CN117887725A - A gene for mitigating tritium-induced visual nervous system damage, DMBX1A - Google Patents

Abstract

The present invention belongs to the field of molecular biology, and specifically relates to a gene for alleviating damage to the visual nervous system caused by tritiated water. The present invention provides a gene dmbx1a for alleviating damage to the visual nervous system caused by tritiated water. After overexpression, the spontaneous movement and hatching of the tail of zebrafish exposed to tritiated water can be improved, the apoptosis of head cells can be alleviated, the apoptotic bodies can be reduced, the maxillofacial angle can be enlarged, and the length of the mandible can be shortened. The results of the embodiment show that dmbx1a can alleviate the toxic effects of the visual nervous system of zebrafish caused by tritium exposure.

Description

A gene for mitigating tritium-induced visual nervous system damage, DMBX1A

Technical Field

The invention belongs to the field of molecular biology, and in particular relates to a gene for alleviating tritium-induced visual nervous system damage.

Background technique

Tritium is an important type of radionuclides in the effluents discharged during the operation of nuclear power plants. Its diffusion, migration and transformation in the environment and the environmental pollution and health hazards it causes to organisms have attracted great attention.

Tritium, also known as super tritium, is an isotope of hydrogen, and its properties are very similar to hydrogen. After entering the body, tritium will appear as tissue free tritium (TFWT) and organically bound tritium (OBT), and participate in the metabolic cycle. Due to the biological half-life of the two, OBT stays in the body longer than TFWT, which will cause more serious harm.

Whether it is OBT or TFWT, once it enters the body, the damage caused to the body is systemic and multi-faceted, and almost all tissues and organs will be affected to varying degrees. Especially in OBT, tritium is fixed in H-containing compounds or even DNA molecules, and the body will continue to be irradiated by beta rays. Ionizing radiation damage is not only related to the type and dose of radionuclides, but also to the sensitivity of tissues and organs. For the nervous system, ionizing radiation affects the nervous system in many ways, such as neuronal migration and dendritic development, by damaging neural stem cells. During embryonic development, active nerve cells that divide and differentiate are more sensitive to radiation. At this time, the effect of ionizing radiation on the body's nervous system is more serious, which can lead to abnormalities in neuronal cell proliferation, differentiation, migration, etc., thereby leading to developmental defects of the nervous system and intellectual disabilities.

As a part of the central nervous system, the optic nerve system is susceptible to the effects of radiation, so a method is needed to reduce the damage to the development of the optic nerve system caused by radionuclides.

Summary of the invention

The object of the present invention is to provide a method for alleviating the damage of the visual nervous system caused by tritium.

In order to achieve the above object, the present invention provides the following technical solutions:

The present invention provides a gene dmbx1a for alleviating tritium-induced visual nervous system damage, and its sequence is shown in SEQ ID No. 1. It can enable DNA binding transcription factor activity, RNA polymerase II specificity and RNA polymerase II transcription regulatory region sequence-specific DNA binding activity, participate in brain development, eye development and negative regulation of DNA template transcription, act upstream or internally in multiple processes, including midbrain development and regulation of neural retina development, and is homologous to human DMBX1 (diencephalon/midbrain homeobox 1).

The present invention provides a method for alleviating tritium-induced visual nervous system damage, which utilizes dmbx1a gene overexpression to achieve the purpose of alleviating visual nervous system damage. Specifically, dmbx1a is intervened, and the endogenous dmbx1a gene of zebrafish is activated using the CRISPRa system to alleviate the damage to the zebrafish visual nervous system caused by tritium water.

Preferably, the method for overexpressing the dmbx1a gene is to inject a mixture of dcas9-VP64 and dmbx1a-sgRNA into the cell, wherein dcas9-VP64 is a plasmid constructed by dcas9-NLS and VP64. The sequence of dcas9-NLS is shown in SEQ ID No.2; the sequence of VP64 is shown in SEQ ID No.3; the sequence of dmbx1a-sgRNA is shown in SEQ ID No.4. Specifically, embryo microinjection is performed at the single-cell stage or the two-cell stage of zebrafish embryonic development.

After overexpression intervention of the dmbx1a gene in zebrafish embryos, qPCR and other technologies were used to examine the changes in genes such as dmbx1a in zebrafish after tritium exposure.

Preferably, the concentrations of dcas9-VP64 and dmbx1a-sgRNA in the mixture of dcas9-VP64 and dmbx1a-sgRNA are 400 ng/μL and 200 ng/μL, respectively.

Preferably, reducing tritium-induced visual nervous system damage comprises reducing head cell apoptosis.

The present invention also provides an application of the dmbx1a gene in alleviating the damage to the nervous system structure of zebrafish caused by tritium exposure.

The present invention also provides application of the dmbx1a gene in improving the hatching rate of zebrafish under tritium exposure.

The present invention also provides the use of dmbx1a gene in alleviating the damage of the nervous system caused by radionuclides.

Beneficial effects:

The dmbx1a gene of the present invention can alleviate the damage to the nervous system structure of zebrafish caused by tritium water exposure. Dmbx1a can also improve the spontaneous movement and hatching of the tail of zebrafish exposed to tritium water, reduce the apoptosis of head cells, reduce the number of apoptotic bodies, increase the maxillofacial angle, and shorten the length of the mandible. At the same time, the dmbx1a gene can regulate the apoptosis of head cells caused by tritium exposure and alleviate the toxic effects of the visual nervous system of zebrafish.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

Figure 1 shows the relative expression level of dmbx1a in the head of zebrafish larvae.

Figure 2 shows the brain development of tritium-exposed zebrafish after dmbx1a overexpression intervention.

Figure 3 shows the quantification of the development of the zebrafish diencephalon, optic tectum and cerebellum after dmbx1a intervention, P < 0.01.

Figure 4 shows the effects of dmbx1a intervention on the nervous system of tritium-exposed zebrafish during development. A: The effects of dmbx1a overexpression intervention on the spontaneous movement of the tail of tritium-exposed zebrafish; B: The effects of dmbx1a overexpression intervention on the hatching rate of tritium-exposed zebrafish; P<0.01.

Figure 5 shows the effect of dmbx1a intervention on apoptosis in the head of zebrafish larvae - apoptosis in the head of larvae.

Figure 6 shows the effect of dmbx1a intervention on apoptosis in the head of zebrafish larvae - fluorescence quantitative analysis of apoptosis results. P<0.01.

Figure 7 shows the improvement of maxillofacial development in tritium-exposed zebrafish after dmbx1a overexpression intervention

Figure 8 shows the development of the lower jaw (LJL) of the juvenile fish, as shown by the yellow arrow in Figure 7; C: The angle between the jaw and face of the zebrafish, as shown by the blue line angle in Figure 7. P<0.05.

Note: HTO in the figure indicates tritiated water exposure only; HTO+VP64 indicates the control group that was injected with dcas9-VP64 alone and then exposed to tritiated water; HTO+dmbx1a sgRNA indicates the experimental group that was co-injected with dmbx1a-sgRNA and dcas9-VP64.

Detailed ways

The present invention provides a gene dmbx1a for alleviating tritium-induced visual nervous system damage, and its sequence is shown in SEQ ID No. 1. Also provided is a method for alleviating tritium-induced visual nervous system damage by overexpressing the dmbx1a gene.

In an embodiment, the experimental animals are bred as:

Wild-type zebrafish (adult AB strain) purchased from the National Zebrafish Resource Center (Wuhan, China) were used. All experimental procedures were approved by the Animal Care and Use Association of Soochow University, in accordance with the Guide for the Care and Use of Laboratory Animals, and were implemented in accordance with the Regulations on Laboratory Animals of Soochow University and the relevant regulations on experimental animals.

The adult zebrafish were 2.0 cm long, male and female were separated, and kept in a constant temperature circulating water system. The water temperature was maintained at 28±1℃, the pH was maintained at 7.0±0.5, and the conductivity was 850-900μs/cm. All water quality indicators met the experimental requirements. The light-dark cycle in the culture room was 14:10h, and the culture room was maintained at 28℃.

Frying zebrafish were raised in still water, and 5 days after fertilization, they were fed with starter feed for fry, and 10 days later, they were fed with an appropriate amount of brine shrimp twice a day, and the culture medium was changed every two days. Adult fish were fed three times a day, with brine shrimp in the morning and evening, and adult fish feed at noon.

The night before the mating, place the adult fish in the spawning tank, separate the male and female fish with a partition in the tank, and keep them overnight. The next morning, remove the partition under bright light to allow the fish to mate freely, and collect the eggs after spawning.

In the embodiment, the reagents involved are prepared as follows:

1) Preparation of E3 solution: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl ₂ , 0.33 mM MgSO ₄ , 0.01% methylene blue and 1 L of deionized water were used to prepare 1 L of culture solution.

2) Preparation of tritium water: dilute the stock solution of tritium water with known activity in E3 solution to reach 3.7×10 ⁴ Bq/mL. Store the prepared tritium water in a -4℃ refrigerator for later use. Take it out of the refrigerator one hour before poisoning and place it at room temperature to warm up to room temperature before use.

3) MS222 solution: Weigh a small amount of MS222 powder, add tap water that has been left to stand for 2 days, dissolve and dilute to a final concentration of 15 μg/mL MS222 solution.

In this example, the steps of tritium water exposure are:

1) Grouping: The collected zebrafish eggs were placed in a culture dish, and 1,000 eggs were randomly selected and randomly divided into two groups, a tritium exposure group and a control group, with 500 eggs in each group. Then, every 50 eggs were placed in the same culture dish for culture.

2) Tritium exposure: The fish eggs in the tritium exposure group were placed in 3.7×10 ⁴ Bq/mL tritiated water (HTO), which was diluted with E3 culture medium. The fish eggs in the control group were cultured in E3 culture medium.

The Elavl3 gene was first discovered by researchers in fruit flies. Studies have shown that the loss of elavl3 function can cause defects in the visual nervous system. It also plays an important role in the early development of the nervous system in zebrafish and is an important developmental gene in the development of the nervous system. Therefore, this experiment uses elavl3 transgenic zebrafish Tg (elavl3:EGFP) to directly observe the neural structure of zebrafish in order to evaluate the changes in the nervous system after exposure to tritium water.

The present invention uses elavl3 transgenic fish of CZ160, knu3Tg, Tg(elavl3:EGFP) (purchased from National Zebrafish Center, Wuhan, China).

The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and/or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

In order to further illustrate the present invention, the dmbx1a gene provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

Example 1: Verification of dmbx1a expression level after dmbx1a overexpression

After mixing dmbx1a-sgRNA at a concentration of 645ng/μL and dcas9-VP64 at a concentration of 4000ng/μL, zebrafish embryos were microinjected. The final concentration of sgRNA after mixing was 200ng/μL, and the concentration of cas9 was about 400ng/μL. Dcas9-VP64 was injected alone as a control to prevent microinjection from interfering with the expression level of the target gene. Zebrafish embryos were then exposed to tritium water to compare the changes in damage.

The experimental results showed that there was no significant difference in dmbx1a expression between the group injected with dcas9-VP64 alone and the group without microinjection, indicating that microinjection itself would not affect the expression of the target gene. Compared with the group injected with dcas9-VP64 alone, the expression level of dmbx1a at different tritium exposure time points was significantly increased in the group injected with dcas9-VP64-sgRNA and dcas9-VP64 (Figure 1).

Example 2: Effect of dmbx1a overexpression on tritium exposure-induced structural damage of the nervous system

Dmbx1a was overexpressed in elavl3 transgenic zebrafish Tg (elavl3:EGFP), and the damage to the zebrafish nervous system was observed after tritium exposure. The results are shown in Figures 2 and 3. Compared with the control group (injected with dcas9-VP64 alone), the developmental length of the diencephalon and optic tectum in the Dmbx1a overexpression group increased significantly after tritium exposure, while there was no significant difference in the developmental length of the cerebellum. The results show that dmbx1a can alleviate the damage to the nervous system structure of zebrafish caused by tritium water exposure.

The zebrafish nervous system damage detection method is as follows: CZ160, knu3Tg, Tg (elavl3: EGFP) elavl3 transgenic fish (purchased from the National Zebrafish Center, Wuhan, China) were used for tritium exposure experiments. The transgenic zebrafish after tritium exposure were placed under a fluorescence microscope for observation and photography.

Example 3: Effect of Dmbx1a overexpression on tritium exposure-induced spontaneous tail movement and hatching rate

The hatching rate of zebrafish was determined by observing and recording the hatching rate of zebrafish at 72 hpf.

Zebrafish movement behavior monitoring includes:

(1) Detection of locomotor activity under L/D stimulation in zebrafish

12 zebrafish were randomly selected from each dish and placed in a 48-well plate, one fish per well. The zebrafish were acclimated for 10 minutes before the test to adapt to the environment, and then the light-dark conversion (L/D) was used to stimulate the movement of the zebrafish. The movement of the zebrafish larvae was detected using ViewPointLifeSciences (Montreal, Canada). The detection content included the movement rate and movement distance of the zebrafish larvae. The experiment was repeated three times.

(2) Autonomous swimming behavior of zebrafish under natural light

Twelve zebrafish were randomly selected from each dish and placed in a 48-well plate, one fish per well. The zebrafish were acclimated for 10 min under natural light, and the movement distance of the zebrafish larvae was measured using ViewPoint LifeSciences (Montreal, Canada).

(3) Zebrafish distance statistics

The data monitored in (1) were divided into the total distance under L/D (light-dark conversion) mode, the total distance under L (light) mode, and the total distance under D (dark room) mode, and the movement distance of each fish was quantified.

(4) Calculation of zebrafish movement speed

The distance traveled by the zebrafish under L/D mode stimulation was compared with the time to calculate the average movement speed of the zebrafish.

The tritium-exposed experimental group and the tritium-exposed control group after dmbx1a overexpression were monitored according to the above method. The results are shown in Figure 4. The tritium-exposed experimental group and the tritium-exposed control group after dmbx1a overexpression showed an increased number of spontaneous tail movements and an increased hatching rate compared with the tritium-exposed control group, but there was no significant difference compared with the control group that was not exposed to tritium.

Example 4: Effects of Dmbx1a overexpression on tritium exposure-induced head cell apoptosis and fronto-facial development

The zebrafish larvae were randomly divided into the dmbx1a overexpression experimental group co-injected with dmbx1a-sgRNA and dcas9-VP64 and the control group injected with dcas9-VP64 alone, and both were exposed to 3.4×10 ⁴ Bq/mL tritium water at the same time. YO-PRO-1 staining was performed on the zebrafish larvae to observe the cell apoptosis after tritium exposure. The results are shown in Figures 5 and 6. The apoptosis of zebrafish head cells after Dmbx1a overexpression intervention was alleviated and the apoptotic bodies were reduced. The maxillofacial development indicators of zebrafish exposed to tritium at 4 dpf were quantified and it was found that the maxillofacial angle of zebrafish in the dmbx1a overexpression group increased and the length of the mandible decreased. The results are shown in Figures 7 and 8.

From the above examples, it can be seen that after overexpression of the gene dmbx1a for reducing the damage of the visual nervous system caused by tritium water provided by the present invention, the spontaneous movement and hatching of the tail of zebrafish exposed to tritium water can be improved, the apoptosis of head cells can be alleviated, the apoptotic bodies can be reduced, the maxillofacial angle can be enlarged, and the length of the mandible can be shortened. This shows that dmbx1a can regulate the toxic effect of apoptosis of head cells caused by tritium exposure on the visual nervous system of zebrafish.

Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims (8)

1. A gene dmbx1a for alleviating tritium-induced visual nervous system damage, characterized in that the sequence of the dmbx1a is shown in SEQ ID No.1. 2. A method for reducing tritium-induced visual nervous system damage, characterized in that the method is to overexpress the dmbx1a gene described in claim 1. 3. The method for reducing tritium-induced visual nervous system damage according to claim 2 is characterized in that the method for overexpressing the dmbx1a gene is to inject a mixture of dcas9-VP64 and dmbx1a-sgRNA into embryonic cells, wherein dcas9-VP64 is a plasmid constructed by dcas9-NLS and VP64, and the gene sequences of dcas9-NLS, VP64 and dmbx1a-sgRNA are shown in SEQ ID No. 2 to 4, respectively. 4. The method for reducing tritium-induced visual nervous system damage according to claim 3, characterized in that the concentrations of dcas9-VP64 and dmbx1a-sgRNA2 in the mixed solution of dcas9-VP64 and dmbx1a-sgRNA2 are 400 ng/μL and 200 ng/μL, respectively. 5. The method for reducing tritium-induced visual nervous system damage according to claim 2, characterized in that reducing tritium-induced visual nervous system damage includes reducing head cell apoptosis or reducing apoptotic bodies. 6. Use of the dmbx1a gene according to claim 1 to alleviate the structural damage of the nervous system of zebrafish caused by tritium exposure. 7. Use of the dmbx1a gene according to claim 1 to improve the hatching rate of zebrafish under tritium exposure. 8. Use of the dmbx1a gene according to claim 1 in alleviating neurological damage caused by radionuclides.