Expression And Role Of Connective Tissue Growth Factor In Premature Rats With Hyperoxia-Induced CLD

prefaceChronic lung disease(CLD)is a major complication of premature who inspired hyperoxia or accepted mechanical ventilation.With the rise of survival rate in low birth weight premature,the incidence of CLD is going up.10%-15%CLD infants die of respiratory failure while the survivors depend on long-time oxygen therapy or mechanical ventilation.The final termination of CLD is pulmonary fibrosis and damaged lung function,which seriously influences the life quality of those infants. Thus the exploration of CLD fibrosis mechanism has become a troublesome problem to solve.Current research has proved that hyperoxia is major danger to the immature lungs, which was confirmed by the animal model of CLD set up with hyperoxia.The deficiency of antioxidant system(including SOD,catalase,glutathione peroxidase and et al)is the major cause of lung injury of the premature animals,immature labor,patent ductus arterious,mechanical ventilation and infection.Cytokines have been paid more attention to in recent research of CLD,such as TNF-α.High level of TNF-αcan initiate chronic inflammatory reaction,while low level of it can decrease the danger and severity.High level of IL-6,IL-8 and TNF-αis the prediction factor to CLD.Otherwise, some cytokines such as IL-4,IL-10,TGFβ1,CTGF and VEGF et al have the action of antiinflammation,profibrosis and regulation of neovascularization.In many cytokines, some key growth factors contribute to the development of CLD.Transforming growth factor and connective tissue growth factor are especially paid attention to.TGF-βis a multifunctional cytokine which is essensial in the development and homeostasis of lung tissue.However it is also related to the lung fibrosis.Out previous research has proved its intimate correlation with CLD.Some research has already tried to resist fibrosis by blocking its function.The effect of cytokine is extensive and its potency is complex.If we intercept its expression completely,the consequence is hard to predict.Thus it is important to search the downstream cytokine which is more specific in fibrosis,and it would be an effective way to prevent CLD.Connective tissue growth factor(CTGF)belongs the CCN family of immediate early genes.Its protein has a cysteine-rich,38KDa secreted peptide.It can promote fibroblast proliferation and the deposition of collagen.More and more researches have proved its important effect on fibrosis disease.However its function in fibrosis is unknown.Based on our previous research,we establish the CLD model of premature rats to observe lung fibrosis and the expression of CTGF by using hematoxylin-eosin stain and immunohistochemistry et al;meanwhile we segregate and culture lung fibroblast from premature rats,use TGF-β1 for intervention and observe the expression of CTGF in fibroblast trying to prove that maybe TGF-β1 is the upstream cytokine of CTGF and plays an important role in hyperoxia-induced CLD in premature rats by inducing the overexpression of CTGF.Materials and Methods1.Animal experiment(1)Animal model and subgroupWe gained premature rats by uterine-incision delivery for Wistar rats which were 21 days gestational age.The premature rats were divided into two groups randomly,the experiment group(hyperoxia group)and the control group(air group).Each group contained 50 premature rats.In the experiment group,the premature 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 food and clean dirty cages.Nursing mothers 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 1,3,7,14,21.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 and immunohistochemistry.2.Extraorgan experiment(1)Lung fibroblast cuture and identificationIsolate and identify lung fibroblast of fetal rats(19 days).Culture primary and passage cells.During the 3^rdpassage,we intervene the cells with TGF-β1 and use the vimentin immunohistochemistry to indentify fibroblast.(2)TGF-β1 stimulation testWhen the conjugation of third passge cells was 70%,serum-free medium was replaced.The subgroup experiment started after 48 hours.In the condition of 21% oxygen concentration,different dose of TGF-β1(0 ng/ml,1 ng/ml,5ng/ml,10 ng/ml) was used to stimulate lung fibroblast of premature rats.After 24 hours,the cells was collected and placed in -80℃refrigerator.We used the 10 ng/ml TGF-β1 to stimulate the isolated fibroblast,collected the cells at different times(12,24,48h) and placed the cells in -80℃refrigerator.The control group(without TGF-β1 stimulation at 12,24,48h)was set.(3)Collect exampleThe fibroblast of TGF-β1 stimulation in different concentration(0ng/ml,1 ng/ml, 5ng/ml,10 ng/ml)was collected.The fibroblasts stimulated by 10 ng/ml TGF-β1 and control cells were collected at different time points(12,24,48h).3.Experiment methods and detection of target (1)Lung histomorphologyâ‘ Observe lung histomorphology using light microscope to invest the pathology of CLD lung.â‘¡Use Ashcroft methods to score fibrosis and assesse the degree of fibrosis.(2)The immunohistochemistry of lung(SABC)10 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 was finded in endochylema,the cell was masculine.American Universal Imagining Porppration system was used to analyse the quantitative determination while Meta Morph software was used to accumulate the mean optical desity value.(3)Vimentin immunohistochemistry(identification of fibroblast)Collect premature lung fibroblast.Stain vimentin by using SABC method.View and identify fibroblast.(4)CTGFmRNA in lung fibroblast detecting using RT-PCR technologyWe used RT-PCR to amplify the gene of lung fibroblast.Analysis of amplification products:the amplification products experienced 2%gel electrophoresis and were stained by ethidium bromide and shot in the ultraviolet light.Kodak1D gelatum imagine system was used to analyse electrophoresis strip.The relative mRNA level was expressed by the ratios of CTGF vsβ—actin.4.Statistical analysisSPSS11.5 was used to perform statistical analysis,with all data expressed as ((?)±s),according to the test of homogeneity.T-test or t'-test was used in two groups and Spearman was used 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 1 of the experiment,it was observed in both room air and hyperoxia group that the alveolar was thick and the alveolar structure was irregular.On day 3,the alveolar septum was thinner and the alveolar structure of the air group was more regular meanwhile in hyperoxia group,there was a few red cells which exuded to alveolus.From 7d to 21d,the alveolar septum was more thinner and the alveolar structure in air group became regular gradually.In hyperoxia group on day 7,it was observed that there was inflammatory response,more interstitial cells,lung septum degraded and lung edema;on day 14,the interstitial cells was increased with the alveolar septum thickened obviously and lung fibrosis emerged gradually;on day 21, the interstitial cells increased obviously with alveolar septum much thicker and there was consol a little or multiplace.2.Lung fibrosis scoresOn day 14 and day 21,there was statistical difference between control and experiment group(p＜0.05).3.The expression of CTGF protein in the lungs(1)The expression location of CTGF proteinIn control group,CTGF was expressed tenuously in vascular endothelial cell, bronchi and bronchiole epithelium.In experiment group,the expression of CTGF protein was similar with the control group on day 1 and 3.However,on day 7,CTGF protein was also expressed tenuously in pulmonary epithelial cells,alveolar septum and fibrolast in experiment group.On day 14,the expression of CTGF protein in pulmonary epithelial cells and interstitial fibroblast increased significantly.On day 21,the expression of CTGF protein increased further in these cells and lung fibroblast became the main expression cell.(2)The expression strength of CTGF proteinOn day 1,3 and 7,there was no significant deviation in the expression strength of CTGF protein between two groups(p＞0.05).On day 14,the expression was signigicantly higher in experiment group than in control group:11.23±2.11,5.42±0.59 individually(p＜0.01)and on day 21:15.91±3.02,5.86±0.73 individually(p＜0.01). 4.Dependablity analysisSpearman method was used for dependablity analysis:the CTGF protein expression of hyperoxia lung tissue was obviously positive correlation with lung fibrosis scores:r=0.892,p＜0.01.5.Identification of fibroblastThe cytoplasm of fibroblast was stained huffy in which there was silkness Vimentin under light microscope(×200).6.The expression of CTGF mRNA in lung fibroblast of premature rats under the stimulation of TGF—β1Under the stimulation of TGF—β1 with the concentration of 0 ng/ml,1 ng/ml, 5ng/ml and 10 ng/ml,the CTGFmRNA expression was 0.26±0.02,0.29±0.03, 0.62±0.09 and 0.88±0.02 individually after 24 hours.Compared with 0 ng/ml group, the p value was less than 0.05 in both 5ng/ml and 10 ng/ml groups but more than 0.05 in 1 ng/ml group.When compared between groups of 1 ng/ml,5ng/ml and 10 ng/ml,p value was less than 0.05 in all.0 ng/ml and 10 ng/ml TGF-β1 was used to stimulate lung fibroblast and the expression of CTGF mRNA was tested after 12,24 and 48 hours. After 12 hours,the expression of CTGF mRNA in 10 ng/ml and 0 ng/ml groups was 0.31±0.02 and 0.17±0.01 individually(p＜0.05).After 24 hours,the expression was 0.79±0.04 and 0.19±0.03(p＜0.05).After 48 hours,the expression was 0.96±0.03 and 0.20±0.02(p＜0.05).Under the stimulation of 10 ng/ml TGF—β1,p value was all less than 0.05 between groups after 12,24 and 48 hours.Conclusion1.The high level expression of CTGF protein in lung tissue of premature rats exposed to hyperoxia had close correlation with lung fibrosis.2.Using TGF—β1 to stimulate the expression of CTGF,it was manifested that CTGF would be one of the downstream cytokines of TGF—β1 which induced premature lung fibrosis.