AIM: To examine the effects of cigarette smo ke extract (CSE) and lipo polysaccharide (LPS) on the production of transforming growth factor-? 1 (TGF- ? 1 ) mRNA and protein in cultured human embryonic lung fibroblasts (HELF). METHODS: The cultured HELF were incubated with CSE, LPS or CSE in com bination with LP S for 24 hours at 37℃, respectively. The total RNA was extracted from the cells . The expression levels of TGF-? 1 mRNA were evaluated by reverse transcription - p olymerase chain reaction (RT-PCR). The mRNA levels of TGF-? 1 were corrected by GAPDH transcripts, and TGF-? 1 protein levels were determined by immunocytoch emistry. RESULTS: CSE stimulated the TGF-? 1 mRNA expression i n HELF at lower concentr ati ons (1∶50 and 1∶25)( P 0.05). LPS enhanced TGF-? 1 mRNA levels in HELF at a ll three doses (0.1 mg/L, 1 mg/L, and 10 mg/L)( P

Background Benign lymphoepithelial lesion of lacrimal gland is not a common orbital disease in clinic,which mainly presented as symmetrical and painless enlargement of bilateral lacrimal glands.However,the etiology and pathogenesis of this disease is still unclear now.Objective This study was to screen the differentially expressed genes between benign lymphoid epithelial lesion of lacrimal gland and orbital cavernous hemangioma and explore the pathogenesis of benign lymphoepithelial lesion of lacrimal gland at the molecular level.Methods Nine patients diagnosed as benign lymphoepithelial lesion of lacrimal gland in Beijing Tongren Hospital,Capital Medical University were enrolled from September 2010 to April 2013,and nine patients with orbital cavernous hemangioma served as control group.The intraorbital tissue was collected during surgery.Whole-genome gene expression microarray was used to detect the expressed genes,and limma algorithm was used to analyze the differentially expressed genes between the benign lymphoepithelial lesion of lacrimal gland and the orbital cavernous hemangioma.Real-time PCR was used to verify differentially expressed genes,Fisher method and gene ontology (GO) functional analysis were performed to realize function and signaling pathways analysis.This study complied with Helsinki Declaration and the protocol was aproved by Institutional Review Board of Beijing Tongren Hospital,and informed consent was obtained.Results Total 5 260 differentially expressed genes were screened between benign lymphoepithelial lesion of lacrimal gland and orbital cavernous hemangioma.The Fisher function and signaling pathways analysis showed that 109 GO terms were significantly upregulated and 101 GO terms were significantly downregulated,and 32 relevant signaling pathways were significantly upregulated and 25 signaling pathways were significantly downregulated in the benign lymphoepithelial lesion of lacrimal gland.GO analysis showed that the expression enrichment of complement receptormediated signaling pathway was high,then following the upregulation of T cell and B cell signaling pathways and downregulation of mitogen-activated protein kinase (MAPK) and transforming growth factor-β (TGF-β) signaling pathways.Real-time PCR results showed that the expressions of TIPRL,TLR7 and TLR10 genes were significantly higher in the benign lymphoepithelial lesion of lacrimal gland than that in the orbital cavernous hemangioma,with significant differences between the two diseases (Z =-2.03,-2.32,-2.32;all at P＜0.05),which was consistent with the microarray data.Conclusions Gene expression profiles are significantly different between benign lymphoepithelial lesion of lacrimal gland and orbital cavernous hemangioma.Those differentially expressed genes play roles in the upregulation of T cell and B cell signaling pathways,downregulation of MAPK and TGF-β signaling pathways and the change of complement system.It is implied that a comprehensive effect of various genes and pathways participates in the pathogenesis of benign lymphoepithelial lesion of lacrimal gland.

Background Idiopathic orbital inflammatory pseudotumor (IOIP) is a common orbital disease, but its etiology is still unclear,so the effect of glucocorticoid treatment is unsatisfied.Objective This study was to investigate the effects of dexamethasone on orbital fibroblasts from IOIP patients and explore the action machanism.Methods Six pieces of IOIP tissues from 6 IOIP patients and 3 pieces of normal orbital connective tissues from lacrimal gland prolapse patients were obtained during the surgery in Beijing Tongren Hospital from November 2011 to January 2012.The orbital fibroblasts were cultured using explant culture method.The morphology of the cells were observed under the optical microscope,and biomarks of the cells were detected by immunochemistry.The growth and proliferation of the cells were assayed using WST-8.The expression of ICAM-1 in the cells in both the control group and the IOIP group was detected by immunochemistry.The fibroblasts were incubated in 96-well plates, and different concentrations of dexamethasone (0,1 × 10-3 , 1 × 10-4 , 1 × 10-5 and 1 × 10-6 mol/L) were respectively added into the medium for 24,48 and 72 hours,and then the proliferation of the cells was detected by WST-8 assay.The contents of ICAM-1 in different concentrations of dexamethasone groups were assayed by ELISA.Results The characteristics of the cells were similar between the control group and the IOIP group with the spindle shape and long protructions.The cells showed the positive response for vimentin and absent response for desmin, S-100, cytokeratin (CK).Compared with the control group,the growth speed of fibroblasts was fast in the IOIP group.The proliferative values of the cells (absorbancy) were gradually reduced with the increase of dexamethasone concentrations (F ion =36.27,P=0.00) and the lapse of acting time (Ftime =3.69 ,P=0.00).In cultured cells without dexamethasone for 24,48 and 72 hours,the mean expression levels of ICAM-1 were 0.298±0.008,0.312±0.003 and 0.319±0.011, showing a gradually increasing trend.However,the expression of ICAM-1 was gradually reduced with the increases of concentrations and the lapse of acting time of dexamethasone (Fconcentration =75.17,P=0.00;Ftime =3.11,P=0.00).Conclusions Occurrence and development of IOIP is probably associated with the over-expression of ICAM-1 in orbital fibroblasts.Dexamethasone plays anti-inflammation and treating effects on IOIP by down-regulating the expression of ICAM-1 and inhibiting the proliferation of orbital fibroblasts.

Objective To summarize the clinical features of 22 probands diagnosed with congenital muscular dystrophy (CMD),and to provide genetic counseling and prenatal diagnosis for 23 fetuses of these pedigrees.Methods Data of 22 CMD patients who were treated in the Pediatric Department of Peking University First Hospital during October 2006 to March 2016 were analyzed.Informed written consents for participation in this study were obtained from the parents or guardians.Prenatal diagnosis was performed using DNA samples extracted from fetal villus cells of 12 cases at 11-13 gestational weeks and amniotic fluid of 11 cases at 18-22 gestational weeks.Direct DNA sequencing by polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) were used to detect CMD-related gene mutations.Linkage analysis of short tandem repeats (STRs) was used to identify maternal blood contamination and biological parents.Results Thirteen out of the 22 probands with CMD were diagnosed with congenital muscular dystrophy type 1 A (MDC1A),and all of them carried compound heterozygous mutations in LAMA2 gene.Prenatal diagnosis of 13 fetuses from these pedigrees found that four fetuses were wild-type,seven were heterozygotes and two carried the same mutations as their proband.Three probands with LMNA-related congenital muscular dystrophy (L-CMD) carried de novo mutations in LMNA gene.In these pedigrees,two fetuses were wild-type and one whose mother was mosaicism carried the same mutations as the proband.One proband with Ullrich congenital muscular dystrophy carried compound heterozygous mutations in COL6A2 gene and the fetus of the same pedigree was wild-type.Five probands were diagnosed with α-dystroglycanopathies.And among them,two cases of muscle-eye-brain disease (MEB) carried compound heterozygous mutations in POMGnT1 gene and the fetuses of the two peidgrees were heterozygotes;one case of congenital muscular dystrophy type 1C (MDC1C) had compound heterozygous mutations in FKRP gene and the fetus carried the same mutations;one patient diagnosed with POMGnT1-related congenital muscular dystrophy with mental retardation (CMD-MR) carried compound heterozygous mutations in POMGnT1 gene,and the fetus was positive for the same mutations;one proband with POMT1-related CMD-MR was positive for compound heterozygous mutations in POMT1 gene and the results of prenatal diagnosis for two fetuses of this pedigree showed that the first fetus had the same mutations as the proband,while the second was heterozygote.Conclusions No effective therapeutic method is available for CMD.Therefore,accurate genetic counseling and prenatal diagnosis are necessary to prevent CMD child from birth.

Cigarette smoking is intimately related with the development of chronic obstructive pulmonary diseases, and alveolar epithelium is a major target for the exposure of cigarette smoke ex- tract. In order to investigate the effect of cigarette smoke extract on the proliferation of alveolar epithelial cell type Ⅱand its relationship with P21WAF1, the alveolar epithelial type Ⅱ cell line (A549) cells were chosen as surrogate cells to represent alveolar epithelial type Ⅱ cells. MTT assay was used to detect cell viability after interfered with different concentrations of cigarette smoke ex-tract. It was observed cigarette smoke extract inhibited the growth of A549 cells in a dose- and time-dependent manner. The morphological changes, involving the condensation and margination of nuclear chromatin, even karyorrhexis, were observed by both Hoechst staining and electronic mi-croscopy. Flow cytometry analysis demonstrated the increased cell percentages in G1 and subG1phases after the cells were incubated with cigarette smoke extract. The expression of p21WAF1 protein and mRNA was also significantly increased as detected by the methods of Western blot or reverse transcription-polymerase chain reaction respectively. In conclusion, cigarette smoke extract inhibits the proliferation of alveolar epithelial cell type Ⅱ and blocks them in G1/S phase. The intracellular accumulation of P21WAF1 may be one of the mechanisms which contribute to cigarette smoke ex-tract-induced inhibition of cell proliferation.

Objective:To explore the effects of conditioned medium of human bone marrow mesenchymal stem cells (BMSCs) on the proliferation, adhesion and differentiation of immortalized human Müller cell line (MIO-M1).Methods:The differentiation was induced in the third-passage BMSCs with osteogenic, chondrogenic and adipogenic medium and identified by alizarin red, alcian blue and oil red O staining, respectively.The expression levels of mesenchymal stem cell markers CD73, CD90 and CD105 and hematopoietic cell markers CD34, CD45 and human leukocyte antigen-DR (HLA-DR) were assayed by flow cytometry.The expressions levels of Müller cell markers SOX9, glutamine synthetase (GS), vimentin and cellular retinaldehyde-binding protein (CRALBP), retinal stem cell markers SOX2, nestin and CHX10, and cell proliferation marker cyclin D3 (CCND3) in MIO-M1 cells were detected by immunofluorescence staining.The MIO-M1 cells were divided into standard medium group, 293T conditioned medium group, and BMSC conditioned medium group and were incubated in the medium according to grouping.The cellular area, circularity, elongation factor and perimeter were analyzed quantitatively.The cell cycle was detected by flow cytometry, and the cell proliferation was determined by neurospora experiment and 5-ethynyl-2'-deoxyuridine (EdU) staining.The expression of vascular cell adhesion molecule 1 (VCAM-1) at protein and mRNA levels in the culture supernatant was detected by enzyme linked immunosorbent assay (ELISA) and quantitative real-time PCR (qRT-PCR), respectively.The expression of retinal neuron markers protein kinase C (PKCα), Rhodopsin, microtubule-associated protein 2 (MAP2) and β-tubulin (Tuj1) was detected by immunofluorescence staining and qRT-PCR.Results:CD73, CD90, CD105 showed an enhanced expression, and CD34, CD45 and HLA-DR showed weakened expression in the BMSCs.The BMSCs differentiated into osteoblasts, chondrocytes and adipocytes.Expression of SOX9, GS, vimentin and CRALBP, SOX2, CHX10, nestin and CCND3 was found in the MIO-M1 cells.Compared with standard medium group and 293T conditioned medium group, MIO-M1 cells cultured in BMSC conditioned medium group changed into an elongated spindle-shaped or multipolar morphology with reduced cell area, increased elongation index and decreased circularity, showing statistically significant differences among them ( F=6.973, 12.370, 6.311; all at P<0.01). There were increased neurospheres formed by MIO-M1 cells in BMSC conditioned medium group compared with standard medium group and 293T conditioned medium group at different time points ( Fgroup=134.300, P<0.001; Ftime=82.910, P<0.001). Compared with the standard medium group and 293T conditioned medium group, the EdU-positive rate and proliferation index of MIO-M1 cells in BMSC conditioned medium group were significantly increased, with statistically significant differences ( F=6.973, 74.110; all at P<0.05); the VCAM-1 protein expression in cell supernatant and the relative expression level of VCAM-1 mRNA in BMSC conditioed medium group were significantly increased ( F=13.720, 7.896; all at P<0.05); the mRNA expression levels of PKCα, Rhodopsin, Tuj1 and MAP2 were higher in MIO-M1 cells of BMSC conditioned medium group under the condition of differentiation ( F=14.490, 5.424, 14.330, 7.405; all at P<0.05). Conclusions:BMSCs conditioned medium can change the morphology of MIO-M1 cells and promote their proliferation, adhesion and differentiation into retinal neurons.

Objective:To explore the anti-inflammatory effect and safety of two non-steroidal anti-inflammatory drugs in the phacoemulsification combined with intraocular lens (IOL) implantation.Methods:A randomized, double-blind, clinical trial was conducted.A total of 90 age-related cataract patients (90 eyes) who were diagnosed in Qingdao Eye Hospital Affiliated to Shandong First Medical University were enrolled from October 2020 to February 2021.The patients were randomized to diclofenac sodium group and bromofenac sodium group by random number table method, with 45 cases (45 eyes) in each group.All patients underwent phacoemulsification combined with IOL implantation, and 0.1% diclofenac sodium eye drops (preservative-free), 4 times a day, and 0.1% pramiphene eye drops, 2 times a day were applied in the perioperative period.The duration of continuous medication treatment and follow-up time were 6 weeks.The subjective symptoms of the patients were scored before and after surgery.The amount of tear fluid secretion was detected by Schirmer I test, and the tear film breakup time was recorded with the Oculus dry eye analyzer.Corneal fluorescein staining was observed under a slit lamp microscope with cobalt blue light.Anterior chamber flash was measured by slit-lamp biomicroscopy.The thickness of central macular area and the presence of macular cystoid edema was measured by optical coherence tomography.Visual acuity, noncontact intraocular pressure (IOP) and the drug safety were examined and evaluated.This study adhered to the Declaration of Helsinki and was approved by the Ethics Committee of Qingdao Eye Hospital (No.[2020]60).All patients were informed about the surgery and postoperative medication and signed the informed consent form.Results:All subjects had no intraoperative complications, and completed treatment and follow-up as required.The preoperative, 1-day postoperative, 1-week postoperative, 6-week postoperative subjective symptom scores were (0.47±0.73), (0.56±0.62), (0.33±0.48), and (0.51±0.66) points in the diclofenac group, and (0.47±0.51), (0.75±0.61), (0.64±0.65), and (0.78±0.77) points in the bromfenac group.There were statistically significant differences in the subjective symptom scores at different time points between the two groups ( Fgroup=5.001, P=0.028; Ftime=2.920, P=0.035), and the subjective symptom scores of diclofenac sodium group were significantly lower than those of bromofenac sodium group (all at P<0.05).The preoperative, 1-week postoperative, 6-week postoperative tear secretion volume were (5.87±2.37), (6.07±2.53), and (6.29±0.25) mm in diclofenac sodium group, and (7.36±2.74), (6.29±3.46), and (5.80±2.76) mm in bromofenac sodium group.There was statistically significant difference in the tear secretion volume between the two groups before surgery ( F=6.910, P=0.012), but there was no significant difference on postoperative weeks 1 and 6 ( F=1.121, 0.772; P=0.729, 0.384).The preoperative, 1-week postoperative, 6-week postoperative non-invasive tear break-up time (NIBUT) were (8.00±6.28), (6.68±5.24), and (6.17±5.00) seconds in diclofenac sodium group, and (6.40±5.28), (4.50±2.46), and (5.39±5.39) seconds in bromofenac sodium group.There was no significant difference in NIBUT between the two groups ( Fgroup=3.415, P=0.068).There was significant difference in NIBUT within groups among different time points ( Ftime=4.358, P=0.020).The 1-day postoperative, 1-week postoperative, 6-week postoperative corneal epithelial staining score were (1.40±0.81), (0.13±0.34), (0.00±0.00) points in diclofenac sodium group, and (1.38±0.89), (0.22±0.47), and (0.00±0.00) points in bromofenac sodium group.There was no statistically significant difference in the corneal epithelial staining score between the two groups after surgery ( Fgroup=0.110, P=0.741).There were statistically significant differences in corneal epithelial staining scores within groups among different time points ( Ftime=175.054, P<0.01).The 1-day postoperative, 1-week postoperative, 6-week postoperative anterior chamber flare classification were 1.13±0.51, 0.13±0.34, and 0.00±0.00 in diclofenac sodium group, and 1.02±0.34, 0.16±0.37, and 0.00±0.00 in bromofenac sodium group.There was no significant difference in the overall anterior chamber flash between the two groups ( Fgroup=0.045, P=0.507).There were statistically significant differences in anterior chamber flash within groups among different time points ( Ftime=322.331, P<0.001).There was no significant difference in the preoperative and 6-week postoperative macular fovea thickness between both groups ( t=-0.221, -0.374; both at P>0.05).The incidence of macular cystoid edema 6 weeks after operation was 0% in both groups.Subjects tolerated the two tested drugs well.Eight adverse events occurred in this study, all of which were mild postoperative IOP elevation, including 3 in diclofenac sodium group with an incidence of 6.67% and 5 in bromofenac group with an incidence of 11.1%.IOP returned to normal in all the patients 1 week after stopping the use of drug. Conclusions:Two nonsteroidal anti-inflammatory drugs are safe and effective for anti-inflammatory treatment after cataract phacoemulsification combined with IOL implantation.The new diclofenac sodium eye drops are more comfortable than bromfenac sodium eye drops.