Objective To observe the effect of epithelial-mesenchymal transdifferentiation (EMT) of human retinal pigment epithelial (RPE) cells induced by vitreous humor in vitro. Methods The third to fifth passage cultured RPE cells were divided into two groups of treatment by 10% serum containing Dulbecco minimum essential medium (DMEM)/F12 medium (group A), or the same medium supplemented with 25% human vitreous (group B). The morphological changes were observed with a phase contrast microscrope. Cell migration, invasion and contractility were tested using a scratch wound assay, Transwell invasion assay and collagen gel contraction analysis. The expression levels of alpha;-smooth muscle actin (SMA) and Snail1 were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Results The RPE cells in group A were flat and gathered together. The RPE cells in group B grew as a fan-shaped projection at one edge and cone-shaped tail at the opposite edge, or spindle-shaped, and appeared to separate. In group A, filamentous actin distributed mainly at the margin of the cells with the distribution an oval shape. In group B, filamentous actin reorganized and formed fan-like flat pseudopodia at one edge of the cells. Compared to group A, the migration and invasion of the cells increased significantly (t=14.190, 22.630; P＜0.05), but contractility decreased remarkably (t=6.221, P＜0.05) in group B. Compared to group A, the expression level of Snail1 mRNA increased significantly (t=3.218, P=0.032), but the expression level of alpha;-SMA mRNA decreased (t=3.990, P=0.016). Conclusions Vitreous humor can induce the EMT of RPE cells. Increasing cell migration, cell invasion, and expression of Snail1 mRNA as well as up-regulated cellsprime; contractility and expression of alpha;-SMA mRNA may be the mechanism.
Objective To observe the autofluorescence （AF） manifestation in related lesions of periphery retinopathy.Methods Sixty eyes of 42 patients with periphery retinopathy underwent the examination of Optomap fundus photograph (200deg;) and fundus fluorescein angiography (FFA). The HRAⅡ melaninrelated nearinfrared fundus autofluorescence (NIA, excitation 795 nm) and lipofuscinrelated fundus autofluorescence (FAF, excitation 488 nm) were measured for all the patients. The AF was recorded with nine images per second, and then a final AF image with 55deg; view and 822times;768 pixel was generated by the HRA. AF images can be valuable or valueless if there was or was not visible blood vessels and related retinal tissues on the image. AF from lesion regions can be normal or abnormal fluorescence comparing to the normal vascular and retinal tissue AF. The abnormal fluorescence was divided into no AF, weak AF and b AF relative to the background grayscale. The grading consistency of abnormal fluorescence based on FAF and NIA examination was comparatively analyzed. Results Valuable AF images were captured in 53/60 eyes （88.33%）and valueless AF images were captured in 7/60 eyes （11.67%）. Among 53 eyes with valuable AF image, NIA showed normal fluorescence in 28 eyes （52.83%），abnormal fluorescence with sheetlike, dotshaped or stripped in 25 eyes （47.17%）; FAF showed normal fluorescence in two eyes （3.77%）, abnormal fluorescence with sheetlike, scattered along vessels or pigments in 51 eyes （96.23%）. Twentyfive eyes with abnormal fluorescence were observed both in two examinations, including same grades in 18 eye （72.00%） and different grades in seven eyes （28.00%）. Conclusion The AF manifestation with different levels exists in related lesions of periphery retinopathy.
Objective To observe the expression of proteins in light-injured retinal pigment epithelial (RPE) cells. Methods ARPE19 cells were exposed to the cool white light at the intensity of (2200plusmn;300) Lx for 6 hours to set up the light injured model. Cellular soluble proteins was extracted and analyzed by means of twodimensional electrophoresis to find out the changes of protein map of lightinjured RPE cells. Results Cellular soluble proteins had (390plusmn;10) spots on the map, in which 11 spots had obvious difference between the light injured group and the normal control group. In the lightinjured cells, the expressio of 8 proteins increased, 1 decreased, and 2 disappeared. Conclusion Twodimensional electrophoresis can find out the difference of expression of proteins in lightinjured and normal RPE cells.
Objective To resolve the tough problem of how to observe the growing cells in an opaque vector. Methods The urethral epithelial cells from a young male New Zealand rabbit were inoculated, and were primarily cultured in vitro and subcultured for 3 passages. Then, the urethralepithelial cells were cultured in the collagen chitosan complex for 3, 7, 14 and 21 days. The cells were dyed with 6-carboxyfluorescein diacetateacetoxymethyl ester and propidium iodine, respectively. Then, Interactive Laser Cytometer was used to detect the growing cells. Results The urethral epithelial cells grew and proliferated very well in the collagen chitosan complex vector. After the urethral epithelial cells grew in the collagen-chitosan complex vector for 3 and 7 days, the fluorescent density amount of the surviving cells were(1.09±0.13)×10.8 and (2.04±0.13)×10.8, respectively. However, after 14and 21 days, the fluorescent density amount of the surviving cells was (0.55± 0.09)×10.8 and (0.47±0.03)×108, respectively. There was a significant difference when compared with the amount of the surviving cells at 3 and 7 days（P＜0.05）.Conclusion Using Interactive Laser Cytometer for measurement of the green and red fluorescent densities of different waves, the activity of the cultured urethral epithelial cells in vitro can be rapidlymeasured with the in situ quantitation method. This method solves a difficult problem of observing the growing cells in an opaque vector. The dynamic growing state of the engineering tissues can be observed.
Objective To study the expression of human Runt-related transcription factor 1 (RUNX1) in rat airway epithelial cells stimulated by cigarette smoking extract (CSE), and explore the role of RUNX1 in regulating epithelial-mesenchymal transition (EMT). Methods Primary rat bronchial epithelial cells were cultured by enzyme digestion and stimulated with different concentrations of CSE. The viability of cells was detected by CCK-8 to explore the appropriate concentration of CSE. After the cells were treated with CSE, the Runx1 interference and overexpression vectors were constructed and transfected into the cells to silence or overexpress the Runx1 gene. Immunocytochemical method was used to detect RUNX1 expression and Western blot analysis was used to detect the expression of RUNX1, nuclear factor-κB (NF-κB), Snail, E-cadherin, and vimentin. Results The survival rate of bronchial epithelial cells could be reduced by CSE, and the degree of reduction was directly positively correlated to the concentration of CSE. After CSE stimulation, the expression level of E-cadherin in primary rat bronchial epithelial cells decreased significantly (P<0.05); the expression levels of RUNX1, NF-κB, Snail and vimentin significantly increased (P<0.05). After interfering with RUNX1 gene, the expression level of E-cadherin was up-regulated (P<0.05), and the expression levels of NF-κB, Snail and vimentin were down-regulated (P<0.05). After overexpression of RUNX1 gene, the expression level of E-cadherin decreased (P<0.05), and the expression levels of NF-κB, Snail and vimentin increased (P<0.05). Conclusions CSE promotes the expression of RUNX1 in rat airway epithelial cells. RUNX1 might regulate EMT process by involving in the regulation of NF-κB /Snail expression.
Objective To explore the effects of prolonged inhalation of Aspergillus fumigatus ( Af)spores on epithelial cell injury and expression of epidermal growth factor receptor( EGFR) in airways of asthmatic rats. Methods 64 male Wistar rats were randomly divided into 8 groups, ie. chronic asthma group ( group A) , chronic asthma plus Af spores inhalation for 1 week ( group B) , 3 weeks ( group C) and 5 weeks ( group D) , chronic asthma plus saline inhalation for 5 weeks ( group E) , OVA-sensitized and salinechallenged group ( group F) , and OVA-sensitized and saline-challenged plus Af spores inhalation for 5 weeks ( group G) ( each n =8) . The airway resistance ( Raw) and change of Raw after acetylcholine provocation were detected using a computerized system. The concentrations of epidermal growth factor ( EGF) andtransforming growth factor alpha( TGF-α) in BALF were measured by ELISA. The extents of epithelial cell injury and goblet cell hyperplasia were evaluated on hematoxylin and eosin-stained( HE) and periodic acidschiff ( PAS) stained lung sections. The expression of EGFR in airway epithelia was demonstrated byimmunohistochemistry, and the level of EGFR protein in the rat lung tissues was measured by western blot.Results The concentration of EGF( pg/mL) ( 51. 72 ±8. 54, 68. 12 ±7. 85, 86. 24 ±9. 12, respectively)and TGF-α( pg/mL) ( 55. 26 ±9. 30, 75. 58 ±11. 56, 96. 75 ±14. 66, respectively) , detached/ inner perimeter of epithelium( % ) ( 11. 25 ±3. 12, 26. 45 ±5. 56, 28. 50 ±7. 50, respectively) , the ratio of goblet cell area to epithelial cell area ( % ) ( 16. 42 ±5. 24, 22. 64 ±6. 82, 36. 38 ±9. 21, respectively) , the integrated optical density ( IOD) of EGFR positive stain in airway epithelial cells ( 82 ±15,120 ±19, 165 ±21, respectively) , and the EGFR protein levels in lung tissues ( 0. 91 ±0. 26, 1. 61 ±0. 52, 2. 52 ±0. 78,respectively) in group B, C, and D were higher than those in group A, E, F and G( P lt; 0. 05 or P lt;0. 01) .The change rates of Raw( % ) ( 61. 91 ±5. 26, 84. 69 ±6. 38) in group C and D were higher than those in group A, E, F and G ( P lt; 0. 05 or P lt;0. 01) . The IOD of EGFR was positively correlated with detached/inner perimeter of epithelium( % ) and the ratio of goblet cell area to epithelial cell area( % ) ( r = 0. 692,P lt;0. 01; r = 0. 657, P lt; 0. 01, respectively) . Conclusion Prolonged inhalation of Aspergillus fumigatus spores can aggravate airway epithelial cell injury, up-regulate the expression of EGFR in airway epithelial cell and induce goblet cell hyperplasia, thus increase the airway responsiveness in rats with chronic asthma.
摘要:目的: 探討聯合LCT和高危型HPV檢測對CIN宮頸治療后的隨訪意義。 方法 :對200例LCT異常,高危型HPV陽性,陰道鏡活檢證實為CIN1~3的患者行LEEP治療或宮頸冷刀錐切,治療后進行嚴格隨訪,包括LCT和高危型HPV檢測,陽性病例行組織學檢查。 結果 :(1)所有病例經治療后均無病變殘留,其治愈率為100%。(2)從治療后3個月起,CIN1組高危型HPV轉陰率為100%。在隨訪的第3個月和6個月,CIN2~3組高危型HPV轉陰率分別為7317%和9085%,顯著低于CIN1組,差異有統計學意義(〖WTBX〗P ＜005)。(3)從隨訪12個月起,一直有2例病例持續HPV陽性,均為CIN3患者,但LCT和陰道鏡檢查未發現細胞學異常,繼續隨訪。 結論 :CIN治療后高危型HPV的轉陰時間及轉陰率與CIN的級別有關;高危型HPV持續陽性,但LCT和陰道鏡檢查無異常者可繼續嚴格隨訪;LCT聯合高危型HPV檢測是CIN治療后臨床追蹤隨訪的有效手段。Abstract: Objective: To investigate the Significance of LCT joint highrisk HPV testing for followup after CIN treatment. Methods : 200 cases that highrisk HPV infection were tested by realtime PCR and CIN1~3 were confirmed with LCT and colposcopy biopsy were considered. The patients were treated with LEEP treatment or cold knife conization. After treatment, all cases were strictly followed up with LCT and HPV test, and the patients with positive results were examined by histology. Results : 1) After treatment, there was no residual disease in all cases, the cure rate was 100%. 2) From 3 months after treatment, highrisk HPV negative rate was 100% in CIN1 cases. While at 3rd and 6th month after treatment, highrisk HPV negative rate in CIN2~3 cases were 7317% and 9085%, which were significantly lower than those in CIN1 cases,the difference was statistically significant. 3) From the 12th monthafter treatment, there are still two cases of sustained highrisk HPV positive but normal with LCT and colposcopy biopsy. All cases are still strictly followedup. Conclusion : After treatment, the negative rate and time of highrisk HPV concerned with the grade of the CIN; the patients with persistent positive highrisk HPV, but without abnormalities detected by LCT and colposcopy biopsy could continue to strictly follow up; LCT joint highrisk HPV detection is an effective clinical means for followup after CIN treatment.
