Bone Marrow Mesenchymal Stem Cells Tracking And Role In The Treatment Of Pulmonary Fibrosis

Background Silicosis is a systemic disease caused by long-term inhalation of a large amount of free crystalline silicon dioxide,with nodular fibrosis changes in lung tissue as the main pathological feature.Because of its insidious onset,the diagnosis of this disease was usually delayed,rendering treatment difficult.Currently,available management strategies are focused on controlling the associated symptoms and complications;there is no proven curative treatment for silicosis.Mesenchymal stem cells were initial cells with self-renewing,high proliferation and multiple-directional differentiation ability.Bone marrow-derived mesenchymal stem cells(BMSCs)are readily available,stably in vitro amplification and culture,with low immunogenicity,which is conveniently for allotransplantation.The administration of BMSCs is currently investigated as a new therapeutic method for pulmonary fibrosis.BMSCs can migrate to injured sites and secrete multiple paracrine factors and then regulate immune function,decrease inflammation,or replace damaged lung cells through differentiation to enhance tissue repair.Although studies have recognized BMSC-based therapy as a new potential treatment option for lung fibrosis,longitudinal,non-invasive methods to trace the cells post-transplantation and to evaluate their biodistribution,function,and final fate are lacking.A clear understanding of the in vivo behavior of the transplanted cells will be not only helpful in determining the possible mechanism of stem cell therapy but also necessary for translation from basic research to clinical practice.So in this work,we want to focus on the study of stem cell tracking and treatment mechanism.We developed a nanomaterial with good biocompatibility and dual-mode imaging ability,which can be used for the labeling of transplanted stem cells and non-invasive long-term tracking in vivo.On this basis,the possible mechanism of stem cell therapy for silicosis fibrosis is discussed.Part Ⅰ Preparation of CT/NIRF dual-modal nanotracer and BMSCs labelingObjective To prepare the Au nanoparticles which are used for Computed Tomography(CT)and near-infrared fluorescence(NIRF)dual-modal imaging.To label the BMSCs with the prepared nanoparticles in vitro and check the safety of nanoparticles labeling.Methods Gold nanoparticles were prepared by reducing chloroauric acid using bovine serum albumin as a template,and modified with indocyanine green(ICG),a nearinfrared dye,to obtain a nano-tracer suitable for dual-modal imaging.The morphology and size of the prepared AA@ICG@PLL were characterized by transmission electron microscopy(TEM).The surface charge property and hydrodynamic size of the NPs were analyzed by dynamic light scatterer(DLS).The ICG loading efficiency of the nanoparticles was detected with the ultraviolet spectrophotometer.Micro-CT and nearinfrared imager were used to observe the imaging capability of nanoparticles.Human BMSCs were obtained and cultured in vitro.The cells were co-cultured with prepared the NPs for cell labeling.In order to determine the optimal conditions for cell labeling with AA@ICG@PLL NPs,dynamic processes of BMSCs uptake of NPs under different treatment times and concentrations were observed using a live cell imaging system.The flow cytometry detection and inductively coupled plasma mass spectrometry(ICP-MS)were used to check the labeling efficiency.To evaluate the safety of AA@ICG@PLL labeling,cell migration,and proliferation and multidifferentiation potential after labeling.Results 1)The synthesized AA@ICG@PLL nanotracer has uniform particle size distribution and good solution stability.Compared with traditional iodine-based contrast agents,AA@ICG@PLL nanotracer has better CT imaging contrast and also has good near-infrared fluorescence imaging ability.2)Co-incubation of AA@ICG@PLL with BMSCs with 200 μg/m L Au content for 24 hours can achieve the best labeling efficiency without affecting cell viability.3)Labeled BMSCs maintain good migration,proliferation,differentiation function,and stable CT and near-infrared imaging ability.Conclusion The AA@ICG@PLL NPs were successfully prepared and used to label the BMSCs without interfering with cellular migration,proliferation,and differentiation.Part Ⅱ In vivo tracing of BMSCs labeled with AA@ICG@PLLObjective To explore the authenticity and reliability of long-term tracing of AA@ICG@PLL labeled BMSCs in silicosis mice.Methods A single high-dose tracheal infusion of silica was used to construct the mice silicosis models.AA@ICG@PLL labeled BMSCs were transplanted into the lungs by intra-tracheal instillation.Micro-CT and NIRF imager was used to monitor the survival and distribution of labeled BMSCs in the lungs.Fluorescence staining of lung tissue sections was used to verify the authenticity of AA@ICG@PLL tracking of BMSCs in vivo;The body weight and pathological changes of mice’s main organs were checked to evaluate the safety of AA@ICG@PLL-labeled BMSCs transplantation.Results 1)AA@ICG@PLL labeled BMSCs can be traced in vivo in the lungs of silicosis model mice for at least 21 days;2)The authenticity of AA@ICG@PLL labeled BMSCs was confirmed by immunofluorescence staining in vivo;3)There was no mice acute death and chronic organ damage occurred after the transplantation of labeled BMSCs.Conclusion This section suggested that the labeled BMSCs could be tracked for up to 21 d after transplantation in vivo by NIRF and CT imaging,and the transplantation was safe and reliable.Part Ⅲ The effect and mechanism of BMSCs transplantation in the treatment of silicosisObjective To evaluate the therapeutic effect of BMSCs transplantation and to explore the mechanism of BMSCs transplantation involved in regulating pulmonary fibrosis.Methods The animals were randomized to the following treatment: Control group mice were intratracheally injected with 100 μL of NS instead of Si O2 suspension.NS was intratracheally administered to the mice again on Day 7.Si O2 group mice received an intratracheal injection with 100μl 50mg/m L Si O2 suspension.On Day 7 after Si O2 injection,NS was intratracheally administered to the mice.Si O2 +labeled BMSCs group mice received 100μl 50mg/m L Si O2 suspension and then intratracheal transplantation of 1×106 AA@ICG@PLL BMSCs on Day 7 after Si O2 injection.Si O2 +unlabeled BMSCs group mice received Si O2 exposure and then intratracheal transplantation of 1×106 normal BMSCs on Day 7.Mice in each group were sacrificed after completing the Micro-CT scanning on Day 28.The histopathology of the left lungs was assessed by HE staining and Sirius staining.The inflammation-related protein concentration of bronchoalveolar lavage fluid(BALF)using ELISA.The expressions of IL6,TNFα,CXCL2 m RNA level were analyzed by RT-PCR.The hydroxyproline in blood was assessed by ELISA.THP-1 monocytes derived macrophages were used to establish an in vitro model of macrophage activation induced by Si O2 exposure.Western blot as well as the cell immunofluorescence staining,were used to detect the expression of M1 and M2 markers in macrophages,confirming the successful construction of the cell models.In order to directly identify the role of BMSCs in macrophage activation,we collected the BMSCs conditioned medium to treat activated macrophages.Then we focused on identifying the secreted factor(s)responsible for the observed effects.ELISA assay was used to detect the s TNFR1 in BMSCs conditioned medium.To further study the role of s TNFR1 secreted by BMSC in inhibiting the activation of macrophages,the concentration of s TNFR1 in BMSC-CM was downregulated by s TNFR1 neutralizing antibody and si RNA interference technology.Results 1)Transplantation of labeled or unlabeled BMSCs significantly decreased the lung inflammation and fibrosis degrees in mice treated with silica.2)BMSCs treatment could inhibit Si O2-induced macrophage activation.3)s TNFR1 in BMSC-CM participated in inhibiting the activation of macrophages caused by Si O2.Conclusion The findings in this section showed that BMSC could participate in the treatment of pulmonary fibrosis by down-regulating macrophage activation through paracrine function.The therapeutic effect may be related to the regulation of M1 macrophage activation by BMSC secreting s TNFR1.Part Ⅳ Zinc finger protein ZC3H4 as a potential target for BMSCs treatment in silicosisObjective To verify the effect of ZC3H4 on macrophage activation;To explore the specific mechanism of ZC3H4 in affecting macrophage activation;To explore the feasibility of ZC3H4 as a target for BMSCs treatment in silicosis.Methods The silicosis mice model and the THP-1 derived macrophages were used to detect the effect of Si O2 stimulation on the expression of ZC3H4 in alveolar macrophages in vivo and in vitro;The CRISPR/Cas9 technology was used to specifically knockdown the expression level of ZC3H4 in alveolar macrophages to verify the relationship between ZC3H4 and macrophage activation;The cell scratch experiment was used to detect the regulation ability of activated macrophages on the migration of fibroblasts;The CRISPR/Cas9 technology along with the pharmacological methods were used to explore the mechanisms by which ZC3H4 regulated macrophage activation;The effect of BMSCs on the expression of ZC3H4 was checked in vivo and in vitro.Results 1)Si O2 stimulation could induce the activation of alveolar macrophages with increased expression of ZC3H4;2)ZC3H4 was involved in regulating Si O2 induced macrophage activation;3)ZC3H4 regulated macrophage activation through the endoplasmic reticulum stress pathway;4)BMSCs transplantation could down-regulate the expression of ZC3H4 in macrophages,thereby inhibiting the activation of macrophages to treat silicosis.Conclusion The findings in this section showed that the zinc finger protein ZC3H4 could affect the activation of macrophages through the endoplasmic reticulum stress pathway.BMSCs down-regulate the expression of ZC3H4 to inhibit macrophage activation,thereby delaying the process of silicosis.