Objective To observe the permeability and stability of the transfection of antisense oligonucleotide (ASODN) hybridized epidermal growth factor receptor (EGFR) to retinal glial cells (RG).Methods Phosphorothioate and unmodified EGFR ASODN conjugated with 5′-isothioc yanate (5′-FITC) were encapsulated with or without lipofectin, and then added into human retinal glial cells culture media. The cellular permeability and stability of the transfection were observed by fluorescence microscopy in fixed cells.Results In the absence of lipofectin, phosphorothioate and unmodified EGFR ASODN were found in a few RG cells at 30 minutes, and in about 50% RG cells at 4 hours. Phosphorothioate EGFR ASODN were kept in RG cells for 3-4 hours and disappeared at about 8 hours. In the presence of lipofectin, phosphoro thioate and unmodified EGFR ASODN were found in a few RG cells at 15 minutes and about 70%-80% RG cells at 4 hours. Phosphorothioate EGFR ASODN were kept in cells for 10-12 hours, and phosphorothioate and unmodified EGFR ASODN were disapp eared at about 14 hours and 4 hours respectively.Conclusion 5′-FITC EGFR ASODN encapsulated with lipofectin could enter RG cells and express stably in RG cells. (Chin J Ocul Fundus Dis，2003，19：52-54)
Objective To explore the effect of membrane surface nucleolin （NCL） on activation of epidermal growth factor receptor （EGFR） signaling. Methods Immunohistochemistry was used to identify the expressions of membrane surface NCL or EGFR in pallilary thyroid carcinoma （PTC） tissues. The level of phosphorlated EGFR in TPC-1 cells was observed by Western blot. TPC-1 cells invasion capacity was detected by Transwell assay. Results The posi-tive expression rates of membrane surface NCL and EGFR in PTC tissues were 100% （56/56） and 80.4% （45/56） respe-ctively， while the expressions of NCL and EGFR were related with lymph node metastasis （P＜0.05）. There was posi-tive correlation between the expressions of NCL and EGFR （r=0.635， P＜0.01）. Western blot showed that anti-NCL or anti-EGFR of TPC-1 cells could inhibit the expression of phosphorlation EGFR （P＜0.01）. Transwell assay showed the number of membrane-invading cells were reduced significantly in anti-NCL group anti-EGFR group （P＜0.01）. Conclusions Membrane surface NCL may be a kind of indispensable component in activation of EGFR signaling， by which EGFR can participate in growth and invasion of tumors. NCL can be used as a target for developing a new field of tumor treatment.
Porpose To investigate the optimal concentration of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on DNA synthesis and their synergism indensity arrested human retinal pigment epithelial (RPE) cells. Methods Growth factor effects in cultured human RPE of the 6th generation were assessed by [3 H]-thymidine incorporation and radioautography. Results EGF and bFGF were potent stimulators when used alone,and their optimal concentrations were 10ng/ml in DMEM and 1ng/ml in 2% serum DMEM.When used in combination (10ng/ml EGF and 10ng/ml bFGF),they caused a significant enhancement of [3 H]-thymidine incorporation about 2.96 times. Conclusion EGF and bFGF were potent stimulators in RPE cells,and demonstrated synergism in their action. (Chin J Ocul Fundus Dis,1998,14:98-100)
Objective To explore the effect of basic fibroblast growth factor（bFGF）and epidermal growth factor（EGF）on the growth of muscle derived stem cells(MDSCs). Methods MDSCs were isolated from hindlimb muscle of 15 new born Kunming mice through serial preplates. 2% fetal bovine serum-containing DMEM was used to induce MDSCs to differentiate into skeletal muscle lineage. The expressions of stem cell marker Sca-1 and skeletal musclecell marker αSarcomeric actin were examined by immunocytochemistry. The effect of bFGF and EGF on the proliferation of MDSCs was determined by MTT colorimetric microassay. The solo effect of bFGF or EGF at different concentrations (6.25,12.50, 25.00, 50.00, and 100.00 ng/ml) was examined at 96 h and the combined effect (100.00 ng/ml) was examined at 24,48,72 and 96 h.Results MDSCs were successfully isolated from the hindlimb of neonatal mice. Over 90% of MDSCs showed Sca-1 positive immunoreactivity. MDSCs could give rise to α Sarcomeric actin positive myotubes in differentiation cultures. The proliferative effect of bFGF and EGF on MDSCs increased with the elevated concentration.bFGF began to show significant proliferative effect at 12.50 ng/ml (P＜0.05). The effect increased significantly when the concentration reached 25.00 ng/ml from 12.50 ng/ml (P＜0.01) and reached a saturation point. The effect at 50.00 ng/ml or 100.00 ng/ml showed no significant increase when compared with thatat 25.00 ng/ml. EGF had a similar effect to bFGF except that the saturation concentration was 50.00 ng/ml. EGF showed significant effect at 72 h and bFGF at 96 h (Plt;0.01). When they were applied together, significant effect was shownat 24 h (Plt;0.01) and much higher effect was observed at 48, 72 and 96 h (Plt;0.05). Conclusion Both bFGF and EGF can promote the proliferation of MDSCs. The combined application reacts faster and ber.
ObjectiveTo detect expressions of epidermal growth factor receptor (EGFR), BRAF, and K-Ras genes in colorectal carcinoma tissues and explore pathogenesis in colorectal carcinoma. MethodThe expressions of EGFR, BRAF, and K-Ras genes were detected in these 136 colorectal carcinoma tissues and their corresponding adjacent normal colorectal tissues by immunohistochemistry. ResultsThe expressions of EGFR and BRAF in the colorectal carcinoma tissues were significantly higher than those in their corresponding adjacent normal colorectal tissues (P<0.05), but the expression of K-Ras had no significant difference between these two tissues (P>0.05). The expression of EGFR gene was related to the TNM stage, lymphatic metastasis, or degree of differentiation. The expression of BRAF gene was related to the TNM stage. The expression of K-Ras gene wasn,t related to the TNM stage, lymphatic metastasis, or degree of differentiation. The correlation analysis results showed that there was no relation among the EGFR, K-Ras, or BRAF expression. ConclusionsUp-regulated of EGFR and BRAF gene expressions might be related to development of colorectal carcinoma, and role of K-Ras is unclear. Anti EGFR and BRAF target therapy might be benefited for patients with colorectal carcinoma.
Objective To assess the efficacy of a kind of new material lipid magnetic particle for isolation and detection of lung cancer circulating tumor cells (CTCs). Methods Immune lipid magnetic particles were prepared with reverse evaporation method and they were assembled into kits with EpCAM and EGFR antibody respectively. Their efficacy were evaluated by detecting A549 cells in group A (A549 cells mixed in phosphated buffer solution) and group B (A549 cells mixed in blood from healthy volunteers). Lung cancer CTCs of hospitalized patients were also detected with both immune magnetic particals. Then the detecting efficacy was compared between EpCAM immune lipid magnetic particles and the conventional CellsearchTM system. Results The immune lipid magnetic particles had high capture efficiency for CTCs isolation and identification. The median of EpCAM immune lipid magnetic particles method in detecting A549 cells in group A was 92%, and EGFR was 90%. The median of EpCAM immune lipid magnetic particles method in detecting A549 cells in group B was 85%, and EGFR was 81%. In 13 patients with lung cancer, CTCs can be detected with both immune lipid magnetic particles methods and both medians were 5; In negative control, the medians of both methods were 0 (P＜0.05). EpCAM immune lipid magnetic particles method can detect more CTCs than conventional CellsearchTM system in 3 lung cancer patients. Conclusions Immune lipid magnetic particles have good efficacy for lung cancer CTCs detection and has promising clinical application value. The EpCAM immune lipid magnetic particles have equal efficiency in detecting lung cancer CTCs with EGFR. There is a trend that EpCAM immune lipid magnetic particles is superior to the conventional CellsearchTM system.
OBJECTIVE This paper aims to explore the new method of continuous delivery of epidermal growth factor to wounds by transfected fibroblasts to promote wound repair. METHODS It was constructed a novel chimeric expression plasmid in which the biologically active portion of the human epidermal growth factor (EGF) gene was fused in-frame to the human granulocyte colony-stimulating factor signal sequence. RESULTS Clonally selected human fibroblasts transfected with this construct could secrete biologically active EGF. After the transplantation of irradiated gene-transfected fibroblasts suspended in fibrin glue to murine full-thickness wounds, EGF could be demonstrated for at least seven days in the wounds, slowly decreasing from initially 470 ng/L to 140 ng/L in 7 days. No EGF was found in the wound at 14 days. CONCLUSION A single application of irradiated EGF gene transfected fibroblasts to wounds can continuously deliver the transgene in vivo and can be used to administer drugs to the wound bed during the crucial first seven days of wound-healing.
ObjectiveTo investigate the value of serum soluble CD146 (sCD146) in determining acquired epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistance in lung adenocarcinoma.MethodsA total of 144 lung adenocarcinoma EGFR sensitive patients in People’s Hospital of Zhengzhou University diagnosed from January 2016 to December 2016 were recruited in the study. According to the different time of taking drugs, the patients were divided into a non-medication group (31 cases), a 1 to 3 month treatment group (25 cases), a 4 to 6 month treatment group (19 cases), a 7 to 12 month treatment group (25 cases), a drug-resistant group (24 cases), and a nonresistant group up to 1 year of treatment (20 cases). The serum levels of sCD146, carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE) were measured by ELISA and chemiluminescence and compared between different period of medication. The relationship of serum sCD146 with tumor markers (CEA, NSE) and tumor related clinical parameters (age, gender, tumor stage, metastasis, tumor diameter, number of the lesions) were analyzed.ResultsThe serum sCD146 level was minimum in the non-medication group that did not receive pioglitazone treatment, highest in the 1 to 3 month treatment group (early treatment period), and declined with duration of medication until resistance occurred without significant difference (P>0.05). The level of sCD146 of the drug-resistant group was significantly lower than that of all nonresistant groups, with significant difference (allP<0.05), but still higher than that of the non-medication group (P<0.05). The serum sCD146 levels in the nonresistant patients with medication over 1 year and within 1 year were similar (P>0.05), and significantly higher than the non-medication group and drug-resistance group (allP<0.05). The serum CEA levels did not differ significantly between 6 groups (P>0.05). The serum NSE level of the 4 to 6 month treatment group was lower than that of the 7 to 12 month treatment group (P<0.05), but both in the normal reference range. The NSE levels did not differ in any other groups (P>0.05). Serum sCD146 was associated with metastasis (P<0.05), but not associated with serum CEA or NSE, nor with sex, age, tumor staging, tumor diameter or lesion number (allP>0.05).ConclusionsCD146 may be involved in the mechanism of TKI killing tumor cells and the mechanism of TKI resistance, and may be a serological marker for monitoring the efficacy of TKI and judging the resistance of TKI.
【Abstract】Objective To investigate the protective effects of epidermal growth factor (EGF) on pancreas of rats with acute pancreatitis(AP). Methods Seventytwo male SpragueDawley rats were randomly divided into 3 groups: Control group, AP group and AP-EGF group. Subcutaneously injection of EGF (0.1 μg/g) were given to animals in the AP-EGF group after the establishment of the model of AP. The other two groups of animals received the same volume of saline. At 6 h, 12 h and 24 h after induction of AP, 8 animals in each group were sacrificed respectively， 4 ml of blood sample was withdrawn from heart，2 ml for the analysis of amylase activity and 2 ml for MDA content in serum. Ascites was sucked with dry gauzes and was weighed thereafter. Changes of pancreas morphology were evaluated at every time point. The same part of pancreas was removed for measurement of MDA content, apoptotic index (AI) and histologic changes. Results Histologic injury of the animals in the APEGF group was milder than that in the AP group. Ascites weight in the AP-EGF group decreased significantly compared with that in the AP group at 12 h and 24 h 〔(4.53±1.29) g vs (6.58±1.47) g, (7.64±1.85) g vs (11.96±2.13) g，P＜0.05，P＜0.01〕. Amylase activity in the APEGF group also decreased significantly compared with that in the AP group at 12 h and 24 h 〔(142.0±8.3) U/L vs (187.9±10.4) U/L, (194.3±10.4) U/L vs (253.3±8.6) U/L, P＜0.05，P＜0.01〕. MDA content in plasm 〔(2.34±0.23) μmol/L vs (3.15±0.38) μmol/L, P＜0.05〕 and in pancreas 〔(5.21±1.46) μmol/g vs (7.68±1.63) μmol/g, P＜0.01〕 in the APEGF group decreased significantly compared with those in the AP group at 24 h. AI of pancreas in the APEGF group increased significantly compared withthatintheAPgroupafteroperation〔(16.22±3.53)%〖KG4vs (7.35±1.04)%, (11.67±2.40)% vs (4.81±0.86)%, (6.38±1.42)% vs (1.97±0.21)%, P＜0.01〕. Conclusion EGF may accelerate the restoration of pathologic injury and alleviate the hemorrhage and edema of pancreas. It may also depress MDA content in plasm and in pancreas so that to lessen oxidative damage. EGF may protect pancreas by inducing cellular apoptosis.
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.