BACKGROUND:Several studies have demonstrated that the number of alveolar epithelial cells decreases gradually as pulmonary fibrosis develops,but the reasons remain unknown.The expression of P16,CyclinD1,and CDK4 might be abnormal and P16-CyclinD1/CDK4 call cycle regulatory pathway may play an important role in the progressive reduction of alveolar epithelial cells during pulmonary fibrosis.OBJECTIVE:To investigate the dynamic expression of P16,CyclinD1,and CDK4 in the pulmonary tissue of bleomycin-inducad pulmonary fibrosis mice.METHODS:Pulmonary fibrosis was induced in 6-week-old KM mice by intratracheal injection of bleomycin(BLM group).The control mice were administered the same volume of physiological saline(control roup).At 3,7,14,and 28 days after modeling,hematoxylin-eosin staining was performed to observe the pathomorphological changes of lung tissue.P16,CydinD1,and CDK4protein expression in lung tissue was detected by immunohistochemistry and Western blot analysis.RESULTS AND CONCLUSION:In the BLM group,typical changes of pulmonary fibrosis were observed,and P16 and CDK4protein expression in the pulmonary tissue increased with time,but CyclinD1 protein expression was decreased with time.P16and CDK4 protein-positive calls in the BLM group were more than in the control group.Compared with the control group,P16protein expression in the BLM group was higher at 14 and 28 days after modeling(P<0.01)and CDK4 protein expression was higher at 7,14 and 28 days after modeling(P<0.05).CydinD1 protein-positive cells and protein expression were greater at 3and 7 days after modeling in the BLM group(P<0,05),but they were less at 28 days after modeling(P<0.05),than in the control group.At 14 days after modeling,CydinD1 protein-positive cells in the BLM group were more,but CyclinD1 protein expression was ess,than in the control group(P<0.05).These findings suggest that P16,CyclinD1 and CDK4 protein expression was abnormal during pulmonary fibrosis and P16-CyclinD1/CDK4 cell cycle regulatory pathway greatly results in progressive reduction of alveolar epithelial calls during this process.

Objective To investigate the expression of interleukin (IL)-34/colony stimulating factor(CSF)-1R in the process of transforming growth factor ( TGF)-β1 inducing epithelial-mesenchymal transition ( EMT) of human alveolar epithelial cells A549 cells.Methods A549 cells were cultured in vitro.CCK 8 was used to test the influence of the proliferative rate of A549 cells which were stimulated by TGF-β1 at different concentrations and time points .A549 cells were stimulated by 5μg/L TGF-β1 at 0 hour, the 12th hour, the 24th hour, and the 48th hour.Western blotting was adopted to detect changes of the following proteins: α-smooth muscle actin (α-SMA ) , E-cadherin ( E-Cad ) , matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1(TIMP-1). Real-time PCR was adopted to detect changes of the following genes: IL-34 mRNA, CSF-1R mRNA, MMP-2 mRNA and MMP-9 mRNA.Results TGF-β1 had no significant influence in the proliferation of A 549 cells compared with the control group(P>0.05).TGF-β1(5μg/L)stimulated A549 cells at different time point (0 hour, 12, 24, 48 hours), compared with the control group .The epithelial phenotype E-Cad protein was gradually down-regulated ( P <0.01 ) , while the mesenchymal phenotype α-SMA protein was gradually up-regulated ( P <0.01 ) and the protein of MMP-2 increased gradually (P<0.01).The protein of MMP-9 increased firstly and then was reduced (P<0.01),the peak was at the 24th hour.The protein of TIMP-1 was firstly transiently increased and then reduced (P<0.01), the minimum was at the 48th hour.Compared with the control group , the gene of IL-34 mRNA increased gradually (P<0.01), and the genes of CSF-1R mRNA, MMP-2 mRNA and MMP-9 mRNA increased firstly and then decreased ( P <0.01), which were peaked respectively at the 24th hour, the 24th hour, the 12th hour, respectively.Conclusion In the process of TGF-β1 inducing A549 cells transition,there is accompanied with the expression of IL-34/CSF-1R.

Objective To investigate the dynamic CT features of peripheral lung cancer (PLC) and pulmonary inflammatory pseudotumor(IPT).Methods 23 patients with PLC and 6 patients with IPT were undergone plain and dynamic enhanced CT scans (beginning at 30 s after the onset of injection,serial scan were obtained at 30 s intervals within 180 s) before surgery or administration of antibiotic.Results The average CT values of plain CT,CT enhanced attenuation at 30 s and 90 s were significantly different between PLC and IPT.There were no significant decline following peak enhancement in IPT but PLC within 180 s.Conclusion The comprehensive analysis of plain CT value,enhanced CT value at 30 s,and the decreasing rate after 90 s of administration of contrast material may be useful in differential diagnosis between PLC and IPT.

Objective To study the relationship between expression of p16 and tumor angiogenesis as well as value of CT enhancement in peripheral lung cancer. Methods twenty-three patients with peripheral lung cancer were examed with dynamic CT scan before surgery.The MVD、protein expression of VEGF and p16 were measured immunohistochemically on speciments of resected tumors.Results There were negative correlation tendency between expressions of p16 and VEGF,but the difference were no significant.During the course of delaying 30 s from injecting contrast dose,the relating coefficient was the highest between enhanced CT value and MVD,P=0.9364.The mean value of MVD and CT enhancement in the course of delaying 30 s in p16 negative expression group were higher than that in the group of p16 positive expression with no significant.Conclusion p16 negative expression may improve angiogenesis in peripheral lung cancer.

Objective To transfect the recombinant mIL-28A adenovirus vector into lung adenocarcinoma cell line LA795 and research its anticancer activity. Methods Transfected the constructed mouse IL-28(mIL-28) recombinant adenovirus vector into LA795 cell line, detected with PCR, immunocytal fluorescence, Tunel, Annexin V and MTT. Results Transfected with rAd-mlL-28A into the LA795 cells, mIL-28A gene expression products mRNA increased obviously, IL-28 expression was detected in cells obviously,apoptosis cell number increased, and the growth of LA795 cells transfected with rAd-mIL-28A were inhibited obviously. Conclusion The recombinant miL-28A adenovirus vector we have constructed, which expresses IL-28 when transfected to lung adenocarcinoma cell line LA795, inhibits growth of carcinoma cell to some extent, and may work by promoting the apoptosis of cancer cells.

Objective:To explore the effect of intratracheal transplantation human umbilical cord blood mesenchymal stem cells on pulmonary interstitial fibrosis by bleomycin in mice,compare the treatment in pulmonary fibrosis of intratracheal transplantation with tail vain injection human umbilical cord blood mesenchymal stem cells.Methods:The second generation of HUCBMSCs were cultured to the fourth generation.Sixty specific pathogen free male Kunming mice were randomly divided into 4 groups:negative control group ( Cont group) ,bleomycin group( BLM group) ,HUCBMSCs transplantation groupⅠ( MⅠgroup) and HUCBMSCs transplantation groupⅡ(MⅡ group),each group 15 mice.Pulmonary fibrosis models were induced by bleomycin via intratracheal perfusion in the latter three groups.Twenty four hours after model establishment,5-bromo-2-deoxyuridine( Brdu) marked HUCBMSCs were poured in trachea in MⅠgroup ,the same were injected into tail vein in MⅡ group.At the 7th,14th,28th day,5 mice in each group were executed re-spectively.The morphological changes of the lung tissues were observed by HE staining and Masson staining.The localization and distribution of human umbilical cord blood mesenchymal stem cells were determined by the method of immunohistochemistry.The hydroxyproline contents were measured by alkali hydrolysis assay.The protein levels of transforming growth factor β-1 ( TGF-β1 ) and smooth muscle alpha-actin(α-SMA) were detected by Western blot.Results:In the two mesenchymal stem cell transplantation groups, there were Brdu marked cells at the 7th,14th,28th days in lung tissue.The alveolitis and fibrosis in lung of the two mesenchymal stem cell transplantation groups were milder than which of the the bleomycin group(P<0.05).Hydroxyproline levels in the bleomycin groups at 3 time points showed a gradually rising trend,the highest level was at 28th day( P<0.01).The protein levels of TGF-β1 andα-SMA in the lung tissues ranked as follows:BLM group>MⅠ,MⅡgroup>Cont group(P<0.05),the difference between MⅠgroup and MⅡgroup was not statistically significant(P>0.05).Conclusion:The colonization of human umbilical cord blood mesenchymal stem cells can be seen in the damaged tissue via intratracheal transplantation which can alleviate pulmonary fibrosis in mice caused by bleomycin.

Objective To express mouse IFN-λ2 stably and study its biological activity. Methods Full-length of mIFN-λ2 cDNA was obtained by using RT-PCR from cells of mouse spleen stimulated by ve-sicular stomatitis virus(VSV) and then subcloned to eukaryotic expressing vector PCAGG-EGFP. The recom-binant was transfected into CHO cells. VSV * GFP-B16 system was used to measure the antivirus activity. The constructed cell line MDBK-Mxp-Luc was used to study the character of Mx1 protein induced by the mIFN-λ2. Results The recombinant pMD18-T-mIFN-λ2 was digested by two kinds of enzyme, Sac I and Xho I, to produce the fragment was of 582 bp, and of which the sequence analysis of sequence shows it was entirely consistent with the nucleotide sequences reported in GenBank. PCAGG-EGFP-mIFN-λ2 eukaryotic expressing vector was constructed successfully and expressed stably in CHO cells, and the mRNA of mIFN-λ2 was verified expressing in CHO-PCAGG-EGFP-mIFN-λ2 cell line by RT-PCR. The antivirus activity of in the supernatant secreted by the CHO-PCAGG-EGFP-mIFN-λ2 cell line was 10~4 AU/ml. The mIFN-λ2 pro-tein can could induce the expression of the antivirus protein Mx1, and the expression of Mx1 protein induced by mIFN-λ2 enhanced with time going, 9 to 12 hours achieved the peak, 24 hours vanished. Conclusion Gene cloning of mIFN-λ2 was successful. The eukaryotic expressing vector of mIFN-λ2 was constructed suc-cessfully and expressed stably in CHO cells, and its product has obvious antivirus activity in vitro. And the antivirus activity of the product was closely correlated with inducing expression of antivirus protein Mx1.

Idiopathic pulmonary fibrosis(IPF)is a chronic interstitial lung disease marked by the significant in-volvement of fibroblasts,macrophages and lymphocytes.The CD27/CD70 axis is pivotal in shaping the immune mi-croenvironment present in the fibrotic aeras of lungs.This mentioned interaction involves molecular pathways that work in tandem with lung immune cells,particularly exerting a suppressive influence in the early phases of pulmo-nary fibrosis.Consequently,the CD27/CD70 axis presents a promising new target for immunotherapy in IPF.

Gene therapy represents a promising treatment for the Alzheimer׳s disease (AD). However, gene delivery specific to brain lesions through systemic administration remains big challenge. In our previous work, we have developed an siRNA nanocomplex able to be specifically delivered to the amyloid plaques through surface modification with both CGN peptide for the blood-brain barrier (BBB) penetration and QSH peptide for -amyloid binding. But, whether the as-designed nanocomplex could indeed improve the gene accumulation in the impaired neuron cells and ameliorate AD-associated symptoms remains further study. Herein, we prepared the nanocomplexes with an siRNA against -site amyloid precursor protein-cleaving enzyme 1 (BACE1), the rate-limiting enzyme of A production, as the therapeutic siRNA of AD. The nanocomplexes exhibited high distribution in the A deposits-enriched hippocampus, especially in the neurons near the amyloid plaques after intravenous administration. In APP/PS1 transgenic mice, the nanocomplexes down-regulated BACE1 in both mRNA and protein levels, as well as A and amyloid plaques to the level of wild-type mice. Moreover, the nanocomplexes significantly increased the level of synaptophysin and rescued memory loss of the AD transgenic mice without hematological or histological toxicity. Taken together, this work presented direct evidences that the design of precise gene delivery to the AD lesions markedly improves the therapeutic outcome.