CN105251018A - Application of NONRATT021972 small interference RNA in preparation of drugs used for treating nervous tissue NONRATT021972 dysfunction and related diseases - Google Patents
Application of NONRATT021972 small interference RNA in preparation of drugs used for treating nervous tissue NONRATT021972 dysfunction and related diseases Download PDFInfo
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Abstract
The invention provides a long non-coding ribonucleic acid NONRATT021972 small interference RNA in preparation of drugs used for treating nervous tissue NONRATT021972 dysfunction and related diseases. According to experiment results, it is observed that the long non-coding NONRATT021972 small interference RNA can inhibit behavior reaction of rats with type w diabetes to pain sensation, change nerve conduction velocity, reduce the content of cell inflammatory factor-tumor necrosis factor (TNF-alpha) in the serum of the rats with diabetes and improve the activity of catalase (CAT) and superoxide dismutase (SOD) in serum of the rats with diabetes. It is proved that the long non-coding NONRATT021972 small interference RNA acts on nervous tissue NONRATT021972 dysfunction and related diseases and is applicable to the drugs, reagents and preparations used for diagnosis, prevention and treatment of nervous tissue NONRATT021972 dysfunction and related diseases.
Description
Technical field
The present invention relates to the application in the medicine of preparation treatment nervous tissue's long non-coding ribonucleic acid NONRATT021972 dysfunction and relevant disease.
Background technology
Along with completing and the application of degree of depth sequencing technologies of new generation of genome project, to it is found that in mammalian cell more than the transcription sequence of 95% to be non-encoding ribonucleic acid (noncodingRNA, ncRNA).Non-coding RNA is a class not coded protein but have the RNA molecule of adjusting function biology, it is by participating in adjustment, the transport of protein, the processing of RNA and the modification of the stable of mRNA and translation skill and affecting the mechanism such as chromosomal structure, the basic vital movement of regulation and control organism is simultaneously relevant to the pathophysiological process of some important diseases.Non-coding RNA comprises short non-encoding ribonucleic acid (comprising siRNA, miRNA, piRNA) and long non-coding ribonucleic acid (longnon-codingRNA, lncRNA).Length is greater than 200 bases (200nt) for long-chain non-coding RNA (lncRNA).The research that current length is less than the non-coding RNA (as microRNA, siRNA and piRNA) of 50 nucleotide etc. has made a breakthrough, but few to the research of the long non-coding RNA with function.
Summary of the invention
The object of the present invention is to provide the novelty teabag of long non-coding ribonucleic acid NONRATT021972 siRNA, the application of long non-coding ribonucleic acid NONRATT021972 siRNA in preparation nervous tissue's long non-coding ribonucleic acid NONRATT021972 dysfunction and relevant disease medicine.
By Bioinformatics Prediction and molecular biology checking, the present invention SOLiD high-flux sequence also determines that rat dorsal root ganglion (DRG) exists long non-coding ribonucleic acid NONRATT021972 [http://www.noncode.org], and finding that diabetic model rats dorsal root ganglion long non-coding ribonucleic acid NONRATT021972 expresses comparatively matched group and obviously increases, the long non-coding ribonucleic acid NONRATT021972 of prompting dorsal root ganglion is relevant with function of nervous system and pathological change.
The present invention by observing the improvement of type 2 diabetes mellitus rat function after the process of long non-coding ribonucleic acid NONRATT021972 siRNA, for long non-coding ribonucleic acid NONRATT021972 siRNA preparation nervous tissue's long non-coding ribonucleic acid NONRATT021972 dysfunction and complication diagnosis, prevention and therapy offer help.
After the small interference ribonucleic acid of injection long non-coding ribonucleic acid NONRATT021972, in type 2 diabetes mellitus model group DRG, the expression of long non-coding ribonucleic acid NONRATT021972 reduces, after injection interference reagent, the bitterly mechanical and temperature-sensitive pain threshold of diabetes rat raises simultaneously, and coccygeal nerve sensory conduction velocity increases, show that long non-coding ribonucleic acid NONRATT021972 siRNA to nervous tissue's long non-coding ribonucleic acid NONRATT021972 dysfunction and complication generation effect, can improve neural damage phenomenon.The content reducing the Cellular inflammatory factor-tumor necrosis factor (TNF-α) in diabetes rat serum can be lowered after the small interference ribonucleic acid process of long non-coding ribonucleic acid NONRATT021972 simultaneously, increase the activity of catalase (CAT) and superoxide dismutase (SOD) in diabetes rat serum.Therefore, long non-coding ribonucleic acid NONRATT021972 siRNA has the improvement result to nervous tissue's long non-coding ribonucleic acid NONRATT021972 dysfunction and relevant disease.
Accompanying drawing explanation
Fig. 1 is the machinery contracting foot reflex changes of threshold figure in type 2 diabetes mellitus rat patternmaking process.Inhibited to the machinery pain behavior of type 2 diabetes mellitus rat after the siRNA process of long non-coding ribonucleic acid NONRATT021972.Experiment grouping: matched group; Type 2 diabetes mellitus model group; The siRNA processed group of type 2 diabetes mellitus model+long non-coding ribonucleic acid NONRATT021972; SiRNA feminine gender (NCsi) matched group of type 2 diabetes mellitus model+out of order.Wherein
*p<0.05,
*p<0.01 represents and to compare with normal group,
##p<0.01 represents and to compare with diabetic model group.
Fig. 2 is the pyrocondensation foot reflex changes of threshold figure in type 2 diabetes mellitus rat patternmaking process.Inhibited to the temperature-sensitive pain behavior of type 2 diabetes mellitus rat after the siRNA process of long non-coding ribonucleic acid NONRATT021972.Wherein test grouping: matched group; Type 2 diabetes mellitus model group; The siRNA processed group of type 2 diabetes mellitus model+long non-coding ribonucleic acid NONRATT021972; The siRNA negative control group of type 2 diabetes mellitus model+out of order.Wherein
*p<0.05,
*p<0.01 represents and to compare with normal group,
##p<0.01 represents and to compare with diabetic model group.
Fig. 3 is the coccygeal nerve sensory conduction velocity variation diagram in type 2 diabetes mellitus rat patternmaking process.The coccygeal nerve sensory conduction velocity of type 2 diabetes mellitus rat can be improved after the siRNA process of long non-coding ribonucleic acid NONRATT021972.Experiment grouping: matched group; Type 2 diabetes mellitus model group; The siRNA processed group of type 2 diabetes mellitus model+long non-coding ribonucleic acid NONRATT021972; The siRNA negative control group of type 2 diabetes mellitus model+out of order.Wherein
*p<0.05,
*p<0.01 represents and to compare with normal group,
##p<0.01 represents and to compare with diabetic model group.
Fig. 4 is reverse transcriptase chain reaction (RT-PCR) the testing result figure of type 2 diabetes mellitus rat dorsal root ganglion (DRG) long non-coding ribonucleic acid NONRATT021972.The long non-coding ribonucleic acid NONRATT021972 level that type 2 diabetes mellitus rat DRG raises can be reduced after the siRNA process of long non-coding ribonucleic acid NONRATT021972.Experiment grouping: matched group; Type 2 diabetes mellitus model group; The siRNA processed group of type 2 diabetes mellitus model+long non-coding ribonucleic acid NONRATT021972; The siRNA negative control group of type 2 diabetes mellitus model+out of order.Fig. 4 (a) is RT-PCR experimental result picture, and Fig. 4 (b) compares block diagram for analysis of experimental data, wherein
*p<0.01 represents and to compare with normal group,
##p<0.01 represents and to compare with diabetic model group.
Fig. 5 is type 2 diabetes mellitus rat blood serum TNF-α change-detection result figure.The TNF-α change level that type 2 diabetes mellitus rat is raised can be reduced after the siRNA process of long non-coding ribonucleic acid NONRATT021972.Experiment grouping: matched group; Type 2 diabetes mellitus model group; The siRNA processed group of type 2 diabetes mellitus model+long non-coding ribonucleic acid NONRATT021972; The siRNA negative control group of type 2 diabetes mellitus model+out of order.Wherein
*p<0.01 represents and to compare with normal group,
##p<0.01 represents and to compare with diabetic model group.
Fig. 6 is type 2 diabetes mellitus rat blood serum catalase activity measurement result figure.Catalase in serum (CAT) activity level that type 2 diabetes mellitus rat is lowered can be increased after the siRNA process of long non-coding ribonucleic acid NONRATT021972.Experiment grouping: matched group; Type 2 diabetes mellitus model group; The siRNA processed group of type 2 diabetes mellitus model+long non-coding ribonucleic acid NONRATT021972; The siRNA negative control group of type 2 diabetes mellitus model+out of order.Wherein
*p<0.01 represents and to compare with normal group,
##p<0.01 represents and to compare with diabetic model group.
Fig. 7 is type 2 diabetes mellitus rat blood serum Determination of erythrocyte superoxide dismutase activity result figure.Serum superoxide dismutases (SOD) activity level increasing type 2 diabetes mellitus rat and lower can be risen after the siRNA process of long non-coding ribonucleic acid NONRATT021972.Experiment grouping: matched group; Type 2 diabetes mellitus model group; The siRNA processed group of type 2 diabetes mellitus model+long non-coding ribonucleic acid NONRATT021972; The siRNA negative control group of type 2 diabetes mellitus model+out of order.Wherein
*p<0.01 represents and to compare with normal group,
##p<0.01 represents and to compare with diabetic model group.
Detailed description of the invention
Contrast accompanying drawing below in conjunction with embodiment the present invention is described in further detail.
Embodiment 1.
Use method well-known in the art, make be applicable to prepare nervous tissue's long non-coding ribonucleic acid NONRATT021972 dysfunction and complication diagnosis, prevention, oral, the injection for the treatment of, buccal tablet or other local or systemic administration dosage form containing long non-coding ribonucleic acid NONRATT021972 siRNA preparation.
One, materials and methods.
1. animal and grouping.
Healthy Sprague-Dawley (SD) rat, medical college Laboratory Animal Science portion of University Of Nanchang provides.Healthy SD male rat 60, about body weight 200g, after adaptability raises 1 week, is divided into 2 groups at random, matched group (Ctrl) 8, feeds with normal feedstuff; All the other rats (52) are Glycemia Decline group, feed with high glucose and high fat feedstuff (high glucose and high fat feed formula: normal feedstuff 66.5%, sucrose 20%, Adeps Sus domestica 10%, cholesterol 2.5%, sodium cholate 1%, do glomeration to be placed in constant temperature blast drying oven 80 DEG C dry roasting 2 days).5th weekend, after all rats survey body weight, fasting glucose and post-prandial glycemia, the modeling group rat limosis lumbar injection STZ30mg/kg (preparation of STZ solution: the pH before use STZ being dissolved in 4 DEG C of refrigerator pre-coolings is in the 0.1mol/L sodium citrate-citric acid buffer of 4.2, be mixed with the STZ solution of 2.5g/L), the 0.1mol/L of control rats lumbar injection same volume, pH are 4.2 sodium citrate-citric acid buffer.6th weekend surveyed blood glucose, and fasting glucose < 7.8mmol/L and the modeling group rat of post-prandial glycemia < 11.1mmol/L give lumbar injection STZ30mg/kg on an empty stomach again.7th weekend surveyed blood glucose, and fasting glucose is greater than 7.8mmol/L, or post-prandial glycemia > 11.1mmol/L is for becoming mould standard.
Long non-coding ribonucleic acid NONRATT021972 tiny RNA interference (siRNA) sequence is provided by Invitrogen (Carlsbad, CA) company, and target sequence is 5 '-GAATGTTGGTCATATCAAA-3 '; Negative control scrambledsiRNA (CNsi) purchases to Invitrogen (Carlsbad, CA) company.At body transfection reagent by Entranster
tMcompany provides.According to Entranster
tMin body transfection description, test the 7th week successful rat of foot couple Glycemia Decline and carry out long non-coding ribonucleic acid NONRATT021972-siRNA and test at body minor interference.
At the 7th weekend, the rat of Glycemia Decline Cheng Mo is divided into 3 groups at random, that is: diabetes model+normal saline group (DM+NS), diabetes model+long non-coding ribonucleic acid NONRATT021972 small interference ribonucleic acid (siRNA) processed group (DM+ long non-coding ribonucleic acid NONRATT021972si), diabetes model+scrambledsiRNA (out of order small interference ribonucleic acid) negative control (negativecontrolsiRNA, NCsi) group (DM+NCsi).The rat of DM+ long non-coding ribonucleic acid NONRATT021972si and DM+NCsi gives sublingual vein injection long non-coding ribonucleic acid NONRATT021972siRNA and NCsiRNA respectively.Ctrl and the DM group sublingual vein injecting normal saline in early stage, becomes saline control group (Ctrl+NS) and diabetes model+normal saline group (DM+NS) respectively.(dorsal root ganglion) is drawn materials after surveying blood glucose after 1 week.
2. medicine and reagent.
Streptozotocin (Sigma company), transfection reagent (Entranster in animal body
tM-invivo).
3. key instrument.
Luo Kang full vigor type blood glucose meter (German Roche Holding Ag); Softron intelligence non-invasive blood pressure measuring-Mus note surveys instrument (Gene & I company); Antiseptic gauze, towel, cotton swab etc., iodine tincture and 75% ethanol, surgical kit: shears, ophthalmology tip-curved forceps, vascular forceps, silk thread, toothed forceps etc.
4. body weight determination.
Measure each group of rat body weight respectively at the the the 1st, 5,7,8 weekend, time and other conditions of each measurement are consistent.
5. the mensuration of blood glucose.
Measure each group of rat fasting blood-glucose and post-prandial glycemia respectively at the the the 1st, 5,7,8 weekend, time and other conditions of each measurement are consistent.When surveying blood glucose, first rat is fixed, with cotton ball soaked in alcohol wiping rat tail, then cut off tail point 2-3cm with sterilization shears, the blood on tail is directly dropped on the off-the-shelf blood sugar test paper be arranged in blood glucose meter.After blood sampling terminates, with iodophor disinfection rat tail wound.
6. rat behavior measures.
Respectively at measuring each group of rat machinery contracting foot reflex threshold value (MWT) and pyrocondensation foot reflex incubation period (TWL) the the the 1st, 5,7,8 weekend, time and other conditions of each measurement are consistent.
(1) detection of machinery contracting foot reflex threshold value: the device of employing is VonFrey filament.Rat is placed in the transparent organic glass box that length, width and height are 20cm × 10cm × 30cm, and is placed on wire gauze, this device, higher than laboratory table, clearly can observe In The Rat Sole, detects after adapting to half an hour.The folding power of VonFrey is respectively 0.008,0.02,0.04,0.07,0.16,0.4,0.6,1.0,1.4,2.0,4.0,6.0,8.0,10.0,15.0,26.0.With the right vola of filament stimulation in rats, every each dynamics of rat tests 10 times, and the interval time of adjacent twice test is at least 30 seconds, just can carry out next time and test after stimulating the reaction (as lick foot, swing one's legs) caused to disappear completely.During measurement from minimum dynamics, until when the number of times that certain dynamics stimulation in rats induces reaction is greater than 5 times, write down this dynamics, be machinery contracting foot reflex threshold value.26.0g is designated as when being greater than 26.0g.
(2) detection of Thermal allodynia contracting foot reflex threshold value: the instrument that this test adopts is the full-automatic burning pain stimulation instrument of BME-410C type.Be placed on by glass case same as described above on the thick glass plate of 3mm, rat is placed in glass case, tests after 30 minutes until adaptation.Adopting irradiated with thermal radiation Rat Right vola, recording from being irradiated to the time occurring lifting lower limb, i.e. TWL.For preventing tissue injury, break time is 30s.
7, the mensuration of rat coccygeal nerve sensory conduction velocity.
Respectively at measuring each group of rat coccygeal nerve sensory conduction velocity the the the 1st, 5,7,8 weekend.With 10% chloral hydrate 300mg/kg intraperitoneal injection of anesthesia before measuring, prostrate fixing, forward method measures coccygeal nerve sensory conduction velocity.Stimulate ring electrode to be positioned at tail far-end, stimulating electrode negative electrode (black) is placed in coccygeal nerve near-end, anode electrode (redness) is placed in far-end, and both are at a distance of 1cm; Record annular electrode is positioned at stimulation point near-end 10cm place.Ground electrode is placed between stimulating electrode and recording electrode, and stimulus intensity is fixed as 1.2mA.Sensory nerve conduction velocity (m/s)=recording electrode and stimulating electrode spacing (10cm) × 10/ incubation period (ms), wave amplitude measures and gets peak-to-peak value.Statistical analysis respectively organizes result.
8, reverse transcriptase chain reaction (RT-PCR).
(1) total serum IgE reverse transcription becomes cDNA is extracted: all apparatuses are all through DEPC process.Draw materials at the 8th weekend, get each group of rat dorsal root ganglion respectively, rinse with the PBS of DEPC process, be placed in RNAStore liquid ﹣ 20 DEG C preservation, being taken out to move to by neuroganglion during extraction RNA is added with in 1mlTrizol homogenizer, transfer in the rnase-free centrifuge tube of 1.5ml after grinding, add 0.2ml chloroform, concuss 15s, leave standstill 3min, , low-temperature centrifugation 12000g × 15min, collect the colourless liquid phase in upper strata, add isopyknic isopropyl alcohol with liquid phase, mixing, room temperature leaves standstill 25min, precipitated rna, 4 DEG C of centrifugal 12000g × 10min afterwards, abandon supernatant, at the bottom of pipe, visible a little white precipitate is RNA, add 75% ethanol of 1ml nuclease free water configuration, abundant washing RNA.After 4 DEG C of centrifugal 5000g × 3min, inhale and abandon supernatant, be inverted 2 minutes, slightly dry (be sure not overdrying, otherwise RNA being water insoluble), adds 20 μ l and precipitates without RNase water dissolution.Adopt 50 μ l reverse transcription reaction systems: nuclease free water 11.75 μ l, 5 × buffer10 μ l, dNTP3 μ l, OligDT2 μ l, MMLV2 μ l, Rnasin1.25 μ l, RNA sample 20 μ l, totally 50 μ l, centrifugal, constant temperature 37 DEG C of water-baths 1 hour.
(2) primer is designed: list of references design long non-coding ribonucleic acid NONRATT021972 primer sequence.The present invention selects β-actin as standard internal reference, and primer sequence is respectively:
(3) polymerase chain reaction reaction system (totally 30 μ l):
(4) polymerase chain reaction reaction condition:
(5) agarose gel electrophoresis: prepare 1.5% agarose gel, get PCR primer with liquid-transfering gun and carry out loading by 10 μ L/ holes, electrophoretic buffer is 1 × TAE, voltage 100V, electric current 300mA, electrophoresis time 40min, employing gel imaging system is taken pictures, analytical data.Mark is carried out as the density of internal reference to long non-coding ribonucleic acid NONRATT021972 with β-actin.
9, enzyme-linked immunosorbent assay (Elisa) measures serum TNFa levels.
(1) serum prepares: at the 8th weekend, after rats by intraperitoneal injection chloral hydrate anesthesia, and dorsal position is fixing gets blood through carotid artery, after centrifuge tube collects blood, room temperature leaves standstill 30min, the centrifugal 10min of 3000g, gets supernatant (i.e. serum) ,-20 DEG C of preservations after subpackage;
(2) detect first 20 minutes from refrigerator taking-up test kit and serum, balance to room temperature.And the lath needed for taking out, be put in 4 DEG C of preservations after remaining lath sealing;
(3) foundation of standard curve: set up 8 gauge orifices, every hole sets up 3 multiple holes as averaging.The sample diluting liquid 100 μ l in test kit is added in every hole, first hole adds standard substance 100 μ l afterwards, taking out 100 μ l after mixing joins in second hole, so repeatedly half-and-half be diluted to the 7th hole, after mixing, from the 7th hole, draw 100 μ l discard, every pore volume is 100 μ l, and the 8th hole is blank;
(4) application of sample: add test serum 100 μ l in remaining cleaned plate cylindrical void, and set up 3 multiple holes in contrast;
(5) Sptting plate is placed in 37 DEG C of reaction 120min;
(6) plate is washed: the cleaning mixture abundant washing reaction plate in use test kit 4-6 time, finally prints dry liquids on filter paper.
(7) in every hole, add 100 μ l first antibody working solutions;
(8) Sptting plate fully mixes and is placed on 37 DEG C of effect 60min;
(9) plate is washed: the cleaning mixture abundant washing reaction plate in use test kit 4-6 time, finally prints dry liquids on filter paper;
(10) in every hole, add 100 μ l enzyme labelled antibody working solutions;
(11) Sptting plate fully mixes and is placed on 37 DEG C of effect 60min;
(12) plate is washed: the same;
(13) in every hole, add 100 μ l substrate solutions, be placed in 37 DEG C of lucifuge effect 10min;
(14) in every hole, add 150 μ l stop buffer mixings;
(15) 450nm place absorbance is surveyed.
10, oxidative stress level determination.
10.1, catalase (CAT) determination of activity.
CAT decomposing H
2o
2reaction can stop rapidly by adding ammonium molybdate, remaining H
2o
2a kind of flaxen complex can be generated with ammonium molybdate effect, measure its growing amount in 405nm place, the vigor of CAT can be calculated, with the H of 1 μm of ol of every milliliter of serum decomposition each second
2o
2amount be a unit of activity.Built up in the CAT test kit that company provides by Nanjing and comprise following reagent:
Reagent one: liquid 100ml1 bottle, 4 DEG C of preservations;
Reagent two: substrate liquid 10ml1 bottle, 4 DEG C of preservations;
Reagent three: colour developing 1 bottle, powder, adds distilled water before use and dissolve to 100ml, 4 DEG C of preservations;
Reagent four: liquid 10ml1 bottle, 4 DEG C of preservations.
Operating procedure is as follows:
CAT vigor computing formula is:
10.2, superoxide dismutase (SOD) determination of activity.
Ultra-oxygen anion free radical (O is generated by xanthine and xanthine oxidase response system
2 -), the latter is oxidized azanol and forms nitrite, in aubergine under the effect of developer, measures its absorbance.When containing SOD in test sample, it has single-minded inhibitory action to ultra-oxygen anion free radical, and the nitrite making it be formed reduces.The SOD vigor obtaining test sample can be calculated by formula.Built up in the SOD test kit that company provides by Nanjing and comprise following reagent:
Reagent one: stock solution 10ml1 bottle, the used time adds distilled water and is diluted to 100ml, 4 DEG C of preservations;
Reagent two: liquid 10ml1 bottle, 4 DEG C of preservations;
Reagent three: liquid 10ml1 bottle, 4 DEG C of preservations;
Reagent four: liquid 350 μ lx2 props up, 4 DEG C of preservations; No. 4 diluent 10ml1 bottles, the used time both presses 1:14 dilution;
Reagent five: 1, powder, the used time adds 70-80 DEG C of distilled water 75ml and dissolves for subsequent use;
Actual six: 1, powder, the used time adds distilled water 75ml and dissolves for subsequent use;
The preparation of developer: by reagent five: reagent six: the volume of glacial acetic acid=3:3:2 matches well developer.
Operating procedure is as follows:
SOD vigor computing formula is:
11, statistical method.
Application SPSS statistical software analyzes experimental data, and result is with mean ± standard deviation
represent, between each group, diversity adopts variance analysis, and compare between group and adopt LSD method, p<0.05 indicates significant difference, and p<0.01 is pole significant difference.
Two, result.
(1) behavioristics's result.
Limb movement disturbance and autotomy phenomenon is showed no in each group of rat modeling.Each group of room machine contracting foot reflex threshold value and the preclinical basic value no significant difference of pyrocondensation foot reflex (p>0.05, F is measured before modeling
3,28=2.15).
1, machinery pain quick (MWT) measurement result.
Giving diabetic diet after 4 weeks, the machinery contracting foot reflex threshold value of diabetic model rats starts to reduce, but it reduces not statistically significant (p>0.05, F compared with matched group
3,28=2.67); To give after STZ lumbar injection 2 weeks, the machinery contracting foot reflex threshold value of diabetic model rats obviously reduces, and has significant difference (p<0.01, F compared with matched group
3,28=21.87); After diabetic model rats gives the process of long non-coding ribonucleic acid NONRATT021972 small interference ribonucleic acid, machinery contracting foot reflex threshold value obviously increases, its no significant difference (p>0.05, F compared with the control
1,14=1.67), significant difference (p<0.01, F is had compared with diabetic model group
1,14=13.54); And model+out of order minor interference processed group has significant difference (p<0.01, F compared with between matched group
1,14=15.36) its no significant difference (p>0.05, F, but compared with diabetic model group
1,14=2.96) (see Fig. 1).
2, Thermal allodynia (TWL) measurement result.
Giving diabetic diet after 4 weeks, the pyrocondensation foot reflex threshold value of diabetic model rats starts to reduce, but it reduces not statistically significant (p>0.05, F compared with matched group
3,28=1.54); To give after STZ lumbar injection 2 weeks, the pyrocondensation foot reflex threshold value of diabetic model rats group obviously reduces, and has significant difference (p<0.01, F compared with matched group
3,28=13.32); After diabetic model rats gives the process of long non-coding ribonucleic acid NONRATT021972 small interference ribonucleic acid, pyrocondensation foot reflex threshold value obviously increases, its no significant difference (p>0.05, F compared with matched group
1,14=2.71), significant difference (p<0.01, F is had compared with diabetic model rats group
1,14=12.36); And give model+out of order minor interference processed group have significant difference (p<0.01, F compared with between matched group
1,14=10..76), but compared with diabetic model group its no significant difference (p>0.05, F
1,14=2.36) (see Fig. 2).
(2) coccygeal nerve sensory conduction velocity result.
Giving diabetic diet after 4 weeks, coccygeal nerve sensory conduction velocity there was no significant difference (p>0.05, F compared with matched group of diabetic model rats
3,28=1.89); To give after STZ lumbar injection 2 weeks, the coccygeal nerve conduction velocity of diabetic model rats obviously reduces, and has significant difference (p<0.01, F compared with matched group
3,28=21.37); After diabetic model rats gives the process of long non-coding ribonucleic acid NONRATT021972RNA siRNA, coccygeal nerve conduction velocity compared with the control its difference does not have statistical significance (p>0.05, F
1,14=1.44), significant difference (p<0.01, F is had compared with diabetic model group
1,14=32.56); And give model+out of order minor interference processed group have significant difference (p<0.01, F compared with between matched group
1,14=26.57) its no significant difference (p>0.05, F, but compared with diabetic model group
1,14=1.16) (see Fig. 3).
(3) reverse transcriptase chain reaction (RT-PCR).
RT-PCR detects long non-coding ribonucleic acid NONRATT021972 and expresses.
The RT-PCR result of gel imaging system software analysis long non-coding ribonucleic acid NONRATT021972 is adopted to show: the middle long non-coding ribonucleic acid NONRATT021972 expression of diabetic model group dorsal root ganglion (DRG) comparatively matched group obviously increases (p<0.01, F
1,18=34.35), diabetes model+between long non-coding ribonucleic acid NONRATT021972 small interference ribonucleic acid group and matched group no significant difference (p>0.05, F
1,18=2.98), and and between diabetic model group difference have statistical significance (p<0.01, F
1,18=43.21).The expression of diabetes model+out of order minor interference processed group long non-coding ribonucleic acid NONRATT021972 is higher than matched group (p<0.01, F
1,18, and and there was no significant difference (p>0.05, F between diabetic model group=15.71)
1,18=3.28) (see Fig. 4).
(4) enzyme-linked immunosorbent assay (ELISA) detects rat blood serum TNF-α.
Concentraton gradient according to kit standard product: the lg value of 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml, 15.625pg/ml, 7.8125pg/ml and its OD value measured do standard curve mapping, carry out analysis by software and draw regression equation Y=0.259Lg (X)-0.315, R
2=0.962 (Y represents the OD value of sample TNF-α, and X represents sample concentration), Y value is brought into, draws each group of TNF-α: the TNF-α concentration of diabetic model group obviously increases (p<0.01, F than matched group
1,14=22.98); And after diabetic model rats gives the process of long non-coding ribonucleic acid NONRATT021972 small interference ribonucleic acid, TNF-α concentration ratio diabetic model group obviously reduces (p<0.01, F
1,14=45.34) there was no significant difference (p>0.05, F, and between matched group
1,14=2.98); Without significant difference (p>0.05, F between the TNF-α concentration of diabetes model+out of order minor interference processed group and diabetic model group
1,14=2.53) (see Fig. 5).
(5) oxidative stress measurement result.
1, catalase activity measurement result.
Interpretation of result shows, the catalatic activity of diabetic model group comparatively matched group obviously reduces, and has statistical significance (p<0.01, F
1,14=26.76); Diabetes model+between long non-coding ribonucleic acid NONRATT021972 small interference ribonucleic acid group and diabetic model group, difference has significant (p<0.01, F
1,14, and and no significant difference (p>0.05, F between matched group=13,46)
1,14=2.97), diabetes model+out of order minor interference processed group its no significant difference (p>0.05, F compared with diabetic model group
1,14=1.84) (see Fig. 6).
2, Determination of erythrocyte superoxide dismutase activity result.
Interpretation of result shows, diabetic model group and diabetes model+out of order minor interference processed group cross superoxide dismutase activity comparatively matched group obviously reduce (p<0.01, F
1,14=57.88), diabetes model+between long non-coding ribonucleic acid NONRATT021972 small interference ribonucleic acid group and diabetic model group, difference has significant (p<0.01, F
1,14, and and no significant difference (p>0.05, F between matched group=21.33)
1,14=3.01), diabetes model+out of order minor interference processed group its no significant difference (p>0.05, F compared with diabetic model group
1,14=2.66) (see Fig. 7).
Inventor studies and finds after the small interference ribonucleic acid of injection long non-coding ribonucleic acid NONRATT021972, in type 2 diabetes mellitus model group DRG, the expression of long non-coding ribonucleic acid NONRATT021972 reduces, machinery pain and the temperature-sensitive pain threshold of observing diabetes rat after injection interference reagent raise simultaneously, and coccygeal nerve sensory conduction velocity increases, show that long non-coding ribonucleic acid NONRATT021972 siRNA to nervous tissue's long non-coding ribonucleic acid NONRATT021972 dysfunction and complication generation effect, can improve neural damage phenomenon.The content reducing the Cellular inflammatory factor-tumor necrosis factor (TNF-α) in diabetes rat serum can be lowered after the small interference ribonucleic acid process of long non-coding ribonucleic acid NONRATT021972 simultaneously, increase the activity of catalase (CAT) and superoxide dismutase (SOD) in diabetes rat serum.Therefore, long non-coding ribonucleic acid NONRATT021972 siRNA has the improvement result to nervous tissue's long non-coding ribonucleic acid NONRATT021972 dysfunction and complication.
Claims (2)
1. the application of long non-coding ribonucleic acid NONRATT021972 siRNA in preparation nervous tissue NONRATT021972 dysfunction and relevant disease medicine.
2. long non-coding ribonucleic acid NONRATT021972 siRNA carries out diagnosis, the prevention and therapy of above-mentioned disease with oral, injection, buccal tablet or other local or systemic administration drug form.
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Non-Patent Citations (4)
| Title |
|---|
| JAE N ET AL: "Long noncoding RNAs in diabetic retinopathy", 《CIRC RES》 * |
| 李欣: "李欣 神经病理痛大鼠背根神经节神经细胞和脊髓背角血管内皮生长因子受体2及嘌呤2X2/3的相互作用研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑 E070-14》 * |
| 汤晓丽 等: "2型糖尿病早期大鼠外周神经节基因表达谱分析", 《遗传》 * |
| 汤晓丽: "II型糖尿病组织基因表达谱构建、分析及相关病理机制探讨", 《中国博士学位论文全文数据库 医学卫生科技辑 E065-9》 * |
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