CN110878330B - Application of human embryo kidney cell strain in drug screening - Google Patents
Application of human embryo kidney cell strain in drug screening Download PDFInfo
- Publication number
- CN110878330B CN110878330B CN201911296185.4A CN201911296185A CN110878330B CN 110878330 B CN110878330 B CN 110878330B CN 201911296185 A CN201911296185 A CN 201911296185A CN 110878330 B CN110878330 B CN 110878330B
- Authority
- CN
- China
- Prior art keywords
- human embryonic
- embryonic kidney
- kidney cell
- cell line
- drug screening
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Urology & Nephrology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Engineering & Computer Science (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Developmental Biology & Embryology (AREA)
- Gynecology & Obstetrics (AREA)
- Reproductive Health (AREA)
- Pregnancy & Childbirth (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an application of a human embryonic kidney cell line in drug screening, the human embryonic kidney cell line is used for drug screening in neuropathic pain and cerebral ischemia caused by hypertension and apoplexy, the human embryonic kidney cell line is a human embryonic kidney cell line HEK 293T-NMDAR-alpha 2 delta 1, which is preserved in the China center for type culture collection, and the preservation address is as follows: wuhan university collection center, accession number: CCTCC NO of C2019189, preservation date: 09 and 05 months in 2019. The human embryonic kidney cell line can stably express three genes of CACNA2D1, GRIN1 and GRIN2B at the same time, and the medicament has an inhibiting effect on current formed by the alpha 2 delta-1-NMDAR. The screening method is simple, the alpha 2 delta-1-NMDAR compound is used as an action target point for drug screening, the current is used as an index for drug screening, a basis is provided for treatment of neuropathic pain and cerebral ischemia, and wide application and research of the alpha 2 delta-1-NMDAR compound in drug screening are promoted.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to application of a human embryonic kidney cell strain in drug screening.
Background
α 2 δ is an accessory subunit of voltage-gated calcium channels (VGCC) CaV1 and CaV2 (i.e., L, P/Q, N and R-type), which subunit (Cav α 2 δ) is composed of a single geneCacna2dEncoded, homologous genes have been found to encode four subtypes of the α 2 δ subunit (α 2 δ -1 to α 2 δ -4). α 2 δ -1 was first identified and was initially isolated in skeletal muscle along with calcium channel complexes, and subsequent studies found α 2 δ -1 to be ubiquitous in cardiac and smooth muscle as well as central and peripheral nervous system and secretory tissues, predominantly at axonal terminals of excitatory neurons.
Numerous studies have consistently shown that peripheral nerve injury leads to upregulation of α 2 δ -1 expression in sensory neurons and to increased transport of α 2 δ -1 to presynaptic terminals in the spinal cord and dorsal root ganglia; mice over-expressing α 2 δ -1 in the absence of nerve injury exhibit a neurogenic phenotype (tactile abnormalities and hyperalgesia), indicating that α 2 δ -1 has a modulatory effect on excitability of dorsal root neurons; in accordance with this, it is possible to,cacna2d1the knockout mice have reduced sensitivity to mechanical stimulation after peripheral nerve injury, and show phenotypes such as delayed onset of neurogenic mechanical hypersensitivity.
GRIN1 and GRIN2B are NMDAR receptor genes encoding GluN1 and GluN2B methylene groups, respectively, of an NMDAR receptor. α 2 δ -1 interacts with the methylene groups of NMDAR (GluN 1 and GluN 2B) through its C-terminus to form a heteromeric complex (α 2 δ -1-NMDAR) that facilitates cell surface transport and localization on the synaptic membrane of NMDAR. Increased α 2 δ -1 expression in nerve-ligated rat spinal cord, triggering increased NMDA receptor (NMDAR) current; overexpression of Cacna2d1 enhanced presynaptic and postsynaptic NMDAR activity of spinal cord dorsal horn neurons, leading to hyperalgesia. On the contrary, the present invention is not limited to the above-described embodiments,Cacna2d1knockdown or disruption normalizes synaptic NMDAR activity with increased nerve damage. Nerve damage enhances the activity of NMDAR at synapses, while gabapentin or C-terminal interfering peptides of α 2 δ -1 restore the activity of NMDAR at synapses to normal. Thus, α 2 δ -1 is a protein that interacts with NMDAR, triggering an increase in NMDAR current, enhancing processes in spinal dorsal horn neuronsPre-and post-synaptic NMDAR activity, increases the dynamic distribution of NMDAR in neuropathic pain at synapses, leading to hyperalgesia.
The alpha 2 delta-1-NMDAR compound intervenes in a plurality of pathological mechanisms and plays an important role in neuropathic pain diseases, but at present, no cell strain can form the alpha 2 delta-1-NMDAR compound, no tool cell which takes the alpha 2 delta-1-NMDAR compound as an action target is used for drug screening research, and the application and the research of the alpha 2 delta-1-NMDAR compound in the drug screening are limited.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: overcomes the defects of the prior art and provides an application of a human embryonic kidney cell strain in drug screening.
The technical scheme adopted by the invention for solving the technical problem is as follows: an application of a human embryonic kidney cell line in drug screening, wherein the human embryonic kidney cell line is used for drug screening in neuropathic pain and cerebral ischemia.
Further, the cerebral ischemia is caused by hypertension and stroke.
Further, the human embryonic kidney cell line is a human embryonic kidney cell line HEK 293T-NMDAR-alpha 2 delta 1, which is preserved in China center for type culture Collection with the preservation address: wuhan university collection center, accession number: CCTCC NO of C2019189, preservation date: 09 and 05 months in 2019.
Furthermore, the human embryonic kidney cell line can stably express three genes of CACNA2D1, GRIN1 and GRIN2B at the same time, and can form an alpha 2 delta-1-NMDAR compound under a simulated pathological condition, wherein the gene sequence of the CACNA2D1 is shown as SEQ ID No.1, the gene sequence of the GRIN1 is shown as SEQ ID No.2, and the gene sequence of the GRIN2B is shown as SEQ ID No. 3.
Further, the drug is a substance having an inhibitory effect on the current formed by the α 2 δ -1-NMDAR.
The drug screening comprises the following steps:
a) culturing a human embryonic kidney cell strain: the method comprises the following steps: a1) recovering and screening; a2) liquid changing; a3) passage; a4) freezing and storing;
b) drug screening: and detecting the current by adopting a patch clamp whole cell recording technology to screen the medicament.
Further, the specific operation steps of the step a1) are as follows: taking out the cryopreserved human embryonic kidney cell strain, rapidly shaking and thawing in a 37 ℃ water bath tank, transferring the cell sap into a centrifuge tube, adding the inhibitor MK-801 and MgCl2The glutamic acid-free DMEM is evenly mixed and centrifuged, the supernatant is poured off, the cells are blown into single cell suspension by the culture medium, the single cell suspension is transferred into a culture bottle and placed at 37 ℃ and 5% CO2Culturing in a constant temperature incubator until the cells adhere to the wall, and adding screening markers Puro, Hygro and Bsd for screening after about 5-6 h.
Further, the specific operation steps of the step a 3) are as follows: when the cell fusion degree reaches 85% -90%, passaging can be carried out, the original culture medium is poured off, the cells are washed by PBS and then digested by pancreatin, when the cell gap is widened and the cells become round and bright, the inhibitor containing AP5, KA and MgCl is added immediately2The glutamic acid-free DMEM is used for stopping digestion, is beaten and evenly mixed, is centrifuged, the supernatant is poured off, the cells are beaten into single cell suspension by the culture medium, and are transferred into a culture bottle and placed at 37 ℃ and 5% CO 2Culturing in a constant temperature incubator.
Further, the specific operation steps of the step a 4) are as follows: washing cells in logarithmic phase with PBS, digesting with 0.25% pancreatin, when cell gaps widen and cells become round and bright, immediately adding a primary screening culture medium to terminate digestion, blowing a culture dish by a suction pipe to make the cells shed, transferring the cells into a centrifuge tube to centrifuge, pouring off supernatant, resuspending the cell sediment by using a prepared frozen stock solution FBS (DMSO): =9:1, adjusting cell density, subpackaging the cell sediment into a freezing tube, performing gradient cooling and freezing for 24 h, and freezing and storing the cell sediment in a liquid nitrogen tank for later use.
Further, the specific operation steps of the step b) are as follows: whole cell recording was performed at 25 ℃, cell supernatant was slowly poured out, extracellular fluid containing the drug to be selected at different concentrations was added, and the intracellular fluid was used in combination to observe the change in cell current. The application of the human embryonic kidney cell strain in drug screening has the beneficial effects that: the screening method is simple, the alpha 2 delta-1-NMDAR compound is used as an action target point for drug screening, the current is used as an index for drug screening, a basis is provided for treatment of neuropathic pain and cerebral ischemia, and wide application and research of the alpha 2 delta-1-NMDAR compound in drug screening are promoted.
Drawings
FIG. 1 is a schematic diagram of cell microscopic imaging of human embryonic kidney cell screening.
Detailed Description
The invention is further illustrated with reference to the following figures and examples, which are not intended to limit the scope of the invention in any way.
Example 1
The application of a human embryonic kidney cell line in drug screening is used for drug screening of neuropathic pain, the human embryonic kidney cell line is a human embryonic kidney cell line HEK 293T-NMDAR-alpha 2 delta 1, and the human embryonic kidney cell line is preserved in the China center for type culture Collection with the preservation address: wuhan university collection center, accession number: CCTCC NO of C2019189, preservation date: in 2019, 09 and 05, the human embryonic kidney cell line can simultaneously and stably express three genes of CACNA2D1, GRIN1 and GRIN2B, and can form an alpha 2 delta-1-NMDAR compound under simulated pathological conditions, the gene sequence of the CACNA2D1 is shown as SEQ ID No.1, the gene sequence of the GRIN1 is shown as SEQ ID No.2, the gene sequence of the GRIN2B is shown as SEQ ID No.3, and the medicine is a substance which has an inhibiting effect on the current formed by the alpha 2 delta-1-NMDAR.
Example 2
The application of a human embryonic kidney cell line in drug screening is used for drug screening of cerebral ischemia caused by hypertension, the human embryonic kidney cell line is a human embryonic kidney cell line HEK 293T-NMDAR-alpha 2 delta 1, and the human embryonic kidney cell line is preserved in the China center for type culture Collection with the preservation address: wuhan university collection center, accession number: CCTCC NO of C2019189, preservation date: in 2019, 09 and 05, the human embryonic kidney cell line can simultaneously and stably express three genes of CACNA2D1, GRIN1 and GRIN2B, and can form an alpha 2 delta-1-NMDAR compound under simulated pathological conditions, the gene sequence of the CACNA2D1 is shown as SEQ ID No.1, the gene sequence of the GRIN1 is shown as SEQ ID No.2, the gene sequence of the GRIN2B is shown as SEQ ID No.3, and the medicine is a substance which has an inhibiting effect on the current formed by the alpha 2 delta-1-NMDAR.
Example 3
The application of a human embryonic kidney cell line in drug screening is used for drug screening of cerebral ischemia caused by stroke, the human embryonic kidney cell line is a human embryonic kidney cell line HEK 293T-NMDAR-alpha 2 delta 1, which is preserved in the China center for type culture Collection with the preservation address: wuhan university collection center, accession number: CCTCC NO of C2019189, preservation date: in 2019, 09 and 05, the human embryonic kidney cell line can simultaneously and stably express three genes of CACNA2D1, GRIN1 and GRIN2B, and can form an alpha 2 delta-1-NMDAR compound under simulated pathological conditions, the gene sequence of the CACNA2D1 is shown as SEQ ID No.1, the gene sequence of the GRIN1 is shown as SEQ ID No.2, the gene sequence of the GRIN2B is shown as SEQ ID No.3, and the medicine is a substance which has an inhibiting effect on the current formed by the alpha 2 delta-1-NMDAR.
Example 4
The method for screening the drug from the human embryonic kidney cell strain specifically comprises two steps of culturing the human embryonic kidney cell strain and screening the drug, wherein the culturing of the human embryonic kidney cell strain specifically comprises the following steps:
(1) recovering and screening: taking out the cryopreserved human embryonic kidney cell strain, rapidly shaking and thawing in a constant temperature water bath box at 37 ℃, transferring the cell sap into a centrifuge tube in a super clean bench, adding inhibitors MK-801 and MgCl 2The glutamic acid-free DMEM is evenly mixed and then centrifuged for 5 min at 1000 rpm, the supernatant is poured off, the cells are blown into single cell suspension by the culture medium, the single cell suspension is transferred into a culture bottle and then placed at 37 ℃ and 5% CO2Culturing in a constant temperature incubator until the cells adhere to the wall, adding screening markers Puro, Hygro and Bsd for screening after about 5-6 hours, and imaging a cell microscope image in the screening; the cell microscopic image is shown in FIG. 1, and the white area in the right image of FIG. 1 is green fluorescence, and it can be seen from FIG. 1 that the human embryonic kidney cell line was successfully screened.
(2) Liquid changing: changing the culture medium every 24 h, and continuing culturing;
(3) passage: when the cell fusion degree reaches 85% -90%, passage can be carried out, the original culture medium is poured off, the cells are washed for 2-3 times by PBS, 0.25% pancreatin is used for digestion, when the cell gap is widened and the cells become round and bright, the cell culture medium containing inhibitors AP5, KA and MgCl is immediately added2The glutamic acid-free DMEM is used for stopping digestion, is beaten and evenly mixed, is centrifuged for 5 min at 1000 rpm, is poured out of supernatant, is used for beating cells into single cell suspension, is transferred into a culture bottle and is placed at 37 ℃ and 5% CO2Culturing in a constant-temperature incubator;
(4) cells in logarithmic growth phase were washed with PBS and digested with pancreatin, as the cell space widened and the cells became round and bright, primary selection medium (DMEM 27 ml, FBS 3 ml, 300 ul diabesin, MK-8011.5 ul, MgCl) was added immediately 2300ul, Puro 4.8ul, Hygro 72 ul, Bsd 6 ul) to stop digestion, transferring the cells into a centrifuge tube after the cells are detached by blowing a culture dish with a pipette, centrifuging, pouring off the supernatant, resuspending the cell sediment by using the prepared frozen stock solution FBS: DMSO =9:1, adjusting the cell density, subpackaging the cell sediment into a freezing tube, and freezing for 24 hours by gradient cooling, and freezing in a liquid nitrogen tank for standby.
The specific operation steps of the drug screening are as follows:
(1) whole-cell recording method for NMDAR current on 293T-alpha 2 delta-1-GluN 1/2B cells
Drawing a glass microtube into a silicic acid glass microelectrode, and filling electrode solution (135 CsF, 2 MgCl)2，0.5 CaCl25 EGTA, 2 Mg-ATP and 10 HEPES, pH 7.25, 285 mOsmol/L, unit is mM) with a resistance of 3-5M omega, and the electrode electrolyte is filled in the electrode to serve as a conductive medium. The perfusion solution is extracellular fluid (160 NaCl, 2.5 KCl, 0.2 CaCl)210 HEPES, pH 7.35, 300-310 mOsmol/L in mM). Whole cell currents were recorded using an EPC-10 amplifier (HEKA Instruments, Lambrrecht, Germany) at a holding potential of-60 mV. After the establishment of the whole cell structure, the cell membrane capacitance and resistance were compensated electronically and NMDAR current was induced using glycine (10 uM) and NMDA (300 uM). In voltage-dependent Mg ⁺ blocking experiment, 2mM MgCl 2The conductance-voltage relationship of NMDARs was evaluated by addition to extracellular fluid.The sensitivity of NMDARs to Mg ⁺ was evaluated by fitting the current-voltage relationship and the boltzmann function. All HEK293T cell recording experiments were performed at 25 ℃.
(2) Drug screening
Medicine 1: CT-Tat: VSGLNPSLWSIFGLQFILLWLVSGSRHYLWYGRKKRRQRRR
Recording the cell current according to the steps, and adding the candidate drug into the extracellular fluid according to different concentrations to form perfusion fluid containing the candidate drug CT-Tat; gabapentin (Gabapentin) was used as a positive control (160 NaCl, 2.5 KCl, 0.2 CaCl)210 HEPES, pH 7.35; 300-310 mOsmol/L in mM, Gabapendin 1 mM); and (4) injecting the perfusion device in parallel. The specific operations in perfusion are as follows:
after the cells are clamped, the perfusion tube containing the agonist is opened for 2 s, and the cell current I is recorded0Switching to a perfusion tube 2 s filled with extracellular fluid and AP-5, and returning the current to the baseline;
perfusing with extracellular fluid for 3 min, perfusing with perfusion tube containing candidate drug for 1 min, opening perfusion tube containing agonist for 2 s, and recording cell current I1Switching to a perfusion tube 2 s filled with extracellular fluid and AP-5, and returning the current to the baseline;
perfusing with perfusion tube containing extracellular fluid only for 3 min, opening perfusion tube containing extracellular fluid and agonist for 2 s, and recording cell current I 0' switching to a perfusion tube 2 s filled with extracellular fluid and AP-5, and returning the current to the baseline;
perfusing with extracellular fluid for 3 min, perfusing with perfusion tube containing gabapentin for 1 min, opening perfusion tube containing extracellular fluid and agonist for 2 s, and recording cell current I2And switching to a perfusion tube containing extracellular fluid and AP-5 for 2 s, returning the current to the baseline, and repeating the above procedures for 6-12 cells to obtain an average value. I is1 < I0 Indicating that the candidate drug is effective; i is0 ≥ I0' indicates that the candidate drug is not effective; (I)0- I1）/（I0’- I2) Characterizing the effect of the drug candidate.
The results of the CT-Tat drug screening are as follows: i is0 = 840 pA，I1 = 510 pA，I0’ = 848 pA，I2= 560 pA, wherein I1 < I0，（I0- I1）/（I0’- I2) =1.15, indicating CT-Tat is effective, and its inhibitory effect on current is slightly stronger than gabapentin.
Medicine 2: tat: YGRKKRRQRRR
Recording cell current according to the steps, and adding the candidate drug into extracellular fluid according to different concentrations to form perfusion fluid containing the candidate drug Tat; gabapentin (Gabapentin) was used as a positive control (160 NaCl, 2.5 KCl, 0.2 CaCl)210 HEPES, pH 7.35; 300-310 mOsmol/L in mM, Gabapendin 1 mM); and (4) injecting the perfusion device in parallel. The specific operations in perfusion are as follows:
after the cells were clamped, the perfusion tube containing the agonist was first opened for 2 s and the cell current (I) was recorded 0= 920 pA), switch to perfusion tube 2 s with extracellular fluid and AP-5, current return to baseline;
perfusing with extracellular fluid for 3 min, perfusing with perfusion tube containing candidate drug for 1 min, opening perfusion tube containing agonist for 2 s, and recording cell current (I)1 = 930 pA), switch to perfusion tube 2 s with extracellular fluid and AP-5, current return to baseline;
perfusing with perfusion tube containing only extracellular fluid for 3 min, opening perfusion tube containing extracellular fluid and agonist for 2 s, and recording cell current (I)0' = 910 pA), switch to perfusion tube 2 s with extracellular fluid and AP-5, current return to baseline;
perfusing with extracellular fluid for 3 min, perfusing with perfusion tube containing gabapentin for 1 min, opening perfusion tube containing extracellular fluid and agonist for 2 s, and recording cell current (I)2= 540 pA), switch to perfusion tube with extracellular fluid and AP-5 for 2 s, return current to baseline, repeat 6-12 cells as above procedure, obtain mean.
The results of Tat drug screening were: i is0 = 920 pA，I1 = 930 pA，I0’ = 910 pA，I2= 540 pA, wherein I1 >I0Indicating that the candidate drug Tat is not effective.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> Hunan agriculture university
<120> application of human embryo kidney cell strain in drug screening
<141> 2019-12-16
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3276
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atggctgctg gctgcctgct ggccttgact ctgacacttt tccaatcttg gctgatcggc 60
ccctcgagcg aggagccctt cccttcgccc gtcactatca agtcatgggt ggataaaatg 120
caggaagacc ttgtcacact ggcaaagaca gcaagtggag tgactcagct tgctgacatt 180
tatgaaaaat accaagattt gtatactgtg gagccaaata atgcacgcca actggttgaa 240
attgcagcta gagacattga gaaacttctg agcaacagat ctaaagccct ggtgcgcctg 300
gctatggaag cagagaaagt ccaagccgcc caccaatgga gggaagattt tgcaagcaac 360
gaagttgtct actacaacgc taaagacgat cttgatcctg aaagaaatga gagtgagtca 420
ggcagccaac ggattaaacc tgttttcatc gaagacgcta attttggacg ccagatatcc 480
tatcaacatg cagcggtcca tattcccacg gacatctatg agggctcaac catagtgtta 540
aacgaactca actggacaag tgccttagat gaagttttca aaagaaatcg agatgaagac 600
cctacattgc tgtggcaagt gtttggcagc gccaccggcc tggcccgata ttatccagct 660
tctccgtggg tggataacag cagaactcca aacaagatcg atctctacga tgtacgcaga 720
agaccatggt acatccaggg agctgcatcc cccaaagaca tgctcattct ggtggacgtg 780
agtggaagtg tgagcggatt gactctgaaa ctcatccgga catctgtctc tgagatgtta 840
gaaacccttt ccgatgatga tttcgtgaat gtagcttcat ttaacagcaa tgctcaggat 900
gtgagttgtt tccagcacct ggttcaagcg aatgtaagga acaagaaggt gctgaaggat 960
gcggtgaata acatcacagc aaaggggata acagattaca agaaaggctt tagcttcgcg 1020
tttgaacagc tacttaatta taatgtttcc agagccaatt gtaataagat catcatgtta 1080
ttcacggatg gaggagaaga gagagcccag gagatatttg ccaagtacaa taaagacaaa 1140
aaagtccgcg tgtttacatt ttctgttggt caacataatt atgacagagg acctattcag 1200
tggatggctt gtgaaaacaa aggttattat tatgagattc cctctattgg tgcaataaga 1260
atcaatactc aggaatatct agatgttctg ggaagaccaa tggttttagc cggggacaaa 1320
gcaaagcaag tccagtggac aaatgtgtat ttggatgctc tggagctggg acttgtcatt 1380
actggaactc taccggtctt caacgtcact ggccagtctg aaaataagac taacttgaag 1440
aaccagttga ttcttggtgt gatgggcgtt gatgtgtctc tggaagatat taagagattg 1500
acaccacgtt ttacactgtg ccccaacggt tactattttg caatcgatcc taatggttat 1560
gtcttactgc atccaaatct tcagccaaag aaccccaaat ctcaggagcc ggtcacactg 1620
gattttctcg atgctgagtt agagaatgat attaaagtgg agattcgaaa taaaatgata 1680
gacggagaaa gtggagaaaa aacattcaga actctggtca aatctcaaga tgagagatat 1740
attgacaaag gaaatcgaac atacacatgg acacctgtca atggcacaga ttacagtttg 1800
gccttggtat tgccaaccta cagtttttac tatataaaag ccaaaataga agagacaata 1860
actcaggcca gatcaaaaaa ggggaaaatg aaggattcag aaaccctgaa gccagacaat 1920
tttgaagaat ctggctacac tttcatagca ccaagggaat actgcaatga cctgaaacct 1980
tcagataata acactgaatt tcttttaaat ttcaatgaat ttattgatag gaaaactcca 2040
aacaaccctt cctgtaatac agatttgatt aatagaatct tgctggatgc gggttttaca 2100
aatgaacttg tccaaaatta ttggagtaaa cagaaaaata tcaaaggagt gaaggcacgc 2160
tttgtggtga ctgatggcgg aattacaaga gtttacccca aagaggctgg agaaaactgg 2220
caagagaacc cagagacgta tgaggacagc ttctacaaac ggagcctaga taacgataac 2280
tacgttttca ctgcgcccta cttcaacaaa agtggacctg gtgcctatga atctggaatt 2340
atggtaagca aagctgtaga actgtacatc caaggaaaac ttcttaagcc tgcagttgtg 2400
ggaattaaaa ttgacgtaaa ctcctggata gaaaatttta ccaaaacttc aatcagggat 2460
ccgtgtgctg gtccagtttg tgactgcaaa agaaacagtg atgtaatgga ctgtgtcatt 2520
ctagatgatg gtggatttct tctgatggcc aatcatgatg attacactaa tcagattgga 2580
cgtttttttg gagagattga cccgagcatg atgagacacc tggttaatat atcactttat 2640
gcattcaata aatcatatga ctatcagtct gtgtgtgatc caggggcagc accaaagcaa 2700
ggagcaggac atcgctcagc ctatgtgcca tcaattacag acatactcca gattggctgg 2760
tgggccactg ctgccgcctg gtctattctc cagcagctac tcttgagttt gacatttcca 2820
cggctccttg aggcagttga aatggaagag gatgacttca cagcttccct gtctaagcag 2880
agctgcatca cagaacaaac tcagtacttc ttcaagaacg atactaaatc attcagtggt 2940
ttactggact gtggaaactg ttccaggatc tttcatgttg agaagcttat gaacaccaac 3000
ctagtgttca taatggtgga gagcaaggga acatgtccgt gtgacacgcg gctgctcatg 3060
caagcagaac agacttctga tggtccagat ccttgcgaca tggttaagca gcccagatac 3120
cgaaaaggac ctgatgtctg ctttgataac aatgtgctgg aggattatac cgactgtggt 3180
ggtgtttctg gattaaaccc ttccttatgg tctatctttg gactccaatt tatactcctt 3240
tggctggtat ctggcagcag acactatcta tggtga 3276
<210> 2
<211> 2817
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgagcacca tgcacctgct gacattcgcc ctgctttttt cctgctcctt cgcccgcgcc 60
gcctgcgacc ccaagatcgt caacatcggc gcggtgctga gcacgcgcaa gcatgaacag 120
atgttccgcg aggcagtaaa ccaggccaat aagcgacacg gctcttggaa gatacagctc 180
aacgccactt ctgtcaccca caagcccaac gccatacaga tggccctgtc agtgtgtgag 240
gacctcatct ctagccaggt ctacgctatc ctagttagcc acccgcctac tcccaacgac 300
cacttcactc ccacccctgt ctcctacaca gctggcttct acagaatccc tgtcctggga 360
ctgactaccc gaatgtccat ctactctgac aagagtatcc acctgagttt ccttcgcacg 420
gtgccgccct actcccacca gtccagcgtc tggtttgaga tgatgcgagt ctacaactgg 480
aaccacatca tcctgctggt cagcgacgac cacgagggac gggcagcgca gaagcgcttg 540
gagacgttgc tggaggaacg ggagtccaag gcagagaagg tgctgcagtt tgacccagga 600
accaagaatg tgacggctct gctgatggag gcccgggaac tggaggcccg ggtcatcatc 660
ctttctgcaa gcgaggacga cgctgccaca gtgtaccgcg cagccgcaat gctgaacatg 720
acgggctctg ggtacgtgtg gctggtcggg gaacgcgaga tctctgggaa cgccctgcgc 780
tacgctcctg atggcatcat cggacttcag ctcatcaatg gcaagaatga gtcagcccac 840
atcagtgacg ccgtgggcgt ggtggcacag gcagttcacg aactcctaga gaaggagaat 900
atcactgacc caccgcgggg ttgcgtgggc aacaccaaca tctggaagac aggaccattg 960
ttcaagaggg tgctgatgtc ttctaagtat gcggacggag tgactggccg tgtggaattc 1020
aatgaggatg gggaccggaa gtttgccaac tatagtatca tgaacctgca gaaccgcaag 1080
ctggtgcaag tgggcatcta caatggtacc catgtcatcc caaatgacag gaagatcatc 1140
tggccaggag gagagacaga gaaacctcga ggataccaga tgtccaccag actaaagata 1200
gtgacaatcc accaagagcc cttcgtgtac gtcaagccca caatgagtga tgggacatgc 1260
aaagaggagt tcacagtcaa tggtgaccca gtgaagaagg tgatctgtac ggggcctaat 1320
gacacgtccc caggcagccc acgccacaca gtgccccagt gctgctatgg cttctgcata 1380
gacctgctca tcaagctggc gcggaccatg aattttacct atgaggtgca cctggtggca 1440
gatggcaagt ttggcacaca ggagcgggta aacaacagca acaaaaagga gtggaacgga 1500
atgatgggcg agctactcag tggccaagcg gacatgattg tggcaccact gaccatcaac 1560
aatgagcgtg cgcagtacat agagttctcc aagcccttca agtaccaggg cctgaccatt 1620
ttggtcaaga aggagattcc caggagcaca ctggactcat ttatgcagcc ttttcagagc 1680
acactgtggt tgctagtagg actgtcagtt catgtggtgg ctgtgatgct gtacctgctg 1740
gaccgcttca gtccctttgg ccgattcaag gtgaacagtg aggaggagga ggaagatgca 1800
ctgaccctgt cctctgccat gtggttttcc tggggcgtcc tgctcaactc cggcattggg 1860
gaaggtgccc cccggagttt ctctgcacgt atcctaggca tggtgtgggc tggtttcgcc 1920
atgatcatag tggcttccta cactgccaac ttggcagctt tcctggtgct ggatcggcct 1980
gaggagcgca tcacgggcat caatgacccc aggctcagaa acccctcaga caagttcatc 2040
tacgcaactg taaagcagag ctccgtggac atctacttcc ggaggcaggt ggagttgagt 2100
accatgtacc ggcacatgga aaaacacaat tacgagagcg cagctgaggc catccaggct 2160
gtgcgggaca acaagctgca cgcctttatc tgggactcgg ccgtgctgga gtttgaggct 2220
tcacagaagt gcgatctggt gaccacgggt gagctgttct tccgctcagg ctttggcatc 2280
ggcatgcgca aggacagccc ctggaagcag aacgtttccc tgtccatact caagtcccat 2340
gagaatggct tcatggaaga tctggataag acatgggttc ggtatcagga atgcgactcc 2400
cgcagcaatg ctcctgcaac cctcactttt gagaacatgg caggggtctt catgctggtg 2460
gctggaggca tcgtagctgg gattttcctc attttcattg agatcgccta caagcgacac 2520
aaggatgccc gtaggaagca gatgcagctg gcttttgcag ccgtgaacgt gtggaggaag 2580
aacctgcagg atagaaagag tggtagagca gagcccgacc ctaaaaagaa agccacattt 2640
agggctatca cctccaccct ggcctccagc ttcaagagac gtaggtcctc caaagacacg 2700
agcaccgggg gtggacgcgg cgctttgcaa aaccaaaaag acacagtgct gccgcgacgc 2760
gctattgaga gggaggaggg ccagctgcag ctgtgttccc gtcataggga gagctga 2817
<210> 3
<211> 4449
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgaagccca gcgcagagtg ctgttccccc aagttctggt tggtgttggc cgtcttggcc 60
gtatcaggca gcaaagctcg ttcccaaaag agccccccca gcatcggcat cgctgtcatc 120
ctcgtgggca cttcagacga agtggccata aaagacgccc acgagaaaga tgacttccat 180
catctctcag tagttccccg ggtggagctg gtagccatga acgaaactga cccaaagagc 240
atcatcaccc gtatctgcga tcttatgtct gaccggaaga tccagggggt ggtgttcgcg 300
gatgacaccg accaagaagc catcgctcag atcctcgact tcatttctgc tcagactctc 360
acccccatcc tgggcatcca tgggggctca tctatgataa tggcggataa ggatgagtcc 420
tccatgttct tccagtttgg cccgtctatc gaacagcaag cttccgtcat gctcaacatc 480
atggaagaat atgactggta catcttttcc atcgtcacca cctacttccc tggctaccag 540
gactttgtga acaagatccg cagtaccatc gagaacagct tcgtgggctg ggagctcgag 600
gaagtcctcc tgctagacat gtctctggac gatggcgact ctaagattca gaatcagctg 660
aagaagctcc aaagccccat cattctcctt tattgcacga aggaggaagc cacctacatt 720
tttgaagtag ctaactcagt tgggctgact ggctacggct acacgtggat tgtgccgagt 780
ctggtggccg gggatacgga cacggtgcct tcagagttcc ccacggggct tatctctgtg 840
tcttatgatg aatgggacta tggccttcct gccagagtga gagatggaat tgccatcatc 900
accactgctg cctcggacat gctgtccgaa cacagtttca tccctgagcc caagagcagt 960
tgctacaaca cccacgagaa gaggatctac cagtctaaca tgttgaatag gtatctgatc 1020
aatgtcactt ttgaagggag aaacctgtcc ttcagcgaag atggctacca gatgcatccg 1080
aagctggtga taatccttct gaacaaggag aggaagtggg agagggtggg gaaatggaag 1140
gacaagtccc tgcagatgaa gtattatgtg tggcctcgga tgtgtcctga gactgaggag 1200
caagaggatg accatctgag cattgtcacc ttggaggagg cgccatttgt cattgtggaa 1260
agcgtggacc ctctcagtgg aacctgcatg aggaatacag tcccgtgcca gaagcgcatc 1320
atctctgaga ataaaacaga tgaggaacca ggctacatca aaaaatgctg caaggggttc 1380
tgtattgaca tccttaagaa aatttctaag tctgtgaagt tcacctatga cctttacctg 1440
gtgaccaatg gcaagcacgg gaagaagatt aatgggacct ggaatggcat gatcggtgag 1500
gtggtcatga agagggccta catggcagtg ggatcactaa ctatcaatga agaacggtca 1560
gaggtggttg acttctctgt acccttcata gaaactggca tcagtgtcat ggtatctcgc 1620
agcaatggga ctgtgtcacc ttctgccttc ttagagccat tcagcgctga cgtgtgggtg 1680
atgatgtttg tgatgctgct cattgtttct gcggtggctg tctttgtctt tgaatacttc 1740
agccctgtgg gttacaacag gtgcctagcc gatggcagag agccaggagg cccatctttc 1800
accatcggca aagcaatttg gttactctgg ggtctggtgt ttaacaactc cgtacctgtg 1860
cagaacccaa aggggaccac ctccaagatc atggtgtcag tgtgggcctt ctttgctgtc 1920
attttcctgg ccagctacac tgccaactta gcagccttca tgatccaaga ggagtatgtg 1980
gaccaggttt ctggcctgag tgacaagaag ttccagagac ctaatgactt ctcaccccct 2040
ttccgctttg ggactgtgcc caatggcagc acagagagga atatccgtaa taactatgca 2100
gaaatgcatg cctacatggg aaagttcaac caaaggggtg tagatgatgc attgctctcc 2160
ctgaaaacag ggaagcttga tgcattcatc tatgatgcag ctgtgctcaa ctacatggct 2220
ggaagggacg aaggctgcaa actggtgacc attggcagtg gcaaggtctt tgcttctacc 2280
ggctatggca ttgctatcca aaaggactcc gggtggaagc gccaggtgga cctggctatc 2340
ctgcagctgt ttggagatgg ggagatggaa gaactggaag ctctctggct cactggcatt 2400
tgccacaatg agaagaatga ggtgatgagc agccagctgg acatcgacaa tatggcaggt 2460
gtcttctata tgttgggggc agccatggcc ctcagcctca tcaccttcat ctgtgagcat 2520
ctgttctatt ggcagttccg gcattgcttc atgggtgtct gttctggcaa gcctggcatg 2580
gtcttctcca tcagcagagg tatctacagc tgtatccatg gggtagccat agaggagcgc 2640
caatccgtga tgaactcccc cactgccacc atgaacaaca cccactccaa catcctacgc 2700
ttgctccgca cggccaagaa catggccaac ctgtctggag taaacggctc ccctcagagt 2760
gccctggact tcatccgccg agagtcctcc gtctacgaca tctctgagca tcgtcgcagc 2820
ttcacgcatt cagactgcaa gtcttacaat aacccaccct gtgaggaaaa cctgttcagt 2880
gactacatta gcgaggtaga gagaacattt ggtaacctgc agctgaagga cagcaatgtg 2940
taccaagacc actatcacca tcaccaccgg ccacacagca tcggcagcac cagctccatt 3000
gatgggctct atgactgtga caacccaccc ttcaccaccc agcccaggtc aatcagcaag 3060
aaacccctgg acatcggcct gccctcctcc aaacatagcc agctcagcga cctgtatggc 3120
aagttctctt tcaagagtga ccgctacagt ggccacgacg acttgattcg atcggatgtc 3180
tccgacatct ccacgcacac tgtcacctat gggaacatcg agggcaacgc agccaagagg 3240
aggaaacagc agtataagga cagtctaaag aagcggccag cctcggccaa atcgaggagg 3300
gagtttgatg aaatcgagct ggcctaccgt cgccgaccac cccgctcccc ggaccacaag 3360
cgctacttca gggacaaaga agggctccga gacttctacc tggaccagtt ccgaacaaag 3420
gagaactcgc ctcactggga gcacgtggac ttgactgaca tttacaaaga acgcagtgac 3480
gacttcaagc gagattcggt cagtggaggt gggccctgta ccaacaggtc tcacctcaaa 3540
cacggaacgg gcgagaagca cggagtggta ggcggggtgc ctgctccttg ggagaagaac 3600
ctgaccaatg tggattggga ggaccggtct gggggcaact tctgccgcag ctgtccttcc 3660
aagctgcaca attactcctc gacggtggca gggcagaact cgggccggca ggcctgcatc 3720
agatgtgagg cctgtaagaa ggctggtaac ctgtacgaca tcagcaagga caactccctg 3780
caggaactgg accagccggc tgcccccgtg gctgtgacat ccaacgcctc cagcaccaag 3840
taccctcaaa gcccgactaa ttccaaggct cagaagaaga atcggaacaa actgcgccgg 3900
cagcattcct acgacacctt cgtggacctg cagaaggagg aggccgcctt ggccccacgc 3960
agcgtgagcc tgaaagacaa gggccgattc atggatggga gcccctacgc ccatatgttt 4020
gagatgccag ctggtgagag ctcctttgcc aacaagtcct cagtgcccac tgccggacac 4080
caccacaaca accccggcag cggctacatg ctcagcaagt cgctctaccc tgaccgggtc 4140
acgcaaaacc ctttcatccc cacttttggg gatgaccagt gcttgcttca cggcagcaaa 4200
tcctacttct tcaggcagcc cacggtggca ggggcgtcaa aaacaaggcc ggacttccgg 4260
gcccttgtca ccaataagcc agtggtgtca gcccttcatg gggctgtgcc aggtcgtttc 4320
cagaaggaca tttgtatagg gaaccagtcc aacccctgtg tgcctaacaa caaaaacccc 4380
agggctttca atggctccag caatggacat gtttatgaga aactttctag tattgagtct 4440
gatgtctga 4449
Claims (8)
1. An application of a human embryonic kidney cell strain in drug screening is characterized in that: the human embryonic kidney cell line is used for screening drugs in neuropathic pain and cerebral ischemia, the cerebral ischemia is caused by hypertension and apoplexy, the human embryonic kidney cell line is classified and named as human embryonic kidney cell line HEK 293T-NMDAR-alpha 2 delta 1, and the human embryonic kidney cell line is deposited in China center for type culture collection, and the deposition address is as follows: china, wuhan university, accession number: CCTCC NO of C2019189, preservation date: 09 and 05 months in 2019.
2. The application of the human embryonic kidney cell line in drug screening as claimed in claim 1, which is characterized in that: the human embryonic kidney cell line can stably express three genes of CACNA2D1, GRIN1 and GRIN2B at the same time, and can simulate an alpha 2 delta-1-NMDAR compound formed under pathological conditions, wherein the gene sequence of the CACNA2D1 is shown in SEQ ID No.1, the gene sequence of the GRIN1 is shown in SEQ ID No.2, and the gene sequence of the GRIN2B is shown in SEQ ID No. 3.
3. The application of the human embryonic kidney cell line in drug screening as claimed in claim 2, which is characterized in that: the drug is a substance which has an inhibitory effect on the current formed by the alpha 2 delta-1-NMDAR.
4. The application of the human embryonic kidney cell line in drug screening as claimed in claim 1, which is characterized in that: the drug screening comprises the following steps:
a) culturing a human embryonic kidney cell strain: the method comprises the following steps: a1) recovering and screening; a2) liquid changing; a3) passage; a4) freezing and storing;
b) drug screening: and detecting the current by adopting a patch clamp whole cell recording technology to screen the medicament.
5. The application of the human embryonic kidney cell line in drug screening as claimed in claim 4, wherein: the specific operation steps of the step a1) are as follows: taking out the cryopreserved human embryonic kidney cell strain, rapidly shaking and thawing in a 37 ℃ water bath tank, transferring the cell sap into a centrifuge tube, adding the inhibitor MK-801 and MgCl2And (3) uniformly mixing the glutamic acid-free DMEM, centrifuging, pouring out supernatant, blowing cells into single cell suspension by using the culture medium, transferring the single cell suspension into a culture bottle, culturing the single cell suspension in a constant-temperature incubator until the cells adhere to the wall, and adding screening markers Puro, Hygro and Bsd for screening after about 5-6 hours.
6. The application of the human embryonic kidney cell line in drug screening as claimed in claim 4, which is characterized in that: the specific operation steps of the step a 3) are as follows: when the cell fusion degree reaches 85% -90%, passage can be carried out, the original culture medium is poured off, and PBS is used for washing cellsDigesting with pancreatin, when intercellular space widens and cell becomes round and bright, immediately adding inhibitor AP5, KA and MgCl2The glutamic acid-free DMEM stops digestion, is blown and evenly mixed, is centrifuged, the supernatant is poured off, the cells are blown and blown into single cell suspension by the culture medium, and the single cell suspension is transferred to a culture bottle and then is placed in a constant temperature incubator for culture.
7. The application of the human embryonic kidney cell line in drug screening as claimed in claim 4, wherein: the specific operation steps of the step a 4) are as follows: washing cells in logarithmic phase with PBS, digesting with pancreatin, when cell gap widens and cell becomes round and bright, immediately adding a primary screening culture medium to terminate digestion, blowing a culture dish by a suction pipe to remove cells, transferring the cells into a centrifuge tube to centrifuge, pouring off supernatant, resuspending cell sediment by using prepared cryopreservation liquid FBS (DMSO =9: 1), adjusting cell density, subpackaging the cells into a cryopreservation tube, and after freezing for 24 h by gradient cooling, freezing and storing in a liquid nitrogen tank for later use.
8. The application of the human embryonic kidney cell line in drug screening as claimed in claim 4, wherein: the specific operation steps of the step b) are as follows: whole cell recording was performed at 25 ℃, cell supernatant was slowly poured out, extracellular fluid containing the drug to be selected at different concentrations was added, and the intracellular fluid was used in combination to observe the change in cell current.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911296185.4A CN110878330B (en) | 2019-12-17 | 2019-12-17 | Application of human embryo kidney cell strain in drug screening |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911296185.4A CN110878330B (en) | 2019-12-17 | 2019-12-17 | Application of human embryo kidney cell strain in drug screening |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110878330A CN110878330A (en) | 2020-03-13 |
| CN110878330B true CN110878330B (en) | 2022-06-10 |
Family
ID=69731297
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911296185.4A Active CN110878330B (en) | 2019-12-17 | 2019-12-17 | Application of human embryo kidney cell strain in drug screening |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110878330B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101012460A (en) * | 2006-12-12 | 2007-08-08 | 浙江大学 | Pig production trait related gene CACNA2D1 order and uses thereof |
| CN102251013A (en) * | 2011-02-22 | 2011-11-23 | 北京市肿瘤防治研究所 | Antibody and antigen for recognizing tumor initiator cell and application thereof |
-
2019
- 2019-12-17 CN CN201911296185.4A patent/CN110878330B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101012460A (en) * | 2006-12-12 | 2007-08-08 | 浙江大学 | Pig production trait related gene CACNA2D1 order and uses thereof |
| CN102251013A (en) * | 2011-02-22 | 2011-11-23 | 北京市肿瘤防治研究所 | Antibody and antigen for recognizing tumor initiator cell and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| Genbank.NM_012574.1.《Genbank》.2019,序列. * |
| Genbank.NM_012919.3.《Genbank》.2019,序列. * |
| Genbank.NM_017010.2.《Genbank》.2019,序列. * |
| Meichun Deng等.Presynaptic NMDA receptors control nociceptive transmission at the spinal cord level in neuropathic pain.《Cell Mol Life Sci.》.2019,第76卷(第10期),1889-1899. * |
| Youfang Chen等.Increased α2δ-1-NMDA receptor coupling potentiates glutamatergic input to spinal dorsal horn neurons in chemotherapy-induced neuropathic pain.《J Neurochem》.2019,第148卷(第2期),252-274. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110878330A (en) | 2020-03-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20210140965A1 (en) | Corneal endothelial cell marker | |
| Straube et al. | EB3 regulates microtubule dynamics at the cell cortex and is required for myoblast elongation and fusion | |
| AU2008333810B2 (en) | Compositions and methods to modulate cell membrane resealing | |
| Zimmerman et al. | Rsp1p, a J domain protein required for disassembly and assembly of microtubule organizing centers during the fission yeast cell cycle | |
| Peterson et al. | Molecular genetic analysis of myoC, a Dictyostelium myosin I | |
| CN107073067A (en) | Use the treatment of the new cornea of laminin | |
| Tian et al. | Higher plant cortical microtubule array analyzed in vitro in the presence of the cell wall | |
| CN110878330B (en) | Application of human embryo kidney cell strain in drug screening | |
| Stricker et al. | Cloning and expression pattern of chicken Ror2 and functional characterization of truncating mutations in Brachydactyly type B and Robinow syndrome | |
| Brown Jr et al. | Molecular biology of Duchenne muscular dystrophy | |
| Sinnecker et al. | Induced pluripotent stem cells in cardiovascular research | |
| Tang et al. | The kic1 kinase of Schizosaccharomyces pombe is a CLK/STY orthologue that regulates cell–cell separation | |
| Liu et al. | LrrA, a novel leucine-rich repeat protein involved in cytoskeleton remodeling, is required for multicellular morphogenesis in Dictyostelium discoideum | |
| CN114763561B (en) | Cell strain capable of stably expressing dopamine 2 type receptor | |
| CN108300763A (en) | A method of screening miR-101-3P target genes | |
| CN114591987B (en) | Genetic code fluorescence biosensor for detecting mTORC1 activity in living cells and construction method thereof | |
| Saway | A Multidimensional Analysis of the Structure and Function of The C-Terminal Region of The Microtubule Polymerase, Minispindles | |
| Tippelt | The First 3D Structure of a Full Length Hox Protein and Evidence for a Conformational Change | |
| Hoff | The Impact of TUBA1A Tubulinopathy Mutations on the Regulation of Microtubule Dynamics | |
| Lowe | Mini Spindles TOG1 Maintains Microtubule Polymerization Rates and Mitotic Spindle Formation | |
| Wang et al. | Structural Determination of the Polycystin-2 Channel by Electron Cryo-Microscopy | |
| Franco et al. | A human mitofusin 2 mutation causes mitophagic cardiomyopathy | |
| Kubo et al. | Identification of proteins that interact with a plant nuclear protein using the yeast split-Trp sensor | |
| 石原悟 et al. | Activation of Integrin-based Cell Adhesion inMouse L Fibroblasts Expressing Stabilized βCatenin in the Nucleus | |
| Olson | Neuronal connexin36 localization and association with zonula occludens-1 in brain, pancreas and adrenal gland |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: 410128 biological science and Technology College of Hunan Agricultural University, Furong district, Changsha, Hunan Applicant after: Hunan Agricultural University Address before: 410128 College of animal science and technology, Hunan Agricultural University, Furong district, Changsha City, Hunan Province Applicant before: Hunan Agricultural University |
|
| CB02 | Change of applicant information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |