CN116751745A - Combined immune cell exosome polypeptide regeneration factor and application thereof - Google Patents

Combined immune cell exosome polypeptide regeneration factor and application thereof Download PDF

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CN116751745A
CN116751745A CN202310993902.9A CN202310993902A CN116751745A CN 116751745 A CN116751745 A CN 116751745A CN 202310993902 A CN202310993902 A CN 202310993902A CN 116751745 A CN116751745 A CN 116751745A
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immune cell
immune
serum
cancer
culture
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杨晓晨
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Beijing Shengmei Cell Life Science Engineering Research Institute Co ltd
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Beijing Shengmei Cell Life Science Engineering Research Institute Co ltd
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Abstract

The application relates to a combined immune extracellular body polypeptide regeneration factor and application thereof, relates to the technical field of cell therapy, has obvious inhibition effect on thyroid cancer, lung cancer, gastric cancer, pancreatic cancer, liver cancer and other tumor cells, has obvious and broad-spectrum anti-tumor activity, provides a new thought for developing broad-spectrum anti-cancer drugs capable of effectively treating various cancers, and has practical application value.

Description

Combined immune cell exosome polypeptide regeneration factor and application thereof
Technical Field
The application relates to the technical field of cell therapy, in particular to a combined immune cell exosome polypeptide regeneration factor and application thereof.
Background
Cancer cells are routinely produced in our body but are constantly destroyed by the healthy immune system, and cancerous tumors form when the immune system fails to destroy these regularly formed diseased cells. Cancer has become a major public health problem worldwide, severely threatening human health, and morbidity and mortality continue to rise. Among them, thyroid cancer, lung cancer, gastric cancer, pancreatic cancer and liver cancer are all high-incidence cancers, and have a tendency to be increasing year by year.
At present, the cancer is treated by adopting the traditional treatment method mainly comprising operation, radiotherapy and chemotherapy, but the treatment effect is not optimistic, and the high treatment cost also becomes a major difficulty for families of patients. The advent of targeted drugs has provided new options for tumor treatment, but clinical resistance issues have emerged. Therefore, it is quite significant to develop a novel broad-spectrum anticancer drug capable of effectively treating various cancers.
Disclosure of Invention
In order to solve the problems, the application provides a combined immune exosome polypeptide regeneration factor and application thereof, wherein the combined immune exosome polypeptide regeneration factor has obvious inhibition effect on thyroid cancer, lung cancer, gastric cancer, pancreatic cancer, liver cancer and other tumor cells, has obvious and broad-spectrum anti-tumor activity, provides a new thought for developing broad-spectrum anti-cancer drugs capable of effectively treating various cancers, and has practical application value.
In a first aspect, the present application provides a combined immune cell exosome polypeptide regenerating factor, and the preparation method of the combined immune cell exosome polypeptide regenerating factor includes the following steps:
adding peripheral blood mononuclear cells into a serum-free immune cell primary culture medium for first-stage amplification culture to obtain primarily amplified immune cells;
adding the immune cells subjected to primary amplification into a serum-free immune cell induction culture medium to perform second-stage induction amplification culture to obtain induction amplified immune cells;
adding the immune cells induced to be amplified into a serum-free immune cell activating culture medium to perform activating amplification culture at a third stage to obtain activated and amplified immune cells;
culturing the immune cells activated and amplified in a fourth stage to obtain a culture solution;
separating and extracting the culture solution to obtain the combined immune cell exosome polypeptide regeneration factor;
wherein, the serum-free immune cell primary culture medium comprises the following components: IL2600-1000U/ml, IL101200-1500U/ml, LIF0.5-2.5ng/ml, gamma-interferon 400-600U/ml and laminin 5-10ug/ml;
the serum-free immune cell induction medium comprises the following components: IL2600-1000U/ml, IL101200-1500U/ml, bFGF2-4ng/ml, BMP-40.5-0.8ug/ml, sarhillin 0.003-0.005KE/ml and laminin 5-10ug/ml;
the serum-free immune cell activation medium comprises the following components: IL2600-1000U/ml, IL101200-1500U/ml, TGF-beta 1-2ng/ml, resveratrol 40-60ng/ml and laminin 5-10ug/ml.
Further, the step of the first-stage amplification culture includes: after the peripheral blood mononuclear cells are added into the serum-free immune cell primary culture medium, the culture medium is placed in a 37 ℃ incubator for 12 hours, and then the adherent cell impurities are removed to complete the subculture.
Further, the step of the second-stage amplification culture includes: and adding the immune cells which are primarily amplified into a serum-free immune cell induction culture medium, and then placing the immune cells in a 37 ℃ incubator for culturing for 10-14 days, wherein the immune cells are passaged every 2 days.
Further, the step of the third-stage amplification culture includes: after the immune cells after induced amplification are added into serum-free immune cell activation culture medium, the immune cells are placed in a 37 ℃ incubator for culture for 10-14 days, and the immune cells are passaged every 2 days.
Further, the step of fourth stage amplification culture includes: and continuously placing the activated and amplified immune cells in a 37 ℃ incubator for monitoring, and supplementing liquid according to the monitored cell morphology until the immune cells are amplified to the standard cell morphology, thereby obtaining the final culture liquid.
Further, the step of separating and extracting the culture solution to obtain the combined immune cell exosome polypeptide regeneration factor comprises the following steps: and (3) sequentially centrifuging the culture solution, then adopting an affinity chromatography column to purify the protein, and collecting eluent to obtain the combined immune cell exosome polypeptide regeneration factor.
Further, the serum-free immune cell primary culture medium comprises the following components: IL2800U/ml, IL101350U/ml, LIF1.5ng/ml, gamma interferon 500U/ml and laminin 8ug/ml;
the serum-free immune cell induction medium comprises the following components: IL2800U/ml, IL101350U/ml, bFGF3ng/ml, BMP-40.7ug/ml, sarhillin 0.004KE/ml and laminin 8ug/ml;
the serum-free immune cell activation medium comprises the following components: IL2800U/ml, IL101350U/ml, TGF-. Beta.1.5 ng/ml, resveratrol 50ng/ml and laminin 8ug/ml.
In a second aspect, the present application provides the use of a combination immune exosome polypeptide regenerating factor according to any one of the first aspects in the manufacture of a medicament for the treatment, and/or prevention, of a neoplastic disease; the tumor disease comprises at least one of thyroid cancer, lung cancer, gastric cancer, pancreatic cancer and liver cancer.
In a third aspect, the present application provides an oral enteric capsule for the treatment of a neoplastic disease, the active ingredients of the oral enteric capsule comprising the combination immune extracellular exosome polypeptide regenerating factor of any one of the first aspects; the tumor disease comprises at least one of thyroid cancer, lung cancer, gastric cancer, pancreatic cancer and liver cancer.
In a fourth aspect, the present application provides a lyophilized powder formulation for use in the treatment of a neoplastic disease, the active ingredients of the lyophilized powder formulation comprising the combination immune extracellular exosome polypeptide regenerating factor of any one of the first aspects; the tumor disease comprises at least one of thyroid cancer, lung cancer, gastric cancer, pancreatic cancer and liver cancer.
Compared with the prior art, the technical scheme provided by the embodiment of the application has at least the following advantages:
the embodiment of the application provides a combined immune cell exosome polypeptide regeneration factor and application thereof, wherein the combined immune cell exosome polypeptide regeneration factor is prepared by adding specific culture components into a first-stage amplification culture, a second-stage induction amplification culture and a third-stage activation amplification culture respectively, and finally separating and extracting the obtained culture solution. The combined immune extracellular exosome polypeptide regeneration factor has obvious inhibition effect on thyroid cancer, lung cancer, gastric cancer, pancreatic cancer, liver cancer and other tumor cells, provides a new thought for developing broad-spectrum anticancer drugs capable of effectively treating various cancers, and has practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the application and together with the description, serve to explain the principles of the application.
In order to more clearly illustrate the embodiments of the application or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, and it will be obvious to a person skilled in the art that other drawings can be obtained from these drawings without inventive effort.
FIG. 1 is a schematic flow chart of a method for preparing a combined immune extracellular exosome polypeptide regenerating factor according to an embodiment of the present application;
FIG. 2 is a graph showing the results of pre-treatment tests for patients with gastric cancer in exemplary case 2 of the present application;
FIG. 3 is a graph showing the results of pre-treatment tests for gastric cancer patients in exemplary case 2 of the present application;
FIG. 4 is a graph showing the results of pre-treatment tests for lung cancer patients in exemplary case 3 of the present application;
FIG. 5 is a graph showing the results of pre-treatment tests for lung cancer patients in exemplary case 3 of the present application.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more apparent, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present application are commercially available or may be prepared by existing methods.
In a first aspect, the present application provides a method for preparing a composite immune extracellular exosome polypeptide regenerating factor, as shown in fig. 1, comprising the following steps:
adding peripheral blood mononuclear cells into a serum-free immune cell primary culture medium for first-stage amplification culture to obtain primarily amplified immune cells;
adding the immune cells subjected to primary amplification into a serum-free immune cell induction culture medium to perform second-stage induction amplification culture to obtain induction amplified immune cells;
adding the immune cells induced to be amplified into a serum-free immune cell activating culture medium to perform activating amplification culture at a third stage to obtain activated and amplified immune cells;
culturing the immune cells activated and amplified in a fourth stage to obtain a culture solution;
separating and extracting the culture solution to obtain the combined immune cell exosome polypeptide regeneration factor;
wherein, the serum-free immune cell primary culture medium comprises the following components: IL2600-1000U/ml, IL101200-1500U/ml, LIF0.5-2.5ng/ml, gamma-interferon 400-600U/ml and laminin 5-10ug/ml;
the serum-free immune cell induction medium comprises the following components: IL2600-1000U/ml, IL101200-1500U/ml, bFGF2-4ng/ml, BMP-40.5-0.8ug/ml, sarhillin 0.003-0.005KE/ml and laminin 5-10ug/ml;
the serum-free immune cell activation medium comprises the following components: IL2600-1000U/ml, IL101200-1500U/ml, TGF-beta 1-2ng/ml, resveratrol 40-60ng/ml and laminin 5-10ug/ml.
The embodiment of the application provides a combined immune cell exosome polypeptide regeneration factor and application thereof, wherein the combined immune cell exosome polypeptide regeneration factor is obtained by adding specific culture components into a first stage of amplification culture, a second stage of induction amplification culture and a third stage of activation amplification culture respectively, performing amplification culture on peripheral blood mononuclear cells through a specific culture medium to obtain DC cells, NK cells, T cells and B cells, continuing to culture for a period of time, and finally separating and extracting the obtained culture solution. The combined immune extracellular exosome polypeptide regeneration factor has obvious inhibition effect on thyroid cancer, lung cancer, gastric cancer, pancreatic cancer, liver cancer and other tumor cells, provides a new thought for developing broad-spectrum anticancer drugs capable of effectively treating various cancers, and has practical application value.
The peripheral blood mononuclear cells can be directly purchased as a commercial product, and can also be prepared from fresh blood collected from a human body according to the existing separation preparation method of the peripheral blood mononuclear cells.
In the first stage amplification culture, the serum-free immune cell primary culture medium is obtained by adding IL2600-1000U/ml, IL101200-1500U/ml, LIF0.5-2.5ng/ml, gamma-interferon 400-600U/ml and laminin 5-10ug/ml into the existing commercial T cell basic culture medium. Preferably, the serum-free immune cell primary culture medium comprises the following components: IL2800U/ml, IL101350U/ml, LIF1.5ng/ml, gamma interferon 500U/ml and laminin 8ug/ml.
In the second stage induction amplification culture, the serum-free immune cell induction culture medium is obtained by adding 'IL 2600-1000U/ml, IL101200-1500U/ml, bFGF2-4ng/ml, BMP-40.5-0.8ug/ml, sarhillin 0.003-0.005KE/ml and laminin 5-10 ug/ml' into the existing commercial T cell basic culture medium. Preferably, the serum-free immune cell induction medium comprises the following components: IL2800U/ml, IL101350U/ml, bFGF3ng/ml, BMP-40.7ug/ml, sarhillin 0.004KE/ml and laminin 8ug/ml.
In the third stage of induced amplification culture, the serum-free immune cell activating culture medium is obtained by adding 'IL 2600-1000U/ml, IL101200-1500U/ml, TGF-beta 1-2ng/ml, resveratrol 40-60ng/ml and laminin 5-10 ug/ml' into the existing commercial T cell basic culture medium. Preferably, the serum-free immune cell activation medium comprises the following components: IL2800U/ml, IL101350U/ml, TGF-. Beta.1.5 ng/ml, resveratrol 50ng/ml and laminin 8ug/ml.
In some embodiments, the step of first stage amplification culture comprises: after the peripheral blood mononuclear cells are added into the serum-free immune cell primary culture medium, the culture medium is placed in a 37 ℃ incubator for 12 hours, and then the adherent cell impurities are removed to complete the subculture.
In some embodiments, the step of second stage amplification culture comprises: and adding the immune cells which are primarily amplified into a serum-free immune cell induction culture medium, and then placing the immune cells in a 37 ℃ incubator for culturing for 10-14 days, wherein the immune cells are passaged every 2 days.
In some embodiments, the step of third stage amplification culture comprises: after the immune cells after induced amplification are added into serum-free immune cell activation culture medium, the immune cells are placed in a 37 ℃ incubator for culture for 10-14 days, and the immune cells are passaged every 2 days.
In some embodiments, the step of fourth stage amplification culture comprises: continuously placing the activated and amplified immune cells in a 37 ℃ incubator for monitoring, and supplementing liquid according to the monitored cell morphology until the immune cells are amplified to the standard cell morphology, so as to obtain a final culture solution; in some embodiments, among others, standard cell morphology may be understood as standard mature T cell morphology.
In some embodiments, the step of separating and extracting the culture solution to obtain the combined immune cell exosome polypeptide regeneration factor comprises the following steps: and (3) sequentially centrifuging the culture solution, then adopting an affinity chromatography column to purify the protein, and collecting eluent to obtain the combined immune cell exosome polypeptide regeneration factor. Specifically, the following procedure may be included:
centrifuging the culture solution to obtain a supernatant; and then, carrying out balancing by using a MabSelectTMStereTM affinity chromatography column and using 5 column volumes of balancing buffer solution, loading the sample subjected to the primary purification in the step 1 onto the affinity chromatography column, eluting by using 5 column volumes of eluting buffer solution, collecting the obtained target protein eluate by using a container with a pre-placed neutralizing solution, and keeping the pH value of the neutralized target protein solution to be 6.5-8.0. Regenerating by using 5 column volumes of regeneration liquid, cleaning the chromatographic column by using 3 column volumes of cleaning liquid, balancing the chromatographic column by using 5 column volumes of balancing buffer liquid, and preserving the chromatographic column by using preserving liquid; wherein, the balance buffer: 20mM Tris-HCl,150mMNaCl,pH7.2; eluting buffer: 20mM Tris-HCl,150mMNaCl,pH7.2; elution buffer: 100mM citrate buffer, 0.1M sucrose, pH3.4; and (3) neutralizing liquid: 1MTris-HCl, pH8.5; regeneration liquid: 100mM HOC; cleaning liquid: 0.1MNaOH; preservation solution: 20% ethanol by volume.
It should be noted that, the component materials involved in the preparation method of the polypeptide regeneration factor of the combined immune exosome provided in the embodiment of the application are not particularly limited or described, and all components such as IL2 (interleukin-2), IL10 (interleukin 10), LIF (leukemia inhibitory factor), gamma-interferon, laminin, bFGF (basic fibroblast growth factor), BMP-4 (bone morphogenetic protein 4), sarbanlin, TGF-beta (transforming growth factor-beta), resveratrol and the like can be directly commercially available products. Meanwhile, the operation steps involved in the preparation method of the polypeptide regeneration factor of the combined immune cell exosome can be carried out according to the conventional operation mode in the field unless special description or specific limitation is provided, and the document of the application is not repeated.
In a second aspect, the present application provides the use of a combination immune exosome polypeptide regenerating factor according to any one of the first aspects in the manufacture of a medicament for the treatment, and/or prevention, of a neoplastic disease; the tumor disease comprises at least one of thyroid cancer, lung cancer, gastric cancer, pancreatic cancer and liver cancer.
The combined immune cell exosome polypeptide regeneration factor provided by the application has obvious inhibition effect on thyroid cancer, lung cancer, gastric cancer, pancreatic cancer, liver cancer and other tumor cells, provides a new thought for developing broad-spectrum anticancer drugs capable of effectively treating various cancers, and has practical application value.
In a third aspect, the present application provides an oral enteric capsule for the treatment of a neoplastic disease, the active ingredients of the oral enteric capsule comprising the combination immune extracellular exosome polypeptide regenerating factor of any one of the first aspects; the tumor disease comprises at least one of thyroid cancer, lung cancer, gastric cancer, pancreatic cancer and liver cancer.
The combined immune cell exosome polypeptide regeneration factor provided by the application can be prepared into an oral enteric capsule, and the preparation method can be carried out according to the existing preparation technology of the oral enteric capsule, and specifically comprises the following steps: directly purchasing enteric hollow capsule, and adding the prepared freeze-dried powder.
In a fourth aspect, the present application provides a lyophilized powder formulation for use in the treatment of a neoplastic disease, the active ingredients of the lyophilized powder formulation comprising the combination immune extracellular exosome polypeptide regenerating factor of any one of the first aspects; the tumor disease comprises at least one of thyroid cancer, lung cancer, gastric cancer, pancreatic cancer and liver cancer.
The combined immune cell exosome polypeptide regeneration factor provided by the application can be prepared into a freeze-dried powder preparation (after being diluted by sterile water or normal saline, injection and reinfusion can be directly carried out), and the preparation method can be carried out according to the existing freeze-dried powder preparation process, and specifically comprises the following steps: the freeze-drying is directly carried out at low temperature by a freeze dryer.
The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedures, which are not specified in the following examples, are generally determined according to national standards. If the corresponding national standard does not exist, the method is carried out according to the general international standard, the conventional condition or the condition recommended by the manufacturer.
Example 1
The example provides a combined immune cell exosome polypeptide regeneration factor, and the preparation method of the combined immune cell exosome polypeptide regeneration factor comprises the following steps:
adding peripheral blood mononuclear cells into a serum-free immune cell primary culture medium, placing the culture medium in a 37 ℃ incubator for 12 hours, then removing adherent cell impurities to finish subculture days, and finishing first-stage amplification culture to obtain primary amplified immune cells;
adding the immune cells subjected to preliminary amplification into a serum-free immune cell induction culture medium, and then placing the immune cells in a 37 ℃ incubator for culture for 12 days after the second-stage induction amplification culture is completed, so as to obtain the immune cells subjected to induction amplification;
adding the immune cells induced to be amplified into a serum-free immune cell activating culture medium, and then placing the immune cells in a 37 ℃ incubator for culturing for 12 days after the third stage of activating and amplifying culture is completed, so as to obtain activated and amplified immune cells;
continuously placing the immune cells activated and amplified in a 37 ℃ incubator for monitoring, and supplementing liquid according to the monitored cell morphology until the immune cells are amplified to the standard cell morphology, and completing the fourth-stage culture to obtain a culture liquid;
adding the culture solution into a centrifugal machine for separation to obtain supernatant; and then, carrying out balancing by using a MabSelectTMStereTM affinity chromatography column and using 5 column volumes of balancing buffer solution, loading the sample subjected to the primary purification in the step 1 onto the affinity chromatography column, eluting by using 5 column volumes of eluting buffer solution, collecting the obtained target protein eluate by using a container with a pre-placed neutralizing solution, and keeping the pH value of the neutralized target protein solution to be 6.5-8.0. Regenerating by using 5 column volumes of regeneration liquid, cleaning the chromatographic column by using 3 column volumes of cleaning liquid, balancing the chromatographic column by using 5 column volumes of balancing buffer liquid, and preserving the chromatographic column by using preserving liquid; obtaining the combined immune cell exosome polypeptide regeneration factor; equilibration buffer: 20mM Tris-HCl,150mMNaCl,pH7.2; eluting buffer: 20mM Tris-HCl,150mMNaCl,pH7.2; elution buffer: 100mM citrate buffer, 0.1M sucrose, pH3.4; and (3) neutralizing liquid: 1MTris-HCl, pH8.5; regeneration liquid: 100mM HOC; cleaning liquid: 0.1MNaOH; preservation solution: 20% ethanol by volume;
wherein, the serum-free immune cell primary culture medium is prepared by adding the following components in the existing T cell basal culture medium (serum-free immune cell culture medium): IL2600U/ml, IL101200U/ml, LIF0.5ng/ml, gamma interferon 400U/ml and laminin 5ug/ml;
the serum-free immune cell induction medium is prepared by adding the following components into the existing T cell basic medium (serum-free immune cell medium): IL2600U/ml, IL101200U/ml, bFGF2ng/ml, BMP-40.5ug/ml, sarhillin 0.003KE/ml and laminin 5ug/ml;
the serum-free immune cell activation medium is prepared by adding the following components into the existing T cell basic medium (serum-free immune cell medium): IL2600U/ml, IL101200U/ml, TGF-. Beta.1 ng/ml, resveratrol 40ng/ml and laminin 5ug/ml.
Example 2
This example provides a combination immune cell exosome polypeptide regenerating factor, the preparation method of which differs from example 1 only in that: the serum-free immune cell primary culture medium is additionally added with the following components: IL21000U/ml, IL101500U/ml, LIF2.5ng/ml, gamma interferon 600U/ml and laminin 10ug/ml; the serum-free immune cell induction medium comprises the following additional components: IL21000U/ml, IL101500U/ml, bFGF4ng/ml, BMP-40.8ug/ml, sarhillin 0.005KE/ml and laminin 10ug/ml; the serum-free immune cell activation medium comprises the following additional components: IL21000U/ml, IL101500U/ml, TGF-. Beta.2 ng/ml, resveratrol 60ng/ml and laminin 10ug/ml; the rest steps and parameters are the same.
Example 3
This example provides a combination immune cell exosome polypeptide regenerating factor, the preparation method of which differs from example 1 only in that: the serum-free immune cell primary culture medium is additionally added with the following components: IL2800U/ml, IL101350U/ml, LIF1.5ng/ml, gamma interferon 500U/ml and laminin 8ug/ml; the serum-free immune cell induction medium comprises the following additional components: IL2800U/ml, IL101350U/ml, bFGF3ng/ml, BMP-40.7ug/ml, sarhillin 0.004KE/ml and laminin 8ug/ml; the serum-free immune cell activation medium comprises the following additional components: IL2800U/ml, IL101350U/ml, TGF-. Beta.1.5 ng/ml, resveratrol 50ng/ml and laminin 8ug/ml; the rest steps and parameters are the same.
Comparative example 1
This example provides a combination immune cell exosome polypeptide regenerating factor, the preparation method of which differs from example 1 only in that: regulating IL2 in the serum-free immune cell primary culture medium, the serum-free immune cell induction culture medium and the serum-free immune cell activation culture medium to IL4 (interleukin-4); the rest steps and parameters are the same.
Comparative example 2
This example provides a combination immune cell exosome polypeptide regenerating factor, the preparation method of which differs from example 1 only in that: adjusting BMP-4 in the serum-free immune cell induction medium to SCF (recombinant human stem cell factor); the rest steps and parameters are the same.
Comparative example 3
This example provides a combination immune cell exosome polypeptide regenerating factor, the preparation method of which differs from example 1 only in that: modulating TGF- β in the serum-free immune cell activation medium to TNF- α (tumor necrosis factor- α); the rest steps and parameters are the same.
Comparative example 4
This example provides a combination immune cell exosome polypeptide regenerating factor, the preparation method of which differs from example 1 only in that: no sartorine is added into the serum-free immune cell activation culture medium; the rest steps and parameters are the same.
Comparative example 5
This example provides a combination immune cell exosome polypeptide regenerating factor, the preparation method of which differs from example 1 only in that: resveratrol is not added into the serum-free immune cell activation culture medium; the rest steps and parameters are the same.
Test case
In this example, the combination immunocyte exosome polypeptide-regenerating factors obtained in examples 1 to 3 and comparative examples 1 to 5 were subjected to in vitro test for the proliferation inhibition effect of various tumor cells.
The testing method comprises the following steps: the proliferation inhibition of compounds on various tumor cells was examined using the MTT assay.
Positive control group: a selective PI3K delta/gamma inhibitor IPI-145 was used.
Blank control group: using 96-well plates, 3 parallel experimental groups were set. A plurality of tumor cells (including pancreatic cancer cells PATU8999T, hepG liver cancer cells, TPC-1 thyroid cancer cells, A549 lung cancer cells and MGC-803 stomach cancer cell lines) are paved on a 96-well plate at the density of 7500 cells per well, after incubation for 72 hours, 10 mu LMTT is added for incubation for 4 hours, 100 mu LMTTBuffer is added for dissolving cell crystallization, and the absorbance detection wavelength is 560 nm.
Experimental group: a total of 8 groups (experiment group 1 added with the combined immune exosome polypeptide regeneration factor obtained in example 1, experiment group 2 added with the combined immune exosome polypeptide regeneration factor obtained in example 2, experiment group 3 added with the combined immune exosome polypeptide regeneration factor obtained in example 3, experiment group 4 added with the combined immune exosome polypeptide regeneration factor obtained in comparative example 1, experiment group 5 added with the combined immune exosome polypeptide regeneration factor obtained in comparative example 2, experiment group 6 added with the combined immune exosome polypeptide regeneration factor obtained in comparative example 3, experiment group 7 added with the combined immune exosome polypeptide regeneration factor obtained in comparative example 4, experiment group 8 added with the combined immune exosome polypeptide regeneration factor obtained in comparative example 5), and a 96-well plate was used, each group was set up for 3 parallel experiments. The concentrations of the combined immunocytosomal polypeptide regeneration factors obtained in examples 1 to 3 and comparative examples 1 to 5 were set to 10. Mu.M, 5. Mu.L was added to each well, incubated with tumor cells for 72 hours, 10. Mu.LMTT was added, incubated for 4 hours, 100. Mu.LMTT buffer was added to lyse the cell crystals, and the absorbance detection wavelength at 560 nm.
The test results are shown in table 1, the threshold value of the activity is set to be 50%, and the result shows that after the 10 mu M action is performed for 72 hours, the combined immune extracellular exosome polypeptide regeneration factor provided by the application has obvious inhibition effect on thyroid cancer, lung cancer, gastric cancer, pancreatic cancer, liver cancer and other tumor cells, and the inhibition effect is obviously better than that of comparative examples 1-5 and positive control groups.
TABLE 1
Typical case 1
Mr 65 years old, found a hard mass in the neck half a year ago, with a size of about an egg, pain, dysphagia, vomiting, shortness of breath, palpitation. A hard lump is visible on the neck, the size is about 6cm x5cm, the texture is hard, the surface is concave-convex, TSH, T3 and T4 are all raised, the WBC is raised to generate secondary infection, the ultrasonic boundary is blurred, and the morphological irregularity is diagnosed as thyroid cancer in three stages.
The treatment method comprises the following steps:
the combined immune extracellular body polypeptide regeneration factors prepared in example 3 are respectively prepared into oral enteric capsules (the content of each combined immune extracellular body polypeptide regeneration factor is mg) and freeze-dried powder preparations. The specific treatment is as follows:
firstly, the enteric capsule is orally taken from 3 days of 2 months in 2020 to 11 days of 2 months in 2020, and the daily dosage is one day.
Under the guidance of CT, the freeze-dried powder is added into sterile water for preparing a solution with the concentration of 2mg/ml and then injected into thyroid tumor, the injection amount is 15ml, the patient complains about vomiting and the vomiting is weakened, and the inflammation index is reduced.
The first round of infusion of the combined immune extracellular exosome polypeptide regeneration factor is performed three times in total on 13 days of 2 months 2020.
The exosome polypeptide regeneration factor composition is infused back for the first time in the first round of 200ml in the period of 13 of 2 months in 2020. The vomiting sense is weakened, the inflammation index is continuously reduced, and the tumor has no obvious change.
The enteric capsule is continuously taken for 14 days to 21 days of 4 months in 3 months 2020, and the daily dosage is one day.
The first round of second infusion combines the immune extracellular exosome polypeptide regeneration factors for 22 days in 4 months 2020, the infusion quantity is 200ml, the dysphagia feeling is relieved, the vomit feeling is obviously weakened, the respiration is normal, the mass texture is softer, and the mass is reduced to 4.3 multiplied by 3.8 cm.
The enteric capsule is continuously taken from 23 days in 4 months in 2020 to 30 days in 5 months in 2020, and the daily dosage is one granule per day.
The third time of the first round of reinfusion combines the immune extracellular exosome polypeptide regeneration factors at 31 days of 5 months 2020, the reinfusion amount is 200ml, blood tests show that TSH, T3 and T4 begin to decline, WBC declines, secondary infection gradually changes well, and the tumor is reduced to 2.3 multiplied by 1.8 cm.
The enteric capsule is continuously taken from 1 day of 2020 to 10 days of 7 days of 2020, and the daily dosage is one day.
And 7.11.2020, performing a second round of infusion of the combined immunocyte exosome polypeptide regeneration factor.
The exosome polypeptide regeneration factor composition is infused back for the first time in the second round at 200ml after 7 months and 11 days in 2020. TSH, T3 and T4 of blood examination continuously decrease, WBC value continuously decreases, and tumor is reduced to 1.3 multiplied by 0.9 cm
The enteric capsule is continuously taken from 7 months 11 days 2020 to 8 months 20 days 2020, and the daily dosage is one day.
The exosome polypeptide regeneration factor composition is infused back for the second time in the period of 21 months in 2020, the infusion quantity is 200ml, the dysphagia of patients complains is eliminated, no vomit is caused, the breathing is recovered to be normal, the mass is soft, and the mass is reduced to 0.3 multiplied by 0.4 cm.
The enteric capsule is continuously taken from 22 days in 2020 to 29 days in 2020, and the daily dosage is one day.
The exosome polypeptide regeneration factor composition is infused back for the third time for the second time of 4 days in 10 months in 2020, the infusion volume is 200ml, TSH, T3 and T4 are checked by blood, the WBC is restored to normal level, no infection is caused, and the tumor is basically disappeared
15ml of a freeze-dried powder preparation sterile aqueous solution with the concentration of 2mg/ml is injected under CT guidance for 11 months 5 days 2020 until thyroid tumors completely disappear, and the physical signs of the patient are restored to normal. The enteric capsule is continuously taken for three weeks, and the daily dosage is one day, so that the recurrence is prevented.
Typical case 2
The male patients with 52 years old nausea, stomach severe pain, central heating, acid regurgitation, abdominal distention, hematochezia, constipation, etc. The lower bend of the stomach body is seen by B ultrasonic, the superficial depression of a sheet-shaped mucous membrane with the size of about 3.3X3.8cm, congestion and rotten nodule of the mucous membrane at the bottom are uneven, the surrounding crease is seen to be entangled and interrupted, lesions are accumulated on the anus side, the stomach angle is affected by the small bend of the stomach body, the surface proliferation of a large irregular erosion kitchen is seen, the mucous membrane at the antrum becomes pale, submucosal blood vessels are seen thoroughly, and the gastric ulcer is scattered. Atrophic gastritis is accompanied by erosion, duodenal bulbar inflammation, reflux esophagitis is diagnosed as gastric cancer.
The treatment method comprises the following steps:
the combined immune extracellular body polypeptide-regenerating factors prepared in example 3 were prepared into oral enteric capsules (the same preparation method as in typical case 1) and freeze-dried powder preparations (the same preparation method as in typical case 1), respectively. The specific treatment is as follows:
first, the enteric capsule is orally taken from 3 days of 6 months in 2020 to 11 days of 6 months in 2020, and the daily dosage is one day.
The freeze-dried powder preparation with the concentration of 2mg/ml is injected into 15ml of a stomach cancer focus under CT guidance in 6 months and 12 days of 2020, so that the patient complains about the vomiting and the stomach pain are reduced.
The first round of infusion was performed three times in total on 13/6/2020.
The first round of freeze-dried powder is infused back for the first time, the infusion quantity is 200ml, at the year of 2020, 6 and 13; the vomiting is reduced, the stomach ache, the abdominal distention and hematochezia are all reduced.
The enteric capsule is continuously taken from 14 days to 30 days of 6 months in 2020, and the daily dosage is one day.
7 months and 1 day in 2020, the freeze-dried powder is infused back for the first time for the second time, and the infusion quantity is 200ml; the patient complains that vomiting is continuously reduced, pain is obviously reduced, acid regurgitation, abdominal distention and hematochezia are continuously reduced, the mass is softened, and the mass is reduced to 2.3 multiplied by 2.8 cm.
The enteric capsule is continuously taken from 7 months in 2020 to 7 months in 2020, and the dosage of the enteric capsule is one day.
31 days of 7 months in 2020, the freeze-dried powder is infused back for the third time in the first round, and the infusion quantity is 200ml; blood tests TSH, T3 and T4 begin to decline, WBC declines, secondary infection gradually turns well, and tumor is reduced to 1.7X1.8 cm.
The enteric capsule is continuously taken from 8 months in 2020 to 8 months in 2020, and the dosage of the enteric capsule is one day per day.
And 8 months and 21 days in 2020, performing a second round of reinfusion.
The first time of the second round of freeze-dried powder is carried out on 21 days of 8 months 2020, and the returned amount is 200ml. The patient is checked to basically avoid vomiting, the pain is continuously weakened, the occasional night pain is caused, and the abdominal distention is eliminated. The mass is soft, and the mass is reduced to 1.1 multiplied by 0.9 cm.
The enteric capsule is continuously taken from 21 days in 8 months in 2020 to 9 months in 2020, and the daily dosage is one granule.
The second round of freeze-dried powder is infused back for the second time for 21 days in 9 months 2020, the infusion quantity is 200ml, no vomit is generated, the pain is basically disappeared, the hematochezia is disappeared, the mass is soft, and the mass is reduced to 0.5 multiplied by 0.4 cm.
The enteric capsule is continuously taken from 22 days of 9 months in 2020 to 29 days of 10 months in 2020, and the daily dosage is one day.
The freeze-dried powder is infused back for the second time for 10 months and 30 days in 2020, the infusion quantity is 200ml, no vomit, no pain and no infection are caused, and the standard of the checked cancer is reduced by 0.13 multiplied by 0.1 cm based on the standard tumor.
On 11 months and 1 day in 2020, 15ml of a freeze-dried powder preparation sterile aqueous solution with the concentration of 2mg/ml is injected under CT guidance until the focus is reached, the tumor completely disappears, and the physical sign of the patient is recovered to be normal. And the enteric capsule is continuously taken for three weeks to prevent recurrence.
From comparison of the graphs of the detection results before and after treatment of gastric cancer patients in fig. 2 and 3, it can be seen that: the gastric cancer symptoms completely disappear and the stomach cancer is cured.
Typical case 3
Men, age 49, cough, with increased pain during coughing, chest pain, and hemoptysis. Chest CT shows: the posterior segment of the upper leaf of the right lung is similar to a round soft tissue nodular shadow, 3.1cm x1.9cm x3.0cm, the edge is rough, small eccentric hollows are formed, and multiple small nodular shadows are formed in the right lung; the right thoracic cavity has little effusion, and is diagnosed as lung cancer.
The treatment method comprises the following steps:
the combined immune extracellular body polypeptide-regenerating factors prepared in example 3 were prepared into oral enteric capsules (the same preparation method as in typical case 1) and freeze-dried powder preparations (the same preparation method as in typical case 1), respectively. The specific treatment is as follows:
first, 2021, 3 months, 2020, 3 months, 11 days, a week of enteric capsules are orally taken, and the daily dosage is one day.
And (3) 2021 and 12 days, 15ml of a sterile aqueous solution of the freeze-dried powder preparation with the concentration of 2mg/ml is injected to a focus of lung cancer under CT guidance.
The first round of infusion was performed three times in total on day 13, 3, 2021.
The first infusion of the freeze-dried powder is 200ml at 13 days of 2021, and the cough and chest pain of the patient are relieved.
The enteric capsule is continuously taken for 2021, 3, 14, 4, 25 days, and the daily dosage is one day.
The dose of the second infusion of the freeze-dried powder is 200ml after 25 days of 4 months of 2021, the patient complains about continuous cough relief, pain reduction, hemoptysis reduction, soft mass texture and mass reduction of 2.7X1.5X12.4 cm.
The enteric capsule is continuously taken from 26 days of 4 months of 2021 to 5 days of 6 months of 2021, and the daily dosage is one day.
The dose of the third infusion of the freeze-dried powder is 200ml after 2021, the cough is relieved, the pain is continuously reduced, and the tumor is reduced to 2.0x1.1x1.2 cm.
The enteric capsule is continuously taken from 5 days of 2021 to 7 days of 2021, and the daily dosage is one day.
The second round of reinfusion was performed at 2021, 7 and 21.
The first time of the second round of freeze-dried powder is infused back for 2021, 7 and 21 days, and the infusion quantity is 200ml. The pain is continuously weakened, the pain is occasional, the bleeding is paroxysmal, the mass is soft, and the mass is reduced to 2.0 multiplied by 0.9 multiplied by 1.2 cm.
The enteric capsule is continuously taken from 21 days of 7 months of 2021 to 15 days of 9 months of 2020, and the daily dosage is one day.
The second round of freeze-dried powder is infused back for 16 days 9 in 2021, the infusion quantity is 200ml, no hemoptysis exists, pain basically disappears, hematochezia disappears, the mass is soft, and the mass is reduced to 1.1x0.4x0.37 cm.
The enteric capsule is continuously taken from 17 days of 9 months in 2020 to 22 days of 10 months in 2020, and the daily dosage is one day.
The freeze-dried powder is infused back for the third time in the second round of 22 days of 10 months of 2022, the infusion quantity is 200ml, no vomit, no pain and no infection are caused, and the standard of the checked cancer is reduced by 0.3 multiplied by 0.1 cm based on the standard of the normal standard.
On 11 months and 1 day in 2020, 15ml of a freeze-dried powder preparation sterile aqueous solution with the concentration of 2mg/ml is injected under CT guidance until the focus is reached, the tumor completely disappears, and the physical sign of the patient is recovered to be normal. The enteric capsule is continuously taken for three weeks, and the daily dosage is one day, so that the recurrence is prevented.
From comparison of the graphs of the detection results before and after treatment of the lung cancer patients in fig. 4 and 5, it can be seen that: the lung cancer symptoms completely disappear and cure.
In summary, the embodiment of the application provides a combined immune exosome polypeptide regeneration factor and application thereof, and the combined immune exosome polypeptide regeneration factor has obvious inhibition effect on thyroid cancer, lung cancer, gastric cancer, pancreatic cancer, liver cancer and other tumor cells, and provides a new thought for developing broad-spectrum anticancer drugs capable of effectively treating various cancers, and has practical application value.
Various embodiments of the application may exist in a range of forms; it should be understood that the description in a range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the application; it is therefore to be understood that the range description has specifically disclosed all possible sub-ranges and individual values within that range. For example, it should be considered that a description of a range from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5, and 6, wherever applicable. In addition, whenever a numerical range is referred to herein, it is meant to include any reference number (fractional or integer) within the indicated range.
In the present application, unless otherwise specified, terms such as "upper" and "lower" are used specifically to refer to the orientation of the drawing in the figures. In addition, in the description of the present specification, the terms "include", "comprising" and the like mean "including but not limited to". Relational terms such as "first" and "second", and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Herein, "and/or" describing an association relationship of an association object means that there may be three relationships, for example, a and/or B, may mean: a alone, a and B together, and B alone. Wherein A, B may be singular or plural. Herein, "at least one" means one or more, and "a plurality" means two or more. "at least one", "at least one" or the like refer to any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (individual) of a, b, or c," or "at least one (individual) of a, b, and c," may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, c may be single or multiple, respectively.
The foregoing is only a specific embodiment of the application to enable those skilled in the art to understand or practice the application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. The preparation method of the combined immune extracellular body polypeptide regeneration factor is characterized by comprising the following steps of:
adding peripheral blood mononuclear cells into a serum-free immune cell primary culture medium for first-stage amplification culture to obtain primarily amplified immune cells;
adding the immune cells subjected to primary amplification into a serum-free immune cell induction culture medium to perform second-stage induction amplification culture to obtain induction amplified immune cells;
adding the immune cells induced to be amplified into a serum-free immune cell activating culture medium to perform activating amplification culture at a third stage to obtain activated and amplified immune cells;
culturing the immune cells activated and amplified in a fourth stage to obtain a culture solution;
separating and extracting the culture solution to obtain the combined immune cell exosome polypeptide regeneration factor;
wherein, the serum-free immune cell primary culture medium comprises the following components: IL2600-1000U/ml, IL101200-1500U/ml, LIF0.5-2.5ng/ml, gamma-interferon 400-600U/ml and laminin 5-10ug/ml;
the serum-free immune cell induction medium comprises the following components: IL2600-1000U/ml, IL101200-1500U/ml, bFGF2-4ng/ml, BMP-40.5-0.8ug/ml, sarhillin 0.003-0.005KE/ml and laminin 5-10ug/ml;
the serum-free immune cell activation medium comprises the following components: IL2600-1000U/ml, IL101200-1500U/ml, TGF-beta 1-2ng/ml, resveratrol 40-60ng/ml and laminin 5-10ug/ml.
2. The combination immune cell exosome polypeptide regenerating factor of claim 1, wherein the step of first stage expansion culture comprises: after the peripheral blood mononuclear cells are added into the serum-free immune cell primary culture medium, the culture medium is placed in a 37 ℃ incubator for 12 hours, and then the adherent cell impurities are removed to complete the subculture.
3. The combination immune cell exosome polypeptide regenerating factor of claim 1, wherein the step of second stage expansion culture comprises: and adding the immune cells which are primarily amplified into a serum-free immune cell induction culture medium, and then placing the immune cells in a 37 ℃ incubator for culturing for 10-14 days, wherein the immune cells are passaged every 2 days.
4. The combination immune cell exosome polypeptide regenerating factor of claim 1, wherein the step of third stage expansion culture comprises: after the immune cells after induced amplification are added into serum-free immune cell activation culture medium, the immune cells are placed in a 37 ℃ incubator for culture for 10-14 days, and the immune cells are passaged every 2 days.
5. The combination immune cell exosome polypeptide regenerating factor of claim 1, wherein the step of fourth stage expansion culture comprises: and continuously placing the activated and amplified immune cells in a 37 ℃ incubator for monitoring, and supplementing liquid according to the monitored cell morphology until the immune cells are amplified to the standard cell morphology, thereby obtaining the final culture liquid.
6. The method according to claim 1, wherein the step of separating and extracting the culture solution to obtain the polypeptide regeneration factor comprises the following steps: and (3) sequentially centrifuging the culture solution, then adopting an affinity chromatography column to purify the protein, and collecting eluent to obtain the combined immune cell exosome polypeptide regeneration factor.
7. The combination immune cell exosome polypeptide regenerating factor according to any one of claims 1-6, wherein the serum-free immune cell primary medium comprises the following components: IL2800U/ml, IL101350U/ml, LIF1.5ng/ml, gamma interferon 500U/ml and laminin 8ug/ml;
the serum-free immune cell induction medium comprises the following components: IL2800U/ml, IL101350U/ml, bFGF3ng/ml, BMP-40.7ug/ml, sarhillin 0.004KE/ml and laminin 8ug/ml;
the serum-free immune cell activation medium comprises the following components: IL2800U/ml, IL101350U/ml, TGF-. Beta.1.5 ng/ml, resveratrol 50ng/ml and laminin 8ug/ml.
8. Use of a combination immune exosome polypeptide regenerating factor according to any one of claims 1-7 in the manufacture of a medicament for the treatment, and/or prevention, of a neoplastic disease; the tumor disease comprises at least one of thyroid cancer, lung cancer, gastric cancer, pancreatic cancer and liver cancer.
9. An oral enteric capsule for treating a neoplastic disease, characterized in that the active ingredients of the oral enteric capsule comprise the combination immune extracellular exosome polypeptide regenerating factor of any one of claims 1-7; the tumor disease comprises at least one of thyroid cancer, lung cancer, gastric cancer, pancreatic cancer and liver cancer.
10. A lyophilized powder formulation for use in the treatment of a neoplastic disease, wherein the pharmaceutically effective components of the lyophilized powder formulation comprise the combination immune extracellular exosome polypeptide regenerating factor of any one of claims 1-7; the tumor disease comprises at least one of thyroid cancer, lung cancer, gastric cancer, pancreatic cancer and liver cancer.
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