Method for tracking transportation of outer fragment protein in vivo

Abstract

The invention relates to a method for tracking transportation of an outer fragment protein in vivo, which comprises the following steps of: dynamically tracking the transportation process of the outer fragment protein in a mouse body and updating kinetics by utilizing photoconversion fluorescent protein Dendra2, and revealing a path and a path for triggering cell death by tracing the source and the destination of ectopic accumulation Rhodopsin. On the research theory, the research result contributes to the development of Rhodopsin transportation and a membrane disc updating theory, and a new transportation mode can be possibly found by further elaborating the Rhodopsin abnormal transportation process and mode of the retinal degenerative disease model. In clinical treatment, a cell death path of a retinal degenerative disease patient in a later disease course can be found, and a new target spot and a new direction are provided for development of drugs of the retinal degenerative disease.

Description

Technical Field

[0001] The invention relates to the field of biological imaging technology, in particular to a method for tracking the transport of outer segment proteins in vivo. Background Art

[0002] Visual information is an important means for humans and animals to survive, and 80% of external information comes from vision. The retina is the first stop for receiving visual information. Light is focused on the retina through the refractive system composed of the cornea, lens, and vitreous body. Photons are captured by photoreceptor cells, which convert light signals into electrical signals and transmit them to the visual cortex of the brain through the optic nerve, ultimately forming vision. Correspondingly, retinal photoreceptor cell degeneration is an important blinding disease. For example, hereditary retinitis pigmentosa has a worldwide incidence of about 1:4000, affecting nearly 300,000 people in my country, and there is currently a lack of effective treatment.

[0003] In order to ensure the efficiency, sensitivity and specificity of photosensitivity, a membrane disc structure that is relatively isolated from the cell body and tightly stacked is formed at the top of the photoreceptor cell to receive photons, which is called the outer segment. Since the outer segment itself does not have the ability to synthesize proteins and lipids, they need to be synthesized by the inner segment and input through the connecting cilium. The connecting cilium is a narrow microtubule structure that not only relatively isolates the outer segment and inner segment structures to prevent the free diffusion of proteins, but also provides a microtubule channel for the transport of proteins and lipids synthesized in the inner segment. The connecting cilium has complex transport-related ciliary proteins that are involved in the transport of various outer segment proteins and lipids. Any gene mutation that affects the transport of outer segment proteins will eventually lead to various retinal degenerative diseases.

[0004] Abnormal transport of outer segment proteins results in a lack of replenishment of membrane disc renewal, and the large accumulation of outer segment proteins and other protein lipids in the inner segment exceeds the processing load of photoreceptor cells and causes cell necrosis. Due to the lack of appropriate tools to trace the source and destination of the outer segment proteins accumulated ectopically due to transport disorders, it is impossible to further understand the signaling pathway that causes cell death.

[0005] Most of the existing research and development technologies for tracking Rhodopsin localization and transport have stopped at static observation of its transport process, and the initiation and dynamic tracking of renewal cannot be effectively monitored. For example, fusion GFP markers can show the basic time and directionality required for mature membrane renewal, but the various processes at each time point of transport cannot be observed in real time. Immunoelectron microscopy can show the basic route of Rhodopsin localization and migration in membrane discs, but it can only be observed statically. Although isotope labeling can achieve in vivo tracking, there are safety risks and real-time tracking cannot be achieved. In addition, compared with the advantage of easy observation of the retina in animal models such as African clawed frogs and zebrafish, the eye structure and retina of mice are more consistent with humans, but they are also more complex and difficult to observe. In vivo observation of the retinal structure of mice often requires the integration of various technologies for attempts. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for tracking the transport of outer segment proteins in vivo, which can effectively solve the problems raised in the above-mentioned background technology.

[0007] To solve the above problems, the technical solution adopted by the present invention is: a method for tracking the transport of outer segment proteins in vivo, comprising the following steps: S1. Select a target gene and clone the target gene into a plasmid vector, wherein the target gene includes a gene encoding a target protein, wherein the target protein is an outer membrane protein, and the target protein is in the form of a fusion marker protein so as to be observed and tracked by a fluorescence microscope; S2. transfecting the plasmid vector into a host cell to achieve expression of the target protein, wherein the host cell is a mammalian cell capable of expressing the target protein; S3. packaging the target gene into a viral vector through a viral vector packaging system, and using the viral vector to infect retinal cells in a mammal, so as to achieve expression of the target protein in the mammal; S4. Observe and record the location and dynamic distribution of the target protein in the cell using a fluorescence microscope, and track the target protein in real time using a fluorescent labeling technique, wherein the fluorescent labeling technique is Dendra2 fluorescent labeling; S5. Collect and analyze the dynamic expression data of the target protein, and perform quantitative analysis through image processing software to evaluate the distribution of the target protein inside and outside the host cell and its dynamic changes.

[0008] As a further preferred embodiment of the present invention, the target protein is Rhodopsin protein, and the target protein is fused with Dendra2 fluorescent marker protein to track the dynamic transport process of the target protein.

[0009] As a further preferred embodiment of the present invention, the plasmid vector is a PRK5 plasmid vector, and the PRK5 plasmid vector is transfected into a host cell to express the target protein.

[0010] As a further preferred embodiment of the present invention, the viral vector is an adeno-associated virus AAV vector, and the AAV vector is of AAV2 / 8 serotype, which is used for highly efficient transduction of mammalian retinal cells.

[0011] As a further preferred embodiment of the present invention, the observation step includes performing dynamic observation using a living imaging system after injecting the viral vector into the mammal to obtain the distribution of the target protein in the mammal in real time.

[0012] As a further preferred embodiment of the present invention, the dynamic changes include the transport speed, distribution range, aggregation of the protein and its localization changes in different cells, and the data analysis includes the calculation of protein transport rate, measurement of distribution density and identification of aggregation points.

[0013] As a further preferred embodiment of the present invention, the distribution range includes the cell membrane, endoplasmic reticulum, and Golgi region, and the aggregation situation is evaluated as the number and size of protein aggregation points.

[0014] Compared with the prior art, the present invention provides a method for tracking the transport of outer segment proteins in vivo, which has the following beneficial effects: The present invention will use the photoconverted fluorescent protein Dendra2 to dynamically track the process and renewal dynamics of outer segment protein transport in mice, and reveal the pathway and path of cell death by tracing the source and destination of ectopic accumulation of Rhodopsin. In terms of research theory, the research results will contribute to the development of the theory of Rhodopsin transport and membrane disk renewal, and further explain the abnormal transport process and mode of Rhodopsin in retinal degenerative disease models, and it is possible to discover new transport modes. In clinical treatment, the cell death pathway of patients with retinal degenerative diseases in the late stage of the disease can be discovered, providing new targets and directions for the development of drugs for them. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a diagram of the photoconversion process of Rho-Dendra2 activated by 405nm; Figure 2 Tracing Rho transport map for Rho-Dendra2 expression; Figure 3 This is a photoconversion diagram of Rho-Dendra2 activated at 405nm; Figure 4 This is a picture of Rho-Dendra2 in mice that can complete fluorescence photoconversion at 405nm; Figure 5 Tracking Rhodopsin transport map for Dendra2 photoconversion; Figure 6 Diagram of the basic transport process of Rhodopsin in mice for Rho-Dendra2 tracking; DETAILED DESCRIPTION

[0016] If "and / or" or "and / or" appears in the full text, its meaning includes three parallel options. Taking "A and / or B" as an example, it includes option A, or option B, or a option in which both A and B are satisfied.

[0017] In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the fact that ordinary technicians in the field can implement it. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0018] The present invention provides a method for tracking the transport of outer segment proteins in vivo, comprising: 1. Construct the C-terminal Rho-Dendra2 to PRK5 vector of Rhodopsin, verify it by sequencing, and select the plasmid with the correct sequence for amplification and extraction.

[0019] 2. Activate Rho-Dendra2 with light of different wavelengths to verify the efficiency of photoconversion. The Rho-Dendra2 plasmid was constructed as described above and transiently transfected into 293T cells by PEI for in vitro verification experiments. During transfection, observe the expression of Rho-Dendra2 fluorescence in cells. Two days after transfection, the efficiency of photoconversion was detected by light of different wavelengths (600nm→555nm→488nm→405nm) of a mercury lamp under a fluorescence microscope.

[0020] 3. The Rho-Dendra2 that has been successfully transformed is repackaged into the AAV-hRK vector to construct the AAV-hRK-Rho-Dendra2 plasmid. After sequencing verification and amplification, the serotype AAV2 / 8 with high efficiency in infecting retinal photoreceptors is selected for packaging, and then transfected, amplified, purified and concentrated into a high-titer AAV virus.

[0021] 4. The above-mentioned AAV-hRK-Rho-Dendra2 virus was injected subretina to infect rod cells. A certain amount of virus was injected subretina into the retina of adult mice by focusing on the mouse fundus using different microscopes combined with simulated lenses.

[0022] 5. The mice injected with Rho-Dendra2 were raised for 10 days. Rho-Dendra2 basically completed a membrane disk renewal cycle after 10 days, and basically filled the entire outer segment. Combined with the above in vitro verification, the 405nm wavelength verified in vitro was selected to activate the fluorescence photoconversion of Rho-Dendra2 in the photoreceptor cells of mice in vivo.

[0023] 6. Optimize the conditions by continuously optimizing the wavelength and intensity of the excitation laser, evaluate the efficiency of the fluorescence light conversion of the above Rho-Dendra2 through real-time recording and other systems, and confirm the feasibility and efficiency of Rho-Dendra2 tracking Rhodopsin transport by immunofluorescence (IF) and Rhodopsin co-staining. Consider the damage to photoreceptor cells by viruses, ultraviolet light, surgery, etc., and ensure the safety of the technology through function and structure. Use OCT, fundus photography (FP), and ERG to track the changes in retinal function and structure after illumination. Use IF, ultrastructural expansion microscopy, qPCR, etc. to observe the changes in photoreceptor cells and RPE.

[0024] 7. Select the best conditions optimized above to describe the dynamic transport process of Rhodopsin in mice with different gene mutations. After Dendra2 has expressed the entire outer membrane disc, photoconversion is performed to clear the green fluorescence, and the transport of Rhodopsin is accurately tracked with the newly converted red fluorescence. Observe its dynamic transport process at different periods (6h, 12h, 24h, 48h, 72h, 5day, 7day, 10day), calculate the transport rate, directionality, and positioning changes to describe the membrane disc renewal process. Select slices and IF for tracking.

[0025] As a specific embodiment 1 of the present invention: (1) Rho-Dendra2 is activated at 405nm to complete the photoconversion process We have completed the construction of Rho-Dendra2 plasmid in the early stage, packaged it into lentivirus, and transfected 293T cells for in vitro verification experiments. Two days after transfection, Rho-Dendra2 fluorescence was expressed on the cell membrane. The efficiency of photoconversion was detected by light of different wavelengths (600nm→555nm→488nm→405nm) of mercury lamp under a fluorescence microscope. It was found that the green-to-red fluorescence increased with the illumination time under 405nm ultraviolet light. After 50s of irradiation, Rho-Dendra2 was basically completely converted, and extending the illumination time would not enhance its red fluorescence intensity. Figure 1 At the same intensity and time, no photoconversion of Dendra2 was observed at other wavelengths.

[0026] exist Figure 1 A shows the basic elements of the Rho-Dendra2 plasmid and the basic process of in vitro verification. B-F'' shows the photoconversion process of Dendra2 after 405nm irradiation. B' to F' show the changes of green fluorescence with illumination time, and B'' to F'' show the changes of red fluorescence with illumination time. G and H show the trend of fluorescence intensity with time, respectively. n=4 (cells in 4 confocal culture dishes).

[0027] (2) Rho-Dendra2 expression tracks Rho transport In order to improve the infection efficiency of Rho-Dendra2 in mice and target rod photoreceptor cells. We selected the serotype AAV2 / 8 and hRK promoter with high efficiency in infecting retinal photoreceptors, and reconstructed the plasmid of the AAV vector. After AAV virus packaging, purification and concentration, a total of 5*108GC of AAV-hRK-Rho-Dendra2 virus was injected subretinaly to infect rod photoreceptor cells. It can be seen that Rho-Dendra2 was co-stained with Rho fluorescent staining, which can mark Rho. The position of its expression is consistent with the self-expressed Rhodopsin, and no mislocalization was observed. Ten days after the injection of Rho-Dendra2, Rho-Dendra2 basically completed a membrane disk renewal cycle and basically filled the entire outer segment. According to the localization of Rho-Dendra2 in the outer segment, it is preliminarily estimated that the time of Rho transport is about 3.85um / day, referring to Figure 2 .

[0028] exist Figure 2 A shows the basic elements of the Rho-Dendra2 plasmid and the basic process of in vivo expression tracking. B-G'' shows the expression and localization of Dendra2 at different times after injection. H shows the length of Dendra2-labeled Rho. I shows the rate of Dendra2-labeled Rho outer segment transport over time (length / days). n=38 (Dendra2 outer segment). J shows a simplified diagram of Rho-Dendra2 tracing Rho transport.

[0029] (3) Rho-Dendra2 in mice can complete fluorescence photoconversion at 405nm After confirming that the AAV-hRK-Rho-Dendra2 virus can specifically infect rod photoreceptors, we started the experiment of fluorescence photoconversion of Rho-Dendra2 in vivo. There are several difficulties in performing photoconversion in mice. First, the mouse eyeball is small and lacks a suitable lens to transmit light to the fundus. Second, it is necessary to observe the area where the mouse is successfully infected, which requires an instrument with fluorescence excitation. Third, the intensity and range of the transduced light focusing on the retina. Considering the effectiveness of the excitation light transmission route, we first tried to transmit light of different wavelengths through optical fiber wires and directly illuminate the retina through the vitreous cavity. Later, it was found that after direct in vivo illumination, the retina was severely inflamed and the photoconversion efficiency was low. Considering the in vitro conversion, it is directly activated by light under a fluorescence microscope, and the light damage caused by illumination in the vitreous body is reduced. Combined with the experience of subretinal injection, after repeated attempts, we used PBS water drops to form a lens on the mouse eye surface to focus on the fundus, and used 488nm wavelength light to excite dendra2 green fluorescence to focus on the area of ​​successful infection. After focusing on the area with successful dendra2 infection, select the strongest 405nm light intensity for preliminary photoconversion experiments, refer to Figure 3 .

[0030] exist Figure 3 A shows the photoconversion process of Rho-Dendra2 after being excited by 405nm. B shows the area of ​​successful infection after excitation of dendra2 green fluorescence by fluorescence microscopy at 488nm wavelength. C shows illumination with 405nm wavelength light. D shows the image of photoconversion after 15min of 405nm illumination. E shows the image taken 2h after 405nm illumination.

[0031] After confirming that Rho-Dendra2 can complete the fluorescence photoconversion process in mice, the conditions were further optimized to find the lowest intensity and shortest time to reduce light damage. Figure 4 As shown in the figure, 4 gradients are set, namely 40lux; 5μv / cm2, 66lux; 18μv / cm2; 120lux; 31μv / cm2, 390lux; 96μv / cm2, and different illumination time (5min, 10min, 15min) to find the most suitable illumination conditions. Through the efficiency trend chart of fluorescence conversion, it can be found that under the laser intensity of level 2 to level 3, the light conversion increases with the intensity and time.

[0032] exist Figure 4Middle A shows the photoconversion process of mice using 4 gradients of 405nm 10 days after Rho-Dendra2 injection. The intensities were 40lux; 5μv / cm2, 66lux; 18μv / cm2; 120lux; 31μv / cm2, 390lux; 96μv / cm2, and the illumination time was 5min, 10min, and 15min. Level I-5min The left figure shows the fundus image before illumination (A-A'), A is the two channels of green and red fluorescence, and A' is the single channel of red fluorescence. The right figure shows the fundus image converted after illumination of corresponding time and intensity (B-B'). The bottom figure shows the trend of fluorescence intensity with intensity and time. n=4 (mouse eyeball).

[0033] (4) Rho-Dendra2 can track the Rho transport process After determining the conditions under which mouse Rho-Dendra2 can be efficiently converted in vivo, we then used Rho-Dendra2 to illuminate the outer segment membrane discs of adult wild-type mice to define the starting point of Rho turnover and re-track its trafficking. Figure 5 As shown in the figure, the transformation zone can be seen in the samples taken 6 hours after illumination, and all Green-Dendra2 is transformed into Red-Dendra2. After 2 days, Red-Dendra2 can be seen transported to the outer segment, followed by the newly formed Green-Dendra2, and no accumulation in the cell body can be seen. A clear boundary can be seen between Red-Dendra2 and Green-Dendra2. This suggests that Dendra2 can accurately track the localization and transport status of Rhodopsin. Of course, in order to ensure the safety and efficiency of subsequent experiments, we still need to continue a series of experiments to further optimize the conditions.

[0034] exist Figure 5 A shows the process of sampling after 405nm photoconversion of Rho-Dendra2-injected mice. 120lux; 31μv / cm2, 5min illumination time. BF shows the images of photoconversion 2h and 2 days after illumination. E shows a simplified diagram of Rho-Dendra2 transport after photoconversion.

[0035] In summary, our previous work not only provides support for the scientific questions and hypotheses of this project, but also provides technical support and lays a theoretical foundation for the subsequent research of the project. Therefore, it can be applied to track the rate, directionality and renewal dynamics of Rhodopsin transport in different diseases, and observe its abnormal localization and accumulation in the inner segment and cell body through indicators such as intracellular phagocytosis, in order to discover the pathway of cell death caused by Rhodopsin accumulation.

[0036] refer to Figure 6 , as a specific embodiment 2 of the present invention: 1. Construct the C-terminal Rho-Dendra2 to PRK5 vector of Rhodopsin, verify it by sequencing, amplify and extract the plasmid.

[0037] 2. The Rho-Dendra2 plasmid was constructed as described above and transiently transfected into 293T cells by PEI for in vitro experiments. Different wavelengths of light were used to activate Rho-Dendra2 and verify the efficiency of photoconversion.

[0038] 3. The in vitro verified Rho-Dendra2 was repackaged into the AAV-hRK vector to construct the AAV-hRK-Rho-Dendra2 plasmid. After sequencing verification and amplification, the serotype AAV2 / 8 with high efficiency in infecting retinal photoreceptors was selected for packaging, and then transfected, amplified, purified and concentrated into a high titer AAV virus.

[0039] 4. The above-mentioned AAV-hRK-Rho-Dendra2 virus was injected subretina to infect rod cells. A certain amount of virus was injected subretina into the retina of adult mice using a surgical microscope (SZX7, Olympus, Japan) combined with a simulated lens to focus on the mouse fundus.

[0040] 5. The mice injected with Rho-Dendra2 were raised for 10 days. Rho-Dendra2 basically completed a membrane disk renewal cycle in 10 days, and basically filled the entire outer segment.

[0041] 6. 405nm wavelength activated the fluorescence photoconversion of Rho-Dendra2 in photoreceptor cells in mice in vivo. The activation and photoconversion process were completed using a fluorescence microscope (Imager.Z2, Zeiss, Germany), which has a stage for placing mice and an X-Cite fluorescent lamp illumination system (X-Cite 120Q, EXCELITAS, USA), containing lasers of different wavelengths (380nm-680nm), which can capture images of Dendra2 in different fluorescent states. 488nm fluorescent light excites green Dendra2, while 568nm fluorescent light excites red Dendra2. In order to convert Dendra2 from green to red, 405nm fluorescent light was used to illuminate the retina. The power of 405nm irradiation ranged from 20 to 100µW, and the irradiation time was 5 to 15 minutes.

[0042] 7. Select the best conditions optimized above to describe the dynamic transport process of Rhodopsin in mice with different gene mutations. After Dendra2 has expressed the entire outer membrane disc, photoconversion is performed to clear the green fluorescence, and the transport of Rhodopsin is accurately tracked with the newly converted red fluorescence. Observe its dynamic transport process at different periods (6h, 12h, 24h, 48h, 72h, 5day, 7day, 10day), calculate the transport rate, directionality, and positioning changes to describe the membrane disc renewal process. Select slices and IF for tracking.

[0043] Figure 6 The middle label numbers are 1, ophthalmic surgical microscope; 2, adult wild-type mice; 3, the fundus of mice focused on by microscope under dilated pupil; 4, the virus is injected subretina into the retina of mice; 5, schematic diagram of the surgical site of virus injection; 6-8, the fluorescence photoconversion of Rho-Dendra2 photoreceptor cells in vivo activated by fluorescence microscope at 405nm wavelength after 10 days of virus injection in mice; 9, schematic diagram of the conversion of green fluorescence into red fluorescence of photoreceptor rod cells infected with dendra2 virus after photoconversion.

[0044] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification and drawings under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A method for tracking the transport of outer segment proteins in vivo, characterized in that The following steps are involved: S1. Select a target gene and clone the target gene into a plasmid vector, wherein the target gene includes a gene encoding a target protein, wherein the target protein is an outer membrane protein, and the target protein is in the form of a fusion marker protein so as to be observed and tracked by a fluorescence microscope; S2. transfecting the plasmid vector into a host cell to achieve expression of the target protein, wherein the host cell is a mammalian cell capable of expressing the target protein; S3. packaging the target gene into a viral vector through a viral vector packaging system, and using the viral vector to infect retinal cells in a mammal, so as to achieve expression of the target protein in the mammal; S4. Observe and record the location and dynamic distribution of the target protein in the cell using a fluorescence microscope, and track the target protein in real time using a fluorescent labeling technique, wherein the fluorescent labeling technique is Dendra2 fluorescent labeling; S5. Collect and analyze the dynamic expression data of the target protein, and perform quantitative analysis through image processing software to evaluate the distribution of the target protein inside and outside the host cell and its dynamic changes.

2. The method for tracking the transport of outer segment proteins in vivo according to claim 1, characterized in that: The target protein is Rhodopsin protein, and the target protein is fused with Dendra2 fluorescent marker protein to track the dynamic transport process of the target protein.

3. The method for tracking the transport of outer segment proteins in vivo according to claim 1, characterized in that: The plasmid vector is a PRK5 plasmid vector, and the PRK5 plasmid vector is transfected into a host cell to express a target protein.

4. The method for tracking the transport of outer segment proteins in vivo according to claim 1, characterized in that: The viral vector is an adeno-associated virus AAV vector, and the AAV vector is of AAV2 / 8 serotype, which is used for efficiently transducing mammalian retinal cells.

5. The method for tracking the transport of outer segment proteins in vivo according to claim 1, characterized in that: The observation step includes using a living imaging system to perform dynamic observation after injecting the viral vector into the mammal, so as to obtain the distribution of the target protein in the mammal in real time.

6. The method for tracking the transport of outer segment proteins in vivo according to claim 1, characterized in that: The dynamic changes include the transport speed, distribution range, aggregation of proteins and changes in their localization in different cells.

7. The method for tracking the transport of outer segment proteins in vivo according to claim 6, characterized in that: The distribution range includes the cell membrane, endoplasmic reticulum, and Golgi apparatus regions, and the aggregation status is evaluated as the number and size of protein aggregation points.

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