Genetic modification to induce CXCR2 overexpression in mesenchymal stem cells enhances

14 Pages·2017·5.84 MB·English

by  Zongshan Shen

Shenetal.CellDeathandDisease (2018) 9:229
DOI10.1038/s41419-018-0310-x Cell Death & Disease
ARTICLE Open Access
fi
Genetic modi cation to induce CXCR2
overexpression in mesenchymal stem cells
fi
enhances treatment bene ts in radiation-
induced oral mucositis
Zongshan Shen 1,2, Jiancheng Wang2, Qiting Huang1, Yue Shi2, Zhewei Wei3, Xiaoran Zhang2, Yuan Qiu2,
Min Zhang2,4, Yi Wang2, Wei Qin1, Shuheng Huang1, Yinong Huang2, Xin Liu2, Kai Xia2,4, Xinchun Zhang5 and
Zhengmei Lin1
Abstract
Radiation-inducedoralmucositisaffectspatientqualityoflifeandreducestolerancetocancertherapy.Unfortunately,
traditional treatments are insufficient for the treatment of mucositis and might elicit severe side effects. Due to their
immunomodulatory and anti-inflammatory properties, the transplantation of mesenchymal stem cells (MSCs) is a
potentialtherapeuticstrategyformucositis.However,systemicallyinfusedMSCsrarelyreachinflamedsites,impacting
their clinical efficacy. Previous studies have demonstrated that chemokine axes play an important role in MSC
targeting. By systematically evaluating the expression patterns of chemokines in radiation/chemical-induced oral
1234567890():,;1234567890():,; mC(MXuSCcCRos2sC.iXtTiCshR,2uw)sef,owfroemuneudxcpotlshoiatrietsdCtrXtehCaetLm2peowntaets.nIthniaidglehtehlyde,eraMxppSerCeusstCsicXeCdbR,2ewnehexfiehtrisbeaiotsefdctuheletnuhtrreaadnncsMepdlSaCntastrangteieogtnilniggoifbalCbyXileiCtxyRp2tr-oeosvtshetreheexinpCflraXemsCsLien2dgrmeMcueScpCotsosar
in radiation/chemical-induced oral mucositis mouse models. Furthermore, we found that MSCCXCR2 transplantation
accelerated ulcer healing by suppressing the production of pro-inflammatory chemokines and radiogenic reactive
oxygenspecies(ROS).Altogether,thesefindingsindicatethatCXCR2overexpressioninMSCsacceleratesulcerhealing,
providing new insights into cell-based therapy for radiation/chemical-induced oral mucositis.
Introduction
swallowing and speaking ability, ultimately leading to mal-
Approximately 80–100% of patients with head and neck nutrition, and can lead to life-threatening bacteremia2,3,
cancers who receive radiation treatment develop oral thereby reducing patient tolerance to cancer therapy and
mucositis, which is the most common complication of this patientsurvival3.Previousstudieshavefoundthatoxidative
treatment1. Oral mucositis affects food intake and stress induced by radiation leads to reactive oxygen species
(ROS)production,whichgreatlyimpactsmucositisbecause
ROSdamageDNA,inducecellapoptosis,andincreasepro-
Correspondence:XinchunZhang([email protected])orZhengmeiLin
inflammatory cytokine release4. However, traditional treat-
([email protected])
1GuangdongProvincialKeyLaboratoryofStomatology,Departmentof ments,suchaspainmanagement,nutritionsupporttherapy,
OperativeDentistryandEndodontics,GuanghuaSchoolofStomatology,Sun andantibioticsadministration,canalleviatethesymptomsof
Yat-senUniversity,Guangzhou,China
2TheKeyLaboratoryforStemCellsandTissueEngineering,CenterforStem mucositis but are not sufficient for the prevention or
CellBiologyandTissueEngineering,MinistryofEducation,SunYat-sen treatment of this condition1,4,5. Moreover, these treat-
University,Guangzhou,China ments elicit severe side effects, such as opportunistic
Fulllistofauthorinformationisavailableattheendofthearticle
ZongshanShen,JianchengWang,andQitingHuangcontributedequallytothiswork.
EditedbyYWang
©TheAuthor(s)2018
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Results
infections and lipid metabolic disorder. Therefore, it is
essential to explore effective treatments with fewer CXCL2 is upregulated in radiation/chemical-induced oral
adverse effects. mucositis
Because mesenchymal stem cells (MSCs) exhibit bene- To systematically investigate the expression of chemo-
ficial immunomodulatory, anti-oxidative, and anti- kines during the inflammatory phase of RIM/CIM, we
inflammatory characteristics, MSC therapy has been evaluatedthemRNAexpressionofchemokinesassociated
reported to be effective for patients with a series of with skin and mucosal inflammation, including CCL2,
inflammatory and radiogenic diseases, including myo- CCL8, CCL17, CCL19, CCL21, CXCL1, CXCL2, CXCL3,
cardial infarction (MI), spinal cord injury, osteomyelitis, CXCL5, CXCL9, CXCL10, and CXCL1216–19. We found
Crohn’s disease, and radiogenic skin inflammation6–9. that the mRNA levels of various CXCR2 ligands, includ-
These studies indicated that MSC transplantation might ing CXCL1, CXCL3, CXCL5, and CXCL2, were upregu-
representapromisingtherapyforradiogenicmucositis.In lated. The CXCL2 mRNA levels were markedly
aclinicalsetting,MSCsaretypicallyadministeredthrough upregulatedafterradiationcomparedwithnormaltissues
two routes: local transplantation and systemic infusion. (Fig. 1a). Furthermore, CXCL2 upregulation was con-
Because radiogenic mucositis is distributed in various firmed by in situ immunofluorescence staining and wes-
parts of the human body, local transplantation is not tern blotting (Fig. 1b, c). Interestingly, the expression of
appropriate. Additionally, local implantation has many CXCL2 mRNA peaked on day 7 after radiation and then
limitations, such as significant morbidity and disruption gradually declined (Supplementary Fig. 2A), which was
of the structure of the local environment10. Thus, intra- consistent with the clinical symptoms. CIM is another
vascular administration is much more appropriate. model applied for studying oral mucositis caused by
However, the low migratory efficiency of MSCs into the cancer therapy20. Similarly, the expression levels of
inflamed mucosa limits this approach and reduces its CXCL2 mRNA and protein were substantially increased
clinical benefits11. Therefore, studies aimed at promoting inCIM(Fig.1d–f).However,thehighestlevelsofCXCL2
MSC migration toward mucositis sites are vital. mRNA expression were observed 5 days after chemical
Chemokine axes control the migratory patterns of induction(SupplementaryFig.2B).Theseresultsindicate
MSCstospecificsites(i.e.,injuredsites)12,13.Chemokines thatCXCL2isthedominantchemokineexpressedinoral
released from inflammatory tissues might activate adhe- mucositis.
sion ligands and promote the transendothelial migration
or subsequent implantation of MSCs in the surrounding CXCR2-overexpressing MSCs retain the characteristics of
tissues14. The targeting of MSCs toward inflamed sites human MSCs
relies on specific chemokine receptors. However, the Chemokine receptors play an essential role in MSC
expression of these receptors in MSCs decreases after targeting to inflamed sites by binding to their corre-
in vitro expansion15. To enhance their migratory ability, sponding chemokines12,21. Therefore, we evaluated the
researchers have attempted to overexpress the corre- expression of chemokine receptors on MSCs. A tran-
sponding receptors in MSCs. In our previous study, scription quantitative real-time PCR (RT-qPCR) analysis
CXCR5-overexpressing MSCs exhibited enhanced tar- demonstrated that cultured MSCs at passage six expres-
geting ability to the inflamed skin in a contact hypersen- sed very low levels of chemokine receptors, including
sitivity (CHS) mouse model, in which CXCL13 was CXCR2 (Fig. 2a; Supplementary Fig. 2c), which is the
notablyupregulated.Moreover,thesegeneticallymodified receptor of the chemokine that is highly expressed in
MSCs with enhanced targeting ability markedly suppress mucositis, CXCL2.
skininflammation13.Therefore,methodsthatre-establish Subsequently, we overexpressed CXCR2 in MSCs by
the interactions between tissue-specific chemokines and infectingthemwithalentiviralvectorencodingCXCR2to
their corresponding receptors on MSCs are promising construct MSCsCXCR2 (Supplementary Fig. 3A) and
strategiesforenhancingthetargetingabilityofMSCsand potentially enhance the targeting of MSCs to inflamed
therebyimprovethetherapeuticbenefitsofMSCtherapy. sites.Moreover,weinfectedMSCswithalentiviralvector
Here, overexpression of the chemokine receptor encoding GFP (referred to as MSCsGFP) to allow the tra-
CXCR2onMSCsimprovedcellmigrationtotheinflamed cing of MSCs after transplantation (Supplementary
mucosa and promoted cell survival in oral radiation/ Fig. 3B). Indeed, the CXCR2 expression levels were sig-
chemical-induced mucositis (RIM/CIM). Furthermore, nificantly upregulated 2 days after infection (Fig. 2b).
CXCR2-overexpressing MSCs (MSCsCXCR2) accelerated Moreover, this upregulation was further confirmed by
ulcer healing, likely by suppressing ROS and pro- western blotting and flow cytometry (Fig. 2c, d). To
inflammatory chemokine production. Thus, this innova- determine whether CXCR2-overexpressing MSCs retain
tivestrategythatenhancesthetherapeuticbenefitsshows their identities, we investigated the expression of MSC-
promise for future clinical applications. specific markers and the multilineage differentiation
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Fig.1(Seelegendonnextpage.)
potential of the transduced MSCs. A flow cytometry lineage differentiation conditions, both MSCsCXCR2 and
analysis indicated that MSCsCXCR2 expressed the same MSCsGFP showed similar trilineage differentiation
pattern of stem cell surface markers as MSCsGFP (Sup- potential and exhibited osteogenic, adipogenic, and
plementary Fig. 3C). Moreover, under mesenchymal chondrogenic lineage phenotypes during the 2-to-4-week
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(seefigureonpreviouspage)
Fig.1CXCL2isupregulatedinradiation/chemical-inducedmucositis.aThemRNAexpressionlevelsofvariouschemokinesinvolvedin
radiation-inducedtonguemucositiswereanalyzedbyRT-qPCR.Sampleswereextractedfromnormalandinflamedtongues.Thefoldchange
representstheexpressionofeachchemokinemRNAininflamedtonguescomparedwithnormaltonguesonday7afterradiation.Allthedataare
presentedasthemeans±standarderrorsofthemeans(SEMs)(n=6)foreachgroup.bImmunofluorescencestainingofCXCL2(red)innormaland
inflamedtonguesonday5afterradiation.Theexperimentswererepeatedthreetimesinindividualmice.NucleiwerevisualizedthroughDAPI
staining(blue).Scalebar=100μm.cTotaltissuelysatesofnormalandinflamedtonguesweresubjectedtowesternblotanalysisofmouseCXCL2
proteinexpression.Theexperimentswererepeatedthreetimes,andarepresentativeblotisshown.dThefoldchangesinthemRNAlevelsofvarious
chemokinesinchemical-inducedmucositiswereanalyzedbyRT-qPCR.Sampleswereextractedfromnormalandinflamedmucosaltissuesonday5
afterchemicalstimulation.Thedataarepresentedasthemeans±SEMs(n=6)foreachgroup.eImmunofluorescencestainingforCXCL2(red)in
normalandinflamedmucosaltissuesonday5afterchemicalstimulation.Theexperimentswererepeatedthreetimesinindividualmice.Nucleiwere
visualizedthroughDAPIstaining(blue).Scalebar=100μm.fTotaltissuelysatesfromnormalandinflamedmucosaltissuesweresubjectedto
westernblotanalysisofmouseCXCL2.RIMradiation-inducedmucositis,CIMchemical-inducedmucositis
differentiation period (Fig. 2e). A cytogenetic analysis (Supplementary Figs. 3A, B), and infected MSCs to allow
demonstratedthatallMSCsCXCR2(atpassageeight)hada their tracing after in vivo transplantation. Biolumines-
normaldiploidchromosomalcomplement(Fig.2f).Taken cence imaging (BLI) revealed a strong linear relationship
together, these results indicate that the transgenic mod- (r2=0.992)betweenthenumberofMSCsandtheaverage
ification of MSCs does not alter the intrinsic character- luminescence intensity (Fig. 4a, b), indicating that the
istics of MSCs. numberofMSCscanbedeterminedbytheluminescence
intensity.Thus,toinvestigateMSCsurvivalaftersystemic
MSCsCXCR2 exhibit enhanced migration potential in vitro injection in mouse models, we examined the average
and in vivo luminescence intensity through BLI. The average lumi-
Chemokine axes mediate MSC migration to inflamed nescence intensity of both groups peaked 1 day after
sites12,21. Hence, we used a chemotaxis assay to assess infusion. Although the luminescence intensity in the
whether CXCR2-overexpressing MSCs exhibited MSCGFP group decreased sharply within 3 days, the
enhanced targeting ability in vitro. The results showed luminescence intensity intheMSCCXCR2groupremained
thatMSCsCXCR2,butnotMSCsGFP,stronglyrespondedto steady for 3 days and was even observed after 7 days
5ng/ml human CXCL2 (hCXCL2) and 50ng/ml murine (Fig. 4c, d). These findings indicated that the over-
CXCL2 (mCXCL2) (Fig. 3a). Additionally, the number of expression of CXCR2 in MSCs not only enhanced their
migrated MSCsCXCR2 increased nearly two-fold when the targetingabilitytomucositissitesbutalsoincreasedtheir
incubation time was extended from 6 to 12h (Fig. 3b). survival rate.
To determine whether MSCsCXCR2 had an enhanced To further verify that the overexpression of CXCR2 in
ability to target radiation-damaged tongues in vivo, we MSCs increased their survival rate, we used an in vitro
injected MSCsCXCR2 and MSCsGFP into RIM model mice hydrogen peroxide (H O ) model as previously repor-
2 2
through the tail vein on day 7 after radiation. Inflamed ted22. Cell Counting Kit-8 (CCK-8) assays indicated that
tongues were collected from each group on days 1 and 3 MSCsCXCR2 exhibited increased survival rates 2 and 4h
post-injection. An immunofluorescence staining analysis after exposure to oxidative stress compared with
demonstrated that MSCsCXCR2 accumulated in the MSCsGFP (P<0.05) (Fig. 4e). The PI3K/Akt and MAPK/
mucositis region in the RIM model. Interestingly, within Erksignalingpathwayshavealargeimpactoncellsurvival
3 days of injection, the number of MSCsGFP declined andproliferation23.Awesternblotanalysisindicated that
sharply,andthenumberofMSCsCXCR2decreasedslightly theP-AktandP-Erk1/2levelsweresignificantlyincreased
(Fig. 3c, d). To further investigate whether MSCsCXCR2 in MSCsCXCR2 (Fig. 4f). These results indicated that
displayed enhanced targeting ability to CIM sites, we CXCR2 enhances cell survival and proliferation, likely by
injectedMSCsCXCR2andMSCsGFPintoCIMmodelmice. activating the PI3K/Akt and MAPK/Erk signaling
Animmunostaininganalysisindicatedthatthenumberof pathways.
MSCsCXCR2wassignificantlyhigherthanthatofMSCsGFP
in the inflamed mucosa (Supplementary Fig. 4A, B). MSCsCXCR2 notably attenuate RIM/CIM
Altogether, these results indicate that CXCR2 over- To investigate whether the enhanced migration of
expression enhances MSC targeting to RIM/CIM sites. MSCsCXCR2 would improve their beneficial effect on oral
mucositis, we injected mice with MSCsGFP and
MSCsCXCR2 exhibit enhanced survival in RIM MSCsCXCR2 on days 7 and 5 after radiation and chemical
We inserted luciferase 2-monooxygenenase (luc) after induction, respectively. Hematoxylin and eosin (HE)
thechemokinereceptorinalentiviralvector,placingboth staining revealed that the group with radiation-induced
constructs under the control of the same promoter ulcers exhibited a loss of filiform papillae, ulcerations, a
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Fig.2CXCR2-overexpressingMSCsretainthecharacteristicsofhumanMSCs.aHistogramsrepresentingthemRNAexpressionlevelsofC–C
andC–X–Cchemokinereceptorsfromthreeindependentsamplesofsixth-passageMSCs.MSCsurfacemarkers(CD44,CD90,CD73,andCD105)were
detectedasapositivecontrol.Thedataarepresentedasthemeans±SEMsforeachgroup(n=3,t-test).bTheexpressionlevelsofCXCR2mRNAin
MSCsGFPandMSCsCXCR2aftergenetransductionwereanalyzedviaRT-qPCR.Glyceraldehyde3-phosphatedehydrogenase(GAPDH)mRNAwas
detectedasaninternalcontrol.Thedataarepresentedasthemeans±SEMsforeachgroup(n=3,t-test).cTotalMSCGFPandMSCCXCR2lysatesfrom
threeindependentsamplesweresubjectedtowesternblotanalysisofCXCL2expression.dAflowcytometryanalysiswasusedtodetectCXCR2
proteinonthesurfaceofMSCsGFPandMSCsCXCR2.Sixindependentsampleswereanalyzed.eRepresentativeimagesshowingthetrilineage
differentiationpotentialofMSCsCXCR2andMSCsGFPintoadipocytes(oilredO),osteocytes(alizarinred),andchondrocytes(alcianblue).Scalebar=20
µm.fCytogeneticanalysisofMSCsCXCR2attheeighthpassage.Theexperimentswererepeatedthreetimes
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Fig.3MSCsCXCR2exhibitenhancedmigrationpotentialinvitroandinvivo.aInvitromigrationofMSCsCXCR2andMSCsGFPtowardhumanCXCL2
(hCXCL2)ormurineCXCL2(mCXCL2).Transwellfilterswerestainedwith0.1%crystalvioletandobservedunderamicroscope.Scalebar=25μm.b
ThemigratedMSCsafterincubationforeither6or12hwerequantifiedineachmicroscopicfield;***P<0.001.cMSCsCXCR2andMSCsGFP,bothof
whichexpressedGFPwheninjectedintoradiation-inducedmucositismodels,wereexaminedbyimmunofluorescencestainingondays1and3
post-injection.Signals:GFP,green;DAPI,blue.Scalebar=100μm.dGFP-positivecellswerequantifiedineachmicroscopicfieldofthemouse
tongue.Thedataarepresentedasthemeans±SEMsforeachgroup(n=6,t-test).***P<0.001fortheRIM+MSCCXCR2groupcomparedwiththeRIM
+MSCGFPgroupontheindicatedday
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Fig.4MSCsCXCR2exhibitenhancedcellsurvivalinradiation-inducedmucositis.aInvitrobioluminescenceimaging(BLI)ofMSCsCXCR2from
groupswithdifferentcellnumbers.Thecolorscalebarrepresentstheopticalfluorescenceintensityinphotons/second/cm2/steradian(photons/s/
cm2/sr).bLinearcorrelationofthecellnumberwiththeluc-inducedluminescenceintensity(r2=0.992).cInvivoBLIrepresentingMSCsurvivalon
days1,3,7,and10afterinjection.Theluminescenceintensityrepresentsthenumberoflivingcells.Scalebar=10mm.dQuantitativeanalysisofBLI
ondays1,3,7,and10afterinjection.Thedataarepresentedasthemeans±SEMs(n=3).***P<0.001.eCCK-8assayshowingtheviabilityof
MSCsCXCR2andMSCsGFP2hand4hafterexposuretooxidativestress.Thedataarepresentedasthemeans±SEMs(n=3)foreachgroupandare
representativeofthreeindependentexperiments.*P<0.05.fTotalMSCGFPandMSCCXCR2lysatesweresubjectedtowesternblotanalysisofAktand
Erk1/2phosphorylation.Theexperimentswererepeatedthreetimes
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disrupted epithelium layer, inflammatory cell infiltration, apoptosiswasmeasuredviaAnnexinV/propidiumiodide
and decreased mucosal thickness. The MSCGFP group (PI) staining and flow cytometry. The apoptosis of pri-
showed an increase in filiform papillae and decreased marytonguecellssubjectedtoco-culturewithMSCswas
ulceration, but inflammatory cell infiltration was still significantly decreased compared with mono-cultured
observed in the submucosal area. However, extensive MSCs (Supplementary Fig. 5A, B). Taken together, these
ulceration was absent in the MSCCXCR2 group, which findingsindicatethatMSCsacceleratemucositisrecovery,
exhibitedthelowestlevelofinflammatorycellinfiltration likely by decreasing radiogenic ROS production and
and increased thickness of the epithelium layer and protecting tongue cells from cell death.
mucosallining(Fig.5a).Wethenexaminedthemaximum
Discussion
diameter of ulceration in RIM/CIM models to assess the
severity of mucositis. Smaller ulceration diameters in the OralRIM is oneof themost common complications of
mouse mucosa were measured in the MSCCXCR2 group radiotherapy among patients with head and neck cancer.
than in MSCGFP group (Fig. 5b). Inflammatory cytokines, However, traditional treatments for severe mucositis are
includingtumornecrosisfactor-α(TNF-α),interleukin-1β limited and cannot prevent ulcer recurrence. Here, we
(IL-1β), IL-6, and IL-10, play important roles in wound reportaninnovateandhighlyefficienttreatment,namely,
repair4,16.Hence,weexaminedthelevelsofinflammatory thesystematictransplantationofMSCswithanenhanced
cytokines after MSC treatment to assess the anti- targetingcapacitytomucositissites.Thisstudyshedsnew
inflammatory effects of MSCs. A RT-qPCR analysis light on the treatment of RIM and provides groundwork
demonstrated that the mRNA expression of pro- for the clinical application of MSC-based therapy for
inflammatory cytokines in the mouse mucosa was sig- mucositis.
nificantlydownregulatedintheMSCCXCR2groupbutonly Duetotheiranti-inflammatoryandimmunomodulatory
slightly reduced in the MSCGFP group (Fig. 5c). These capabilities, MSCs have therapeutic potential for the
results indicate that MSCsCXCR2 can markedly attenuate treatment of inflammatory diseases6,9. However, systemi-
oral mucositis, in part due to their anti-inflammatory callyinfusedMSCsrarelyhometoinflamedmucosainthe
effects. orofacial region. MSC targeting depends on the interac-
tions between chemokine receptors and specific chemo-
MSCs accelerate mucositis recovery by decreasing kines from inflamed sites12,21. A RT-qPCR analysis
radiation-induced ROS production revealed that CXCL2 was the most highly expressed
RIM is initially caused by radiogenic ROS4,24. Because chemokine in the inflamed mucosa in oral RIM mouse
MSCs have great anti-oxidation potential25,26, we won- models. Recent studies have reported that the over-
dered whether MSCs promoted ulcer healing by pro- expression of corresponding chemokine receptors in
tecting tissue cells from ROS-induced damage. The MSCscanenhancetheirtargetingabilitytoinflamedsites.
staining of inflamed tongue tissues with the superoxide- For example, CCR1, CXCR4, and CCR7 overexpression
sensitivefluorescentdyedihydroethidium(DHE)revealed has been shown to enhance MSC targeting to injured
that both MSCGFP and MSCCXCR2 treatment exerted myocardium29, ischemic myocardium30, and secondary
benefits by reducing the cellular ROS levels. However, lymphoid organs31, respectively. Notably, our chemotaxis
MSCCXCR2treatmenthadthegreatesteffectondecreasing assays demonstrated that MSCs overexpressing CXCR2
thecellularROSlevelsamongallgroupsinvivo(Fig.6a). had enhanced migratory ability when exposed to CXCL2
We further applied CellROXTM for detection of the in vitro. Furthermore, immunostaining and BLI analyses
radiogenic ROS levels in primary tongue epidermal and revealedthatMSCsCXCR2exhibitedenhancedtargetingto
fibroblast cells after treatment with 4Gy of radiation the inflamed mucosa. Interestingly, we also observedthat
in vitro. Based on immunofluorescence staining, the CXCR2 overexpression prolonged MSC survival in the
radiogenic ROS levels in primary tongue cells were atte- inflamed mucosa, and this effect is likely mediated by P-
nuated by co-culture with MSCsCXCR2 or MSCsGFP (P< Akt and P-Erk1/2 upregulation.
0.05) (Fig. 6b). Furthermore, a flow cytometry analysis of Subsequently, we exploited the therapeutic potential of
the intracellular redox status confirmed that primary MSCCXCR2 transplantation. A HE staining analysis
tongue epidermal or fibroblast cells co-cultured with demonstrated that mice that received MSCsCXCR2 dis-
MSCs exhibited less intracellular ROS generation than played enhanced epithelial integrity in the mucosa com-
mono-cultured cells (Fig. 6c). In addition, an oxidative pared with those that received MSCsGFP. Furthermore,
environment contributes to cell death, which is also the ulceration diameters in the MSCCXCR2-treated group
responsible for ulcer healing delays22,27,28. Therefore, we were significantly smaller than those in the MSCGFP-
used an H O -induced cell death model to determine treatedcontrolgroup.Together,theseresultssuggestthat
2 2
whether MSCs protect tongue epidermal or fibroblast MSCCXCR2 transplantation accelerates ulcer recovery.
cells from cell death in an oxidative environment. Cell Conventional treatments, such as pain management and
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Fig.5MSCsCXCR2markedlyattenuateradiation/chemical-inducedmucositis.aHematoxylinandeosin(HE)-stainedtonguesamplesfromfour
groups:thenormalgroup,theRIM/CIMgroup,theRIM/CIM+MSCGFPgroup,andtheRIM/CIM+MSCsCXCR2group.Sampleswerecollectedat72h
post-injection,cryosectioned,andstainedwithHE.Alltheexperimentswererepeatedthreetimes.Scalebar=100μm.bThemaximumulcer
dimensionineachgroupwasexaminedwithVerniercalipers.Thedataarepresentedasthemeans±SEMsforindividualmice;n=6mice/group
fromthreeindependentexperiments.*P<0.05;**P<0.01;***P<0.001.cThelevelsofTNF-α,IL-1β,IL-6,andIL-10mRNAwereanalyzedbyRT-qPCR.
mRNAsampleswereextractedfromthetonguesofmiceineachgroupatday3post-injection.GAPDHmRNAwasdetectedasaninternalcontrol.
Theresultsarerepresentativeofthreeexperiments;*P<0.05;**P<0.01;***P<0.001
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Fig.6(Seelegendonnextpage.)
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