| Radiation-induced lung injury (RILF) is a common major obstacle in thoraciccancer radiotherapy. RILF develops via a complex pathological process, includingacute pneumonia injury early and late pulmonary fibrosis damage. Pulmonaryfibrosis, which is irreversible severely impact the patient’s quality of life, and evenlead to respiratory failure. However, there is no known effective therapeutic strategyfor RILF. Ad-MSC as a member of the family of MSC has been used for damagerepair of tissues and organs due to their multi-lineage differentiation to fat, cartilageand lung epithelial cells, and also the effect of immunoregulation. Compared withother sources of MSC, Ad-MSC are a potential candidate for autologous transplantfree from ethical in the clinical application due to their ease of isolation, abundantdistribution and low immunogenicity. Therefore, Ad-MSC show some superioritywhether in basic science study or in clinical study. The therapeutic effects ofAd-MSC delivery in RILF treatment has not been studied. It is of great significanceto study the effects of Ad-MSC on RILF in clinic. Here, we studied the effects of IVdelivery Ad-MSC on RILF in the Sprague-Dawley rat model and explored itsspecific mechanism. This study provided experimental basis for guiding clinical ofAd-MSC treatment on RILF. The study was divided into three parts:1.Establishment and evaluation of RILF model in Sprague-Dawley ratsTo find out the best model dosage for RILF model, We evaluated therelationship between the ionizing dosages and lung tissue damage in this part. RILFwas related to irradiation animal strains, irradiation site and irradiation category,especially the irradiation dosage. Female SD rats were enrolled in this study and theright-side chest of rats were irradiated by a high single doses of X-ray. The rats wererandomly divided into four groups and were respectively irradiated with7.5,15,22.5and30Gy to their right thorax. General condition and the survival rate of the ratsamong four groups were evaluated within28days after radiation. Besides, histological changes were also analyzed using HE and Masson staining. Our resultsshowed that rats in7.5Gy group have high survival rate (93.33%), but with notypical lesions of RILI. On the contrary, there was no survival rats receiving30Gyirradiation on day28. However, there were more or less death rates of rats in the15Gy group (66.67%) and22.5Gy group (26.67%). And pathological results showedthat the both group rats had typical lung injury including alveolar structure damage,pulmonary interstitial edema, alveolar walls thickening, pulmonary interstitialgathering a lot of collagen, alveolar space smaller and bronchi and vessels around alarge number of lymphocyte infiltration. However, It is not suitable to choose22.5Gy dosage for this experiment model due to their survival rate so low that cannot guarantee the model of quality. Therefore, We chose15Gy of X rays as the bestdosage to produce RILF model in SD rats.2.The identification of Ad-MSC biological characterization in vitroIt is of great importance to obtain a sufficient number of high purity and activityAd-MSC for the further study. The technology of density gradient centrifugationcombining with adherent method was used to isolate and culture stem cells derivingfrom adipose tissue of3-day age rats in sterile condition. We subsequently madecharacterization of Ad-MSC by observing cell general growth, detecting the surfacemarkers using flow cytometer and identifying differentiation potential of Ad-MSC toadipogenesis, osteogenesis and chondrogenesis, respectively. Our results showed:①The isolated rat Ad-MSC grew with adherence and presented aspindle-likemorphology with the typical form of MSC;②Flow cytometry revealed that ratAd-MSC were positive for CD29and CD44, and were negative for CD11b andCD45.③T he multipotency of Ad-MSC was confirmed by differentiation intoadipocytes, osteoblasts and chondrocytes after21days in specific culture media. Allof these results are consistent with the minimal criteria for MSC identification.Ad-MSC could be used as ideal seed cells to go on the further study.3.Therapeutic effects of adipose-derived mesenchymal stem cells onearly radiation-induced lung fibrosis in ratsAccording to the results of Part1and Part2, we chose15Gy of X rays as thebest dosage to build RILF model by evaluating the relation between the degree oflung damage and the ionizing dosages. And we also isolated, cultured and characterized of Ad-MSCs successfully. SD rats were given a single dose of15Gyof X-irradiation to the right thorax. We established SD rats model of RILFsuccessfully. Ad-MSC were harvested at passage3and delivered by the tail veinswithin2h after thorax irradiation. Histopathologic changes, levels of inflammatorycytokines (IL-1, IL-6, IL-10and TNF-α), pro-fibrotic factors (TGF-β1, α-SMA andCol1al), pro or anti-apoptotic mediators (Bcl-2, Bax and caspase-3), andmultifunctional factor HGF were evaluated after Ad-MSC transplant. Our resultsshowed:①Ad-MSC therapy improved the survival rate of rats after irradiation onday28compared to the irradiated group(P<0.05).②Ad-MSC treatment alleviatedsignificantly pathological tissues damage including the integrated alveolar structure,fewer amount of fibroblasts and lymphocytes infiltration and fewer collagendeposition in lung interstitium, compared to the Radiation group (P<0.05).③Furtherstudies showed that Ad-MSC had anti-inflammation as indicated by reduced serumlevels of the pro-inflammatory cytokines IL-1, IL-6and TNF-α, increased levels ofthe anti-inflammatory cytokine IL-10.④Ad-MSC had anti-fibrotic effects asindicated by downregulated TGF-β1, α-SMA, and Col1al levels in irradiated lungtissues, upregulated the expression of the multifunctional mediator HGF.⑤A d-MSCalso regulated the expression of pro and anti-apoptotic mediators (Bcl-2, Bax andcaspase-3) to protect lung cells from apoptosis.⑥Ad-MSC could repair the alveolarepithelium by directly differentiating to type Ⅱalveolar epithelial cells in theirradiated lung tissue on day7.In conclusions, IV Ad-MSC delivery improved the survival rate of rats afterirradiation and attenuated early RILF by anti-inflammation, anti-fibrosis,anti-apoptosis and differentiation potential. |
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