearing tumor with the polypeptide of Caerin1.1 in different treatment times;
FIG. 10 is a drawing showing131I-the tumor weight of the Caerin1.1 polypeptide tumor-bearing nude mice after treatment;
FIG. 11 shows the HE staining results of the tumor tissues of each group after treatment.
Detailed Description
The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The Caerin1.1 polypeptide in this application was purified from the secretions of the dorsal glands of wood frogs in the coastal rainforest region of the east Australia, and the control polypeptide was a randomly designed P3 polypeptide, both of which were synthesized by Qiang Yao Bio Inc. of China, dissolved in PBS solution (GIBCO) to 10mg/mL, 1mg/mL, and 0.1mg/mL, respectively, and stored in a refrigerator at-20 ℃.
Experimental example 1 cell line and cell culture
B-CPAP human thyroid carcinoma cells B-CPAP (papillary) and CAL-62 human thyroid carcinoma cells CAL-62 (undifferentiated), both provided from the Chinese academy of Stem cell Bank, were cultured according to the methods described in the product literature. Wherein, the formula of the B-CPAP cell culture solution is as follows: 87% of RPMI Medium 1640(GIBCO), 10% of heat-inactivated fetal bovine serum from New Zealand, 0.1% of Penicilin-Streptomyces, liquid (GIBCO), 1% of MEM Non-essential amino Acids (NEAA, GIBCO), 1% of Glutamax (GIBCO), 1% of Sodium Pyruvate (GIBCO); the CAL-62 cell culture solution comprises the following components in percentage by weight: 90% DMEM Mediun (GIBCO), 10% heat-inactivated fetal bovine serum from New Zealand, 0.1% Penicilin-Streptomyces, liquid (GIBCO); both cells were incubated at 37 ℃ with 5% CO2Culturing in an incubator.
Experimental example 2 Caerin1.1 polypeptide cell proliferation experiment
The activity of B-CPAP cells and CAL-62 cells was assayed by 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid phenyl) -2H-tetrazole monosodium salt (CCK-8, DOJINDO) as described in the instructions: B-CPAP cells, CAL-62 cells, in logarithmic growth phase at 5X 10 per well3Per 100. mu.L cell concentration was plated in 96-well cell culture plates at 37 ℃ in 5% CO2Culturing for 24h until the cell aggregation rate is about 60-70%. The Caerin1.1 polypeptide and the control peptide P3 were added in different concentrations in groups to give final concentrations in the range of 0-17.5. mu.g/mL (0, 1.25, 2.5, 5, 7.5, 10, 12.5, 15, 17.5. mu.g/mL) in each well, while setting the zeroing well with 4 secondary wells per group and 5% CO at 37 ℃ in 5%2Culturing for 24h under the condition. The experiment was stopped by adding 10. mu.L of CCK-8 to each well and incubating for an additional 4-6 hours. Cell survival was determined using an enzyme linked immunosorbent assay to measure absorbance (OD) at 450 nm.
As shown in FIG. 2, the Caerin1.1 polypeptide starts to inhibit the proliferation of B-CPAP cell and CAL-62 cell (P < 0.01) at the concentration of 2.5. mu.g/mL and 5. mu.g/mL respectively compared with the concentration of 0. mu.g/mL, the survival rate is (84.98 +/-2.95)%, and (86.84 +/-4.99)%, respectively, and the inhibition effect is more and more obvious with the increase of the concentration of the Caerin1.1 polypeptide (FIGS. 2A-B); the inhibition effect of the Caerin1.1 polypeptide on B-CPAP cells and CAL-62 cells is different (P is less than 0.05) at the same concentration when the Caerin1.1 polypeptide is in the range of 2.5 mu g/mL-15 mu g/mL (FIG. 2C), and the proliferation inhibition effect of the Caerin1.1 polypeptide on B-CPAP cells is more obvious at the same concentration. However, both cells were substantially inactivated at 17.5. mu.g/mL of Caerin1.1 polypeptide, and there was no statistical difference between B-CPAP cells and CAL-62 cells (P > 0.05) (FIG. 2C), and the survival rates were (0.55. + -. 0.23)%, (4.81. + -. 1.09)%, respectively. The control P3 polypeptide had no significant inhibitory effect (P > 0.05) on B-CPAPcell and CAL-62 cell even at high concentrations (15. mu.g/mL, 17.5. mu.g/mL) (FIGS. 2A-B), and the survival rates were (98.18. + -. 3.39%) and (95.88. + -. 2.68%) respectively.
Experimental example 3 Caerin1.1 polypeptide IC50Measurement of
Logarithmic growth of B-CPAP cells and CAL-62 cells at 5X 10/well3 Per 100. mu.L cell concentration was plated in 96-well cell culture plates at 37 ℃ with 5% CO2The cells were cultured for 24h under conditions of (1) until they attached to the wall. The Caerin1.1 polypeptide was added to each well in a concentration gradient of fold relationship (0, 1.25, 2.5, 5, 10, 20. mu.g/mL) with zero-adjustment wells set, 4 secondary wells per group, and 5% CO at 37 deg.C2Culturing for 24h under the condition. The experiment was stopped by adding 10. mu.L of CCK-8 to each well and incubating for an additional 4-6 hours. Using enzyme linked immunosorbent assayThe cell survival was determined by measuring the absorbance (OD) at 450 nm. IC calculation Using GraphPad software50(half inhibitory concentration).
The results are shown in FIG. 3, from which the IC of B-CPAP is known504.038. mu.g/mL, CAL-62 IC509.856 μ g/mL. Caerin1.1 polypeptide IC from B-CPAP cells50This is lower than CAL-62 cells, which also demonstrates that the Caerin1.1 polypeptide has a more pronounced inhibitory effect on B-CPAP cells, consistent with the results described above for CCK-8.
Experimental example 4 confocal laser imaging
To observe that the Caerin1.1 polypeptide could be taken up by adenocarcinoma cells, Caerin1.1-FITC, P3-FITC, synthesized by Wuxi Myotu Biotech, Inc., was assigned to the polypeptide, and FITC green fluorescence was added thereto, followed by observation by laser confocal microscope (LSM 880Basic Operation). B-CPAP cells and CAL-62 cells were seeded onto 8-well chamber culture slides, respectively, in FBS-free medium at 37 ℃ in 5% CO2The cells were cultured for 24h under conditions until they attached. The Caerin1.1-FITC and the control P3-FITC were determined based on the Caerin1.1 IC of each cell50The final concentration (5. mu.g/mL for B-CPAP cells and 10. mu.g/mL for CAL-62 cells) was added to each well and incubated for 2h in the dark. The supernatant was aspirated, and each well was gently washed with a suitable Wash Buffer (90% PBS + 10% FBS-free medium) for a total of 5 times. The supernatant was removed and dried, each well was covered with a suitable Mounting Medium (with DAPI) (solarbio), and after the chamber frame was removed, the slide was dried and covered with a cover slip. In confocal imaging, wavelengths of 405nm and 488nm are respectively used as exciting light, fluorescence signals of FITC and DAPI are detected, and confocal laser scanning imaging is carried out on cells to detect whether the Caerin1.1 polypeptide can enter thyroid cancer cells.
The results are shown in FIG. 4, from which it can be seen that at 2h, a green fluorescence signal of Caerin1.1-FITC is observed in the cytoplasm of B-CPAP cells and CAL-62 cells, wherein DAPI is a blue fluorescence signal bound to the nucleus. Whereas no significant fluorescence signal was seen in the cytoplasm and only a very weak fluorescence signal was seen in the control group P3-FITC.
Experimental example 5131Obtaining of I-tagged Caerin1.1 Polypeptides
The above-mentioned131The preparation process of the I-labeled Caerin1.1 polypeptide comprises the following steps:
s1, taking 40 mu L of Caerin1.1 polypeptide solution with the mass concentration of 1mg/mL into a 0.5mL EP tube, adding 100 mu L of Na131Solution I (1 mci/3.7X 10)4kBq)), then adding 100 μ L chloramine-T solution (with the mass concentration of 1mg/mL) to prepare 240 μ L system to obtain reaction solution; the primary structure of the Carein1.1polypeptide is as follows: GLLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH 2;
s2, placing the reaction solution prepared in the step S1 in a vortex mixer to shake and react for 30min at the temperature of 25 ℃ to obtain a reaction mixture;
s3, separating the reaction mixture obtained in the step S2 by using a paper chromatography131And (3) the Caerin1.1 polypeptide of the I.
Experimental example 6131I mark rate determination
Mixing 3 μ L131I-Caerin1.1 spotting on stationary phase Xinhua No. I chromatographic paper (10 x 120mm) with the length of 12cm, placing the stationary phase Xinhua No. I chromatographic paper in mobile phase physiological saline for development after air drying, taking out the stationary phase Xinhua No. I chromatographic paper for air drying after the development, cutting the stationary phase Xinhua No. I chromatographic paper at intervals of 1cm, measuring radioactivity gamma section by section, and counting; radioactivity gamma counting curves were plotted using GraphPad software and the labeling rate was calculated. Labeling rate-integration of radioactive peak area at zero/total radioactive peak area. This experiment was repeated three times.
The results are shown in FIG. 5, from which it can be seen that in the development system in which the stationary phase was Xinhua No. I chromatography paper and the mobile phase was physiological saline, the free phase was released131The specific implantation (RF) of I is about 0.9-1.0,131RF of I-Caerin1.1 is about 0.0-0.1, so131The marking rate of I is 92.25-98.25%.
Experimental example 7131Stability assay for I-carein1.1 Polypeptides
Measurement of131I-Caerin1.1 stability at room temperature (25 ℃) and 37 ℃ for 72h, respectively. At the same time, measuring131I-Caerin1.1 stability of FBS and NS at 72h in room temperature (25 ℃) and 37 ℃ respectively. Respectively taking 120 mu L131Placing the I-Caerin1.1 in a water bath kettle at room temperature and 37 ℃; respectively taking 20 mu L131The I-Caerin1.1 is placed in 100 mu LFBS (room temperature, 37 ℃ C.), 100. mu. LNS (room temperature, 37 ℃ C.). Small samples were taken at different time points of 0h, 12h, 24h, 48h and 72h, and the respective radiochemical purity (RCP) was determined by paper chromatography, and the stability was evaluated by the change in the radiochemical purity. Radiochemical purity and curve drawing were calculated using GraphPad software.
The results of the experiment are shown in FIG. 6, and the measurement is carried out131I-Caerin1.1 was placed in 25 ℃/37 ℃ for 72h of RCP, and in 25 ℃/37 ℃ FBS/NS for 72h of RCP to evaluate its stability. Similarly, we measured RCP using paper chromatography. Within 72h at room temperature (25 ℃),131RCP of I-Caerin1.1 is (85.76 +/-14.80)%; within 72 hours at the temperature of 37 ℃,131RCP of I-Caerin1.1 is (77.35 +/-13.24)%; in 72h of room temperature (25 ℃) FBS,131RCP of I-Caerin1.1 is (92.01 +/-8.768)%; in 72h at 37 ℃ of FBS,131RCP of I-Caerin1.1 is (90.24 + -8.588)%; in 72h at room temperature (25 ℃) in NS,131RCP of I-Caerin1.1 is (91.41 + -8.762)%; within 72h at 37 ℃ NS,131RCP of I-Caerin1.1 was (91.33. + -. 5.399)%. As described above, in both room temperature (25 ℃ C.), 37 ℃ C., and room temperature (25 ℃ C.), 37 ℃ FBS or NS,131I-Caerin1.1 has better stability (FIGS. 6A-B).
EXAMPLE 8 fat and Water partition coefficient (logP)
The water and fat distribution coefficient experiment can judge131I-Caerin1.1 is hydrophilic or lipophilic and predicts its excretion pathway in vivo. The operation process is as follows: 500. mu.L of n-octanol, 500. mu.LNS and 40. mu.L of the mixture were added to a 1.5mL centrifuge tube131I-Caerin1.1, sealing, shaking for 2min, and centrifuging for 5min (4000rpm) to make the two phases of n-octanol and NS reach an equilibrium state. 100. mu.L of each of the organic and aqueous phases was sampled and placed in two gamma counting tubes for gamma counting.
Calculating the formula: LogP ═ Log [ (n-octanol γ count-background mean γ count)/NS γ count-background mean γ count) ]
The results are shown in Table 1.
TABLE 1131Lipid-water partition coefficient of I-Caerin1.1 polypeptide
As can be seen from table 1, the fat-water distribution coefficient Log P is 0.827 ± 0.036(n is 3), which is illustrated131I-Caerin1.1 exhibits lipophilicity.
Experimental example 9131I-Caerin1.1 polypeptide cell proliferation assay
Logarithmic growth of B-CPAP cells and CAL-62 cells at 5X 10/well3 Per 100. mu.L cell concentration was plated in 96-well cell culture plates at 37 ℃ with 5% CO2Culturing for 24h under the condition until the cell aggregation rate is 70%. Will be provided with131I-Caerin1.1 and free131I was added in different concentration groups to each well (0, 2000, 4000, 8000, 16000kBq/mL) while setting the zeroing well, 3 secondary wells each, and 5% CO at 37 deg.C2Culturing for 24h under the condition. The experiment was stopped by adding 10. mu.L of CCK-8 to each well and incubating for an additional 5 hours. Cell survival was determined using an enzyme linked immunosorbent assay to measure absorbance (OD) at 450 nm.
As shown in FIG. 7, it was found that, when the radioactive concentration was compared with 0kBq/mL,131I-Caerin1.1 started to inhibit proliferation of B-CPAP cells and CAL-62 cells (P < 0.001) at concentrations of 2000kBq/mL and 4000kBq/mL, respectively, with survival rates of (84.17 + -4.48)%, (64.24 + -3.23)%, respectively, and with increasing concentrations131The inhibitory effect was also more and more evident with increasing I-Caerin1.1 radioactive concentration (FIGS. 7A-B). When in use131I-Caerin1.1 in the range of 2000kBq/mL-4000kBq/mL, the inhibition of B-CPAP cells and CAL-62 cells was different at the same concentration (P < 0.01) (FIG. 7C),131the proliferation inhibition effect of the I-Caerin1.1 on B-CPAP cells is more obvious under the same concentration. But do not131At 8000kBq/mL of I-Caerin1.1, B-CPAP cells and CAL-62 cells were not substantially viable, survival rates were (8.96. + -. 0.21)%, (9.16. + -. 2.23)%, respectively, and there was no statistical difference between cells (P > 0.05) (FIG. 7C). Control group free131I has obvious inhibition effect (P < 0.01) on the B-CPAP cell and CAL-62 cell only at 8000-.
Experimental example 10131Thyroid cancer tumor-bearing mouse animal experiment of I-Caerin1.1 polypeptide
10.1 Experimental animals
BALB/c female nude mice were purchased from Guangdong province medical laboratory animal center (license number: SCYK (Yue) 2018-. The total 12 mice are aged for 3-4 weeks, the weight is about 9-12 g, and all the mice are raised in an experimental animal center (license number: SYXK (Guangdong) 2017-0124) according to the cleaning-grade animal management standard. All experiments were approved and performed as directed by the ethical committee on animal experiments. The treatment of the mice in the experimental process meets the standard of guidance opinions about animals to be tested, which is published by the science and technology department of the people's republic of China.
10.2 establishment of CAL-62 tumor-bearing nude mouse subcutaneous model
The tumor nude mouse model is established by using a human undifferentiated thyroid cancer CAL-62 cell strain. Culturing CAL-62 cells to logarithmic growth phase, washing with PBS buffer solution for 2 times, digesting with 0.25% trypsin and collecting cells, washing with PBS for 2 times, and diluting with PBS to obtain cell suspension 12.5 × 106And (4) placing the seeds/mL on an ice box for storage. Feeding BALB/c female to 5-6 weeks, weighing about 14-16g, inoculating CAL-62 cells subcutaneously into right axilla of nude mice with 1mL syringe, each nude mouse inoculating about 2.5X 10 cells6200 μ L. The ordinary feeding water is changed into KI feeding water with the concentration of 0.1 percent, and the thyroid gland is sealed until the experiment is finished.
10.3 CAL-62 tumor-bearing nude mouse tumor growth inhibition experiment
After solid tumors grew on day 4, tumor growth inhibition experiments were performed.
12 tumor-bearing nude mice were randomly divided into 4 large groups (experimental group:131I-Caerin1.1 group; control group one: group Caerin 1.1; control group two: na (Na)131Group I; control group three: PBS group), were all intratumorally injected with disposable insulin needles.
Injection for experimental group131I-Caerin1.1350mci/120 mL/one control group, Caerin1.120 mu g/120 mL/one control group is required to be injected according to the amount of the experimental group, and Na with equal activity is injected into the control group131I350mci/120 mL/bodyAnd three control groups are injected with 120 mL/body of PBS buffer solution with the same volume. After the first injection, injections were repeated every other day for a total of 3 injections. On day 7 after the start of the treatment, each group of tumor-bearing nude mice was sacrificed by cervical dislocation, and tumor tissues were completely dissected and separated, and weighed.
After the first intratumoral injection, the state and the activity form of the nude mice were observed every day. The body weight and tumor size of the nude mice were measured and recorded before each injection, and the long and short diameters of the tumors were measured with a vernier caliper for 7 days. The method for calculating the tumor volume is as follows: v is long diameter x short diameter2×1/2。
10.4 weight changes of CAL-62 tumor-bearing nude mice before and after treatment
The body weight changes of the tumor-bearing nude mice in each group are shown in Table 2 and FIG. 8. Before treatment, PBS group, Na131Group I,131The weights of tumor-bearing nude mice in the I-Caerin1.1 group and the Caerin1.1 group are respectively (15.73 +/-0.38) g, (15.03 +/-0.96) g, (15.07 +/-0.12) g and (15.97 +/-0.71) g, and the difference between the groups has no statistical significance (P is more than 0.05); the change in body weight of each group was not obvious with the treatment time, and by day 7, PBS group and Na were added131Group I,131The weights of tumor-bearing nude mice in the I-Caerin1.1 group and the Caerin1.1 group are respectively (17.23 + -0.46) g, (16.10 + -0.78) g, (17.07 + -0.38) g and (17.83 + -0.91) g, and the difference between each group and the PBS group is not statistically significant (P is more than 0.05).
TABLE 2 CAL-62 tumor-bearing nude mice treated for different periods of time for weight change (g)
10.5 CAL-62 tumor growth in nude mice bearing tumor
The growth of tumor in each group of tumor-bearing nude mice is shown in Table 3 and FIG. 9. Before treatment, PBS group, Na131Group I,131The tumor volumes of the I-Caerin1.1 group and the Caerin1.1 group of tumor-bearing nude mice are respectively (39.77 +/-6.78) mm3，(46.21±14.52)mm3，(67.87±27.45)mm3And (40.67. + -. 14.82) mm3The difference between the groups has no statistical significance (P is more than 0.05); PBS group and Na with increasing treatment time131The tumor volume of the tumor-bearing nude mice in the group I is gradually increased,131the tumor volumes of the I-Caerin1.1 group and the Caerin1.1 group tumor-bearing nude mice gradually decrease until the 7 th day, the PBS group and Na131Group I,131The tumor volumes of the I-Caerin1.1 group and the Caerin1.1 group of tumor-bearing nude mice are respectively (59.36 +/-11.37) mm3，(66.29±5.94)mm3，(8.79±2.94)mm3And (22.75. + -. 2.18) mm3，131The I-Caerin1.1 group had statistical significance (P < 0.01) in comparison with the PBS group,131I-Caerin1.1 group with Na131The group I contrast has statistical significance (P is less than 0.001), the Caerin1.1 contrast with the PBS contrast has statistical significance (P is less than 0.05), Na131Group I PBS group comparison, the difference was not statistically significant (P > 0.05). In FIG. 9, it indicates that the difference is statistically significant (P < 0.05); indicates that the difference is statistically significant (P < 0.01); indicates that the difference is statistically significant (P < 0.001).
TABLE 3 CAL-62 tumor-bearing nude mice treated for different time periods for tumor volume change (mm)3)
Note: indicates that the difference was statistically significant (P < 0.05) compared to the PBS group; indicates that the difference was statistically significant (P < 0.01) compared to the PBS group.
10.6 tumor weight status of CAL-62 tumor-bearing nude mice after treatment
The tumor weight of each group of tumor-bearing nude mice after complete dissection and separation is shown in Table 4 and FIG. 10. PBS group, Na131Group I,131The tumor weights of the tumor-bearing nude mice in the I-Caerin1.1 group and the Caerin1.1 group were (21.60 + -3.86) g, (21.93 + -4.23) g, (16.43 + -6.55) g, and (17.57 + -4.90) g, respectively, and the difference between the groups was not statistically significant (P > 0.05).
TABLE 4 weight (g) of tumors in CAL-62 tumor-bearing nude mice after treatment
| Group of | Weight (g) (mean. + -. standard deviation) |
| PBS | 21.60±3.86 |
| Na131I | 21.93±4.23 |
| 131I-Caerin1.1 | 16.43±6.55 |
| Caerin1.1 | 17.57±4.90 |
The tumor growth results of tumor-bearing nude mice show that PBS group and Na group are added along with the prolonging of the treatment time131The tumor volume of the group I was gradually increased,131the tumor volumes of the I-Caerin1.1 group and the Caerin1.1 group of tumor-bearing nude mice are gradually reduced,131the tumor volume of the group I-Caerin1.1 is reduced obviously. When the treatment is finished, the medicine is put into the oral cavity,131the I-Caerin1.1 group had statistical significance (P < 0.01) in comparison with the PBS group,131I-Caerin1.1 group with Na131Group I comparison shows that the difference is statistically significant (P is less than 0.001). The experimental results show that the high-temperature-resistant steel,131I-Caerin1.1 has obvious inhibition effect on the growth of undifferentiated thyroid cancer tumor, and131the treatment effect of the I-Caerin1.1 is more obvious than that of the group Caerin1.1.
10.7CAL-62 tumor tissue HE staining
The results of HE staining of tumor tissue after treatment in each group are shown in fig. 11. Fig. 11 (a):131the tumor cells of the I-Caerin group have large sheet necrosis, lose normal cell morphology and tissue structureThe damage and the infiltration of the neutrophils are obvious. Fig. 11 (B): the Caerin group can be seen in tumor cell necrosis, obvious chromatin condensation and neutrophil infiltration; FIG. 11 (C-D): na (Na)131In the group I and the PBS, the tumor tissue structure is compact, the cells are generated diffusely, the cell nucleuses are different in size, and a small amount of neutrophil infiltration can be seen. The results of HE staining of the tumor tissue show that,131compared with other treatment groups, the I-Caerin tumor tissue has obvious cell necrosis, loses normal cell morphology, has damaged tissue structure and obvious infiltration of neutrophils, and prompts that tumor cells are obviously damaged and die; the inhibition effect of Caerin group tumor is better131The I-Caerin group is small, which shows131The treatment effect of the I-Caerin on tumor tissues is more obvious; and Na131No significant tumor cell destruction was seen in group I and PBS.
In summary, the experimental results in this section show that,131the I-Caerin has tumor targeting property, can be combined with thyroid cancer cells in a targeted manner, is retained in cells, has a cytotoxic killing effect, and has obvious inhibition and treatment effects on the tumor growth of thyroid undifferentiated cancer.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Sequence listing
<110> Zhongao biomedical technology (Guangzhou) Co., Ltd
<120> Caerin1.1 polypeptide containing 131I mark and application thereof
<130>2020.2.27
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>29
<212>PRT
<213> Amino acid sequence of Carein1.1polypeptide (Amino acid sequence of Carein1.1polypeptide)
<400>1
Gly Leu Leu Ser Val Leu Gly Ser Val Ala Lys His Val Leu Pro His
1 5 10 15
Val Leu Pro His Val Val Pro Val Ile Ala Glu His Leu
20 25
Claims (4)
1. A kind of131The peptide of the Caerin1.1 marked I is characterized in that the preparation process comprises the following steps:
s1, 40. mu.L of Caerin1.1 polypeptide solution was put in a 0.5mL EP tube, and 100. mu.L of Na was added131Adding 100 mu L of chloramine-T solution into the solution I to prepare a 240 mu L system to obtain a reaction solution;
s2, placing the reaction solution prepared in the step S1 in a vortex mixer to shake and react for 30min at the temperature of 25 ℃ to obtain a reaction mixture;
s3, separating the reaction mixture obtained in the step S2 by using a paper chromatography131And (3) marked Caerin1.1 polypeptide of I.
2. The method of claim 1131The I-labeled Caerin1.1 polypeptide is characterized in that the amino acid sequence information of the Carein1.1polypeptide is shown as SEQ ID NO. 1.
3. The method of claim 1131I-labeled Caerin1.1 polypeptide, characterized in that131The preparation process of the I-labeled Caerin1.1 polypeptide, wherein the mass concentration of the Caerin1.1 polypeptide solution in the step S1 is 1 mg/mL; the Na is131When the ionizing radiation activity of the solution I is 1mci, the decay number of the nucleus is 3.7X 104kBq; the chloramine-T solution needs to be prepared just before use, and the mass concentration is 1 mg/mL.
4. A process as claimed in any one of claims 1 to 3131Tumor-associated peptide of Caerin1.1 labeled IApplication in diagnosis and treatment integration.
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