Effect And Role Of TGF-β1 On The Cell Cycle Of Lung Fibroblasts In Neonatal Rats

PrefaceChronic lung disease (CLD) is the most common and serious complication of newborn, especially premature, who suffered from heart and lung disease and inspired hyperoxia or accepted mechanical ventilation. In recent years, with the rise of improvement of medical care, the survival rate of premature with low birth weight is going up, and the incidence of CLD is also going up. The serious CLD infant die of respiratory failure while the survivors depend on long-time (several months or years) oxygen therapy or mechanical ventilation. The final termination of CLD is pulmonary fibrosis and damage lung function, which seriously influences the life quality of those infants. Because the pathogenesis is not clear and there is still no satisfactory treatment, the exploration of CLD fibrosis mechanism has become a problem to be resolved.Recent study have indicated that the pathological basis of pulmonary fibrosis is the abnormal proliferation of lung fibroblast (LF), resulting in unbalanced synthesis and degradation of extracellular matrix (ECM), which eventually led to the latest irreversible pulmonary interstitial fibrosis. Study on the mechanism of fibrosis is more concentrated on cytokines. Among them, the role of transforming growth factorβ1 (TGF-β1) is more clearly, and specially paid attention to.A lot of study found that the pathogenesis of pulmonary fibrosis is abnormal proliferation of lung fibroblast. In many cell cytokines, TGF-β1 is has close relationship with fibrosis, which has been paid great attention to.The change of cell cycle regulates cell proliferation. The normal cell cycle include G1, S, G2, M phase. Many checkpoints work on it. Among them, G1 phase to S phase is the most important checkpoint. A variety of proteins involve in cell cycle changes. In the field of renal fibrosis and liver fibrosis, TGF-β1 regulates the expression of cell cycle regulatory molecules (Cyclin, CDK, CDKI) and involved in cell proliferation and fibrosis. Cyclin-dependent kinase-2 (CDK2) and cyclin-dependent kinase inhibitors (P27) play an important regulator of conversion. But the role in CLD is unclear. Therefore, based on our previous research, we establish the CLD model of neonatal rats and culture lung fibroblast cells in vitro, trying to prove the mechanism of TGF-β1 regulate LF proliferation, by using immunohistochemistry and Real-Time PCR et al, with a view to finding new ways to lay the experimental basis for prevention and treatment.Materials and methods1. Animal experiment(1) Animal model and subgroupWe gained neonatal rats by natural delivery for Wistar. The neonatal rats were divided two groups randomly, the experiment group(hyperoxia group) and the control group(air group). Each group contained 32 neonatale rats. In the experiment group, the rats were placed in Plexiglas chamber into which oxygen was continuously delivered to achieve a constant level of 90% oxygen concentration. CO2 was absorbed by sodalime to keep CO2 level below 0.5%. Temperature and humidity were maintained at 25℃-27℃and 50%-70% respectively. Champer was opened for 0.5h daily to change water, add and clean dirty cages. Nursing mother were rotated between oxygen exposed and room air litters everyday to avoid oxygen toxicity. The control group was placed in air conditions(21% oxygen concentration). Methods and control factors were same with the experiment group.(2) Tissue preparationPups from each group were killed on days 3,7,14,21. Thoracic cavity was opened after anesthesia with 10% chloral hydrate. Lungs were removed, and the left lungs were placed in 4% paraformaldehyde for hematoxylin-eosin stain, immunohistochemistry and real-time PCR, and the right were cryopreservationed in-80℃Refrigerator2. Extraorgan experiment(1)Animal experimentNeonatal rats of Natural c pregnant, male or female.(2)Lung fibroblast cuture and identificationThoracic cavity of pups was opened after anesthesia with 10% chloral hydrate and soaked in 75% alcohol. Isolate and identify lung fibroblast of rats. Culture primary and passage cells. During the third passage, we intervene the cell with TGF-β1 and use the vimentin immunohistochemistry to indentify fibroblast.(3)TGF-β1 simulation test and drawn①When the conjugation of third passage cells was 70%, theserm-free medium was replaced. The subgroup experiment stared after 24 hours. In the condition of 21% oxygen concentration, different dose of TGF-β1 (Ong/ml, lng/ml,5ng/ml,10ng/ml), was used to stimulate lung fibroblast of neonatal rats. After 24 hours, the cells was collected and stored on condition.②We used the 10ng/ml TGF-β1 to stimulate the isolated fibroblast, collected the cells at different times (12,24,48h) and stored on condition. The control group (without TGF-β1 stimulation at 12,24,48h) was set.3.Experiment methods and detection of target(1)Lung histomorphologyObserve lung histomorphology using light microscope to invest the pathology of CLD lung.(2)Lung fibrosis scoresUse Ashcroft methods to score fibrosis and assess the degree of fibrosis.(3)The immunohistochemistry of lung(SP)By SP immunohistochemistry,8 slices were selected randomly at different time points and five fields of every slice was selected randomly using light microscope. Windows were fixed. If buffy grains were found in cytoplasm and/or nucleus, the cell was masculine. American Universal Imagining Porppration system was used to analyse the quantitative determination, Meta Morph software was used for the mean Average gray value.(4)Detect CDK2 and P27mRNA in lung using real-time PCR technologyReal-time PCR, first the RNA standard of was constructed target gene and housekeeping gene. Second, making the standard curve, using the standard curve samples quantitative target gene and housekeeping gene. Third, detect the expression of CDK2 and P27 mRNA.(5)Vimentin immunohistochemistry (identification of fibroblast)Collect lung fibroblast, stain vimentin by using SP and immunofluorescence method, view and identify fibroblast. (6)Cell Counting Kit-8 detect cell proliferationTake third-generation lung fibroblast cell, then regulate the concentration to 5000/ 100μL. 100μL were seeded in 96 well plates per hole, respectively. We used TGF-β1 to stimulate the cell, then seeded CCK8 10μL in every hole. Incubate in 37℃5% CO2 for 1 hour. Detect the absorbance at 450nm wavelength.(7)Flow cytometry detect cell cycleFibroblast cell was stimulated by TGF-β1, fixed at 70% ethanol and 4℃, added RNA enzyme, then digested in a water bath at 37℃for 30min. Place in dark for 30 min, then detect in flow cytometry.(8)The immunohistochemistry of fibroblast cell(SP)By SP immunohistochemistry,4 slices were selected randomly at different time points and five fields of every slice was selected randomly using light microscope. Windows were fixed. If buffy grains were found in cytoplasm and/or nucleus, the cell was masculine. American Universal Imagining Porppration system was used to analyse the quantitative determination, Meta Morph software was used for the mean Average gray value.(9)Detect CDK2 and P27mRNA in cell using real-time PCR technologyReal-time PCR, first the RNA standard of was constructed target gene and housekeeping gene. Second, making the standard curve, using the standard curve samples quantitative target gene and housekeeping gene. Third, detect the expression of CDK2 and P27 mRNA.4.Statical analysisAll data was expressed as(x±S)and use SPSS 14.0 perform statistical analysis, according the test of homogeneity, t-test or t'-test was used in two groups, and Spearman was use to analyse the correlation, ANOVA was used to analyse multigroups, and it had significance when P<0.05.Results1.The histmorphology findings of the lung tissueOn day 3 of the experiment, it was observed in both room air and hyperoxia group that the alveolar septum was thinner, the alveolar structure of the two groups was more regular, but in hyperoxia group, there was a few red cells which exude to alveolus. From 7d to 21d, the alveolar septum was more thinner, the alveolar structure in air group became regular gradually. In hyperoxia group, on day 7, it was observed that there was inflammatory response and more interstitial cells, lung septum degraded and lung edema; On day 14, the interstitial cells was increased the alveolar septum thicken obviously, lung fibrosis emerge gradually; On day 21, the interstitial cell increased obviously, alveolar septum was more thicker, there was consol a little or multiplace.2.Lung fibrosis scoresOn day 14 and day 21, there were statical difference between control and experiment groups (P<0.05).3.The expression of CDK2 and P27 protein in the lungs(1)The expressed locationIn control group, CDK2 was expressed tenuously in vascular endothelial cell, bronchi and bronchiole epithelium. In hyperoxia group, the expression of CDK2 protein was similar with the control group on day 3. On day 7, pulmonary epithelial cells, alveolar septum, and fibrolast expressed CDK2 protein tenuously. On day 14, the expression of CDK2 protein in pulmonary epithelial cells and interstitial fibroblast increased significantly. On day 21, in these cells, CDK2 protein increased greatly, lung fibroblast was the main expression cells.In control group, P27 was expressed in vascular endothelial cell, bronchi and bronchiole epithelium. In hyperoxia group, the expression of P27 protein was similar with the control group on day 3. On day 7, in pulmonary epithelial cells and fibrolast P27 protein expressed decreased. On day 14, the expression of P27 protein in pulmonary epithelial cells and interstitial fibroblast decreased significantly. On day 21, in these cells, P27 protein decreased greatly.(2)The expression strength of CDK2 and P27 proteinOn day 3 and 7, there was not significant deviation in the expression strength of CDK2 and P27 protein between two groups (P>0.05).The CDK2 expression increased and the P27 decreased. Conpared day 14 and 21, P<0.05, respectively.4.The expression of CDK2 and P27 mRNAOn day 3 and 7, there was not significant deviation in the expression strength of CDK2 and P27 mRNA between two groups (P>0.05). On day 14, the CDK2 expression of experiment and control group were 16.05±6.17 and 6.94±3.42(P<0.05), respectively; P27 were 33.16±7.61 and 51.51±8.56 (P<0.05), respectively. On day 21, the CDK2 expression were 27.06±10.65 and 8.24±3.60 (P<0.05), respectively; P27 were 14.82±3.34 and 41.36±13.05 (P<0.05), respectively.5.Dependability analysisSpearman method was used for dependability analysis:the CDK2 expression of hyperoxia lung tissue was obviously positive correlation with lung fibrosis scores: r1=0.784,r2=0.702, P<0.05; the P27 expression of hyperoxia lung tissue was obviously negative correlation with lung fibrosis scores:rl=-0.819, r2=-0.765, P<0.05.6.Identification of fibroblastThe fibroblast was like spindle under inverted microscope(×100). The cytoplasm of fibroblast was stained buffy in which there was silk vimentin under light microscope (×400). The cytoplasm of fibroblast was stained green using immunofluorescence (×400).7.The proliferation of lung fibroblast under the stimulation of TGF-β1Under the stimulation of TGF-β1 with the concentration increased, the OD were 0.251±0.023,0.318±0.023,0.418±0.028 and 0.470±0.014 individually after 24 hours. Compared with Ong/ml group, the P value of 5ng/ml and 10ng/ml group were less than 0.05 in all, but the P value of 1ng/ml group was more than 0.05; Compared with each group, the P value was less than 0.05 in all. Under the stimulation of TGF-β1 with the time extended, the OD were 0.362±0.048,0.470±0.014 and 0.684±0.119 individually after 12,24,48 hours. Compared with control group, the P value was less than 0.05 in all; Compared with each group, the P value was less than 0.05 in all.8.The cell cycle of lung fibroblast under the stimulation of TGF-β1Under the stimulation of TGF-β1 with the concentration of Ong/ml,1ng/ml, 5ng/ml and 10ng/ml, the proportion G0/G decreased gradually; the proportion S increased gradually; Compared with Ong/ml group, P<0.05 in 5ng/ml and 10ng/ml group, P<0.05 in 1ng/ml group; Compared with each group, P<0.05 in all. 10ng/ml and Ong/ml were used to stimulate lung fibroblast and the expression of cell cycle was tested after 12,24 and 48 hours. Compared with control group and between groups, P<0.05. 9.The expression of CDK2 and P27 protein in lung fibroblast under the stimulation of TGF-β1Under the stimulation of TGF-β1 with the concentration of Ong/ml,1ng/ml, 5ng/ml and 10ng/ml, the CDK2 protein expression was positive correlation with concentration, and P27 protein was negative correlation with concentration after 24 hours. Compared with Ong/ml group, the P value was less than 0.05 in both 5ng/ml and 10ng/ml groups, but more than 0.05 in 1ng/ml group. When compared between groups of lng/ml,5ng/ml and 10ng/ml, P<0.05.10ng/ml and Ong/ml were used to stimulate lung fibroblast and the expression of CDK2 and P27 protein was tested after 12,24 and 48 hours. The expression of CDK2 protein was positive correlation with concentration, and P27 protein was negative correlation with concentration. Compared with control group P<0.05. Under the stimulation of 10ng/ml TGF-β1, P value was less than 0.05 between groups after 12,24 and 48 hours.10.The expression of CDK2 and P27 mRNA in lung fibroblast under the stimulation of TGF-β1Under the stimulation of TGF-β1 with the concentration of Ong/ml,1ng/ml, 5ng/ml and 10ng/ml, the CDK2 mRNA expression was positive correlation with concentration, and P27 mRNA was negative correlation with concentration after 24 hours. Compared with Ong/ml group, the P value was less than 0.05 in both 5ng/ml and lOng/ml groups, but more than 0.05 in 1ng/ml group. When compared between groups of lng/ml,5ng/ml and 10ng/ml, P<0.05.10ng/ml and Ong/ml were used to stimulate lung fibroblast and the expression of CDK2 and P27 mRNA was tested after 12,24 and 48 hours. After 12 hours, the expression of CDK2 mRNA was positive correlation with concentration, and P27 mRNA was negative correlation with concentration. Compared with control group.P<0.05. Under the stimulation of 10ng/ml TGF-β1, P value was less than 0.05 between groups after 12,24 and 48 hours.Conclusion1.The expression of CDK2 and P27 in lung tissue of neonatal rats exposed to hyperoxia increased gradually; The expression of P27 decreased gradually.2.Using TGF-β1 to stimulate lung fibroblast in vitro, it was manifested that TGF-β1 could promote proliferation by regulating the expression of CDK2 and P27.