1.Recombinant tetra-cell adhesion motifs supports adhesion, migration and proliferation of keratinocytes/fibroblasts, and promotes wound healing.
Mi Yeon JUNG ; Narendra THAPA ; Jung Eun KIM ; Jung Duk YANG ; Byung Chae CHO ; In San KIM
Experimental & Molecular Medicine 2007;39(5):663-672
An extracellular matrix protein plays an important role in skin wound healing. In the present study, we engineered a recombinant protein encompassing the 9th and 10th type III domains of fibronectin, and 4th FAS1 domain of beta ig-h3. This recombinant protein, in total, harbors four known-cell adhesion motifs for integrins: Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD) in 9th and 10th type III domains of fibronectin, respectively, and Glu-Pro-Asp-Ile-Met (EPDIM) and Try-His (YH) in 4th FAS1 domain of big-h3, were designated to tetra-cell adhesion motifs (T-CAM). In vitro studies showed T-CAM supporting adhesion, migration and proliferation of different cell types including keratinocytes and fibroblasts. In an animal model of full-thickness skin wound, T-CAM exhibited excellent wound healing effects, superior to both 4th FAS1 domain of beta ig-h3 or 9th and 10th type III domains of fibronectin. Based on these results, T-CAM can be applied where enhancement of cell adhesion, migration and proliferation are desired, and it could be developed into novel wound healing drug.
Amino Acid Motifs
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Animals
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Cell Adhesion/*drug effects
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Cell Line
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Cell Movement/*drug effects
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Cell Proliferation/*drug effects
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Extracellular Matrix Proteins/chemistry/genetics/pharmacology
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Fibroblasts/cytology/drug effects/physiology
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Fibronectins/chemistry/genetics/*pharmacology
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Humans
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Keratinocytes/cytology/drug effects/physiology
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Mice
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NIH 3T3 Cells
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Rabbits
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Recombinant Fusion Proteins/chemistry/genetics/pharmacology
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Transforming Growth Factor beta/chemistry/genetics/pharmacology
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Wound Healing/*drug effects/physiology
2.Effect of Shenluotong on secretion of ECM and expression of TGF-beta1 in rats mesangial cells of rats.
Ya-li WANG ; Yu-yong ZHAO ; Zhi-qiang CHEN ; Qing-you XU ; Hua-zhou XU ; Tong-zhong LI ; Xian-ming HOU
China Journal of Chinese Materia Medica 2005;30(3):201-203
OBJECTIVETo investigate the effect of Shenluotong on the expression of transforming growth factor-beta1 (TGF-beta1) and extracellular matrix (ECM) in Ang II-induced MCs.
METHODFibronectin (FN) and collagen type IV (Col IV) of extracellular matrix were detected by enzyme-linked immunosorbent assay; the expression of TGF-beta1 mRNA were measured by semi-quantitative reverse transcription-polymerase chain reaction.
RESULTA positive correlation between TGF-beta1 and ECM were found in the present study. FN, Col IV and TGF-beta1 mRNA were inhibited by Shenluotong significantly.
CONCLUSIONShenluotong can decrease the accumulation of ECM and inhibit the expression of TGF-beta1, suggesting further that shenluotong can be used to prevent and treat various glomerular diseases and delay glomerular sclerosis.
Animals ; Astragalus membranaceus ; chemistry ; Cells, Cultured ; Collagen Type IV ; metabolism ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Extracellular Matrix ; metabolism ; Female ; Fibronectins ; metabolism ; Glomerular Mesangium ; cytology ; Male ; Materia Medica ; isolation & purification ; pharmacology ; Oligochaeta ; chemistry ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Salvia miltiorrhiza ; chemistry ; Transforming Growth Factor beta ; biosynthesis ; genetics ; Transforming Growth Factor beta1
3.Effects of siRNAs silencing transforming growth factor-beta1 on fibronectin in SD rat masangial cells.
Hua-xiong MAO ; Zhu-wen YI ; Xiao-chuan WU ; Xi-qiang DANG ; Xiao-jie HE ; Yan CAO ; Shuang-hong MO
Chinese Journal of Pediatrics 2007;45(7):494-497
OBJECTIVETo investigate fibronectin synthesis in SD rat mesangial cells after transforming growth factor-beta1 (TGF-beta1) is silenced by the short interfering RNA (siRNA) expressed by reconstructed pGEFP-C1 vectors.
METHODSDepending upon the 538th - 556th (A) and 895th - 913th (B) nucleotides of rat TGF-beta1 gene, a nucleotide (A or B) was constructed into a small hairpin nucleotide which was separately (A or B) or together (A plus B) inserted into a pGEFP-C1 vector with three reconstructed pGEFP-C1 vectors separately expressing the siRNAs for A or/and B. TGF-beta1 and fibronectin were dynamically investigated for their interrelationship by ELISA in the supernatant and RT-PCR in their extracted total RNA.
RESULTSThe siRNA hairpin-like molecules were constructed according to the 538th - 556th nucleotides of rat TGF-beta1 gene were able to markedly silence the expression of TGF-beta1 mRNA (P < 0.01) and protein (P < 0.01) at 48 h. Lipfectamin 2000 transfection stimulated the peak secretion of fibronectin at 24 h in the control and the experimental group whose TGF-beta1 was not silenced, but the silence of TGF-beta1 in both experimental groups delayed the top values of fibronectin to 48 h (P < 0.01).
CONCLUSIONThe silence of TGF-beta1 by siRNA decreased the fibronectin expression, but the latter was possibly not completely TGF-dependent.
Animals ; Cells ; Cells, Cultured ; Fibronectins ; metabolism ; Mesangial Cells ; drug effects ; metabolism ; RNA Interference ; drug effects ; physiology ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; chemistry ; genetics
4.Influence of ginsenoside Rg1, a panaxatriol saponin from Panax notoginseng, on renal fibrosis in rats with unilateral ureteral obstruction.
Xi-Sheng XIE ; Man YANG ; Heng-Cuang LIU ; Chuan ZUO ; Zi LI ; Yao DENG ; Jun-Ming FAN
Journal of Zhejiang University. Science. B 2008;9(11):885-894
Total saponins of Panax notoginseng (PNS) have been shown to ameliorate renal interstitial fibrosis. Ginsenoside Rg1, a panaxatriol saponin, is one of the major active molecules from PNS. The present study was undertaken to investigate the effect of ginsenoside Rg1 on renal fibrosis in rats with unilateral ureteral obstruction (UUO). The rats were randomly divided into 3 groups: sham-operation (n=15), UUO (n=15) and UUO with ginsenoside Rg1 treatment (n=15, 50 mg per kg body weight, intraperitoneally (i.p.) injected). The rats were sacrificed on Days 7 and 14 after the surgery. Histological examination demonstrated that ginsenoside Rg1 significantly inhibited interstitial fibrosis including tubular injury as well as collagen deposition. alpha-smooth muscle actin (alpha-SMA) and E-cadherin are two markers of tubular epithelial-myofibroblast transition (TEMT). Interestingly, ginsenoside Rg1 notably decreased alpha-SMA expression and simultaneously enhanced E-cadherin expression. The messenger RNA (mRNA) of transforming growth factor-beta1 (TGF-beta1), a key mediator to regulate TEMT, in the obstructed kidney increased dramatically, but was found to decrease significantly after administration of ginsenoside Rg1. Further study showed that ginsenoside Rg1 considerably decreased the levels of both active TGF-beta1 and phosphorylated Smad2 (pSmad2). Moreover, ginsenoside Rg1 substantially suppressed the expression of thrombospondin-1 (TSP-1), a cytokine which can promote the transcription of TGF-beta1 mRNA and the activation of latent TGF-beta1. These results suggest that ginsenoside Rg1 inhibits renal interstitial fibrosis in rats with UUO. The mechanism might be partly related to the blocking of TEMT via suppressing the expression of TSP-1.
Actins
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biosynthesis
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Animals
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Cadherins
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biosynthesis
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Collagen Type I
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genetics
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metabolism
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Fibronectins
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genetics
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metabolism
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Ginsenosides
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pharmacology
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Immunohistochemistry
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Male
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Nephritis, Interstitial
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drug therapy
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genetics
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metabolism
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pathology
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Panax notoginseng
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chemistry
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RNA, Messenger
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biosynthesis
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genetics
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Random Allocation
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Rats
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Rats, Sprague-Dawley
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Reverse Transcriptase Polymerase Chain Reaction
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Smad2 Protein
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biosynthesis
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Thrombospondin 1
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biosynthesis
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genetics
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Transforming Growth Factor beta1
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biosynthesis
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genetics
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Ureteral Obstruction
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metabolism
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pathology
5.Effect of ligustrazine nanoparticles nano spray on transforming growth factor-β/Smad signal pathway of rat peritoneal mesothelial cells induced by tumor necrosis factor-α.
Shuai YAN ; Lan YANG ; Yin-Zi YUE ; Wen-Lin LI ; Li ZENG ; Jin YUE ; Chun-Qin MAO
Chinese journal of integrative medicine 2016;22(8):629-634
OBJECTIVETo study the effect of ligustrazine nanoparticles nano spray (LNNS) on transforming growth factor β (TGF-β)/Smad signal protein of rat peritoneal mesothelial cells (RPMC) induced by tumor necrosis factor α (TNF-α), and the anti-adhesion mechanism of LNNS in the abdominal cavity.
METHODSThe primary culture and subculture of rat peritoneal mesothelial cells (RPMC) was processed by trypsin digestion method in vitro. The third generation was identifified for experiment and divided into 5 groups: a blank group: RPMC without treatment; a control group: RPMC stimulated with TNF-α; RPMC treated by a low-dosage LNNS group (2.5 mg/L); RPMC treated by a medium-dosage LNNS group (5 mg/L); and RPMC treated by a high-dosage LNNS group (10 mg/L). Reverse transcription-polymerase chain reaction was applied to test the expression of fifibronectin, collagen I (COL-I), TGF-β mRNA, and Western blot method to test the Smad protein 7 expression of RPMC.
RESULTSCompared with the blank group, a signifificant elevation in fifibronectin (FN), COL-I and TGF-β mRNA expression of RPMC were observed in the control group (P<0.05). Compared with the control group, LNNS suppressed the expressions of FN, COL-I and TGF-β mRNA in a concentrationdependent manner (P<0.05). The expression of Smad7 protein of RPMC was down-regulated by TNF-α stimulation, and up-regulated with the increase of LNNS dose (P<0.05).
CONCLUSIONSTNF-α may induce changes in RPMC's viability, leading to peritoneal injury. LNNS could reverse the induction of fifibrosis related cytokine FN, COL-I and TGF-β, up-regulating the expression of Smad7 by TNF-α in RPMC, thus attenuate peritoneal injury by repairing mesothelial cells.
Animals ; Collagen Type I ; genetics ; metabolism ; Epithelium ; drug effects ; metabolism ; Fibronectins ; metabolism ; Male ; Nanoparticles ; chemistry ; ultrastructure ; Particle Size ; Peritoneal Cavity ; cytology ; Pyrazines ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Smad Proteins ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology
6.Renal protective effect of Shenkang pill on diabetic rats.
Wei XIAO ; Lian-Bo WEI ; Yun MA ; Hai-Bo LONG ; Guo-Bao CHEN
China Journal of Chinese Materia Medica 2006;31(12):1006-1009
OBJECTIVETo investigate the effects of Shenkang pill on renal function and extracellular matrix secretion on the diabetic rats.
METHODThe diabetic rat models were induced by intraperitoneal injection of streptozotocin (STZ) and randomly divided into 3 groups' model control group; Capoten group and Shenkangwan group. Some normal other rats were used as normal control group. All rats were treated with corresponding drugs for 8 weeks. During and after the treatment, the general state, blood and urine glucose levels, excretion rate of the 24 hour urine protein and albumin, serum creatinine and blood urea nitrogen contents, kidney weight and relative kidney weight were measured. The mRNA of fibronectin(FN) in the kidney also detected by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR).
RESULTDiabetes mellitus and renal lesions occurred in the three model groups. The expression of FN mRNA of the kidney in diabetic rats increased obviously. Shenkang pill could improve the general state and renal function of the diabetic rats, decrease the blood glucose levels and the excretion rate of the 24 hour urine protein and albumin, reduce the expression of FN mRNA in kidney.
CONCLUSIONShenkang pill has a certain protective effect on the diabetic kidney.
Animals ; Blood Glucose ; metabolism ; Blood Urea Nitrogen ; Diabetic Nephropathies ; chemically induced ; metabolism ; pathology ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Fibronectins ; biosynthesis ; genetics ; Glycated Hemoglobin A ; metabolism ; Kidney ; metabolism ; Male ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Wistar ; Streptozocin
7.Effect of a novel EZH2 inhibitor GSK126 on prostate cancer cells.
Weiren LIN ; Yatian CHEN ; Linghui ZENG ; Rongbiao YING ; Feng ZHU
Journal of Zhejiang University. Medical sciences 2016;45(4):356-363
To investigate the effect of a novel EZH2 inhibitor GSK126 on cell growth, apoptosis and migration of prostate cancer cells.Prostate cancer PC-3 and DU145 cells were treated with GSK126 at different doses. Cell growth was detected by sulforhodamine assay. Cell apoptosis was assayed by Annexin V-/PI kit. Transwell chamber and wound healing assays were conducted to detect cell migration. The mRNA level was detected by quantitative PCR, and protein expression was detected by Western blot analysis.GSK126 showed significant effect on cell growth and apoptosis when the dose was higher than 50 μmol/L. Wound healing assay revealed that scratch space in PC-3 cells was significantly increased in a dose-dependent manner in GSK126-treated groups[(247.2±24.4),(347.2±19.2) and (410.5±18.1) μm in low, medium and high dose (5.0, 20.0, 50.0 μmol/L), respectively] as compared with the control group[(171.3±17.8) μm](all<0.05). Transwell assay showed that migrated PC-3 cells in control group was 322.0±17.9,while those in GSK126-treated groups were 198.3±15.4 (low),82.7±6.2 (medium) and 30.2±4.1 (high), and the differences between the control group and GSK126-treated groups were significant(all<0.05). In addition, GSK126 up-regulated E-cadherin mRNA expression and down-regulated N-cadherin and Vimentin mRNA expression, whereas had no significant effect on Snail, Fibronectin and VEGF-A mRNA expression. The protein expression of E-cadherin was elevated but VEGF-A protein did not change in GSK126-treated groups. Similar results were exhibited in DU145 cell.GSK126 can significantly inhibit cell migration and invasion in prostate cancer PC-3 and DU145 cells, which may be resulted from its effect on epithelial-mesenchymal transition. GSK126 may be used as a potential anti-prostate cancer dug in clinic.
Apoptosis
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drug effects
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Cadherins
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analysis
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drug effects
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metabolism
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Cell Line, Tumor
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drug effects
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Cell Movement
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drug effects
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Cell Proliferation
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drug effects
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Down-Regulation
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drug effects
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Drug Screening Assays, Antitumor
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methods
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Enhancer of Zeste Homolog 2 Protein
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analysis
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drug effects
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metabolism
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Fibronectins
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analysis
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drug effects
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metabolism
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Humans
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Indoles
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pharmacology
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Male
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Prostatic Neoplasms
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chemistry
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genetics
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physiopathology
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Pyridones
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pharmacology
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RNA, Messenger
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Up-Regulation
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drug effects
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Vascular Endothelial Growth Factor A
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analysis
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drug effects
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Vimentin
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analysis
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drug effects
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metabolism