CN1947711A - Application of glutamine for preparing medicine for treating and preventing cellular sugar deficiency injury - Google Patents
Application of glutamine for preparing medicine for treating and preventing cellular sugar deficiency injury Download PDFInfo
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- CN1947711A CN1947711A CN 200510030435 CN200510030435A CN1947711A CN 1947711 A CN1947711 A CN 1947711A CN 200510030435 CN200510030435 CN 200510030435 CN 200510030435 A CN200510030435 A CN 200510030435A CN 1947711 A CN1947711 A CN 1947711A
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Abstract
An application of L-glutamine in preparing the medicines for preventing and treating the saccharide-defficiency injury of cells and treating the ischemic diseases is disclosed.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of new purposes of condition essential amino acids-glutamine, be specifically related to the application of L-glutaminate in preparation control cellular sugar deficiency injury medicine.
Background technology
Cellular sugar deficiency injury is to be accompanied by cell ischemia injury and cell hypoxia damage and a kind of common, the serious cell injury type that takes place, cerebral ischemia, myocardial ischemia all be common clinically with cellular sugar deficiency injury with the cell hypoxia damage be pathologic basis disease, severe patient causes death; Document reports that also the disease of some nerve retrograde affections such as Alzheimer, amyotrophic lateral sclerosis etc. are also relevant with the damage that scarce sugar causes.Therefore intervention and the molecular mechanism thereof of studying the cellular sugar deficiency damage are one of present focuses of paying close attention to both at home and abroad, and particularly seeking effectively, the intervention medicine has important theoretical significance and application value.
Glutamine (Glutamine, Gln) be a kind of condition essential amino acids, when organizing major injury and disease, show as essential amino acids, participate in repair process (Byrne TA, RL Persinger, the LS Young of damaged tissue, TR Ziegler, DW Wilmore, 1995, Ann Surg.222 (3): 243-255; Ziegler, R Thomas, Bazargan, Niloofar, Leader, M Lorraine, Martindale, G Robert, 2000, Current Opinion in Clinical Nutrition﹠amp; Metabo-lic Care. (5): 355-362).In recent years Gln be applied to more and more as the medicine of a high security clinical, as the application of glutamine in oncotherapy, to the treatment of cardiac insufficiency, to insulin resistant influence, antifatigue effect or the like.There is report to show that all glutamine can be by strengthening heat shock protein (heat shock protein, hsp) family member's expression and bring into play its pharmacological action in recent years.Sanders and Kon find that glutamine can strengthen expression (Sanders MM, C Kon.1991, the J Cell Physiol.146:180 of hsp70 under the condition that heat is replied; 1992, J Cell Ph.150 (3): 620-631), it is injected directly into Chinese hamster ovary celI matter, confirm also that through Northern and Western experiment hsp70 expresses enhancing (Wischmeyer PE, J Riehm, KD Singleton, H Ren, MW Musch, M Kahana, EB Chang.2003, Glutamine Attenuates TumorNecrosis Factor-_Release and Enhances Heat Shock Protein 72in Human Peripheral BloodMononuclear Cells Nutrition.19 (1): 1-6; Paul E, MD Wischmeyer.2002, Glutamine and HeatShock Protein ExpressionNutrition 18:225-228).Wischmeyer report glutamine can cause also that the expression of hsp70 and hsp72 strengthens in the IEC-18 cell after the heat shock (Wischmeyer PE.Glutamine inducesheat shock protein expression.[J] .Nutrition, 2002,18:225-228.; Musch MW, Hayden D, Sugi K, Straus D, Chang EB.Cell-specific induction ofhsp72-mediated protection by glutamine againstoxidant injury in IEC18 cells.[J] .Proc Assoc Am Phys, 1998,110:136).Also have simultaneously and studies show that exogenous glutamine can strengthen the expression of rat intestine epithelial cell hsp72; (the Chow A thereby the damage of the enterocyte that oxidant and heat shock are caused plays a protective role; Zhang R.Glutamine reduces heatshock-induced cell death on rat intestinal epithelial cells.[J] .J Nutr; 1998,128:1296).This potentiation of glutamine is than glutamic acid---and the precursor substance and the metabolite of glutamine are eager to excel 100 times, analog 6-diazonium-5-oxygen-left-handed nor-leucine (6-diazo-5-oxo-L-norleucine) replacement glutamine with glutamine acts on cultured cell in vitro and finds to have same effect (Paul E, Wischmeyer, Jacob, Riehm et al.Glutamine attenuates tumor necrosis factor-alpha release and enhances heat shock protein 72 inhuman peripheral blood mononuclear cells.[J] .Nutrition, 2003,19 (1): l-6), confirmed that therefore this effect of glutamine and its metabolic process are irrelevant.Though showing glutamine, existing a large amount of reports the cell injury that multiple factor causes is had protective effect, the rarely seen report of protective effect of glutamine pair cell sugar deficiency injury by the report that raises heat shock protein.
Glucose regulated protein 75 (grp75) is Cytoplasm and Intramitochondrial a kind of important molecular chaperones, lacking sugar, under the stressed conditions such as radiation, grp75 presents mileometer adjustment and reaches, especially the irritant reaction of the scarce sugar of pair cell is the most obvious, to play protected protein matter, assist protein (particularly mitochondrial protein) transhipment, improve the effect (YanLIU of cell to the stress toleration, WenLIU, XiaodongSONG, JiZUO.Effect ofgrp75overexpression on intracellular ATP level, mitochondrial membrane potential and ROSaccumulation following glucose deprivation in PC 12 cells[J] .Molecular andCellularBiochemistry2005, (268): 45-51; Zuo J, Xia Bl, Massa SM, et al.grp75 is upregulated in ischemic brain.[J] .J.Neurochem.1996,67:S33A.).This biological characteristics of grp75 and the protective effect of pair cell sugar deficiency injury provide a kind of new thinking for the treatment of diseases of various pathologic basis with cellular sugar deficiency injury clinically.Yet the factor of inducing grp75 to raise mostly is injurious factor, as anoxia, and Denatured protein, ray etc., the up-regulated expression of the grp75 that excites thus and the protective effect of the pair cell that produces also is very limited; And, utilize transgenic to realize that its clinical treatment effect also has high difficulty with regard to present technology, lack operability.Therefore seek a kind of safe grp75 expression facilitator and realize that the protection of its pair cell sugar deficiency injury will have suitable medical application and be worth.
Summary of the invention
The new purposes that the purpose of this invention is to provide condition essential amino acids-glutamine is specifically related to the application of L-glutaminate in preparation control cellular sugar deficiency injury medicine.
Grp75 is Cytoplasm and Intramitochondrial a kind of important molecular chaperones, and its N end 1-23 site is the mitochondrial film signal of wearing, and can help albumen correctly to fold, assist proteinic transhipment.Grp75 can help the biomacromolecule relevant with energy metabolism to pass the various reactions that mitochondrion participates in energy metabolism in the cellular energy metabolic process; so the enhancing of its expression can be played the protective effect of pair cell sugar deficiency injury; because glutamine can raise the expression of hsp family member hsp70, hsp72; if also can raise the expression of grp75; therefore and the cell of sugar deficiency injury is produced protective effect; then the molecule protection mechanism can be changed into and have clinical workable means, it is significant that pair cell lacks the control of sugar damage
The present invention has carried out the influence of glutamine to the expression of grp75, and the pair cell sugar deficiency injury that therefore produces and be protection, the therapeutical effect experiment of the animal ischemic brain injury of pathologic basis with the cellular sugar deficiency injury.
It is object of study that the present invention adopts the PC12 cell, cultivates by DMEM sugar-free culture fluid and sets up the cellular sugar deficiency injury model.With the proteic expression of grp75 under SABC, western blotting contrast normal cell, sugar deficiency injury model and the glutamine effect; The effect of at mRNA horizontal detection glutamine grp75 being expressed with the RT-PCR method.Detect the influence of glutamine with mtt assay to the survival rate of sugar deficiency injury cell; The fluidic cell method detect cell lack under the sugared condition and the glutamine protection under the variation, the change of mitochondrion transmembrane potential of apoptosis rate.And make the animal cerebral ischemic model with middle cerebral artery line bolt legal system, observe the therapeutical effect of glutamine to animal pattern.The result shows that glutamine is expressed stress gene grp75 regulating and controlling effect; Glutamine can produce protective effect by the expression pair cell sugar deficiency injury that strengthens grp75; Glutamine also has therapeutical effect to the animal ischemia injury.The present invention is for seeking cellular sugar deficiency injury protectiveness medicine, providing foundation for the treatment of ischemic diseases clinically; Also lay a good foundation for the mechanism of further analyzing the grp75 expression regulation.
The present invention realizes by following method and step
1, cell culture
The PC12 cell routine is cultivated,
PC12 cell sugar-free is cultivated (setting up the cellular sugar deficiency injury model),
The variable concentrations glutamine acts on conventional cultured cell and sugar-free cultured cell respectively;
2, immunocytochemical method detects the expression of grp75 in the cell
Cell culture and grouping,
Immunocytochemistry detects;
3, Western blotting (Western blot) detects the expression of grp75 in the cell
Cell culture and grouping,
The protein immunoblotting experiment
Detect the influence of glutamine respectively to the expression of grp75 in conventional cultured cell and the sugar-free cultured cell,
Detect of the influence of variable concentrations glutamine to the expression of the grp75 of conventional cultured cell,
Detect the influence that glutamine was expressed conventional cultured cell grp75 under different action times;
4, reverse transcriptase polymerase chain method (RT-PCR) detects grp75mRNA content in the cell
Cell culture and grouping,
The design primer:
The amplimer of grp75 gene
sense：5′-TCAACTGATGCCTTGCA-3′
antisense：5′-AACCGTACATACGACCT-3′
The product fragment is 406bp,
The amplimer of β-actin gene
sense：5′-AACCGTGAAAAGATGACCCAG-3′
antisense：5′-CTCCTGCTTGCTGATCCACAT-3′
The product fragment is 741bp;
The RT-PCR experiment;
5, antisense grp75 transfection PC12 cell
The pcDNA3/antigrp75 expression vector establishment,
The target gene PCR amplimer that has EcoR I and BamH I restriction enzyme site
sense：5’AGTGAATTCaggtgccagaactacccctt3’
antisense：5’GATTAagctttacttggggctctg3’，
Expression vector plasmid amplification, extraction and evaluation,
The cell transfecting of antigrp75 and evaluation;
6, MTT detects the cell motility rate
Cell culture and grouping,
MTT detects the influence of glutamine pair cell motility rate
Detect the influence of glutamine to the motility rate of conventional cultured cell,
Detect the influence of glutamine to sugar-free cultured cell motility rate,
Detect glutamine sugar-free is cultivated the influence that grp75 loses the express cell motility rate;
7, cell hypodiploid rate detects
Cell grouping and cultivation,
The fluidic cell method detects the variation of apoptosis rate under the glutamine effect;
8, the mitochondrion transmembrane potential detects
Cell culture and grouping,
Flow cytometer detects the variation of cell mitochondrial transmembrane potential;
9, animal brain ischemia model (MCAO) is made
The MCAO rat model is set up,
Glutamine is to the treatment of animal pattern;
10, the glutamine therapeutical effect is observed
Experimentize continuously after the operation neurological deficits score of animal,
Animal brain's sections observation,
Infarction kitchen range area is calculated in brain sheet cresyl viollet dyeing back.
Material of the present invention is:
Rat is had a liking for chromium oncocyte system (PC12 cell line) available from Shanghai cell institute of the Chinese Academy of Sciences; The e. coli jm109 strain; Taq archaeal dna polymerase (worker bio-engineering corporation is given birth in Shanghai); 4 * dNTPs (worker bio-engineering corporation is given birth in Shanghai); The pcDNA3:pcDNA3 expression vector is enough in American I nvitrofen company; HindIII restricted enzyme and reaction buffer (Huamei Bio-Engrg Co.); EcoR I restricted enzyme and reaction buffer (Huamei Bio-Engrg Co.); T4 dna ligase and buffer (Promega company); DNA reclaims test kit (Clontech company); .lipofectamine 2000 (invitrogen companies); G418 (Lnvitrogen company) DMEM high glucose medium and DMEM sugar-free culture-medium (GiBco company), hyclone, horse serum (GiBco company); Glutamine (Sigma company); Sheep anti mouse grp75 polyclonal antibody (Santa Craz company); Immunocytochemistry test kit (doctor's moral company); Anti-sheep two anti-, the ECL test kits (the general company that flies) of HRP-donkey; Reverse transcriptase M-MLVRT, 10 * M-MLVRT Buffer (Promega company), TRIZOL (magnificent Shun's reagent company), Oligo d (T)
16, 10 * PCR reaction buffer (Shanghai give birth to worker bio-engineering corporation); MTT, DMSO DiOC6 (3) (Sigma company); Cresyl viollet (Sigma company).
Description of drawings
Fig. 1 is grp75 change of Expression immunocytochemical stain result (200 *) in the PC12 cell under the glutamine effect
Wherein A:PC12 is through conventional culture fluid effect 24h, and B:PC12 cell sugar-free is cultivated 24h, and brown particle is a grp75 albumen in the kytoplasm, and the C:PC12 cell is through the conventional culture fluid effect of glutamine 24h, and D:PC12 is through containing glutamine sugar-free culture-medium effect 24h.
Fig. 2 is that immunoblotting detects grp75 expression in the PC12 cell under the glutamine effect
A wherein: sugar-free is cultivated 24h, B, C: the conventional 24h that cultivates, and D:PC12 is through the conventional culture fluid effect of glutamine 24h, and E:PC12 is through containing glutamine sugar-free culture-medium effect 24h.
Fig. 3 is the expression of grp75 in the PC12 cell under the effect of variable concentrations glutamine
A:0.2mmol/L B:0.4mmol/L C:4mmol/L D:8mmol/L wherein.
Fig. 4 is glutamine expression of grp75 in the PC12 cell under different action times
A:6h B:12h C:18h D:24h wherein.
Fig. 5 is the variation of grp75mRNA level in the PC12 cell under the glutamine effect
Wherein A:PC12 is through containing glutamine sugar-free culture-medium effect 24h, and through conventional culture fluid effect 24h, D:PC12 is through sugar-free culture-medium effect 24h normal cell through the conventional culture fluid effect of glutamine 24h C:PC12 for B:PC12,
Fig. 6 is the influence of glutamine to the low express cell sugar deficiency injury motility rate of grp75
Fig. 7 is the influence of glutamine to sugar deficiency injury cell mitochondrial transmembrane potential
Wherein the A::PC12 cell routine is cultivated 24h, and B:PC12 cell sugar-free is cultivated 24h, and C, D:PC12 cell contain glutamine sugar-free culture fluid and cultivate 24h.
Fig. 8 respectively organizes the rat neurological deficits score
Fig. 9 respectively organizes rat cerebral tissue's section to show infarct size
A wherein: normal cerebral tissue's cortex, B: ischemia 14d cortex, C: glutamine is handled the back cortex, D: the normal cerebral tissue Hippocampus, E: ischemia 14d Hippocampus, F: glutamine is handled the back Hippocampus.
The specific embodiment
The experiment of embodiment 1 isolated cells
Glutamine is regulated cultured cell grp75 and is expressed experiment; The protective effect of glutamine pair cell sugar deficiency injury
Experiment
1 cell culture
The normal cultivation of PC12 cell
10ml culture bottle or culture plate 0.1mg/ml poly-D-lysine, PC12 cell culture are put 37 ℃, 5%CO in the DMEM high glucose medium
2Incubator changes liquid in good time, goes down to posterity.
The sugar-free of PC12 cell is cultivated
Treat that cell grows into 1 * 10
5Density is inhaled and is gone normal culture medium, washes cell twice with DMEM sugar-free culture fluid, adds the DMEM sugar-free culture-medium, and 37 ℃, 5%CO
2Cultivate in the incubator.
The glutamine effect
The PC12 cell culture is in the DMEM high glucose medium, treat that cell is paved with after, change to DMEM high glucose medium that contains the variable concentrations glutamine and the DMEM sugar-free culture-medium that contains the variable concentrations glutamine respectively.
2 immunocytochemical methods detect the expression of grp75 in the cell
Cell culture and grouping
Cell culture is divided into negative control group in 96 orifice plates, normal control group, sugar-free cultivation group, L-glutamine group (4mmol/L), each 4 multiple holes of glutamine sugar-free (4mmol/L) group.Each carries out the immunocytochemistry experiment after organizing cell culture 24h.
Immunocytochemistry detects
Cultured cell is with the fixing 15min of methanol, and PBS embathes.. 37 ℃ of sealings of normal rabbit serum 30min, grp75 antibody is hatched 1h with (1: 50) 37 ℃, and PBS embathes 15min, and negative control group replaces one to resist with PBS.Biotinylation two anti-, SABC mixed liquors are respectively hatched 1h for 37 ℃; DAB colour developing, microscopy.
3, Western blotting (Western blot) detects the expression of grp75 in the cell
Cell culture and grouping
Cell culture is divided into negative control group in the 10ml culture bottle, the normal control group, and sugar-free cultivation group, L-glutamine group (4mmol/L), 37 ℃ of CO2 gas incubator of glutamine sugar-free (4mmol/L) group are cultivated 24h.
Cell culture is in the 10ml culture bottle, wait to grow to certain density after, change to the glutamine DMEM culture fluid that contains variable concentrations, 37 ℃ of CO
2Incubator is cultivated 24h.
Cell culture is in the 10ml culture bottle, wait to grow to certain density after, change to and contain 4mmol/L glutamine DMEM culture fluid, 37 ℃ of CO
2Incubator is cultivated 6h, 12h, 18h and 24h respectively.
The protein immunoblotting experiment
The above-mentioned cell extraction protein of respectively organizing carries out the SDS-PAGE electrophoresis, and half dry type changes the film instrument and changes film 15min (source current≤0.8mA/cm
2).By dilution in 1: 100,37 ℃ were shaken and hatch 1h the antibody of grp75 gently with pBS.4 ℃ are spent the night.TTBS adds two anti-working solutions (1: 1000) after washing film, and room temperature is shaken and hatched 45min.Press ECL test kit description exposure imaging.
4, reverse transcriptase polymerase chain method (RT-PCR) detects grp75mRNA content in the cell
Cell culture and grouping
Cell culture is divided into negative control group in the 10ml culture bottle, normal control group, sugar-free cultivation group, L-glutamine group (4mmol/L) and glutamine sugar-free group (4mmol/L), 37 ℃ of CO
2Incubator is cultivated 24h.
Design of primers
The amplimer of grp75 gene
sense：5′-TCAACTGATGCCTTGCA-3′
antisense：5′-AACCGTACATACGACCT-3′
The product fragment is 406bp,
The amplimer of β-actin gene
sense：5′-AACCGTGAAAAGATGACCCAG-3′
antisense：5′-CTCCTGCTTGCTGATCCACAT-3′
The product fragment is 741bp,
The RT-PCR experiment
Total RNA measures OD in the Trizol extracting cell
260And OD
280Value is to calculate purity.4ugRNA adds oligdT1 μ l, adds DEPC water to 13 μ l, 70 ℃ of heating 10min, ice bath 1min.Add 5 * buffer, 5 μ l, dNTPS 5ul, M-MLVRT1 μ l, Rasin (inhibitor) 1 μ l, 37 ℃ of water bath with thermostatic control 1h of mixing, 90 ℃ of 2-3min synthesize the synthetic of cDNA first chains.The 50ulPCR reaction system comprises cDNA, grp75 primer, Actin primer, dNTP, PCR buffer, Taq enzyme, water.The PCR loop parameter is set: pre-94 ℃ of 7min of degeneration, 94 ℃ of 60s of degeneration, 55 ℃ of 60s of renaturation extend 72 ℃ of 60s, extend 72 ℃ of 5min after 35 circulations.Sepharose electrophoresis, Eagle Eye gel imaging instrument testing result.
5, antisense grp75 transfection PC12 cell
The grp75 antisense expression vector makes up
The structure of antisense expression vector oppositely inserts the pcDNA3 carrier with it again by the segment of one section 986bp of amplification grp75 coding region.
Amplimer
sense：5’AGTGAATTCaggtgccagaactacccctt3’
antisense：5’GATTAagctttacttggggctctg3’
The PCR reaction system comprises primer, grp75plasmid, 10 * buffer, dNTPs, Taq enzyme etc.The PCR loop parameter is: pre-94 ℃ of 90s of degeneration, 94 ℃ of 45s of degeneration, 60 ℃ of 45s of annealing, extend 72 ℃ of 60s, extend 72 ℃ of 300s again, cycle-index is 32 times.PCR product and pCDNA3 carrier are with EcoR I and BamH I enzyme action, and DNA reclaims test kit and reclaims DNA; 2 μ l5 * reaction buffers, 1 μ lT4 ligase, 20ng PCR enzyme action reclaims product and 60ngpCDNA3 enzyme action product, adds ddH
2O to 10 μ l, 4 ℃ are spent the night, so that genes of interest is connected with pCDNA3 enzyme action product.
Expression vector plasmid amplification, extraction and evaluation
The preparation competent escherichia coli cell makes plasmid pcDNA/antigrp75 pass bacterial cell membrane and enters escherichia coli, and grow into bacterium colony in selective medium.The single bacterium colony of picking is seeded in the LB fluid medium that contains ampicillin 60 μ g/ml, and plasmid amplification, extraction back are carried out enzyme action with HindIII restriction endonuclease and EcoRI restriction endonuclease and identified.
The cell transfecting of antigrp75 and evaluation
PC12 is incubated in 24 orifice plates, to stand density be 80%, liposome (.lipofectamine 2000)-target DNA complex slowly is added dropwise in the culture plate.Renew bright culture fluid after cultivating 6h, continue cultured cell 48h, changing liquid and adding G418 is that 400 μ g/ml screen to final concentration, to obtaining cell clone.Western blotting is identified the low grp75 of expression cell clone.
6, MTT detects the influence of glutamine to no born of the same parents' motility rate
Cell culture and grouping
The PC12 cell culture is in 96 orifice plates, and inoculum density is 1 * 10
5Individual/ml; Treat about cell density to 70%, change respectively with DMEM high sugared culture fluid, DMEM sugar-free culture fluid and the DMEM sugar-free culture fluid that contains the variable concentrations glutamine, every group of each 6 multiple holes.6 every group multiple holes of the low express cell of grp75, totally 5 groups are inoculated in 96 orifice plates with same density, treat about cell density to 70%, change to contain the DMEM sugar-free culture fluid (0.2mM, 0.4mM, 4mM, 8mM and 40mM) of variable concentrations glutamine (0.2mM, 0.4mM, 4mM, 8mM and 40mM).
MTT detects the cell motility rate
Above-mentioned each porocyte is cultivated 24h, and every hole adds 10 μ lMTT dye solutions, 37 ℃ of 5%CO
2Cultivate 4h in the incubator.After above-mentioned solution is abandoned in suction, add 100 μ l DMSO dissolution precipitations, room temperature 30min.The 570nm place measures the OD value on the microplate reader.
7, cell hypodiploid rate detects
The PC12 cell culture is in the 10ml culture bottle, and stand density is 10
6Individual/ml.Change respectively with DMEM high sugared culture fluid, DMEM sugar-free culture fluid and the DMEM sugar-free culture fluid that contains glutamine (4mM), effect 24h, the fluidic cell method detects apoptosis rate.
8, the mitochondrion transmembrane potential detects
Cell culture detects with cell hypodiploid rate with processing, trypsin digestion cell, and the corresponding culture fluid piping and druming of 0.25ml cell becomes cell suspension, adds DiOC
6(3), to final concentration of cells be 2nM, 37 ℃ of lucifuges are hatched 15min, flow cytometer detects DiOC
6(3) fluorescence intensity, excitation wavelength 484nm, emission wavelength 501nm.
The result shows:
Immunochemistry dyeing shows that glutamine raises grp75 and expresses
Cell living cells quantity behind sugar deficiency injury 24h reduces, and cell space diminishes, cell shrinkage is justified in change.And cell quantity is similar to normal cell with character behind the 24h that grows in containing the sugar-free culture fluid of glutamine.
The cellular immunization chemical results shows that grp75 albumen mainly is distributed in the Cytoplasm, grp75 lack present after sugar stimulates mileometer adjustment reach (with matched group than P＜0.05=; The grp75 expressing quantity is higher after the glutamine effect, and the cell no matter result's demonstration grows in normal culture fluid or sugar-free culture fluid is through the effect of glutamine, and grp75 all presents mileometer adjustment and reaches, through gray analysis, and with SPSS software statistics P＜0.01.The cell of growing in sugar-free culture fluid up-regulated expression after the effect of glutamine more obvious (Fig. 1), table 1 are grp75 expression immunocytochemistry gray value analyses as a result in the PC12 cell under the glutamine effect.
Table 1
| Group | n | Gray value |
| Matched group sugar-free cultivation group Gln group Gln sugar- | 10 10 10 10 | 0.0580±0.096 0.218±0.088 * 0.3117±0.024 ** 0.3745±0.037 ** |
*With matched group than P＜0.05
*With matched group than P＜0.01
Protein immunoblotting shows that glutamine raises grp75 and expresses
The PC12 cell after DMEM, DMEM sugar-free culture-medium, the DMEM that contains glutamine and DMEM sugar-free culture-medium are cultivated 24h, extracts the expression of protein grp75 in protein immunoblotting experiment detection cell respectively.The result shows grp75 albumen after sugar-free is cultivated 24h (model group), expresses obviously to raise; And after the glutamine effect, grp75 is tangible up-regulated expression, and this up-regulated expression is to the cell of sugar deficiency injury more obvious (Fig. 2).
The PC12 cell is cultivated 24h with the glutamine sugar-free culture-medium of variable concentrations respectively, extracts protein and carries out the immunoblotting detection, the relation between the dosage of observation glutamine and the effect of its rise grp75.Western blotting shows when the grp75 band of expression was obviously low than concentration when glutamine concentration was 4mmol/L and 8mmol/L obvious (Fig. 3).
Cultivate the PC12 cell with the DMEM that contains 4mmol/L concentration glutamine, extract cell protein respectively at harvesting after changing to 6h, 12h behind the glutamine culture fluid, 18h, 24h, identify glutamine difference action time by western blotting, grp75 expression in the cell.The expression of grp75 in the experimental result showed cell is along with the prolongation of glutamine action time also strengthens (Fig. 4).
RT-PCR measures glutamine and improves the grp75mRNA level
The present invention expresses at mRNA horizontal detection cell grp75 under the glutamine effect with the reverse transcriptase polymerase chain method, has verified the expression influence of glutamine to grp75.As internal reference, the result shows that the interior grp75mRNA amount of cell obviously increases under the glutamine effect with β-actin, and this increasing acts on more remarkable (Fig. 5) under the sugar-free condition.
The transfection of antisense grp75 expression vector reduces grp75 expression in the PC12 cell
The antisense expression vector of grp75 the band of 5.4kb and 986bp occurs meeting through HindIII and EcoRI enzyme action behind the agarose gel electrophoresis, the sequence and the direction of insertion of antisense vector all meet the requirements.
Behind antisense grp75 transfectional cell, extract protein and carry out the protein immunoblotting experiment, the grp75 expression in the visible cell reduces.
Mtt assay detects the influence of glutamine to sugar deficiency injury cell motility rate
Whether have toxicity for observing the glutamine pair cell, handle cell 24h, detected of the influence of variable concentrations glutamine the normal cell motility rate with mtt assay with the DMEM high glucose medium of the glutamine that contains variable concentrations.The OD of each concentration group compares P＞0.05 with matched group as a result, shows that glutamine to not influence of Normocellular growth, has shown the high security of glutamine as medicine.Table 2 is influences of glutamine pair cell motility rate.
Table 2
| Group | n | OD | |
| x | SD | ||
| Matched group sugar-free group 0.2mM 0.4mM | 6 6 6 6 6 6 6 | 0.509 0.167 0.577* 0.422* 0.483* 0.521* 0.475* | 0.119 0.034 0.042 0.032 0.101 0.039 0.067 |
*With matched group than P＞0.05 sugar-free group than P＜0.01
Cell is after sugar-free is cultivated 24h; the MTT value only is 26% of a normal cultured cell; and through its MTT value of cell of glutamine effect apparently higher than model cell, and in concentration protective effect has to a certain degree appearred during only for 0.2mmol/L, and along with the concentration of glutamine rises and strengthens.Table 3 is that glutamine is to lacking the influence of sugared cell motility rate.
Table 3
| Group | n | OD | ％ | |
| x | SD | |||
| Control group sugar-free cultivation group 0.2mmol/L 0.4mmol/L 4mmol/L 8mmol/L 40mmol/ | 6 6 6 6 6 6 6 | 0.684 0.178 △ 0.341* 0.398* 0.576* 0.543* 0.479* | 0.142 0.058 0.081 0.132 0.178 0.095 0.254 | 100％ 26.0％ 49.9％ 58.2％ 84.2％ 79.3％ 70.0％ |
△ and normal control group are than P＜0.01
*With model group than P＜0.01
The present invention detects the low motility rate of cell in the sugar-free culture fluid of the glutamine that contains variable concentrations of expressing of grp75 with mtt assay.The result shows that the low expression of grp75 compares with cellular control unit; the survival rate of two groups of cells all increases under the glutamine effect; but compare with matched group; after grp75 loses and expresses; the protective effect of glutamine pair cell obviously reduces (Fig. 6), confirms that glutamine is an expression role by regulating grp75 to the protective effect of sugar deficiency injury cell really.
Cell hypodiploid rate testing result
Among the present invention, cellular sugar deficiency 24h, the cultured cell apoptosis rate reaches 33.42%, and cell is grown in containing the sugar-free culture fluid of 4mM the same time, apoptosis rate only is 7.97% (Fig. 7).
Mitochondrion transmembrane potential testing result
Cell is after lacking sugared 24h, and mitochondrial transmembrane potential obviously reduces, and the cell of growing in containing glutamine (4mM) sugar-free culture fluid is not then seen the situation (Fig. 7) of this mitochondrion transmembrane potential collapse.
The experiment of embodiment 2 whole animal
Glutamine is to the protective effect experiment of rat ischemia brain injury
1, sets up rat cerebral ischemia model (MCAO)
The animal grouping: experiment is all raised in 22 ℃ of room temperatures with rat, the Animal House of relative humidity 55%, and the 12h circadian rhythm, ad lib and drinking-water begin experiment after 1 week that conformed.The equal fasting 12h of all laboratory animals before the art, rat is divided into matched group, model group (MCAO) and glutamine processed group at random.
Set up the MCAO model; Model group and glutamine processed group rat all select left side middle cerebral artery (MCA) district as the infarction side, 10% chloral hydrate (3ml/kg) intraperitoneal anesthesia.; The medisection skin of neck, expose common carotid artery and upwards isolate internal carotid artery and external carotid artery trunk and branch thereof, bipolar radio frequency electrocoagulator electricity coalescence is cut off occipital artery and the superior thyroid artery that external carotid artery is told, at lingual artery and jaw aortic bifurcation place's ligation external carotid artery, the bulldog clamp folder closes the crotch of external carotid artery and internal carotid artery.Cut an osculum near ligation place of external carotid artery far-end, insert nylon wire and loose ligation, slowly withdraw from nylon wire, skin suture behind the 2h.
2, glutamine treatment
Glutamine processed group animal begins behind the 24h to handle before hands, and it is heavy with 0.75g/kg dosage lumbar injection, once a day to sacrifice of animal to press animal body.
3, the neurological deficits score of glutamine treatment back animal
The operation back is the neurological deficits score of evaluation laboratory animal continuously, once a day to sacrifice of animal.Animal was put to death in operation in back 14 days.
Standards of grading: 0 minute: normal; 1 minute: contralateral limbs flexing 2 minutes: when holding the tail post-tensioning, contralateral limbs was unable; 3 minutes: hold the Mus tail, turn-take to sick side; 4 minutes: be sent to the disease side certainly and turn-take; 5 minutes: can not autonomic movement (Zea-longa E, Weinstein PR, Carlson, et al.[J] .Stroke, 1989,20 (1): 84-91).
4, brain tissue slice infarction kitchen range area calculates
The brain sheet is made: put to death animal in 4,7,14 days respectively at the operation back.0.9% normal saline intracardiac perfusion flushing, 4% paraformaldehyde is fixed about 30min, and broken end is got brain, 4% paraformaldehyde fixative spends the night, and gets brain and does the continuous coronal section, and section begins from rat bregma mouth side 1.4mm, cut to bregma tail side 4.8mm, every thickness is 40um.And observation brain tissue impairment situation.
Infarction kitchen range area is calculated in brain sheet cresyl viollet dyeing back:
Get one every 10 after the serial section and do cresyl viollet dyeing, calculate the cerebral ischemia Infarction volume, measure the cerebral infarct size of section behind the photo scanning with the Leica image processing system, utilize formula (Ekdahl CT, MohapelP, Elmer E, et al.Eur J Neurosci, 2001; 14 (6): 937-45) calculate infarct and ischemia offside brain sheet volume, utilize infarct volume percentage ratio (infarct volume/offside brain sheet volume * 100%) expression degree of injury.
The result shows:
The neurological deficits score of glutamine treatment back animal
The normal control treated animal is not seen neurologic impairment.Neurologic impairment to a certain degree appears in operation group rat postoperative, As time goes on the rat neurological functional deficit reduces gradually, function of nervous system's symptom after the 7th day obviously alleviated than the 1st day, but have neurologic impairment to a certain degree all the time, the 14th day neurological deficits score still is 1 minute.Function of nervous system's symptom of glutamine processed group rat rose promptly to have significantly and alleviates in the 2nd day after surgery, and its functional impairment scoring in 2-6 days all is lower than compares model group; Rose in the 7th day and promptly be reduced to 0 minute (Fig. 8).
Glutamine is to the influence of MCAO rat cerebral infarction volume
The section of normal cerebral tissue is compared, and the damage of 14 days cerebral tissue ishemic part of ischemia is serious, and 14 days the cortex of organizing forebrain ischemic area in the brain tissue slice of ischemia, striatum are all obviously downright bad; Damage side hippocampus badly broken, Hippocampus is the cavity shape.The cortex that 14 days identical time model groups with ischemia of hindbrain tissue ischemia are compared forebrain ischemic area in the brain tissue slice behind the injection glutamine only has the small part necrosis, the striatal damage degree is lighter, breakage appears in hippocampus damage side tape measure obscurity boundary but degree of injury obviously alleviates; And Hippocampus form complete substantially (Fig. 9).
The neuronal quantity of cresyl viollet dyeing visible damage tissue reduces, and form changes, and Nissl body reduces, and the glutamine protection down, the similar normal cortex of form; Cresyl viollet dyeing shows that the infarction kitchen range appears in rats with cerebral ischemia left side brain.Performed the operation back 14 days, the cerebral infarction volume of glutamine treatment group is significantly less than model group.Table 4 is Gln influences (%) to MCAO rat cerebral infarction volume.
Table 4
| Group | n | The MCAO group | The Gln group |
| 4th day 7th day | 6 6 6 | 38.21±5.64 35.88±7.61 36.42±4.23 | 28.71±3.02 22.13±6.17 21.87±6.52 |
Claims (5)
1, the application of L-glutaminate in preparation control sugar deficiency injury cell drug.
2, application according to claim 1, wherein said glutamine is by strengthening the proteic expression control of grp75 sugar deficiency injury cell.
3, the application of L-glutaminate in preparation grp75 expression facilitator.
4, L-glutaminate is prevented and treated application in the ischemic diseases medicine in preparation.
5, the application of L-glutaminate in preparation control ischemic brain injury medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103203026A (en) * | 2012-01-14 | 2013-07-17 | 复旦大学 | The applications of gene Grp75 in the preparation of drugs for the treatment of liver fibrosis |
| WO2020036371A1 (en) * | 2018-08-13 | 2020-02-20 | 울산과학기술원 | Composition for preventing or treating nerve damage disorders comprising grp75 as active ingredient |
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2005
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103203026A (en) * | 2012-01-14 | 2013-07-17 | 复旦大学 | The applications of gene Grp75 in the preparation of drugs for the treatment of liver fibrosis |
| WO2020036371A1 (en) * | 2018-08-13 | 2020-02-20 | 울산과학기술원 | Composition for preventing or treating nerve damage disorders comprising grp75 as active ingredient |
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