CN105251018B - NONRATT021972 siRNA is applied in preparation its dysfunction of nerve fiber and related disease drug - Google Patents
NONRATT021972 siRNA is applied in preparation its dysfunction of nerve fiber and related disease drug Download PDFInfo
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Abstract
Application of the long non-coding ribonucleic acid NONRATT021972 siRNA in preparation nerve fiber NONRATT021972 dysfunction and related disease drug.Germicidal efficacy can inhibit the pain sensation behavior reaction of diabetes B rat to long non-coding ribonucleic acid NONRATT021972 small interference ribonucleic acid, change nerve conduction velocity, the Cellular inflammatory factor-tumor necrosis factor (TNF-α) content in diabetes rat serum is reduced, the activity of catalase (CAT) and superoxide dismutase (SOD) in diabetes rat serum is increased.Show that long non-coding ribonucleic acid NONRATT021972 siRNA has effect in nerve fiber NONRATT021972 dysfunction and complication, can be applied in its diagnosis, the drug prevented and treated, reagent, preparation.
Description
Technical field
The present invention relates to preparation treatment nerve fiber long non-coding ribonucleic acid NONRATT021972 dysfunction and correlations
Application in the drug of disease.
Background technique
With the completion of genome project and the application of deep sequencing technology of new generation, it has been found that mammal
Transcription sequence in cell more than 95% is non-encoding ribonucleic acid (noncoding RNA, ncRNA).Non-coding RNA is a kind of
Not coding protein but the RNA molecule with biology adjusting function, it can be by participating in the stabilization and translation skill of mRNA
The mechanism such as adjusting, the transport of protein, the processing of RNA and modification and the structure for influencing chromosome, regulate and control the basic of organism
Vital movement, at the same it is related to the pathophysiological process of some important diseases.Non-coding RNA includes short non-encoding ribonucleic acid
(including siRNA, miRNA, piRNA) and long non-coding ribonucleic acid (long non-coding RNA, lncRNA).Length is big
In 200 bases (200nt) be long-chain non-coding RNA (lncRNA).Non-coding RNA of the length less than 50 nucleotide at present
The research of (such as microRNA, siRNA and piRNA) has made a breakthrough, but to the functional long non-coding RNA of tool
Research is few.
Summary of the invention
The purpose of the present invention is to provide the new applications of long non-coding ribonucleic acid NONRATT021972 siRNA, long
Non- encoding ribonucleic acid NONRATT021972 siRNA is in preparation nerve fiber long non-coding ribonucleic acid
Application in NONRATT021972 dysfunction and related disease drug.
Present invention SOLiD high-flux sequence simultaneously verifies determining rat back by Bioinformatics Prediction and molecular biology
There are long non-coding ribonucleic acid NONRATT021972 [http://www.noncode.org] for root neural section (DRG), and find
Diabetic model rats dorsal root ganglion long non-coding ribonucleic acid NONRATT021972 expression is obviously increased compared with control group, is mentioned
Show that long non-coding ribonucleic acid NONRATT021972 and the nervous function of dorsal root ganglion are related with pathological change.
The present invention is big by diabetes B after observation long non-coding ribonucleic acid NONRATT021972 siRNA processing
The improvement of mouse nervous function is long non-coding ribonucleic acid NONRATT021972 siRNA in the long non-volume of preparation nerve fiber
Diagnosis, the prevention and treatment of code ribonucleic acid NONRATT021972 dysfunction and complication provide help.
After the small interference ribonucleic acid of injection long non-coding ribonucleic acid NONRATT021972, diabetes B model group
The expression of long non-coding ribonucleic acid NONRATT021972 reduces in DRG, while the diabetes rat after reagent is interfered in injection
Mechanical pain and temperature-sensitive pain threshold increase, and nervus coccygeus sensory conduction velocity increases, and shows long non-coding ribonucleic acid
NONRATT021972 siRNA can be to nerve fiber long non-coding ribonucleic acid NONRATT021972 dysfunction and concurrent
Disease generation effect, improves the damage phenomenon of nerve.The small interference ribonucleic acid of long non-coding ribonucleic acid NONRATT021972
Can lower simultaneously after processing reduces the Cellular inflammatory factor-tumor necrosis factor (TNF-α) content in diabetes rat serum,
Increase the activity of catalase (CAT) and superoxide dismutase (SOD) in diabetes rat serum.Therefore, long non-coding
Ribonucleic acid NONRATT021972 siRNA has the function of to nerve fiber long non-coding ribonucleic acid NONRATT021972
The improvement result of exception and related disease.
Detailed description of the invention
Fig. 1 is the mechanical paw withdrawal reflex threshold variation diagram in diabetes B rat patternmaking process.Long non-coding ribonucleic acid
It is inhibited to the mechanical pain behavior of diabetes B rat after the siRNA processing of NONRATT021972.Experiment point
Group: control group；Diabetes B model group；The small interference of diabetes B model+long non-coding ribonucleic acid NONRATT021972
RNA processing group；Negative (NCsi) control group of diabetes B model+random ordering siRNA.Wherein*P < 0.05,**The table of p < 0.01
Show and compare with normal group,##P < 0.01 is indicated compared with diabetic model group.
Fig. 2 is the pyrocondensation foot reflex changes of threshold figure in diabetes B rat patternmaking process.Long non-coding ribonucleic acid
It is inhibited to the temperature-sensitive pain behavior of diabetes B rat after the siRNA processing of NONRATT021972.It is wherein real
Test grouping: control group；Diabetes B model group；Diabetes B model+long non-coding ribonucleic acid NONRATT021972 is small
RNA interfering processing group；Diabetes B model+random ordering siRNA negative control group.Wherein*P < 0.05,**P < 0.01 is indicated
Compare with normal group,##P < 0.01 is indicated compared with diabetic model group.
Fig. 3 is the nervus coccygeus sensory conduction velocity variation diagram in diabetes B rat patternmaking process.Long non-coding ribose core
The nervus coccygeus sensory conduction velocity of diabetes B rat can be improved after the siRNA processing of sour NONRATT021972.Experiment
Grouping: control group；Diabetes B model group；Diabetes B model+long non-coding ribonucleic acid NONRATT021972 is small dry
Disturb RNA processing group；Diabetes B model+random ordering siRNA negative control group.Wherein*P < 0.05,**P < 0.01 indicate and
Normal group compares,##P < 0.01 is indicated compared with diabetic model group.
Fig. 4 is the reverse of diabetes B rat dorsal root ganglion (DRG) long non-coding ribonucleic acid NONRATT021972
Record-polymerase chain reaction (RT-PCR) testing result figure.The siRNA of long non-coding ribonucleic acid NONRATT021972
The long non-coding ribonucleic acid NONRATT021972 that diabetes B rat DRG up-regulation can be reduced after processing is horizontal.Experimental group:
Control group；Diabetes B model group；Diabetes B model+long non-coding ribonucleic acid NONRATT021972 siRNA
Processing group；Diabetes B model+random ordering siRNA negative control group.Fig. 4 (a) is RT-PCR experimental result picture, Fig. 4
(b) compare histogram for analysis of experimental data, wherein**P < 0.01 is indicated and normal group is compared,##P < 0.01 is indicated and diabetes
Model group compares.
Fig. 5 is that diabetes B rat blood serum TNF-α changes testing result figure.Long non-coding ribonucleic acid
The Serum TNF-α change level of diabetes B rat up-regulation can be reduced after the siRNA processing of NONRATT021972.Experiment
Grouping: control group；Diabetes B model group；Diabetes B model+long non-coding ribonucleic acid NONRATT021972 is small dry
Disturb RNA processing group；Diabetes B model+random ordering siRNA negative control group.Wherein**P < 0.01 is indicated and normal group ratio
Compared with,##P < 0.01 is indicated compared with diabetic model group.
Fig. 6 is diabetes B rat blood serum catalase activity measurement result figure.Long non-coding ribonucleic acid
The catalase in serum (CAT) that the downward of diabetes B rat can be increased after the siRNA processing of NONRATT021972 is living
Property it is horizontal.Experimental group: control group；Diabetes B model group；Diabetes B model+long non-coding ribonucleic acid
The siRNA processing group of NONRATT021972；Diabetes B model+random ordering siRNA negative control group.Wherein**
P < 0.01 is indicated and normal group is compared,##P < 0.01 is indicated compared with diabetic model group.
Fig. 7 is diabetes B rat blood serum Determination of erythrocyte superoxide dismutase activity result figure.Long non-coding ribonucleic acid
It can be risen after the siRNA processing of NONRATT021972 and increase the serum superoxide dismutases that diabetes B rat is lowered
(SOD) activity level.Experimental group: control group；Diabetes B model group；Diabetes B model+long non-coding ribonucleic acid
The siRNA processing group of NONRATT021972；Diabetes B model+random ordering siRNA negative control group.Wherein**p
< 0.01 indicates to compare with normal group,##P < 0.01 is indicated compared with diabetic model group.
Specific embodiment
Below with reference to embodiment and compares attached drawing invention is further described in detail.
Embodiment 1.
With method well-known in the art, it is made and is suitable for preparation nerve fiber long non-coding ribonucleic acid
The diagnosis of NONRATT021972 dysfunction and complication, oral, the injection, lozenge or other local or complete prevented, treated
The NONRATT021972 siRNA preparation of ribonucleic acid containing long non-coding of body drug formulation.
One, material and method.
1. animal and grouping.
Healthy Sprague-Dawley (SD) rat, Laboratory Animal Science portion of medical college of University Of Nanchang provide.Healthy SD is male
Property rat 60, weight 200g or so, adaptive feeding 1 week after, be randomly divided into 2 groups, control group (Ctrl) 8 is fed with basis
Feed；Remaining rat (52) are Glycemia Decline group, are fed with high glucose and high fat feed (high glucose and high fat feed formula: basal feed
66.5%, sucrose 20%, lard 10%, cholesterol 2.5%, sodium taurocholate 1% does glomeration and is placed on 80 in constant temperature blast drying oven
DEG C dry roasting 2 days).5th weekend, after all rats survey weight, fasting blood-glucose and postprandial blood sugar, the intraperitoneal injection of modeling group rat limosis
STZ (preparation of STZ solution: is dissolved in the 0.1mol/L lemon that the pH of 4 DEG C of refrigerators pre-cooling is 4.2 before use by STZ 30mg/kg
In sour sodium-citrate buffer solution, it is configured to the STZ solution of 2.5g/L), same volume is injected intraperitoneally in control rats
0.1mol/L, pH are 4.2 sodium citrate-citric acid buffers.6th weekend surveyed blood glucose, fasting blood-glucose < 7.8mmol/L and postprandial
The modeling group rat of blood glucose < 11.1mmol/L gives intraperitoneal injection STZ 30mg/kg on an empty stomach again.7th weekend surveyed blood glucose, empty
It is into mould standard that abdomen blood glucose, which is greater than 7.8mmol/L or postprandial blood sugar > 11.1mmol/L,.
Long non-coding ribonucleic acid NONRATT021972 tiny RNA interferes (siRNA) sequence by Invitrogen
(Carlsbad, CA) company provides, target sequence 5 '-GAATGTTGGTCATATCAAA-3 '；Negative control scrambled
SiRNA (CNsi) is purchased to Invitrogen (Carlsbad, CA) company.In body transfection reagent by EntransterTMCompany provides.
According to EntransterTMSpecification is transfected in body, the successful rat of the 7th week foot couple Glycemia Decline of experiment carries out long non-coding core
Ribosomal ribonucleic acid NONRATT021972-siRNA is in the small interference experiment of body.
The rat of Glycemia Decline Cheng Mo is randomly divided into 3 groups at the 7th weekend, it may be assumed that diabetes model+physiological saline group
(DM+NS), diabetes model+long non-coding ribonucleic acid NONRATT021972 small interference ribonucleic acid (siRNA) processing group
(DM+ long non-coding ribonucleic acid NONRATT021972 si), diabetes model+scrambled siRNA (out-of-order small interference core
Ribosomal ribonucleic acid) negative control (negative control siRNA, NCsi) group (DM+NCsi).DM+ long non-coding ribonucleic acid
The rat of NONRATT021972si and DM+NC si gives sublingual vein injection long non-coding ribonucleic acid respectively
NONRATT021972siRNA and NC siRNA.Ctrl the and DM group sublingual vein injecting normal saline of early period, respectively becomes life
Manage saline control group (Ctrl+NS) and diabetes model+physiological saline group (DM+NS).(Dorsal root is refreshing for materials after surveying blood glucose after 1 week
Warp knuckle).
2. drug and reagent.
Streptozotocin (Sigma company), transfection reagent (Entranster in animal bodyTM-in vivo)。
3. key instrument.
The full vigor type blood glucose meter of Luo Kang (German Roche Holding Ag)；Softron intelligence non-invasive blood pressure measuring-mouse note surveys instrument (Gene&
I company)；Antiseptic gauze, towel, cotton swab etc., the tincture of iodine and 75% alcohol, surgical kit: scissors, ophthalmology tip-curved forceps, vessel forceps,
Silk thread, pincers etc..
4. body weight determination.
Each group rat body weight is measured respectively at the 1st, 5,7,8 weekend, the time and other conditions measured every time is consistent.
5. the measurement of blood glucose.
Measure each group rat fasting blood-glucose and postprandial blood sugar respectively at the 1st, 5,7,8 weekend, the time measured every time and its
He is consistent condition.When surveying blood glucose, first rat is fixed, rat tail is wiped with cotton ball soaked in alcohol, is then cut with disinfection scissors
Tail point 2-3cm is removed, the blood on tail is directly dripped on the blood sugar test paper being mounted in blood glucose meter being ready for.Blood sampling terminates
Afterwards, with iodophor disinfection rat tail wound.
6. rat behavior measures.
It is latent respectively at the 1st, 5,7,8 weekend measurement each group rat machinery paw withdrawal reflex threshold (MWT) and pyrocondensation foot reflex
Phase (TWL), the time and other conditions measured every time are consistent.
(1) detection of mechanical paw withdrawal reflex threshold: the device used is Von Frey filament.It is a height of that rat is placed in length and width
It in 20cm × 10cm × 30cm transparent organic glass box, and is placed on wire netting, this device is higher than experimental bench, so as to clearly
In The Rat Sole is observed to Chu, is detected after adapting to half an hour.The folding power of Von Frey 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 filament stimulation Rat Right foot
Bottom, each dynamics of every rat are tested 10 times, and the adjacent interval time tested twice is at least 30 seconds, reaction caused by stimulating
(such as lick foot, swing one's legs) can just carry out next test after completely disappearing.When measurement since minimum dynamics, until some dynamics
When stimulation rat causes the number of reaction to be greater than 5 times, this dynamics is write down, as mechanical paw withdrawal reflex threshold.Remember when greater than 26.0g
For 26.0g.
(2) detection of Thermal allodynia paw withdrawal reflex threshold: the instrument that this test uses is that BME-410C type full-automatic heat pain is pierced
Swash instrument.Glass case same as described above is placed on the glass plate of 3mm thickness, rat is placed in glass case, after adapting to 30 minutes
It is tested.Using irradiated with thermal radiation Rat Right vola, record from the time for being irradiated to appearance lift leg, i.e. TWL.To prevent tissue
Damage, break time 30s.
7, the measurement of rat nervus coccygeus sensory conduction velocity.
Each group rat nervus coccygeus sensory conduction velocity is measured respectively at the 1st, 5,7,8 weekend.With 10% hydration chlorine before measurement
Aldehyde 300mg/kg intraperitoneal injection of anesthesia, prostrate are fixed, and forward method measures nervus coccygeus sensory conduction velocity.Stimulation ring electrode is located at
Tail distal end, stimulating electrode cathode (black) is placed in nervus coccygeus proximal end, anode electrode (red) is placed in distal end, and the two is apart
1cm；Record annular electrode is located at the 10cm of stimulation point proximal end.Grounding electrode is placed between stimulating electrode and recording electrode, stimulation
Intensity is fixed as 1.2mA.Sensory nerve conduction velocity (m/s)=distance (10cm) × 10/ is latent between recording electrode and stimulating electrode
Volt phase (m s), wave amplitude measurement take peak-to-peak value.Statistical analysis compares each group result.
8, reverse transcriptase chain reaction (RT-PCR).
(1) extract total serum IgE and reverse transcription into cDNA: all instruments are handled through DEPC.It draws materials at the 8th weekend, takes respectively
Each group rat dorsal root ganglion is rinsed with the processed PBS of DEPC, 20 DEG C of ﹣ preservations in RNA Store liquid is placed in, when extracting RNA
Neuromere taking-up is moved to added in 1ml Trizol homogenizer, is transferred to after grinding in the rnase-free centrifuge tube of 1.5ml, is added
0.2ml chloroform, acutely shakes 15s, stands 3min, low-temperature centrifugation 12000g × 15min collects the colourless liquid phase in upper layer, be added with
The isometric isopropanol of liquid phase mixes, and room temperature stands 25min, precipitates RNA, later 4 DEG C of centrifugation 12000g × 10min, in abandoning
Clearly, it is RNA in the visible a little white precipitate of tube bottom, 75% ethyl alcohol of 1ml nuclease free water configuration is added, sufficiently washing RNA.4℃
It after being centrifuged 5000g × 3min, inhales and abandons supernatant, be inverted 2 minutes, slightly dry (being sure not overdrying, otherwise RNA is not soluble in water),
Add 20 μ l to dissolve without RNase water to precipitate.Using 50 μ l reverse transcription reaction systems: 11.75 10 μ of μ l, 5 × buffer of nuclease free water
3 μ l, Olig DT of l, dNTP, 22 1.25 μ l, RNA sample of μ l, Rnasin of μ l, MMLV, 20 μ l, totally 50 μ l, is centrifuged, constant temperature 37
DEG C water-bath 1 hour.
(2) design primer: bibliography designs long non-coding ribonucleic acid NONRATT021972 primer sequence.The present invention
β-actin is selected to be used as standard internal reference, primer sequence is respectively as follows:
(3) polymerase chain reaction reaction system (totally 30 μ l):
(4) polymerase chain reaction reaction condition:
(5) agarose gel electrophoresis: 1.5% Ago-Gel of preparation takes PCR product to carry out by 10 holes μ L/ with liquid-transfering gun
Loading, electrophoretic buffer are 1 × TAE, voltage 100V, electric current 300mA, and electrophoresis time 40min is clapped using gel imaging system
According to analysis data.β-actin is used to be standardized as density of the internal reference to long non-coding ribonucleic acid NONRATT021972.
9, enzyme-linked immunosorbent assay (Elisa) measures serum TNFa levels.
(1) serum prepares: after the 8th weekend, rats by intraperitoneal injection chloral hydrate anesthesia, dorsal position fixation takes through arteria carotis
Blood, centrifuge tube are stored at room temperature 30min after collecting blood, and 3000g is centrifuged 10min, takes supernatant (i.e. serum), -20 DEG C of guarantors after packing
It deposits；
(2) it detects first 20 minutes and takes out kit and serum, balance to room temperature from refrigerator.And the lath needed for taking out, it remains
4 DEG C of preservations are put in after remaining lath sealing；
(3) foundation of standard curve: setting up 8 gauge orifices, and every hole is set up 3 multiple holes conducts and is averaged.Every Kong Zhongjia
Enter the 100 μ l of sample diluting liquid in kit, 100 μ l of standard items is added in the first hole later, and 100 μ l are taken out after mixing and are added to the
In two holes, it is half-and-half diluted to the 7th hole repeatedly, after mixing, 100 μ l is drawn from the 7th hole and are discarded, every pore volume is
100 μ l, the 8th hole are blank control；
(4) it is loaded: 100 μ l of test serum being added in remaining clean lath hole, and set up 3 multiple holes as control；
(5) reaction plate is placed in 37 DEG C of reaction 120min；
(6) with cleaning solution abundant washing reaction plate 4-6 times in kit, dry liquids finally board-washing: are printed on filter paper.
(7) 100 μ l first antibody working solutions are added in every hole；
(8) reaction plate, which mixes well, is placed on 37 DEG C of effect 60min；
(9) with cleaning solution abundant washing reaction plate 4-6 times in kit, dry liquids finally board-washing: are printed on filter paper；
(10) 100 μ l enzyme labelled antibody working solutions are added in every hole；
(11) reaction plate, which mixes well, is placed on 37 DEG C of effect 60min；
(12) board-washing: ibid；
(13) 100 μ l substrate solutions are added in every hole, is placed in 37 DEG C and is protected from light effect 10min；
(14) mixing of 150 μ l terminate liquids is added in every hole；
(15) absorbance at 450nm is surveyed.
10, oxidative stress measures.
10.1, catalase (CAT) determination of activity.
CAT decomposing H2O2Reaction can by be added ammonium molybdate stop rapidly, remaining H2O2It is acted on ammonium molybdate
A kind of flaxen complex compound can be generated, its production quantity is measured at 405nm, the vigor of CAT can be calculated, with every milliliter
The H for 1 μm of ol that serum each second decomposes2O2Amount be a unit of activity.It is built up by Nanjing in the CAT kit of company's offer
Include following reagent:
Reagent one: 1 bottle of liquid 100ml, 4 DEG C of preservations；
Reagent two: 1 bottle of substrate liquid 10ml, 4 DEG C of preservations；
Reagent three: 1 bottle of pulvis of colour developing, before use plus distilled water is dissolved to 100ml, and 4 DEG C save；
Reagent four: 1 bottle of liquid 10ml, 4 DEG C of preservations.
Operating procedure is as follows:
CAT vigor calculation formula are as follows:
10.2, superoxide dismutase (SOD) determination of activity.
Ultra-oxygen anion free radical (O is generated by xanthine and xanthine oxidase reaction system2 -), the latter aoxidizes hydroxyl
Amine forms nitrite, is in aubergine under the action of color developing agent, measures its absorbance.When containing SOD in test sample,
There is single-minded inhibiting effect to ultra-oxygen anion free radical, the nitrite formed it into is reduced.It can be calculated and be found out by formula
The SOD vigor of test sample.It is built up in the SOD kit of company's offer by Nanjing comprising following reagent:
Reagent one: 1 bottle of stock solution 10ml, the used time adds distilled water to be diluted to 100ml, 4 DEG C of preservations；
Reagent two: 1 bottle of liquid 10ml, 4 DEG C of preservations；
Reagent three: 1 bottle of liquid 10ml, 4 DEG C of preservations；
Reagent four: 350 μ l x2 branch of liquid, 4 DEG C of preservations；No. 41 bottle of dilution 10ml, both used times are diluted by 1:14；
Reagent five: 1, pulvis, the used time adds 70-80 DEG C of distilled water 75ml dissolution spare；
Practical six: 1, pulvis, the used time adds distilled water 75ml dissolution spare；
The preparation of color developing agent: press reagent five: reagent six: glacial acetic acid=3:3:2 volume ratio matches color developing agent.
Operating procedure is as follows:
SOD vigor calculation formula are as follows:
11, statistical method.
Experimental data is analyzed using SPSS statistical software, as a result with mean ± standard deviationIt indicates, each group
Between otherness use variance analysis, comparison among groups use LSD method, and p < 0.05 indicates significant difference, and p < 0.01 is extremely to show
Write difference.
Two, result.
(1) behaviouristics result.
Limb movement disturbance and autotomy phenomenon are showed no in each group rat modeling.It is anti-that each group room machine paw withdrawal is measured before modeling
Penetrate threshold value and preclinical basic value no significant difference (p > 0.05, the F of pyrocondensation foot reflex3,28=2.15).
1, mechanical quick (MWT) measurement result of pain.
After giving diabetic diet 4 weeks, the mechanical paw withdrawal reflex threshold of diabetic model rats starts to reduce, but with it is right
Not statistically significant (p > 0.05, F are reduced compared to it according to group3,28=2.67)；It gives after STZ is injected intraperitoneally 2 weeks, diabetes model
The mechanical paw withdrawal reflex threshold of rat is substantially reduced, and has significant difference (p < 0.01, F compared with the control group3,28=
21.87)；After diabetic model rats give the processing of long non-coding ribonucleic acid NONRATT021972 small interference ribonucleic acid, machine
Tool paw withdrawal reflex threshold obviously increases, compared with the control its no significant difference (p > 0.05, F1,14=1.67), and with sugar
Urine disease model group, which is compared, significant difference (p < 0.01, F1,14=13.54)；And model+random ordering it is small interference processing group with it is right
There are significant difference (p < 0.01, F according to comparing between group1,14=15.36) its difference is without statistics, but compared with diabetic model group
Learn meaning (p > 0.05, F1,14=2.96) (see Fig. 1).
2, Thermal allodynia (TWL) measurement result.
After giving diabetic diet 4 weeks, the pyrocondensation foot reflex threshold value of diabetic model rats starts to reduce, but with compare
Group reduces not statistically significant (p > 0.05, F compared to it3,28=1.54)；Give after STZ is injected intraperitoneally 2 weeks, diabetes model
The pyrocondensation foot reflex threshold value of rat group is substantially reduced, and has significant difference (p < 0.01, F compared with the control group3,28=
13.32)；After diabetic model rats give the processing of long non-coding ribonucleic acid NONRATT021972 small interference ribonucleic acid, heat
Paw withdrawal reflex threshold obviously increases, compared with the control group its no significant difference (p > 0.05, F1,14=2.71), with glycosuria
Disease model rat group, which is compared, significant difference (p < 0.01, F1,14=12.36)；And give the small interference processing group of model+random ordering
Have significant difference (p < 0.01, F compared between control group1,14=10..76), but it is poor compared with diabetic model group
Different not statistically significant (p > 0.05, F1,14=2.36) (see Fig. 2).
(2) nervus coccygeus sensory conduction velocity result.
After giving diabetic diet 4 weeks, the nervus coccygeus sensory conduction velocity of diabetic model rats is compared with the control group
There was no significant difference (p > 0.05, F3,28=1.89)；Give after STZ is injected intraperitoneally 2 weeks, the nervus coccygeus of diabetic model rats passes
It leads speed to be substantially reduced, there is significant difference (p < 0.01, F compared with the control group3,28=21.37)；Diabetic model rats
After giving the processing of long non-coding ribonucleic acid NONRATT021972RNA siRNA, nervus coccygeus conduction of velocity is compared with the control
Its difference is not statistically significant (p > 0.05, F1,14=1.44), have compared with diabetic model group significant difference (p <
0.01, F1,14=32.56)；And give the small interference processing group of model+random ordering has a significant difference (p compared between control group
< 0.01, F1,14=26.57) its no significant difference (p > 0.05, F, but compared with diabetic model group1,14=1.16)
(see Fig. 3).
(3) reverse transcriptase chain reaction (RT-PCR).
RT-PCR detects long non-coding ribonucleic acid NONRATT021972 expression.
Using the RT-PCR result table of gel imaging system software analysis long non-coding ribonucleic acid NONRATT021972
Bright: long non-coding ribonucleic acid NONRATT021972 expression is compared with control group in diabetic model group dorsal root ganglion (DRG)
Obviously increase (p < 0.01, F1,18=34.35), the small interference core of diabetes model+long non-coding ribonucleic acid NONRATT021972
No significant difference (p > 0.05, F between ribosomal ribonucleic acid group and control group1,18=2.98), and between diabetic model group
Difference statistically significant (p < 0.01, F1,18=43.21).The small interference processing group long non-coding ribose of diabetes model+random ordering
The expression of nucleic acid NONRATT021972 is higher than control group (p < 0.01, F1,18=15.71) nothing, and between diabetic model group
Significant difference (p > 0.05, F1,18=3.28) (see Fig. 4).
(4) enzyme-linked immunosorbent assay (ELISA) detects rat blood serum TNF-α.
According to the concentration gradient of kit standard product: 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml,
The lg value of 31.25pg/ml, 15.625pg/ml, 7.8125pg/ml and its measurement OD value make standard curve mapping, by software into
Row analysis obtains regression equation Y=0.259Lg (X) -0.315, R2=0.962 (the OD value of Y expression sample TNF-α, X expression
Sample concentration), Y value is brought into, obtain each group Serum TNF-α: the TNF-α concentration of diabetic model group obviously increases than control group
(p < 0.01, F1,14=22.98)；And diabetic model rats give the small interference of long non-coding ribonucleic acid NONRATT021972
After ribonucleic acid processing, TNF-α concentration is substantially reduced (p < 0.01, F than diabetic model group1,14=45.34), with control group it
Between there was no significant difference (p > 0.05, F1,14=2.98)；The TNF-α concentration and sugar of the small interference processing group of diabetes model+random ordering
It urinates between disease model group without significant difference (p > 0.05, F1,14=2.53) (see Fig. 5).
(5) oxidative stress measurement result.
1, catalase activity measurement result.
Interpretation of result shows that the activity of diabetic model group catalase is substantially reduced compared with control group, there is statistics meaning
Justice (p < 0.01, F1,14=26.76)；Diabetes model+long non-coding ribonucleic acid NONRATT021972 small interference ribonucleic acid
Difference has significant (p < 0.01, F between group and diabetic model group1,14=13,46), and between control group difference without
Statistical significance (p > 0.05, F1,14=2.97), the small interference processing group of diabetes model+random ordering compared with diabetic model group its
No significant difference (p > 0.05, F1,14=1.84) (see Fig. 6).
2, Determination of erythrocyte superoxide dismutase activity result.
Interpretation of result show diabetic model group and the small interference processing group of diabetes model+random ordering superoxide dismutase
The activity of enzyme is substantially reduced (p < 0.01, F compared with control group1,14=57.88), diabetes model+long non-coding ribonucleic acid
Difference has significant (p < 0.01, F between NONRATT021972 small interference ribonucleic acid group and diabetic model group1,14=
21.33) no significant difference (p > 0.05, the F, and between control group1,14=3.01), diabetes model+random ordering is small dry
Disturb processing group its no significant difference (p > 0.05, F compared with diabetic model group1,14=2.66) (see Fig. 7).
Inventor studies discovery after the small interference ribonucleic acid of injection long non-coding ribonucleic acid NONRATT021972, and 2
The expression of long non-coding ribonucleic acid NONRATT021972 reduces in patients with type Ⅰ DM model group DRG, while observing dry in injection
The mechanical of diabetes rat is increased with temperature-sensitive pain threshold bitterly after disturbing reagent, and nervus coccygeus sensory conduction velocity increases, and shows to grow non-
Encoding ribose nucleic acid NONRATT021972 siRNA can be to nerve fiber long non-coding ribonucleic acid NONRATT021972 function
Energy exception and complication generation effect, improve the damage phenomenon of nerve.Long non-coding ribonucleic acid NONRATT021972's is small
Can lower simultaneously after disturbance ribonucleic acid processing reduces the Cellular inflammatory factor-tumor necrosis factor in diabetes rat serum
The content of (TNF-α) increases the activity of catalase (CAT) and superoxide dismutase (SOD) in diabetes rat serum.
Therefore, long non-coding ribonucleic acid NONRATT021972 siRNA has to nerve fiber long non-coding ribonucleic acid
The improvement result of NONRATT021972 dysfunction and complication.
Claims (1)
1. the medicine that long non-coding ribonucleic acid NONRATT021972 siRNA improves diabetes complicated neurotrosis in preparation
Application in object, small interference of the long non-coding ribonucleic acid NONRATT021972 as shown in SEQ ID NO:3, described
RNA is as shown in SEQ ID NO:1 and SEQ ID NO:2.
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Non-Patent Citations (4)
| Title |
|---|
| 2型糖尿病早期大鼠外周神经节基因表达谱分析;汤晓丽 等;《遗传》;20120229;第34卷(第2期);198-207 * |
| 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;汤晓丽;《中国博士学位论文全文数据库 医学卫生科技辑 E065-9》;20150115(第1期);1104-1107 * |
| Long noncoding RNAs in diabetic retinopathy;Jae N et al;《Circ Res》;20150327;第116卷(第7期);E065-9 * |
| 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;李欣;《中国优秀硕士学位论文全文数据库 医药卫生科技辑 E070-14》;20120415(第4期);E070-14 * |
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