WO2014180272A1

WO2014180272A1 - Method for isolating and preparing retinal nerve cell layer in vitro

Info

Publication number: WO2014180272A1
Application number: PCT/CN2014/076547
Authority: WO
Inventors: 彭广华
Original assignee: 郑州大学第一附属医院
Priority date: 2013-05-06
Filing date: 2014-04-30
Publication date: 2014-11-13
Also published as: CN103234774B; CN103234774A

Abstract

Disclosed is a method for isolating and preparing a retinal nerve cell layer in vitro, comprising the following steps: providing eyeballs from experimental animals or donated bodies; cleaning the eyeballs; fixing the eyeballs; providing a retinal extractor; cutting all layers of the eyeball wall all around the eyeball along the margin of the corneosclera or the pars plana using the blade of the retinal extractor, forming an eye cup; placing a retinal-extracting spoon into the eye cup, and taking the crystalline lens and vitreous body out; isolating the inner edge of the eyeball wall all around along the incision of the eyeball wall, and then inserting the retinal-extracting spoon into the subretinal cavity along the radian of the eyeball wall, and taking out the complete retinal nerve cell layer. The method can rapidly extract the retinal nerve cell layer while maintaining the activity of the retina nerve cells, and this improves the efficiency, and can correctly reflect the experimental results.

Description

Method for separating and preparing retinal nerve cell layer in vitro

The invention relates to the field of biotechnology, and in particular to a method for separating and preparing a retinal nerve cell layer in vitro.

Background technique

According to statistics from the World Health Organization (WHO), there are about 160 million visually impaired people and 40 million legally blind people in the world. China is the country with the most blind people in the world, with about 7 million blind people, and about 450,000 people are blind every year in China. , add a blind person every minute. If the rapid increase in the number of blind people in China cannot be effectively controlled, the number of blind people in China is expected to increase to 20 million by 2020. The damage caused by blind eye diseases and the heavy burden on society will be incalculable. Retinal membrane disease is the most common cause of blindness in developed countries (more than 50%). Nearly 200,000 (45%) of the patients with such diseases are blind in China each year. It is a serious blinding eye disease that has no effective treatment in the world. Therefore, finding a treatment for blinding retinopathy with high incidence and serious harm is a major demand for China's economy, society and people's health. Therefore, it is extremely urgent to vigorously carry out basic and clinical research on the retina and to find effective treatment measures.

In the field of experimental ophthalmology, in the research of retinal basic and clinical research, the research on the development of the retina and its function, the pathogenesis of various retinal diseases, it is necessary to separate the retina in the eyeball of the eye source of different species or the donor source of the human body. After removal, the levels of various related cells, molecules, genes, nucleic acids or proteins are detected.

The retina is an important part of the ocular structure. It is a transparent film located in the inner layer of the eye wall. It consists of the retinal nerve layer and the retinal pigment epithelial layer. The retinal nerve layer and pigment. There is a cavity between the epithelial layers, which is the subretinal space, and the retinal pigment epithelial layer is closely connected with the choroid. At present, the commonly used methods for removing the retinal nerve cell layer at home and abroad are obtained by enzymatic digestion and microdissection, and generally take 15-25 minutes. The biggest disadvantage of enzymatic digestion is that the retinal pigment epithelial cells or choroidal pigment cells in the adjacent parts of the retinal nerve cell layer are digested, causing contamination between cells and reducing the purity of retinal nerve cells. The method of microdissection and separation takes a long time. The longer the retina is exposed to the air, the more nerve cells die, and the skilled micromanipulation skills require long training. Since the number of deaths of retinal nerve cells increases with the prolongation of ex vivo time, retinal nerve cells generally account for 40-60% of retinal neuronal cell death after 15-25 minutes of in vitro, which can cause serious errors in the experimental results. How to solve the rapid separation of the retinal nerve cell layer and obtain the active retinal nerve cell layer has been a problem faced by scientists in the ophthalmology field for many years, and it is also a key problem that we urgently need to solve.

Summary of the invention

The object of the present invention is to provide a method for isolating and preparing a retinal nerve cell layer in vitro, which can overcome the shortcomings of the common separation of the retinal nerve cell layer and achieve rapid separation of the retinal nerve cell layer. The invention provides a method for preparing a retinal nerve cell layer in vitro, comprising the following steps:

A. Providing eyeballs or remains of experimental animals to donate eyeballs;

B. Cleaning the eyeball described in step A;

C. Fixing the eyeball described in step B on the eyeball holder;

D. Providing a retina extractor, the retina extractor provided, including a handle, one end of the handle is provided with a retina removal spoon, and the other end is provided with a blade, and the retina removal spoon is flat The surface is inclined at an angle with the horizontal axis of the handle, and the front end of the blade is provided with a cutting edge;

E. Under a dissecting microscope, use the blade of the retina extractor described in step D to cut the entire wall of the eyeball along the corneoscleral limb or flat portion to form an eye cup;

F. Using the retina removal spoon described in step D, enter the eye cup, and take out the lens and the vitreous;

G. Separate the inner layer of the wall of the eyeball along the incision along the wall of the eyeball, and then remove the sclera along the arc of the eyeball into the subretinal space with the retina of the eyeball in step D, and take out the intact retinal nerve cell layer.

Preferably, the layer of retinal nerve cells taken out of step G is placed in a pre-cooled centrifuge tube for the next experiment.

Preferably, the method of cleaning in the step B comprises the following steps: firstly rinsing the eyeball with the pre-cooled phosphate buffer of pH 7.4, and assessing whether there is bacterial contamination or infection according to the source of the eyeball, and selecting the corresponding concentration. Antibiotics are added to the pre-cooled saline to fully flush the eyeballs. Generally, the infection is prevented. Gentamicin is used to contain 20 IU per milliliter of normal saline. The washed eyeballs are placed on the eyeball holder with ice cubes, and finally the eyeballs are removed. All blood-like appendages and other accessory tissues on the wall surface retain four rectus muscles.

Preferably, the pre-cooling is to pre-bury the bottle containing the pH buffer of the pH 7.4 and the physiological saline in the ice, and maintain the temperature of the pH buffer of the pH 7.4 and the physiological saline at about 0 degrees. The cleaning step is carried out in an environment where the temperature is between 0 and 4 degrees.

Preferably, the four rectus muscles are fixed on an eyeball holder equipped with ice cubes corresponding to the size of the eyeball.

6-1. 5mm, the depth of the whole layer of the eye wall is 360 degrees, the depth of the wall of the eyeball is 0. 6-1. 5mm, the depth of the whole wall of the eye wall is 360 degrees. Remove the anterior segment of the eye and preserve the tissue structure inside the eye cup.

Optimum solution: Before cutting the whole layer of the eye wall, use the marker pen to draw the part to be cut in advance to avoid misalignment.

Preferably, the separation in the step G is performed by gently gripping the inner edge of the eye wall with a gray tooth, and the front end of the iris separator is fully separated and separated along the entire circumference of the eye wall incision. The depth is about 2-5mm, the average mouse is 2_2. 5mm, the large animal is 3_4mm, and the human eye is mostly 5 or so.

Beneficial effects: Quickly remove the retinal nerve cell layer, make the experimental results reflect the authenticity, solve the problems that have plagued the scientists in the ophthalmology field for many years; quickly take out the retinal nerve cell layer, simply take out the method, preserve the activity of the retinal nerve cells, and improve The key to efficiency is to accurately reflect the experimental results and create solid and favorable basic conditions for the study of retinal pathogenesis, retinal development and gene regulation mechanisms.

Using the retina extractor developed in this patent, the retinal nerve cell layer is quickly and completely removed from the eyeball or animal donated eyeball in 5 seconds to ensure the activity of retinal nerve cells, and can accurately detect the origin of different species. The level of RNA, protein expression, and gene expression of retinal neurons in vertebrates, eyeballs or remains of different developmental stages donated to the eye, which makes the results of various experiments the most authentic, for the study of retinal development and early retinal lesions. Provide technical support.

This patented method is compared with conventional enzymatic digestion and anatomical separation of the retina: quantitative analysis of mice 14 days after birth, Rho and Mop two opsin, ganglion cells in photoreceptor cells in each pair of retinal neurons The detection of Thyl protein content showed that: the expression levels of the three proteins in the retinal nerve cells obtained by the patent method were 90%, respectively. 80%, 75%; enzymatic digestion method is 35%, 31%, 26%; anatomical separation method is 36%, 30%, 25%; the overall level of the three proteins is more than 2 times higher than the existing methods; A qualitative analysis of the levels of Rho, Mop and Thyl in a pair of retinal neurons showed that the levels of the three proteins in the retinal neurons obtained by this patent method were significantly higher than the other two methods; Quantification of total RNA levels in the retina showed 98% of the patented method, 40% for enzymatic digestion, and 35% for microdissection.

DRAWINGS

Figure 1. This patent and the conventional enzymatic digestion, anatomical separation of three methods to obtain the retinal nerve layer, three retinal nerve cell protein level quantitative analysis comparison chart;

Figure 2. Qualitative analysis of Rho, Mop and Thyl levels in the three methods of enzymatic digestion and anatomical separation;

Figure 3. Comparison of quantitative analysis of total RNA levels in retinal neurons in the three methods of enzymatic digestion and anatomical separation;

detailed description

Example 1: A method for in vitro separation preparation of a retinal nerve cell layer of an experimental animal mouse eyeball, the following steps were taken as follows:

(1) Cleaning the eyeball: Firstly rinse the mouse-derived eyeball with pre-cooled phosphate buffer (pH 7.4), and then fully flush the eyeball with pre-cooled physiological saline containing 20 IU of gentamicin solution per ml; Place on the eyeball holder with ice cubes, remove all blood sample attachments and other accessory tissues on the surface of the eye, and retain the four rectus muscles. The precautions in this step are as follows: Because there are many blood sample attachments and other ancillary tissues on the surface of the eyeball from the animal (or the eyeball donated by the remains), in order to prevent contamination between the tissue cells, carefully cut the eye carefully with a curved scissors. Tissue and blood sample attachment on the surface of the ball, retaining four rectus muscles. 0-1. 5mm, remember not to take the eyeball, because the thickness of the eyeball wall is about 0. 6-0. 8mm, the wall thickness of the large animal eye is about 0. 8_1. 2mm, the thickness of the human eye wall is about 1. 0-1. 5mm, remember not to put the eyeball The wall was cut. This procedure requires accurate and rapid action, ensuring operation at low temperatures of 0 °C - 4 °C to ensure the activity of various cells in the retinal nerve layer. For each eyeball, a disposable cleaning film for the eyeball holder is required to prevent contamination.

(2) Fixing the eyeball: Fix the four rectus muscles of the cleaned mouse eyeball on the eyeball holder with ice cubes corresponding to the size of the eyeball. Note: In order to prevent contamination, replace one eye protection film with a disposable cleaning film after each eyeball is rinsed. When fixing the eyeball, be careful not to forcibly pull the four rectus muscles, because the scleral thickness of the osseous sclera attachment point is about 0. 2-0. 3mm, so as not to cause the eyeball to rupture. Choose an eyeball holder of the appropriate size and select it based on the diameter of the eyeball. If the size of the eyeball holder is not suitable, when the wall of the eyeball is cut, severe cases may cause the contents of the eye to escape, which may also cause inconvenience in operation.

(3) Incision of the eye wall: Under a dissecting microscope, the blade of one end of the retina extractor is used on the eyeball of the mouse, and the entire wall of the eyeball is cut 360 degrees along the limbus after lmm. Note: The blade must ensure the sharpness of its blade to ensure that unnecessary pressure is applied to the eyeball during surgery to avoid repeated use to prevent contamination. According to the thickness of the eye wall, the depth of the cut wall is well controlled. 5毫米。 The small animal eye wall thickness of about 0. 6-0. 8mm. This step must be carefully performed under the microscope, and should not be cut too shallow or too deep, both of which can cause damage to the cells of the eye tissue. The small animal's eye wall is cut open along the limbus lmm, and the marked area is pre-cut with a marker to avoid misalignment.

(4) Clearing the contents of the eye: Remove the anterior segment of the eye and take the retina at the end of the retina extractor Take out the spoon, enter the eye along the incision in the wall of the eyeball, and gently lift the lens and the vitreous. Precautions: Do not apply any pressure to the eye cup during operation. Small animal use the retina removal spoon to enter from the arc of the eye wall along the arc of the ball wall, gently pull the lens together with the vitreous body; The movement must be gentle, remember not to use it to stir the eye, so as not to pull the retina, causing the artificial retinal detachment.

(5) Remove the retinal nerve layer: Gently grasp the gray-white inner layer edge with a toothless sputum, and use the front end of the iris separator to fully separate along the entire circumference of the eye wall incision. The separation depth is 2- 2. About 5mm, replace the removal spoon and enter the subretinal space along the separated inner layer of the ball wall, and take out the intact retinal nerve cell layer. Note: As the eyeball is isolated, the longer the in vitro time, the easier the retinal nerve layer is to detach from the retinal pigment epithelial layer. The action must be gentle during the operation of the step, and enter the natural arc of the subretinal space. The retinal nerve layer is removed. According to the size of the eyeball, select the retina removal spoon of the appropriate diameter. Adjust the retina to take out the spoon at a proper angle and then enter the subretinal space. Remember to do not rude the operation. This sputum is most likely to cause incomplete retinal removal, or the retina is fragmented.

(6) Treatment after removal of the retinal nerve layer: After removing the retinal nerve layer, immediately place it in a pre-cooled centrifuge tube for the next experiment.

In order to confirm the superiority and reliability of the patented method, the retinal nerve cell layer separation preparation and the conventional enzymatic digestion and anatomical separation method are used to obtain the retinal nerve cell layer of the mouse 14 days after birth, and the retinal nerve cell is obtained. The quantitative levels of Rho, Mop and Thyl were quantitatively and qualitatively analyzed. After RNA extraction from the retina obtained by the three methods, total RNA levels of retinal neurons were quantitatively detected.

Figure 1. Extraction of retinal neural cell layer with conventional enzymatic digestion and dissection using this patented method The method of isolation was compared: the retinal nerve cells of 14-day-old mice were taken out, and the levels of Rho, Mop and Thyl were quantitatively analyzed. The content of three proteins in retinal neurons obtained by this method was significantly higher than that. 2 times the other two methods.

Figure 2. Comparison of methods for extracting retinal nerve cell layers with conventional enzymatic digestion and anatomical separation: Retinal nerve cells from 14-day-old littermates were removed, and three retinal nerve layers, Rho, Mop, and Thyl, were performed. Qualitative analysis of the main protein levels in photoreceptor cells and ganglion cells. The content of three proteins in retinal neurons obtained by this method was significantly higher than the other two methods. The protein commonly present in β-Actin was used as a positive control group. There was no significant difference in the retinal nerve layer extraction method compared to the protein. The experiment visually shows that rapid removal of retinal neural layer cells can correctly reflect the content of various related proteins in the retinal nerve layer.

Figure 3. Quantitative detection of total RNA levels in retinal neurons: Total RNA levels in a pair of retinas per mouse were quantified by 98% for the patented method, 40% for enzymatic digestion, and 35% for microdissection.

Example 2: A method for in vitro separation and preparation of a retinal nerve cell layer for donating eyeballs to human remains, the same procedure as in Example 1 is not repeated, except that: Under a dissecting microscope, the full-thickness of the eye wall was cut 360 degrees on the human donor eyeball 4 mm behind the corneoscleral edge with a blade at one end of the retina extractor. To remove the contents of the eye, first remove the lens with the retina removal spoon along the eye wall incision horizontally, and then gently enter the glass body along the arc of the ball wall. When the inner layer of the eyeball incision is separated by the iris restorer, the separation depth is generally about 5 mm.

Claims

claims

1. A method for isolating and preparing the retinal nerve cell layer in vitro, including the following steps:

A. Provide eyeballs from experimental animals or donated eyeballs from dead bodies;

B. Clean the eyeball as described in step A;

C. Fix the eyeball mentioned in step B on the eyeball holder;

D. Provide a retina extractor. The retina extractor provided includes a handle. One end of the handle is provided with a retina extraction spoon, and the other end is provided with a blade. The plane where the retina extraction spoon is located forms an oblique angle with the horizontal axis of the handle, so The front end of the blade is provided with a tip;

E. Under a dissecting microscope, use the blade of the retinal extractor described in step D to incise the entire thickness of the eyeball wall along the corneoscleral edge or flat part of the eyeball to form an eye cup;

F. Use the retinal removal spoon described in step D to enter the eye cup and remove the lens and vitreous body;

G. Separate the inner layer of the eyeball wall along the entire circumference of the incision, then use the retinal removal spoon described in step D to enter the subretinal space along the curvature of the eyeball wall, and remove the complete retinal nerve cell layer.

2. The method for in vitro separation and preparation of the retinal nerve cell layer according to claim 1, further comprising: placing the retinal nerve cell layer taken out in step G into a pre-cooled centrifuge tube.

3. The method for in vitro separation and preparation of the retinal nerve cell layer according to claim 1, characterized in that: the cleaning of the isolated eyeball in step B includes the following steps, first using pre-cooled phosphate buffer of ΙΉ7.4 Fully flush the eyeball with the solution, and then evaluate whether there is bacterial contamination or infection based on the source of the eyeball. Select antibiotics of corresponding concentration and add them to pre-cooled saline to flush the eyeball. Place the flushed eyeball on an eyeball holder filled with ice cubes. Finally, all blood sample attachments and other attached tissues on the surface of the eyeball are removed, leaving the four rectus muscles.

4. The method for in vitro separation and preparation of retinal nerve cell layer according to claim 3, characterized in that: the pre-cooling is to bury bottles containing phosphate buffer solution of pH 7.4 and physiological saline in ice in advance , keep the temperature of the phosphate buffer solution of pH 7.4 and physiological saline at about 0 degrees, and the cleaning steps are carried out in an environment with a temperature of 0-4 degrees.

5. The method for in vitro separation and preparation of the retinal nerve cell layer according to claim 3, characterized in that: the four rectus muscles are fixed on an eyeball holder equipped with ice cubes corresponding to the size of the eyeball.

6. The method for in vitro separation and preparation of the retinal nerve cell layer according to claim 1, characterized in that: in the step E, the entire thickness of the eyeball wall is incised 1-4 mm behind the scleral limbus of the eye corner, and the depth of the incision of the eyeball wall is 0. 6-1. 5mm, the circumference of the full-thickness incision on the eyeball wall is 360 degrees.

7. The method for in vitro separation and preparation of the retinal nerve cell layer according to claim 6, characterized in that: before incising the entire thickness of the eyeball wall, use a marker pen to draw the part to be incised in advance to avoid misalignment.

8. The method for in vitro separation and preparation of the retinal nerve cell layer according to claim 1, characterized in that: in the separation in step G, use a pair of toothless tweezers to clamp the gray-white inner edge of the eyeball wall, and use The front end of the iris restorer is separated along the entire circumference of the incision on the eyeball wall, with a separation depth of 2-5mm.