| ALI/ARDS was generally accepted as a kind of clinical syndrome in the1960s, itperforms as hyoxemia and acute severe respiratory failure with bilateral pulmonaryinfiltrates. The causes of ALI/ARDS are complex, and main cause of this disease isdiffuse lung infection, especially gram-negative bacterial infection, while LPS is themost important pathogenic factor of gram-negative bacterial infection. The currenttreatment of ALI/ARDS is mainly supportive care, lung protective ventilation anddrugs conservative treatment. However, of the broad array of pharmacologic therapiesevaluated in clinical trials to date, none has proved effective, and none can becurrently recommended as standard therapy for ALI. The mortality of ALI/ARDS isstill high at40%. Thus the development of creative treatments for ALI/ARDS isextremely necessary.Stem cells have been the hot issues of research at present both in the domesticand overseas, and also mesenchymal stem cells (MSCs) have been the main resourceof cells in clinical research and application because of their superior safety. Thus, thisstudy was to establish the model of endotoxin-induced acute pulmonary injury firstly,and obtained human umbilical cord mesenchymal stem cells secondly, in the end thisstudy aimed to discuss the treatment of human umbilical cord mesenchymal stem cellson endotoxin-induced acute lung injury, the practical application research andcomparison with the general drug in clinical treatment, also the possible therapeuticmechanism.Thus,we developed the following experimental contents and methods:1.Establishment of endotoxin-induced acute lung injury: C57BL/6male mice,8-10week old, intratracheal instillation of LPS with doses of10mg/kg,20mg/kg,30mg/kg,40mg/kg separately, and observe mortality and lung pathological inflammationseparately under different doses.2. Obtain of the mesenchymal stem cells without heterogeneity protein and can be used in clinical therapy: Mesenchymal stem cellswere obtained by wash and trypsinization of fresh umbilical cord tissue. Primary cellswere cultured using serum-free medium, surface markers and differentiationidentification were undertaken after cell passages to obtain mesenchymal stemcells.3.Application of hUC-MSCs in the treatment of acute lung injury andcomparison of hUCMSCs and methylprednisolone in treatment:1). mice weretransplanted with2×105hUC-MSCs via tail-vein after establishment of model ofendotoxin-induced acute lung injury, and effective time point of cell transplantationwas confirmed by using different transplantation time(0.5h,4h,24h).2). The lethal doseof50%was given to the mice model of LPS-induced acute lung injury, subsequentlydifferent doses of hUC-MSCs (2×105,5×105,1×106) were transplanted to mice viatail-vein, and then the most appropriate dose could be obtained.3). Comparison oftreatment of hUC-MSCs and methyprednisolone on acute lung injury:The therapeuticeffect of cell and medicine was compared by these indicators including pathologicalinjury (pathological score), lung wet-to-dry ratio, total cell counts and neutrophilcounts in BALF, content of total protein in BALF (BCA protein kit), content ofcytokines in BALF(CBA inflammation kit),flow cytometry of whole lung cellscontent etc.The model of intratracheal instillation of LPS-induced acute lung injury wasbuild successfully. The establishment of model was easy and stability of lunginjury,they both accorded with pathological and physical changes of infective acutelung injury in clinic.The mortality and pulmonary pathological injury score wereincreased as the increment of dose of LPS, in addition, these values were stable andhomogeneous. Hence relevant experiments could be designed according to differentrequirements of animal mortality and extent of pulmonary pathological injury.We had used collagenase digestion to obtain single human umbilical cordmesenchymal stem cell, by human mesenchymal serum-free culture medium.The cellsnumber was amplification folded, and after the generation to generaton, the purity,morphology and stability of the cells, still had the MSC typical characters. They canexpress typical MSC cell surface antigen such as CD44, CD73, CD90, CD105andHLA-ABC, no hematopoietic or endothelial cell markers such as CD31，CD34, CD45and HLA-DR detected. In vitro culture through specific hUC-MSCs osteogenic, adipogenic induction medium, results suggested that hUC-MSCs still has strongability of differentiation in P4generation. From the analysis of morphology, surfacemarkers and differentiation ability,the hUC-MSCs we had obtained accorded with thebiological characteristics of adult mesenchymal stem cells.After the sucess of mouse model of endoxin-induced acute lung injury, micewere transplanted with2×105hUC-MSCs via tail-vein at time points of0.5h,4h,24h,and extent of pathological injury of animals was observed after48h. The resultsshowed that pulmonary pathological injury could be alleviated at0.5h,4h,24h bytransplantation of hUC-MSCs.Besides,after high dose of LPS given to the mice, mice were transplanted PBSand different doses of hUC-MSCs(2×105,5×105,1×106)via tail-vein.The mortalityand extent of lung pathological injury of animals were observed after48h. The resultsdemonstrated that under the situation of equal dose of LPS and cells transplantationsimultaneously given to the model, the mortality was the lowest and lung injury waslighter at the dose of2×105hUC-MSCs. However, as a increase dose of hUC-MSCs,therapeutic effect was weakened, the injury score even mortality was increased.After modeling of24h mice were given PBS, hUC-MSCs, methylprednisolone(Drug), hUC-MSCs+methylprednisolone (MSC+Drug) via intravenoustransplantation. Observation of animals lung injury degree after48h, the mice givenhUC-MSCs,methylprednisolone and combined treatment of hUC-MSCs andmethylprednisolone were effectively reduce the injury score of lung tissue. From theinjury score, the best was hUC-MSCs in combination with methylprednisolonetreatment, its injury score was the lowest, but compared with hUC-MSCs alone ormethylprednisolone group was not statistically significant, the effect is not better thanthe other two groups.In mice given hUC-MSC, methylprednisolone and hUC-MSC in combinationwith methylprednisolone treatment, the lung wet to dry weight ratio were lower thanthose in PBS group, there were significant difference in hUC-MSC group andcombined treatment group compared with PBS group,but the methylprednisolonegroup did not reach statistical difference.After given hUC-MSCs, methylprednisolone and hUC-MSCs in combination with methylprednisolone treatment,compared with the PBS treatment group, the totalcells, the percentage of neutrophils and the neutrophil count in BALF weresignificantly decreased, all the therapy groups can reduce endotoxin induced acutelung injury.After treatment with hUC-MSCs, methylprednisolone,methylprednisolone+hUC-MSC, the total protein content of BALF decreasedsignificantly in all the three treatment groups, but there was no statistical differencenin the three groups.TNF, IL-6content in BALF decreased significantly in all the three treatmentgroups,compared with PBS group. IFN-γ, IL-10, MCP-1, IL-12did not changesignificantly in all groups.Given treatment with PBS,hUC-MSCs and methylprednisolone after lunginjury,we observed CD45-CD31+cells (lung endothelial cells), CD45+cells (lungwhite blood cells), CD31-CD45-EpCAM+cells (lung epithelial cells),CD31-CD45-EpCAM-Sca1+cells (lung mesenchymal cells),CD31-CD45-CD24lowEpCAMhicells (lung epithelial stem/progenitor cells) about thecell component changes at the time point of2d,7d,14d,28d.The results show that,compared with PBS group, methylprednisolone group at2d the CD45+cells (lungwhite blood cells) decreased significantly, while hUC-MSC group did not induce anycell components change. Except the CD45+cells, there was no obvious change in allgroups of any cells, and at the time point of7d,14d,28d,either. Consider from this,part of the mechanism of drug was to reduce the CD45+cells to alleviate lunginflammation, while the hUC-MSC did not take effect by changing the cellcomponents.Based on the animal model and experimental methods we used before,we havealso participated in Dr.Xie’s research of NO and H2in the treatment of acute lunginjury.Thus we have got the following conclusions：1. The mouse model of LPS-induced acute lung injury was establishedsuccessfully and stable.2. The hUC-MSCs were obtained successfully without heterogeneity protein and they had lower immunogenicity. These factors can supply more beneficial andsecureapplications for clinic and tissue engineering and other.3.1) Based on the clinical applications, effective therapeutic time point and themost appropriate dose of cell transplantation were found out, yet there have been norelevant findings in equal area.2) In the relevant research of treatment on endoxin-induced acute lung injury,this is the very first time to compare therapeutic effect of cell and clinical commonmedicine methyprednisolone, the results specifically demonstrate that both thehUS-MSCs and methyprednisolone have remarkable effect on acute lung injury,whilethere is no difference between the two groups in statistics.Moreover,there is nosynergistic effect of combination of hUC-MSC with methyprednisolone.3)In the study of treating acute lung injury, this is the first time to use flowcytometry to analyse the whole lung cells content of each treatment group, and theresults clearly show that hUC-MSCs have therapeutic effect on acute lung injury notby changing cell components. This finding lays foundation to study the mechanism ofhUC-MSCs treating acute lung injury in future.4. NO and/or H2provide therapeutic efficacy for ALI by reducing inflammationand apoptosis, which might be through inhibition of NF-κB activation. |
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