Effects Of Adipose-derived Stem Cells On The Apoptosis Of Keratinocytes Induced By Thermal Injury In Burn Wounds

Background and ObjectiveAs the development of society and improvement of medical technology, togetherwith the aesthetic standards of people increasing, the standards of treatment of burnwounds have been improving. Therefore, the timely and correct treatments of burnwounds are particularly important at the early stage. The interactions of keratinocytes,fibroblasts and cytokines/tissue factors in wound or local majority determine the qualityof wound repair. The final outcome of wound repair is re-epithelialization. Keratinocytesare particularly important in the process of wound healing. If lack or poor activity ofkeratinocytes in the burn wound, the repair process of wound would delayed completedor even couldn’t completed. Thermal injury or burn often causes apoptosis, necrosis orloss of keratinocytes in burn wounds. There are a small number of survivingkeratinocytes and other kinds of cells in the burn wound at the early stage. What’s more,the outcomes of these surviving cells partly determine the prognosis of burn wounds.Mesenchymal stem cells (MSCs) are kinds of adult stem cells, which derived fromthe interstitial tissues in body. MSCs not only have the stem cell-related skills ofself-renewal, multi-directional differentiation and potential of trans-differentiation, butalso have a strong paracrine and immune regulation function. While they have significantly roles in promoting wound healing and improvement of tissue repair.Adipose-derived stem cells (ADSCs) are derived from adipose tissues, which are kind ofMSCs, with the relevant characteristics of MSCs. To investigate the effect of ADSCs onthe apoptosis of keratinocytes induced by thermal injury or burn and the relatedmechanism of the action, we treat burn wounds with the stem cell-related features ofADSCs at the early stage. With these effects, we want to provide a certain theoreticalbasis for treating burn wounds withADSCs in the future.Methods1. ADSCs were isolated, cultured and purified from bilateral inguinal adiposetissues of5BALB/c mice by collagenase digestion in vitro. The3rd passage ADSCswere detected surface markers with flow cytometry, and were induced into adipocytesand osteocytes by adipogenic and osteogenic differentiation medium in vitro.2. Keratinocytes were isolated, cultured and purified from cutaneous tissues of3new-born BALB/c mice by trypsin digestion in vitro. HaCaT cells were cultured innormal conditions. Keratinocytes or HaCaT cells were incubated at51.5℃for35seconds to reproduce thermal injury model. The apoptosis of cells were detected byacridine orange/ethidium bromide (AO/EB) staining and the apoptotic rates of cells wereimmediately detected by flow cytometry.3. HaCaT cells were incubated at0.9%sodium chloride solution for24h and thenwere randomly divided into control group (cultured with serum-free DMEM) andexperimental group (cultured with100%ADSCs-CM), respectively. After12h, theapoptosis of cell were detected by AO/EB staining in2groups. HaCaT cells wereincubated at51.5℃for35seconds to reproduce thermal injury model. Thermallyinjured cells were randomly divided into normal group (cultured with DMEM containing10%FBS), serum-free group (cultured with serum-free DMEM),50%ADSCs-CM group(cultured with DMEM containing50%ADSCs-CM), and100%ADSCs-CM group(cultured with100%ADSCs-CM) according to the random number table. The apoptosisof cell were detected by AO/EB staining in each group at3h,6h,12h,24h after cultured,respectively. After12h, the apoptotic rates and cell cycles of each group were detected by flow cytometry, the levels of Bcl-2, caspase-3mRNA and protein were detected byreal-time polymerase chain reaction (TR-PCR) technique and Western-blot.4. After depilation of the back hair,20healthy and female BALB/c mice wererandomly divided into burn group and sham group, which were administrated with93±2℃steam and37℃water for5s, respectively. After treatment, the back cutaneoustissues were harvest at post-injury0h,3h,6h,12h,24h and48h, the histopathologicalchanges were detected by H&E staining, respectively. And the apoptosis of keratinocyteswithin the wounds or local were respectively detected by TUNEL staining at post-injury0h,3h,6h,12h,24h.5. After depilation of the back hair,18healthy and female BALB/c mice wererandomly divided into control group, DMEM group and ADSCs-CM group, and thenadministrated with93±2℃steam for5s. The mice in each group were treated andinjected with the same amount of sterile PBS, serum-free DMEM,100%ADSCs-CM onthe surface and local of wound at post-injury0h,2h,4h, respectively. The back cutaneoustissues were harvest at post-injury6h, the apoptosis of keratinocytes within the woundsor local were respectively detected by TUNEL staining, and the expression levels ofBcl-2, caspase-3in the wound and local skin were respectively detected byimmunohistochemistry staining. The levels of Bcl-2, caspase-3mRNA and protein in theskin were detected by real-time polymerase chain reaction (TR-PCR) technique andWestern-blot, respectively.Results1. The3rd passage of cultured ADSCs proliferated well with fusiform shapesimilar to fibroblasts. The positive expression rates of CD3l, CD34, CD45were less than10%, while those of CD90and CDl05were all above90%. The cells were induced intothe phenotypic characteristics of the adipocytes and osteocytes, respectively.2. The primary cultured keratinocytes proliferated well with cobblestone-like shapein morphology, but the3rd to5th passages of cultured keratinocytes are easily to aging.HaCaT cells cultured and proliferated well in the normal condition. The3rd passage ofkeratinocytes and HaCaT cells were incubated at51.5℃for35s, the keratinocytes mostly died after thermal injury, the apoptotic rate of keratinocytes was uncertain, andthere was no statistically significant difference (P>0.05). Immediately after injury, theapoptotic rate of HaCat cells was (9.8±0.45)%(P＜0.05).3. ADSCs-CM can significantly inhibit the apoptosis of HaCaT cell induced bystarvation in vitro. The numbers of apoptotic cells of the thermally injured HaCaT cellsreached to the peak at post-injury12h with AO/EB staining.100%ADSC-CM cansuppress the apoptosis of HaCaT cells induced by thermal injury though regulating theexpression of Bcl-2and caspase-3, accelerate their cell cycle progressions byameliorating the blockage of cell in G2/M phase.4. With depilation of the back hair, it can reproduce a representative deep Ⅱ°burnwound in the mice back, which were administrated with93±2℃steam for5s. Thenumber of apoptotic keratinocytes within wound or local and surviving appendagesreached to the peak at post-injury6h with TUNEL staining.5. Treatment the burn wound with100%ADSCs-CM at the early stage, which cansignificantly increase the mRNA and protein expression levels of the Bcl-2and reducethe expression levels of caspase-3in the skin. With these effects,100%ADSCs-CM canobviously reduce the number of apoptotic keratinocytes in burn wound.ConclusionThe results proved that ADSCs can significantly suppress apoptosis of keratinocytesinduced by thermal injury through regulating the expression of Bcl-2and caspase-3bythe paracrine function in vitro and vivo. With these effects, ADSCs might have somepotential in treatment of burn wounds at early stage.