CN103234774A - Method for in vitro separation and preparation of retina nerve cell layer - Google Patents
Method for in vitro separation and preparation of retina nerve cell layer Download PDFInfo
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- CN103234774A CN103234774A CN2013101621809A CN201310162180A CN103234774A CN 103234774 A CN103234774 A CN 103234774A CN 2013101621809 A CN2013101621809 A CN 2013101621809A CN 201310162180 A CN201310162180 A CN 201310162180A CN 103234774 A CN103234774 A CN 103234774A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention provides a method for in vitro separation and preparation of a retina nerve cell layer. The method comprises the following steps of providing an experimental animal eyeball or a body donation eyeball; cleaning the eyeball, and fixing the eyeball; providing a retina extractor; cutting open a full layer of eyeball wall around the eyeball by a blade of the retina extractor along a corneosclera edge or a flat section to form an eye cup; causing a retina extracting ladle to enter the eye cup; taking out a crystalline humor and a vitreous body; separating the edge of the inner layer of the eyeball wall along the complete cycle of an incision of the eyeball wall; and entering a lower cavity of the retina by the retina extracting ladle along the radian of the eyeball wall, and taking out an intact retina nerve cell layer. By adopting the method, the retina nerve cell layer can be rapidly taken out; the activity of the retina nerve cell is stored; the efficiency is improved; and the experimental result can be correctly reflected.
Description
Technical field:
The present invention relates to biological technical field, particularly relate to a kind of method of retinal neuronal cell layer in-vitro separation preparation.
Background technology:
The World Health Organization's statistical data shows that the whole world has 1.6 hundred million people with visual impairment approximately, 4,000 ten thousand legal blind persons, and China is the maximum country of whole world blind person, and 7,000,000 blind persons are arranged approximately, and China has 450,000 people blind every year approximately, per minute increases a routine blind person newly.If the trend that China blind person increases fast can't be controlled effectively, will be increased to 2,000 ten thousand to the Chinese blind person of the year two thousand twenty expection, the injury that causes because of blinding illness in eye and can't estimate to the heavy burden that society causes.Retinal disease is the common cause (surpassing 50%) of developed country's blinding, and blind nearly 200,000 (the accounting for 45%) of patient of the annual therefore class disease of China are the serious blinding illness in eye of still not having effective treatment means at present in the world.Therefore, seeking treatment means for incidence of disease height, the heavy blinding retinal disease of harm, is the great demand of China's economy, society and people ' s health.Therefore, carry out amphiblestroid basis and clinical research energetically, the research of seeking therapy measure is extremely urgent.
The experimental eye scientific domain, in retina basis and clinical research, Study on Pathogenesis for retinal development and function thereof, various retinal diseases, all need to carry out the detection of the levels such as cell, molecule, gene, nucleic acid or protein of all kinds of correlativitys again after the intraocular retina separation taking-up in the eyeball of different genera animal origin or human body donation source.
Retina is the important component part of eye structure, it is the layer of transparent film that is positioned at the wall of eyeball internal layer, formed by cerebral stratum of retina and layer of retina,pigment epithelium, one lacuna is arranged between cerebral stratum of retina and the pigment epithelial layer, be the retina cavity of resorption, layer of retina,pigment epithelium closely links to each other with choroid.At present, the method for taking-up retinal neuronal cell layer commonly used is to separate to obtain with microdissection with enzymic digestion both at home and abroad, and generally needing the time is 15-25 minute.The disadvantage of enzymic digestion is that retinal pigment epithelium or the pigment cell of choroid of retinal neuronal cell layer adjacent regions are digested, and causes the pollution between the cell, and retinal neuronal cell purity is descended; The method that microdissection separates is consuming time longer, and it is more long that retina is exposed in the air time, and the mortality of neurocyte is more many, and consummate micromanipulation skill needs training for a long time.Because retinal neuronal cell is along with the dead quantity of the prolongation of isolated time increases, general retinal neuronal cell exsomatized after 15-25 minute, and the retinal neuronal cell mortality ratio accounts for 40-60%, can cause the gross error of experimental result.How solving the quick separation of retinal neuronal cell layer, obtain having active retinal neuronal cell layer, is the puzzlement that the field of ophthalmology scientist faces for many years, also is the emphasis difficult problem that we are badly in need of solving.
Summary of the invention:
The object of the invention provides a kind of method of retinal neuronal cell layer in-vitro separation preparation, can overcome the long shortcoming of used time of separation of retinal neuronal cell layer commonly used, realizes the quick separation of retinal neuronal cell layer.The invention provides a kind of method of retinal neuronal cell layer in-vitro separation preparation, comprise the steps:
A., animal used as test eyeball or Remains Donation eyeball are provided;
B. eyeball described in the cleaning A;
C. eyeball described in the step B is fixed on the ophthalmostat;
D., the retina extractor is provided, and the retina extractor that provides comprises handle, described handle one end is provided with retina and takes out spoon, the other end is provided with blade, and described retina takes out spoon plane, place and the handle horizontal axis is slanted angle, and the front end of described blade is provided with point of a knife;
E. under dissecting microscope, use the blade of retina extractor described in the step D to cut the eyeball wall of eyeball holostrome in full week along corneoscleral junction or par, form an eyecup;
F. take out spoon with retina described in the step D and enter eyecup, crystalline lens and vitreum are taken out;
G. separate the internal layer of wall of eyeball along the full week of otch of wall of eyeball, take out spoonful radian along wall of eyeball with retina described in the step D again and enter the retina cavity of resorption, take out complete retinal neuronal cell layer.
Preferred version: comprise that also the retinal neuronal cell layer that step G is taken out is placed in the centrifuge tube of precooling, is used for next step experiment.
Preferred version: the method that cleans among the described step B comprises the following steps, eyeball fully washes with the phosphate buffer of the PH7.4 of precooling in elder generation, originate according to eyeball, whether assessment has bacterial contamination or infection, select the interior fully flushing of the physiological saline eyeball of the microbiotic adding precooling of respective concentration, general prevention is infected, use gentamicin then every ml physiological saline contains 20IU, the eyeball that washed is placed on the ophthalmostat that ice cube is housed, remove all blood sample adjuncts and other attached tissues on wall of eyeball surface at last, keep four rectus.
Preferred version: described precooling, be that handle is equipped with the phosphate buffer of PH7.4 and the bottle of physiological saline is embedded in the ice in advance, the temperature of the phosphate buffer of PH7.4 and physiological saline is remained on about 0 degree, and the step of cleaning is in temperature under the environment of 0-4 degree carries out.
Preferred version: described four rectus are fixed on the eyeball size are equipped with accordingly on the ophthalmostat of ice cube.
Preferred version: in the described step e, 1-4mm cuts the holostrome of wall of eyeball behind the canthus limbus of sclera, and the degree of depth of cutting wall of eyeball is 0.6-1.5mm, and the wall of eyeball holostrome otch circumference number of degrees are 360 degree, remove anterior chamber of eye tissue, keeps the interior institutional framework of eyecup;
Preferred version: cut before the wall of eyeball holostrome, the position of drawing and will cut with marker pen in advance is in order to avoid misplace.
Preferred version: the separation among the described step G, clamp wall of eyeball gently with a Smooth forceps and be linen internal layer edge, the sufficient separation that gives in full week with the front end edge wall of eyeball otch of iris separation vessel, separate the degree of depth about 2-5mm, general mouse 2-2.5mm, large animal 3-4mm, human eye mostly is about 5mm.
Beneficial effect: take out the retinal neuronal cell layer fast, make experimental result reflect authenticity, solved the problem that perplexs the field of ophthalmology scientist for many years; Take out fast the retinal neuronal cell layer, easy removing method, the activity of having preserved retinal neuronal cell, improved efficient, key is energy correct response experimental result, is retina pathogenesis, retinal development and gene regulation Study on Mechanism, has created solid favourable basic condition.
Adopt the retina extractor of developing in this patent, in 5 seconds fast complete from animal origin eyeball or the eyeball of Remains Donation take out the retinal neuronal cell layer, guarantee the activity of retinal neuronal cell, can accurately detect rna level, protein expression level, gene expression dose of the retinal neuronal cell of the vertebrate, different developmental phases animal eyeball or the Remains Donation eyeball that derive from different genera etc., make various experimental results possess authenticity, for the research of retinal development and the research of retina early lesion provide technical support.
This patent method and conventional enzymic digestion and amphiblestroid three kinds of method comparative studies of anatomical isolation: the quantitative test back 14 days mouse that is born, each is to Rho in the photoreceptor cell in the retinal neuronal cell and two kinds of opsins of Mop, the detection of Thy1 protein content in the ganglion cell, the result shows: in the retinal neuronal cell that this patent method obtains, the expression of three kinds of albumen is respectively 90%, 80%, 75%; Enzyme digestion is respectively 35%, 31%, 26%; The anatomical isolation method is 36%, 30%, 25%; The aggregate level of three kinds of albumen improves more than 2 times than existing method; Each is to Rho, Mop in the retinal neuronal cell and three kinds of protein level qualitative analyses of Thy1, and the result shows that the level of three kinds of albumen is apparently higher than other two kinds of methods in the retinal neuronal cell that this patent method obtains; Every a pair of amphiblestroid total rna level of mouse is shown that quantitatively this patent method is 98%, and enzyme digestion is 40%, and microdissection is 35%.
Accompanying drawing and description of drawings
Fig. 1. this patent obtains cerebral stratum of retina, protein level quantitative test comparison diagrams in three kinds of retinal neuronal cells with three kinds of methods of enzymic digestion, anatomical isolation of routine;
Fig. 2. Rho, Mop and three kinds of protein level qualitative analyses of Thy1 comparison diagram in three kinds of methods of enzymic digestion, anatomical isolation of this patent and routine;
Fig. 3. the total rna level quantitative test of retinal neuronal cell comparison diagram in three kinds of methods of enzymic digestion, anatomical isolation of this patent and routine.
Embodiment:
Embodiment 1: the method that a kind of retinal neuronal cell layer in-vitro separation that the animal used as test eyeball of mouse is carried out prepares, the step of taking is as follows:
(1) cleaning eyeball: at first use the phosphate buffer (pH7.4) of precooling fully to wash the eyeball that mouse is originated, fully wash eyeball with every milliliter of pre-cold saline that contains the gentamicin liquid of 20IU again; Eyeball is placed on the ophthalmostat that ice cube is housed, and removes all blood sample attachments and other affiliated groups of eyeball surface, keeps four rectus.Points for attention are as follows in this step: because from animal (or eyeball of Remains Donation) eyeball surface many blood sample attachments and other attached tissues are arranged, in order to prevent the pollution between the histocyte, carefully carefully wipe out tissue and the blood sample attachment of eyeball surface with the curved scissors cutter, keep four rectus.Because wall of eyeball is thinner, the about thickness 0.6-0.8mm of toy wall of eyeball, the about 0.8-1.2mm of large animal wall of eyeball thickness, people's the about 1.0-1.5mm of wall of eyeball thickness makes sure to keep in mind wall of eyeball not to be broken.This step requires action accurately rapidly, guarantees 0
0-4
0Operate under the cryogenic conditions, to guarantee the various cell activity of cerebral stratum of retina.Each eyeball, needs are changed the disposable film of keeping a public place clean of an ophthalmostat, with anti-pollution.
(2) fixing eyeball: four rectus of the eyeball of mouse that cleaning is crossed are fixed on the eyeball size and are equipped with accordingly on the ophthalmostat of ice cube.Points for attention: in order to prevent polluting, each eyeball is changed a disposable film of keeping a public place clean of opening one's eyes the ball fixator after flushing.When fixing eyeball, ten million notes not four rectus of tractive by force, because the scleral thickness of rectus sclera attachment point is approximately 0.2-0.3mm, in order to avoid cause eyeball rupture.Selecting sizeable ophthalmostat, serves as according to selecting with the diameter of eyeball.If the ophthalmostat size is not suitable for, when cutting wall of eyeball, severe patient can cause tolerant the deviating from of intraocular, also can bring the inconvenience of operation.
(3) cut wall of eyeball: under dissecting microscope, on eyeball of mouse, 1mm cuts wall of eyeball 360 degree of holostrome behind the corneoscleral junction with the blade of retina extractor one end.Points for attention: blade must be guaranteed the sharp of its blade, avoids eyeball is applied unnecessary pressure when guaranteeing to perform the operation, and avoids reusing, and pollutes preventing; According to the thickness of different walls of eyeball, grasp the degree of depth of cutting the ball wall, the toy wall of eyeball thickness of generally growing up is approximately 0.6-0.8mm.This step must carefully be operated at microscopically, and the mistake of Qie Kaiing is not shallow or dark excessively, and the two all can cause the eyeball tissue cells injury.It is that 1mm cuts along corneal limbus outside that the toy wall of eyeball cuts, and the position of drawing and will cut with marker pen in advance is in order to avoid misplace.
(4) it is tolerant to remove intraocular: remove anterior chamber of eye, take out spoon with the retina of retina extractor one end, enter intraocular along the otch of wall of eyeball, crystalline lens and vitreum are held out gently.Points for attention: eyecup is not applied any pressure during operation, toy takes out the arc of spoon from the otch of wall of eyeball along the ball wall with retina and enters, and gently crystalline lens and vitreum is held out together; When retina taking-up spoon entered the intraocular operation, action must be soft, makes sure to keep in mind to use it to stir the intraocular thing, in order to avoid the tractive retina causes artificial detachment of retina.
(5) take out cerebral stratum of retina: with one Smooth forceps is clamped wall of eyeball gently and be linen internal layer edge, the sufficient separation that gives in full week with the front end edge wall of eyeball otch of iris separation vessel, separate the degree of depth about 2-2.5mm, change to take out under the interior layer tissue that spoon separated along the ball wall and enter the retina cavity of resorption, take out complete retinal neuronal cell layer.Points for attention: because after eyeball exsomatized, isolated time was more long, cerebral stratum of retina is more easy to break away from layer of retina,pigment epithelium, and action must be soft during this step operation, enters along the natural radian of retina cavity of resorption, and the holostrome cerebral stratum of retina is taken out.According to the size of eyeball, the retina of the diameter of selecting to adapt takes out spoon.Adjust and enter the retina cavity of resorption after retina takes out the certain appropriate angle of spoon, make sure to keep in mind operational motion not roughly, this step, the easiest retina that causes took out not exclusively, or retina is the fragment shape.
(6) processing behind the taking-up cerebral stratum of retina: after taking out cerebral stratum of retina, be placed on immediately in the centrifuge tube of precooling, carry out next step experiment.
For superiority and the reliability that confirms this patent method, utilize this patent method retinal neuronal cell layer to separate enzymic digestion and the anatomical isolation method of preparation and routine, obtain the retinal neuronal cell layer of back 14 days mouse of birth, Rho, Mop and the Thy1 three kind protein levels contained to retinal neuronal cell wherein carry out quantitatively and qualitative analysis; And the retina that three kinds of methods obtain carried out carrying out the total rna level of retinal neuronal cell and quantitatively detecting after RNA extracts.
Fig. 1. adopt this patent method to extract the retinal neuronal cell layer and compare research with the enzymic digestion of routine and the method for anatomical isolation: take out the retinal neuronal cell of giving birth to back 14 days brood mouse, carry out three kinds of protein level quantitative test of Rho, Mop and Thy1, the content of three kinds of albumen is apparently higher than 2 times of other two kinds of methods in the retinal neuronal cell that this method is obtained.
Fig. 2. adopt this method to extract the retinal neuronal cell layer and compare research with the enzymic digestion of routine and the method for anatomical isolation: take out the retinal neuronal cell of giving birth to back 14 days brood mouse, carry out the horizontal qualitative analysis of major protein in three kinds of cerebral stratum of retina photoreceptor cells of Rho, Mop and Thy1 and the ganglion cell, the content of three kinds of albumen is apparently higher than other two kinds of methods in the retinal neuronal cell that this method is obtained; Ubiquitous albumen is as positive controls in retina for β-Actin, and three kinds of cerebral stratum of retina removing methods contrast this albumen no significant difference.This experiment shows intuitively, takes out the retina neural confluent monolayer cells fast, the content of various associated protein in the reaction cerebral stratum of retina that can be correct.
Fig. 3. the total rna level of retinal neuronal cell quantitatively detects: every a pair of amphiblestroid total rna level of mouse shows that quantitatively this patent method is 98%, and enzyme digestion is 40%, and microdissection is 35%.
Embodiment 2: be the method that a kind of retinal neuronal cell layer in-vitro separation that people's Remains Donation eyeball carries out prepared, the step identical with embodiment 1 no longer repeats, difference is: cut in the wall of eyeball step, under dissecting microscope, cut wall of eyeball 360 degree of holostrome with blade 4mm after human body is contributed eyeball upper edge corneoscleral junction of retina extractor one end.Remove in the tolerant step of intraocular, take out spoon with retina earlier and enter along wall of eyeball otch level crystalline lens is held out, enter along the arc of ball wall then vitreum is held out gently.When separating the internal layer in full week of eyeball otch with iris repositor, generally separating the degree of depth is about 5mm.
Claims (8)
1. the method for a retinal neuronal cell layer in-vitro separation preparation comprises the steps:
A., animal used as test eyeball or Remains Donation eyeball are provided;
B. eyeball described in the cleaning A;
C. eyeball described in the step B is fixed on the ophthalmostat;
D., the retina extractor is provided, and the retina extractor that provides comprises handle, described handle one end is provided with retina and takes out spoon, the other end is provided with blade, and described retina takes out spoon plane, place and the handle horizontal axis is slanted angle, and the front end of described blade is provided with point of a knife;
E. under dissecting microscope, use the blade of retina extractor described in the step D to cut the eyeball wall of eyeball holostrome in full week along corneoscleral junction or par, form an eyecup;
F. take out spoon with retina described in the step D and enter eyecup, crystalline lens and vitreum are taken out;
G. separate the internal layer of wall of eyeball along the full week of otch of wall of eyeball, take out spoonful radian along wall of eyeball with retina described in the step D again and enter the retina cavity of resorption, take out complete retinal neuronal cell layer.
2. the method for retinal neuronal cell layer in-vitro separation preparation according to claim 1 is characterized in that: comprise that also the retinal neuronal cell layer that step G is taken out is placed in the centrifuge tube of precooling.
3. the method for retinal neuronal cell layer in-vitro separation according to claim 1 preparation, it is characterized in that: the in-vitro eyeball cleaning comprises the following steps among the described step B, eyeball fully washes with the phosphate buffer of the PH7.4 of precooling in elder generation, originate according to eyeball again, whether assessment has bacterial contamination or infection, select the interior flushing of the physiological saline eyeball of the microbiotic adding precooling of respective concentration, the eyeball that washed is placed on the ophthalmostat that ice cube is housed, remove all blood sample attachments and other attached tissues of eyeball surface at last, keep four rectus.
4. the method for retinal neuronal cell layer in-vitro separation according to claim 3 preparation, it is characterized in that: described precooling, be that handle is equipped with the phosphate buffer of PH7.4 and the bottle of physiological saline is embedded in the ice in advance, the temperature of the phosphate buffer of PH7.4 and physiological saline is remained on about 0 degree, and the step of cleaning is in temperature under the environment of 0-4 degree carries out.
5. the method for retinal neuronal cell layer in-vitro separation preparation according to claim 3 is characterized in that: described four rectus are fixed on the eyeball size are equipped with accordingly on the ophthalmostat of ice cube.
6. the method for retinal neuronal cell layer in-vitro separation according to claim 1 preparation, it is characterized in that: in the described step e, 1-4mm cuts the wall of eyeball holostrome behind the canthus limbus of sclera, and the degree of depth of cutting wall of eyeball is 0.6-1.5mm, and the wall of eyeball holostrome otch circumference number of degrees are 360 degree.
7. the method for retinal neuronal cell layer in-vitro separation according to claim 6 preparation is characterized in that: cut before the wall of eyeball holostrome, the position of drawing and will cut with marker pen in advance is in order to avoid misplace.
8. the method for retinal neuronal cell layer in-vitro separation according to claim 1 preparation, it is characterized in that: the separation among the described step G, clamp wall of eyeball with a Smooth forceps and be linen internal layer edge, with separating in full week of the front end edge wall of eyeball otch of iris repositor, the separation degree of depth is 2-5mm.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310162180.9A CN103234774B (en) | 2013-05-06 | 2013-05-06 | Method for in vitro separation and preparation of retina nerve cell layer |
| PCT/CN2014/076547 WO2014180272A1 (en) | 2013-05-06 | 2014-04-30 | Method for isolating and preparing retinal nerve cell layer in vitro |
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| CN201310162180.9A CN103234774B (en) | 2013-05-06 | 2013-05-06 | Method for in vitro separation and preparation of retina nerve cell layer |
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Cited By (2)
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| WO2014180272A1 (en) * | 2013-05-06 | 2014-11-13 | 郑州大学第一附属医院 | Method for isolating and preparing retinal nerve cell layer in vitro |
| CN111925988A (en) * | 2020-08-29 | 2020-11-13 | 郑州大学 | Method for preparing single cell suspension based on retina tissue/organoid-retina tissue |
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| CN111925988A (en) * | 2020-08-29 | 2020-11-13 | 郑州大学 | Method for preparing single cell suspension based on retina tissue/organoid-retina tissue |
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| CN103234774B (en) | 2015-06-03 |
| WO2014180272A1 (en) | 2014-11-13 |
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