The Initiation Investigation Of Human Umbilical Cord Mesenchymal Stem Cells On Acute Lung Injury Model

PARTⅠ THE ISOLATION、CULTURE ANDIDENTIFICATION OF HUMAN UMBILICAL CORDMESENCHYMAL STEM CELLSObjective：To establish stable and efficient method to isolate andculture human umbilical cord mesenchymal stem cell（sHUC-MSCs），to laythe foundation for the acute lung injury treatment.Method：HUC-MSCs were isolated and cultured by direct adherentmethod. The umbilical cord tissue of normal healthy full-term delivery fetuswas taken, cut off thread on both ends, purified from the umbilical cordtissue with sterile D-Hanks balanced salt solution for several times. Theumbilical cord was cut, strip veins, arteries, and outer membrane with thesize of1mm×1mm, move the tissue into75cm2culture flasks，and added thecomplete medium5ml. Cells were incubated in incubator with5%CO2at37°C, then afer24h and added medium to12ml. the medium were replacedevery three days and the large pieces of tissue not adherent were removed. When cells reached80%confluent,0.25%trypsin was used to detach thecells, at the ratio of2:1for sub-culture cells. The third passage cells, thephenotypic markers CD34,CD45,HLA-DR,CD73,CD90and CD105wasanalyzed with the flow cytometer. To observe the differentiation ability ofHUC-MSCs, the p3cell were treated with the osteogenesis inductionmedium for20days to show typical osteoblast illustrated by alizarin redstaining, meanwhile, with the adipogenic induction medium for15days toshow adipocyte illustrated by oil red O staining.To verify chondrogenicdifferentiation through Alcian Blue staining following the protocols, thepellets were embedded in paraffin and cut into sectionsResults: The primary isolations of HUC-MSCs were heterogeneous,fibroblast-like cells possessing short and long processes as well as smallround and triangle cells. After several cell subculture, the morphologyHUC-MSCs was similar to the parent cells, even the p10cell didn’t showaged. HUC-MSCs from passage3were characterized by flow cytometry.HUC-MSCs were stained positively for CD73(99.96%)，CD90(99.10%)，CD105(99.42%)， and negative CD34(0.19%)， CD45(0.19%)，HLA-DR0.19(0.10%). The calcified nodules and lipid-filled vesicles werestained alizarin red and the O oil red were stained in red under theosteogenic and adipogenic differentiation conditions stimulated. Cultureunder chondrogenic differentiation conditions stimulated chondrogenicpellet, after Alcian Blue staining present blue. Conclusion: The adherent methods of culture HUC-MSCs is stableand reliable method to produce high purity HUC-MSCs, which havemulti-directional differentiation ability. PART Ⅱ THE METHOD OF ESTABLISHING THE MODELOF ACUTE LUNG INJURY(ALI) AND THE EFFECT OFHUC-MSCS ON ALIObjective：To establish the ALI mouse model induced by LPS，and toobserve the effects of HUC-MSCs on ALI mouse model.Method： BALB/c mice were divided into four groups(PBS+PBS，PBS+MSCs，LPS+PBS，LPS+MSCs). The mouse ALI model was inducedby intratracheal LPS instillation, anesthetized with an intraperitonealinjection of10%chloral hydrate. One-hours after LPS instillation, micewere intratracheally administered with HUC-MSCs or PBS. The cells andtotal protein in bronchoalveolar lavage (BAL) were evaluated at1,3, and5days post-injury. Light microscopic evaluations were performed toevaluate the lung injury scores and calculate the W/D ratio.Results: The intratracheal LPS instillation can establish ALI modelfor mice. The pathological alterations and wet/dry ratio were confirmed the ALI mice model. The mortality of ALI mice were decreased afterHUC-MSCs transplantation. The histology of lung in ALI+MSCs groupshowed the significiant decreased congestion and cellular infiltration atpost-injury days3and5（P＜0.05). The wet/dry ratio was decresed afterHUC-MSCs instillation intratracheally, suggesting that it can alleviatepulmonary edema significiantly.Conelusion: ALI mouse model was successfully established throughinstilling LPS intratracheally. The pathological changes of lung tissue, thealleviate pulmonary edema，MP0activity of lung tissues, inflammatoryfactor level of IL-6, IFN-γ were decreased ALI mice intratrachealtransplantation of HUC-MSCs, suggesting that HUC-MSCs have protectiveeffects on ALI mice. PART Ⅲ THE INITIAL MECHANISMS OF HUC-MSCS ONALI MICEObjective：To explore the effect of HUC-MSCs on ALI mice, and toillustrate the molecular mechanism of HUC-MSCs treat acute lung injurymodel.Method：HUC-MSCs were labeled with DAPI.HUC-MSCs transferand residence time in pulmonary were detected through instilling the labeling cells intratracheally. BALB/c mice were divided into fourgroups(PBS+PBS，PBS+MSCs，LPS+PBS，LPS+MSCs). BALF werecollected at the day of1，2，3after hUC-MSCs instillation, to detect themyeloperoxidase （MPO） activity， BALF protein concentration, proteinlevels of interleukin (IL-6) and interferonγ(INF-γ). The differences ofsecreted factors were screened by protein microarray and confirmed byELISA in BALF between mice treated LPS+PBS and LPS+MSC groups.Results：HUC-MSCs could be labeled by DAPI with high efficiency.The distribution of HUC-MSCs was even in the lung tissue afterintratracheal5hours, while the distribution was decreased post-intratracheal24hours. The protein concentration in BALF, a marker ofendothelial and epithelial permeability, which is increased in inflammatorylung tissue, was significantly reduced in the mice treated with HUC-MSCs.the secreted level of INF-γ, TLR4, MCP-1, GM-CSF, IL-6, TNF-α, IL-1βwere decreased, while increased VEGF in the protein microarray withtotal308targets in the mice treated with LPS+MSCs. The level ofinflammatory cytokines IL-6and IFN-γwere reduced in BALF of micetreated with HUC-MSCs in comparison with LPS+PBS.Conelusion: HUC-MSCs could reside in lung tissue shortly. Theeffect of HUC-MSCs on ALI mice was through releasing paracrine solublefactors to suppress inflammation factor secretion and to protect ALI.