CN104764624B - The materials and fixing means of animal lung tissue - Google Patents
The materials and fixing means of animal lung tissue Download PDFInfo
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- CN104764624B CN104764624B CN201410006085.4A CN201410006085A CN104764624B CN 104764624 B CN104764624 B CN 104764624B CN 201410006085 A CN201410006085 A CN 201410006085A CN 104764624 B CN104764624 B CN 104764624B
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Abstract
The present invention relates to the processing method of animal tissue, and in particular to the materials and fixing means of experimental animal lung tissue, and the lung tissue being prepared according to described materials and fixing means or lung tissue section.The method of the present invention is easy to operate, solves the artificial artefact such as alveolar collapse and expansion；The histotomy prepared using this method can keep the physiology of lung tissue, pathological state, and keep the natural extension state of alveolar and blood vessel.The method of the present invention can be widely applied to the histology of lungs and the histopathology of pneumonopathy with pathomechanism research, also having important practical value in medicine non-clinical study.
Description
Technical field
The present invention relates to the processing method of animal tissue, and in particular to the materials and fixing means of experimental animal lung tissue,
And the purposes of methods described.
Background technology
In injury of lungs, pneumonopathy and medicine nonphosphorylated neurofilament H lung tissue structure and normal lung tissue's structure etc.
In research, often need to carry out histology film-making, and it is each by H.E dyeing or histochemistry, immunohistochemistry, in situ hybridization etc.
Kind specific stain, carries out observation by light microscope and research.Due to lung tissue mainly by the bronchial branches at different levels of intrapulmonary and its
A large amount of alveolars composition of terminal, is presented the particularity such as the spongelike structure containing a large amount of gases.If as most of internal organs
Fixed using fixation is soaked after taking-up, it will because to the people of lung tissue during the forfeiture and film-making of alveolar inside and outside differential pressure
For extruding, large stretch of collapse of lung image is presented in the lung tissue for causing to observe under light microscopic, to observation alveolar septa fine structure and
The research such as function of various cells brings considerable hurdle in further investigation lung tissue.
To solve problem above, OECD recommends to use cardiac perfusion fixation and Intratracheal instillation fixation at present（OECD
Guidance Document on Histopathology for Inhalation Studies,
28September2009Draft1-36）.Although both approaches part solves alveolar collapse problem, cardiac perfusion is fixed
Method makes intrapulmonary blood vessel especially alveolar wall capillary overdistension, and blood vessel is in non-physiological state after perfusion, original lesion（Such as
Haemocyte side collection, detain）Destroyed, can also cause pulmonary capillaries to rupture as perfusion pressure is excessive；Intratracheal instillation is fixed
Method irrigates fixer from tracheal strips, inevitably will produce excessive compression to alveolar wall, alveolar is lost under physiological status
Characteristics of organizational structure.This external cause various pulmonary lesions remains in IA material, and such as inflammatory exudate, albumen edematous fluid all will
Its distribution is washed away or changes by fixer.The defects of above method, brings deviation very to the pathological diagnosis and research of lung tissue
To difficulty.
The content of the invention
In order to solve the above problems, the present inventor gropes by many experiments and repeatedly, finally found that one kind
It is easy to operate at the same can make lungs be in natural extension state lung tissue materials and fixing means, this completes the present invention.
First aspect present invention is related to the materials and fixing means of animal lung tissue, and it comprises the following steps：
（1）Ligature the tracheae of dead animal, the distal end detachment tracheae at ligation, by whole lungs and the tracheae of ligation
Take out together；
（2）By step（1）The whole lungs obtained are fixed, the lung tissue being fixed；Preferably, the fixation side
Method is that whole lungs are completely soaked in fixer.
The materials and fixing means of any one according to a first aspect of the present invention, wherein the animal is mammal, such as
For mouse, rat, cavy, dog, monkey, rabbit, sheep, horse, pig etc..
In the present invention, the execution method of the animal is preferably capable the method for making lungs keep natural extension state,
Such as thoracic cavity will not be exposed, or because of excessive struggle blood will not be caused to enter lung tissue（Such as alveolar）.The present invention's
In embodiment, the execution method is abdominal aorta sacrificed by exsanguination after anesthesia.
The materials and fixing means of any one, wherein step according to a first aspect of the present invention（1）Before tracheae is ligatured, animal
The negative pressure state in thoracic cavity is not destroyed.
In the present invention, the negative pressure state in the thoracic cavity is not destroyed refers to not expose or injure thoracic cavity, although or sudden and violent
Reveal or injure thoracic cavity, but the negative pressure state in thoracic cavity is maintained by lung ventilator.
The materials and fixing means of any one according to a first aspect of the present invention, wherein described by step（1）What is obtained is whole
Any achievable method that the method that lungs are completely soaked in fixer can be known in the art, such as including solid
The object of lung tissue emersion can be prevented, weight is connected with tracheae or in order to keep away by determining liquid fluid level or the covering of lung tissue surface
Exempt from the destruction to lung tissue, can select lung tissue and other adjacent tissues（Such as heart etc.）Take out in the lump, toy can
Lung tissue is set to be completely soaked in fixer with the weight of its adjacent tissue, weight can be also connected by big animal with heart.
In the present invention, the weight refers to that the weight of the object is enough to make lung tissue be completely soaked in fixer, and
And the weight will not be interfered with the fixation of lung tissue, will not be destroyed the original physiology of lung tissue or pathology by fixed corrosion
State.
In embodiments of the invention, the weight is connected with tracheae.
The materials or fixing means of any one according to a first aspect of the present invention, wherein what the fixer was known in the art
Tissue fixative solution, for example, aldehydes solution or alcohol solution, be selected from formalin, paraformaldehyde solution, ethanol solution or
The mixed solution of the above-mentioned solution of person, it is preferable that the formalin or the concentration of paraformaldehyde solution are about 4%.
In embodiments of the invention, the fixer is 4% neutral formalin solution.
In the present invention, the method for the fixed lung tissue is well known in the art, such as can use chemical method, such as adopts
Fixed liquid with various chemical solutions.In embodiments of the invention, the method for the fixed lung tissue is using fixer
Method.
In the present invention, the time of the fixed lung tissue is well known in the art, such as can be according to the fixer of selection
Difference determines the corresponding set time；In embodiments of the invention, the time of fixed lung tissue is 24 hours.
In the present invention, the lung tissue is healthy lung tissue or the lung tissue in pathological state.
Second aspect of the present invention is related to the animal lung tissue being prepared according to the method for any one of first aspect present invention.
Third aspect present invention is related to the preparation method of lung tissue section, and it includes taking for any one of first aspect present invention
Material or fixing means, and it is optional the step of lung tissue after fixation is carried out into film-making.
In the present invention, the method that the lung tissue after fixation is carried out to film-making is well known in the art, such as can use and incite somebody to action
Lung tissue after fixation is embedded film-making again, or the lung tissue after fixation is carried out to the method for frozen section.
In the present invention, the method lung tissue after fixation embedded is well known in the art, for example, paraffin bag
Bury or plastic embedding.
In embodiments of the invention, using the method for FFPE.
In embodiments of the invention, the FFPE includes the steps such as dehydration, transparent, waxdip.
In the present invention, the method that the tissue block after embedding is carried out to film-making (or section) is well known in the art, described
The thickness of section determines according to experiment purpose.In embodiments of the invention, cut into slices using paraffin slicing machine, slice thick
Spend for 3 μm.
In the present invention, the method dyed to histotomy is well known in the art.Such as HE dyeing,
Trichrome is dyed, sirius red stains, Van Gieson ' s dyeing, SABC etc..In embodiment of the present invention
In, to histotomy using HE dyeing (hematoxylin eosin staining).
Fourth aspect present invention is related to the lung tissue that the preparation method of any one according to a third aspect of the present invention is prepared
Section.
The invention further relates to the materials of any one of first aspect present invention or fixing means to be used to determine in vitro lung tissue
The purposes of histological type or pathological state, or the purposes of the non-clinical study experiment for medicine.
The beneficial effect of invention
The invention provides the materials of animal lung tissue and fixing means, its is easy to operate, solves alveolar collapse and expansion
Etc. artificial artefact；Compared with routinely immersing fixation, Intratracheal instillation fixation, cardiac perfusion fixation, there is alveolar structure
It is complete clear, the advantages that red blood cell non-spill phenomenon in alveolar wall capillary；The histotomy energy prepared using this method
Original physiology, the pathological state of lung tissue are enough kept, and keeps the natural extension state of alveolar and blood vessel, is various pulmonary lesions
Diagnosis（Such as pulmonary edema, bullae, atelectasis and inflammatory cell ooze out）Good morphological base has been established with research.This hair
Bright method can be widely applied in the histology of lungs and the histopathology of pneumonopathy and pathomechanism research, in medicine
Also there is important practical value in thing non-clinical study.
Brief description of the drawings
Dorsal position is placed on autopsy table after Fig. 1 rat anesthesias, exposure abdominal cavity, abdominal aorta sacrificed by exsanguination.Cut off chest neck
Skin, blunt separation escape pipe.Pay attention to that Chest Wall Structure must not be injured, in order to avoid destroy intrathoracic negative pressure.
Fig. 2 ligatures tracheae under rat thyroid cartilage, pays attention to pricking extremely, it is impossible to have gas leakage.
The veutro that Fig. 3 completely separates lungs is seen, and because ligaturing tracheae before exposure thoracic cavity, intrapulmonary fills gas, therefore lung is in
Bulge shape.Lungs separation process is as follows：Distal end cuts tracheae at ligation, and thymus gland and heart are simultaneously removed in careful exposure thoracic cavity,
Lifting tracheae with tweezers, from top to bottom blunt separation goes out full lung（Pay attention to avoiding injuring each lobe of the lung）, esophagus is cut from diaphragm,
Take out lungs.Connect a weight in the tracheae broken ends of fractured bone.
The dorsal part that Fig. 4 completely separates lungs is seen, and it is in bulge shape to pay attention to lungs.
Fig. 5 is a weight at tracheae, lungs snorkeling is fixed in 4% neutral formalin liquid.
Fig. 6 lungs tissue paraffin section des, HE dyeing.Show the distant view photograph for including all 5 leaf lungs sections, it is seen that each
Lobe of the lung alveolar is in natural extension state, and a small lattice are 2.5mm in figure.
Fig. 7 Fig. 6 partial enlargements are seen, and show lung tissue alveolars at different levels（Alveolar sac, breathing and alveolar）Structure is in natural extension
State, have no that alveolar is subside or overdistension, a small lattice are 0.25mm in figure.
Fig. 8 Fig. 7 further amplifies sight, shows alveolar natural extension, and alveolar septa is belittled, and a small lattice are 100 μm in figure.
Dorsal position is placed on autopsy table after Fig. 9 mouse anesthesias, exposure abdominal cavity, abdominal aorta sacrificed by exsanguination.Cut off chest neck
Skin, blunt separation escape pipe.Pay attention to that Chest Wall Structure must not be injured, in order to avoid destroy intrathoracic negative pressure.
Figure 10 ligatures tracheae under thyroid cartilage, pays attention to pricking extremely, it is impossible to have gas leakage.
The veutro of the complete separating mouse lungs of Figure 11 is seen, and because ligaturing tracheae before exposure thoracic cavity, intrapulmonary fills gas, therefore lung is in
Bulge shape.Lungs separation process is as follows：Distal end cuts tracheae at ligation, and thymus gland and heart are simultaneously removed in careful exposure thoracic cavity,
Lifting tracheae with tweezers, from top to bottom blunt separation goes out full lung（Pay attention to avoiding injuring each lobe of the lung）, esophagus is cut short from diaphragm,
Take out lungs.Connect a weight in the tracheae broken ends of fractured bone.
The dorsal part of the complete separating mouse lungs of Figure 12 is seen, and it is in bulge shape to pay attention to lungs.
Figure 13 is a weight at tracheae, mouse lung snorkeling is fixed in 4% neutral formalin liquid.
Figure 14 mouse lung tissue paraffin section des, HE dyeing.Show the distant view photograph for including all 5 leaf lungs sections, it is seen that
Each lobe of the lung alveolar is in natural extension state, and a small lattice are 2.5mm in figure.
Figure 15 Figure 14 partial enlargements are seen, and show lung tissue alveolars at different levels（Alveolar sac, breathing and alveolar）Structure expands in nature
State, have no that alveolar is subside or overdistension, a small lattice are 100 μm in figure.
Figure 16 utilizes lung tissue section's HE colored graphs prepared by the method for embodiment 1, shows that alveolar structure is completely clear, alveolar wall
Red blood cell non-spill phenomenon in capillary, a small lattice are 25 μm in figure.
Lung tissue section's HE colored graphs that Figure 17 is prepared using conventional immersion fixation, show that most alveolar walls are local thicker,
The level of resolution technology is not easy, a small lattice are 25 μm in figure.
Figure 18 utilizes lung tissue section's HE colored graphs prepared by cardiac perfusion fixation, shows alveolar wall caused by pressure is big
Telangiectasis, alveolar is relative to diminish, and a small lattice are 25 μm in figure.
Figure 19 utilizes lung tissue section's HE colored graphs prepared by Intratracheal instillation fixation, shows that alveolar is of uneven density, alveolar wall
Red blood cell in capillary overflows, and a small lattice are 25 μm in figure.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is unreceipted specific in embodiment
Condition person, the condition suggested according to normal condition or manufacturer are carried out.Agents useful for same or the unreceipted production firm person of instrument, it is
The conventional products of acquisition purchased in market can be passed through.
The materials and fixing means of the lung tissue of rats of embodiment 1
（1）Rat（Sprague Dawley）Dorsal position is placed on autopsy table after anesthesia, along median line from pubic symphysis extremely
Lower jaw part is cut off and peels off skin, exposure abdominal cavity, abdominal aorta sacrificed by exsanguination.Cut off chest skin of neck, blunt separation escape pipe.
Pay attention to that Chest Wall Structure must not be injured, in order to avoid destroy intrathoracic negative pressure（See Fig. 1）.
（2）Tracheae is ligatured under rat thyroid cartilage with suture, pays attention to pricking extremely, it is impossible to have gas leakage（See Fig. 2）.
（3）Lungs separation process is as follows：Distal end cuts tracheae at ligation, and thymus gland and the heart are simultaneously removed in careful exposure thoracic cavity
Dirty, lifting tracheae with tweezers, from top to bottom blunt separation goes out full lung（Pay attention to avoiding injuring each lobe of the lung）, food is cut from diaphragm
Road, take out lungs（See Fig. 3,4）.
（4）It is a weight at tracheae, lungs snorkeling is fixed on 4% neutral formalin liquid（40% formaldehyde 100ml,
Sodium dihydrogen phosphate 4g, disodium hydrogen phosphate 6.5g, distilled water 900ml, pH7.0）In, fix 24 hours（See Fig. 5）.
（5）The lung tissue fixed is repaiied into blanking method per leaf according to document（OECD Guidance Document on
Histopathology for Inhalation Studies,28September2009Draft1-36）Repair and be cut into thickness about
0.2mm or so blocks, every animal is repaiied altogether cuts five pieces.It is conventional to be dehydrated transparent waxdip（Dehydration：The second of the ethanol of 85% ethanol → 95% → 95%
The ethanol of the ethanol of alcohol → 100% → 100%, often walks 60min.It is transparent：1/2+1/2 dimethylbenzene of ethanol → dimethylbenzene → dimethylbenzene, is often walked
30min.Infiltration：Paraffin → paraffin → paraffin → paraffin, often walks 30min）.FFPE, cycle type microtome, thickness are
3 μm, HE dyeing, and observe under the microscope.Fig. 6 shows the distant view photograph for including all 5 leaf lungs sections, it is seen that each lobe of the lung lung
Bubble is in natural extension state.Fig. 7 sees for Fig. 6 partial enlargements, shows lung tissue alveolars at different levels（Alveolar sac, breathing and alveolar）
Structure is in natural extension state, has no that alveolar is subside or overdistension.Fig. 8 is that Fig. 7 parts further see by amplification, and display alveolar is certainly
So expansion, alveolar septa are belittled.
The materials and fixing means of the mouse lung tissue of embodiment 2
（1）Mouse（KM）Dorsal position is placed on autopsy table after anesthesia, exposure abdominal cavity, abdominal aorta sacrificed by exsanguination.Cut off chest
Skin of neck, blunt separation escape pipe.Pay attention to that Chest Wall Structure must not be injured, in order to avoid destroy intrathoracic negative pressure（See Fig. 9）.
（2）Tracheae is ligatured under mouse thyroid cartilage with suture, pays attention to pricking extremely, it is impossible to have gas leakage（See Figure 10）.
（3）Lungs separation process is as follows：Distal end cuts tracheae at ligation, and thymus gland and the heart are simultaneously removed in careful exposure thoracic cavity
Dirty, lifting tracheae with tweezers, from top to bottom blunt separation goes out full lung（Pay attention to avoiding injuring each lobe of the lung）, food is cut from diaphragm
Road, take out lungs（See Figure 11,12）.
（4）It is a weight at tracheae, mouse lung snorkeling is fixed in 4% neutral formalin liquid, fixes 24 hours
（See Figure 13）.
（5）The lung tissue fixed is repaiied into block method per leaf according to document（OECD Guidance Document on
Histopathology for Inhalation Studies,28September2009Draft1-36）Repair and be cut into thickness about
0.2mm blocks or so, every animal is repaiied altogether cuts five pieces.It is conventional to be dehydrated transparent waxdip, specimens paraffin embedding slices（Method is the same as embodiment 1）,
Slice thickness is 3 μm, HE dyeing, and in Microscopic observation.
Figure 14 shows the distant view photograph for including all 5 leaf lungs sections, it is seen that each lobe of the lung alveolar is in natural extension state.
Figure 15 sees for Figure 14 partial enlargements, shows lung tissue alveolars at different levels（Alveolar sac, breathing and alveolar）Structure is in natural extension shape
State, have no that alveolar is subside or overdistension.
The rat fixing means of embodiment 3 contrasts.
Lung tissue of rats section is prepared according to the method for embodiment 1, while is fixed according to conventional immersion fixation, Intratracheal instillation
Method, cardiac perfusion fixation fix lung tissue respectively（Fixing means reference literature：OECD Guidance Document on
Histopathology for Inhalation Studies,28September2009Draft1-36）, the lung that will fix
Tissue, which per leaf according to document is repaiied block method and repaiied, is cut into about 0.2mm or so blocks, and every animal is repaiied altogether cuts five pieces.Conventional dehydration embedding is cut
Piece（Method is the same as embodiment 1）, slice thickness is 3 μm, HE dyeing, and paired observation is carried out under mirror.
Wherein Figure 16 is the lung tissue section's HE colored graphs prepared using the method for embodiment 1, it can be seen that alveolar structure is complete
It is whole clear, the red blood cell non-spill phenomenon in alveolar wall capillary；
Figure 17 is to utilize the conventional lung tissue section's HE colored graphs for immersing fixation and preparing, it can be seen that alveolar collapse is more
Number alveolar wall is local thicker, is not easy the level of resolution technology；
Figure 18 is the lung tissue section's HE colored graphs prepared using cardiac perfusion fixation, it can be seen that alveolar wall is because of pressure
Telangiectasis caused by big, alveolar is relative to diminish；
Figure 19 is the lung tissue section's HE colored graphs prepared using Intratracheal instillation fixation, it can be seen that alveolar density is not
Even, the red blood cell in alveolar wall capillary overflows.
Although the embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.Root
According to disclosed all teachings, those details can be carried out with various modifications and replacement, these change in the guarantor of the present invention
Within the scope of shield.The four corner of the present invention is provided by appended claims and its any equivalent.
Claims (10)
1. the materials and fixing means of animal lung tissue, it comprises the following steps：
(1) ligature dead animal tracheae, the distal end detachment tracheae at ligation, by whole lungs and ligation tracheae together
Take out；
(2) the whole lungs that step (1) obtains are fixed, the lung tissue being fixed；
Wherein before tracheae is ligatured, the negative pressure state in animal thoracic cavity is not destroyed step (1).
2. the materials and fixing means of claim 1, wherein, in step (2), the fixing means is that whole lungs are complete
It is soaked in fixer.
3. the materials and fixing means of claim 1, wherein the animal is mammal.
4. the materials and fixing means of claim 3, wherein the animal is mouse, rat, cavy, dog, monkey, rabbit, sheep, Ma Huo
Pig.
5. the materials and fixing means of claim 2, wherein the whole lungs that step (1) is obtained are completely soaked in fixation
Method in liquid includes, and the object of lung tissue emersion can be prevented, by weight in fixer fluid level or the covering of lung tissue surface
It is connected with tracheae or takes out lung tissue in the lump with heart.
6. the materials and fixing means of claim 2, wherein the fixer is selected from formalin, paraformaldehyde solution, ethanol
The mixed solution of solution or above-mentioned solution.
7. the animal lung tissue that the materials and fixing means according to any one of claim 1-6 are prepared.
8. the preparation method of lung tissue section, it includes any one of claim 1-6 materials and fixing means, and will be fixed
Lung tissue afterwards carries out the step of film-making.
9. the lung tissue section that the preparation method according to claim 8 is prepared.
10. any one of claim 1-6 materials and fixing means are used for the purposes that the non-clinical study of medicine is tested.
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