CN114306607A - Primary ciliary regulation of biological rhythms and related applications thereof - Google Patents
Primary ciliary regulation of biological rhythms and related applications thereof Download PDFInfo
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- CN114306607A CN114306607A CN202111659840.5A CN202111659840A CN114306607A CN 114306607 A CN114306607 A CN 114306607A CN 202111659840 A CN202111659840 A CN 202111659840A CN 114306607 A CN114306607 A CN 114306607A
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Abstract
The invention relates to the field of biotechnology, in particular to a primary cilium-regulated biological rhythm and related application thereof. Specifically, the invention provides the application of the cilium generation regulator in the regulation of cilium generation, the preparation of products for regulating biological rhythm and the preparation of products for treating diseases related to biological rhythm; preferably, the modulator of ciliation comprises FTY720, S1P, Ki16425, LPA, LY294002, Wortmannin, AKTiIV, PIA23, GSK690693, Perifosine, PHA-680632, Tubacin and pharmaceutically acceptable salts thereof, modulators of ciliogenesis genes.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a primary cilium-regulated biological rhythm and related application thereof.
Background
The primary cilia are an immobile microtubule-like structure that is present on the surface of most mammalian cells. The antenna-like organelle can integrate external mechanical, physical, chemical and other stimuli to be transmitted to the inside of cells, and plays an important role in early embryonic development.
The behaviors of the living beings such as sleeping/waking, eating and the like and various physiological, biochemical and metabolic processes follow rhythmic changes of about 24 hours, commonly called biological clocks which are closely related to the normal physiological functions of the human body. Research shows that the biological rhythm is closely related to various physiological indexes of individual sleep, diet, cognition, emotion, behavior, metabolism and the like, influences the core body temperature, muscle strength, flexibility, cell damage and oxidative stress level of the individual and the secretion process of various hormones, is related to the coordination and orderly performance of the whole organism and is adaptive to the environment, and if the normal biological rhythm is disturbed, the abnormality of the psychological and physiological functions of the organism can be caused, and the functional disorder of various tissues and organs of the organism can be caused. In humans, changes in the biological clock cause many problems, such as sleep disorders, depression, metabolic disorders, aging, blood disorders, diabetes, and obesity. The circadian rhythm system plays a crucial role in regulating human physiology, and recent studies have further revealed the relationship of disruption of circadian rhythms with sleep disorders, cancer, decreased immune competence, obesity, alzheimer's disease, and aging. There is increasing research evidence showing that shift workers and night workers have a high probability of developing tumors, cardiovascular diseases, metabolic syndrome and there is no currently accepted treatment regimen.
At present, there is no report about the involvement of cilia in the regulation of biological rhythm.
FTY720 is an immunosuppressant newly developed in recent years, the medicine selectively reduces the number of peripheral circulating lymphocytes, obviously prolongs the survival of transplanted organs of experimental animals, does not damage the immune response and the immunological memory function to viruses, has low toxic or side effect, shows good synergistic effect with CsA, FK506, RAD and other clinical first-line immunosuppressive drugs, has good effect on first-phase clinical trial of kidney transplant patients, and has wide clinical application prospect.
Disclosure of Invention
The invention discloses that the primary cilia of the suprachiasmatic nucleus (SCN) of the thalamus region have obvious circadian variation for the first time; the cilia in the SCN region are specifically knocked out to directly cause the abnormal rhythm of the mouse, and the specific expression is that the behavioural rhythm period of the mouse is prolonged, and the adaptive capacity of the inversion difference is enhanced; we found that lysophospholipid S1P (sphingosine-1-phosphate) and its related drug FTY720 could regulate ciliogenesis and verify the effect of the drug on the rhythm of brain tissue, and the experimental results found that: the drug FTY720, which affects ciliogenesis, is capable of directly disrupting the biological rhythm of brain tissue.
The cilium generation regulator provided by the invention provides a potential scheme for treating special diseases caused by rhythm disorder.
Applications of
In one aspect, the invention provides the use of a cilium production modulator for modulating cilium production, modulating biological rhythms, and treating diseases associated with biological rhythms;
more specifically, the use of a ciliary production modulator in the manufacture of a product for modulating ciliary production, modulating a biological rhythm, or treating a disease associated with a biological rhythm.
Preferably, the modulator comprises an inhibitor; the modulation includes promoting ciliogenesis or inhibiting ciliogenesis.
The "biological rhythm related diseases" of the present invention include sleep disorders, jet lag, depression, metabolic disorders, aging, blood diseases, diabetes, obesity, and the like.
The cilia are structurally and functionally divided into moving cilia and static cilia; preferably, the modulated ciliation of the present invention is directed to a static cilium (also referred to as "primary cilia" in the present invention).
Preferably, said modulating biorhythm aspects include enhancing the adaptation of the moveout (increasing the adaptation to changes in photoperiod); for example, the backward difference adaptability can be shown as that the new environmental factors can be adapted in less time relative to the control under the condition that the environmental factors such as the illumination period, the illumination intensity, the oxygen concentration and the like are changed; the less time may be one-half, one-third, one-fourth, one-fifth, one-sixth, or less of the control time.
Preferably, the photoperiod change of the present embodiment is an advance/retard of 8 hours.
Preferably, the term "photoperiod" is also referred to herein as "photoperiod" where 12 hours of light and 12 hours of darkness are present in 24 hours under a standard photoperiod, such as: the lamp is turned on at the early 7 th, and turned off at the late 7 th, and the illumination intensity is 50 lux.
Preferably, the modulator of ciliation comprises FTY720, S1P, Ki16425, LPA, LY294002, Wortmannin, AKTiIV, PIA23, GSK690693, Perifosine, PHA-680632, Tubacin and the like and pharmaceutically acceptable salts thereof, modulators of ciliogenesis genes.
The "FTY 720" of the present invention, Fingolimod, is translated into Fingolimod; FTY720 is an immunosuppressant newly developed in recent years, and is prepared by modifying a component ISP-I with immunosuppressive effect in Chinese caterpillar fungus extract; has the advantage of little toxic and side effects. The invention provides a new application of the compound in regulating biological rhythm. The structural formula of FTY720 is as follows:
the "pharmaceutically acceptable salts" of the present invention are non-toxic in the amounts and concentrations administered. The preparation of such salts may facilitate pharmacological applications by altering the physical properties of FTY720 without preventing FTY720 from exerting a physiological effect.
Preferably, the pharmaceutically acceptable salt may be an acid addition salt, a base addition salt.
Preferably, the acid addition salts include, but are not limited to, any one of or a combination of at least two of hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, tosylate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate, and salicylate, malonate, adipate, hexanoate, arginate, fumarate, nicotinate, phthalate, or oxalate salts of FTY 720.
Preferably, the base addition salts include, but are not limited to, lysine or histidine salts of FTY 720.
Preferably, the ciliogenic genes include Ift88, Ift 20.
Preferably, the inhibitor comprises a reagent used by gene editing technologies such as siRNA interference technology, CRISPR technology, TALEN technology, ZFN technology, Cre-loxP gene recombination technology and the like.
Preferably, the inhibitor is a reagent used in gene editing techniques such as Cre-loxP gene recombination technique.
Preferably, said use occurs in vitro;
preferably, the use is for non-therapeutic purposes.
Preferably, the product comprises a pharmaceutical composition.
Preferably, the pharmaceutical composition may be administered by any mode of administration, such as: oral or parenteral.
Preferably, the parenteral form comprises intravenous, intramuscular, intraperitoneal or subcutaneous injection.
Preferably, the pharmaceutical composition can be pills, tablets, capsules, granules, oral liquid, suspensions, injections, microspheres or liposomes prepared from the ciliation modulator and pharmaceutically acceptable excipients by a conventional method.
Method
In another aspect, the invention provides a method of modulating cilia, modulating a biological rhythm, the method comprising administering a cilia modulator.
In another aspect, the present invention provides a composition for modulating cilia occurrence, modulating a biological rhythm, the composition comprising a cilia occurrence modulator.
In another aspect, the present invention provides a method of treating a biorhythm-related disease, the method comprising administering a ciliogenesis modulator according to the present invention.
In another aspect, the present invention provides a pharmaceutical composition for treating a biorhythm-related disease, said composition comprising a ciliation modulator.
Preferably, the method occurs in vitro or in vivo.
More preferably, the method is performed in vitro on cells.
Preferably, the method may also be performed in vivo in a subject.
The term "subject" as used herein refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, rodents, etc., which will be the particular recipient of the methods of the invention.
Preferably, the rodents include the family of the cricotidae (e.g. mouse-like hamsters), the family of the cricotidae (e.g. hamsters, new world rats and mice, voles), the general family of the rats (true mice and rats, gerbils, spiny rats, crowned rats), the family of the marmoraceae (climbing mice, rock mice, tailed rats, madagaska rats and mice), the family of the spiny muridae (e.g. spiny mice), and the spale (e.g. mole rats, bamboo rats and zokors) animals.
Animal model
In another aspect, the invention provides a method of constructing an animal model of biorhythm abnormality, the method comprising knocking out Ift88 or Ift 20;
more specifically, the method specifically knocks out the Ift88 or Ift20 of the SCN region; that is, the SCN region of the animal model is specifically deficient for cilia, while other regions are normal for cilia.
Preferably, the other regions include the hippocampus of the brain (hippocampus, which may be abbreviated as Hippo), the subthalamic nucleus (PVN), the cerebellum, and body organs (e.g., kidney, lung, liver, etc.).
Preferably, the animal model (or referred to as model animal) includes all animals;
preferably, the animal model comprises a non-human mammal.
Preferably, the non-human mammal includes orangutan, monkey, horse, cow, sheep, pig, donkey, camel, dog, rabbit, cat, rat, mouse, fish, bird, and the like.
Preferably, the animal model is a mouse.
Preferably, the method used for knockout comprises any one of: siRNA interference technology, CRISPR technology, TALEN technology, ZFN technology, Cre-loxP gene recombination technology and the like;
preferably, the method comprises the steps of:
1) obtaining an animal model of the specific expression Cre of the SCN region;
2) obtaining an animal model of Ift88-loxp or Ift 20-loxp; the Ift88-loxp contains a loxp sequence at each end of the encoding gene or part of the encoding gene of the Ift 88; the Ift20-loxp contains a loxp sequence at each end of the encoding gene or part of the encoding gene of the Ift 20;
3) animal models of 1) and 2) were mated.
Preferably, the animal model obtained in step 3) further needs to be propagated for multiple generations.
Preferably, the terms "hypothalamic suprachiasmatic nucleus", "scn (suprachiamatic nucleus)" "suprachiasmatic nucleus" and "thalamic suprachiasmatic nucleus" are all the same meaning and are used interchangeably. SCN is the central structure of the mammalian circadian rhythm regulation system, and produces and regulates a plurality of biological rhythms such as sleep-wake, hormone, metabolism and reproduction; on the one hand, SCN has autonomous circadian rhythms such as electrophysiological properties, utilization of sugars, protein synthesis, and the like; on the other hand, SCN accepts integration of light information of the external environment, synchronizing the intrinsic rhythm of the organism with the external environment.
On the other hand, the invention provides the animal model constructed by the construction method of the animal model with abnormal biological rhythm and the application thereof.
Preferably, said use comprises studying cilia function, use of cilia in the mechanism of modulation of biological rhythms.
Detecting the amount of cilia
In another aspect, the invention provides a method of detecting the amount of cilia, the method comprising detecting the amount of expression of a ciliogenic gene.
Preferably, the ciliogenic genes include Ift88, Ift 20.
Preferably, the number of cilia is also expressed in the proportion of ciliated cells.
Preferably, the expression amount is an expression amount of a protein.
More preferably, the method is to detect the expression level of ciliogenic genes in the SCN region.
When the expression level of the ciliogenic gene is reduced, the proportion of ciliated cells is reduced.
Drawings
FIG. 1 is a graph of the results of SCN zone cilia detection under standard light cycles; FIG. 1A is a graph showing representative results of fixed section staining, FIG. 1B is a graph showing statistics of the number of cilia of SCN region and the expression level of Cry1, and FIG. 1C is a graph showing statistics of the length of cilia of SCN region.
FIG. 2 is a graph showing the protein expression level and cilia detection result of SCN region cilia-deficient mice; FIG. 2A shows the effect of the Western Blot on the knockdown of Ift88, FIG. 2B shows the effect of the Western Blot on the knockdown of Ift20, FIG. 2C shows the ciliary detection in SCN region, and FIG. 2D shows the ciliary detection in other regions.
FIG. 3 is a graph of biorhythmic changes in SCN-zone ciliated mice; FIG. 3A is a representation of the results of a running wheel behavior placed in a continuous dark environment (DD) after two weeks of standard light cycle (LD) acclimation; FIG. 3B is a statistical result of the movement period of FIG. 3A under the DD environment; FIG. 3C shows representative results of the running wheel behavior of mice when the light schedule is changed; FIG. 3D is the phase shift statistics of FIG. 3C; fig. 3E is a graph of the phase shift 50% time statistic of fig. 3C. The mouse wheel-race behavior is shown in the figure with black marks, white background and gray background representing light and dark, respectively.
FIG. 4 is a graph of the results of measurements of ciliary and rhythmic changes following FTY720 administration; FIG. 4A is a representation of cilia occurrence, FIG. 4B is the statistical result of 4A, FIG. 4C is the statistical result of FTY720 in regulating cilia occurrence, and FIG. 4D is the oscillatory change of the rhythmic gene of SCN region; fig. 4D shows the statistical results of fig. 4C.
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.
Materials and reagents for use in the invention
1. Per2: the product is given to the second-class teacher of Beijing institute of Life sciences. That is, as described in "Ju et al, chemical Properties, derived vertical, RUVBL2 regulations, the circumferadian Phase in Mammals, Sci, Transl, Med.12, eaba0769 (2020)", the public is available from the Applicant and is available for use in the experiments to replicate the invention, and not for use elsewhere; ift88-loxp mice were purchased from Jackson lab, cat # 022409; ift20-loxp mice were purchased from Jackson lab, cat # 012565; NMS-Cre mice were purchased from Jackson lab under cat number 027205.
2. Reagent
TABLE 1 reagents used in the invention
| Reagent | Manufacturer(s) | Goods number |
| ACII antibodies | Santa Cruz Co | sc-588 |
| IFT88 antibody | Proteitech Corp | 3967-1-AP |
| IFT20 antibody | Proteitech Corp | 13615-1-AP |
| alpha-Tubulin antibodies | MBL Co Ltd | PM054 |
| Goat anti-Rabbit Alexa Fluor 488 antibody | Thermo Fisher Scientific Co | A11034 |
| Luciferin | Promega Corp | E1602 |
| DNA dye Hoechst | Invitrogen corporation | H3570 |
| 4% Paraformaldehyde | Macgene Corp Ltd | P1110 |
| Encapsulating tablet Mount Medium | China fir Jinqiao Co Ltd | ZLI-9556 |
| DMEM medium and HBSS buffer solution | Gibco Corp | |
| Anti-drop slide and tissue embedding agent OCT | SAKURA Corp Japan |
General experimental method
1.Western
blob detection knock-out effect
Different tissues of ciliated mice were collected, ground into single cells and cellular holoproteins were extracted for Western blot experiments to detect the knockdown effect.
(1) Preparing SDS-PAGE gel: preparing glue with corresponding concentration according to the molecular weight of the protein to be detected;
(2) loading: loading 30 μ g protein per well;
(3) electrophoresis: concentrating the gel at constant pressure of 80V; separating gel, constant pressure 120V. The front edge of the bromophenol blue runs to the edge of the glue and the glue is placed;
(4) transfer printing: the required filter paper (4 pieces per gel), fiber pad, nitrocellulose membrane, SDS-PAGE gel were placed in a 1 XTransfer Buffer and equilibrated for 10 minutes. (PVDF, a hydrophobic membrane, requires special treatment before use: methanol soak for about 10 s). And (4) putting the prepared transfer sandwich into a transfer electrode box (paying attention to the positive and negative electrodes), and adding a transfer buffer solution and an ice box to start transfer. In the transfer process, the whole transfer groove is placed in an ice bath, and 400mA is used for transfer printing for 2 hours;
(5) and (3) sealing: after the transfer printing is finished, taking out the transfer printing sandwich, putting the transfer printing nitrocellulose membrane into a sealing solution (5% skim milk) prepared in advance by using a pair of tweezers, and incubating and sealing for 1 hour at room temperature;
(6) a first antibody: incubate with Ift88, Ift20, alpha-tubulin primary antibody, respectively, overnight at 4 ℃, followed by washing the membrane 3 times with TBST, 5 minutes each time;
(7) secondary antibody: incubating with a secondary antibody corresponding to the primary antibody, incubating for 1 hour at room temperature, and washing the membrane with TBST for 5 minutes three times;
(8) and (3) developing: taking out the washed nitrocellulose membrane, draining TBST carried on the membrane as much as possible, putting the nitrocellulose membrane on a preservative film with the right side facing upwards, uniformly mixing equal amounts of ECL reagent A, B liquid which are taken out in advance respectively, and spreading the solution on the membrane dropwise for developing;
2. mouse tissue frozen section preparation
Fixing: placing the separated tissue in 4% paraformaldehyde, and standing overnight at 4 ℃;
washing: PBS wash 2 times;
and (3) dehydrating: 30% sucrose (PBS formulation) overnight at 4 ℃ until the tissue sinks to the bottom;
embedding: selecting a tissue embedding box with a proper tissue size, adding OCT, slowly pouring liquid nitrogen, and storing in a refrigerator at-80 ℃ after the liquid nitrogen is condensed into a solid;
slicing: slicing according to the standard procedure of frozen slice, and storing in a refrigerator at-80 deg.C and a thickness of 10 μm.
3. Immunofluorescence of frozen sections
Dissolution of OCT: taking out the slices, quickly placing the slices in ice PBS, and placing the slices at room temperature for 10-15 minutes;
and (3) circling: organizing the periphery of the slice, and drawing a hydrophobic ring with hydrophobic strokes;
and (3) sealing: 3% BSA (in 0.1% PBST) for 1 hour at room temperature;
a first antibody: removing the blocking solution, and adding primary antibody (AC3 or ARL13B) dropwise at 4 deg.C overnight;
washing: PBS was washed three times for 5 minutes each;
secondary antibody: adding secondary antibody (such as Alexa Fluor 488) at room temperature for 1 hr, and keeping away from light;
washing: washing with PBS for three times, 5 minutes each time, and keeping out of the sun;
dyeing the core: the preparation concentration of Hoechst is 1: 1000, preparing PBS and keeping out of the sun;
washing: washing with PBS for three times, 5 minutes each time, and keeping out of the sun;
sealing: dripping the sealing tablet, covering the tissue with a cover glass, drying in the dark, and keeping at 4 deg.C;
imaging: after the tissue sections were air dried, images were taken with a Zeiss LSM 880 microscope.
RNAscope multichannel fluorescent staining
Baking slices: the slices were baked in a dry oven at 60 ℃ for 1 hour.
Dewaxing:
1. preparing two cleaning agent vessels containing fresh dimethylbenzene and two dye vats containing 100% ethanol in a fume hood;
2. placing the glass slide into a glass slide rack, immersing the glass slide into a first vessel containing dimethylbenzene, incubating the glass slide in the dimethylbenzene for 5 minutes at room temperature, and moving the glass slide rack up and down at intervals;
3. taking out the slide glass rack, immediately putting the slide glass rack into a second dye vat containing dimethylbenzene, incubating for 5 minutes at room temperature, and moving the slide glass rack up and down occasionally;
4. immediately placed in a dish containing 100% ethanol, incubated at room temperature for 2 minutes, and the slide holder was moved up and down from time to time.
5. Immediately placed in a second dish of 100% ethanol and incubated at room temperature for 2 minutes, the slide rack was moved up and down occasionally, and the slides were air dried at room temperature until completely dry.
Pretreatment: place dewaxed slides in HybEZTMDripping 5-8 drops of RNAscope hydrogen peroxide on the glass slide rack to cover the whole slice, and incubating the glass slide for 10 minutes at room temperature; flicking the glass slide, removing RNAscope hydrogen peroxide on the glass slide, immediately inserting the glass slide into a Tissue-Tek glass slide rack immersed in a Tissue-Tek dish filled with distilled water, moving the Tissue-Tek glass slide rack in the distilled water up and down, and cleaning the glass slide for 3-5 times.
Repairing:
1. adding 1 bottle (70mL) of 10X target repairing reagent into a beaker, then adding 630mL of distilled water, and uniformly mixing to prepare 700mL of fresh 700mL
1X target repair agent. Placing the beaker on a hot plate, covering the beaker with aluminum foil, adjusting the hot plate to a high grade for 10-15 minutes, and adjusting the hot plate to be blocked after the 1X target repairing reagent reaches a slow boiling state (98-102 ℃) so as to maintain the slow boiling state;
2. immersing the Tissue-Tek slide glass rack in a boiling 1X target repairing reagent very slowly by using a pair of tweezers, pretreating the slide glass for 15 minutes, and moving the slide glass rack up and down at intervals;
3. immediately transferring the heat-carried glass slide rack from the 1X target repairing reagent to a staining dish containing distilled water by using tweezers, and moving the heat-carried glass slide rack up and down in the distilled water
Cleaning the glass slide for 3-5 times;
4. the slides were transferred to 100% ethanol, incubated for 3 minutes, spun off excess ethanol and dried at room temperature.
Drawing a hydrophobic ring:using ImmedgeTMThe hydrophobic pen draws a hydrophobic circle 2-4 times around each section according to the following template, and completely dries the hydrophobic circle for about 1 minute or overnight at room temperature.
RNAscope protease Plus treatment:
1. place the slide in HybEZTMDropping about 2-3 drops of RNAscope protease Plus reagent on the glass slide rack to completely cover the section;
2. from HybEZTMTaking out HybEZ from hybridization ovenTMAnd a humidity control tray for placing the slide glass rack in the tray. Covering the cover, sealing, horizontally putting the humidity control tray back to the hybridization furnace again, and incubating for 30 minutes at 40 ℃;
3. taking out HybEZ from the hybridization furnaceTMAnd (3) taking the glass slide rack out of the tray, immediately putting the glass slide into a Tissue-Tek staining dish immersed in distilled water, moving the Tissue-Tek glass slide rack in the distilled water, and cleaning the glass slide for 3-5 times.
Hybridization probes: excess liquid was removed from the slide and placed in HybEZTMDropping 20 μ l of probe mixture on the slide holder to completely cover the whole section, and placing the slide holder with the slide into HybEZTMThe hybridization oven was incubated at 40 ℃ for 2 hours, and the slides were washed with 1 XWash buffer for 2 minutes at room temperature. This step was repeated with fresh 1 × wash buffer.
Hybrid AMP 1: excess liquid was removed from the slide and placed in HybEZTMDripping 20 μ l of RNAscope multichannel fluorescent second-generation Amp1 on the slide holder to completely cover the whole section, and placing the slide holder with the slide into HybEZTMThe hybridization oven was incubated at 40 ℃ for 30 minutes, the slides were washed with 1 XWash buffer, washed at room temperature for 2 minutes, and the procedure repeated with fresh 1 XWash buffer.
Hybrid AMP 2: excess liquid was removed from the slide and placed in HybEZTMDripping 20 μ l of RNAscope multichannel fluorescent second-generation Amp2 on the slide holder to completely cover the whole section, and placing the slide holder with the slide into HybEZTMThe hybridization oven was incubated at 40 ℃ for 30 minutes, the slides were washed with 1 XWash buffer, and washed at room temperatureWash for 2 min and repeat this step with fresh 1X wash buffer.
Hybrid AMP 3: excess liquid was removed from the slide and placed in HybEZTMDripping 20 μ l of RNAscope multichannel fluorescent second-generation Amp3 on the slide holder to completely cover the whole section, and placing the slide holder with the slide into HybEZTMThe hybridization oven was incubated at 40 ℃ for 15 minutes, the slides were washed with 1 XWash buffer, washed at room temperature for 2 minutes, and the procedure was repeated with fresh 1 XWash buffer.
Formation of HRP-C1 Signal: excess liquid was removed from the slide and placed in HybEZTMDripping 20 μ l of RNAscope multichannel fluorescent second-generation HRPC1 on the slide holder to completely cover the whole section, and placing the slide holder with the slide into HybEZTMThe hybridization oven was incubated at 40 ℃ for 15 minutes, the slides were washed with 1 XWash buffer, at room temperature for 2 minutes, and this step was repeated with fresh 1 XWash buffer; removing excess liquid from the slide and adding 40. mu.l of diluted solution
Plus fluoroscein, incubated in a hybridization oven at 40 ℃ for 30 minutes, washing the slides with 1X wash buffer, washing at room temperature for 2 minutes, and repeating this step with fresh 1X wash buffer; removing the excess liquid on the glass slide, dripping 20 mu l of RNAscope multichannel fluorescent secondary HRP blocking agent, placing the glass slide in a hybridization oven at 40 ℃ for incubation for 15 minutes, washing the glass slide with 1X washing buffer solution, washing the glass slide for 2 minutes at room temperature, and repeating the step with fresh 1X washing buffer solution.
Antibody staining: reference was made to the immunofluorescent portion of the frozen sections.
5. Mouse SCN brain tissue section separation and culture
1. Mouse SCN brain tissue section isolation
A. Anesthetizing the mice for 8-10 weeks, and when the mice lose consciousness but do not stop breathing, quickly cutting off the heads by scissors;
B. the eyes of the mice are removed by scissors to prevent the optic nerve from being activated in the subsequent process and further destroy SCN;
C. the skull of the mouse was cut with scissors along both sides and all bones were removed until they were visible in the olfactory bulb. The brain is prevented from being extruded, and the SCN on the ventral side is prevented from being damaged;
D. the joint of the olfactory bulb and the optic nerve is cut off by using dissecting scissors, so that the optic nerve is completely cut off;
E. the head was inverted and the intact brain was allowed to fall to fill with pre-cooled HBSS buffer (1 XHBSS, 10mM HEPES, 4.5mM NaHCO)31% Penicilin-Streptomyces) in a 10cm petri dish, the brain was kept at HBSS for 30-60 seconds to ensure cooling of the brain;
F. transferring the brain into a new culture dish by using a spoon, and cutting off the cerebellum by using a sterile scalpel blade;
G. coating super glue on a cutting platform of a Vibratome vibration slicer;
H. carefully wiping off excess HBSS at the incision of the brain with sterile filter paper;
I. fixing the brain tissue on a cutting platform with the incision facing downwards, transferring the brain tissue to a cutting table, and pouring pre-cooled HBSS buffer solution;
J. to reach the SCN region quickly, the 800 μm thickness was cut quickly, the speed was reduced when hypothalamus was first reached, and the cut was continued until the 300 μm thickness was reached when the optic chiasm was seen;
K. after SCN became visible, the layer was cut at 300 μm, brain sections were transferred with a soft brush to a petri dish containing pre-cooled HBSS buffer, and the SCN region was observed under a microscope;
l. SCN area was dissected under a stereomicroscope to remove the optic chiasmatic nerve (OC) and isolate SCN brain slices of 1x 1mm minimal area.
In vitro culture of SCN brain tissue slices
A. Transferring the separated SCN into a Millicell plug-in cell culture dish for culture;
B. DMEM medium 1.2ml (100. mu.M luciferin, 1% B27 serum free additive, 1% Penicillin-Streptomyces) was added and recorded in a LymiCycle apparatus.
6. Experiment of reciprocal time difference of ciliated mice
A. Female mice, 8 weeks old, were individually housed in cages equipped with self-propelled running wheels, given sufficient food and drinking water;
b.12 hours of illumination: acclimating the mice for 14 days in a 12-hour dark (lights turned on 07: 00 early and lights turned off 19:00 late) light cycle, and recording the movement of the mice;
C. on day 15, turn on the light for 8 hours in advance, simulate the condition of the time difference of inversion (turn on light 23:00 late and turn off light 11:00 early), evaluate the time length of the mouse adapting to the new light cycle;
D. after 14 days of recording at the new photoperiod, the photoperiod was adjusted to the original time (lights turned on 07: 00 early and 19:00 late), and the length of time the mice acclimated to the original photoperiod was evaluated.
Example 1 central SCN zone cilia exhibit significant rhythmic changes
Adult female mice (6-8 weeks) were cultured for two weeks under standard light cycle (light on at 7:00 am, light off at 19:00 pm, light intensity 50lux), and then brain tissue samples were taken every 4h after anesthesia and decapitation, with ZT0 at 7:00 am and ZT12 at 19:00 pm, 3-4 mice per group.
FIG. 1A is a representative result of brain tissue stained by fixed section, wherein AC III is a primary ciliary marker and Cry1 is a core rhythm gene, as a control; FIG. 1B is the statistics of the number of cilia in SCN region and the expression level of Cry 1; figure 1C shows statistics of cilia length of SCN region. The paraffin section fluorescence staining result shows that primary cilia of nucleus SCN region on thalamus region optic chiasmatic nerve present obvious rhythmical change no matter the number or the length, the specific change is that the primary cilia gradually decrease in the daytime and gradually increase in the evening, and the change rule is just opposite to the change rule of rhythm gene Cry 1.
Example 2 construction of thalamus region suprachiasmatic nucleus SCN-specific ciliated mice
NMS-Cre is a specific expression Cre tool mouse of an SCN region, Ift88 and Ift20 are important regulatory proteins participating in cilium generation, and NMS-Ift 88-/-and NMS-Ift 20-/-mice are obtained by mating NMS-Cre mice with Ift88-loxp and Ift20-loxp mice respectively and reproducing for multiple generations.
FIGS. 2A and 2B show the effect of detecting the knockdown of Ift88 and Ift20 by Western Blot; FIG. 2C shows the SCN cilia detection result; figure 2D is the results of other zonal cilia assays, where SCN represents the suprachiasmatic nucleus and PVN represents the paraventricular nucleus of the hypothalamus. The above results demonstrate the successful construction of a mouse model of nuclear SCN specific ciliary defect on the optic chiasm in the thalamus region.
Example 3 biological rhythm disorder in SCN-zone cilia-deficient mice
Adult female mice of 8 weeks old were individually housed in a cabinet equipped with a race wheel cage, and the race wheel behavior of the mice was recorded and analyzed in real time using ClockLab (Actimetrics).
FIG. 3A is a representative result of a running wheel behavior in a continuous dark environment (DD) after a control mouse and a SCN region cilia-deficient mouse are acclimated for two weeks by a standard light cycle (LD); FIG. 3B is a statistical result of the running wheel behavior period of the control mice and SCN region cilium-deficient mice in a continuous dark environment; the above results demonstrate that in the case of abnormal light cycle (continuous darkness), the model mice have prolonged movement cycle.
FIG. 3C shows a representative result of mouse rotation behavior after a mouse is acclimated for two weeks in a standard illumination period (7: 00 lights on in the morning and 19:00 lights off in the evening), an illumination schedule is advanced by 8h (23: 00 lights on in the evening and 11:00 lights off in the morning), and the illumination schedule is returned to an initial state after 15 days of observation; FIGS. 3D and 3E are statistical results of phase shift kinetics in the reciprocal time difference experiments for control mice and SCN-zone cilia-deficient mice. The above results demonstrate that in the case of abnormal light cycles (8 h earlier in the light schedule), the model mice can adapt rapidly to the changed light cycle in a short time (1-2 days).
Example 4 ciliary transport drug FTY720 influences the process of the oscillatory change of rhythmic genes
FIG. 4A is a representative graph of S1P (sphingosine-1-phosphate) inhibition of starvation induced ciliation in hTERT-RPE1 cells (human retinal pigment epithelial cells); FIG. 4A shows the left statistical results of FIG. 4A; FIG. 4B is a statistical result of S1P antagonist FTY720 inducing cilia of RPE1 cells in normal culture environment; FIG. 4C shows the result of FTY720 affecting the oscillatory change of the circadian gene in SCN region at tissue level, the arrow represents the time of changing and adding the drug, Per2, the Luc mouse is a tool mouse for observing the oscillatory change of the expression level of the core circadian gene Per 2; FIG. 4D is the statistical result of the oscillation variation period of the rhythm gene Per2 in FIG. 4C. The results show that the cilia generation regulation medicament is possibly applied to the regulation of the biological rhythm of the organism and the treatment of diseases related to the biological rhythm.
Claims (10)
1. Use of a modulator of ciliogenesis of any of:
1) regulating ciliation;
2) preparing a product for regulating biological rhythm;
3) preparing a product for treating diseases related to biological rhythm;
preferably, the biological rhythm-related diseases include sleep disorders, jet lag, depression, metabolic disorders, aging, hematologic diseases, diabetes, and obesity.
2. The use of claim 1, wherein the modulator of ciliation comprises a modulator of FTY720, S1P, Ki16425, LPA, LY294002, Wortmannin, AKTiIV, PIA23, GSK690693, Perifosine, PHA-680632, tunacin and pharmaceutically acceptable salts thereof, a modulator of ciliogenesis gene;
preferably, the cilium modulating agent is FTY 720;
preferably, the ciliogenic genes include Ift88, Ift 20.
3. The use of claim 2, wherein said pharmaceutically acceptable salt comprises an acid addition salt, a base addition salt;
preferably, said acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, tosylate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate and salicylate, malonate, adipate, hexanoate, arginate, fumarate, nicotinate, phthalate or oxalate salts of FTY 720;
preferably, the base addition salt includes, but is not limited to, lithium salt, sodium salt, potassium salt, barium salt, calcium salt, magnesium salt, aluminum salt, iron salt, ferrous salt, copper salt, zinc salt of FTY720, or salt of FTY720 with morpholine, diethylamine, triethylamine, isopropylamine, trimethylamine, lysine or histidine.
4. The use of claim 2, wherein said modulator comprises an agent used in any of siRNA interference technology, CRISPR technology, TALEN technology, ZFN technology, Cre-loxP gene recombination technology;
preferably, the regulatory agent is an agent used in Cre-loxP gene recombination technology.
5. The use of claim 1, wherein the product comprises a pharmaceutical composition;
preferably, the mode of administration of the pharmaceutical composition includes oral or parenteral;
preferably, the parenteral form comprises intravenous, intramuscular, intraperitoneal or subcutaneous injection.
6. The use of claim 5, wherein the pharmaceutical composition is a pill, tablet, capsule, granule, oral liquid, suspension, injection, microsphere or liposome prepared from the ciliogenesis modulator and a pharmaceutically acceptable excipient by conventional methods.
7. A method of modulating ciliogenesis in vitro, the method comprising administering a ciliogenesis modulating agent;
preferably, the ciliogenesis modulator comprises FTY720 and pharmaceutically acceptable salts thereof, modulators of ciliogenesis genes;
preferably, the ciliogenic genes include Ift88, Ift 20.
8. A method for constructing an animal model of biological rhythm abnormality, which comprises specifically knocking out Ift88 or Ift20 of an SCN region of the model animal;
preferably, the model animal comprises a non-human mammal;
preferably, the non-human mammal includes orangutan, monkey, horse, cow, sheep, pig, donkey, camel, dog, rabbit, cat, rat, mouse, fish, bird, etc.;
preferably, the animal model is a mouse;
preferably, the method comprises the steps of:
1) obtaining an animal model of the specific expression Cre of the SCN region;
2) obtaining an animal model of Ift88-loxp or Ift 20-loxp;
3) animal models of 1) and 2) were mated.
9. An animal model constructed by the method of claim 8, or its use in studying cilia function, the mechanism of cilia regulation of biological rhythms.
10. A method of detecting the amount of cilia, the method comprising detecting the expression of cilia genesis genes, the cilia genesis genes comprising Ift88, Ift 20.
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| AU2001268973A1 (en) * | 2000-04-18 | 2001-10-30 | Ipf Pharmaceuticals Gmbh | Method for using transfectable constructions from luciferase and from the promotor of the human period 1 gene for identifying novel active substances that influence the day/night rhythm and sleep |
| AU2010300713A1 (en) * | 2009-09-29 | 2012-04-26 | Dart Neuroscience (Cayman) Ltd | Genes, methods, and compositions related to neurogenesis and its modulation |
| CN103386119A (en) * | 2012-05-08 | 2013-11-13 | 中国科学院上海生命科学研究院 | Application of SIRT1 in adjusting rhythm and improving rhythm disorder |
| CN112062827B (en) * | 2020-09-16 | 2022-10-21 | 中国人民解放军军事科学院军事医学研究院 | Application of CEP55 protein in regulation of cilia de-assembly and preparation of cilia-related disease model |
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