| Background:Acute lung injury (ALI) is acute pathological process that a variety of internal and external pathogenic factors lead to pulmonary alveolar-capillary membrane diffuse damage lung tissue congestion, edema.The mortality of acute respiratory distress syndrome (ARDS) is as high as40%. ARDS seriously affect gas exchange in lung function. Clinical manifestations of ARDS is progressive dyspnea and refractory hypoxemia, its pathogenesis has not been fully understood, many studies show that the imbalance of inflammation-inflammatory reaction is the most important pathophysiological mechanism of ALI/ARDS occurrence and development. Recent years, some scholars study on drugs and methods on the treatment of ALI/ARDS, and it has been confirmed that the treatment of respiratory support is still the current main treatment methods. However, there has not been a breakthrough for the researches of drug treatment yet.MSCs are a class of undifferentiated cells with different self-renewal and differentiation potential which home to the site of injury area involved in the process of lung repairing. Either MSCs endotracheal drip or exogenous input MSCs can reduce lung injury in rats caused by mechanical ventilation. Although MSCs therapy for ALI has achieved ideal effect in animal models, the mechanism is not entirely clear. In addition, MSCs as a whole-cell therapy for lung injury may exist risk to patients. Many researches demonstrate that the function of MSCs is not through multi-differentiation in lung cells, since MSCs can rarely be found in the lung and the proportion of MSCs differentiating into lung cells is quite rare. Meanwhile, a series of studies suggest that hypoxia can stimulate the secretion of KGF in MSCs which can promote the lung injury. Based on these researches, discussions about whether MSCs-derived conditioned medium(MSCs CM) and hypoxia stimulated MSCs CM(H+ MSCs CM) has the same therapeutic effect is very meaningful. In this study, we use the model of mechanical ventilation causing lung injury in rats to observe the therapeutic effects of MSCs CM and explore its mechanism elementarily.AIM:(1) To isolate, culture and identify rat bone marrow mesenchymal stem cells in vitro.(2) To prepare big tidal volume ventilation-induced lung injury model in rats.(3)To study the treatment of MSCs CM and H+ MSCs CM for VILI and the influence of MSCs CM and H+ MSCs CM on the inflammatory response of VILI.Methods:1) In order to study the method of culturing MSCs in vitro and the effect of hypoxia on MSCs, we performed the following experiments.(1) Collection and culture of MSCs:Adult male SD rats were purchased from the Fourth Military Medical University experimental Animal Center, weighing approximately180-200g. SD rats were sacrificed, and soaked in75% alcohol for5min. After that, three generation of cells in good condition were then selected for indentification. When the bone marrow cells growing well, we selected the good condition of3generation of cells for identification.(2) The collection of MSCs CM and hypoxic treatment of MSCs(H+MSCs CM): The medium was collected from normoxia and hypoxia-treated bone marrow mesenchymal stem cells’s supernatant, the supernatant was poured into a ultrafiltration tube (3000Da) and centrifuged (4℃,3000×g,2h). Then the ultrafiltrate (15ml liquid was concentrated to200μl) was collected and placed in4℃for later use.(3) Identification of rat bone marrow mesenchymal stem cells:Collecting P3generation of cells in good condition into three tubes, adding the monoclonal antibody CD45, CD90, and then incubating the tubes in dark place at4℃for3045min. Cells were washed twice with PBS to remove the unbound antibody. Finally, cells resuspending in PBS were detected by flow cytometry instrument.(4) Effect of hypoxia on bone marrow mesenchymal stem cells:the proliferation of P3generation of cells and P3generation of cells treated by hypoxia were determined by MTT method.2) To explore therapy effect of MSCs CM and H+MSCs CM for VILI and the effect of inflammatory cells of VILI. Healthy male SD rats weigh250-300g were provided by the Experimental Animal Center of the Fourth Military Medical University. Rats were randomly divided into4groups(A, B, C, D), each of which share10rats. Group A: Control group; Group B:VILI; Group C:MSCs CM group,; Group D:H+MSCs CM group. SD rats were anaesthetised, orotracheally intubated and subjected to injurious mechanical ventilation (B, C, D were at room temperature, respiratory rate was80beats/min, tidal volume was30ml/kg). After3h of high-volume ventilation (or spontaneous breathing in the control group) the rats were injected with PBS or MSCs CM or H+MSCs CM by tail vein. Another injection of the above medium was after24h. The rats were sacrificed by exsanguinations after48h, The timeline for these experiments is depicted in the figure below. The lung wet/dry ratio and lung homogenates value of MPO and HE staining of the left lung tissue of rat were measured. The level of BALF of the protein content, tumor necrosis factor-α (TNF-α), interleukin -6(IL-6), interleukin -10(IL-10) and keratin growth factor (KGF) expression of the right lung tissues were measured.3) To measure the effect of MSCs CM and H+MSCs CM to A549alveolar epithelial cells. We tested the mRNA expression of KGF of MSCs CM and H+MSCs CM by fluorescent quatitative PCR method.WST-1method was used to measure the proliferation of A549. Cell scratch test was utilized to measure the effect of MSCs CM and H+MSCs CM to the ability of A549migration.Results:(1) MSCs could be isolated from SD rats and the whole bone marrow adherent method produced high purity MSCs in vitro. The environment of low oxygen(3%O2) could stimulate the proliferation of MSCs. Flow cytometry instrument testing showed that the positive expression rate of surface moleculate markers expression of P3MSCs CD45:2.5%, CD90:96%. MTT results indicate that the MSCs proliferation ability under hypoxic training is better than that under normal incubate condition. The cell state and refraction are also better than the normal condition.(2) Bone marrow mesenchymal stem cell conditioned medium can reduce lung injury and the infiltration of inflammatory cells.1)We successfully copied VTLI models of rats: HE staining found that lung tissue significantly increased congestion and edema and a large number inflammatory cell infiltration in VTLI group. Compared to the control group,the left lung W/D ratio,lung tissue MPO values, lavage fluid protein content, expressing of TNF-a and IL-6were all significantly higher (P<0.05).2) H+MSCsCM and MSCsCM therapeutic effect of VTLI:In VILI group, we found that lung tissue significantly increased congestion and edema and a large number inflammatory cell infiltration, while in MSCs CM and H+MSCs CM group, lung structures were nearly close to normal lung tissue structure. Meanwhile, W/D ratio of MSCs CM group decreased by14.1%compared with VILI group, W/D ratio in H+MSCs CM group decreased by12.2%compared with VILI group (P<0.05). For MPO activity, VILI group significantly increased73.21%compared with the control group (P<0.01). MSCs CM group decreased59.02%(P<0.01) compared with the VTLI group, H+MSCs CM group decreased63.14%(P<0.01) compared with the VTLI group. Lavage fluid protein content in MSCs CM Group decreased0.23mg/ml (P<0.01) compared with the VILI group, H+MSCs CM group decreased0.25mg/ml (P<0.01) compared with the VILI group. The expression of IL-10and KGF of BALF were significantly different from the control group (P<0.05).3) The changes of IL-6, TNF-a and IL-10:Compared with VILI group, IL-6and TNF-a in MSCs CM group were reduced by20.07%and34.74%(P<0.05) respectively, while IL-6and TNF-a in H+MSCs CM group were reduced by23.50%and37.11%(P<0.05) respectively. IL-10in MSCs CM group increased by21.16ng/ml (P <0.05), IL-10in H+MSCs CM group reduced by18.79ng/ml (P<0.05).4) KGF: Compared with VILI group, KGF was increased by16.21pg/ml (P<0.01) in MSCs CM group, KGF was increased by22.31pg/ml (P<0.01) in H+MSCs CM group.(3) The results showed that MSCs CM and H+MSCs CM groups could promote cell proliferation and the migration ability of A549alveolar epithelial cells.Conclusion:(1) MSCs can be separated and cultured in vitro from SD rats by the whole bone marrow adherence method, and hypoxia can stimulate the proliferation of MSCs;(2) MSCs CM and H+MSCs CM infusion reduce the infiltration of inflammatory cells and lung damage caused by VILI;(3) MSCs CM and H+MSCs CM promotes the proliferation and migration of A549alveolar epithelium.(4) Hypoxia can stimulate the expression of KGF mRNA in MSCs. |
PDF Full Text Request