| Objectives By replicating rat silicosis model by instilling silica in vivo and culturing cellin vitro to investigate:1. Whether the alveolar macrophage of silicosis rats have autophagyactivation;2. The effects of BMSCs on autophagy activation of AM;3. The relationshipbetween the effects of BMSCs on autophagy of AM and PI3K-AKT-mTOR-P70S6Ksignaling pathways.Methods1. Experimental animals and groups: The SPF male SD rats (180~220gweight) were randomly divided into three groups: saline control group, silicosis modelgroup, BMSCs treatment group. BMSCs were harvested, cultured and passaged from3~5weeks-old SPF male SD rats. The rats model of silicosis were made by method ofone-time infusion of silica dust suspension, using the non-exposed trachealintubation(50mg/ml and1.0ml/only). The control group were given the same amount ofsterile saline infusion. BMSCs treatment group were injected BMSCs suspension1.0ml(3×106/ml) by tail vein, at24h after model establishment successfully.2. Detection ofindex and method:①The expressions of BMSCs surface markers CD29, CD45, CD54and CD106were detected by flow cytometry;②The morphology of AM were observedby HE staining;③The expressions and distributions of LC3, Beclin-1, mTOR, P70S6Kwere detected by immunocytochemistry;④The protein expression levels of LC3,Beclin-1, mTOR, P70S6K were detected by Western Blot;⑤The formation ofautophagesomes were observed by TEM.3. Quantitative assay and Statistical method:The immunocytochemistry staining sections were analyzd with the average positive cellsdetected using Image-ProPlus6.0digital medical image analysis system. PVDFmembrane will be for semi-quantitative analysis by Image J medical image analysissystem, using Integral Optical Density values. The average optical density wasdetermined by the medical CMIAS true color image analysis system at the sameconditions. For all statistical analysis, we used one-way ANOVA to define which groupcontributited to these differences with SPSS16.0. There was statistically significant iflateral P value<0.05. All datas were presented as mean±SD.Results1. Morphological observation of BMSCs: Primary adherent cells proliferatedby a decentralized clone type with high refractivity, most cells were fusiform or round,only some were polygonal. And95%confluenced about10d later, then passaged, withthe increase in the number of passages, cell morphology is more uniform, the cells werearranged parallel to the growth or swirling growth.2. The identification of BMSCssurface marker: The results showed the positive expression rate of CD29and CD106were75.04%and75.49%respectively, were positive; the positive expression rate ofCD45and CD54were4.07%and4.85%respectively, were negative.3. Themorphological observation of AM: HE staining showed that the control group AM wereround or oval, had a clear structure and uniform shape, had no silica dust engulfed particles. Compared with the control group, the model group AM had larger volume andabundant cytoplasm, the phagocytic silica dust particles were observed in some cells, themorphology of AM were not uniform. Compared with model group, the extent of AMdamage was reduced for BMSCs treatment group.4. The results ofimmunocytochemistry: the products of LC-3, Beclin-1positive expression were browngranules, distributed in the cytoplasm. In the control group, the expression of LC-3,Beclin-1were low. Compared with control group, the positive expresstion increased inmodel group at each time point (P<0.05), and the expression change presented a timedependence, the peak was14d (P<0.05), and decreased in28d, but still higher than that incontrol group(P<0.05). At the corresponding time, the level in BMSCs transplantationgroup was significantly lower than that in the model group (P<0.05), but the expressiontrend did not change; mTOR, P70S6K positive expression were brown granules,distributed in the cytoplasm. mTOR, P70S6K had a certain amount of expression incontrol group. Compared with the control group, the positive expression of model groupwere decreased at each time point (P<0.05), at14d was lowest (P<0.05), and increased in28d, but still lower than that in control group(P<0.05). Compared with model group, thelevel in BMSCs treatment group was increased (P<0.05) at each time point, but theexpression trend has not changed.5. The results of western blot: In control group LC-3,Beclin-1had a basic amount of expression, and the amount of expression no change at alltime points. In model group and BMSCs transplantation group western blot resultsconsist with immunocytochemistry results; In control group mTOR, P70S6K have acertain amount of expression, and the amount of expression no change at all time points.In model group and BMSCs transplantation group western blot results consist withimmunocytochemistry results.6. TEM Results: In control group AM had abundantorganelles, uniform extracellular matrix, rich lysosomes and complete mitochondrialstructure, clear nucleolus, normal distribution of nuclear chromatin. In model group, SiO2particles were observed; autophagosomes which had bilayer membrane were observedaround the nucleus, near the mitochondria, circular or oval; and autophagosomeprecursors, crescent-shaped or cup.Conclusions1. Autophagy was activated in AM of the silicosis model, and AMautophagy involved in the pathological process of silicosis rats;2. BMSCs transplantationcould block activation of autophagy in AM of silicosis rats;3. BMSCs transplantationcould regulate the PI3K-AKT-mTOR-P70S6K signaling pathway, inhibit autophagy inAM in the end. |
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