Effect Of HUMSCs Transplantation On Oxidative Stress Of Acute Lung Injury Induced By LPS

Background:ALI(acute lung injury) is a critical illness caused by internal and/or external factors of lung,its basic feature is the formation of pulmonary micro thrombosis and a large number ofinflammatory cell infiltration,its mechanism has not been fully clarified,so currently there is noeffective treatment,their mortality rate close to 40%.Lima confirmed, such as oxidative stress inacute lung injury model of the structure,function and plays an important role in theinflammatory response,which prompted oxidative stress plays an important role in the lunginjury.Mesenchymal stem cells (MSCs) are a kind of adult stem cells that can be self-renewal andhave the ability of multipotential differentiation.MSCs were first found in bone marrow.Subsequently,MSCs are also found in fat and muscle.The intriguing biological characteristic ofMSCs makes them become one of the major seed cells in tissue engineering and regenerationmedicine.Currently,MSCs are mainly derived from bone marrow. However, aspirating bonemarrow is an invasive procedure.The number and differentiating potential of bone marrow alsodecrease with ages. All the reasons constrain us from applying MSCs in biological engineering.However,many researches have proved that MSCs also appear in the human umbilicalcord.Furthermore,comparing to the MSCs derived from bone marrow,MSCs derived fromhuman umbilical cord have characteristics of easier vitro expansions and low immunogenicity,So it has been gradually become the most promising cells in tissue engineering. The finding andisolating of HUMSCs may bring a new dawn to the treatment of ALI.So far,study about effectof HUMSCs transplantation on oxidative stress of acute lung injury induced by LPS reportedrarely.Objection:This study was designed to isolate,cultivate and identify human mesenchymal stem cellsderived from umbilical cord.Furthermore, to explore the effects of HUMSCs transplantation onoxidative stress of acute lung injury induced by LPS.Methods:1、Isolation and cultivation of HUMSCs.Human umbilical cords were collected from full term deliveries under aseptic conditions.Umbilical cord blood was washed by PBS, then cut into pieces the size of 1mm×lmm.Tissuefragments were trypsinized by collagenaseⅡ, and then resuspended in DMEM/F12 mediumincluding 10% fetal bovine serum and penicillin/streptomycin in culture flask. When adherentcells reached nearly 80% confluence, they were re-fed and passaged. Observe the cells’ morphological changes under inverted phase contrast microscope and taken photos.2、Identification of HUMSCs.The surface markers：CD29、CD34、CD45、CD105 were analysed by flow cytometry.3、Vertification differentiation of HUMSCs into adipocyte and osteoblast by Oil red O andalizarin Red staining respectively.4、Observation of human umbilical cord mesenchymal stem cells transplantation on acutelung injury induced by lipopolysaccharide in rat lung tissue pathology.5、Determination W/D of HUMSCs transplantation on ALI induced by LPS in rat lungtissue.6、Explore the effects of HUMSCs transplantation on oxidative stress of acute lung injuryinduced by LPS.30 Wistar rats were divided into 3 groups randomly:control group (group A)，LPS group(group B) and HUMSCs transplantation group (group C),(n=10).After 6h of transplantation,rats were sacrificed and lung tissue were collected and stored with correct method.Observationof pathological changes in lung tissue.Lung W／D ratio were measured.Indexs of SOD andMDA were assessed.Result:1、Adherent cells could be found after incubation in 24 hours in vitro,they were small andhave no evident nucleus with different appearance in morphology, and most undigested tissuesuspended on culture medium. As time went on, adherent cells were increasing constantly.About 4 days later, some adherent cells took on bipolar flat or long-shuttle shape.10 days later,the increasing adherent cells looked likes fibroblast cells and grew in whirlpool way. Thosecells reached 80% confluence in about 2 weeks.The passaged cells were gradually purified, andmaintained their biological characteristic after at least 30 passages in vitro.2、Flow cytometry analysis revealed that the HUMSCs were positive for CD29、CD44 andCD105, but were negative for CD34.3、HUMSCs can be induced into adipocyte and osteoblas.4、Transplantation of HUMSCs can significantly reduce the level of inflammatory cell sinfiltration and lung tissue damage in ALI.5、Transplantation of HUMSCs can reduce the degree of edema of the lung tissue in acutelung injury.6、Transplantation of HUMSCs can attenuates LPS-induced acute lung injury and decreasethe content of MDA and increase the activity SOD in rats.Conclusion:The experimental results show that group B produce significant lung injury after 6h injection of LPS, and marked reduce the body’s ability to resist oxidative stress. MDA in group B issignificantly higher than group A in lung tissue, and the activity of SOD is significantly lowerthan group A. Which improved that LPS-induced lung damage had strong oxidative stress.Under normal circumstances, antioxidant system can remove an excess of oxygen free radicals,so that the body will not be oxygen-derived free radical damage.When LPS injury, the lungs hasstrong oxidative stress but lack of ability to resist oxidative stress which may cause lung tissuedamage.HUMSCs transplantation can significantly improve ALI induced by LPS in rats pathologicalchanges in lung tissue and lung damage, and to some extent correct oxidative stress ofLPS-induced acute lung injury in rats.