| Background Inhalation of high concentrations of oxygen has been used to treat avariety of diseases to ensure that the oxygen demand of the organization, butprolonged oxygen inhalation at the same time will give rise to the abnormal structureand function of lung, which affects the quality of life,some even life-threatening; Asto the developing lung tissue, inhalation of high concentrations of oxygen may leadto irreversible malformation. Bronchopulmonary dysplasia (BPD) is a chronic lungdisease that occurs in very premature infants and is characterized by decreasedalveolarization and abnormal vascularization and associated with surfactantdeficiency, oxidative stress, barotrauma, inflammation, alveolar fibrin deposition,nutrition, and genetic background. Complexity in studying alveologenesis arises fromthe fact that the process is coordinated by multiple interactions through paracrinemechanisms between the fibroblastic, epithelial, and microvascular lung components,and with extracellular matrix (ECM), no effective therapies to reverse or retard thecourse of the disease are available.Severe,long lasting hyperoxia causes an inflammatory reaction with an influx of inflammatory cells, cell proliferation and hypertrophy, an increase of cytokines,apoptotic activity and subsequent morphologic evidence of lung injury. TNF alpha, aninitiation factor of cytokine network, which plays a very important role in inflammatorytriggered by hyperoxia and corresponds to the severity of the pathological findings,Inhibition of TNF alpha in vivo prevents hyperoxia-mediated activation of caspase 3 intypeⅡcells. Transforming growth factor-beta (TGF-beta) is necessary for normal lungdevelopment,and myofibroblast accumulation and activation at sites of injury andrepair are driven in part by TGF-β1,but the sustained excess in TGF-βsignaling thatplays a key role in the subsequent alveolar hypoplasia and fibrosis.Gap junction channels enable the direct flow of signaling molecules andmetabolites between cells. Alveolar epithelial cells show great variability in theexpression of gap junction proteins (connexins) as a function of cell phenotype andcell state. Differential connexin expression and control by alveolar epithelial cellshave the potential to enable these cells to regulate the extent of intercellular couplingin response to cell stress and to regulate surfactant secretion. Cx proteins had alaobeen thought as active players in cell survival. Insights from what is known aboutroles for gap junctions in other systems in the context of the connexin expressionpattern by lung cells can be used to predict potential roles for gap junctionalcommunication between alveolar epithelial cells.Mesenchymal stem cells (MSCs) are multipotent progenitor cells representing anattractive therapeutic tool for regeneratiwe medicine. Several recent studies havedemonstrated that stem cells derived from adult tissues can home to and/or participatein the development of lung tissue, raising the possibility that stem cell-based therapiesmay be developed for effective intervention of lung diseases. several previous reportsthat applyed SCID-NOD rats or an immunoincompetent host duringxenotransplantation of stem cells. Because of their immaturity of immunologicsystem, new born rats might be used as the host of xenotransplantation. As respect tothe way of administration of hMSCs, the way by intravenously transplantation wasmost applied, but administration of hMSCs by intraperitoneal was thought easiertransplantation than that by intravenously.In our present study, we systemically administered hMSCs into new bornrecipients whom were exposed seven-days 95% O2 in vivo, We investigated whether hMSCs would engraft in the lung tissue of normal neonatal rats when administratedintraperitoneally, and if there was any sign of graft-versus-host disease(GVHD). andevaluated whether hMSCs administration altered the clinical course of hyperoxia-induced lung injury and the fate of administrated hMSCs. We also investigated thechanges of alveolar epithelial typeⅡcells suffered hyperoxia when cocultured withhMSCs in vitro.Chapter One Cultivation of human marrow mesenehymal stem cells in vitroObjeetiv To observe the biological features of human marrow mesenchymalstem cells(hMSCs) in vitro.Methods hMSCs were harvested from residual human ribsand separated by a density gradient centrifuge, hMSCs were cultured in vitro andtheir biological features were observed under phase contrast microscope, and culturedcells were labeled with the following anti-humanantibodies: CD33, CD34, CD44, CD45, HLA-DR, CD90, Mouse isotype antibodiesserved as respective controls. Ten thousand labeled cells were acquired and analyzedusing a FACS. In this study, passage 3 cells were also induced by BHA and DMSOrespectively. The morphological changes of induced hMSCs were observed indifferent culture period and the neuronal specific markers, neurofilement(NF) andNestin were evaluated by indirect immunocytochemistry Staining. Result FACSanalysis showed the cells from clones were strongly positively for CD44'CD90, butnegative for the hemopoietic markers CD33, CD34, CD45 and the HLA markerHLA-DR; hMSCs contain great proliferative capacity in vitro and exhibit obviousmrophology changes afte treated with BHA and DMSO in 6 hours,the differentiatedcells showed typical neuronal morphorlogy and expressed NF and Nestin.Conclusion Cultured cells showed some cell phenotypes of MSCs,and the capacity ofself-renewal and multiple potentiality in vitro, and they can also be induced intoneurons by BHA and DMSO.Chapter Two The influence of human mesenehymal stem cells on typeⅡalveolar epithelial cells of new born rat suffered from hyperoxiaObjective To analyze the effects of hyperoxia on typeⅡalveolar epithelial cellsisolated from new born rat lungs in vitro and changes when cocultured with humanmesenchymal stem cells(hMSCs). Methods The lung tissue of 3-day rats was digested by trypsin and collagenasel, then resuspended in the plastic culture flaskcoated with rat IgG for 1h, AECⅡs and lung fibroblasts were isolated and purified atdifferent centrifugal force and different adherence, then cultured for 23h. The natureof the cultures was identified by transmission electron micrography, hMSCs werefrom resuscitated hMSCs as previously described. Four groups were dvided, whichwere group A(AECⅡ+hyperoxia+hMSCs), group B(AECⅡ+hyperoxia), group C(AECⅡ+air+hMSCs) and group D(AECⅡ+air) respectively; as followingdescribed,AECⅡs were cultured for 24 hours after in the condition of 95% O2-5%CO2 or 95% air-5%CO2, then they were cultured alone or cocultured with hMSCsfor 24h by Transwell-Clear Inserts.Apoptosis of AECⅡwere analyzed with HoechstStaining Kit, the levels of TNFαand TGFβ1 in culture supernatant were detected byELASA,and Immunocytochemistry was used to detect the SP-A expression of hMSCsin the inserts. Results Excellent yields of highly purified, cultuarble AECⅡs could beobtained from new born rat lungs, Lamellar bodies in purified AECⅡs were revealedby transmission electron micrography. Apoptosis of AECⅡcould be detected ingroup A and group B(4.9817±0.78617%, 7.3183±0.71692%, t=5.380,p=0.0000),butno of apoptosis cell in group C and group D; The levels of TNFαin fourgroups(A,B,C and D)were 51.797±10.815,55.708±11.475, 21.168±3.835, 21.4594±4.481 respectively before cocultrue, while 29.501±2.169, 41.458±9.765, 20.822±2.605, 21.429±3.448 respectively (F statistic of main effectwere 55.068, 28.253 respectively, and P value were both 0.000; F statistic and P valueof crossover of effect were 19.362, 0.000 respectively); The levels of TGFβ1 ingroup A, B, C and D were 2289.296±1104.337,2339. 0411±125.291, 1337.835±149.936, 1371.835±108.178 respectively before cocultrue, while 1683.296±112.384, 2172.375±123.198, 1409. 441±111.994, 1342.527±105.288 aftercocultrue (F statistic of main effect were 45.840, 129.695 respectively, and P valuewere both 0.000; F statistic and P value of crossover of effect were 30.711, 0.000respectively); When group A was compared with group B, there existed moresignificance between two groups after coculture both the level of TNFαand the levelof TGFβ1 (p=0.001,0.000 respectively), but no significance before coculture(p=0.430,0.077 respectively). There was no expression of SP-A among hMSCs in group andgroup. Conclusion AECⅡs changed their secretion function after suffered hyperoxia,and hMSCs may have a protective effect.Chapter Three The influence of hyperoxia on the expression of connexin 26 inthe lung of neonatal ratsObjective To observe the changes of the tissue structure and connexin 26 (Cx26)expression in the lung of neonate rats suffered hyperoxia. Methods Sixteen 3-day SDrats were randomly divided into two groups; according to different exposurecondition, after 7 days hyperoxia exposure (the level of oxygen was above 95percent) or air exposure, all of rats were recorded their weght and length before andafter the experiment, value of radical alveolar counts (RAC) and microstructure of thelung tissue were observed by hematoxylon-eosin(HE) staining under light andelectron microscope respectively; and immunohistochemistry was used to detect theexpression of Cx26 protein in the lung tissue,while Cx26 mRNA by in situhybridization. Result Compared to the group of air exposure, both weight and lengthwere decreased obviously(13.525±1.270,17.373±0.888,t=7.024,p=0.000; 6.756±0.314,7.310±0.361,t=4.457,p=0.000),and RAC was also decreased(9.600±0.670,14.820±1.080;t=11.620,p=0.000),but the expression of Cx26 protein and the theexpression of Cx26 mRNA were both increased (13.68±1.28%, 17.82±1.72%, t=5.45,p=0.000; 10.335±1.694%, 7.369±1.487%, t=3.721, p=0.002). Conclusiou Hyperoxiadamaged the lung of neonatal rats,and the alteration of the expression of Cx26 mightplay an important role in acute lung injury and the recovery of injury.Chapter Four The Experiment of Transplanting Human Marrow-DerivedMesenchymal Stem Cells into New Born RatsObjective To investigate the influence of transplantation of human marrow-derived MSCs into new born rats. Methods Human Marrow-derived MSCs wereseparated, cultured, amplificated and labeled with BrdU; twenty-four ten-day-old SDrats were randomly divided into group A, group B and group C,they were injectedintraperitoneally with 1×104 MSCs, 5×104 MSCs and PBS respectively, general clinical signs of GHVD were observated after transplantation, three weeks later, thelevels of blood serum of TNFαwere detected with ELASA, Immunocytochemistywas used to determine the expression of BrdU, pathologic grade of livers andintestines were given for the histopathological study under light microscope. ResultsGeneral signs of GVHD were not found in three groups; BrdU-positive cells had beendetected in the lungs, livers and spleens in both group A and group B, but none ofBrdU-positive cells was found in the group C; there existed and significant varianceamong three groups in the grade of the expression of BrdU in lungs,(0.261±0.023,0.215±0.0197,0.000±0.000, F=500.347, p=0.000), there also existedsignificant variance between group A and group B; but there existed no significantvariance among three groups in the fields of the levels of blood serum ofTNFα(21.03±4.19,19.78±1.95,18.64±3.03,F=1.122, p=0.344, pathologic grade oflivers(x2=0.793, p=0.673),intestines(x2=1.080, p=0.583), animal weight after theexperiment(32.04±2.16, 31.15±1.89, 31.01±2.74, F=0.415, p=0.666). Conclusion BrdUlabeled human marrow-derived MSCs can implant organs of new bron rats, and thereexisted no evidence of GVHD in the new born rats injected by hMSCs.Chapter Five The Influence of Human Marrow-Derived Mesenchymal StemCells on the Lung of New Born Rats Suffered HyperoxiaObjective To investigate the influence of human marrow-derived MesenchymalStem Cells(hMSCs) on the lung of new born rats suffered hyperoxia. MethodshMSCs were separated,cultured, amplificated, identified and labeled with BrdU;thirty-two three-day-old Sprague-Dawley (SD) rats were randomly divided into fourgroups according to their exposure condition and with or without administration ofhMSCs. they were suffered with seven-day 95% oxygen or air, then they were injectedfrom tail vein with MSCs of 1×105 or phosphate buffered solution (PBS)respectively; 72h later, all of them were killed,Immunocytochemisty was used todetermine the expression of BrdU,RT-PCR was used for the detection of AluSequences, value of radical alveolar counts (RACs) of the lungs of the rats werecounted for the histopathological study under light microscope, and the levels ofTNFαand TGFβ1 in the lung homogenates were detected by ELISA. ResultsHyperoxia resulted in a significant increase in the levels of both TNFαand TGFβ1 in lung homogenates (142.933±24.017, 79.033±11.573,224.088±41.915,76.500±10.373, F=59.970, P=0.000; 1726.484±91.086, 1530.359±173.441,2047.717±152.057,1515.777±131.049, F=24.977, P=0.000)and asignificantdecrease in the RAGs (11.145±1.331, 13.941±0.985, 9.595±0.672, 14.819±1.080, F=43.234, P=0.000), and all of which might be obviously improved by theadministration of hMSCs. BrdU-positive cells by immunocytochemisty and specificstraps by RT-PCR were only found in the two administration of hMSCs goups, andthere were more BrdU-stained cells in the lung of hyperoia-exposed rats than that inair-exposed ones. Conclusion hMSCs administration by intraperitoneal injection canimplant the lungs of new born rats, and they have effective protection for the damagedlungs suffered hyperoxia.
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