| Lung deveiopment-reiatea diseases, sucn as respiratory distress syndrome bronchopulmonary dysplasia (BPD), are common but refractory diseases of neonatology, which are the two most important causes of death in premature infants and very low birth weight infants in clinic. RDS is due to lack of pulmonary surfactant (PS), the manifestations of which mainly include progressive dyspnea, cyanosis, and respiratory failure within hours after birth. On the basis of immature lung, BPD usually develops with high oxygen, infection and other acute lung injuries, followed by abnormal repair. The functional maturation of the type II alveolar epithelial cell is the understratum of PS secretion and lung injury repair. Exploration of the mechanism of AECII mature and functional regulation is especially important on seeking new therapeutic targets for neonatal lung disease.AECII is a major component of the mammalian alveolar epithelial cells, which is closely related to lung development related diseases. Functionally, type II epithelial cell reduces the collapsing forces at the air-liquid interface of the alveoli to maintain pulmonary function by synthesizing and secreting pulmonary surfactant lipids and proteins; Meanwhile, AECII is believed to serve as stem cell which is able to self-renew sufficiently and to transdifferentiate to alveolar epithelial type I cells, repairing the injured alveolar epithelium. Furthermore, AECII also plays a key role in the maintenance of alveolar fluid balance and the host immune. Therefore, AECII developmental immaturity or functional defects could result in a variety of lung diseases, such as respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD). Currently, normal AECII cell line for long-term studies has not been established, while the function of A549 cell from lung adenocarcinoma can not completely replace the normal AECII, hence, the AECII for research is mostly isolated from adult rat lung tissue. Our study focuses on lung disease of neonate, and the biological characteristics of the rat lung tissue from 19 days fetal rat is in line with the late human fetal lung development. Thus this tissue satisfies our research. The tirst part of this paper was mainly to study the method of AECII separation and culture from the 19-day fetal rat lung tissue, and to perform a series of identification and analysis on the primary AECII, identifying the optimum culture period of the primary AECII for study in vitro.MiRNA (microRNA) is an important group of short endogenous RNA which was discovered recently. In terms of functionality, it could regulate gene expression in post-transcription level through either the direct degradation of the target mRNA or the suppression of their translation. At present, which is known is that it plays a key role in many biological processes, such as the cell proliferation, signal transduction, stem cell differentiation, occurrence and metastasis of the tumor, etc. Among these miRNAs, miR-146b is highly conserved in vertebrate which has been reported to play an important role in the inhibition of tumor development, cell differentiation, proliferation, and other biological processes. The expression of miR-146b has a significant difference at the different late stages of fetal lung development when AECII begin mature and functional. Therefore, we hypothesized that miR-146b might have a certain impact on the mechanism of maturation and function of AECII. In order to verify the hypothesis, we constructed miR-146b overexpression in primary AECII cells by adenovirus vector, then detected the influence of miR-146b overexpression on AECII proliferation, transdifferentiation, parts of surfactant proteins (SPs) expression, and further explored the potential target gene, providing a new theoretical basis for revealing the mechanism of the occurrence of neonatal lung development-related diseases.Part1:Prenminary stuay of the ietai rat alveolar type Ⅱ epithelial cell growth and ABCA3 expression changesObjective:To observe the growing conditions of type Ⅱ alveolar epithelial cell (AECII) and dynamic expression of AECII specific protein ATP binding cassette transporter A3 (ABCA3) in vitro.Methods:Lung tissues of 19-day fetal rats were digested with trypsin and collagenase, then purified for AECII with different centrifugal force and repeated attachment. AECII was identified by electron microscopy. Growth status and shape of attached cells at different time points were observed with inverted phase contrast microscope and the cell growth curve was drawn by MTT assay. The dynamic expression of ABCA3 in AECII was detected by immunofluorescence.Results:The AECII characteristic lamellar bodies with varying size and amount were observed under electron microscopy. The MTT OD values at 24h,36h,48h,72h,96h, and 120h were 0.177±0.009,0.193±0.011,0.212±0.019,0.253±0.019,0.243±0.012, 0.192±0.011 respectively. Cell morphology under microscope was gradually elongating, flattening and the intracellular particles were also gradually reducing. Simultaneously, immunofluorescence showed that ABCA3 expression gradually decreased with time.Conclusions:Primary cultured AECII has the best growth status and the most abundant ABCA3 expression in the early culture time, therefore this period (cultured for 72 hours or less) is the optimal time point to research the mechanism of diseases associated with lung development.Part Ⅱ:MiR-146b reduces proliferation of rat’s type Ⅱ alveolar epithelial cells and surfactant protein-B expression by repressing epidermal growth factor receptorObjective:To verify whether miR-146b plays any roles in the physiological functions of AECII and the possible mechanisms involved.Methods:Firstly, we constructed adenoviral vector pAd-GV275 CMV-rno-mir-146b which contained rat miR-146b precursor sequence and flanking sequences. The empty vector without a miRNA insert was used as a vector control. Then adenovirus was produced by transfecting the adenoviral vector into HEK 293A cells and amplified by transducing HEK 293A cells, followed by infecting primary cultured AECII. The AECIIs transfected with the same amount of virus-free transfection reagent were as blank control. After verification of the overexpression using real-time PCR, the differences of cell proliferation, transdifferentiation, part of the surfactant proteins (SPs) and epidermal growth factor receptor (EGFR) expression among those three groups were detected respectively using the MTT assay, immunofluorescence, real-time PCR and western blot method.Results:When AECIIs were cultured for 48h or 72h, a decreased growth of cells was found in overexpressing miR-146b group compared with the control cells (p<0.05). However, the expression of ABC A3 and AQP5 in AECII cultured for 5 days was no difference between the blank control group and overexpressing miR-146b one. The mRNA expression of SP-B was found significantly lower than the control group when the AECIIs were cultured for 3 days (p<0.01), but no difference was found of the expression of SP-C mRNA and protein among those three groups. Furthermore, the expression of EGFR mRNA and protein in the AECII overexpressing miR-146b were lower than the control group (p<0.05).Conclusion:This study shows that miR-146b negatively regulates cell proliferation and inhibits SP-B expression in primary AECII. Moreover, it is speculated that the possible mecnainsm of this ncgative ieguiation is that MIR-1400 down-ieguiaies EGFR. |
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