CN116270766A - Application of parainfluenza virus type 5 as oncolytic virus in resisting tumor - Google Patents
Application of parainfluenza virus type 5 as oncolytic virus in resisting tumor Download PDFInfo
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Abstract
The application discloses an application of parainfluenza virus type 5 serving as oncolytic virus in resisting tumors, in particular relates to an application of parainfluenza virus type 5 (PIV 5) in treating glioma and ovarian cancer, and belongs to the technical field of medicines. The invention proves that PIV5 can kill in-vivo and in-vitro tumor cells in anti-tumor application, inhibit in-vivo tumor growth, reduce in-vivo tumor volume or eliminate in-vivo tumor, improve invasion and metastasis capability of tumor cells, and induce anti-tumor immune cell response. Therefore, PIV5 has different degrees of oncolytic activity on different tumor cells, and PIV5 can be used for specific treatment of tumors and is used as an effective anti-tumor medicament of oncolytic viruses.
Description
Technical Field
The application relates to the technical field of medical medicines, in particular to application of parainfluenza virus type 5 serving as oncolytic virus in resisting tumors.
Background
In recent years, the morbidity and mortality of tumors are both obviously increased, and the tumor is also one of serious diseases threatening human health. Surgical treatment is one of the traditional treatment means, although the visible focus can be excised, it cannot be guaranteed that all tumor cells can be cleared, and the residual cancer cells can cause recurrence and metastasis of the tumor. Radiotherapy and chemotherapy are used as powerful auxiliary treatment means for operation treatment, and are beneficial to eliminating residual cancer cells and preventing tumor recurrence and metastasis, but the clinical application is greatly limited due to serious toxic and side effects. Oncolytic viruses do not cause serious adverse effects to cancer patients because they selectively target cancer cells. Thus, the advent of oncolytic viral therapy has provided more possibilities for cancer treatment.
Oncolytic Viruses (OV) are a class of replicable viruses that selectively infect and kill tumor cells without damaging normal cells. Oncolytic virus therapy is an innovative tumor targeting therapeutic strategy, which utilizes natural or genetically engineered viruses to selectively infect tumor cells and replicate in the tumor cells, achieving the effects of targeting lysis and killing the tumor cells, but without damaging normal cells. To date, only four OVs-based drugs have been approved for cancer treatment. However, due to limitations in the type of cancer, not all patients have response after administration.
Parainfluenza virus type 5 (Parainfluenza virus type, piv 5) belongs to a member of the paramyxoviridae family, mumps genus, is a single-stranded negative strand RNA virus, and can be isolated from a variety of mammalian and cell cultures. PIV5 infection alone often does not cause clinical symptoms, is less pathogenic, and is therefore safer to use as an oncolytic virus. At present, due to the advantages of relatively stable genome, easiness in culture, genetic operation and the like, PIV5 is often used as a live vaccine carrier for preventing and controlling human and animal epidemic diseases. Notably, at present, the oncolytic activity of PIV5 on tumor cells is not reported, and the invention tries to find the specificity of PIV5 for treating tumors through research, thereby providing a safer and more effective solution for anti-tumor medication.
Disclosure of Invention
In view of the above-mentioned drawbacks or shortcomings of the prior art, it is desirable to provide a parainfluenza virus type 5 as a novel oncolytic virus for use in anti-tumor applications, to provide a novel oncolytic virus capable of producing direct oncolytic effects on a variety of tumor cell lines, and to be used in therapeutic applications for brain gliomas and ovarian cancers, to prepare effective antitumor drugs, to improve the conditions of brain gliomas and ovarian cancer patients.
The application of parainfluenza virus type 5 serving as an oncolytic virus in resisting tumors comprises the application of parainfluenza virus type 5 serving as an oncolytic virus in preparing antitumor drugs.
Preferably, the tumor comprises a brain glioma or an ovarian cancer.
Preferably, the brain glioma cells are selected from the group consisting of the U87 MG cell line (human glioma) and the C6 cell line (murine glioma).
Preferably, the ovarian cancer cells are selected from the group consisting of a2780 cell line (human ovarian cancer) and a2780PPX cell line (paclitaxel resistant breast cancer).
Preferably, parainfluenza virus type 5 is capable of killing tumor cells in vivo and inhibiting tumor growth in vivo.
Preferably, parainfluenza virus type 5 is capable of reducing the volume of or causing the disappearance of a tumor in vivo.
Preferably, parainfluenza virus type 5 is capable of improving invasive metastatic capacity of tumor cells.
Preferably, parainfluenza virus type 5 mediated tumor cell killing mechanisms include direct oncolytic mechanisms and mechanisms that induce anti-tumor immune cell responses.
Preferably, the medicine takes parainfluenza virus type 5 as an active ingredient, and is prepared into a pharmaceutically acceptable dosage form by adding pharmaceutically acceptable auxiliary materials, auxiliary ingredients or carriers, singly or in combination with other medicines.
Preferably, the dosage form of the medicine is oral preparation, mucous membrane administration preparation and injection.
Preferably, the pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field.
Compared with the prior art, the beneficial effects of the application are as follows:
(1) According to the invention, through research and screening, PIV5 is discovered for the first time to be used as an oncolytic virus to generate oncolytic effects of different degrees on human and mouse brain glioma cell lines (U87 MG and C6) and ovarian cancer and drug-resistant ovarian cancer cell lines (A2780 and A2780 PPX), and PIV5 is discovered to be used as an oncolytic virus to have excellent effect in anti-tumor application;
(2) According to the invention, the application and effect of PIV5 as oncolytic virus in resisting tumor are examined for the first time by establishing cytopathic effect (CPE) experiment, MTS cell viability analysis experiment, cell scratch experiment, in vivo antitumor effect experiment and immunohistochemical experiment, and effective conclusion is obtained, so that the application field of oncolytic virus in antitumor treatment is widened, experimental basis is provided for carrying out antitumor treatment by using PIV5 as oncolytic virus, and the method has good application prospect.
It will be appreciated that pharmaceutically acceptable adjuvants, auxiliary ingredients or carriers in the present invention refer to diluents, adjuvants, excipients or vehicles that are co-administered with the active ingredient and which are suitable for contacting the tissues of humans and/or other animals without undue toxicity, irritation, allergic response, complications, or other problem, commensurate with a reasonable benefit/risk ratio, and within the scope of sound medical judgment.
It should be understood that, in the present invention, the term "treating" is used to include alleviation, inhibition or amelioration of symptoms or conditions of the disease; inhibiting the production of complications, improving or preventing potential metabolic syndrome; inhibiting the occurrence of a disease or condition, such as controlling the progression of a disease or condition; alleviating a disease or symptom; causing the disease or symptom to subside; alleviating complications caused by diseases or symptoms, or preventing or treating signs caused by diseases or symptoms. As used herein, administration may result in an improvement in a disease, symptom, or condition, particularly an improvement in severity, a delay in onset, a slowing in progression, or a reduction in duration of the condition.
It should be understood that the description in this summary is not intended to limit key or critical features of embodiments of the present application, nor is it intended to be used to limit the scope of the present application. Other features of the present application will become apparent from the description that follows.
Drawings
Other features, objects and advantages of the present application will become more apparent upon reading of the detailed description of non-limiting embodiments, made with reference to the following drawings, in which:
FIG. 1 is a graph showing changes in cell morphology of various cells in PIV5 oncolytic spectroscopy experiments according to embodiments of the present invention;
FIG. 2 is a graph showing cell viability of various cells under PIV5 infection at various concentrations in an MTS cell viability assay according to an embodiment of the present invention;
fig. 3 is a graph showing growth of tumor cells under the action of PIV5 at different concentrations in a cell scratch test according to an embodiment of the present invention, wherein, panels a and C: wound healing images of U87 MG and C6 cells after PIV5 infection at different concentrations; b and D: percentage of wound healing area of U87 MG and C6 cells after different concentrations of PIV5 infection, *** P<0.05。
fig. 4 is a graph showing the effect of PIV5 on tumor in vivo according to the embodiment of the present invention, wherein, fig. a: tumor growth profiles for mice in different dose PIV5 treatment groups; b, drawing: tumor growth in different groups of tumor-bearing mice; c, drawing: representative of mice in the control group and high dose PIV5 treated group.
FIG. 5 is a graph showing the effect of PIV5 on immune cell induction in immunohistochemical assay according to the embodiment of the present invention.
Detailed Description
The present application is described in further detail below with reference to the drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the application and not limiting of the application. It should be noted that, for convenience of description, only the portions related to the application are shown in the drawings.
It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available. The present application will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.
Embodiment one:
1. cytopathic Effect (CPE) experiments
MA cells, HUVEC cells, U87 MG, C6, A2780 and A2780PPX cells are respectively inoculated into a 6-hole culture plate, PIV5 viruses are respectively inoculated after the culture is carried out for 24 hours, then pathological changes of tumor cells in 72 hours are observed, and when the viruses infect for 24 hours and 72 hours, an inverted phase contrast microscope is used for collecting tumor cell pictures and recording cytopathic effects caused by the PIV 5.
2. Experimental results and conclusions
By performing an oncolytic spectrum experiment on the two normal cells and four tumor cells, observing the pathological changes of CPE cells, and performing photographing recording at 24 hours and 72 hours respectively. The experimental results show that the two normal cells, MA cells and HUVEC cells, do not produce obvious lesions. However, the cell lines A2780, A2780PPX, U87 MG and C6 all can generate obvious pathological changes under the influence of PIV5 as oncolytic virus, and compared with a control group, the cell number of tumor cells is reduced, cell lysis and cracked fragments appear after PIV5 is inoculated (figure 1), so that the PIV5 has obvious killing capability on brain glioma cells and ovarian cancer cells. Moreover, PIV5 has the strongest killing ability to U87 MG and C6 cells through comparison.
Embodiment two:
MTS cell viability assay
After seeding MA cells, HUVEC cells, U87 MG, C6, a2780 and a2780PPX cells in 96-well plates, respectively, and culturing for 24 hours, PIV5 was allowed to infect normal cells and tumor cells, respectively, at different concentrations of MOI values, 3 multiplex wells were set per experimental group. MTS assay was performed 72 hours after inoculation.
2. Experimental results and conclusions
Compared with the MTS method, after the PIV5 with different concentrations is infected, the MA cells and the HUVEC cells have no obvious killing effect. However, PIV5 had an in vitro killing effect on U87 MG, C6, a2780 and a2780PPX cells after infection with PIV5 at different concentrations. The experimental results show that with the increase of the infection quantity of PIV5, the activity of tumor cells shows a remarkable gradual reduction trend and has a certain dose dependence (figure 2), which proves that PIV5 can reduce the activity of brain glioma cells and ovarian cancer cells and has remarkable in-vitro killing capacity of tumor cells.
Embodiment III:
1. cell scratch assay
U87 MG and C6 cells were inoculated into 6-well plates, respectively, and after culturing for 24 hours, the cells were scratched on the cell plane with a 200. Mu.l sterile gun head, and then washed 3 times with sterile PBS to remove debris. Cells were exposed to PIV5 at indicated fold infections and images of the wound healing process were taken under an optical microscope at various time points after infection.
2. Experimental results and conclusions
The invasive metastatic capacity of tumor cells was examined by scratch experiments. Experimental results show that PIV5 infection significantly reduced the migration ability of U87 MG and C6 cells to the scored area, compared to the control group, and had a dose-dependence (fig. 3), demonstrating that PIV5 was able to reduce or attenuate the invasive metastatic ability of tumor cells.
Embodiment four:
1. in vivo anti-tumor Effect experiment
By establishing a mouse subcutaneous tumor model on a C6 cell line, the mice are randomly grouped according to body weight and tumor size, and the mice are divided into 4 groups: the in vivo anti-tumor effect of PIV5 was evaluated in the control group and in the PIV5 treatment group with three different doses of administration, high, medium and low, using tumor volume change and tumor tissue HE staining as indicators.
2. Experimental results and conclusions
The in vivo anti-tumor effect results of fig. 4A, 4B and 4C show that PIV5 has a good inhibitory effect on in vivo growth of tumor cells as an oncolytic virus compared to the control group; experimental results show that with the increase of the infection coefficient of the oncolytic virus PIV5, the anti-tumor effect is gradually enhanced, and the anti-tumor effect has certain dose dependency. Fig. 4D is a HE staining pattern of tumor tissue, and HE staining results show that compared with the control group, tumor cells of tumor tissue under PIV5 infection are obviously reduced, and obvious necrotic lesions exist, which proves that PIV5 can reduce tumor volume and make tumor disappear.
Fifth embodiment:
1. immunohistochemical experiments
Dewaxing paraffin sections, and then carrying out antigen retrieval; blocking endogenous peroxidase, and then adopting serum for blocking; after adding the primary antibody, slicing and placing the slices in a wet box for incubation at 4 ℃ overnight; then adding a secondary antibody: placing the slide in PBS (pH 7.4), shaking and washing for 3 times on a decolorizing shaking table for 5min each time, slightly drying slices, dripping secondary antibody (HRP mark) corresponding to the primary antibody in the circles to cover tissues, and incubating for 50min at room temperature; DAB color development: placing the glass slide in PBS (pH 7.4), shaking and washing for 3 times on a decolorizing shaking table for 5min each time, dripping freshly prepared DAB color development liquid into the circle after slicing and spin-drying, controlling the color development time under a microscope, and washing the slices with tap water to terminate the color development; counterstaining the nuclei with hematoxylin; finally, removing the water sealing sheet; and then the sample was subjected to a white light microscope for interpretation.
2. Experimental results and conclusions
The possible mechanism of PIV5 in anti-glioma was investigated by immunohistochemical staining studies. Experimental results showed that the control group had fewer immune-related cells (cd3+ and cd8+ cells) than the dosing group, consistent with immune silencing or "cold" tumors, and that PIV5 showed rapid infiltration of cd3+ and cd8+ cells after treatment (fig. 5), indicating immune-related cells immunoreactive with PIV5 and metastasize to immune "hot" tumors, demonstrating PIV5 was able to induce an anti-tumor immune cell response.
The application and effect of PIV5 as oncolytic virus in resisting tumor are examined by establishing cytopathic effect (CPE) experiment, MTS cell viability analysis experiment, cell scratch experiment, in vivo antitumor effect experiment and immunohistochemical experiment, and it is concluded that parainfluenza virus type 5 has obvious oncolytic effect on ovarian cancer and glioma, especially has more obvious oncolytic effect on glioma cells. Parainfluenza virus type 5 has the potential to act as an oncolytic virus, thus, hopefully providing a safer and more effective solution for anti-tumor dosing regimens.
In the description of the present specification, the terms "one embodiment," "some embodiments," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing is merely a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and variations may be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims (8)
1. The application of parainfluenza virus type 5 as oncolytic virus in preparing antitumor is characterized by comprising the application of parainfluenza virus type 5 as oncolytic virus in preparing antitumor drugs.
2. Use of parainfluenza virus type 5 as oncolytic virus according to claim 1, wherein the tumor comprises brain glioma and ovarian cancer.
3. The use of parainfluenza virus type 5 as oncolytic virus according to claim 1, wherein said parainfluenza virus type 5 is capable of killing tumor cells and inhibiting tumor growth in vivo and in vitro.
4. Use of parainfluenza virus type 5 as oncolytic virus according to claim 1, wherein the parainfluenza virus type 5 is capable of reducing the volume of or vanishing a tumor in vivo.
5. The use of parainfluenza virus type 5 as oncolytic virus according to claim 1, wherein the parainfluenza virus type 5 is capable of improving invasive metastatic capacity of tumor cells.
6. The use of parainfluenza virus type 5 as oncolytic virus according to claim 1, wherein the parainfluenza virus type 5 mediated tumor cell killing mechanism comprises a direct oncolytic mechanism and an induced anti-tumor immune cell response mechanism.
7. The use of parainfluenza virus type 5 as oncolytic virus according to claim 1, wherein the medicament is prepared into pharmaceutically acceptable dosage forms by taking parainfluenza virus type 5 as an active ingredient, and adding pharmaceutically acceptable auxiliary materials, auxiliary ingredients or carriers, alone or in combination with other medicaments.
8. The use of parainfluenza virus type 5 as oncolytic virus according to claim 7, wherein the dosage form of the medicament is oral, mucosal and injectable.
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