CN105002178B - Suppress STAT3 expression and prevent and treat shRNA and the application of chronic graft versus host disease - Google Patents

Abstract

It can effectively suppress STAT3 gene expressions the present invention relates to one and prevent and treat the shRNA of chronic graft versus host disease, and provide STAT3 shRNA to mice spleen CD4⁺CD62L⁺ The influence of cell differentiation, act on mouse bone marrow cells CD117⁺The security of early stage stem/progenitor cells, and the therapeutic action to murine chronic graft versus host disease(GVH disease) (cGVHD) model：Influence after being acted on including STAT3 shRNA to Th17/Treg ratios, cGVHD clinical scores and target organ pathological score.Research for the progress preventing and treating mankind cGVHD in living animal provides experimental basis.For developing, new anti-cGVHD medicines and raising cGVHD therapeutic effect have great importance.

Description

Suppress STAT3 expression and prevent and treat shRNA and the application of chronic graft versus host disease

Technical field

The present invention relates to a kind of shRNA, more particularly to one kind can suppress STAT3 expression and preventing and treating chronic graft versus host The shRNA small molecules targeted drug of disease and its application.

Background technology

Chronic graft versus host disease (chronic graft versus host disease, cGVHD) is that allogene is made Hemocytoblast transplants (allogeneic hemapoietic stem cells transplant, allo-HSCT) long-term surviving The major complications of patient, not only have a strong impact on the life quality of patient, are also to cause the main cause of non-Recurrent death.With Allo-HSCT extensive development, about 30%~70% patient middle position can occur in various degree for 4~6 months after the transfer CGVHD, seriously limits allo-HSCT application and development.GVHD is mainly due to there is immunocompetent donor source T cell The result reacted with receptor tissue.Research is thought at present, activation institutes of the cGVHD mainly due to donor autoreactive T cell The immunological rejection of initiation, existing immunosuppressive therapy is more to suppress T cell activation as main starting point, although these measures can Graft rejection is reduced to a certain extent, but still suffers from the recurrence such as leukaemia, or even because excessive immune suppresses and triggers and causes The complication such as the sexy dye of life, secondary second tumour, and suppressing deficiency then causes cGVHD to be difficult to control to.Find more effective, more accurate The new strategy of regulation and control immunity of organism inhibition is the focus of fields of implantation research, is also probably to solve graft rejection complication Fundamental solution.

In recent years, using RNA perturbation techniques, by the bob of chemical synthesis press from both sides RNA interfering (short hairpin RNA, ShRNA) transduction is to specific cell, and the expression of silence related gene is one of research maximally effective means of gene function, is also One of most attractive method of target gene therapy.Select specific target gene to synthesize effective shRNA, and select to close Suitable rotaring transfecting mode, makes it efficiently be acted in host cell, is the emphasis for implementing this targeted therapy measure.Effective targeting is controlled Treatment is at present both at home and abroad constantly in the treatment method of searching.Inmature CD4⁺Th cells can be activated and differentiation and maturation is to have difference The responsiveness Th cells of function.Th17 cells are one secretions of new discovery in the research of autoimmune disease pathogenesis IL-17, but its differentiation is independent of the cell factor and the CD4 of transcription factor needed for Th1 or Th2 differentiation⁺T cell subgroup. Th17 cells have wide research in autoimmune disease and graft rejection field, but relevant Th17 develops in GVHD In effect, just start to cause focus of attention in recent years.Our early-stage Study application chip gene expression profile find STAT3 and IL-17A, IL-21 are expressed in patient cGVHD and raised, and it was found that there is Th17 ↑/Treg ↓ unbalance in cGVHD peripheral blood in patients. Recently research is found：JAK/STAT signal transduction pathways are the important paths of Th polarizations, and wherein signal transduction and transcription swashs The factor 3 (signal transducer and activator of transcription 3) STAT3 living is Th17 cells point The core of the signal transduction pathway of change, is that T cell breaks up " fulcrum " to Th17 or Treg, dynamic between regulation and control Th17 and Treg State is balanced.

We combine the immune newest focus in field on the basis of early-stage Study, propose first by blocking STAT3, right Th17/Treg balances are regulated and controled, so as to reach the Research Thinking for suppressing cGVHD.Inquire into and block STAT3 signals to induction Th17, Treg break up and its effect mutually regulated and controled and mechanism, illustrate Th17/Treg " drift " and occur evolution in cGVHD In effect, provide experimental basis for the immunization therapy novel targets of further seeking cGVHD.Targeting is for STAT3 genes The shRNA sequences anti-cGVHD genomic medicine new for developing and raising cGVHD therapeutic effect have important meaning, with wide Wealthy application prospect and economic value.

The content of the invention

The primary and foremost purpose of the present invention is that STAT3 expression and preventing and treating chronic graft versus host can efficiently be suppressed by providing one kind The shRNA of disease.

The purpose of the present invention is achieved through the following technical solutions：One kind suppresses STAT3 expression and preventing and treating chronic graft resists The shRNA of host disease, composition sequence is as follows：

Positive-sense strand：

5 '-TCGACTTTGATTTCAACTACAACTCGAGTTGTAGTTGAAATCAAAGTCGTTTTTTC -3 ' antisense strands：

5’-TCGAGAAAAAACGACTTTGATTTCAACTACAACTCGAGTTGTAGTTGAAATCAAAGTCGA-3’

Described shRNA is applied to suppress the expression of STAT3 genes (NM_213659).Targetting sequence is ACTTTGATTTCAACTACAA。

Another object of the present invention is to provide a kind of can efficiently suppress or treat the medicine of chronic graft versus host disease.

The medicine contains shRNA as described above as the active component of medicine.The medicine is by slow virus carrier sense ShRNA medicines are integrated into target gene group by dye host cell, are resisted so as to suppress STAT3 gene expressions and preventing and treating chronic graft Host disease.

Prepare the above-mentioned pharmaceutical procedures that can efficiently suppress or treat chronic graft versus host disease as follows：

For mouse STAT3 gene orders, (RNAi) sequences Design principle is disturbed using the RNA provided in public website, For different 3 RNA interference sequences of shot design and RNAi negative control sequences, optimal kinetic parameter target spot is selected to enter Enter subsequent experimental flow.The single stranded DNA oligo containing interference sequence is synthesized, then annealed pairs produce double-strand.Pass through slow disease Malicious host cells infected reaches is integrated into target gene group by shRNA medicines, and its preparation process is：By the double-strand shRNA of synthesis, Directly it is connected on the slow virus carrier after digestion by restriction enzyme site contained by its two ends；Connection product is transferred to the bacterium prepared After competent cell, PCR identification positive recombinants, sequence verification is sent；Sequencing result confirms that correct clone is structure through comparing Build successful target gene RNAi and negative control slow virus carrier plasmid.Two kinds of Prepare restructuring slow virus plasmid are aided in simultaneously Original paper vector plasmid is packed, three kinds of vector plasmids carry out high-purity endotoxin-free extracting respectively, by Invitrogen companies The operation instructions of Lipofectamine 2000 carry out cotransfection 293T cells, collect the cell supernatant rich in lentiviral particle, The slow virus concentrate of high titre is obtained after being concentrated to it, is determined in 293T cells and demarcates virus titer.Immunomagnetic beads point Select mice spleenCell, activation 72h postoperative infections STAT3-shRNA and negative control slow virus, 96h are stayed Each group cell is taken, RNA, reverse transcription synthesis cDNA, the STAT3 interference of quantitative fluorescent PCR (dye method) checking mRNA level in-site is extracted Effect, filtering out the shRNA described in claim 1 is used to formally test.

The present invention has the following advantages and effect relative to prior art：

1st, the shRNA of suppression STAT3 of the invention expression is by slow-virus infection mice spleen Cell, after testing interference effect screening is obtained；The expression of STAT3 genes can efficiently be suppressed, compared with negative control group, mRNA The downward degree of level reaches 52 times or so, such as Fig. 1,2, shown in table 3.

2nd, the shRNA of suppression STAT3 of the invention expression acts on mice spleen by slow virus carrierAfter cell, each subpopulations direction (such as table 4) is influenceed, wherein Th17 ratios are reduced, and Treg ratios increase High (as shown in Figure 3,4), Th17/Treg ratios reduction is compared, the statistically significant (P of difference with negative control group<0.05).

3rd, the shRNA of suppression STAT3 of the invention expression acts on mouse bone marrow cells CD117 by slow virus carrier⁺In early days After stem/progenitor cells (Fig. 5), fluorescence quantitative PCR detection STAT3-shRNA slow-virus infection mouse bone marrow cells CD117⁺Early stage, dry ancestral was thin The relative expression quantity (internal reference gene is GAPDH) of STAT3 genes, is compared with negative control group, blank control group during born of the same parents 96h, Difference statistically significant (P of the STAT3-shRNA interference group STAT3 genes in the relative expression quantity mean of mRNA level in-site< 0.001, P<0.001)；Negative control group is compared with blank control group, relative expression quantity mean of the STAT3 genes in mRNA level in-site Difference not statistically significant (P=0.063) (Fig. 6).Interference group cell proliferation activity (such as table 5, Fig. 7), early apoptosis rate are (such as Table 6, Fig. 8) and vitro differentiation be each system's hematopoietic colonies ability (such as table 7, Fig. 9), compared with control group, difference that there are no significant (P>0.05) its security, is illustrated.

4th, the shRNA of suppression STAT3 of the invention expression is applied to vivo therapeutic mouse cGVHD models, and cGVHD is clinical Scoring (such as table 8, Figure 10) and target organ pathological score (such as table 9, Figure 11-12) are reduced, and are compared with negative control group, difference has Statistical significance (P<0.05).

5th, it is related to the shRNA sequences of STAT3 genes for the new anti-cGVHD genomic medicines of exploitation and improves controlling for cGVHD Therapeutic effect has important meaning, has broad application prospects and economic value.

The shRNA of the present invention can be used for the expression for suppressing STAT3 genes, for preparing treatment chronic graft versus host disease Small molecule targeted drug, infected by slow virus carrier and be integrated into aim cell genome, the table of silence STAT3 genes Reach, play therapeutic action.

Brief description of the drawings

Fig. 1 is by fluorescence microscope STAT3-shRNA slow-virus infection mouse in embodiment 1Cell, wherein (a) is blank control group carried out in the case of 200 times of white lights (Bright) it is aobvious Micro mirror is observed；(b) it is micro- sem observation that blank control group is carried out in the case of 200 times of fluorescence (GFP)；(c) it is blank pair The micro- sem observation carried out according to group in the case of 200 times of white fluorescent fusions (Merge)；(d) it is shRNA2 groups in 200 times of white lights (Bright) the micro- sem observation carried out in the case of；(e) it is that shRNA2 groups are carried out in the case of 200 times of fluorescence (GFP) Micro- sem observation；(f) it is micro- sem observation that shRNA2 groups are carried out in the case of 200 times of white fluorescent fusions (Merge).

Fig. 2 is by the slow virus of fluorescence quantitative PCR detection STAT3-shRNA1,2,3 and negative control virus in embodiment 1 Infecting mouse spleenThe mRNA level in-site relative expression quantity of STAT3 genes during cell 96h.

Fig. 3 is infected by Flow cytometry STAT3-shRNA2 slow virus and negative control virus in embodiment 2 Mice spleenCD4 during cell 96h⁺CD25⁺FOXP3⁺Treg cell subsets, wherein (a) represents gating, it is purple Color represents interference group CD4+ cell mass, and (b) represents interference group Treg cell subset Isotype controls, and (c) represents interference group Treg Cell subset, (d) represents gating, and purple represents negative control group CD4+ cell mass, and (e) represents negative control group Treg subgroups Cell Isotype control, (f) represents negative control group Treg cell subsets.

Fig. 4 is infected by Flow cytometry STAT3-shRNA2 slow virus and negative control virus in embodiment 2 Mice spleenCD4 during cell 96h⁺IL-17A⁺Th17 cell subsets, wherein (a) represents gating, purple generation Surface drying disturbs group CD4+ cell mass, and (b) represents interference group Th17 cell subset Isotype controls, and (c) represents interference group Th17 subgroups Cell；(d) gating is represented, purple represents negative control group CD4+ cell mass, and (e) represents negative control group Th17 cell subsets Isotype control (f) represents negative control group Th17 cell subsets.

Fig. 5 is the shRNA2 slow virus senses for applying suppression STAT3 expression of the invention in fluorescence microscope embodiment 3 Contaminate mouse bone marrow cells CD117⁺Early stage stem/progenitor cells 96h figure, wherein (a) represents 200 times of white light, (b) represents 200 times of fluorescence, (c) 400 times of white light is represented, (d) represents 400 times of fluorescence.

Fig. 6 is by fluorescence quantitative PCR detection STAT3-shRNA2 slow-virus infection mouse bone marrow cells CD117 in embodiment 3⁺ The mRNA level in-site relative expression quantity of STAT3 genes during early stage stem/progenitor cells 96h, is from left to right blank control group, shRNA respectively Interference group and negative control group.

Fig. 7 is infection 96h CCK8 method detection each group mouse bone marrow cells CD117⁺Cell OD values, are from left to right blank respectively Control group, shRNA interference group and negative control group.

Fig. 8 is Flow cytometry infection 96h each groups CD117⁺Stem/progenitor cells apoptosis rate, wherein (a) represents blank pair According to group, (b) represents shRNA2 groups, and (c) represents negative control group.(mean of 3 testing results of digitized representation in figure bracket ± Standard deviation)

Fig. 9 is mouse CD117⁺Stem/progenitor cells vitro differentiation is each system's hematopoietic colonies figure (14d inverted microscopes 100-400 Times), it is from left to right blank control group, shRNA interference group and negative control group respectively, is CFU-E, CFU- respectively from top to bottom GM,CFU-T。

Figure 10 is the mean schematic diagram of each time point difference group clinical score.

Figure 11 is shRNA2 treatment groups and negative control group pathology schematic diagram.

Figure 12 is the cGVHD pathological scores of the different target organs of each group.

Figure 13 is STAT3-shRNA slow virus carrier structure chart.

Figure 14 is negative control RNA slow virus carrier structure chart.

Figure 15 is two kinds of auxiliary packaging original paper carrier structure figures, and wherein a is the carriers of pHelper 1.0, and b is pHelper 2.0 carrier.

Embodiment

With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited In this.

Embodiment 1

This example is to use STAT3-RNAi slow-virus infection mouseCell, is filtered out optimal STAT3-RNAi。

First, the preparation of RNAi slow virus carriers

1st, shRNA shot designs

Gene Name：STAT3 species：Mouse

For STAT3 gene orders, using the RNA interference sequence design principles provided in public website, multiple RNA are designed Interfered target sequence, 3 optimal kinetic parameter target spots of selection enter subsequent experimental flow.

Shown in its result such as following table (table 1).

The mouse STAT3-shRNA interfered target sequences of table 1

While generally acknowledged sequence in some RNA interference experiments of use, such as RNAi negative controls (Negative Control, NC) Scramble sequences (TTCTCCGAACGTGTCACGT) are used as negative control.

Gene information：Scramble sequences

Core sequence：TTCTCCGAACGTGTCACGT

It is compared by NC sequences in gene pool, as a result two genes of the NC sequences only with Zebrafish have continuously The homology of 16 bases, in addition to two above gene, any sequence in the sequence and genome does not have continuous 16 alkali The homology of base, therefore general type negative control can be used as.

2nd, 3 STAT3-shRNA and negative control RNA are synthesized

The single stranded DNA oligo containing the interference sequence of shRNA1,2,3 or negative control RNA sequence is synthesized first, is then moved back Fire pairing produces double-strand (synthesis of Shanghai Ji Kai gene technology Co., Ltd), then is directly connected into by restriction enzyme site contained by its two ends GV118 or GV112 carriers after digestion (carrier is that Shanghai Ji Kai gene technology Co., Ltd provides).

3rd, purpose and negative control slow virus carrier are built：Clone prepares and identification

Container name：GV118, GV112, wherein GV118 are used to build STAT3-shRNA slow virus carriers, and GV112 is used for Build negative control slow virus carrier.

GV118 element orders：U6-MCS-Ubi-EGFP GV112 original papers order：(MCS contains digestion to U6-MCS-CMV-uro Site)

Connection product in 2 is transferred to after the bacterium competent cell prepared, PCR identification positive recombinants, send sequencing to test Card；Sequencing result is through comparing the target gene RNAi and negative control slow virus carrier that confirm that correct clone as successfully constructs (Shanghai Ji Kai gene technology Co., Ltd).The experimental procedure reference molecule cloning experimentation guide second edition, will not be repeated here.

4th, RNAi and negative control slow virus packaging are detected with titre

Prepare the recombinant virus plasmid of coding lentiviral particle and its two kinds of auxiliary packaging original paper vector plasmids in 3 (carriers of pHelper 1.0, the carriers of pHelper 2.0 are provided by Shanghai Ji Kai gene technology Co., Ltd), three kinds of plasmids are carried Body carries out high-purity endotoxin-free extracting respectively, is total to by the operation instructions of Invitrogen companies lipofectamine 2000 293T cells are transfected, 8h is replaced by after complete medium, culture 48h after transfection, collects the cell conditioned medium rich in lentiviral particle Liquid, obtains the slow virus concentrate of high titre after being concentrated to it, determined in 293T cells and demarcate virus titer.

2nd, mice spleenSorting, the activation of cell.

1st, experimental animal

B10D2(Hc¹H2^d H2-T18^c) mouse, male, 8~10 week old are for experimental study.Plant mouse for SPF grades and be purchased from the U.S. Jackson laboratory, are raised in Experimental Animal Center SPF grades of barrier environment of Zhongshan University.This research institute is equal using animal Meet《Guangdong Province's management of laboratory animal regulations》It is required that, and pass through the examination batch of medical research Ethics Committee of Guangdong People's Hospital It is accurate.

2nd, experimental procedure：

(1) 12 orifice plates are coated with：1 μ L anti-mouse CD3e are added in 12 orifice plates containing 1mL PBS (1 μ g/mL), 5%CO₂, be incubated coating in 37 DEG C of incubators and remove PBS after 2 hours, 2 hours.

(2) cervical dislocation puts to death B10D2 mouse, is immediately placed in 3~5min of immersion in 75% ethanol；Sterile taking-up spleen It is dirty, it is careful to remove mesenteric tissue around spleen, move into and washed in plate with RPMI 1640 culture mediums 3 times, be subsequently placed in sterile 70 Gently milled with 2mL plungers in μm filter screen, while RPMI 1640 culture mediums, which are added dropwise, makes splenocyte by mesh, Collect cell flushing liquor and be made single cell suspension, 400-500 × g, 10 DEG C centrifuge 5 minutes, go supernatant to collect cell.

(3) 3mL erythrocyte cracked liquids, room temperature 3-5 minutes are added.

(4) 27mLRPMI1640 complete mediums (containing 10% hyclone and 1% mycillin) are added and terminate cracking.

(5) 400-500 × g, 10 DEG C centrifuge 5 minutes, remove supernatant.

(6) cell is resuspended in 30mL magnetic bead sortings buffer, counts,10 DEG C centrifuge 10 minutes, remove supernatant.

(7) every 10⁸Cell, every 10 is resuspended using 400 μ L magnetic bead sortings buffer in total cell number⁸Total cell number adds 100 μLCD4⁺T Cell Biotin-Antibody Cocktail II, mix rearmounted 4-8 DEG C and are incubated 10 minutes.

(8) every 10⁸Total cell number adds 300 μ L magnetic bead sortings buffer and 200 μ LAnti-Biotin MicroBeads。

(9) after mixing, put 4-8 DEG C and be incubated 15 minutes.

(10) 10mL magnetic bead sortings buffer is added, 4-8 DEG C, 300 × g is centrifuged 10 minutes, removes supernatant.Every 10⁸Cell number Cell is resuspended using 500 μ L magnetic bead sortings buffer.

(11) gone using LS posts unless CD4⁺T cell：The magnetic support that 75% ethanol disinfection is crossed is placed in superclean bench, LS Magnetic pole is installed on magnetic support, and 15mL centrifuge tubes are placed under magnetic pole, and 3mL Beads enrichments buffer is slowly flowed across into magnetic pole, rinses magnetic Post one time.

(12) appropriate total cell suspension is slow transitted through into magnetic pole, slowly rinsed with 3mL Beads enrichments buffer, repeat two Time.The liquid stream passed through is collected, wherein including unlabelled CD4⁺T cell part.

(13) 300 × g are centrifuged 10 minutes, and supernatant is removed completely；Cell is resuspended in 800 μ L Beads enrichments buffer.

(14) 200 μ L CD62L (L-selectin) MicroBeads are added.

(15) after mixing, put 4-8 DEG C and be incubated 15 minutes.

(16) 10mL magnetic bead sortings buffer is added, 4-8 DEG C, 300 × g is centrifuged 10 minutes, and supernatant is removed completely.Every 10⁸Carefully Cell is resuspended using 500 μ L magnetic bead sortings buffer in born of the same parents' number.

(17) MS magnetic poles are used, and change the centrifuge tube under magnetic pole.500 μ L Beads enrichments buffer are slowly flowed across into magnetic Post, rinses magnetic pole one time.

(18) cell suspension is slow transitted through into magnetic pole, slowly rinsed with 500 μ L Beads enrichments buffer, repeated twice.

(19) magnetic pole is removed, magnetic support, and the centrifuge tube more renewed is left.1mL Beads enrichments buffer is added into magnetic pole, used Propeller exerts oneself to pressurize and (press moderate), collects CD4⁺CD62L⁺NaiveT cells are simultaneously counted.Take 1 × 10⁵It is thin that cell is used for streaming Born of the same parents' art grouping system purity.

(20) 300 × g of remaining cell suspension is centrifuged 10 minutes, removes supernatant；RPMI1640 complete mediums are washed 1 time, then Add appropriate RPMI1640 complete mediums and kind of a plate (being coated with anti-mouse CD3e) is resuspended, per hole 1-2 × 10⁶/mL.And Final concentration of 10 μ g/mL anti-mouse CD28 are added, 5%CO is inserted₂, 48-72h in 37 DEG C of incubators.3rd, STAT3- RNAi slow-virus infection mouseCell.

(1) mice spleen is activatedAfter cell 48-72h, each hole cell is harvested, is counted, centrifugation is gone Clearly.

(2) with 1640 complete mediums containing IL-2 (final concentration 40ng/mL) (containing 10% hyclone and 1% blue or green chain Mycin) cell is resuspended, 5 holes are averagely added, per hole cell number 3-4 × 10⁶, volume is 500 μ L during infection.

(3) it is grouped：It is divided into 5 groups, including blank control group, negative control group, STAT3-shRNA1 groups, STAT3- 3 groups of shRNA2 groups, STAT3-shRNA.

(4) infect：Polybrene working solutions (the μ g/ml of final concentration 5) are added per hole, in addition to blank control group, every group equal Add correspondence viral (MOI is 30), sealed membrane shrouding；800 × g removes sealed membrane after centrifuging 90 minutes, inserts 5%CO₂、37℃ In incubator.

(5) 300 × g is centrifuged 10 minutes after 8-12h, changes liquid.Every 24 hours observation cell states, GFP expressions, midway Partly amount liquid can be changed, keep cytoactive.Harvesting during 96h, 300 × g is centrifuged 10 minutes, stays supernatant；PBS washs cell one It is secondary, remove supernatant, often pipe add 1mL TRIzol fully dissolve, put -80 DEG C it is standby.

4th, mRNA level in-site checking STAT3 interference effects, filter out optimal STAT3-RNAi

(1) RNA is extracted：From -80 DEG C of taking-up samples, put on ice, 0.2mL chloroforms added after 5 minutes, are fully mixed, 4 DEG C, 12000rpm centrifuges 30min.Careful upper liquid of drawing adds 0.5mL isopropanols and mixed to another new 1.5mL centrifuge tubes, It is stored at room temperature after 10min, 4 DEG C, 12000rpm centrifugations 30min.Supernatant is removed, and is washed with 75% ethanol (- 20 DEG C) of 1mL precoolings Twice (2~8 DEG C, 10000rpm), mix every time after 30s, centrifuge 10min.Supernatant is abandoned, then centrifuges 2~3min, careful remove remains Remaining ethanol, is placed in 1~1.5h of natural drying on super-clean bench.Add ddH2O (20~50 μ L) dissolvings.The RNA for taking 2 μ L to dissolve Solution detects RNA quality and integrality through 0.8% agarose gel electrophoresis.

(2) cDNA is synthesized：Often synthesizing 20 μ LcDNA reaction systems is：RNase Free dH₂O 4μL、5X PrimeScript Buffer 4μL、Random 6mers and Oligo dT 4μL、PrimerPrimeScript RT Enzyme Mix I 1μL、RNA 7μL；Reaction condition is：37 DEG C of 30min, 85 DEG C of 5sec, 4 DEG C.

(3) fluorescence real-time quantitative PCR method (dye method) detection each group internal reference GAPDH and purpose STAT3 gene mRNA tables Up to level：

1) design, synthetic primer：Primer sequence be see the table below, and primer is synthesized by Invitrogen companies.

The internal reference of table 2 and target gene primer sequence

2) primer working solution is configured：By internal reference and the primer dry powder dH of target gene₂O is diluted to storing liquid (100 μ Mol/L), 10 μ L are further taken out with 90 μ L dH₂O is diluted to 10 μm of ol/L working solutions.- 20 DEG C of refrigerators are saved backup.

3) fluorescence real-time quantitative PCR total reaction volume is：RNase Free dH₂O 3μL、SYBR Premix Ex Taq (Tli RNaseH Plus)(2X)5μL、ROX Reference Dye II(50X)0.2μL、PCR Forward Primer(10 μM) 0.4 μ L, the μ L of 0.4 μ L, cDNA templates of PCR Reverse Primer 1.Reaction system：95 DEG C of 30sec, (95 DEG C of 5sec, 60 DEG C 34sec) x40cycles, 95 DEG C of 15sec, 60 DEG C of 1min, 95 DEG C of 15min.

4) calculating of relative mRNA expression levels：Using relative quantification formula：2^{- △ Ct}× 100%, calculate each group sample STAT3 target gene is with respect to internal reference GAPDH mRNA expressions, wherein △ Ct=Ct (target gene)-Ct (internal reference bases Cause).

5th, statistical analysis

The statistical analysis of data is carried out using the softwares of SPSS 20.0.Normal distribution data are with mean ± standard deviationDescription.Using more multigroup data of One-Way ANOVA, using Levene variance test homogeneous；Each group variance is neat When, the comparison selection between group can carry out the S-N-K (q inspections) that mean compares two-by-two between multiple sample averages；If each group variance is not Qi Ze, which uses to compare between the Welch statistics of correction, group, uses Tamhane ' s T2 methods to carry out Multiple range test.The conspicuousness of inspection Level is set to α=0.05.

Following table and Fig. 2 are by fluorescence quantitative PCR detection STAT3-shRNA slow-virus infection mice spleens in embodiment 1The relative expression quantity of STAT3 genes during cell 96h (internal reference gene is GAPDH)

The relative expression quantity of each group STAT3mRNA levels of table 3

^aWelch statistics

From upper table it can be seen that, STAT3-shRNA2 groups [shRNA2 (22253-1) group i.e. in table 1] have optimal suppression The interference effect of STAT3 gene expressions processed, therefore, the optimal STAT3-RNAi that this group obtains for screening.

Embodiment 2

This example is using the distribution of Flow cytometry Th subgroups.

(1) using STAT3-shRNA2 (22253-1) slow-virus infection mouse of optimum jamming effectCell；Ditto.

(2) each group cell is harvested when infecting 96h, takes 100 μ Lx2 to manage after being mixed per hole, 1 manages for being marked after stimulating；Separately One 300 × g of pipe is centrifuged 5 minutes, and supernatant, streaming dye solution FBS (configurations are removed completely：PBS+0.1%NaN3+5% tire ox bloods It washed once clearly), 300 × g is centrifuged 5 minutes, and supernatant is removed completely；Packet marking streaming antibody after FBS is resuspended；

(3) detection of Treg cells：

1) 1 μ L Anti-MouseCD4-PE, 1.5 μ L Anti-MouseCD25Alexa-Fluor700 are added, room temperature is kept away Light is incubated 15 minutes.

2) vortex vibration is resuspended after cell, and often pipe adds Foxp3/Transcription Factor Staining Buffer fixes/rupture of membranes working solution (Fresh, fixation/rupture of membranes dilution #00-5223 dilution fixation/ruptures of membranes of 3 times of volumes Concentrate #00-5123) 1ml, vortex mixing again, the incubation 50 minutes of 4 DEG C of lucifuge.

3) without washing, rupture of membranes buffer solution working solution #00-8333 (deionized waters 1 are directly added into:9 are diluted to working solution) 2mL, 300 × g are centrifuged 5 minutes, remove supernatant, repeated washing 1 time.Often pipe adds 100 μ L FBS and is resuspended.

4) 1.5 μ LAnti-Mouse FOXP3PE-CY5.5 and the Isotype control of same volume are separately added into.Mix, room temperature is kept away Light is incubated 20 minutes.

5) rupture of membranes buffer solution working solution #00-8333 (deionized waters 1 are added:9 are diluted to working solution) 2mL, 300 × g centrifugations 5 minutes, remove supernatant, repeated washing 1 time.

6) often pipe adds 400 μ L FBS resuspensions, and lucifuge, upper machine testing is simultaneously analyzed.It such as can not immediately detect, use 0.4mL 1% paraformaldehyde is resuspended, and 4 DEG C of lucifuge preserves upper machine analysis in 24 hours.

(4) detection of Th1/2/17 cells：

1) it is grouped：Preliminary experiment sets multiple holes, and stimulant and blocking agent ratio are respectively 0.5:1、1:1、2:1；It is thin according to reflection The CD69 expressions of results of born of the same parents' state of activation, it is determined that stimulant and the concentration of blocking agent that formal experiment is used.Stimulation group：Cell hangs Liquid+stimulant PMA/Ionomycin+ blocking agent BFA/Monensin Mixture；Do not stimulate control group：Cell suspension+blocking Agent BFA/Monensin Mixture；

2) stimulate：Insert 5%CO₂, in 37 DEG C of incubators, per rocking within 1-2 hours, mix cell suspension once；4 hours Afterwards, appropriate cell suspension mark Mouse CD69PE are drawn per hour, by flow cytomery CD69 expression, are understood thin The activation degree of born of the same parents, it is determined that the stimulation time formally tested.

3) streaming antibody is marked：1 μ L Anti-MouseCD4-PE are added, room temperature lucifuge is incubated 15 minutes.Add FIX＆ The μ L of PERM Reagent A 100 are incubated at room temperature 15 minutes, add 3mL streaming dye solutions, and 300 × g is centrifuged 5 minutes, gone Take back collection cell completely, liquid feed is clean, should not remain；It is vortexed and mixes precipitation, adds the μ L of FIX＆PERM Reagent B 100 With intracellular antibody (1.5 μ L Anti-Mouse IFN gamma PE-eFluor 610,1.5 μ L Anti-Mouse IL-4APC, 2.5 μ L Anti-Mouse/Rat IL-17A PE-Cy7), Isotype control pipe adds the antibody of Isodose, mixes, and room temperature is kept away Light is incubated 20 minutes；3mL streaming dye solutions are added, 300 × g is centrifuged 5 minutes, goes supernatant to collect cell；With 400 μ L FBS It is resuspended, lucifuge, upper machine testing is simultaneously analyzed.It such as can not immediately detect, be resuspended with the paraformaldehydes of 0.4mL 1%, 4 DEG C of lucifuge preserves 24 Upper machine analysis in hour.

Table 4 is by Flow cytometry STAT3-shRNA2 slow-virus infection mice spleens in embodiment 2Each cell subset ratio during cell 96h.

Table 4

Remarks：*P<0.05shRNA2vs NC

Embodiment 3

This example is detection STAT3-shRNA2 (22253-1) to mouse bone marrow cells (c-kit) CD117⁺Cell propagation, differentiation Influence.

First, semisolid culturemedium is prepared

2.2% methocel solution：Configured according to literature method.Methylcellulose pulvis 1.1g is weighed, it is white with cleaning Paper bag, is added in the 25mL distilled waters boiled, with magnetic stirrer 2 hours, is allowed to fully dissolving.Treated after 120 DEG C of sterilizations 37 DEG C or so are cooled to, 2 × RPMI1640 liquid 25ml are added, and continue stirring 4 hours.Put 4 DEG C overnight, foam is all disappeared Lose, transparent shinny glue state.4 DEG C of preservations after packing.When configuring cultivating system, directly drawn with 1mL syringes.

2nd, configure, dispense cell factor

SCF stock concentrations：100 μ g/mL working solution concentration：10 μ L add 990 μ L 0.1%BSA dilutions, often the μ L of pipe 200 Dispense (1 μ g/mL)；Add 50 μ L (100ng/mL) in the 0.5mL final volumes of every hole when using.

IL-3100 μ L sterilized waters dissolve 10 μ g, 100 μ g/mL；Stock concentration：(10 μ L add 490 μ L 0.1%BSA dilutions Liquid) x10 pipes (2 μ g/mL)；Working solution concentration：1 pipe is taken, (every 50 μ L+450 μ L 0.1%BSA dilutions) x10 pipes (200ng/ mL)；Add 50 μ L (20ng/mL) in the 0.5mL final volumes of every hole when using.

EPO stock concentrations：10000IU/mL, working solution concentration：3 μ L are taken to add 997 μ L RPMI1640 culture mediums (30IU/ ML), 50 μ L (3IU/mL) are added in the 0.5mL final volumes of every hole when using.

IL-6100 μ L sterilized waters dissolve 10 μ g, 100 μ g/mL；Storage/working solution concentration：10 μ L add 990 μ L 0.1% BSA dilutions, often the μ L of pipe 200 packing (1 μ g/mL)；Add 50 μ L (100ng/mL) in the 0.5mL final volumes of every hole when using.

The μ L sterilized waters of GM-CSF 200 dissolve 20 μ g, 100 μ g/mL；Stock concentration：(10 μ L add 490 μ L 0.1%BSA Dilution) x10 pipes (2 μ g/mL)；Working solution concentration：1 pipe is taken, (every 50 μ L+450 μ L 0.1%BSA dilutions) x10 pipes (200ng/mL)；Often add 50 μ L (10ng/mL) in the 0.5mL final volumes of hole with 1640 pair of half dilution again when using.

The μ L sterilized waters of TPO 100 dissolve 10 μ g, 100 μ g/mL；Storage/working solution concentration：10 μ L add 990 μ L 0.1% BSA dilutions, often the μ L of pipe 200 packing (1 μ g/ml)；Often added again with 1640 pair of half dilution in the 0.5mL final volumes of hole when using 50uL(50ng/mL)。

3rd, flow cytometry sorting mouse bone marrow cells (c-kit) CD117⁺Cell

(1) cervical dislocation, which is put to death, takes out mouse double lower limb shin under mouse (5 B10D2 ♂ 6-8weeks), aseptic condition Bone, femur and ilium, RPMI1640 rush marrow, and supernatant is removed in centrifugation；FBS 2mL are washed one time；

(2) cell is resuspended in 1mL FBS, take 100 μ L blank controls, 100 μ L marks RatIgG2bK PE Isotype controls, 800ul mark CD117 (C-KIT)-PE, 4 DEG C, lucifuge be incubated 30 minutes；

(3) 1X erythrocyte cracked liquids (1：3BM) 5mL is fully mixed, and cracks 5min；Supernatant is removed in centrifugation, and meter is resuspended in 20mLFBS Number, 300x g centrifuge 5 minutes, remove supernatant；

Control group is resuspended in (4) 400 μ L FBS, and sorting group, upper machine sorting is resuspended in 800 μ L.Prepare to contain 5% hyclone, 1% The RPMI1640 culture mediums of mycillin receive cell.

4th, STAT3-shRNA (22253-1) slow-virus infections mouse bone marrow cells (c-kit) CD117⁺Cell

(1) 300xg, is centrifuged 5 minutes, goes supernatant to collect cell, and 15% complete RPMI1640 culture mediums are resuspended cell, planted Plate, divides 3 groups, and add cell factor (SCF:50ng/mL、GM-CSF:20ng/mL、IL3:20ng/mL、IL6：20ng/mL、 EPO：3U/mL) proliferative induction.

(2) 48h is induced, every group adds Polybrene working solutions (the μ g/ml of final concentration 5), in addition to blank control group, often Group adds correspondence virus, and (negative control virus, STAT3-shRNA22253-1 slow virus, MOI are 30) sealed membrane shrouding； 800 × g removes sealed membrane after centrifuging 90 minutes, inserts 5%CO₂, in 37 DEG C of incubators.

(3) 300 × g is centrifuged 10 minutes after 8-12h, changes liquid.Every 24 hours observation cell states, GFP expressions, midway Partly amount liquid can be changed, keep cytoactive.Harvesting when infecting 72h, 300 × g is centrifuged 10 minutes, stays supernatant；A part of PBS is washed Wash cell once, remove supernatant, often pipe add 1mL TRIzol fully dissolve, put -80 DEG C it is standby.A part is used for the detection of CCK8 methods Propagation.A part is planted in 2.2% methylcellulose semisolid culturemedium, is separately added into different cytokines, induction differentiation.

5th, interference group and control group mice marrow (c-kit) CD117⁺Cell STAT3mRNA relative expression levels detect

Extract RNA, synthesis cDNA, quantitative fluorescent PCR (dye method) detection：Ditto.

6th, CCK8 methods detection interference group and control group mice marrow (c-kit) CD117⁺Cell proliferative conditions

(1) 72h is infected, inoculation each group cell liquid is in 96 well culture plates, per the μ L of pore volume 100 (about~10⁴Cell number)； And the blank control wells of condition of culture are set up, every group is all provided with 3 parallel holes.10 μ L CCK8 liquid are added into every hole respectively, then are put Enter 37 DEG C, saturated humidity, 5%CO₂CMC model 3h；

(2) 96 orifice plates are taken out, A450 values (OD values), indirect reaction living cells quantity are determined at ELIASA 450nm wavelength.

7th, Flow cytometry interference group and control group mice marrow (c-kit) CD117⁺Apoptosis situation

(1) 96h is infected, cell is collected and is resuspended in Binding Buffer, add the Annexin V and PI of APC marks, Room temperature lucifuge is incubated 5 minutes；

(2) flow cytomery.

8th, 2.2% methylcellulose semisolid culturemedium and different cytokines induction interference group and control group mice bone Marrow (c-kit) CD117⁺Cell differentiation

(1) plate is planted：24 well culture plates, every group of 3 holes are respectively used to induction erythroid cell colonies BFU-E, granular leukocyte macrophage Colony CFU-GM and megakaryocyte colony CFU-Meg (multiple holes are set when cell number is more).

Red system hole：Per hole 0.5mL systems：Sequentially add 75 μ L hyclone (final volume percentage 15%), 50 μ L IL3 (final concentration 20ng/mL), 50 μ L SCF (final concentration 100ng/mL), 50 μ L EPO (final concentration 30U/mL), 100 μ L The methylcellulose of RPMI1640 culture mediums, 125 μ L, mixes, bubble is avoided as far as possible；The last cell liquid that 50 μ L are added per hole (about 1 × 10⁵)。

Grain is hole：Per hole 0.5mL systems：Sequentially add 75 μ L hyclone (final volume percentage 15%), 50 μ L IL3 (final concentration 20ng/mL), 50 μ L IL6 (final concentration 20ng/mL), 50 μ L SCF (final concentration 100ng/mL), 50 μ L GM-CSF (final concentration 10ng/mL), 50 μ L RPMI1640 culture mediums, 125 μ L methylcellulose, mix, gas are avoided as far as possible Bubble；The last cell liquid (about 1 × 10 that 50 μ L are added per hole⁵)

Macronucleus system hole：Per hole 0.5mL systems：Sequentially add 75 μ L hyclone (final volume percentage 15%), 50 μ L IL6 (final concentration 100ng/mL), 50 μ L SCF (final concentration 100ng/mL), 50 μ L TPO (final concentration 50ng/mL), 100 μ The methylcellulose of L RPMI1640 culture mediums, 125 μ L, mixes, bubble is avoided as far as possible；The last cell liquid that 50 μ L are added per hole (about 1 × 10⁵)

(2) count：Gently mix after be placed in 37 DEG C, saturated humidity, 5%CO₂Under the conditions of cellar culture；Micro- after 14 days Each group BFU-E (50 cells of ＞ are 1 colony), CFU-GM (40 cells of ＞ are 1 colony), CFU-Meg (＞ are counted under mirror 3 cells are 1 colony) quantity.

9th, statistical analysis

The statistical analysis of data is carried out using the softwares of SPSS 20.0.Normal distribution data are with mean ± standard deviationDescription；Non-normal data is described with median.When each group variance is neat, compared using two sample t-tests between two groups Mean；If each group heterogeneity of variance, the rank test (Mann-Whitney Test) compared using two samples.That examines is notable Property level is defined as α=0.05.

The infection 96h CCK8 method detection each group mouse bone marrow cells of table 5 CD117⁺Cell OD values

^aWelch statistics

The infection 96h flow cytometer detection each group mouse bone marrow cells of table 6 CD117⁺Early apoptosis of cells rate

^aWelch statistics

The colony number that each group is broken up after the STAT3-shRNA of table 7 effects

Embodiment 4

This example is the implementation that STAT3-shRNA2 (22253-1) slow virus is applied to vivo therapeutic mouse cGVHD models Example.The shRNA of suppression STAT3 of the invention expression is applied to vivo therapeutic mouse cGVHD models in embodiment 4, and cGVHD faces Bed scoring (table 8 and Figure 10) and lungs target organ pathological score (table 9) are reduced, and are compared with negative control group, difference has system Meter learns meaning (P<0.05).Figure 11,12 show each group target organ pathomorphism and scoring.

The mean of difference group clinical score of each time point of table 8

The mean of the different group lungs pathological scores of table 9

First, murine chronic Graft-versus-host Disease Model is prepared

1st, experimental animal

It is B10D2 (Hc for mouse¹H2^d H2-T18^c) mouse, male, 8~10 week old, purchased from U.S. Jackson laboratories； It is inbred strais BALB/c (H2 by mouse^d) mouse, female, 8~10 week old, purchased from Zhongshan University's Experimental Animal Center, are SPF grades Animal.

2nd, pre-processed and be grouped by mouse

20 BALB/c mouses start to drink addition antibiotic (erythromycin 250mg/L, gentamicin 32 for first 1 week in transplanting Ten thousand U/L) aqua sterilisa carry out INTESTINAL CLEANSING, and maintain to transplant after 2 weeks.Packet transaction：A, blank control group, it is synchronous to raise, Without intervention；B, radiocontrast group：The liquid of RPMI 1640 is transfused after radiation；C, transplanting control group：BMC liquid is transfused after radiation；D、 CGVHD experimental groups：BMC and SpC mixed liquors are transfused after radiation.The same day (0d) is transplanted, B, C, D group are received linear accelerator by mouse (Sweden's medical courses in general reach Precise 151337) SSD100,30x30 irradiation fields, 700cGy, 698 jump, and single full-body exposure (TBI) is pre- Processing, ource-skin Distance is 100cm.After irradiation immediately supplement drinking-water, rest 4~6 hours after through tail vein injection final volume be 0.4mL Transplanted cells liquid.Animal ethics standard is met to the disposal of animal in experimentation.

3rd, bone-marrow transplantation

B10D2 is as follows for the preparation method of mouse BMC suspensions：Put to death with cervical dislocation for after mouse, being put into 75% ethanol Soak and femur and shin bone are peeled off under 3~5min, aseptic condition, ossis is rinsed with RPMI 1640 culture mediums, collection contains marrow Flushing liquor single cell suspension is made.Red blood cell is abolished with erythrocyte cracked liquid after single cell suspension filtering, RPMI 1640 is washed Wash, count and cell is resuspended with RPMI 1640, adjustment BMC counts up to 4 × 10⁷/ mL is standby.For the preparation method of mouse SpC suspensions It is as follows：Put to death for after mouse, being put into 75% ethanol and taking out spleen under 3~5min of immersion, aseptic condition, removed with cervical dislocation Mesenteric tissue around spleen, moves into and is washed, milled with RPMI 1640 culture mediums in culture dish, collects cell flushing liquor and list is made Cell suspension, abolishes red blood cell with erythrocyte cracked liquid after filtering, RPMI1640 washings are counted and are resuspended carefully with RPMI 1640 Born of the same parents, adjustment SpC counts up to 4 × 10⁷/ mL is standby.Every mouse of control group and cGVHD experimental groups is transplanted respectively through tail vein injection 8×10⁶BMC, or 8 × 10⁶BMC+8×10⁶SpC cell suspension.

4th, general state is observed

Mouse general state, including weight, fash, depilation, the back of a bow, diarrhoea etc. are observed after transplanting daily, every 3 after 14d It carries out a clinical score, and standard is shown in Table 10：

The clinical score standard of the chronic GVHD of table 10

Note：Ear, tail, sole often locate 0.3 point of note of peeling or form a scab, and skin clinical manifestation is minimum to obtain 0 point, and highest is obtained 3.9 point；Skin score is considered as generation cGVHD more than 0.6 point；Even if resolution of symptoms, mouse natural death or human factor are caused Death is also considered as occurring cGVHD；The asymptomatic death of mouse is considered as no cGVHD.

5th, leukocyte counts and judgement is lapsed to

Every group of mouse in after transplanting 7,10 and 14d docking blood sampling count peripheral white blood cells (white blood cell, WBC), to monitor hematopoietic reconstitution situation.Method of counting：Docking takes the μ L of blood 10, adds the white 2% glacial acetic acid cell diluents of 190 μ L It is middle to mix, then it is added drop-wise on cell counting count board, cell number (N), WBC=(N in 4 block plaids is counted after horizontal rest 1min ÷20)×10^9/L.2, smear, Microscopic observation WBC Appearance and classifies after Wright's staining simultaneously.The criterion lapsed to： (1) hematopoietic reconstitution：WBC≥1×10^9/L；(2) hematopoiesis function exhaustion：WBC before dead<1×10^9/L；(3)cGVHD：Occur The clinical manifestation such as depilation, skin peeling or incrustation, Body weight loss, and WBC >=1 × 10^9/L, small intestine, lung, skin, liver organization There is cGVHD pathological changes Deng pathological examination；(4) associated death is transplanted：In addition to cGVHD and Haematopoietic failure caused by reason WBC >=1 × 10^9/L before death, including the reason such as infection, bleeding and organ failure, dead mouse, but it is clinical without cGVHD Performance and pathological change.

6th, cGVHD pathological score

Each group mouse is put to death in dying mouse or experimental endpoints dislocation of cervical vertebra, dissection, take skin (scapular region or diseased region), The organs such as liver, small intestine, lung.

(1) it is fixed：Take each organs and tissues to be cut into small pieces, 24 hours are fixed in 10% neutral formalin solution；

(2) following procedure is completed in the full-automatic enclosed dewaterers of Shandon：The tissue specimen fixed is existed respectively 80% ethanol 50min, 90% ethanol 50min, 95% ethanol III each 40min, 100% each 40min of ethanol III；It will be marked after dehydration This places 30min in dimethylbenzene I, II, III respectively；Each 25min is placed in low melt point paraffin I, II, III, and (temperature setting is 62 degrees Celsius)；High melting point paraffin I cylinders 25min (temperature setting is 64 degrees Celsius)；

(3) after tissue handling procedure terminates, sample is taken out, is placed in the wax pan of embedding machine, embedded that (temperature setting is 62 degrees Celsius)；

(4) cut into slices：4 μm of sections are cut on slicer, are floated on clean water face, are opened up in 50-57 degrees Celsius of warm water Open, then histotomy is picked up；In 70 degrees Celsius of oven for baking 15-20min；

(5) section is taken out from baking box, room temperature slightly cools down, section is put on overflow dyeing machine and carries out full-automatic HE dyeing, contaminated Color program is as follows：Each 7min, 100% ethanol III, 95% ethanol III, 80% ethanol, distillation are placed in dimethylbenzene I, II, III Each 1min of water.15 minutes in haematine dye liquor, washing, 0.5% hydrochloride alcohol 10s；2% lithium carbonate aqueous solution 1min；Flowing water is rushed Wash 10min, 80% ethanol, 95% ethanol III, 80% ethanol, 100% ethanol IIIIII, each 1min of dimethylbenzene I, II；

(6) section is taken out, section is hung up into Shandon mountings machine carries out mounting.

(7) optical microphotograph Microscopic observation HE staining tissue slides.Microscopic observation simultaneously scores, and standard is shown in Table 11.The pathology of lung Scoring determined standards of grading (table 12) in 1996 with reference to Cooke KR.

The cGVHD of table 11 pathological score standard

Note：Score is considered as generation cGVHD more than 2.

Table 12 transplants mouse lung tissue pathological change

The ratio that the scope of damage is involved according to lung tissue is come (the 5%-25%=1 that scores；>25%-50%=2；>50% =3)

Total score is the summation that the infiltration of chamber week and pneumonia score.

Infiltration refers to macrophage, neutrophil leucocyte, lymphocyte, is mixed with fibrin, or edematous fluid.

2nd, the shRNA vivo therapeutic cGVHD mouse expressed using the suppression STAT3 of the present invention：

1st, treatment time and method：

Morbidity state mouse (cGVHD clinical scores reach 0.6 point or more) is randomly assigned, shRNA treatment groups, feminine gender are right Each 6 according to group and blank control group.ShRNA treatment groups：Every mouse passes through the cold 0.3-0.4mLRPMI of tail vein injection 1640 liquid [contain 1x10⁷TU STAT3-shRNA2 (22253-1) slow virus]；Negative control group：Every mouse passes through tail vein The cold liquid of 0.3-0.4mLRPMI 1640 of injection (contains 1x10⁷TU negative control slow virus)；Blank control group：Every mouse is led to Cross the cold liquid of 0.3-0.4mLRPMI 1640 of tail vein injection.Operation is rapid, keep virus activity.

2nd, clinical observation after treating：Ditto.It is+58d after transplanting, treatment+30d or so to observe terminal.Compare each group treatment Variation Features of clinical score afterwards.

After transplanting in 7d, occurred perpendicular hair, vigor decline, weight loss by mouse；+ 7~+10d, marrow implantation, hematopoiesis is extensive Multiple, by mouse vitality restoration, body weight is gone up.+ 17d, is started the related clinical manifestations for cGVHD progressively occur by mouse, and weight differs；Extremely + 28d, 18 experimental subjects are fallen ill, and are scored 0.6 or more；Using+28d as treatment site, it will be tested according to clinical score Object be divided into gently (0.6~2 point), in (2.3 points~) two degree, be randomly assigned to shRNA treatment groups, negative control group, sky White control group, every group 6, every group has slight 3, moderate 3.With 30d after treatment ,+58d Germicidal efficacies the most are whole after transplanting Point, puts to death all study subjects.

+ 28d to+50d, each group experimental subjects clinical manifestation is aggravated, and the mean of cGVHD scorings is lasting in rising trend.+ 50d starts, and depilation mitigation, shortness of breath mitigation, vitality restoration, body weight rising, cGVHD scorings occurs in shRNA treatment groups experimental subjects Mean decline；Negative control group and blank control group experimental subjects still have depilation, skin incrustation, and vigor is poor, shortness of breath weight, the back of a bow Influence motion, body weight is light, into the plateau of clinical manifestation severe, and cGVHD scorings continue in higher level.(table 8, Figure 10)

+ 28d starts treatment, to observation terminal+58d, using the variance analysis of Repeated Measurements, as a result shows：Time Effect statistically significant (F=19.886, P<0.001), illustrate that cGVHD clinical scores are changed over time；Time hands over processing Mutual effect also statistically significant (F=2.256, P<0.001), illustrate in shRNA treatment groups and negative control group, the object of observation The trend that changes over time of cGVHD clinical scores it is different.Negative control group and blank control group compare, and Treatment Effects have no system Meter learns meaning (F=0.171, P=0.844), illustrates that the trend that two groups of cGVHD clinical scores are changed over time is consistent.Observation is eventually Point+58d, through two sample t-tests, by the level of α=0.05, it is believed that shRNA treatment groups and negative control group cGVHD clinical scores are total The difference of body mean is statistically significant (t=2.370, P=0.039, n=6).(table 8, Figure 10)

3rd, observation terminal puts to death mouse, takes target organ (skin, lung, liver, enteron aisle) row cGVHD pathological scores：Ditto.

Pathology is pointed out：There is different degrees of Pulmonary Vascular/peribronchial inflammation cellular infiltration in each group cGVHD target organs, Hepatic portal area under control inflammatory cell；Enteron aisle mild inflammation, no ulcer；Skin follicle is reduced, focal inflammation, and fat cell is reduced, collagen Deposit (Figure 11).Further Analysis interference group and negative control, the target organ pathological score of blank control group, point out interference group lung Dirty pathological score is significantly lower than negative control group (t=2.449, P=0.034, n=6) (table 9)；Interference group skin, enteron aisle disease Reason scoring is compared with control group has no notable sexual clorminance (Figure 12)；Except negative control group has a small number of mouse slight hepatic portal area under control occur Inflammatory cell, each group liver has no obvious liver cGVHD specific findings.

3rd, statistical analysis

This data carries out statistical analysis using SPSS20.0 statistics softwares.Meet the measurement data of normal distribution UsingRepresent.CGVHD experimental groups are compared with transplanting the data of control group, using two sample t-tests；Different time points are continuous The sample of measurement, using the variance analysis of Repeated Measurements.Show that difference is statistically significant with P ＜ 0.05.

Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

Claims (4)

1. a kind of suppression STAT3 gene expressions and the shRNA for preventing and treating chronic graft versus host disease, it is characterised in that：It is described The nucleotide sequence of shRNA positive-sense strand is as shown in sequence table 1, and the nucleotide sequence of its antisense strand is as shown in sequence table 2.

2. the medicine for preventing and treating chronic graft versus host disease, it is characterised in that make containing the shRNA described in claim 1 For the active component of medicine.

3. the medicine according to claim 2 for being used to prevent and treat chronic graft versus host disease, it is characterised in that：Described use It is that shRNA medicines are integrated into by mesh by slow virus carrier host cells infected in the medicine for preventing and treating chronic graft versus host disease Genome, so as to suppress STAT3 gene expressions and prevent and treat chronic graft versus host disease.

4. the medicine according to claim 2 for being used to prevent and treat chronic graft versus host disease, it is characterised in that described use Include in the medicine forming steps for preventing and treating chronic graft versus host disease：

(1) STAT3 gene orders are directed to, designs and filters out RNA interference sequences；

(2) the single stranded DNA oligo containing RNA interference sequences is synthesized, then annealed pairs produce double-strand shRNA；

(3) by the double-strand shRNA of synthesis, directly it is connected on the slow virus carrier after digestion by restriction enzyme site contained by its two ends；

(4) two kinds of auxiliary packaging original paper vector plasmids of Prepare restructuring slow virus plasmid, three kinds of vector plasmids carry out high-purity respectively Endotoxin-free extracting is spent, cotransfection 293T cells collect the cell supernatant rich in lentiviral particle, high titre is obtained after concentration STAT3-shRNA slow virus concentrates.