CN110384076A - The construction method of Caenorhabditis elegans anaesthetic biological model, building biological model and its detection method - Google Patents
The construction method of Caenorhabditis elegans anaesthetic biological model, building biological model and its detection method Download PDFInfo
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- 229960004739 sufentanil Drugs 0.000 claims description 184
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- 206010002091 Anaesthesia Diseases 0.000 description 1
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Abstract
The present invention discloses a kind of construction method of Caenorhabditis elegans anaesthetic biological model, it is related to biological model technical field, the following steps are included: being placed in Caenorhabditis elegans in the NGM culture medium I containing various concentration anaesthetic after culture, through cracking, centrifuged deposit, and it is plated on the NGM culture medium II containing anaesthetic after precipitating is resuspended, it cultivates to first phase larva, Caenorhabditis elegans anaesthetic biological model is established, the present invention also provides the Caenorhabditis elegans anaesthetic biological models and its detection method that are constructed by above-mentioned construction method.The beneficial effects of the present invention are: the construction method of Caenorhabditis elegans anaesthetic biological model disclosed by the invention and the biological model of building provide new technical support for anaesthetic safety evaluatio.
Description
Technical field
The present invention relates to biological model technical fields, and in particular to a kind of Caenorhabditis elegans anaesthetic biological model
Construction method, the biological model of building and its detection method.
Background technique
Caenorhabditis elegans (Caenorhabditis elegans, C.elegans) is a kind of line that can independently survive
Worm, individual is small, adult only 1.5mm long, is hermaphroditic, and the 0.2% of male Jin Zhan group, it can self-fertilization or double property
Reproduction, the average history of life is 3.5 days at 20 DEG C, and average reproductive capacity is 300-350, if but mate with male worm, it can produce more
Up to 1400 or more offsprings.
As social population's aging increasingly aggravates, undergoing surgery person-time is sharply increased, and anaesthetic is clinical operation
Drug must be used, refers to that Central nervous has anesthetic effect, continuous use, abuse or unreasonable use are also easy to produce physiological dependence
Property and psychic dependence, can be at the drug of addiction.Such as Propofol is widely used to each after the development after more than ten years
The anesthesia induction and maintenance of class operation, and due to its have the characteristics that it is rapid-action, hold time short, the drug has become at present
The first-line drug of the sedation treatment Intensive Care Therapy (ICU).Organism physiology system normal function can be damaged using anaesthetic, such as nerve
Toxicity and genotoxicity.It is more at present to use mammal mouse or rat studies anaesthetic side effect, but model foundation institute
Take time length, heavy workload, cannot achieve quickly screening and evaluation.Caenorhabditis elegans has many excellent as model organism
Gesture, individual is small, and reproductive development is fast, and culture is simple, and genetic background is clear, is the effective carrier as high flux screening.
Summary of the invention
Present invention solves the technical problem that one of be to provide a kind of structure of Caenorhabditis elegans anaesthetic biological model
Construction method.
The present invention adopts the following technical solutions solves above-mentioned technical problem:
The present invention provides a kind of construction method of Caenorhabditis elegans anaesthetic biological model, comprising the following steps: will
Caenorhabditis elegans is placed in the NGM culture medium I containing various concentration anaesthetic after culture, through cracking, centrifuged deposit, and
It is plated on the NGM culture medium II containing anaesthetic after precipitating is resuspended, cultivates to first phase larva, that is, establish beautiful hidden bar
Nematode anaesthetic biological model.
Preferably, the anaesthetic includes sufentanil, Oxycodone, Remifentanil or Propofol.
Preferably, the preparation method of the anaesthetic are as follows: dissolve anaesthetic by solvent of LB liquid medium, prepare
The anaesthetic of various concentration.
Present invention solves the technical problem that two be to provide a kind of Caenorhabditis elegans constructed by above-mentioned construction method
Anaesthetic biological model.
Present invention solves the technical problem that three be to provide a kind of inspection of Caenorhabditis elegans anaesthetic biological model
Survey method.
Preferably, the detection method of the Caenorhabditis elegans anaesthetic biological model, comprising the following steps:
(1) cut from culture medium II agar block containing first phase larva to number be A, B, C, D, E, F, G seven training
It supports in ware, is grown after hatching to fourth phase larva, the nematode in culture dish is labeled as F0 for nematode at this time；
(2) F0 grows to the manhood for nematode, selects several into new culture dish, every other day transfers them to new culture dish
In, until oviposition terminates；
(3) F0 in A, B, C, D, E, F, G culture dish is observed and recorded during step (1) to (2) under stereomicroscope
For the index of correlation of nematode.
Preferably, index of correlation of the F0 for nematode are as follows:
(1) F0 is measured for the influence of nematode reproductive system；
(2) F0 is measured for the influence of the motor behavior of nematode；
(3) F0 is measured for the influence of the growth and development of nematode；
(4) F0 is measured for the influence of the nervous function of nematode.
Preferably, fourth phase children is only entered the following steps are included: selecting 20 on the F0 measurement influenced for nematode reproductive system
The Caenorhabditis elegans of worm 36h detects the laying in 2h and generation time into new culture dish；The generation time is F0 generation
The time interval of its F1 generation Adult worms producting eggs is arrived in nematode oviposition.
Preferably, to F0 for the measurement of the motor behavior of nematode the following steps are included:
(1) frequency of head oscillation: the number of adult of the same period head oscillation in 1min is recorded；
(2) the curved frequency of body: adult of the same period curved number in 30s is recorded.
Preferably, to F0 for the measurement of the growth and development of nematode the following steps are included:
(1) service life: polypide is touched with syringe in the 0th day for being denoted as life starting with nematode egg hatching, reactionless to be considered as line
The termination of worm life records the nematode service life, i.e., from starting to the number of days of termination；
(2) body is long: Caenorhabditis elegans and film-making of the measurement into 4 period of larva 36h after microscope is taken pictures, calculate line
Length of the worm from head to tail portion central axes.
Preferably, to F0 for the nervous function of nematode measurement the following steps are included: serotonin autofluorescence measurement:
Caenorhabditis elegans and film-making of the measurement into fourth phase larva 36h, are taken pictures by fluorescence microscope and calculate beautiful hidden bar line
Worm serotonin expression.
The beneficial effects of the present invention are:
(1) construction method of Caenorhabditis elegans anaesthetic biological model disclosed by the invention and the biological mould of building
Type provides new technical support for anaesthetic safety evaluatio；
(2) Caenorhabditis elegans anaesthetic biological model disclosed by the invention has the safety of assessment anaesthetic
Significance, can simple and quick influence of the evaluation anaesthetic to growth and development and nervous function, and it is short to construct the time, building side
Method and detection method are simple and quick, application value with higher.
Detailed description of the invention
Fig. 1 is influence of the sufentanil of various concentration in the embodiment of the present invention 2 to each generation Caenorhabditis elegans laying
Result figure；
Fig. 2 is shadow of the sufentanil to each generation Caenorhabditis elegans generation time of various concentration in the embodiment of the present invention 2
Ring result figure；
Fig. 3 is influence knot of the sufentanil to each generation Caenorhabditis elegans service life of various concentration in the embodiment of the present invention 2
Fruit figure；
Fig. 4 is influence knot of the sufentanil to each generation caenorhabditis elegans body length of various concentration in the embodiment of the present invention 2
Fruit figure；
Fig. 5 is the sufentanil of various concentration in the embodiment of the present invention 2 to each generation Caenorhabditis elegans head oscillation frequency
Influence result figure；
Fig. 6 is the sufentanil of various concentration in the embodiment of the present invention 2 to each generation Caenorhabditis elegans body corner frequency
Influence result figure；
Fig. 7 is that the sufentanil of various concentration in the embodiment of the present invention 2 expresses each generation Caenorhabditis elegans serotonin
Influence result figure.
Specific embodiment
The present invention is described in further details below with reference to Figure of description and embodiment.
Test material and reagent as used in the following examples etc., unless otherwise specified, commercially obtain.
In the examples where no specific technique or condition is specified, can according to the literature in the art described technology or
Condition is carried out according to product description.
Embodiment 1
(1) preparation of experiment reagent:
(1) preparation of NGM culture medium: according to the method in wormbook, being separately added into 3g NaCl in 2L conical flask,
975mL distilled water, high pressure sterilization is added in 17g agar and 2.5g peptone.20min is balanced at 55 DEG C after sterilizing, then
It is separately added into 1mL 1M CaCl2, 1mL 5mg/mL cholesterol, 1mL 1M MgSO4With 25mL KPO4Buffer pours into after mixing
In sterile petri dish, overnight after spread Escherichia coli OP50 bacterium, be put into after drying 4 DEG C it is spare.
(2) preparation of M9 culture medium: 3g KH2PO4、6g Na2HPO4, 5g NaCl and 1mL 1M MgSO4, distilled water constant volume
It is spare after high pressure sterilization to 1L.
(3) preparation of anaesthetic:
It prepares the storing liquid of anaesthetic: dissolving anaesthetic, concentration 10mM by solvent of LB liquid medium.
(4) preparation of Escherichia coli OP50 bacterium NGM culture medium is spread
1. preparing 1.0mM anaesthetic sufentanil+OP50 20mL: take respectively 2mL anaesthetic stock solution (10mM) and
The OP50 bacterium solution of 18mL fresh cultured, bed board after mixing, 4 DEG C of placement is spare after drying；
2. preparing 0.1mM anaesthetic sufentanil+OP50 20mL: taking 2mL anaesthetic application liquid (1.0mM) respectively
With the OP50 bacterium solution of 18mL fresh cultured, bed board after mixing, 4 DEG C of placement is spare after drying；
3. preparing 0.01mM anaesthetic sufentanil+OP50 20mL: taking 2mL anaesthetic application liquid (0.1mM) respectively
With the OP50 bacterium solution of 18mL fresh cultured, bed board after mixing, 4 DEG C of placement is spare after drying；
4. preparing 0.001mM anaesthetic sufentanil+OP50 20mL: taking 2mL anaesthetic application liquid respectively
The OP50 bacterium solution of (0.01mM) and 18mL fresh cultured, bed board after mixing, 4 DEG C of placement is spare after drying；
5. preparing 0.0001mM anaesthetic sufentanil+OP50 20mL: taking 2mL anaesthetic application liquid respectively
The OP50 bacterium solution of (0.001mM) and 18mL fresh cultured, bed board after mixing, 4 DEG C of placement is spare after drying；
6. preparing 0.00001mM anaesthetic sufentanil+OP50 20mL: taking 2mL anaesthetic application liquid respectively
The OP50 bacterium solution of (0.0001mM) and 18mL fresh cultured, bed board after mixing, 4 DEG C of placement is spare after drying.
(2) building of Caenorhabditis elegans anaesthetic biological model
(1) the 3CM culture dish of the NGM culture medium I containing Caenorhabditis elegans is prepared, and is divided into 7 groups, first group is control
Group, Escherichia coli OP50300 μ L needed for being coated with Caenorhabditis elegans normal growth above (are labeled as A), and second group is coated with
OP50+0.00001mM anaesthetic sufentanil (is labeled as B), and third group is coated with OP50+0.0001mM anaesthetic Shu Fentai
Buddhist nun (is labeled as C), and the 4th group is coated with OP50+0.001mM anaesthetic sufentanil (labeled as D), and the 5th group is coated with OP50+
0.01mM anaesthetic sufentanil (is labeled as E), and the 6th group is coated with OP50+0.1mM anaesthetic sufentanil and (is labeled as
F), it is coated with OP50+1.0mM anaesthetic sufentanil for the 7th group (labeled as G)；
(2) it obtains synchronized Caenorhabditis elegans first phase larva (L1 phase): being trained using M9 culture medium from 3-5 NGM
It supports and collects a large amount of hermaphroditic wild-type adults on ware to 1.5mL centrifuge tube, 2000rpm is discarded supernatant after being centrifuged 2min, added
Enter M9 culture medium 1mL cleaning precipitating 3 times, be resuspended, be plated in the culture dish of culture medium II of only NGM with M9 culture medium afterwards,
It is placed in incubator culture 12h；The present embodiment is by taking sufentanil anaesthetic as an example, other anaesthetics such as Oxycodone, Rui Fen
Too Buddhist nun, Propofol, since their pharmacology, drug effect and sufentanil are close, those of ordinary skill in the art are according to this implementation structure
The biological model built is equally applicable to Oxycodone, Remifentanil, propofol drug.
Embodiment 2
(1) detection method of Caenorhabditis elegans anaesthetic biological model:
(1) agar block of the picking embodiment 1 containing first phase larva culture medium is to seven groups of culture dishes of A, B, C, D, E, F, G
In, every group of 4 Duplicate Samples are respectively labeled as: A1-A4, B1-B4, C1-C4, D1-D4, E1-E4, F1-F4 and G1-G4；
(2) when growing to fourth phase larva after seven culture dish hatchings of A-G, the insect in every group of each culture dish is checked
Number, it is ensured that insect number is 20 in each ware.At this point, Caenorhabditis elegans is denoted as F0 generation in this batch cultivation ware；
(3) F0 grows to adulthood for nematode, 20 is selected at random into corresponding new culture dish, every other day by its turn
New culture dish is moved to, until oviposition terminates.Index of correlation is carried out under stereomicroscope to this 20 nematodes in the process
Measurement and record.
(2) measuring method that A1-G1 F0 is influenced for Caenorhabditis elegans reproductive system:
Select 20 fourth phase larvas, continue cultivate 36h after, be transferred in new culture dish, detect 2h in laying and
Generation time；Generation time is the time interval that F0 arrives its F1 generation Adult worms producting eggs for nematode oviposition.
(3) measuring method that A1-G1 F0 is influenced for Caenorhabditis elegans motor behavior:
(1) frequency of head oscillation: the number of adult of the same period head oscillation in 1min is recorded；
(2) the curved frequency of body: adult of the same period curved number in 30s is recorded.
(4) to A1-G1 F0 for the measuring method of Caenorhabditis elegans growth and development:
(1) service life: polypide is touched with syringe in the 0th day for being denoted as life starting with nematode egg hatching, reactionless to be considered as line
The termination of worm life records the nematode service life, i.e., from starting to the number of days of termination；
(2) body is long: Caenorhabditis elegans and film-making of the measurement into 4 period of larva 36h after microscope is taken pictures, calculate line
Length of the worm from head to tail portion central axes.
(5) measuring method that A1-G1 F0 is influenced for Caenorhabditis elegans nervous function:
The measurement of serotonin autofluorescence: Caenorhabditis elegans and film-making of the measurement into fourth phase larva 36h pass through
Fluorescence microscope takes pictures and calculates Caenorhabditis elegans serotonin expression, Caenorhabditis elegans serotonin expression
Measuring method be the prior art.
(6) replication
In above-mentioned seven groups of culture dishes adult to the egg-laying season when chosen respectively to 2h of laying eggs on corresponding new culture dish after put back to original
Culture dish continues the measurement of each index.The batch newly laid eggs is denoted as F1 generation, repeats the above steps, and detects the impacted journey of F1 generation
Degree.
Former culture dish is put back to after being chosen respectively when F1 generation adult is to the egg-laying season to 2h of laying eggs on corresponding new culture dish, is continued each
The measurement of index.The batch newly laid eggs is denoted as F2 generation, repeats the above steps, and detects the degree of susceptibility of F1 generation.
F2 for adult to the egg-laying season when chosen respectively to 2h of laying eggs on corresponding new culture dish after put back to former culture dish, continue each
The measurement of index.The batch newly laid eggs is denoted as F3 generation, repeats the above steps, and detects the degree of susceptibility in F3 generation.
(7) experimental result
As shown in Figure 1, the sufentanil of various concentration is to F0, F1, F2 and F3 for the shadow of Caenorhabditis elegans reproductive system
Sound is roughly the same, i.e., as sufentanil concentration increases, nematode laying is reduced.In F0 generation, control group nematode laying is
279.55 ± 11.12,0.00001nM sufentanil processing group nematode laying are 274.75 ± 12.43,0.0001mM Shu Fentai
Buddhist nun's processing group nematode laying be 267.55 ± 17.91,0.001mM sufentanil processing group nematode laying be 263.35 ±
13.59,0.01mM sufentanil processing group nematode layings are the production of 260.15 ± 16.69,0.1mM sufentanil processing group nematode
Ovum number is that 253.7 ± 11.98,1.0mM sufentanil processing group nematode laying is 248.2 ± 15.41.
In F1 generation, control group nematode laying is the production of 279.85 ± 11.76,0.00001nM sufentanil processing group nematode
Ovum number is that 278.25 ± 12.76,0.0001mM sufentanil processing group nematode laying is that 278.7 ± 13.76,0.001mM relaxes
Fentanyl processing group nematode laying be 270.45 ± 17.16,0.01mM sufentanil processing group nematode laying be 262.3 ±
20.36,0.1mM sufentanil processing group nematode layings are the production of 266.55 ± 22.56,1.0mM sufentanil processing group nematode
Ovum number is 255.5 ± 19.34.
In F2 generation, control group nematode laying is the oviposition of 277.85 ± 13.9,0.00001nM sufentanil processing group nematode
Number is that 281.9 ± 11.80,0.0001mM sufentanil processing group nematode laying is 277.9 ± 12.53,0.001mM Shu Fentai
Buddhist nun's processing group nematode laying be 274.6 ± 10.43,0.01mM sufentanil processing group nematode laying be 261.9 ±
15.54,0.1mM sufentanil processing group nematode layings are the oviposition of 256.9 ± 17.81,1.0mM sufentanil processing group nematode
Number is 249.35 ± 17.99.
In F3 generation, control group nematode laying is 279 ± 11.33,0.00001nM sufentanil processing group nematode laying
It is 282.05 ± 9.61,0.001mM sufentanil for 282.2 ± 11.12,0.0001mM sufentanil processing group nematode laying
Processing group nematode laying is that 267.8 ± 22.62,0.01mM sufentanil processing group nematode laying is 261.6 ± 23.02,
0.1mM sufentanil processing group nematode laying is that 242.15 ± 17.87,1.0mM sufentanil processing group nematode laying is
241.5±17.07。
As shown in Fig. 2, the sufentanil of various concentration is to F0, F1, F2 and F3 for the shadow of Caenorhabditis elegans generation time
Sound is roughly the same, i.e., as sufentanil concentration increases, nematode generation time is reduced.
In F0 generation, control group nematode generation time is 68.8 ± 4.38h, 0.00001nM sufentanil processing group nematode generation
It is 69 ± 3.73h for the time, 0.0001mM sufentanil processing group nematode generation time is 66.85 ± 3.95h, and 0.001mM relaxes
Fentanyl processing group nematode generation time is 69.1 ± 4.11h, and 0.01mM sufentanil processing group nematode generation time is 65.8
± 4.45h, 0.1mM sufentanil processing group nematode generation time are 64.25 ± 3.77h, 1.0mM sufentanil processing group nematode
Generation time is 61.65 ± 5.78h.
In F1 generation, control group nematode generation time is 67.8 ± 3.71h, 0.00001nM sufentanil processing group nematode generation
It is 67.35 ± 4.9h for the time, 0.0001mM sufentanil processing group nematode generation time is 69.05 ± 3.96h, 0.001mM
Sufentanil processing group nematode generation time is 68.4 ± 4.63h, and 0.01mM sufentanil processing group nematode generation time is
67.1 ± 4.67h, 0.1mM sufentanil processing group nematode generation time are 63.6 ± 4.49h, 1.0mM sufentanil processing group
Nematode generation time is 63.9 ± 5.36h.
In F2 generation, control group nematode generation time is 67.85 ± 4.83h, 0.00001nM sufentanil processing group nematode generation
It is 67.2 ± 3.79h for the time, 0.0001mM sufentanil processing group nematode generation time is 67.25 ± 3.99h, 0.001mM
Sufentanil processing group nematode generation time is 66.7 ± 4.94h, and 0.01mM sufentanil processing group nematode generation time is
66.25 ± 5.01h, 0.1mM sufentanil processing group nematode generation time are 63.35 ± 4.92h, the processing of 1.0mM sufentanil
Group nematode generation time is 58.9 ± 5.99h.
In F3 generation, control group nematode generation time is 66.85 ± 4.48h, 0.00001nM sufentanil processing group nematode generation
It is 65.95 ± 5.02h for the time, 0.0001mM sufentanil processing group nematode generation time is 66.55 ± 4.75h, 0.001mM
Sufentanil processing group nematode generation time is 66.45 ± 4.52h, and 0.01mM sufentanil processing group nematode generation time is
66.55 ± 4.92h, 0.1mM sufentanil processing group nematode generation time are 63.6 ± 4.48h, 1.0mM sufentanil processing group
Nematode generation time is 58.85 ± 6.04h.
As shown in figure 3, the sufentanil of various concentration to F0, F1, F2 and F3 for Caenorhabditis elegans aging effects substantially
Identical, i.e., as sufentanil concentration increases, the nematode service life is reduced.
In F0 generation, the control group nematode service life is 8.5 ± 1.24 days, and the 0.00001nM sufentanil processing group nematode service life is
8.4 ± 1.11 days, the 0.0001mM sufentanil processing group nematode service life was 7.5 ± 1.36 days, 0.001mM sufentanil processing group
The nematode service life is 7.85 ± 1.8 days, and the 0.01mM sufentanil processing group nematode service life is 7.95 ± 1.69 days, 0.1mM Shu Fentai
Buddhist nun's processing group nematode service life is 7.7 ± 1.9 days, and the 1.0mM sufentanil processing group nematode service life is 7.5 ± 1.8 days.
In F1 generation, the control group nematode service life is 8.8 ± 1.17 days, and the 0.00001nM sufentanil processing group nematode service life is
8.35 ± 1.11 days, the 0.0001mM sufentanil processing group nematode service life was 7.65 ± 1.56 days, the processing of 0.001mM sufentanil
The group nematode service life is 7.75 ± 1.76 days, and the 0.01mM sufentanil processing group nematode service life is 7.9 ± 1.84 days, 0.1mM Shu Fen
Too Buddhist nun's processing group nematode service life is 7.45 ± 1.72 days, and the 1.0mM sufentanil processing group nematode service life is 7.5 ± 1.69 days.
In F2 generation, the control group nematode service life is 8.75 ± 1.04 days, and the 0.00001nM sufentanil processing group nematode service life is
8.75 ± 0.99 days, the 0.0001mM sufentanil processing group nematode service life was 7.7 ± 1.42 days, the processing of 0.001mM sufentanil
The group nematode service life is 8.2 ± 1.5 days, and the 0.01mM sufentanil processing group nematode service life is 7.7 ± 1.52 days, 0.1mM Shu Fentai
Buddhist nun's processing group nematode service life is 7.25 ± 1.7 days, and the 1.0mM sufentanil processing group nematode service life is 7.85 ± 1.24 days.
In F3 generation, the control group nematode service life is 8.05 ± 1.77 days, and the 0.00001nM sufentanil processing group nematode service life is
7.15 ± 1.31 days, the 0.0001mM sufentanil processing group nematode service life was 7.85 ± 1.71 days, the processing of 0.001mM sufentanil
The group nematode service life is 7.45 ± 1.71 days, and the 0.01mM sufentanil processing group nematode service life is 7.05 ± 1.86 days, 0.1mM Shu Fen
Too Buddhist nun's processing group nematode service life is 6.05 ± 1.66 days, and the 1.0mM sufentanil processing group nematode service life is 6.35 ± 1.15 days.
As shown in figure 4, the sufentanil of various concentration is big for the influence of caenorhabditis elegans body length to F0, F1, F2 and F3
Cause identical, i.e., as sufentanil concentration increases, nematode body is long to be reduced.
In F0 generation, a length of 978.42 ± 38.9 μm of control group nematode body, 0.00001nM sufentanil processing group nematode body is long
It is 961.63 ± 43.53 μm, a length of 936.42 ± 62.70 μm of group nematode body of 0.0001mM sufentanil processing, 0.001mM Shu Fen
Too a length of 921.72 ± 47.57 μm of Buddhist nun's processing group nematode body, 0.01mM sufentanil processing group nematode body a length of 910.53 ±
58.43 μm, a length of 887.95 ± 41.93 μm of group nematode body of 0.1mM sufentanil processing, 1.0mM sufentanil processing group nematode
A length of 868.7 ± 53.94 μm of body.
In F1 generation, a length of 979.47 ± 41.18 μm of control group nematode body, 0.00001nM sufentanil processing group nematode body
A length of 973.88 ± 44.66 μm, a length of 975.45 ± 48.18 μm of group nematode body of 0.0001mM sufentanil processing, 0.001mM relaxes
A length of 946.57 ± 60.07 μm of group nematode body of fentanyl processing, 0.01mM sufentanil processing group nematode body a length of 918.05 ±
71.26 μm, a length of 932.92 ± 78.95 μm of group nematode body of 0.1mM sufentanil processing, 1.0mM sufentanil processing group nematode
A length of 894.25 ± 67.69 μm of body.
In F2 generation, a length of 972.48 ± 48.65 μm of control group nematode body, 0.00001nM sufentanil processing group nematode body
A length of 986.65 ± 41.29 μm, a length of 972.65 ± 43.87 μm of group nematode body of 0.0001mM sufentanil processing, 0.001mM relaxes
A length of 961.1 ± 36.5 μm of group nematode body of fentanyl processing, 0.01mM sufentanil processing group nematode body a length of 916.65 ±
54.4 μm, a length of 899.15 ± 62.36 μm of group nematode body of 0.1mM sufentanil processing, 1.0mM sufentanil processing group nematode body
A length of 872.72 ± 62.95 μm.
In F3 generation, a length of 976.5 ± 39.66 μm of control group nematode body, 0.00001nM sufentanil processing group nematode body is long
It is 987.7 ± 38.9 μm, a length of 987.18 ± 33.63 μm of group nematode body of 0.0001mM sufentanil processing, 0.001mM Shu Fentai
A length of 937.3 ± 79.18 μm of group nematode body of Buddhist nun's processing, a length of 915.6 ± 80.56 μ of 0.01mM sufentanil processing group nematode body
A length of 847.52 ± 62.53 μm of group nematode body of m, 0.1mM sufentanil processing, 1.0mM sufentanil processing group nematode body are a length of
845.25±59.75μm。
As shown in Figure 5 and Figure 6, the sufentanil of various concentration is moved and is gone for Caenorhabditis elegans to F0, F1, F2 and F3
For influence it is roughly the same, i.e., as sufentanil concentration increases, nematode head oscillation frequency and body corner frequency are reduced.
In F0 generation, control group nematode head oscillation number is 60.85 ± 4.22 times, 0.00001nM sufentanil processing group
Nematode head oscillation number is 58.3 ± 5.33 times, and 0.0001mM sufentanil processing group nematode head oscillation number is 58.95
± 3.46 times, 0.001mM sufentanil processing group nematode head oscillation number is 53.5 ± 4.57 times, at 0.01mM sufentanil
Reason group nematode head oscillation number is 50.35 ± 5.75 times, and 0.1mM sufentanil processing group nematode head oscillation number is 48.3
± 6.13 times, 1.0mM sufentanil processing group nematode head oscillation number is 45.9 ± 4.63 times.
In F1 generation, control group nematode head oscillation number is 60 ± 3.81 times, 0.00001nM sufentanil processing group nematode
Head oscillation number be 59.35 ± 4.16 times, 0.0001mM sufentanil processing group nematode head oscillation number be 60.4 ±
3.72 times, 0.001mM sufentanil processing group nematode head oscillation number is 56.7 ± 3.77 times, the processing of 0.01mM sufentanil
Group nematode head oscillation number is 52.85 ± 5.83 times, and 0.1mM sufentanil processing group nematode head oscillation number is 50.05
± 4.15 times, 1.0mM sufentanil processing group nematode head oscillation number is 50.3 ± 7.22 times.
In F2 generation, control group nematode head oscillation number is 60.1 ± 3.87 times, 0.00001nM sufentanil processing group line
Worm head oscillation number be 58.4 ± 4.12 times, 0.0001mM sufentanil processing group nematode head oscillation number be 59.75 ±
3.52 times, 0.001mM sufentanil processing group nematode head oscillation number is 56.1 ± 4.56 times, the processing of 0.01mM sufentanil
Group nematode head oscillation number is 55.15 ± 4.93 times, and 0.1mM sufentanil processing group nematode head oscillation number is 52.45
± 4.31 times, 1.0mM sufentanil processing group nematode head oscillation number is 49.4 ± 4.47 times.
In F3 generation, control group nematode head oscillation number is 58.55 ± 4.36 times, 0.00001nM sufentanil processing group
Nematode head oscillation number is 60.25 ± 5.36 times, and 0.0001mM sufentanil processing group nematode head oscillation number is 59.1
± 4.4 times, 0.001mM sufentanil processing group nematode head oscillation number is 57.25 ± 4.79 times, at 0.01mM sufentanil
Reason group nematode head oscillation number is 55.2 ± 4.33 times, and 0.1mM sufentanil processing group nematode head oscillation number is 54.7
± 4.3 times, 1.0mM sufentanil processing group nematode body head oscillation number is 51.8 ± 4.46 times.
In F0 generation, control group nematode body number of bends is 22.65 ± 3.15 times, 0.00001nM sufentanil processing group
Nematode body number of bends is 20.45 ± 3.54 times, and 0.0001mM sufentanil processing group nematode body number of bends is 21.15
± 2.43 times, 0.001mM sufentanil processing group nematode body number of bends is 17.6 ± 3.91 times, at 0.01mM sufentanil
Reason group nematode body number of bends is 16.8 ± 2.52 times, and 0.1mM sufentanil processing group nematode body number of bends is 17.2
± 2.62 times, 1.0mM sufentanil processing group nematode body number of bends is 16.75 ± 2.90 times.
In F1 generation, control group nematode body number of bends is 23.05 ± 3.19 times, 0.00001nM sufentanil processing group
Nematode body number of bends be 22 ± 2.9 times, 0.0001mM sufentanil processing group nematode body number of bends be 22.8 ±
3.73 times, 0.001mM sufentanil processing group nematode body number of bends is 21.35 ± 2.46 times, at 0.01mM sufentanil
Reason group nematode body number of bends is 19.1 ± 1.34 times, and 0.1mM sufentanil processing group nematode body number of bends is 17.7
± 2.9 times, 1.0mM sufentanil processing group nematode body number of bends is 17.45 ± 2.29 times.
In F2 generation, control group nematode body number of bends is 22.15 ± 3.24 times, 0.00001nM sufentanil processing group
Nematode body number of bends is 23.35 ± 2.93 times, and 0.0001mM sufentanil processing group nematode body number of bends is 22.05
± 3.4 times, 0.001mM sufentanil processing group nematode body number of bends is 22.85 ± 3.18 times, at 0.01mM sufentanil
Reason group nematode body number of bends is 21.35 ± 2.92 times, and 0.1mM sufentanil processing group nematode body number of bends is
19.85 ± 3.26 times, 1.0mM sufentanil processing group nematode body number of bends is 17.25 ± 2.26 times.
In F3 generation, control group nematode body number of bends is 22.75 ± 3.26 times, 0.00001nM sufentanil processing group
Nematode body number of bends be 22.85 ± 3.12 times, 0.0001mM sufentanil processing group nematode body number of bends be 22 ±
3.19 times, 0.001mM sufentanil processing group nematode body number of bends is 21.45 ± 3.11 times, at 0.01mM sufentanil
Reason group nematode body number of bends is 21.4 ± 3.04 times, and 0.1mM sufentanil processing group nematode body number of bends is 18.75
± 2.93 times, 1.0mM sufentanil processing group nematode body number of bends is 17.95 ± 2.40 times.
As shown in fig. 7, the sufentanil of various concentration expresses F0, F1, F2 and F3 for Caenorhabditis elegans serotonin
Influence it is roughly the same, i.e., with sufentanil concentration increase, serotonin expression reduce.
In F0 generation, control group nematode serotonin relative expression quantity is the processing of 1.0 ± 0.14,0.00001nM sufentanil
Group nematode serotonin relative expression quantity is 0.90 ± 0.16,0.0001mM sufentanil processing group nematode serotonin with respect to table
It is 0.93 ± 0.11,0.001mM sufentanil processing group nematode serotonin relative expression quantity is 0.77 ± 0.17 up to amount,
0.01mM sufentanil processing group nematode serotonin relative expression quantity is 0.74 ± 0.11,0.1mM sufentanil processing group line
Worm serotonin relative expression quantity is that 0.76 ± 0.12,1.0mM sufentanil processing group nematode serotonin relative expression quantity is
0.74±0.13。
In F1 generation, control group nematode serotonin relative expression quantity is the processing of 1.02 ± 0.14,0.00001nM sufentanil
Group nematode serotonin relative expression quantity is 0.97 ± 0.13,0.0001mM sufentanil processing group nematode serotonin with respect to table
It is 1.0 ± 0.11,0.001mM sufentanil processing group nematode serotonin relative expression quantity is 0.94 ± 0.11 up to amount,
0.01mM sufentanil processing group nematode serotonin relative expression quantity is 0.84 ± 0.06,0.1mM sufentanil processing group line
Worm serotonin relative expression quantity is that 0.78 ± 0.13,1.0mM sufentanil processing group nematode serotonin relative expression quantity is
0.77±0.1。
In F2 generation, control group nematode serotonin relative expression quantity is the processing of 0.97 ± 0.14,0.00001nM sufentanil
Group nematode serotonin relative expression quantity is 1.03 ± 0.13,0.0001mM sufentanil processing group nematode serotonin with respect to table
It is 0.97 ± 0.15,0.001mM sufentanil processing group nematode serotonin relative expression quantity is 1.0 ± 0.14 up to amount,
0.01mM sufentanil processing group nematode serotonin relative expression quantity is 0.94 ± 0.12,0.1mM sufentanil processing group line
Worm serotonin relative expression quantity is that 0.84 ± 0.16,1.0mM sufentanil processing group nematode serotonin relative expression quantity is
0.83±0.13。
In F3 generation, control group nematode serotonin relative expression quantity is the processing of 1.0 ± 0.14,0.00001nM sufentanil
Group nematode serotonin relative expression quantity is 1.0 ± 0.14,0.0001mM sufentanil processing group nematode serotonin with respect to table
It is 0.97 ± 0.14,0.001mM sufentanil processing group nematode serotonin relative expression quantity is 0.94 ± 0.14 up to amount,
0.01mM sufentanil processing group nematode serotonin relative expression quantity is 0.94 ± 0.13,0.1mM sufentanil processing group line
Worm serotonin relative expression quantity is that 0.84 ± 0.16,1.0mM sufentanil processing group nematode serotonin relative expression quantity is
0.83±0.15。
To sum up, Caenorhabditis elegans sensitive can respond anaesthetic sufentanil, and neuromotor system is that anaesthetic is made
Target organ, Caenorhabditis elegans be can high flux screening and evaluation anaesthetic neurotoxicity effective carrier.
The above is only the preferred embodiment of the present invention, protection scope of the present invention is not limited merely to above-described embodiment,
It is within the scope of the invention with present inventive concept without the various process programs of substantial differences.
Claims (10)
1. a kind of construction method of Caenorhabditis elegans anaesthetic biological model, it is characterised in that: the following steps are included: by elegant
Beautiful hidden rhabditida is placed in the NGM culture medium I containing various concentration anaesthetic after culture, through cracking, centrifuged deposit, and will
Precipitating is plated on the NGM culture medium II containing anaesthetic after being resuspended, and is cultivated to first phase larva, that is, is established beautiful hidden bar line
Worm anaesthetic biological model.
2. the construction method of Caenorhabditis elegans anaesthetic biological model according to claim 1, it is characterised in that: institute
Stating anaesthetic includes sufentanil, Oxycodone, Remifentanil or Propofol.
3. the construction method of Caenorhabditis elegans anaesthetic biological model according to claim 1, it is characterised in that: institute
State the preparation method of anaesthetic are as follows: dissolve anaesthetic by solvent of LB liquid medium, prepare the arcotic of various concentration
Object.
4. using the Caenorhabditis elegans arcotic of construction method as claimed in any one of claims 1-3 building
Object biological model.
5. detecting the detection method of Caenorhabditis elegans anaesthetic biological model as claimed in claim 4, it is characterised in that:
The following steps are included:
(1) cut from culture medium II agar block containing first phase larva to number be seven culture dishes of A, B, C, D, E, F, G
In, it is grown after hatching to fourth phase larva, the nematode in culture dish is labeled as F0 for nematode at this time；
(2) F0 grows to the manhood for nematode, selects several into new culture dish, every other day transfers them in new culture dish,
Until oviposition terminates；
(3) F0 is observed and recorded during step (1) to (2) in A, B, C, D, E, F, G culture dish under stereomicroscope for line
The index of correlation of worm.
6. the detection method of Caenorhabditis elegans anaesthetic biological model according to claim 5, it is characterised in that: institute
F0 is stated for the index of correlation of nematode are as follows:
(1) F0 is measured for the influence of nematode reproductive system；
(2) F0 is measured for the influence of the motor behavior of nematode；
(3) F0 is measured for the influence of the growth and development of nematode；
(4) F0 is measured for the influence of the nervous function of nematode.
7. the detection method of Caenorhabditis elegans anaesthetic biological model according to claim 6, it is characterised in that: right
F0 is measured for what nematode reproductive system influenced the following steps are included: selecting the 20 beautiful hidden bar lines for only entering fourth phase larva 36h
Worm detects the laying in 2h and generation time into new culture dish；The generation time is F0 for nematode oviposition to its F1 generation
The time interval of Adult worms producting eggs.
8. the detection method of Caenorhabditis elegans anaesthetic biological model according to claim 6, it is characterised in that: right
F0 for the motor behavior of nematode measurement the following steps are included:
(1) frequency of head oscillation: the number of adult of the same period head oscillation in 1min is recorded；
(2) the curved frequency of body: adult of the same period curved number in 30s is recorded.
9. the detection method of Caenorhabditis elegans anaesthetic biological model according to claim 6, it is characterised in that: right
F0 for the growth and development of nematode measurement the following steps are included:
(1) service life: polypide is touched with syringe in the 0th day for being denoted as life starting with nematode egg hatching, reactionless to be considered as nematode life
The termination of life records the nematode service life, i.e., from starting to the number of days of termination；
(2) body is long: measurement into 4 period of larva 36h Caenorhabditis elegans and film-making, after microscope is taken pictures, calculate nematode from
The length of head to tail portion central axes.
10. the detection method of Caenorhabditis elegans anaesthetic biological model according to claim 6, it is characterised in that:
To F0 for the nervous function of nematode measurement the following steps are included: serotonin autofluorescence measurement: measurement enter the fourth phase
The simultaneously film-making of the Caenorhabditis elegans of larva 36h is taken pictures by fluorescence microscope and calculates the expression of Caenorhabditis elegans serotonin
It is horizontal.
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