CN115927198A - Drug screening model for inhibiting NAAA activity and construction method thereof - Google Patents
Drug screening model for inhibiting NAAA activity and construction method thereof Download PDFInfo
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Abstract
The invention relates to a drug screening model for inhibiting NAAA activity and a construction method thereof, wherein the drug screening model comprises HEK293T cells transfected by NAAA lentiviruses with reporter gene labels, a reporter group is an anti-biological resistance gene, a plasmid carrying the reporter gene is pLV-EF1-puro cs2.0-C-Flag, the position of an insert fragment is 3345bp-4451bp, and the insert fragment comprises SEQ ID NO:1.
Description
Technical Field
The invention relates to the field of drug screening, in particular to a drug screening model for inhibiting NAAA activity.
Background
Palmitoylethanolamide (PEA, N-hexadecanoylethanolamide) is a bioactive endogenous lipid mediator with pain relieving and anti-inflammatory effects. It can inhibit the release of inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta) and interleukin 6 (IL-6) by activating peroxisome proliferator-activated receptor alpha (PPAR-alpha), thereby achieving the purpose of anti-inflammation and analgesia. While the signaling activity of the PEA-PPAR-alpha pathway can be terminated by degradation of PEA by N-acylglycolaminic acid amidase (NAAA). NAAA is a cysteine hydrolase that is expressed at high levels in immune cells. The active pharmacological inhibitor thereof plays a far-reaching analgesic and anti-inflammatory role in rodent models. It follows that inhibitors of this enzyme are potential targets for therapeutic drug discovery.
N-acylethanolamine amidase (NAAA) is a specific hydrolase of PEA and catalyzes the degradation of the lipid-derived messenger Palmitoylethanolamide (PEA) to palmitic acid and ethanolamine. In the organism, NAAA is mainly distributed in organs such as lung, spleen, thymus and the like, and also distributed in a small amount in brain, small intestine and kidney, and has the highest expression in macrophages, monocytes, T cells, microglia and the like. Subsequent experiments showed that NAAA is highly expressed mainly in lysosomes.
NAAA is a cysteine hydrolase, belonging to the family of N-terminal nucleophilic hydrolases. It has no structural homology to FAAH, but has 35% sequence homology to acid ceramide hydrolase (ASAH 1), and is therefore classified as a cholylglyceridehydrolase (cholesylglycine hydrolase) family and is capable of selectively hydrolyzing amide bonds. NAAA has a 362 amino acid (rat and mouse) and 359 amino acid (human) sequence with molecular weights of 40.3kDa (rat) and 40.1kDa (mouse and human), respectively. Among these amino acid sequences, the homology between rat and mouse is 90.1%, the homology between rat and human is 76.5%, and the homology between mouse and human is 76.7%. Studies have shown that the entire protein structure of NAAA is an inactive precursor (complete form around 52 kDa) that needs to be activated by autocatalytic peptide bond cleavage to generate a heterodimer consisting of a β (35 kDa) subunit fragment and another α subunit fragment that remain physically linked. The self-activation reaction of NAAA renders the enzyme biologically active by exposing the active site (human Cys126, mouse and rat Cys 131) that catalyzes the nucleophile. To date, the crystal structure of NAAA has not been investigated and reported, and thus the spatial composition of NAAA-catalyzed hydrolysis is not fully understood. However, there is currently a presumption that there are four possible steps for the hydrolysis catalysis of NAAA: (1) Transfer of a proton from the thiol group of Cys126 to the alpha-amino group of the same residue, resulting in the formation of a sulfate anion; (2) Nucleophilic attack of sulfate on PEA carbonyl carbon and stable generation of oxyanions through hydrogen bonding with Glu195 and Asn 287; (3) Protonation dissociation of the ethanolamine moiety of PEA, with formation of an acylase intermediate; (4) regeneration of the initial enzyme. The research shows that the catalytic site mutation can cause the NAAA enzymatic hydrolysis activity to be reduced or even disappear. The glycosylation site plays an important role in the structural stability and activity of the enzyme.
The most well-known substrate, PEA, of NAAA is a member of the Fatty Acid Ethanolamide (FAE) lipid messenger family, which also includes N-arachidonoylethanolamide (AEA) and Oleoylethanolamide (OEA). PEA and OEA play a key role in controlling the cellular level of PEA by activating the peroxisome proliferator-activator receptor alpha (PPAR- α) to regulate energy balance, pain and inflammation NAAA. In healthy tissues, this lipid mediator is produced by the action of a structurally distinct phospholipase D (N-acylphosphatidylethanolamine phospholipase D, NAPE-PLD), which cleaves the glycerophospholipid precursor N-palmitoylphosphatidylethanolamine to produce FAEs and phosphatidic acid. Increased NAAA expression and activity will promote inflammation in two ways: (1) Blocking PEA-PPAR-alpha signal channel, eliminating PEA protection, and promoting inflammatory reaction. (2) The product of PEA hydrolysis product, namely palmitic acid, is increased, and the expression of a transcription coactivator PGC 1-alpha can be inhibited to promote inflammatory response. Therefore, by inhibiting the activity of NAAA, the degradation inactivation of PEA can be prevented, thereby playing the roles of analgesia and anti-inflammation.
The current reports of screening models for N-acylglycolamic acid amidase (NAAA) inhibitors are mainly TLC-based radioactive assays; the LC-MS method and the molecular-based fluorescence detection method are three methods, and the difference is mainly on the selection of a substrate and the detection method of a hydrolysate. (I) radioactive detection method based on TLC
The carbon atom radioactivity of the PEA substrate is labeled, after the reaction is finished, the PEA substrate is extracted and purified by ethyl acetate, methanol and citric acid, then the PEA substrate is spread on a silica gel plate, and the PEA substrate is scanned by an instrument for the change of the radioactivity after the reaction to screen the enzyme inhibitor. The method has large limitation on substrates and operation places, and relates to radioactivity, sample treatment is complicated, and sample screening is limited. And (II) an LC-MS method, wherein a fatty acid is used as an internal standard substance through liquid phase mass spectrum combination, and changes of the hydrolysate are detected so as to screen the enzyme inhibitor. The method has the disadvantages of certain requirements on sample treatment, complex operation and low efficiency. But the method has higher sensitivity and wide application. And (III) using a connecting product N- (4, methylcoumarin) -Palmitamide (PAMCA) of PEA and coumarin as a substrate by a molecular-based fluorescence detection method, wherein the substrate can generate 7-amino-4-methylcoumarin (AMC) with fluorescence property under the hydrolysis action of NAAA, and the enzyme inhibitor is screened by detecting the change of the fluorescence of a hydrolysate after the reaction. The stronger the enzyme inhibitor activity, the lower the fluorescence intensity of the hydrolysate. Compared with the former two methods, the method has the advantages of high screening speed and high flux. However, the extract of natural products is often complex in components or contains some fatty acids, so that the interference on the whole reaction system is large, false positive results are easy to generate, and the accuracy is low.
However, the existing screening models are not suitable for screening complex traditional Chinese medicine extracts, especially NAAA (ananas) inhibitory activity. Therefore, a NAAA inhibitor screening model suitable for a traditional Chinese medicine complex system needs to be established to accelerate the discovery of NAAA inhibitors from natural sources.
Disclosure of Invention
Therefore, the invention provides a drug screening model for inhibiting NAAA activity, which can solve the technical problem of accelerating the discovery of NAAA inhibitors from natural sources and screening the NAAA inhibitory activity by establishing an NAAA inhibitor screening model suitable for a complex system of traditional Chinese medicines.
To achieve the above objects, in one aspect, the present invention provides a drug screening model for inhibiting NAAA activity, comprising:
the drug screening model is HEK293T cells transfected by NAAA lentivirus with a reporter gene label.
Further, the reporter gene is an antibiotic resistance gene.
Furthermore, the plasmid carrying the reporter gene is pLV-EF1-puro cs2.0-C-Flag, wherein the position of the inserted fragment is 3345bp-4451bp.
Further, the insert comprises SEQ ID NO. 1.
Further, the insert fragment comprises an upstream vector SEQIDNO:2 and a downstream vector SEQIDNO:3, wherein the molar ratio of the insert fragment to the plasmid is 2:1-4:1.
on the other hand, the invention provides a method for constructing a drug screening model for inhibiting the activity of NAAA, which comprises the steps of S1, constructing LV239-pLV-EF1-puro cs2.0-C-Flag-hNAAA recombinant plasmid;
s2, culturing HEK293T cells, wherein in the culturing of the HEK293T cells, a central control unit adjusts the shaking frequency of a shaking mechanism for shaking the culture device in a stopping process according to the cell mass in a preset time after pancreatin digestive juice is added, and the blowing force and the blowing angle of a blowing mechanism for injecting liquid into the culture device are adjusted to enable the cell mass to meet the standard, wherein the shaking mechanism is arranged on a supporting plate and comprises a manipulator for grabbing a culture bottle and a first power mechanism for controlling the rotation frequency of the manipulator, the blowing mechanism is arranged on the supporting plate, and the blowing mechanism comprises a replaceable injector, a second power mechanism for controlling the blowing force of the liquid in the injector and a supporting mechanism for controlling the activity angle of the culture device so as to adjust the blowing angle;
s3, detecting the density of the cultured HEK293T cells, and if the cell density does not meet the standard, judging that the standard parameters of the cell quality and the blowing strength of the blowing mechanism are secondarily adjusted by the central control unit so as to enable the culture of the next HEK293T cell to meet the standard;
s3, injecting a mixed solution formed by mixing the prepared DNA solution and a transfection reagent into HEK293T cells for transfection, performing suction filtration on the transfected HEK293T cell supernatant to form a filtrate, performing concentration and chromatographic purification on the filtrate, and detecting titer;
and S4, adding an NAAA substrate-buffer solution into the transfected HEK293T cells, and determining and establishing an NAAA inhibitor screening model according to the fluorescence value of the hydrolysate.
Further, in the step S2, the central control unit determines the cell state S according to the cell roundness d after a preset time after adding the pancreatin digestive juice, sets S = (d 1+ d2+ \8230; dn)/n, where d1 is the first cell roundness, d2 is the second cell roundness \8230 \ dn is the nth cell roundness, and n is the total number of cells in the visual field, selects the cell evaluation parameter according to the comparison between the acquired cell state and the preset cell state S,
when S is less than or equal to S1, the central control unit selects a first preset evaluation parameter A1 as a cell evaluation parameter;
when S1 is larger than S and smaller than S2, the central control unit selects a second preset evaluation parameter A2 as a cell evaluation parameter;
when S is larger than or equal to S2, the central control unit selects a third preset evaluation parameter A3 as a cell evaluation parameter;
the central control unit presets a cell state S, sets a first preset cell state S1 and a second preset cell state S2, presets an evaluation parameter A1, sets a first preset evaluation parameter A1, a second preset evaluation parameter A2 and a third preset evaluation parameter A3.
Further, the central control unit determines a cell mass m according to the cell number n and cell evaluation parameters at a preset time after adding pancreatin digestive juice, sets m = n × Ai, compares the determined cell mass with a preset cell mass, determines whether to adjust the shaking frequency of the shaking mechanism and the blowing force and the blowing angle of the blowing mechanism during the suspension process, wherein,
when M is less than or equal to M1, the digestion time is prolonged by the central control unit;
when M1 is larger than M and smaller than M2, the central control unit improves the shaking frequency of the shaking mechanism, improves the beating force of the beating mechanism and improves the beating angle of the beating mechanism;
when M is larger than or equal to M2, the central control unit does not adjust the shaking frequency of the shaking mechanism, shakes the cell culture device at a preset shaking frequency, and reduces the blowing force of the blowing mechanism;
the central control unit presets a cell mass M, and sets a first preset cell mass M1 and a second preset cell mass M2.
Further, the supporting mechanism comprises a transverse actuator arranged on the supporting plate and a longitudinal actuator arranged on the transverse actuator and used for adjusting the supporting height of the cell culture device, when the supporting mechanism is started to support the cell culture device, the transverse supporting position of the cell culture device is adjusted through the transverse actuator, the longitudinal actuator adjusts the longitudinal position of the cell culture device to adjust the blow-beating angle of the blow-beating mechanism, the blow-beating angle W is preset by the central control unit, the regulated blow-beating angle is compared with the preset blow-beating angle by the central control unit, and the longitudinal position and the transverse position of the cell culture device are adjusted, wherein,
when theta 1 is less than or equal to W1, the central control unit does not adjust the longitudinal height and the transverse distance of the cell culture device;
when W1 is more than or equal to theta 1 and less than or equal to W2, the central control unit increases the longitudinal height of the cell culture device;
when theta 1 is larger than or equal to W2, the central control unit increases the longitudinal height of the cell culture device and increases the transverse distance;
the central control unit presets a blow-beating angle W, sets a first preset blow-beating angle W1 and a second preset blow-beating angle W2.
Further, in the step S3, the central control unit obtains the density k of the cultured cells, compares the density k with a preset cell density, adjusts the preset cell quality, selects an adjustment parameter to adjust the preset blow-beating force Q0 and the preset blow-beating angle θ 0 of the blow-beating mechanism, wherein,
when K is less than or equal to K1, the central control unit improves the quality of the preset cells, improves the preset blowing force and improves the preset blowing angle;
when K1 is more than K and less than K2, the central control unit judges that the relevant parameters are not adjusted;
when K is more than or equal to K2, the central control unit reduces the quality of the preset cells;
the central control unit presets a cell density K, and sets a first preset cell density K1 and a second preset cell density K2.
Compared with the prior art, the method has the beneficial effects that the current report on the screening model of the N-acylglycollic acid amidase (NAAA) inhibitor mainly comprises a radioactive detection method based on TLC; the LC-MS method and the molecular-based fluorescence detection method are mainly distinguished in the selection of a substrate and the detection method of a hydrolysate, but the models screened by the methods are not suitable for screening complex traditional Chinese medicine extracts and NAAA inhibitory activity, so that the establishment of a NAAA inhibitor screening model suitable for a complex system of traditional Chinese medicines can accelerate the discovery of NAAA inhibitors from natural sources. The invention provides a method for constructing a drug screening model for inhibiting NAAA activity, which comprises the steps of transfecting HEK293T cells with constructed LV239-plv-EF1-purocs2.0C-Flag-hNAAA lentivirus, screening by puromycin to obtain surviving positive cell clones, using the positive cell clones as an NAAA inhibition drug screening model after passage, and naming the NAAA inhibition drug screening model cells as HEK293-hNAAA inhibition agent screening model cells, and simultaneously adopting specific equipment to improve the process efficiency of cell digestion of cell culture so as to guarantee the cell culture quality and further screen out the NAAA inhibition agent with good quality.
Particularly, the cell state is parameterized according to the roundness of cells in a visual field, the cell state obtained through calculation is compared with preset cell state parameters, the evaluation parameters are selected as cell evaluation parameters for objectively determining the cell quality, the condition that the cell quality is not objectively evaluated due to human influence is avoided, the obtained cell quality is compared with the preset cell quality, whether the parameters of a shaking mechanism and a blowing mechanism are adjusted is determined, wherein if the cell quality is smaller than or equal to the first preset cell quality, the cell quality after current pancreatin digestion is not good and does not reach a stopping condition, a central control unit prolongs digestion time to wait for improving the cell quality, if the cell quality is between the first preset cell quality and a second preset cell quality, the cell quality after current pancreatin digestion is better, the central control unit improves the shaking frequency of the shaking mechanism, so that injected stopping liquid is fully contacted with adherent cells in a cell culture device, and improves the blowing force and the blowing angle of the blowing mechanism to fully break adherent cells to form single cells, the agglomeration is avoided, the subsequent influence is larger than the influence on the cell quality, and the cell quality is reduced if the cell quality is larger than the preset cell blowing control unit.
Particularly, the cell culture device is provided with a specific supporting mechanism, the transverse supporting position and the longitudinal supporting height of the cell culture device are adjusted through a transverse actuator and a longitudinal actuator on the supporting mechanism to control the inclination angle of the cell culture device, and the cell culture device is matched with a blowing and beating mechanism to realize adjustment of a blowing and beating angle, wherein if the adjusted blowing and beating angle is smaller than or equal to a first preset blowing and beating angle, the longitudinal supporting height and the transverse distance of the cell culture device are not adjusted, if the adjusted blowing and beating angle is between the first preset blowing and beating angle and a second preset blowing and beating angle, the central control unit only improves the blowing and beating angle of the cell culture device by improving the longitudinal height of the cell culture device, and if the adjusted blowing and beating angle is larger than or equal to the second preset blowing and beating angle, the central control unit simultaneously improves the longitudinal height and the transverse distance to greatly improve the blowing and beating angle.
Particularly, the invention divides the preset cell density into two standards, and compares the cell density after cell culture with the two standards of the preset cell density to judge whether the current cell culture meets the standards, wherein if the cell density after cell culture is less than or equal to the first preset cell density, the current cell culture does not meet the standards, the reason that the current cell culture is poor is that the cell quality parameters preset by the central control unit are too low, so the central control unit improves the parameters for evaluating the cell quality and simultaneously improves the preset blow-beating force and the blow-beating angle to break up more adherent cells, if the cell density after cell culture is between the first preset cell density and the second preset cell density, the current cell culture process meets the standards, and if the cell density after cell culture is greater than or equal to the second preset cell density, the current cell culture density is too high to be beneficial to subsequent operation, and the reason that the cell quality parameter standards are set to be too high, so the central control unit reduces the preset cell quality parameters to be matched with the cell culture density.
Drawings
FIG. 1 is a schematic structural diagram of a blow-beating device according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a rocking mechanism according to an embodiment of the present invention;
FIG. 3 is a schematic structural diagram of a blow striking mechanism and a supporting mechanism according to an embodiment of the invention;
FIG. 4 is a schematic diagram of a plasmid according to an embodiment of the present invention;
FIG. 5 is a graph showing the relationship between the number of cells and the degradation of a substrate by NAAA in the examples of the present invention;
FIG. 6 is a schematic diagram of high expression cells obtained by cell transfection and puromycin screening according to an embodiment of the present invention;
FIG. 7 is a graphical representation of the relationship between concentration of different substrates and inhibition of NAAA activity in an example of the invention; .
Detailed Description
In order that the objects and advantages of the invention will be more clearly understood, the invention is further described below with reference to examples; it should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only for explaining the technical principle of the present invention, and do not limit the scope of the present invention.
It should be noted that in the description of the present invention, the terms of direction or positional relationship indicated by the terms "upper", "lower", "left", "right", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, which are only for convenience of description, and do not indicate or imply that the device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.
Furthermore, it should be noted that, in the description of the present invention, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
Fig. 1 is a schematic view of a blow-beating device according to an embodiment of the present invention, including,
the shaking mechanism 3 is arranged on the supporting plate, is connected with one end of the cell culture device 4 and shakes the cell culture device to ensure that adherent cells are fully contacted with the injected liquid;
the blowing and beating mechanism 1 is arranged on the supporting plate, arranged at the other end of the cell culture device and used for blowing and beating adherent cells into the cell culture device through a suction pipe of the blowing and beating mechanism;
and the supporting mechanism 2 is arranged on the supporting plate and is used for adjusting the inclination angle of the cell culture device by changing the supporting position.
Please refer to fig. 2, which is a schematic structural diagram of a shaking mechanism according to an embodiment of the present invention, including a third motor 37 disposed on the support plate for controlling the telescopic length of a third telescopic rod 36, a fourth motor 33 disposed on the third telescopic rod for controlling the rotation speed of the manipulator, and a manipulator for gripping the cell culture device, where the manipulator includes a gear 35 for adjusting the rotation direction of the manipulator, a rotating shaft 32 for connecting the gripping portions, and a first gripping unit 31 and a second gripping unit 34 connected to the rotating shaft, and during use, the manipulator of the shaking mechanism grips one side of the cell culture device, preferably a bottle cap of the cell culture device, and when the central control unit starts the shaking mechanism to shake the cell culture device, the fourth motor controls the manipulator to rotate so as to achieve slow rotation of the cell culture device, so as to fully contact the liquid in the cell culture device with adherent cells.
Please refer to fig. 3, which is a schematic structural diagram of a beating mechanism and a supporting mechanism according to an embodiment of the present invention, wherein the beating mechanism includes a first motor 13 for controlling the expansion and contraction of a first telescopic rod 12, a connecting pipe 14 disposed on the first telescopic rod, a replaceable suction pipe 15 for drawing liquid, and a pump body 11 for controlling a suction force, in a use process, the liquid to be injected is injected into the cell culture device through the connecting pipe, when the central control unit starts the shaking device, the beating mechanism does not work, when the central control unit starts the beating mechanism to beat the parietal cells in the cell culture device, the pump body sucks the liquid in the cell culture device, and the beating position of the suction pipe in the cell culture device is controlled by adjusting the expansion and contraction length of the first telescopic rod, so as to achieve automatic beating of the parietal cells in the cell culture device; the supporting mechanism comprises a transverse slider and a longitudinal slider, the transverse slider comprises a first limiting block 21, a sliding rod, a first sleeve 22 arranged on the sliding rod and a second motor 23 used for controlling the moving position of the first sleeve on the sliding rod, the longitudinal slider comprises a second telescopic rod 25 and a supporting rod 24 connected with the second telescopic rod, in use, the cell culture device is placed on the supporting rod, the sleeve is controlled by the second motor to move on the sliding rod, the change of the transverse supporting point of the cell culture device is realized, and meanwhile, the change of the longitudinal supporting point of the cell culture device is realized through the adjustment of the height of the second telescopic rod.
Specifically, the blowing and beating device of the embodiment of the invention is applied to any operation steps requiring injection, blowing and beating, for example, in an injection operation, liquid is injected into a cell culture device through a connecting pipe and a suction pipe of a blowing and beating mechanism, a manipulator of the shaking mechanism grabs the cell culture device during shaking, and rotates the cell culture device under the driving of a fourth motor, in the embodiment of the invention, in order to achieve full contact between the injected liquid and cells adhered to the wall in the cell culture device, the shaking frequency of the shaking mechanism is kept low, wherein the manipulator drives the cell culture device to rotate within a unit time for 5-8 times/min, in the blowing and beating process, a pump body is started to suck the liquid in the cell culture device, the height of a first telescopic rod of the blowing and beating mechanism is changed, the pump body injects the sucked liquid into the cell culture device, the inner wall of the cell culture device is blown and beaten by the pipe of the cell culture device, so as to achieve scattering of the cells adhered to the cells in the cell culture device, the blowing and beating force is 6-15kpa, and the supporting mechanism adjusts the transverse distance and the longitudinal height while the blowing and beating mechanism adjusts the horizontal angle of the cells, so as to achieve the cell culture device and the cell culture device to achieve the cell culture device, wherein the cell culture device, and the cell culture device are formed by the cell culture device, and the cell culture device synchronously.
Specifically, the embodiment of the invention provides a method for constructing a drug screening model for inhibiting the activity of NAAA, which comprises the following steps,
s1, constructing an LV239-pLV-EF1-puro cs2.0-C-Flag-hNAAA recombinant plasmid;
s2, culturing HEK293T cells, wherein in the culturing of the HEK293T cells, a central control unit adjusts the shaking frequency of a shaking mechanism for shaking the culture device in a stopping process according to the cell mass in a preset time after pancreatin digestive juice is added, and the blowing force and the blowing angle of a blowing mechanism for injecting liquid into the culture device are adjusted to enable the cell mass to meet the standard, wherein the shaking mechanism is arranged on a supporting plate and comprises a manipulator for grabbing a culture bottle and a first power mechanism for controlling the rotation frequency of the manipulator, the blowing mechanism is arranged on the supporting plate, and the blowing mechanism comprises a replaceable injector, a second power mechanism for controlling the blowing force of the liquid in the injector and a supporting mechanism for controlling the activity angle of the culture device so as to adjust the blowing angle;
s3, detecting the density of the cultured HEK293T cells, and if the cell density does not meet the standard, judging that the standard parameters of the cell quality and the blowing strength of the blowing mechanism are secondarily adjusted by the central control unit so as to enable the culture of the next HEK293T cell to meet the standard;
s3, injecting a mixed solution formed by mixing the prepared DNA solution and a transfection reagent into HEK293T cells for transfection, performing suction filtration on the transfected HEK293T cell supernatant to form a filtrate, performing concentration and chromatographic purification on the filtrate, and detecting titer;
and S4, adding an NAAA substrate-buffer solution into the transfected HEK293T cells, and determining to establish an NAAA inhibitor screening model according to the fluorescence value of the hydrolysate.
The current reports of screening models for N-acylglycolamic acid amidase (NAAA) inhibitors are mainly TLC-based radioactive assays; the LC-MS method and the molecular-based fluorescence detection method are mainly distinguished in the selection of a substrate and the detection method of a hydrolysate, but the models screened by the methods are not suitable for screening complex traditional Chinese medicine extracts and NAAA inhibitory activity, so that the establishment of a NAAA inhibitor screening model suitable for a complex system of traditional Chinese medicines can accelerate the discovery of NAAA inhibitors from natural sources. The invention provides a construction method of a drug screening model for inhibiting NAAA activity, which comprises the steps of transfecting HEK293T cells by using constructed LV239-pLV-EF1-puro cs2.0-C-Flag-hNAAA lentiviruses, screening by puromycin to obtain surviving positive cell clones, carrying out passage to obtain an NAAA inhibition drug screening model, naming the NAAA inhibition drug screening model cells as HEK293-hNAAA inhibitor screening model cells, and simultaneously improving the process efficiency of cell digestion of cell culture by adopting specific equipment to guarantee the cell culture quality so as to ensure that an NAAA inhibitor with good quality is screened out.
In the step S2, the central control unit determines the cell state S according to the roundness d of each cell after a preset time after adding the pancreatin digestive juice, sets S = (d 1+ d2+ \8230; dn)/n, wherein d1 is the roundness of the first cell, d2 is the roundness of the second cell 8230; \8230;/dn is the roundness of the nth cell, and n is the total number of cells in the visual field, and selects the cell evaluation parameters according to the comparison between the acquired cell state and the preset cell state S,
when S is less than or equal to S1, the central control unit selects a first preset evaluation parameter A1 as a cell evaluation parameter;
when S1 is more than S and less than S2, the central control unit selects a second preset evaluation parameter A2 as a cell evaluation parameter;
when S is larger than or equal to S2, the central control unit selects a third preset evaluation parameter A3 as a cell evaluation parameter;
the central control unit presets a cell state S, sets a first preset cell state S1 and a second preset cell state S2, presets an evaluation parameter A1, sets a first preset evaluation parameter A1, a second preset evaluation parameter A2 and a third preset evaluation parameter A3.
The central control unit determines the cell mass m according to the cell number n and cell evaluation parameters within a preset time after adding pancreatin digestive juice, sets m = n multiplied by Ai, compares the determined cell mass with the preset cell mass, and determines whether to adjust the shaking frequency of the shaking mechanism and the blowing and beating force and angle of the blowing and beating mechanism in the suspension process, wherein,
when M is less than or equal to M1, the central control unit prolongs the digestion time t0 to t1, and t1= t0 x (1 + (M1-M)/M1) is set;
when M1 < M2, the central control unit increases the shaking frequency g0 to g1 of the shaking mechanism, sets g1= g0 × (1 +0.5 × (M2-M) × (M-M2)/(M1 × M2)), and increases the blow-beating force Q0 to Q1 of the blow-beating mechanism, sets Q1= Q0 × (1 +0.75 × (M2-M) × (M-M2)/(M1 × M2)), and simultaneously increases the blow-beating angle θ 0 to θ 1 of the blow-beating mechanism, sets θ 1= θ 0 × (1 = θ 0.45 × (M2-M) × (M-M2)/(M1 × M2));
when M is larger than or equal to M2, the central control unit does not adjust the shaking frequency of the shaking mechanism, shakes the cell culture device at a preset shaking frequency g0, reduces the blowing force Q0 to Q2 of the blowing mechanism, and sets Q2= Q0 x (1- (M-M2)/M2);
the central control unit presets a cell mass M, and sets a first preset cell mass M1 and a second preset cell mass M2.
Specifically, the cell state is parameterized according to the roundness of cells in a visual field, the cell state obtained through calculation is compared with preset cell state parameters, evaluation parameters are selected as the cell evaluation parameters to objectively determine the cell quality, the condition that the cell quality is not objectively evaluated due to human influence is avoided, the obtained cell quality is compared with the preset cell quality, whether the parameters of a shaking mechanism and a blowing mechanism are adjusted is determined, wherein if the cell quality is smaller than or equal to the first preset cell quality, the cell quality after current pancreatin digestion is not good and does not reach a stopping condition, a central control unit prolongs digestion time to wait for improving the cell quality, if the cell quality is between the first preset cell quality and a second preset cell quality, the cell quality after current pancreatin digestion is better, the central control unit improves the shaking frequency of the shaking mechanism, so that injected stopping liquid is fully contacted with adherent cells in a cell culture device, and improves the blowing force and the blowing angle of the blowing mechanism to fully break adherent cells to form single cells, the cluster is avoided, the subsequent influence is larger than the influence of cell quality, and the cell quality is reduced if the cell quality is larger than the preset cell blowing control mechanism, and the cell quality is reduced.
The supporting mechanism comprises a transverse actuator arranged on a supporting plate and a longitudinal actuator arranged on the transverse actuator and used for adjusting the supporting height of the cell culture device, when the supporting mechanism is started to support the cell culture device, the transverse supporting position of the cell culture device is adjusted through the transverse actuator, the longitudinal actuator adjusts the longitudinal position of the cell culture device so as to adjust the blow-beating angle of the blow-beating mechanism, the blow-beating angle W is preset by the central control unit, the adjusted blow-beating angle is compared with the preset blow-beating angle by the central control unit, and the longitudinal position and the transverse position of the cell culture device are adjusted, wherein,
when theta 1 is less than or equal to W1, the central control unit does not adjust the longitudinal height and the transverse distance of the cell culture device;
when W1 < θ 1 < W2, the central control unit raises the longitudinal height h0 to h1 of the cell culture device, setting h1= h0 × (1 + (W2- θ 1) × (θ 1-W1)/(W1 × W2));
when θ 1 is greater than or equal to W2, the central control unit raises the longitudinal height h0 to h2 of the cell culture device, sets h2= h0 x (1 + (θ 1-W2)/W2), and raises the transverse distance L to L1, sets L1= L x (1 +0.2 x (θ 1-W2)/W2);
the central control unit presets a blow-beating angle W, sets a first preset blow-beating angle W1 and a second preset blow-beating angle W2.
Specifically, the longitudinal height of the cell culture device in the embodiment of the present invention is the distance between the contact point of the longitudinal actuator and the cell culture device and the support plate, and the lateral distance is the distance between the lateral position of the cell culture device and the limit block of the lateral actuator, as shown in fig. 2, the longitudinal height is the distance between the point a and the support plate, and the lateral distance is the distance between the point B and the first limit block.
The cell culture device is provided with a specific supporting mechanism, the transverse supporting position and the longitudinal supporting height of the cell culture device are adjusted through a transverse actuator and a longitudinal actuator on the supporting mechanism to control the inclination angle of the cell culture device, and the cell culture device is matched with a blowing and beating mechanism to realize adjustment of a blowing and beating angle, wherein if the adjusted blowing and beating angle is smaller than or equal to a first preset blowing and beating angle, the longitudinal supporting height and the transverse distance of the cell culture device are not judged to be adjusted, if the adjusted blowing and beating angle is between the first preset blowing and beating angle and a second preset blowing and beating angle, a central control unit only improves the longitudinal height of the cell culture device to improve the blowing and beating angle, and if the adjusted blowing and beating angle is larger than or equal to the second preset blowing and beating angle, the central control unit simultaneously improves the longitudinal height and the transverse distance to greatly improve the blowing and beating angle.
Wherein, in the step S3, the central control unit obtains the density k of the cultured cells and compares the density k with the preset cell density, adjusts the preset cell quality, selects the adjusting parameters to adjust the preset blow-beating force Q0 and the preset blow-beating angle theta 0 of the blow-beating mechanism, wherein,
when K is less than or equal to K1, the central control unit increases preset cell masses Mj to Mj1, sets Mj1= Mj x (1 + (K1-K)/K1), simultaneously increases preset blow-off forces Q0 to Q01, sets Q01= Q0 x (1 +0.5 x (K1-K)/K1), simultaneously increases preset blow-off angles θ 0 to θ 01, sets θ 01= θ 0 x (1 +0.2 x (K1-K)/K1);
when K1 is more than K and less than K2, the central control unit judges that the relevant parameters are not adjusted;
when K is larger than or equal to K2, the central control unit reduces the preset cell mass Mj to Mj2, and Mj2= Mj x (1-0.75 x (K-K2)/K2) is set;
the central control unit presets a cell density K, sets a first preset cell density K1, and sets a second preset cell density K2, j =1,2.
Specifically, the preset cell density is divided into two standards, and whether the current cell culture meets the two standards is determined by comparing the cell density after the cell culture with the two standards of the preset cell density, wherein if the cell density after the cell culture is smaller than or equal to the first preset cell density, the current cell culture does not meet the standards, and the reason that the current cell culture is poor is that the preset cell quality parameters of the central control unit are too low, so that the central control unit improves the parameters for evaluating the cell quality and simultaneously improves the preset blow-beating force and the blow-beating angle to break up more adherent cells, if the cell density after the cell culture is between the first preset cell density and the second preset cell density, the current cell culture process meets the standards, and if the cell density after the cell culture is larger than or equal to the second preset cell density, the current cell culture density is too high, which is not beneficial to subsequent operations, and the reason that the current situation is that the cell quality parameter standards are set too high, the preset cell quality parameters are reduced by the central control unit to be matched with the cell culture density.
Please continue to refer to fig. 4, which is a schematic diagram of a plasmid according to an embodiment of the present invention, wherein the reporter gene is an antibiotic resistance gene, the embodiment of the present invention employs a puromycin resistance gene (puro), wherein the position of the insert fragment is 3345bp-4451bp, the gene sequence of the insert fragment includes seq id No. 1, the insert fragment further includes an upstream vector seq id No. 2 and a downstream vector seq id No. 3, and the insert fragment is circular DNA of hnaa.
Specifically, the molar ratio of the hNAAA circular DNA to the plasmid 194plv-EF1-purocs2.0C-Flag is 2:1-4:1; the molar ratio of the hNAAA circular DNA to the plasmid plv-EF1-purocs2.0C-Flag is 2:1-4:1.
in particular, the present invention provides a preferred embodiment, a method for constructing a drug screening model for inhibiting NAAA activity, comprising,
step S1, constructing NAAA expression cells;
the step S1 comprises a step S11 of culturing HEK293T cells, and the step S11 comprises a step S111 of constructing LV239-plv-EF1-purocs2.0C-Flag-hNAAA recombinant plasmids; step S112, placing the culture device of the HEK293T cells in the logarithmic growth phase on a supporting device, removing the culture solution in the culture device, injecting PBS buffer solution, and starting a shaking mechanism to shake the culture device; step S113, adding pancreatin digestive juice into the culture device, taking a quantitative culture solution to detect the cell state after a preset time, determining the shaking frequency of the shaking mechanism, the blowing force and the blowing angle of the blowing mechanism according to the cell number and the state (shrinking and rounding), adjusting (avoiding poor cell state caused by prolonged time and improving the suspension of the next step), adding a stop solution into the culture device, and starting the shaking mechanism and the blowing mechanism according to the determined shaking frequency, blowing force and blowing angle to enable the stop solution to be fully contacted with cells in the culture device so as to break up adherent cells;
step S114, collecting cell suspension in the cell culture device, injecting the cell suspension into a centrifugal tube for centrifugation, and counting;
step S115, removing supernatant in the cell culture device, adding a culture medium into the cell culture device, placing the cell culture device on a support rod, and starting a blowing mechanism to resuspend cells in the cell culture device;
step S116, inoculating the resuspended cells on a culture plate, culturing for a certain time, detecting the cell density, and if the cell density does not meet the standard, judging that the secondary adjustment is carried out on the standard parameters of the cell quality and the blowing and beating force of a blowing and beating mechanism by a central control unit;
step S117, when the density of the cultured cells reaches the standard, replacing the culture medium 1h before transfection, preparing a DNA solution, and uniformly mixing the DNA solution with a transfection reagent with a corresponding volume to form a mixed solution, wherein the ratio of pMD to VSVG to Rev = 5;
step S12, carrying out transfection on cultured HEK293T cells, wherein the step S12 comprises the step S121 of slowly dripping the mixed solution prepared in the step S117 into the culture solution of the HEK293T cells, uniformly mixing, and carrying out 5-percent CO conversion at 37 DEG C 2 Culturing in a cell culture box, removing a culture medium containing the transfection mixture after culturing for 12h, adding 1mL of PBS (phosphate buffer solution) for cleaning once, slightly shaking a culture dish to wash residual transfection mixture, and then pouring out;
step S122, slowly adding 3mL of cell culture medium containing 10% of serum, continuously culturing for 48-72h in a 5-percent CO2 incubator at 37 ℃, respectively collecting HEK293T cell supernatants of 48h and 72h (the transfection content is 0 h) after transfection according to the cell state, and storing at 4 ℃;
step S123, transferring the collected supernatant to a 0.22 μm filter for suction filtration to remove cell debris and the like; enabling the collected sample to flow through a tangential flow filtration system, concentrating viruses, simultaneously removing DNA and protein residues to the maximum extent, further purifying the sample by using an AKTA anion-cation chromatography system, collecting the sample, and storing at 4 ℃; loading the recovered virus sample into an ultrafiltration concentration tube, centrifuging at 5500rpm at 4 ℃, and adjusting the time of each round of centrifugation according to the concentration speed of the venom until the virus sample is concentrated to a target volume; collecting the concentrated virus liquid in a 1.5mL centrifuge tube, and centrifuging for 5min at 11000 rpm; the supernatant was aspirated through a 2mL syringe, purified through a 0.22 μm filter (PVDF), and the titer was detected.
S2, establishing an NAAA inhibitor screening model;
step S21, 3-10 ten thousand/well HEK293-hNAAA (DMEM +10% FBS +1% P-S +2ug/ml puromycin) cells were seeded in 96-well plates at 37 ℃ and 5% CO 2 Cultured overnight in the medium.
In step S22, samples (0.1 ml per well) were added at different concentrations, respectively, and a control group was established.
Step S23, after the medicine acts for 12-24 hours, the culture medium is discarded, and PBS is used for washing once; adding 0.1% TritonX100-PBS solution into each well, shaking for 5min, and freezing at-80 deg.C for 1 hr;
s24, after the mixture is rapidly melted at 37 ℃, the mixture is frozen and stored for 1 hour at minus 80 ℃;
step S25, preparing NAAA substrate-buffer solution (the NAAA substrate is N- (4-)
Methlcoumarin) -Palmitamide (PAMCA); the final concentration was 10uM, and the temperature was preheated at 37 ℃. )
And S26, taking out the cells, rapidly melting at 37 ℃, continuously incubating to 37 ℃, and rapidly adding 150uL of preheated NAAA substrate-buffer solution. After stable shaking, incubating for 2h at 37 ℃;
step S27, transferring 100ul of the hydrolysate into a 96-hole opaque black enzyme label plate, and measuring the fluorescence value of the hydrolysate by using an enzyme label instrument (wherein the Excitation is 355 nm/the emulsion is 460 nm);
please refer to fig. 5, which is a diagram illustrating the relationship between the number of cells and the degradation of NAAA to the substrate in the examples of the present invention, which shows that the degradation of NAAA to the substrate is proportional to the number of cells and has a very good correlation.
Please refer to fig. 6, which is a schematic diagram of high-expression cells obtained by cell transfection and puromycin screening according to the embodiment of the present invention, and the embodiment of the present invention verifies that the constructed NAAA inhibitor screening model can screen out high-expression NAAA inhibitors.
S3, verifying a NAAA inhibitor screening model;
step S31, 5 ten thousand/well of HEK293-hNAAA (DMEM +10% FBS +1% P-S +2ug/ml puromycin) cells were seeded into 96 well plates and incubated overnight at 37 ℃ in 5% CO2.
Step S32, adding 1.25-20 mu M positive samples F96 and AM9053 (0.1 ml per well) respectively, and setting up a control group.
Step S33, removing the culture medium after the medicine acts for 24 hours, and washing with PBS once; adding 0.1% TritonX100-PBS solution 150ul per well, shaking for 5min, and freezing at-80 deg.C for 1h;
step S34, after the mixture is rapidly melted at 37 ℃, the mixture is frozen and stored for 1 hour at minus 80 ℃;
step S35, taking out the cells, rapidly melting at 37 ℃, then continuously incubating to 37 ℃, rapidly adding 150uL of preheated NAAA substrate-buffer solution (N- (4-Methylocumarin) -palmitamide with the final concentration of 10 uM), stably shaking uniformly, and then incubating for 2h at 37 ℃;
step S36, transferring 100ul of the sample into a 96-hole opaque black enzyme label plate, and measuring the fluorescence value (Excitation: 355 nm/emulsion: 460 nm) of a hydrolysate by using an enzyme label analyzer, wherein the NAAA inhibition activity is calculated by the inhibition rate% = [100- (administration group-BK value)/(Vehicle group-BK value) ]%.
FIG. 7 is a graph showing the relationship between the concentration of various substrates and the inhibition rate of NAAA activity according to the present invention.
See Table I for the inhibition of NAAA at various concentrations administered
So far, the technical solutions of the present invention have been described in connection with the preferred embodiments shown in the drawings, but it is easily understood by those skilled in the art that the scope of the present invention is obviously not limited to these specific embodiments. Equivalent changes or substitutions of related technical features can be made by those skilled in the art without departing from the principle of the invention, and the technical scheme after the changes or substitutions can fall into the protection scope of the invention.
Claims (10)
1. A drug screening model for inhibiting NAAA activity is characterized in that the drug screening model is HEK293T cells transfected by NAAA lentiviruses with reporter gene labels.
2. The drug screening model for inhibiting NAAA activity of claim 1, wherein the reporter gene is an antibiotic resistance gene.
3. The model of claim 2, wherein the reporter gene-carrying plasmid is pLV-EF1-puro cs2.0-C-Flag, and the insertion fragment is 3345bp-4451bp.
4. The drug screening model for inhibiting NAAA activity of claim 3, wherein the insert comprises SEQ ID No. 1.
5. The model for screening drugs that inhibit NAAA activity of claim 2, wherein the insert comprises an upstream vector SEQ ID NO:2 and a downstream vector SEQ ID NO:3, wherein the molar ratio of insert to plasmid is 2:1-4:1.
6. a method for constructing a drug screening model for inhibiting the activity of NAAA, the drug screening model adopts the drug screening model for inhibiting the activity of NAAA in claims 1-5,
s1, constructing an LV239-pLV-EF1-puro cs2.0-C-Flag-hNAAA recombinant plasmid;
s2, culturing HEK293T cells, wherein in the culturing of the HEK293T cells, a central control unit adjusts the shaking frequency of a shaking mechanism for shaking the culture device in a stopping process according to the cell mass in a preset time after pancreatin digestive juice is added, and the blowing force and the blowing angle of a blowing mechanism for injecting liquid into the culture device are adjusted to enable the cell mass to meet the standard, wherein the shaking mechanism is arranged on a supporting plate and comprises a manipulator for grabbing a culture bottle and a first power mechanism for controlling the rotation frequency of the manipulator, the blowing mechanism is arranged on the supporting plate, and the blowing mechanism comprises a replaceable injector, a second power mechanism for controlling the blowing force of the liquid in the injector and a supporting mechanism for controlling the activity angle of the culture device so as to adjust the blowing angle;
s3, detecting the density of the cultured HEK293T cells, and if the cell density does not meet the standard, judging that the standard parameters of the cell quality and the blowing strength of the blowing mechanism are secondarily adjusted by the central control unit so as to enable the culture of the next HEK293T cell to meet the standard;
s3, injecting a mixed solution formed by mixing the prepared DNA solution and a transfection reagent into HEK293T cells for transfection, performing suction filtration on the transfected HEK293T cell supernatant to form a filtrate, performing concentration and chromatographic purification on the filtrate, and detecting titer;
and S4, adding an NAAA substrate-buffer solution into the transfected HEK293T cells, and determining to establish an NAAA inhibitor screening model according to the fluorescence value of the hydrolysate.
7. The method for constructing the drug screening model for inhibiting the NAAA activity according to claim 6, wherein in the step S2, the central control unit determines the cell state S according to the roundness d of each cell after a preset time after adding pancreatin digestive juice, sets S = (d 1+ d2+ \8230; dn)/n, wherein d1 is the first cell roundness, d2 is the second cell roundness \8230; \8230, dn is the nth cell roundness, and n is the total number of cells in the visual field, and selects the cell evaluation parameter according to the comparison between the acquired cell state and the preset cell state S, wherein when S is less than or equal to S1, the central control unit selects the first preset evaluation parameter A1 as the cell evaluation parameter;
when S1 is more than S and less than S2, the central control unit selects a second preset evaluation parameter A2 as a cell evaluation parameter;
when S is larger than or equal to S2, the central control unit selects a third preset evaluation parameter A3 as a cell evaluation parameter;
the central control unit presets a cell state S, sets a first preset cell state S1 and a second preset cell state S2, presets an evaluation parameter A1, and sets a first preset evaluation parameter A1, a second preset evaluation parameter A2 and a third preset evaluation parameter A3.
8. The method of claim 5, wherein the central control unit determines the cell mass m according to the cell number n and the cell evaluation parameter at a predetermined time after the pancreatin digestive juice is added, sets m = n × Ai, compares the determined cell mass with a predetermined cell mass, and determines whether to adjust the shaking frequency of the shaking mechanism and the force and angle of the blow-beating mechanism during the suspension process, wherein,
when M is less than or equal to M1, the digestion time is prolonged by the central control unit;
when M1 is larger than M and smaller than M2, the central control unit improves the shaking frequency of the shaking mechanism, improves the beating force of the beating mechanism and improves the beating angle of the beating mechanism;
when M is larger than or equal to M2, the central control unit does not adjust the shaking frequency of the shaking mechanism, shakes the cell culture device at a preset shaking frequency, and reduces the blowing force of the blowing mechanism;
the central control unit presets a cell mass M, and sets a first preset cell mass M1 and a second preset cell mass M2.
9. The method of claim 8, wherein the support mechanism comprises a lateral actuator disposed on the support plate, and a longitudinal actuator disposed on the lateral actuator for adjusting the support height of the cell culture device, when the support mechanism is activated to support the cell culture device, the lateral support position of the cell culture device is adjusted by the lateral actuator, the longitudinal actuator adjusts the longitudinal position of the cell culture device to adjust the blow-beating angle of the blow-beating mechanism, the central control unit presets the blow-beating angle W, the central control unit compares the adjusted blow-beating angle with the preset blow-beating angle, and adjusts the longitudinal position and the lateral position of the cell culture device, wherein,
when theta 1 is less than or equal to W1, the central control unit does not adjust the longitudinal height and the transverse distance of the cell culture device;
when W1 is more than theta 1 and less than W2, the central control unit increases the longitudinal height of the cell culture device;
when theta 1 is larger than or equal to W2, the central control unit increases the longitudinal height of the cell culture device and increases the transverse distance;
the central control unit presets a blow-beating angle W, a first preset blow-beating angle W1 and a second preset blow-beating angle W2.
10. The method for constructing a drug screening model for inhibiting NAAA activity of claim 9, wherein in step S3, the central control unit obtains the cell density k after culture, compares the cell density k with a preset cell density, adjusts the preset cell quality, selects an adjusting parameter to adjust the preset blowing and striking force Q0 and the preset blowing and striking angle theta 0 of the blowing and striking mechanism, wherein,
when K is less than or equal to K1, the central control unit improves the quality of the preset cells, improves the preset blowing force and improves the preset blowing angle;
when K1 is more than K and less than K2, the central control unit judges that the relevant parameters are not adjusted;
when K is more than or equal to K2, the central control unit reduces the quality of the preset cells;
the central control unit presets a cell density K, and sets a first preset cell density K1 and a second preset cell density K2.
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