CN101235364B - Newcastle disease virus D90 strain and application thereof - Google Patents
Newcastle disease virus D90 strain and application thereof Download PDFInfo
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- CN101235364B CN101235364B CN2007100030469A CN200710003046A CN101235364B CN 101235364 B CN101235364 B CN 101235364B CN 2007100030469 A CN2007100030469 A CN 2007100030469A CN 200710003046 A CN200710003046 A CN 200710003046A CN 101235364 B CN101235364 B CN 101235364B
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Abstract
The invention discloses a Newcastle Desease Virus, NDV D90 virus strain, the microorganism preservation number is CGMCC No: 1921. The invention also discloses the application of the virus strain in antineoplastic. The invention researches the antineoplastic effect of the Newcastle Desease Virus D90 through adopting a series of means such as cell culture, acridine orange staining, electron microscope, flow cytometry and the like. The result shows that Newcastle Desease Virus D90 can inhibit the growth of tumour cell, which can lead tumour cell to produce apoptosis and necrosis, the Newcastle Desease Virus, NDV D90 virus strain can be applied in the treatment of anti tumor.
Description
Technical field
The present invention relates to a kind of virus stain, relate in particular to newcastle disease virus D 90 strain and the application in antitumor thereof, belong to the biotherapy field of tumour.
Background technology
(Newcastle Desease Virus NDV) belongs to paramyxovirus to Avian pneumo-encephalitis virus, can cause the newcastle disease of bird.This disease is fatal for bird, mainly shows as respiratory inflammation, brain and gastrointestinal tract inflammation also occur.Newcastle disease also can infect the mankind, but it only causes influenza-like symptom, conjunctivitis or the laryngitis of human moderate.In addition, the duplicating efficiency of Avian pneumo-encephalitis virus in cancer cell be in normal cell 10,000 times this make people with it as the anticancer factor of potential (Schirrmacher V, Haas C, Bonifer R, et al.1999.Human tumor cell modification by virus infection:an efficient and safe way toproduce cancer vaccine with pleiotropic immune stim μ latory properties when usingNewcastle disease virus.Gene Ther 6 (1): 63-73.).Because it is nontoxic that common NDV is considered to the mankind, so Avian pneumo-encephalitis virus has been used as the pharmacological agent of CAM, and obtain extensive studies (Bar-Eli N, Giloh H, Schlesinger M, et al.1996.Preferential cytotoxic effect of Newcastle diseasevirus on lympHoma cells.J Cancer Res Clin Oncol 122 (7): 409-15.).
At present, the NDV strain that is widely used in most cancer therapy has molten cancer strain of 73-T, MTH-68 and non-molten cancer strain (the Haas C of Mlster, Ertel C, Gerhards R, et al.1998.Introduction of adhesive andcostim μ latory immune functions into tumor cells by infection with Newcastle DiseaseVirus.Int J Oncol 13 (6): 1105～1115).Main NDV73 T and two kinds of solvability strains of MTH-68 of adopting, all types of tumour cells of NDV73 T energy infected person, and duplicate therein, cause the fusion and the coenocytic formation of cell and cell, finally cause death of neoplastic cells.Because NDV can not duplicate in normal cell, it should be noted that selective killing effect (the Haas C of NDV to tumour cell, Schirrmacher is Inc rease of Autologous Tumor Cell Vaccines by Virus infectionand Attachment of Bispecific Antibodies[J V.1996.Immunogenicity] .Cancer ImmunoI Immunother, 43 (3): 190～194).NDV has many different strains, can be divided into molten cancer strain and non-molten cancer strain (Moss RW.1996.Alternative pHarmacological and biological treatmentsfor cancer:ten promising approaches.J Naturopathic Med 6 (1): 23-32 according to them to the effect of human cancer cell; Tzadok-David Y, Metzkin-Eizenberg M, Zakay-Rones be effect of a mesogenic and a lentogenicNewcastle disease virus strain on Burkitt lympHoma Daudi cells.J Cancer Res ClinOncol 121 (3) Z.1995.The: 169-74).According to the main difference of molten cancer strain and non-molten cancer strain, developed three kinds of different anticancer methods at present: (1) uses the molten cancer of NDV virus strain infection cancer patient; (2) application of oncolysis product, for example, the tenuigenin fragment that will contain the NDV cells infected is as anti-cancer vaccine; (3) the complete infected particle that forms of non-molten cancer virus strain infection cancer cells is as whole-cell vaccines.
Another main difference of molten cancer strain and non-molten cancer strain is that the mechanism of their kill cancer cells is different.The infectious virus filial generation particle that is produced by molten cancer strain is kill cancer cell quickly.Sprouting of progeny virus contains activated HN, F albumen can cause that the plasmalemma of infected cell and adjacent cells plasmalemma merge, and produce big not reproducible fused cell-synplasm then.Molten cancer strain can more effectively duplicate in host cell, kills host cell quickly.Molten cancer strain is transformed into cytolysis by the ability of preferential kill cancer cell; Therefore the NDV strain that has oncolysis becomes molten cancer strain.On the contrary, it is slower that non-molten cancer strain kills cells infected, and its death is owing to the Normocellular metabolism of virus infection causes.In acridine orange dyeing, the morphological change of typical apoptosis has not only appearred, and synplasm has also appearred simultaneously, and molten cancer strain can produce not reproducible fused cell-synplasm.
Apoptosis (apotosis) claims that also (program cell death PCD), is a cell under certain physiological condition to programmed death, the orderly self-digestion process by the active of gene regulating.It is meant the cellular change of a succession of successive without inflammatory reaction, finally causes necrocytosis.It is different from the pathological death-necrocytosis of cell, can be induced by multiple factor such as ionizing rays, antitumor drug etc., has special form and biochemical character
[12] [13] [14]Apoptosis has vital role in tumour forms and treats.Recently progress of research shows, in the generation of tumour, evolution, in a single day the balance between tumor cell proliferation and the apoptosis is destroyed, and apoptosis is suppressed, and tumour just increases rapidly.Therefore select the ideal method, a large amount of apoptosis of inducing tumor cell have important practical significance with the purpose that reaches tumor regression or reverse.In recent years, people utilize the FCM technology to come the detection by quantitative apoptosis, and it and can confirm apoptosis and downright bad difference from many aspects except that having advantages such as analysis of cells quantity is many, susceptibility is high, quick.At present, it is one of direct that the FCM technology has become the apoptotic important means of research.
Investigators have found much genes of abnormal expression when apoptosis, gene participating in apoptosis can roughly be divided into two classes, promote gene and suppressor gene, they play respectively and promote apoptosis and suppress effect of apoptosis, and wherein suppressor gene mainly contains bcl-2, ced-9, bcl-XL, McL1, MyD116, the expression level that detects these specific genes also becomes the apoptotic a kind of common method of detection.What wherein main, present research was maximum is the bcl-2 gene.It is a kind of of oncogene, and is also comparatively sure about its role in apoptosis.Its overexpression in the tumour of human body variform, can suppress apoptosis (the Wang HG that causes by multiple stimulation, MiyashitaT, Takayama S, etal.1994.Apoptosis regulation by interaction of Bcl-2 proteion and Raf-1kinase.Oncogene, 9 (9): 2751).
Summary of the invention
(its microbial preservation number is: CGMCC No:1921 for Newcastle Desease Virus, NDV) D90 strain to the invention provides a kind of Avian pneumo-encephalitis virus that can killing tumor cell; The preservation time is: on January 17th, 2007; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica.
The separation method or the source of newcastle disease D90 strain of the present invention: separated the goose body of falling ill in 2006, identify, prove and called after newcastle disease D90 strain through hemagglutination test, hemagglutination-inhibition test from the infection new city eqpidemic disease of Heilongjiang Province.And then, by cell cultures, clone purification, obtained this strain.This strain is the newcastle disease virulent strain, and HA neuraminidase hemagglutinin gene presents the characteristic sequence of virulent strain, and its F gene nucleotide series is shown in the SEQ IDNO:1.
The present invention has adopted a series of means such as cell cultures, acridine orange dyeing, Electronic Speculum, flow cytometer to study Avian pneumo-encephalitis virus (Newcastle Desease Virus, NDV) antitumous effect of D90 strain.In acridine orange dyeing, can see typical apoptotic morphological change with the tumour cell that newcastle disease virus D 90 strain is handled; By electron microscopic observation virus as seen, find apoptotic body in the tumour cell under the newcastle disease virus D 90 strain effect, also seen typical non-viable non-apoptotic cell simultaneously.Use the analytical results of flow cytometer to show, in the time period of 24h, the mechanism of action of virus mainly shows as apoptosis through the cell of D90 strain effect; In time period, the mechanism of action of virus mainly shows as necrosis at 48h, 72h
In vitro tests shows, the newcastle disease virus D 90 strain can optionally act on tumour cell, suppress the growth of tumour cell, cause tumour cell generation apoptosis and necrosis, the mechanism that newcastle disease D90 strain acts on tumour cell is mainly apoptosis and combines with necrosis, be a kind of effective antitumour biotechnological formulation, can be used for treatment and comprise various malignant tumours such as lung cancer, liver cancer, cancer of the stomach, mammary cancer, leukemia, nasopharyngeal carcinoma, cervical cancer, carcinoma of endometrium or ovarian cancer.
In the safety testing of newcastle disease virus D 90 strain to human normal cell line, the newcastle disease virus D 90 strain does not have any damaging action to fetal liver cells.
Description of drawings
Fig. 1 is without the acridine orange coloration result of control group A 549 lung carcinoma cells of virus function.
Fig. 2 D90 acts on the acridine orange coloration result of A549 lung carcinoma cell.
Fig. 3 D90 acts on the acridine orange coloration result of A549 lung carcinoma cell.
The electron microscopic observation result of Fig. 4 control cells.
The electron microscopic observation result of control cells under Fig. 5 high power.
The electron microscopic observation result of the typical apoptotic body of Fig. 6.
The electron microscopic observation result of the typical non-viable non-apoptotic cell of Fig. 7.
The electron microscopic observation result of the early stage cell of Fig. 8 apoptosis.
The electron microscopic observation result of the early stage cell of Fig. 9 apoptosis.
Figure 10 D90 effect is down in the detection of 24h viable apoptotic cell and non-viable non-apoptotic cell.
ML represents the upper left corner, is the cell quantity of physical abuse; UR represents the upper right corner, is the cell of death; LL represents the lower left corner, is the quantity of viable cell; LR represents the lower right corner, is the cell of early apoptosis.The negative control cells of order from top to bottom, D90 dilution 1 * 10
-1, 1 * 10
-3, 1 * 10
-5Doubly, negative control cell.
Figure 11 D90 effect is down in the detection of 48h viable apoptotic cell and non-viable non-apoptotic cell.
ML represents the upper left corner, is the cell quantity of physical abuse; UR represents the upper right corner, is the cell of death; LL represents the lower left corner, is the quantity of viable cell; LR represents the lower right corner, is the cell of early apoptosis.The negative control cells of order from top to bottom, D90 dilution 1 * 10
-1, 1 * 10
-3, 1 * 10
-5Doubly, negative control cell.
Figure 12 suppresses apoptogene BCL-2 detected result.
Figure 13 light microscopic is observed the effect of newcastle disease virus D 90 strain to fetal liver cells down.
In order further to set forth the present invention, provide a series of embodiment below.These embodiment are illustrative fully, and they only are used for the present invention is specifically described, and not should be understood to limitation of the present invention.
Embodiment
1, material method
1.1 main raw
1.1.1 plant malicious newcastle disease virus D 90 strain (preserving number: CGMCC No:1921);
1.1.2 cell strain lung cancer A549 cell system, fetal liver cells are given by professor Song Chun of Harbin Medical University;
1.1.3 reagent RPMI1640 cell culture fluid, foetal calf serum are all available from HYCLONE company; Acridine orange, Paraformaldehyde 96, Triton X-100, pentanediol, pancreatin, EDTA are all available from the Shanghai China biological company limited of Shun; BECKMAN KULT Annexin V-ECFP apoptosis test regent box is available from BECKMAN company; FITC mark goat anti-mouse IgG is available from Bioisystech Co., Ltd of China fir Golden Bridge in Beijing;
1.1.4 consumptive material 6 holes, 12 holes, 96 porocyte plates, 75cm culturing bottle, plate are all available from the Shanghai China biological company limited of Shun;
1.1.5 instrument JEM-1220 type Electronic Speculum (OLYMPASS), fluorescent microscope (LEICA), flow cytometer (BECTON DICKINSON FACSORT).
1.2 acridine orange dyeing: the A549 lung carcinoma cell that will be in logarithmic phase is prepared into 5 * 10 through the digestion of the EDTA of 0.25% trypsinase and 0.02% mixed solution
4The cell suspension of/ml after the piping and druming evenly, adds in the 12 porocyte culture plates; After treating that cell is individual layer, on the basis of preliminary experiment, with D90 virus strain 1 * 10
-1Doubly dilution in 1/10 ratio inoculating cell, and is established contrast with the every hole of viral liquid, continues cultivation in 37 ℃, 5%CO2 incubator; Behind the effect 24h, PBS washes, and directly drips 1～3 of 0.01% acridine orange on culture, and fluorescent microscope is observed down behind the 5min.
The acridine orange coloration result: observe under fluorescent microscope, without control group A 549 lung carcinoma cells of virus function, nucleus DNA sends uniform yellow-green fluorescence, and periphery is a tenuigenin, presents orange or the orange haloing; D90 acts under the A549 lung carcinoma cell 24h fluorescent microscope and observes, and in the same visual field, as seen presents identical dyeing form with control group.Can know that also tenuigenin and the nucleus of seeing cell are fine and close dense yellow-green colour of dying, minority yellow-green colour fragment is also arranged, chromatin is concentrated to nuclear limit, nuclear fragmentation, the yellow-green colour in the visible cell matter or the non-viable non-apoptotic cell of safran fluorescent weakening or disappearance, and the cell that has forms synplasm; The results are shown in Figure 1, Fig. 2, Fig. 3.
1.3 electron microscopic section is observed: the A549 lung carcinoma cell that will be in logarithmic phase is prepared into 5 * 10 through the digestion of the EDTA of 0.25% trypsinase and 0.02% mixed solution
4The cell suspension of/ml, piping and druming evenly back add in the 50ml culturing bottle.After treating that cell is individual layer, be 1 * 10 with 10 times of doubling dilutions of D90 virus strain
-1, 1 * 10
-3, 1 * 10
-53 concentration, every bottle of different viral liquid that drip poison are in 1/10 ratio inoculating cell, and establish contrast, at 37 ℃, 5%CO
2Continue to be cultured to 24h in the incubator.Through the EDTA of 0.25% trypsinase and 0.02% mixed solution digestion, pH7.4PBS washed cell, and carrying out cell counting, to make cell quantity be 1 * 10
5/ ml, 3000rmp is centrifugal, makes the agglomerating Eppendorf of being gathered in of cell pipe bottom, slowly adds 2% pentanediol along centrifugal tube wall then, and 4 ℃ are carried out slicing treatment, electron microscopic observation after spending the night.
The electron microscopic observation result: the A549 lung carcinoma cell of D90 strain effect comes in every shape, and is most of rounded about big or small 10 μ m, also as seen has the cell of elongated plasma membrane projection; Nucleus is big, and karyoplasmic ratio is big, and kernel is very obvious, and is multinuclear benevolence; Organoid is positioned at a nuclear side, the plastosome densification, and mitochondrial cristae is thick.The results are shown in Figure 4, Fig. 5.
1.4 the detection of early apoptosis and non-viable non-apoptotic cell: with the adherent cell of 0.25% pancreatin 0.02%EDTA digestion, make single cell suspension, place the 6 porocyte plates of placing cover glass to continue to cultivate and hatch into individual layer with culturing bottle.Changing liquid is the REMI-1640 substratum of 2% foetal calf serum, with 1 * 10 of D90
-1, 1 * 10
-3, 1 * 10
-53 concentration virus liquid connect poison in culturing bottle, establish one bottle of contrast, hatch 24h, 48h.Flow cytometer detects, and operates with reference to specification sheets.
The detected result of early apoptosis and non-viable non-apoptotic cell:
As seen from the figure, D90 1 * 10
-1The apoptosis of concentration reaches 8.67%, and along with the quantity that reduces apoptotic cell of concentration is tending towards normal cell gradually; As time passes, during to 48h, the quantity of control cells group viable cell has increased by 7% than 24h, and the increase by 43%～53% suddenly of the quantity of the necrocytosis cell after the processing of D90 virus strain does not wait, the amount of apoptotic cell also increases to some extent, but the amount that increases is less with respect to the cell of necrosis, that is to say that necrosis accounts for leading behind the 48h.Experimental result is seen Figure 10, Figure 11.
1.5 the detection of apoptosis suppressor BCL-2: get well-grown A549 cell, go down to posterity in culturing bottle, put 37 ℃, 5%CO2 incubator 24h to individual layer.With D90 dilution 1 * 10
-1, 1 * 10
-3, 1 * 10
-5Doubly, insert in the culturing bottle, establish blank in 1/10 ratio.Put 37 ℃, 5%CO2 incubator 24h.Flow cytometer detects: collecting cell, and it is inferior to give a baby a bath on the third day after its birth with pH7.4PBS, and suspension contains 2 * 10
6Cell, 2% Paraformaldehyde 96,200 μ l are 15min fixedly, add 1ml pH7.4PBS, the centrifugal 5min of 1500rpm abandons supernatant, add 0.5%Triton X-100 100 μ l effect min, add 1: 100 mouse anti BCL-2 antibody 10 μ l, 37 ℃ of water-bath 30min add 1ml pH7.4PBS, the centrifugal 5min of 1500rpm abandons supernatant.Add 1: 20 fluorescent-labeled antibody 20 μ l, the room temperature lucifuge adds 1ml pH7.4PBS, and the centrifugal 5min of 1500rpm abandons supernatant, and after pH7.4PBS washed, the upflowing cell instrument detected.
, detect through flow cytometer and to obtain the result of each group as maker with the normal cell that stimulates without virus through the cell BCL-2 of virus function genetic expression.Order from top to bottom is D901 * 10
-1, 1 * 10
-3, 1 * 10
-5, control cells, the shared per-cent of cell that control cells is expressed the BCL-2 gene is 56.23%; The shared per-cent of cell of D90-1 virus treated group cell expressing BCL-2 gene is 1.83%; D90-3 is 0.95%; D90-5 is 1.88%.
1.6 virus is to the effect of fetal liver cells
The fetal liver cells for preparing is sub-packed in the culturing bottle of 50ml, with the newcastle disease virus D 90 strain with ten times doubly amount be inoculated in fetal liver cells and in 24h, 48h, 72h, 96h.Observation of cell form under the light microscopic.Found that as time passes, the fetal liver cells of control group fetal liver cells and virus treated all presents same form, promptly cell mellow and full, full, stereoscopic sensation (Figure 13) arranged.
Sequence table
＜110〉Harbin Veterinary Medicine Inst., China Academy of Agriculture
＜120〉newcastle disease virus D 90 strain and application thereof
<130>p089
<160>1
<170>PatentIn?version?3.1
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<211>504
<212>DNA
<213>Newcastle?Desease?Virus
<400>1
atagtcaagt?tgctcccgaa?tatgcccaga?gataaggagg?cgtgtgcaaa?agccccatta 60
gaggcatata?acagaacact?gactactttg?ctcactcctc?ttggcgactc?catccgcaag 120
atccaagggt?ctgtgcccac?gtctggaaga?aggagacaaa?aacgctttat?aggtgctgtt 180
attggcagtg?tagctcttgg?ggttgcaacg?gcggcacaga?taacagcagc?tgcggcccta 240
atacaagcca?accagaatgc?cgccaacatc?ctccggctta?aggagagcat?tgctgcaacc 300
aatgaagctg?tgcatgaagt?caccgacgga?ttatcacaac?tatcagtggc?agttgggaag 360
atgcagcaat?ttgtcaatga?ccagtttaat?aatacagcgc?gagaattgga?ttgtataaaa 420
atcacacaac?aggttggtgt?agaactcaac?ctatacctaa?ctgaattgac?tacagtattc 480
gggccacaaa?tcacttcccc?ctgc 504
Claims (4)
1. Avian pneumo-encephalitis virus (Newcastle Desease Virus) D90 strain, its microbial preservation number are: CGMCC No.1921.
2. according to Avian pneumo-encephalitis virus (Newcastle Desease Virus) the D90 strain of claim 1, it is characterized in that: its F gene nucleotide series is shown in the SEQ ID NO:1.
3. pharmaceutical composition, it is characterized in that: newcastle disease virus D 90 strain and pharmaceutically acceptable carrier by claim 1 are formed.
4. the newcastle disease virus D 90 strain of claim 1 is in the purposes of preparation in the anti-lung-cancer medicament.
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102952783A (en) * | 2011-08-18 | 2013-03-06 | 广西医科大学 | Newcastle disease virus NDV PY strain and application thereof |
| CN103451198B (en) * | 2013-08-20 | 2015-11-25 | 中国农业科学院哈尔滨兽医研究所 | The full-length infectious CDNA of oncolytic type newcastle disease virus D 90 strain and construction process thereof and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1463747A (en) * | 2002-06-12 | 2003-12-31 | 北京华兴辰光生物技术有限公司 | A Newcastle disease vaccine and method for preparing the same |
| CN1565629A (en) * | 2003-06-18 | 2005-01-19 | 沧州市兴济动物药厂 | Novel pestilence DNA vaccine and its usage |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1463747A (en) * | 2002-06-12 | 2003-12-31 | 北京华兴辰光生物技术有限公司 | A Newcastle disease vaccine and method for preparing the same |
| CN1565629A (en) * | 2003-06-18 | 2005-01-19 | 沧州市兴济动物药厂 | Novel pestilence DNA vaccine and its usage |
Non-Patent Citations (2)
| Title |
|---|
| 符芳 等.新城疫病毒D90毒株致肺癌A549细胞死亡的方式.中国肿瘤生物治疗杂志13 6.2006,13(6),422. |
| 符芳等.新城疫病毒D90毒株致肺癌A549细胞死亡的方式.中国肿瘤生物治疗杂志13 6.2006,13(6),422. * |
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