CN114946768A - Method for constructing peripheral lung cancer animal model under ultrasonic guidance - Google Patents
Method for constructing peripheral lung cancer animal model under ultrasonic guidance Download PDFInfo
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- 208000020816 lung neoplasm Diseases 0.000 title claims abstract description 37
- 206010058467 Lung neoplasm malignant Diseases 0.000 title claims abstract description 36
- 201000005202 lung cancer Diseases 0.000 title claims abstract description 36
- 238000010171 animal model Methods 0.000 title claims abstract description 31
- 230000002093 peripheral effect Effects 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 26
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 85
- 238000002604 ultrasonography Methods 0.000 claims abstract description 21
- 210000004072 lung Anatomy 0.000 claims abstract description 17
- 239000000725 suspension Substances 0.000 claims abstract description 16
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 11
- 230000009278 visceral effect Effects 0.000 claims abstract description 9
- 239000012528 membrane Substances 0.000 claims abstract description 7
- 238000002592 echocardiography Methods 0.000 claims abstract description 6
- 230000003601 intercostal effect Effects 0.000 claims abstract description 6
- 201000003144 pneumothorax Diseases 0.000 claims abstract description 6
- 210000001519 tissue Anatomy 0.000 claims description 68
- 238000010008 shearing Methods 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 19
- 241000283977 Oryctolagus Species 0.000 claims description 17
- 206010002091 Anaesthesia Diseases 0.000 claims description 10
- 230000037005 anaesthesia Effects 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 10
- 229960001412 pentobarbital Drugs 0.000 claims description 9
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 claims description 7
- 238000010186 staining Methods 0.000 claims description 7
- 210000004177 elastic tissue Anatomy 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 5
- 230000000249 desinfective effect Effects 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
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- 210000003205 muscle Anatomy 0.000 claims description 3
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 claims 2
- 210000001835 viscera Anatomy 0.000 claims 1
- 230000005855 radiation Effects 0.000 abstract description 2
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 8
- 210000000038 chest Anatomy 0.000 description 8
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/12—Animals modified by administration of exogenous cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/107—Rabbit
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
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- Animal Behavior & Ethology (AREA)
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Abstract
The invention discloses a method for constructing a peripheral lung cancer animal model under the guidance of ultrasound, which relates to the technical field of peripheral lung cancer animal models, and comprises the following steps: s1, preparing VX2 tumor blocks; s2, anaesthetizing; s3, implanting VX2 tumor blocks: ultrasonically observing rabbit lung and pleural echoes, selecting intercostal gaps with good acoustic windows, obliquely advancing the needle under the chest membrane of the visceral layer under the real-time guidance of ultrasound with the needle tip submerged in lung tissue, injecting tumor tissue suspension, rapidly withdrawing the needle after rotating the puncture needle, pressing by gauze, and ultrasonically observing whether the needle is in a bleeding-free and pneumothorax position; s4, observing; the method provided by the invention utilizes ultrasonic real-time guidance to accurately construct the animal model of the peripheral lung cancer, and has the advantages of high accuracy, good reliability, small wound and no radiation.
Description
Technical Field
The invention relates to the technical field of peripheral lung cancer animal models, in particular to a method for constructing a peripheral lung cancer animal model under the guidance of ultrasound.
Background
The degree of pleura invasion of peripheral lung cancer has important significance on TMN staging and treatment decision of tumors. Pleural invasion is classified into 4 categories: PL0, tumor localized under visceral pleura elastic fiber layer; PL1, tumor breaks through fibrous layer, but does not reach visceral pleura full layer; PL2, tumor breaks through the entire visceral pleura, without affecting parietal pleura; PL3, tumor involvement of parietal pleura or chest wall tissue. In the 8 th edition of TMN staging, if lung cancer is accompanied by PL1/PL2, the staging is upgraded from T1 to T2 a. The NCCN guideline of 2021 recommends that patients with stage IB lung cancer who are accompanied by PL1/PL2, even if the margin is negative, still benefit from postoperative chemotherapy. Lung cancer has different surgical patterns with different degrees of pleural invasion. If PL3 is reached, lung cancer, pleura, along with extensive chest wall tissue, need to be resected; if PL2 or less is reached, only lung cancer and the corresponding pleura are removed. Therefore, accurate assessment of the degree of pleural invasion is a difficult problem that needs to be solved clinically. At present, in clinical practice, CT is the most common imaging method, and the related research on the application value of CT in the degree of pleural invasion is more, but the accuracy is still insufficient.
The ultrasonic imaging method is a real-time imaging, safe and cheap imaging method, is high in resolution and various in technology, can evaluate pleural affected conditions from multiple aspects such as acoustic features, hardness and blood perfusion, and has great potential in accurately evaluating pleural invasion degree. A suitable animal model is the basis of the experiment, and most of tumor models constructed under the guidance of CT or surgical incision are non-adherent lung cancers and are not suitable for ultrasonic imaging.
At present, a method for constructing a peripheral lung cancer animal model under the guidance of ultrasound does not exist.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the method for constructing the peripheral lung cancer animal model under the ultrasonic guidance.
The invention aims to protect a method for constructing a peripheral lung cancer animal model under the guidance of ultrasound, which comprises the following steps:
s1, preparing VX2 tumor blocks: resuscitating VX2 tumor tissue, shearing the resuscitated VX2 tumor tissue to obtain the sheared VX2 tumor tissue, injecting the sheared VX2 tumor tissue to the lower limb muscle layer of a rabbit by using an injector, culturing to obtain a fresh tumor block, removing a necrotic part, reserving fish-like and tough tumor tissue, placing the tumor tissue in a normal saline solution, and shearing again to obtain a tumor tissue suspension;
s2, anesthesia: taking a New Zealand rabbit, establishing an ear edge vein channel, injecting a 3% sodium pentobarbital solution, fixing the new Zealand rabbit on a laboratory table in a left lateral lying position after anesthesia, and depilating and disinfecting the right chest;
s3, implanting VX2 tumor blocks: ultrasonically observing rabbit lung and pleural echoes, selecting intercostal gaps with good acoustic windows, obliquely advancing the needle under the chest membrane of the visceral layer under the real-time guidance of ultrasound with the needle tip submerged in lung tissue, injecting tumor tissue suspension, rapidly withdrawing the needle after rotating the puncture needle, pressing by gauze, and ultrasonically observing whether the needle is in a bleeding-free and pneumothorax position;
s4, observation: and ultrasonically observing the pulmonary nodule formation condition 7, 10 and 14 days after the tumor tissue suspension is injected, and taking the tumor tissue to perform HE staining and elastic fiber staining.
Preferably, in step S1, the shearing is to 1mm long; the second shearing was to 1mm long.
Preferably, in step S1, the syringe is a 5ml syringe, and the needle of the syringe is an 18G needle.
Preferably, in step S1, the culturing period is 10 days, and the size of the fresh tumor mass is 1-2 cm.
Preferably, in step S1, the ratio of the amount of the fish-like, tough tumor tissue to the physiological saline solution is 1g to 10 ml.
Preferably, in step S2, the gender of the New Zealand rabbit is male and female, and the weight of the New Zealand rabbit is 2-3 kg.
Preferably, in step S2, the injection amount of the 3% sodium pentobarbital solution is 1 ml/kg.
Preferably, in step S3, the rotating puncture needle is a rotating puncture needle 3 rings.
Preferably, in step S3, the gauze is pressed for 30 seconds.
Preferably, in step S3, the needle is obliquely advanced under real-time ultrasound guidance to a depth of 0.3-0.5cm below the pleural layer of the heart and the needle tip is immersed in the lung tissue.
The invention has the following beneficial effects:
(1) the method provided by the invention can be used for accurately constructing the animal model of the peripheral lung cancer, and has high accuracy and good reliability.
(2) The method provided by the invention adopts ultrasonic real-time guidance, has small wound and no radiation.
Drawings
In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.
FIG. 1 is a flowchart of a method for constructing a peripheral lung cancer animal model under ultrasound guidance according to embodiments 1-3 of the present invention;
FIG. 2 is an ultrasound image of the peripheral lung cancer animal model obtained in example 1;
FIG. 3 is a 200-fold optical microscope photograph of the peripheral lung cancer animal model obtained in example 1 after HE staining and spandex staining.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.
Example 1
The embodiment provides a method for constructing a peripheral lung cancer animal model under the guidance of ultrasound, as shown in fig. 1, the specific steps are as follows:
s1, preparing VX2 tumor blocks: reviving VX2 tumor tissue, shearing the revived VX2 tumor tissue to prepare the sheared VX2 tumor tissue, injecting the tumor tissue to the lower limb muscular layer of the rabbit by using an injector, culturing to obtain a fresh tumor block, removing a necrotic part, reserving the fish-like and tough tumor tissue, putting the tumor tissue into a normal saline solution, and shearing again to prepare a tumor tissue suspension;
s2, anesthesia: taking a New Zealand rabbit, establishing an ear edge vein channel, injecting a 3% pentobarbital sodium solution, fixing the new Zealand rabbit on a laboratory bench in a left lateral lying position after anesthesia, and depilating and disinfecting the right chest;
s3, implanting VX2 tumor blocks: ultrasonically observing rabbit lung and pleural echoes, selecting intercostal gaps with good acoustic windows, obliquely advancing the needle under the chest membrane of the visceral layer under the real-time guidance of ultrasound with the needle tip submerged in lung tissue, injecting tumor tissue suspension, rapidly withdrawing the needle after rotating the puncture needle, pressing by gauze, and ultrasonically observing whether the needle is in a bleeding-free and pneumothorax position;
s4, observation: and ultrasonically observing the pulmonary nodule formation condition 7, 10 and 14 days after the tumor tissue suspension is injected, and taking the tumor tissue for HE staining and elastic fiber staining.
In step S1, the shearing is to shear to 1mm long; the second shearing was to 1mm long.
In step S1, the syringe is a 5ml syringe, and the needle of the syringe is an 18G needle.
In step S1, the culturing time is 10 days, and the size of the fresh tumor mass is 1 cm.
In step S1, the ratio of the amount of the fish meat-like, tough tumor tissue to the amount of the physiological saline solution is 1g to 10 ml.
In step S2, the new zealand rabbit is sex-independent and has a body weight of 2 kg.
In step S2, the injection amount of the 3% sodium pentobarbital solution is 1 ml/kg.
In step S3, the rotary puncture needle is a 3-turn rotary puncture needle.
In step S3, the gauze is pressed for 30 seconds.
In step S3, the needle is obliquely advanced under ultrasound real-time guidance to a depth of 0.3cm below the pleural layer of the visceral layer and the needle tip is immersed in the lung tissue.
Example 2
The embodiment provides a method for constructing a peripheral lung cancer animal model under the guidance of ultrasound, as shown in fig. 1, the specific steps are as follows:
s1, preparing VX2 tumor blocks: resuscitating VX2 tumor tissue, shearing the resuscitated VX2 tumor tissue to obtain the sheared VX2 tumor tissue, injecting the sheared VX2 tumor tissue to the lower limb muscle layer of a rabbit by using an injector, culturing to obtain a fresh tumor block, removing a necrotic part, reserving fish-like and tough tumor tissue, placing the tumor tissue in a normal saline solution, and shearing again to obtain a tumor tissue suspension;
s2, anesthesia: taking a New Zealand rabbit, establishing an ear edge vein channel, injecting a 3% sodium pentobarbital solution, fixing the new Zealand rabbit on a laboratory table in a left lateral lying position after anesthesia, and depilating and disinfecting the right chest;
s3, implanting VX2 tumor blocks: ultrasonically observing rabbit lung and pleural echoes, selecting intercostal gaps with good acoustic windows, obliquely advancing the needle under the chest membrane of the visceral layer under the real-time guidance of ultrasound with the needle tip submerged in lung tissue, injecting tumor tissue suspension, rapidly withdrawing the needle after rotating the puncture needle, pressing by gauze, and ultrasonically observing whether the needle is in a bleeding-free and pneumothorax position;
s4, observation: and ultrasonically observing the pulmonary nodule formation condition 7, 10 and 14 days after the tumor tissue suspension is injected, and taking the tumor tissue for HE staining and elastic fiber staining.
In step S1, the shearing is to shear to 1mm long; the second shearing was to 1mm long.
In step S1, the syringe is a 5ml syringe, and the needle of the syringe is an 18G needle.
In step S1, the culturing time is 10 days, and the size of the fresh tumor mass is 1.6 cm.
In step S1, the ratio of the amount of the fish meat-like, tough tumor tissue to the amount of the physiological saline solution is 1g to 10 ml.
In step S2, the gender of the New Zealand rabbit is male and female, and the weight of the New Zealand rabbit is 2.4 kg.
In step S2, the injection amount of the 3% sodium pentobarbital solution is 1 ml/kg.
In step S3, the rotary puncture needle is a 3-turn rotary puncture needle.
In step S3, the gauze is pressed for 30 seconds.
In step S3, the needle is obliquely advanced under ultrasound real-time guidance to a depth of 0.3cm below the pleural layer of the visceral layer and the needle tip is immersed in the lung tissue.
Example 3
The embodiment provides a method for constructing a peripheral lung cancer animal model under the guidance of ultrasound, as shown in fig. 1, the specific steps are as follows:
s1, preparing VX2 tumor blocks: reviving VX2 tumor tissue, shearing the revived VX2 tumor tissue to prepare the sheared VX2 tumor tissue, injecting the tumor tissue to the lower limb muscular layer of the rabbit by using an injector, culturing to obtain a fresh tumor block, removing a necrotic part, reserving the fish-like and tough tumor tissue, putting the tumor tissue into a normal saline solution, and shearing again to prepare a tumor tissue suspension;
s2, anesthesia: taking a New Zealand rabbit, establishing an ear edge vein channel, injecting a 3% sodium pentobarbital solution, fixing the new Zealand rabbit on a laboratory table in a left lateral lying position after anesthesia, and depilating and disinfecting the right chest;
s3. implantation of VX2 tumor mass: ultrasonically observing rabbit lung and pleural echoes, selecting intercostal gaps with good acoustic windows, obliquely advancing the needle under the chest membrane of the visceral layer under the real-time guidance of ultrasound with the needle tip submerged in lung tissue, injecting tumor tissue suspension, rapidly withdrawing the needle after rotating the puncture needle, pressing by gauze, and ultrasonically observing whether the needle is in a bleeding-free and pneumothorax position;
s4, observation: and ultrasonically observing the pulmonary nodule formation condition 7, 10 and 14 days after the tumor tissue suspension is injected, and taking the tumor tissue for HE staining and elastic fiber staining.
In step S1, the shearing is to shear to 1mm long; the second shearing was to 1mm long.
In step S1, the syringe is a 5ml syringe, and the needle of the syringe is an 18G needle.
In step S1, the culturing time is 10 days, and the size of the fresh tumor mass is 2 cm.
In step S1, the ratio of the amount of the fish meat-like, tough tumor tissue to the amount of the physiological saline solution is 1g to 10 ml.
In step S2, the New Zealand rabbits are sex-independent and have a body weight of 3 kg.
In step S2, the injection amount of the 3% sodium pentobarbital solution is 1 ml/kg.
In step S3, the rotary puncture needle is a 3-turn rotary puncture needle.
In step S3, the gauze is pressed for 30 seconds.
In step S3, the needle is obliquely advanced under ultrasound real-time guidance to a depth of 0.3cm below the pleural membrane of the visceral layer and the needle tip is immersed in the lung tissue.
Test examples
(1) The results of the ultrasonic testing of the peripheral lung cancer animal model obtained in example 1 are shown in fig. 2.
(2) Tumor tissues were collected from the peripheral lung cancer animal model obtained in example 1, and observed under a 200-fold optical microscope after HE staining and spandex staining, and the results are shown in fig. 3.
As can be seen from FIGS. 2-3, the peripheral lung cancer animal model obtained by the method of the present invention has high accuracy and good reliability.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.
Claims (10)
1. A method for constructing a peripheral lung cancer animal model under the guidance of ultrasound is characterized by comprising the following steps: the method comprises the following steps:
s1, preparing VX2 tumor blocks: resuscitating VX2 tumor tissue, shearing the resuscitated VX2 tumor tissue to obtain the sheared VX2 tumor tissue, injecting the sheared VX2 tumor tissue to the lower limb muscle layer of a rabbit by using an injector, culturing to obtain a fresh tumor block, removing a necrotic part, reserving fish-like and tough tumor tissue, placing the tumor tissue in a normal saline solution, and shearing again to obtain a tumor tissue suspension;
s2, anesthesia: taking a New Zealand rabbit, establishing an ear edge vein channel, injecting a 3% sodium pentobarbital solution, fixing the new Zealand rabbit on a laboratory table in a left lateral lying position after anesthesia, and depilating and disinfecting the right chest;
s3, implanting VX2 tumor blocks: ultrasonically observing rabbit lung and pleural echoes, selecting intercostal gaps with good acoustic windows, obliquely advancing the needle under the chest membrane of the visceral layer under the real-time guidance of ultrasound with the needle tip submerged in lung tissue, injecting tumor tissue suspension, rapidly withdrawing the needle after rotating the puncture needle, pressing by gauze, and ultrasonically observing whether the needle is in a bleeding-free and pneumothorax position;
s4, observation: and ultrasonically observing the pulmonary nodule formation condition 7, 10 and 14 days after the tumor tissue suspension is injected, and taking the tumor tissue for HE staining and elastic fiber staining.
2. The ultrasound-guided method of constructing a peripheral lung cancer animal model according to claim 1, wherein: in step S1, the shearing is to shear to 1mm long; the second shearing was to 1mm long.
3. The ultrasound-guided method of constructing a peripheral lung cancer animal model according to claim 1, wherein: in step S1, the syringe is a 5ml syringe, and the needle of the syringe is an 18G needle.
4. The ultrasound-guided method of constructing a peripheral lung cancer animal model according to claim 1, wherein: in step S1, the culturing time is 10 days, and the size of the fresh tumor mass is 1-2 cm.
5. The ultrasound-guided method of constructing a peripheral lung cancer animal model according to claim 1, wherein: in step S1, the ratio of the amount of the fish meat-like, tough tumor tissue to the amount of the physiological saline solution is 1g to 10 ml.
6. The ultrasound-guided method of constructing a peripheral lung cancer animal model according to claim 1, wherein: in step S2, the New Zealand rabbits have sex-restricted sex and a body weight of 2-3 kg.
7. The ultrasound-guided method of constructing a peripheral lung cancer animal model according to claim 1, wherein: in step S2, the injection amount of the 3% sodium pentobarbital solution is 1 ml/kg.
8. The ultrasound-guided method for constructing a peripheral lung cancer animal model according to claim 1, wherein: in step S3, the rotary puncture needle is a 3-turn rotary puncture needle.
9. The ultrasound-guided method of constructing a peripheral lung cancer animal model according to claim 1, wherein: in step S3, the gauze is pressed for 30 seconds.
10. The ultrasound-guided method of constructing a peripheral lung cancer animal model according to claim 1, wherein: in step S3, the needle is obliquely advanced under real-time ultrasound guidance to a depth of 0.3-0.5cm below the pleural layer of the visceral organ and the needle tip is immersed in the lung tissue.
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| 张东升: "兔VX2 肺癌模型的建立及鉴定", vol. 36, no. 2, pages 171 - 173 * |
| 张火俊: ""射波刀治疗100问"", 第二军医大学出版社, pages: 104 * |
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