Study On Biological Characteristics Of Human Amnion Cells And The Effect Of Human Amnion Epithelial Cells To Hyperoxia-Lnduced Lung Injury

ObjectiveIsolation and culture human amnion epithelial cells and human amnionmesenchymal stem cells, identification their biological characteristics. Exploring therole of human amnion epithelial cells in hyperoxia-induced lung injury in neonatalrats.Method1.Cell separation and identificationThe human amnion tissues were collected from full term placentas, which obtainedfrom cesarean section. Using low speed-trypsin-collagenase digestion method toisolate hAECs and hAMSCs from human amnion tissue. Observing the cellsmorphology and drawing the growth curve to assess the proliferation. Indirectimmunofluorescence to detect the expression of CK19and vimentin. FACS to analysethe cell surface antigen protein, such as CD29, CD73, CD44, CD166, CD90, CD105,CD49e and so on. Pluripotent stem cell surface sign Oct-4and Nanog were examinedby PT-PCR. To examine differerntiation potential of the cells, Oil Red O and Alizarinred staining were used to detect the ability of adipogenic and osteogenic.2. The effect of human amnion epithelial cells to hyperoxia-induced lung injury8nest(8-12/nest) specific pathogen free(SPF) neonatal SD rat were randomlyassigned to normoxia group, hyperoxia group, hyperoxia+PBS group or hyperoxia+hAECs group, within24hours of birth. The neonatal rats of normoxia group wereexposed to normal air with conventional breeding for12days. The hyperoxia group,hyperoxia+PBS group and hyperoxia+hAECs group were exposed to90%oxygen concentration with conventional breeding within24hours of birth. Hyperoxia+hAECs group receivd5×105hAECs in40μL PBS intratracheally, hyperoxia+PBSgroup receivd40μL PBS intratracheally, after exposed to hyperoxia environment for8days. Normoxia group and hyperoxia group were not given any treatment. Hyperoxiagroup, hyperoxia+PBS group and hyperoxia+hAECs group were change fromhyperoxia environment to normal air on10day of birth. All neonatal were humanelykilled to collect blood and lung tissues to test on12day of birth.Flourescencemicroscope to track the labeled hAECs. Lung tissue sections were stained withhematoxylin and eosin, to contrast pathliogical of lung tissue from each group andassese the lung injury scores. Detection of serum and lung tissue inflammatorycytokines IL-6, IL-10levels, oxidative stress indicators SOD activity, MDAconcentration and lung tissue MPO activity from each group.Result1. The biological characteristics of hAECs and hAMSCs1.1The shape of hAECs were round or oval, which growing like cobblestone, maximumspread5generations. HAMSCs were fibroblast-like spindle length, radial or spiralgrowth distribution, could transmitted to about30generations.1.2CK19was expressed in hAECs, while a few vimentin were positive. HAMSCsexpression of vimentin, while CK19was negative. HAECs and hAMSCs expressedCD29, CD73, CD44, CD166, CD90and Oct-4, Nanog and so on. HAECs did notexpress CD49e, CD105.1.3HAECs and hAMSCs Oil Red O and Alizarin red staining were positive.2. The effect of human amnion epithelial cells to hyperoxia-induced lung injury2.1Lung tissue from hyperoxia+hAECs had improved, to reduce thickness of thealveolar septa and increase radial alveolar count. There were a small number ofmarked hAECs in the lung tissue from hyperoxia+hAECs.2.2Compared with hyperoxia group, inflammatory IL-6levels significantly decreasedin hyperoxia+hAECs group, while compared with hyperoxia+PBS group, there wasno statistically differences.The level of IL-10in the lung tissue of each group werequite different. Compared with normoxia group, serum IL-10levels significantly increased in hyperoxia group. There was no significantly different between othergroups.2.3Compared with hyperoxia+PBS group, the activity of SOD, MPO in lung tissue ofhyperoxia+hAECs significantly increased,while MDA concentrations significantlydecreased. Compared with hyperoxia group, the activity of serum SOD fromhyperoxia+hAECs significantly increased, while MDA concentrations significantlydecreased. There was no significantly difference between hyperoxia+hAECs andhyperoxia+PBS group.Conclusion1. HAECs and hAMSCs with stem cell properties.2. HAECs could partiallyimprove hyperoxia-induced lung injury in neonatal rats.3. Oxidative stress may playa leading role in hyperoxia-induced neonatal rats lung injury. HAECs couldsignificant regulate oxidative stress, so reducing oxidative stress may become aneffective way to prevent hyperoxia-induced lung injury and BPD.