CN104922700A - Experimental method for applying Sirt1-Nrf2 novel oxidation resisting pathway in oxidative damage treatment - Google Patents

Abstract

The invention discloses an experimental method for applying a Sirt1-Nrf2 novel oxidation resisting pathway in oxidative damage treatment. The method comprises external experiment and internal experiment, an external experiment result shows that under an oxidative stress state, the protein stability and the protein expression quantity of the Nrf2 are lowered, and the acetylating level is increased; under the oxidative stress state, protein and gene expression levels of the Sirt1 are obviously lowered. An internal experiment result shows that under an oxidative damage condition, expressions of the Nrf2 and the Sirt1 are obviously lowered; up regulation can be carried out on an Nrf2 level by an activating agent of the Sirt1, expressions of oxidation resisting molecules of an organism are increased, and oxidative lung damage is lowered; when the up regulation is carried out on a genetic expression of the Sirt1, the expressions of oxidation resisting molecules of an organism can be increased, and the oxidative lung damage is lowered.

Description

The experimental technique of the new antioxidation path of Sirt1-Nrf2 application in oxidative damage treatment

Technical field

The invention belongs to medical research field, particularly relate to the experimental technique of the new antioxidation path of a kind of Sirt1-Nrf2 application in oxidative damage treatment.

Background technology

Body oxidative and anti-oxidative is unbalance, and caused oxidative damage is the core mechanism that senescence and disease occurs.Verified, the generation comprising acute coronary syndrome, diabetes, liver cirrhosis and the acute and chronic diseases such as poisoning is all relevant with oxidative damage.In view of this, inside and outside many scholars attempt to adopt the polyphenoils treatment oxidative damages such as supplementary superoxide dismutase (SOD), vitamin C, vitamin E, glutathione, melatonin.But exogenous polyphenoils molecular weight is large, and more difficult permeate through cell membranes enters in cell, and rapidly by metabolism, so really cannot effectively correct Cellular Oxidation-antioxidation imbalance state, it also falls flat to the treatment of body oxidative damage.

Nuclear factor E2-related factor 2 transcription factor (nuclear factor E2-related factor-2, Nrf2) belongs to Cap ' n ' Collar family, is to regulate the key factor that in cell, numerous polyphenoils is expressed.Under physiological condition, Nrf2 is combined the state being in disactivation, easily degrading in Cytoplasm with Keap1.At oxidative stress original work with rear (as poisonous substance, smoking, damage etc.), Nrf2 transcription factor and Keap1 activation of dissociating enters nucleus, and combine with Antioxidant responsive element ARE (antioxidant response element), start genetic transcription and the expression such as II phase detoxication enzyme, antioxidation albumen in downstream, increase cell to the resistance of oxidative damage.Mercado etc. study discovery, and deacetylase inhibitor can significantly inhibited oxidation stress Nrf2 protein expression and activity afterwards, and the table of downstream antioxidant molecule reduces, and prompting deacetylation plays an important role in the stability maintaining Nrf2 albumen and activity.Silent message regulatory factor 1 (silent information regulator 1; Sirt1) be the deacetylase that NAD relies on; regulatory factor can be total to transcription factor and transcription factor to interact, regulate target protein activity, genetic transcription by deacetylation effect.In view of this, understand Nrf2 Acetylation status in oxidative stress situation in depth, analyze Sirt1-Nrf2 antiopxidant effect, the treatment for oxidative damage relevant disease is significant.

Summary of the invention

The object of the present invention is to provide the experimental technique of the new antioxidation path of a kind of Sirt1-Nrf2 application in oxidative damage treatment, be intended to understand in depth Nrf2 Acetylation status in oxidative stress situation, analyze Sirt1-Nrf2 antiopxidant effect.

The present invention is achieved in that the experimental technique of the new antioxidation path of a kind of Sirt1-Nrf2 application in oxidative damage treatment comprises experiment in vitro and in vivo test;

Described experiment in vitro comprises:

The separation and purification of step one, mice AEC-II;

Step 2, the epithelial qualification of mice alveolar type;

Step 3, RT-PCR detect time, the dose-effect relationship that PQ exposes Sirt1, Nrf2 gene expression change and PQ effect in mice AEC-II;

Step 4, Western-bolt method detect time, the dose-effect relationship that PQ exposes the change of Sirt1, Nrf2 protein expression and PQ effect in mice AEC-II;

Step 5, immuno-precipitation detect the Acetylation Level that PQ exposes mice AEC-IINrf2;

Step 6, chemical colorimetry detect SOD, CAT, GSH, MDA;

Step 7, ELISA detect HO-1 and express;

Step 8, CO-IP method detect the interaction between Sirt1 and Nrf2 albumen;

Described experiment in vivo comprises:

Step one, mouse primary MSC extract and cultivate;

The identified by immunofluorescence of step 2, BMSC;

Step 3, slow-virus transfection BMSC cell;

Step 4, PQ poisoning mice experiment grouping and model are set up;

Step 5, PRC method, Western-blot method detect the expression of Nrf2, Sirt1 albumen;

Step 6, chemical colorimetry detect MDA, SOD, GSH, CAT and change;

Step 7, ELISA method detect IL-1 β, TNF-α, IL-10, TGF-β 1 protein content;

The detection of step 8, lung injury.

Further, described RT-PCR detects PQ and exposes Sirt1, Nrf2 gene expression in mice AEC-II and change and with the PCR primer in the time of PQ effect, dose-effect relationship be:

Sirt1, upstream sequence is 5 '-acgctgtggcagattgttatta-3 ', and downstream sequence is 5 '-ttgaagaatggtcttgggtctt-3 ';

Nrf2, upstream sequence is 5 '-attctttcagcagcatcctctc-3 ', and downstream sequence is 5 '-acacttccaggggcactatcta-3 ';

β-actin, upstream sequence is 5 '-atatcgctgcgctggtcgtc-3 ', and downstream sequence is 5 '-aggatggcgtgagggagagc-3 '.

Further, described PQ poisoning mice experiment is divided into 8 groups: blank group, NS group, PQ group, PQ+NS group, PQ+LV-MSC-GFP group, PQ+LV-MSC-Nrf2 group, PQ+LV-MSC-Sirt1 group, PQ+LV-MSC-Nrf2/Sirt1 group;

The process often organizing mice is as follows:

Animal fasting 8h before experiment, prohibits water 4h;

PQ+LV-MSC-Nrf2/Sirt1 group: through left side lumbar injection 20%PQ solution, after contamination in 1min through tail vein injection concentration be 10 × 10 ⁶/ ml LV-MSC-Nrf2 cell suspension and each 0.05ml of LV-MSC-Sir1 cell suspension;

PQ+LV-MSC-Nrf2 group: through left side lumbar injection 20%PQ solution, after contamination in 1min through tail vein injection concentration be 5 × 10 ⁶/ ml LV-MSC-Nrf2 cell suspension 0.1ml;

PQ+LV-MSC-Sirt1 group: through left side lumbar injection 20%PQ solution, after contamination in 1min through tail vein injection concentration be 5 × 10 ⁶/ ml LV-MSC-Sirt1 cell suspension 0.1ml;

PQ+LV-MSC-GFP group: through left side lumbar injection 20%PQ solution, after contamination in 1min through tail vein injection concentration be 5 × 10 ⁶ml LV-MSC-GFP cell suspension 0.1ml;

PQ+ normal saline group: through abdominal cavity, left side disposable injection 20%PQ solution, after contamination, 1min is interior through right side lumbar injection;

PQ group: through left side lumbar injection 20%PQ solution;

NS group: through the isopyknic normal saline of left side lumbar injection;

Blank group: do not give any process.

Accompanying drawing explanation

Fig. 1 is the method flow diagram of the new antioxidation path of Sirt1-Nrf2 experiment in vitro of application in oxidative damage treatment that the embodiment of the present invention provides;

Fig. 2 is the method flow diagram of the new antioxidation path of Sirt1-Nrf2 in vivo test of application in oxidative damage treatment that the embodiment of the present invention provides;

Fig. 3 is Sirt1, Nrf2 protein expression level figure that the embodiment of the present invention provides;

Fig. 4 is relative expression's spirogram of Sirt1, Nrf2 albumen that the embodiment of the present invention provides;

Fig. 5 is that the PQ that the embodiment of the present invention provides stimulates mice AEC-II HO-1 change figure.

Detailed description of the invention

For summary of the invention of the present invention, Characteristic can be understood further, hereby exemplify following examples, and coordinate accompanying drawing to be described in detail as follows: the present invention does not exist the innovation of software or method.

The present invention is achieved in that the experimental technique of the new antioxidation path of a kind of Sirt1-Nrf2 application in oxidative damage treatment comprises experiment in vitro and in vivo test;

Experiment in vitro described as shown in Figure 1 comprises:

The separation and purification of S101, mice AEC-II;

S102, the epithelial qualification of mice alveolar type;

S103, RT-PCR detect time, the dose-effect relationship that PQ exposes Sirt1, Nrf2 gene expression change and PQ effect in mice AEC-II;

S104, Western-bolt method detects time, the dose-effect relationship that PQ exposes the change of Sirt1, Nrf2 protein expression and PQ effect in mice AEC-II;

S105, immuno-precipitation detect the Acetylation Level that PQ exposes mice AEC-IINrf2;

S106, chemical colorimetry detect SOD, CAT, GSH, MDA;

S107, ELISA detect HO-1 and express;

S108, CO-IP method detects the interaction between Sirt1 and Nrf2 albumen;

1. experiment in vitro:

1.1 methods:

1.1.1 the separation and purification of mice AEC-II

(1) the healthy ICR mice of cleaning grade takes off neck execution, be placed in volume fraction 75% ethanol and soak 5min, super-clean bench operation after taking out, completely as early as possible take off lung tissue, in the PBS liquid (containing Dnase I0.01g/L) of pre-cooling, remove the tissue such as trachea-bronchial epithelial cell, blood vessel, PBS liquid rinses until washing liquid is limpid as far as possible.

(2) 10mL centrifuge tube is moved to after lung tissue being cut into 1mm × 1mm × 1mm fritter, add 1g/L trypsin containing Dnase I 0.01g/L according to 2mL/ mice) ratio add trypsin, suction pipe piping and druming mixing, 37 DEG C of digestion 5min, the cell suspension digested is removed, adds and stop digesting with the M199 containing 10%FBS of Digestive system equivalent.

(3) the same trypsinization lung tissue again using 1g/L, shifts out the cell suspension that digested and stops digestion after 5min.

(4) ratio adding 1g/L type i collagen enzyme (containing Dnase I0.01g/L) according to 2mL/ mice in residue lung tissue adds type i collagen enzyme, suction pipe piping and druming mixing, 37 DEG C, digestion 15min, cell suspension is shifted out, add with Digestive system equivalent be that the M199 of 10%FBS stops to digest containing volume fraction.

(5) by the cell suspension mixing in above-mentioned steps, suction pipe fully blows and beats mixing, and 200 eye mesh screens filter, and 1000r/min, 5min, centrifugal 2 times, abandon supernatant, by the resuspended precipitation of M19910mL containing volume fraction being 10%FBS.

(6) purification: mouse IgG bag is by culture dish, by pneumonocyte suspension inoculation wherein, in 37 DEG C, volume fraction is hatch 40min in 5%CO2 incubator, liquid sucking-off containing non-attached cell is inoculated in another bag by the culture dish of mouse IgG, continuation like this bag by the culture dish of mouse IgG in 37 DEG C hatch 40min, continuous 2 times.

(7) cultivate: the non-adherent cell of sucking-off, 1000r/min, 5min × 2 are time centrifugal, abandon supernatant, by the resuspended precipitation of M199 containing 10%FBS, adjustment cell concentration is 1 × 109L-1, and be inoculated in 90cm2 batch cultur ware and 6 well culture plates by 4 × 105/cm2 density, 37 DEG C, volume fraction is cultivate in 5%CO2 incubator, changes liquid after 24h, now adherently be Thorium Lung Burden, after this every 48-72h changes liquid 1 time.

1.1.2 the epithelial qualification of mice alveolar type

(1) in 6 well culture plates, add the coverslip of 4 pieces little, the mice Thorium Lung Burden of cancellationization joins (each 1 ~ 2) on coverslip

(2) three times are washed with PBS after covering with coverslip, each 5min until cell attachment

(3) fix twice, each 15min with 4% paraformaldehyde (PBS preparation), rear PBS washes three times, each 5min

(4) with 0.2%TritonX100 and 5%BSA close and rupture of membranes after 30 minutes PBS wash three times, each 5min

(5), after primary antibodie (1: 1000) 4 DEG C of overnight incubation, PBS washes three times, each 5min

(6) two anti-(1: 100) incubated at room are after 2 hours, and PBS washes three times, each 5min

(7) coverslip (cell faces up) to be placed on microscope slide and to observe under being placed in fluorescence microscope

1.1.3 RT-PCR detects time, the dose-effect relationship that PQ exposes Sirt1, Nrf2 gene expression change and PQ effect in mice AEC-II

(1) PCR primer

According to design of primers principle, adopt Primer Premier5.0 software design primer, refer to following table

Table 1 PCR primer

(2) sample preparation

The mice AEC-II of purification is inoculated in 6 porocyte culture plates with 0.5 × 105/ hole.In 37 DEG C, cultivate 24h in 5%CO2 incubator.One group of PQ solution effects 24h adding variable concentrations (0,400,800,1200,1600 μMs), another group adds 800 μMs of PQ solution effects different times (0h, 6h, 12h, 24h, 48h).

(3) according to TRNzol Plus total RNA extraction reagent, Total RNAs extraction illustrates that carrying out chloroform-isopropanol one-step method extracts cell RNA, and concrete steps are as follows:

1) culture fluid in 6 porocyte culture plates after above-mentioned PQ effect is poured out, 1 × PBS rinses 2 times, every hole adds 1ml RNAiso Plus, room temperature places a moment, it is made to come off with liquid-transfering gun piping and druming cell, be transferred to by lysate containing cell debris after abundant cracking in Ep pipe and repeatedly blow and beat until without obvious sediment, room temperature leaves standstill 5min.

2) add chloroform 0.2ml (be 1: 5 with RNAisoPlus volume ratio) to above-mentioned lysate, thermal agitation 15sec, after the fully emulsified mixing of solution, room temperature leaves standstill 5min, makes its natural phase-splitting.

3) 12,000rpm/min, 4 DEG C, centrifugal 15min, sample is divided into three layers, is respectively the organic layer of colourless supernatant, middle white albumin layer and yellow.Absorption supernatant is transferred to new EP and manages.

4) add and supernatant equal-volume isopropyl alcohol at supernatant, room temperature leaves standstill 10min, 4 DEG C, 12,000rpm/min, centrifugal 10min, and supernatant discarded, RNA is deposited at the bottom of pipe.

5) in RNA precipitation, add 1ml 75% ethanol (preparation of DEPC water), gentle vibration washing centrifuge tube, suspend precipitation, and every 1ml RNAiso Plus adds 1ml 75% ethanol.

6) 4 DEG C, 12000rpm/min, centrifugal 5min, discards ethanol.Room temperature is placed, drying precipitated 5min.

7) in precipitation, suitable RNase-free water is added, dissolution precipitation.

8) getting 1 μ lRNA solution adds in 99 μ lDEPC water, after surveying the concentration of RNA and purity, saves backup in-80 DEG C.

(4) total serum IgE reverse transcription becomes cDNA

1) using the total serum IgE extracted as template, reverse transcription reaction is carried out.Reaction system is as follows:

2) reverse transcription reaction condition is as follows:

37 DEG C of 15min (reverse transcription reaction)

85 DEG C of 5sec (inactivation reaction of reverse transcription)

4 DEG C of preservations

(5) polymerase chain reaction (PCR)

1) walk the cDNA of synthesis more than as template, carry out polymerase chain reaction.Reaction system is as follows:

2) PCR reaction condition is as follows:

(6) 2% agarose gel preparations

Take 2g agarose, be dissolved in 100ml 0.5 × tbe buffer liquid, moderate heat heating 3min in microwave oven, agarose pours into glue plate after melting transparent shape completely.Extract comb after 30min, glue is cut into suitable size for subsequent use.

(7) electrophoresis

Getting PCR primer 5 μ l adds in gel pore, 220V electrophoresis, 30min.Gel imaging system video picture.

1.1.4 Western-bolt method detects time, the dose-effect relationship that PQ exposes the change of Sirt1, Nrf2 protein expression and PQ effect in mice AEC-II

(1) preparation of the same RT-PCR method 2.1.2 sample prepared by sample, and cell culture is in 25cm2 Tissue Culture Flask.

(2) PBS of protein extraction pre-cooling washes the cell after three PQ effects, adds appropriate lysate (containing protease inhibitor) and covers cell, place 30min on ice, and sleaker scrapes in cell to new EP pipe.Ultrasonication, places 10min on ice, 4 DEG C, 12000g × 10min high speed centrifugation, collects supernatant ,-80 DEG C of preservations.

(3) BCA surveys protein concentration

1) standard working solution (SWR) is prepared: regent A and reagent B mixes by the ratio in 50: 1.

2) get 0.5mg/ml protein standard solution 0,1,2,4,8,12,16, the 20 μ L prepared in advance to be added in the standard sample wells of 96 orifice plates, add normal saline and supply 20 μ L (standard curve is used).

3) in the sample well of 96 orifice plates, add 2 μ L testing samples, normal saline is mended to 20 μ L.

4) every hole adds 200 μ L standard working solution.Hatch 30min for 37 DEG C.Microplate reader measures the absorbance at 540nm place.

5) according to the numerical value production standard curve that BSA standard sample records, utilize Origin software to carry out quadratic equation curve fitting, obtain the protein concentration of calculation sample after equation.

(4) SDS-PAGE electrophoresis

1) detergent cleaning glass plate, after distilled water flushing is clean, loads shelf, and is installed by shelf.

2) prepare 8% separation gel, add TEMED after having configured and shake up immediately, along glass plate, glue is poured into gently, avoid producing bubble.The adding distil water fluid-tight of glue surface.

3) separation gel removes upper water after fully solidifying, and blots remaining liquid with filter paper.

4) prepare the concentrated glue of 4%, after shaking up immediately after adding TEMED, get final product encapsulating, insert comb, avoid being mixed into bubble.

5) until after concentrated gelling admittedly, extract comb.

6) albuminous degeneration and loading

According to the protein concentration calculated, adjustment loading total protein quality is 30 μ g, and add appropriate 5 × sample-loading buffer to make it be finally 1 ×, 100 DEG C, 10min Denatured protein.Microloader loading.

7) electrophoresis

80V runs to concentrated glue lower floor separation gel upper strata, voltage is risen to 110V race and has just run out of can stop electrophoresis to bromjophenol blue.

(5) transferring film

1) open electricity and turn folder, spread a foam-rubber cushion in its black side, roll away the bubble of the inside.Turn at electricity the three metafiltration paper soaked in liquid in advance on foam-rubber cushion upper berth.

2) pry open glass plate, wipe concentrated glue off, cut the glue scope of molecular weight size.Its length and width measured by ruler, are placed on filter paper by the separation gel cut.Get pvdf membrane, cut the film of suitable size according to the length and width measured, cut certain angle and mark.The pvdf membrane sheared is immersed in methanol and activates 5min, turn in liquid at electricity and soak several seconds balance.

3) by membrane cover on glue, remove bubble after completely whole glue be covered.

4) be placed on after on film remove bubble by turning at electricity three filter paper soaked in liquid in advance.Finally cover another foam-rubber cushion, close clip.

5) install electrotransfer instrument, 300mA turns 60min.

(6) close

After film TBS immersion is wet after transferring film terminates, move to containing in 5% defatted milk powder (TBST solution preparation) plate, on decolorization swinging table, room temperature closes 90min.

(7) immunoreation

1) after having closed, by primary antibodie (Nrf2, Sirt1 1: 1000 dilutes, and β-tublin 1: 3000 dilutes) the 4 DEG C of overnight incubation after film and corresponding dilution.

2) unnecessary primary antibodie is washed away.TBST × 2 time, 10min; TBS × 1 time, 10min.

3) primary antibodie two anti-binding.Two anti-(1: 8000 dilutions) that washed film is placed in goat antirabbit HRP labelling hatch 90min in shaking table.

4) wash away unnecessary two to resist.TBST × 2 time, 10min; TBS × 1 time, 10min.

(8) fluorescence imaging

A, B liquid in 1: 1 mixing ECL luminescence reagent box, is evenly applied on film, the imaging of ImageQuant LAS 4000 chemiluminescence imaging analyser.

(9) result is quantitative

Use Bio-Rad Labworks image acquisition and analysis software image analysis software to protein band quantitative analysis integral optical density value.Destination protein expression represents with the ratio of destination protein band optical density value with internal reference band optical density value.

1.1.5 immuno-precipitation detects the Acetylation Level that PQ exposes mice AEC-IINrf2

(1) the same Western-bolt method prepared by sample

(2) co-immunoprecipitation gets above-mentioned cell pyrolysis liquid adds 1 μ g Nrf2 antibody and 50 μ l Protein A sepharose 4Bs ratio mixing according to 1ml lysate, 4 DEG C of slow overnight incubation of shaking table.

(3) co-precipitation complex collects that to get immunoprecipitation complex centrifugal, and 4 DEG C, 3000g × 5min, by centrifugal for Protein A sepharose 4B at the bottom of pipe, carefully suck supernatant, 1ml lysis buffer washes Protein A sepharose 4B 4 times.With 60 μ l 2 × SDS sample-loading buffers by resuspended for sepharose 4B-antigen antibody complex, mix gently.100 DEG C are boiled 10 minutes, carry out Western-bolt.

(4) Western-bolt primary antibodie selects acetylated lysine antibody 1: 1000 to dilute, the same 1.1.3 of remaining step.1.1.6 chemical colorimetry detects SOD, CAT, GSH, MDA

1.1.4.1 sample prepares the mice AEC-II turned then to be inoculated in 6 orifice plates with 0.5 × 105/ hole.In 37 DEG C, cultivate 24h in 5%CO2 incubator.Add 400 μMs of PQ solution effects 24h.Collecting cell precipitation or supernatant.

1.1.4.2 Indexs measure

1.SOD detects

(1) operating procedure

Reagent	Measure pipe	Control tube
			Reagent one (ml)	1.0	1.0
Sample to be tested (ml)	0.05
			Distilled water (ml)		0.05
Reagent two (ml)	0.1	0.1
			Reagent three (ml)	0.1	0.1
Reagent four (ml)	0.1	0.1

After swirl mixing device fully shakes mixing, 37 DEG C of water bath with thermostatic control 40min

Developer (ml)

2.0

2.0

Mixing, incubated at room 10min, in wavelength 550nm place, uses the cuvette of 1cm optical path, and with distilled water zeroing, colorimetric surveys absorption photometric value.

(2) SOD vigor is calculated

1) unit definition: SOD amount corresponding when every milligram of histone SOD suppression ratio in 1ml reactant liquor reaches 50% is called a SOD unit of activity (U).

2) formula:

2.CAT detects

(1) measuring principle: the reaction of CAT decomposing H 2O2 is by adding ammonium molybdate and Quick stop, and remaining H2O2 and ammonium molybdate effect produce a kind of flaxen complex, and 405nm place measures its growing amount calculates CAT vigor with this.

(2) operating procedure:

After abundant mixing, 37 DEG C of accurate response 1min

Reagent three (ml)	1.0	1.0
			Reagent four (ml)	0.1	0.1
Sample to be tested (ml)	0.05

Mixing, 405nm place, 0.5cm optical path cuvette, with distilled water zeroing, measures each pipe absorbance.

(3) CAT vigor is calculated

1), unit definition: the amount of decomposing 1 μm of ol H2O2 with every milligram of histone each second is a CAT unit of activity.

2), computing formula:

Wherein, 271* is the inverse of slope.

3.GSH detects

(1) measuring principle: GSH can react with dithiobis-nitrobenzoic acid (DTNB), generates a kind of yellow compound, can carry out the content that colorimetric assay measures GSH under 405nm.

(2) operating procedure:

1) supernatant preparation: get 10% tissue homogenate 0.1ml, add 0.1ml reagent one mixing, 3500 revs/min, centrifugal 10 minutes, get supernatant to be measured.

2) chromogenic reaction:

Mixing, leave standstill 5 minutes, 405nm place, microplate reader measures each hole absorbance.

3) GSH content is calculated:

4.MDA detects

(1) measuring principle: the MDA in lipid peroxide catabolite can with thiobarbituricacidα-(TBA) condensation, form red compound, the latter has maximum absorption band at 532nm place.

(2) operating procedure:

Mixed by above-mentioned mixed liquor swirl mixing device, antistaling film tightens test tube mouth, stings an aperture thereon with syringe needle, boiling water bath 40 minutes, after taking out flowing water cooling, 3500 revs/min, centrifugal 10 minutes, get supernatant, 532nm place, 1cm optical path cuvette, with distilled water zeroing, surveys each pipe absorbance.

(3) MDA content is calculated:

1.1.7 ELISA detects HO-1 expression

(1) standard substance liquid is prepared:

In standard substance, add the mixing of 0.5ml distilled water can be mixed with standard solution.Get standard pipe 8, each pipe adds sample diluent 200 μ l.The 2nd pipe is moved to sample injector sucking-off 200 μ l again after the standard solution 200 μ l added by 1st pipe fully mixes.Two-fold dilution so repeatedly, in last 7th pipe, sucking-off 200 μ l discards.8th pipe is as blank.10 × sample diluent distilled water does 1: 10 times of dilution.Cleaning mixture distilled water 1: 20 dilutes.

(2) application of sample:

The accurate sample wells of bidding, testing sample hole, blank control wells, multiple hole is set up in every hole.Wherein do not add sample and enzyme marking reagent in blank control wells.Add 100 μ l standard substance or testing samples to the every hole on enzyme reaction plate, Sptting plate is fully mixed rearmounted 37 DEG C and hatch 120min.

(3) plate is washed:

Discard liquid, dry Sptting plate, it is fully washed 5 times by rear cleaning mixture, gets rid of cleaning mixture, dry by filter paper print.

(4) in every hole, primary antibodie working solution 100 μ l is added.Sptting plate is fully mixed and is placed on 37 DEG C and hatches 60min.

(5) plate is washed: the same.

(6) enzyme labelled antibody working solution 100 μ l adds every hole.Sptting plate is placed in 37 DEG C and hatches 30min.

(7) plate is washed: the same.

(8) every hole adds substrate working solution 100 μ l, is placed in 37 DEG C of dark place reaction 15min.

(9) 100 μ l stop buffers are added and mix homogeneously in every hole.

(10) measure:

Microplate reader is used to survey each hole absorbance in 450nm place in 30min.All OD values give calculating again after subduction blank value.

(11) drawing standard curve:

Using standard concentration as abscissa, OD value, as vertical coordinate, graph paper is mapped, and draws standard curve and calculates the linear regression equation of standard curve.

(12) OD value finds corresponding HO-1 content on canonical plotting per sample, then is multiplied by the concentration that extension rate can calculate sample.

1.1.8 CO-IP method detects the interaction between Sirt1 and Nrf2 albumen

(1) according to the experimental result of this research team Lee holt etc., determine that employing time and dosage are respectively 24h, the PQ solution of 800 μMs stimulates ACE-II;

(2) with the PBS washed cell 2 times of pre-cooling, PBS is blotted for the last time;

(3) every hole adds the protein lysate (mild) of 1ml pre-cooling, is scraped by cell with cell sleaker from culture plate, ensures that cell number is 1 × 10 ⁷left and right;

(4) transferred to by cell suspension in 1.5ml EP pipe, 4 DEG C are slowly shaken 15min;

(5) 4 DEG C, 12000r/m × 10min, transfers in new EP pipe by supernatant;

(6) with PBS, Protein A/G PLUS-Agarose is mixed with the liquid of concentration 50%, and rifle head tip is cut;

(7) add 50 μ l Protein A/G PLUS-Agarose (50%) in every 1ml total protein, 4 DEG C of shaking tables slowly shake 3h and carry out pre cleaning, to remove non-specific foreign protein, reduce background;

(8) 12000r/m × 30s, transfers in new EP pipe by supernatant, BCA method measures protein concentration, and drawing standard curve, is diluted to 1 μ g/ μ l by protein concentration, to reduce the impact of detergent with PBS.

(9) get 50 μ l and be used as input, remaining supernatant is divided into A, B two groups, each group liquid adds PBS to 1ml;

(10) wherein A group adds 10 μ gNrf2 antibody, and B group adds equivalent negative control antibody, gentle shake 2h on 4 DEG C of shaking tables;

(11) often organize and add 50 μ l Protein A/G PLUS-Agarose (50%), 4 DEG C of incubator overnight;

(12) 4 DEG C, 14000r/m × 30s is centrifugal abandons supernatant, washes 4 times with PBS, removes liquid as far as possible with No. 4 syringe needles;

(13) hanged by ProteinA-antibody-protein complexes with 20 μ l 2 × albumen sample-loading buffers, jog mixes;

(14) respectively by input, complex in 95 DEG C, boil 5min, with dissociate Pro tein A/G PLUS-Agarose, antibody, albumen;

(15) 4 DEG C, 12000r/m × 10min, get supernatant in-20 DEG C of preservations, the protein electrophoresis western-blot for next step is qualitative.

Vitro Experimental Results:

1. om observation extracts mice AEC-II

After mice AEC II inoculates, 12 ~ 18h starts to stretch adherent, and rounded or cube, has a large amount of fine particle in Cytoplasm, island mode grows.After cultivating 24h, merge gradually, after 48h, cell is open and flat in polygon, is interconnected to cell monolayer.

2. specific proteins SP-C identified by immunofluorescence extracts mice AEC-II

Mice AEC-II specific proteins SP-C is expressed in endochylema, the visible SP-C protein expression in green particles shape under fluorescence microscope, confirm to extract cell be mice AEC-II.

3.PQ exposes the impact on Sirt1, Nrf2 gene expression

3.1 RT-PCR detect 800 μMs of PQ stimulates Sirt1, Nrf2 gene expression after mice AEC-II different times.Result shows, and 800 μMs of PQ can raise mice AEC-II Nrf2, Sirt1 gene expression, and this effect is the most obvious at 12h.But extend with PQ stimulation time, Nrf2, Sirt1 express and are tending towards declining gradually.After result shows 800 μMs of PQ effect different times equally, Nrf2, Sirt1 are consistent in the expression variation tendency of gene level.

3.2 Sirt1, Nrf2 gene expression after RT-PCR detection variable concentrations PQ stimulation mice AEC-II 24h.Experimental result shows, and the PQ effect of low dosage can raise Nrf2, Sirt1 gene expression, and this effect is the most obvious at 800 μMs.But after PQ Dosages strengthens, Nrf2, Sirt1 gene expression is tending towards declining gradually.After result shows variable concentrations PQ effect 24h equally, Nrf2, Sirt1 are consistent in the expression variation tendency of gene level.

PQ exposes the impact on Sirt1, Nrf2 protein expression

4.1 Western-bolt detect 800 μMs of PQ and act on Sirt1, Nrf2 protein expression after mice AEC-II different time.Result shows, and 800 μMs of PQ can raise mice AEC-II Nrf2, Sirt1 protein expression, and this rise effect peaks when 12h, and PQ stimulation time extends subsequently, and Nrf2, Sirt1 protein expression declines all gradually.After showing 800 μMs of PQ effect different times by destination protein band optical density value with the ratio of internal reference band optical density value, the expression change of Nrf2, Sirt1 protein level is consistent, and sees Fig. 3 A, 4A

4.2 Sirt1, Nrf2 protein expression after Western-bolt detection variable concentrations PQ stimulation mice AEC-II 24h.Result show, low dosage PQ effect can raise Nrf2, Sirt1 protein expression, and this effect peaks when PQ 800 μMs, and subsequently with the rising of PQ dosage, Nrf2, Sirt1 protein expression declines gradually, and the downward trend of Nrf2 comparatively Sirt1 is faster.Destination protein band optical density value is consistent in protein expression level variation tendency with Nrf2, Sirt1 after the ratio result display variable concentrations PQ effect 24h of internal reference band optical density value.See Fig. 3 B, 4B

5.PQ exposes the impact on Nrf2 protein acetylation level

Co-IP detects the Acetylation Level of Nrf2 albumen in cell; result confirms: after PQ stimulates; the Acetylation Level of Nrf2 albumen reduces within the specific limits gradually; after PQ effect is greater than 800 μMs (24h) more than 12h (800 μMs) and concentration, Nrf2 protein acetylation level starts to raise.This variation tendency presents contrary trend substantially with the expression in the Sirt1 albuminous cell that we detect.

6.PQ exposes the impact on mice AEC-II oxidative stress index

Experimental result shows: after 800 μMs of PQ act on mice AEC-II, in cell, the vigor of SOD, CAT presents downward trend with PQ extended durations of action, this downward trend stops after PQ effect 24h, and the vigor short period PQ effect of the display of 48h result SOD, CAT raises.After PQ (400 μMs) the effect 24h of the test results display low dosage of different PQ mass action 24h, in cell, the vigor of SOD, CAT declines, but after μM PQ effect 24h of 800 afterwards, its vigor gos up.After larger dosage (1200 μMs, 1600 μMs) effect, the vigor of SOD, CAT presents downward trend again.

After acting on mice AEC-II different time with 800 μMs of PQ, in cell, GSH content occurs declining with PQ extended durations of action, but after PQ effect 24h, the bottom out of GSH content.With the experimental group of different PQ mass action 24h, after the PQ (400 μMs) that we observe low dosage acts on 24h, in cell, GSH content declines, but afterwards after 800 μMs of PQ effect 24h, its content gos up again to some extent.After larger dosage (1200 μMs, 1600 μMs) effect, GSH content presents downward trend again.

Experimental result shows, and PQ extended durations of action and Dosages strengthen, and in cell, MDA content presents concentration dependent and time dependence decline.In table 2

Table 2 PQ stimulates mice AEC-II oxidative stress index to change (± s, n=4)

7.PQ exposes the change of mice AEC-II HO-1 content

After this experiment have detected the PQ stimulation of different activity and different action time, the expression change of HO-1 in mice AEC-II born of the same parents.Result shows: when identical PQ mass action, the content of HO-1 raises gradually with the prolongation of action time, raises slowly after 24h.The result display of variable concentrations PQ effect 24h, the content of HO-1 raises when low dose of (400 μMs, 800 μMs) effect, and along with the rising of PQ dosage after peaking at 800 μMs, HO-1 content reduces gradually.See Fig. 5

Described experiment in vivo comprises:

S201, mouse primary MSC extract and cultivate;

The identified by immunofluorescence of S202, BMSC;

S203, slow-virus transfection BMSC cell;

S204, PQ poisoning mice experiment grouping and model are set up;

S205, PRC method, Western-blot method detect the expression of Nrf2, Sirt1 albumen;

S206, chemical colorimetry detect MDA, SOD, GSH, CAT and change;

S207, ELISA method detect IL-1 β, TNF-α, IL-10, TGF-β 1 protein content;

The detection of S208, lung injury.

2. experiment in vivo

2.1 methods:

2.1.1 mouse primary MSC extracts and cultivates

1. ICR mice in male 6 week age is taken off neck to put to death, 75% alcohol-pickled 5min, proceeds to super-clean bench after slightly drying;

2. aseptically, from the root back side, afterbody nearly heart side, eye scissors cuts off skin, subcutaneous slightly do blunt separation after, Corium Mus is torn, makes it whole body and press-off;

3. along tibia body surface projection line, insert tibia by eye scissors passivity, to the muscle insertion blunt separation muscle of both sides and joint, completely take off tibia;

4. tibia is cut off in the middle of key, draw DMEM culture medium with 2ml syringe and repeatedly rinse medullary cavity, after the medullary cell gone out is blown and beaten into single cell suspension gently, proceed to centrifuge tube;

5. centrifugal abandon supernatant after, with containing the DMEM culture medium re-suspended cell of 10% hyclone, and add the erythrocyte that 3% glacial acetic acid solution cracking is mixed into, after cell counting, by 1 × 10 ⁵/ cm ²be inoculated in culture bottle in 37 DEG C, 8%CO ₂cultivate in cell culture incubator;

6. change liquid after cultivating 48h, discard non-attached cell, every 3d changes liquid 1 time afterwards, and Growth of Cells to 90% is used 0.25% trypsinization, just can be obtained the higher Marrow Mesenchymal Stem Cells of purity about the third generation that repeatedly goes down to posterity after merging.

2.1.2 the identified by immunofluorescence of BMSC

With reference to Part I experimental technique 1.1.2

2.1.3 slow-virus transfection BMSC cell

1. be inoculated in by the BMSC being in logarithmic (log) phase growth conditions on 6 porocyte culture plates, every hole is placed on an aseptic coverslip in advance, makes cell climbing sheet, and according to preliminary result in early stage, determines that MOI is 50;

2. Dual culture 3d after transfection, takes out cell climbing sheet and is placed on microscope slide, observe luciferase expression situation, the expression of Western-blot testing goal albumen.

2.1.4 PQ poisoning mice experiment grouping and model are set up

1. divide into groups:

Experiment divides 8 groups: blank group, NS group, PQ group, PQ+NS group, PQ+LV-MSC-GFP group, PQ+LV-MSC-Nrf2 group, PQ+LV-MSC-Sirt1 group, PQ+LV-MSC-Nrf2/Sirt1 group.

2. process:

Animal fasting 8h before experiment, prohibits water 4h.1. PQ+LV-MSC-Nrf2/Sirt1 group: through left side lumbar injection 20%PQ solution, after contamination in 1min through tail vein injection concentration be 10 × 10 ⁶/ ml LV-MSC-Nrf2 cell suspension and each 0.05ml of LV-MSC-Sir1 cell suspension; 2. PQ+LV-MSC-Nrf2 group: through left side lumbar injection 20%PQ solution, after contamination in 1min through tail vein injection concentration be 5 × 10 ⁶/ ml LV-MSC-Nrf2 cell suspension 0.1ml; 3. PQ+LV-MSC-Sirt1 group: through left side lumbar injection 20%PQ solution, after contamination in 1min through tail vein injection concentration be 5 × 10 ⁶/ ml LV-MSC-Sirt1 cell suspension 0.1ml; 4. PQ+LV-MSC-GFP group: through left side lumbar injection 20%PQ solution, after contamination in 1min through tail vein injection concentration be 5 × 10 ⁶mlLV-MSC-GFP cell suspension 0.1ml; 5. PQ+ normal saline group: through abdominal cavity, left side disposable injection 20%PQ solution, after contamination, 1min is interior through right side lumbar injection; 6. PQ group: through left side lumbar injection 20%PQ solution; 7. NS group: through the isopyknic normal saline of left side lumbar injection; 8. blank group: do not give any process.

3. observe each group of mice behavior to change

The glossy degree of fur, spontaneous activity, action flexibility ratio etc. that PQ contamination observes each group of mice afterwards poisoning rear every day.

2.1.5 the expression of PRC method, the detection of Western-blot method Nrf2, Sirt1 albumen

Experimental procedure is with reference to Part I experimental technique 1.1.3 and 1.1.4

2.1.6 chemical colorimetry detects MDA, SOD, GSH, CAT change

1. the preparation of lung homogenate

Get lung tissue 200mg, add the normal saline of 1.8ml pre-cooling after shredding, glass homogenizer makes the lung homogenate of 10%; 4 DEG C, 3000r/min × 10min, leaves and takes supernatant, and-80 DEG C of cryopreservation are to be measured.

2. more than, experimental procedure is with reference to Part I experimental technique 1.1.6

2.1.7 ELISA method detects IL-1 β, TNF-α, IL-10, TGF-β 1 protein content

1. preparation:

(1) in advance Elisa test kit is taken out balance to room temperature;

(2) with distilled water, concentrated cleaning solution is diluted by 1: 20;

(3) standard substance are prepared: get in 0.5ml reagent dilutions to lyophilizing standard substance and make standard concentration be 1000pg/ml, room temperature leaves standstill 15min, after it fully dissolves, mixes gently, carries out doubling dilution as required.

(4) biotinylated antibody working solution: with reagent dilutions by 1: 100 dilution concentrated biological elementization antibody, be mixed with biotinylated antibody working solution, matching while using.

(5) enzyme conjugates working solution: with reagent dilutions by the concentrated enzyme conjugates of 1: 100 dilution, be mixed with enzyme conjugates working solution, matching while using.

2. in ELISA Plate, set up blank control wells, standard sample wells, testing sample hole, multiple hole is set up in every hole.Standard substance also add variable concentrations standard substance and testing sample 100 μ l respectively with testing sample hole, seal reacting hole, hatch 90min in 37 DEG C with shrouding gummed paper;

3. every hole adds cleaning mixture 350 μ l, gets rid of liquid in most hole, absorbent paper pats dry, washes plate 5 times after each standing 30s;

4., except blank control wells, every hole adds biotinylated antibody working solution 100 μ l, seals reacting hole, hatch 60min in 37 DEG C with shrouding gummed paper;

5. wash plate 5 times;

6., except blank control wells, every hole adds enzyme conjugates working solution 100 μ l, seals reacting hole with shrouding gummed paper, hatches 30min in 37 DEG C of lucifuges;

7. wash plate 5 times;

8. every hole adds 100 μ l chromogenic substrates, and 37 DEG C of lucifuges hatch 15min;

9. every hole adds 100 μ l stop buffers, in 450nm place after fully mixing, measures OD value in 10min;

10. be abscissa with standard concentration, OD value is vertical coordinate, drawing standard curve, and the linear regression equation calculating standard curve;

11. OD value can calculate corresponding protein content according to formula per sample, then be multiplied by the concentration that extension rate is testing sample.

2.1.8 the detection of lung injury

Often organize random selecting 2 mices, with 10% chloral hydrate intraperitoneal injection of anesthesia, after eyeball gets blood, open thoracic cavity rapidly, get the left lung tissue of mice and fix in 4% paraformaldehyde, HE dyes, and makes pathological examination; Superior lobe of right lung apex pulmonis portion is placed in 2.5% glutaraldehyde solution and fixes, make transmission electron microscope observing after adopting microscope slide squeezing and pressing method to extrude discharge bubble gently.

1. Pathologic specimen makes

(1) carry out paraffin embedding by Pathology Deparment personnel, carry out tissue slice;

(2) paraffin section de-waxing, conventional dewaxing is to aquation: dimethylbenzene I, dimethylbenzene II, 100% ethanol I, 100% ethanol II, each 10 minutes.Each 10 minutes of 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, distillation washing.

(3) 0.1% haematoxylin dyeings 1 minute, distilled water flushing 5min;

(4) 1% hydrochloride alcohol differentiation 3-5s, distilled water flushing is about 10min;

(5) 0.5% eosin stains 1-2 minute;

(6) successively through 70%, 80%, 90%, 95% ethanol ladder dehydration, each 2 minutes.Each 5 minutes of anhydrous alcohol I, anhydrous alcohol II, dimethylbenzene is transparent;

(7) section is taken out, guarantee to drip appropriate neutral gum rapidly before the non-bone dry of dimethylbenzene, then add coverslip sealing, row om observation.

2. transmission electron microscope preparation of specimen

(1) specimen is taken out from 2.5% glutaraldehyde fixative, with PBS rinsing 15min × 3 time, remove residual glutaraldehyde as far as possible;

(2) specimen is moved in 1% osmic acid fixative, continue to rock 5min, fixing 1h;

(3) with PBS rinsing 15min × 2 time, ddH is used ₂o rinsing 15min × 2 time;

(4) specimen is placed in 1% acetic acid uranium block dye 2h;

(5) specimen is immersed successively in 70%, 80%, 90% acetone the 15min that respectively dewaters, pure acetone dewaters 2 times, each 20min;

(6) specimen is placed in acetone successively: embedding liquid=1: 1h in 1,37 DEG C of baking ovens; Acetone: embedding liquid=1: spend the night in 4,37 DEG C of baking ovens; Pure embedding liquid 45 DEG C of baking oven 1h;

(7) ready embedded block bar code and embedded block are together placed in embedding mould, 45 DEG C of baking oven 6h, 65 DEG C of baking oven 2d;

(8) embedded block is made into semithin section location AEC-II, then is made into ultrathin section by Electron Microscopy Room professional, row electron microscopic observation.

1, process LAN slow virus carrier transfection BMSC verifies

Infected by LV-Nrf2-GFP, LV-Sirt1-GFP, determine that MOI value is 50 according to preliminary experiment in early stage, the efficiency of infection of virus approximately reaches more than 80%, and Western-blot detection Nrf2, Sirt1 protein expression obviously raises, and meets requirement of experiment.

2, mice behavior changes

Experimental result shows, and blank group mice ordinary circumstance is good, and physical agility is breathed well-balanced, hair color gloss, without uncomfortable reaction; NS group and blank mice are without obvious difference.Occur being slow in action after the contamination of PQ group mice, dyspnea, feed reduces, and hair color is sent out dim, and about 3d is the most obvious.If dead in 3d, then intoxication conditions will take a turn for the better to some extent subsequently, meal situation comparatively before improve, hair color recovers gradually, but time have mouth breathing, compare with blank group mice, body weight entirety increases not obvious; PQ+NS group, PQ+LV-MSC-GFP group and PQ group mice are without obvious difference.PQ+LV-MSC-Sirt1 group ordinary circumstance comparatively PQ group is light, breathes in new line sample, how drowsiness, but poisoning symptom occurs more late.PQ+LV-MSC-Nrf2/Sirt1 group ordinary circumstance is better, and action comparatively PQ group is quick, and poisoning symptom occurs the latest.The performance of PQ+LV-MSC-Nrf2 group mice is between PQ+LV-MSC-Sirt1 group and PQ+LV-MSC-Nrf2/Sirt1 group.The feed in the 7th day after contamination of each intervention group mice starts to increase, frequent activity.

The process LAN slow virus of 3, carrying genes of interest intervenes PQ poisoning mice to the impact of lung tissue Nrf2, Sirt1 albumen

In blank group and NS group mouse lung tissue, Nrf2, Sirt1 protein content is in reduced levels; Compare with blank group, after contamination, 3d, PQ group, PQ+NS group and PQ+LV-MSC-GFP group Nrf2, Sirt1 protein expression obviously raise (P < 0.05), during 21d, with normal group zero difference; Compare with PQ group, 3d after contamination, in PQ+LV-MSC-Nrf2 group, PQ+LV-MSC-Sirt1 group, PQ+LV-MSC-Nrf2/Sirt1 group, Nrf2, Sirt1 protein expression all has increasing (P < 0.05) in various degree, to 21 time, in each group, Nrf2, Sirt1 protein expression is still in higher level (P < 0.05), wherein PQ+LV-MSC-Nrf2/Sirt1 group the highest (P < 0.05).

The process LAN slow virus of 4, carrying genes of interest intervenes PQ poisoning mice to the impact of lung tissue Nrf2, Sirt1 gene

In blank group and NS group mouse lung tissue, Nrf2, Sirt1 gene expression is all in reduced levels; Compare with blank group, after contamination, 3d, PQ group, PQ+NS group and PQ+LV-MSC-GFP group Nrf2, Sirt1 gene expression obviously raise (P < 0.05); Compare with PQ group, 3d after contamination, in PQ+LV-MSC-Nrf2 group, PQ+LV-MSC-Sirt1 group, PQ+LV-MSC-Nrf2/Sirt1 group, Nrf2, Sirt1 gene expression all has increasing (P < 0.05) in various degree, PQ+LV-MSC-Nrf2/Sirt1 group the highest (P < 0.05).

5, the impact of oxidative stress level

Compare with blank group, PQ group MDA content obviously raises, and SOD, CAT activity is all lower, and GSH content declines; Compare with corresponding time point PQ group, PQ+LV-MSC-Nrf2 group, PQ+LV-MSC-Sirt1 group, PQ+LV-MSC-Nrf2/Sirt1 group MDA content obviously decline, and SOD, CAT are active, GSH content obviously raises.

Table 3 for regulation and control Nrf2/ARE/Sirt1 signal path on each oxidation factor in the damage of paraquat poisoning mouse lung impact

Table 3

6, the change of mice serum proinflammatory factor and anti-inflammatory factors

Blank group, mice serum TNF-α, IL-1 β, IL-10, TGF-β 1 content are all in normal reduced levels; Compare with blank group mice serum, during PQ group 3d, TNF-α, IL-1 β, IL-10, TGF-β 1 obviously raise (P < 0.05), and reaching peak value simultaneously, the amplitude that TNF-α, IL-1 β raise is overall higher than IL-10, TGF-β 1.As time goes on, each Inflammatory Factors Contents declines gradually; Compare with PQ group, same time point, PQ+LV-MSC-Nrf2 group, PQ+LV-MSC-Sirt1 group, PQ+LV-MSC-Nrf2/Sirt1 group TNF-α, IL-1 β content lower (P < 0.05), and IL-10, TGF-β 1 content obviously raises (P < 0.05), wherein PQ+LV-MSC-Nrf2/Sirt1 group intervention effect is best.

Table 4 is for regulation and control Nrf2/ARE/Sirt1 signal path is on the impact of each inflammatory factor in the damage of paraquat poisoning mouse lung

Table 4

7, mouse lung tissue pathological change

7.1 pathological change

Blank group mice is dissected and sees that lung tissue is pink colour, and thoracic cavity is without transudate.Under light microscopic, alveolar structure is complete, has no congested, edema in alveolar space.The two pulmonary congestion of 3d, PQ group mice after PQ contamination, volume slightly becomes large.Alveolar septum fracture under light microscopic, part alveolar collapse, is full of the edematous fluid of homogenizing powder dye, with diffuse inflammatory cellular infiltration in alveolar.PQ+LV-MSC-Nrf2 group, PQ+LV-MSC-Sirt1 group, PQ+LV-MSC-Nrf2/Sirt1 group alveolar septum all have fracture, but comparatively PQ group degree is comparatively light, and lung internal hemorrhage point is less, and edema is not obvious, and wherein PQ+LV-MSC-Nrf2/Sirt1 group degree of injury is the lightest.

7.2 transmission electron microscope results

In blank group, there is a large amount of microvilluss on AEC-II surface, and cellularity is complete, karyon ovalize, and nuclear membrane is complete, and Distribution of chromatin is even, and endochylema includes abundant mitochondrion and osmiophilic multilamellar body.PQ contaminates after 3d, and AEC-II surface microvillus reduces, cellular edema, expansion, and form edema band, nuclear membrane gap enlargement, lamellar body is loosened, and emptying phenomenon appears in part, forms pouch-shaped or strip cavity, mitochondrion pyknosis, reticulum dilatation; PQ+LV-MSC-GFP group shows similar to PQ group.And PQ+LV-MSC-Nrf2 group PQ+LV-MSC-Sirt1 group, PQ+LV-MSC-Nrf2/Sirt1 group are compared with PQ group, cellularity is substantially complete, mitochondrial swelling and reticulum dilatation degree have alleviating in various degree, and microvillus is substantially complete, and lamellar body has compensatory hypertrophy in various degree.

The above is only to preferred embodiment of the present invention, not any pro forma restriction is done to the present invention, every according to technical spirit of the present invention to any simple modification made for any of the above embodiments, equivalent variations and modification, all belong in the scope of technical solution of the present invention.

Claims (3)

1. an experimental technique for the new antioxidation path of Sirt1-Nrf2 application in oxidative damage treatment, is characterized in that, the experimental technique of the new antioxidation path of described Sirt1-Nrf2 application in oxidative damage treatment comprises experiment in vitro and in vivo test;

Described experiment in vitro comprises:

The separation and purification of step one, mice AEC-II;

Step 2, the epithelial qualification of mice alveolar type;

Step 3, RT-PCR detect time, the dose-effect relationship that PQ exposes Sirt1, Nrf2 gene expression change and PQ effect in mice AEC-II;

Step 4, Western-bolt method detect time, the dose-effect relationship that PQ exposes the change of Sirt1, Nrf2 protein expression and PQ effect in mice AEC-II;

Step 5, immuno-precipitation detect the Acetylation Level that PQ exposes mice AEC-IINrf2;

Step 6, chemical colorimetry detect SOD, CAT, GSH, MDA;

Step 7, ELISA detect HO-1 and express;

Step 8, CO-IP method detect the interaction between Sirt1 and Nrf2 albumen;

Described experiment in vivo comprises:

Step one, mouse primary MSC extract and cultivate;

The identified by immunofluorescence of step 2, BMSC;

Step 3, slow-virus transfection BMSC cell;

Step 4, PQ poisoning mice experiment grouping and model are set up;

Step 5, PRC method, Western-blot method detect the expression of Nrf2, Sirt1 albumen;

Step 6, chemical colorimetry detect MDA, SOD, GSH, CAT and change;

Step 7, ELISA method detect IL-1 β, TNF-α, IL-10, TGF-β 1 protein content;

The detection of step 8, lung injury.

2. the experimental technique of the new antioxidation path of Sirt1-Nrf2 as claimed in claim 1 application in oxidative damage treatment, it is characterized in that, the PCR primer that described RT-PCR detection PQ exposes in the time of Sirt1, Nrf2 gene expression change and PQ effect in mice AEC-II, dose-effect relationship is:

Sirt1, upstream sequence is 5 '-acgctgtggcagattgttatta-3 ', and downstream sequence is 5 '-ttgaagaatggtcttgggtctt-3 ';

Nrf2, upstream sequence is 5 '-attctttcagcagcatcctctc-3 ', and downstream sequence is 5 '-acacttccaggggcactatcta-3 ';

β-actin, upstream sequence is 5 '-atatcgctgcgctggtcgtc-3 ', and downstream sequence is 5 '-aggatggcgtgagggagagc-3 '.

3. the experimental technique of the new antioxidation path of Sirt1-Nrf2 as claimed in claim 1 application in oxidative damage treatment, it is characterized in that, described PQ poisoning mice experiment is divided into 8 groups: blank group, NS group, PQ group, PQ+NS group, PQ+LV-MSC-GFP group, PQ+LV-MSC-Nrf2 group, PQ+LV-MSC-Sirt1 group, PQ+LV-MSC-Nrf2/Sirt1 group;

The process often organizing mice is as follows:

Animal fasting 8h before experiment, prohibits water 4h;

PQ+LV-MSC-Nrf2/Sirt1 group: through left side lumbar injection 20%PQ solution, after contamination in 1min through tail vein injection concentration be 10 × 10 ⁶/ ml LV-MSC-Nrf2 cell suspension and each 0.05ml of LV-MSC-Sir1 cell suspension;

PQ+LV-MSC-Nrf2 group: through left side lumbar injection 20%PQ solution, after contamination in 1min through tail vein injection concentration be 5 × 10 ⁶/ ml LV-MSC-Nrf2 cell suspension 0.1ml;

PQ+LV-MSC-Sirt1 group: through left side lumbar injection 20%PQ solution, after contamination in 1min through tail vein injection concentration be 5 × 10 ⁶/ ml LV-MSC-Sirt1 cell suspension 0.1ml;

PQ+LV-MSC-GFP group: through left side lumbar injection 20%PQ solution, after contamination in 1min through tail vein injection concentration be 5 × 10 ⁶ml LV-MSC-GFP cell suspension 0.1ml;

PQ+ normal saline group: through abdominal cavity, left side disposable injection 20%PQ solution, after contamination, 1min is interior through right side lumbar injection;

PQ group: through left side lumbar injection 20%PQ solution;

NS group: through the isopyknic normal saline of left side lumbar injection;

Blank group: do not give any process.