Objective To investigate the effects of quercetin and X-ray on collagen synthesis of cultured human keloid-derived fibroblast and the mechanism.Methods Collagen synthesis of cultured human keloid and normal fibroblast...Objective To investigate the effects of quercetin and X-ray on collagen synthesis of cultured human keloid-derived fibroblast and the mechanism.Methods Collagen synthesis of cultured human keloid and normal fibroblasts were detected by hydroxyproline colorimetric analysis. Immunocytochemical staining was used to investigate collagen I and III expression. mRNA expression of collagen I and III, and transforming growth factor (TGF)-β1 were assayed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Results Quercetin inhibited the collagen synthesis of both keloid and normal fibroblasts in a dose-dependent manner. Immunocytochemical staining indicated that collagen I and III were down-regulated by quercetin and X-ray (P<0.05), particularly collagen I (P<0.05). mRNA expression of both collagen I and III in quercetin groups significantly decreased compared with that in control group (P<0.05), especially in the group treated with both quercetin and X-ray (P<0.01). mRNA level of TGF-β1 gene was down-regulated by quercertin (P<0.05). Conclusions Quercetin will probably be one of the new medicines which could effectively treat keloid. Quercetin combined with X-ray could reduce the dose of radiation.展开更多
AIM: To study the expression of SMAD3 and SMAD7 of transforming growth factor β1 (TGF β1)on keloid derived fibroblasts. METHODS: The expression of SMAD3 (at 1,2,4,24,48 h)and SMAD7(at 0.5 1,1.5, 2,4,24 h) were detec...AIM: To study the expression of SMAD3 and SMAD7 of transforming growth factor β1 (TGF β1)on keloid derived fibroblasts. METHODS: The expression of SMAD3 (at 1,2,4,24,48 h)and SMAD7(at 0.5 1,1.5, 2,4,24 h) were detected by using methods of Western blot after 500 pmol/L TGF β1 were added to the monolayer culture system.RESULTS: The level of SMAD3 were down regulated at 24 h and the SMAD7 were maximum up regulated at 4 h when TGF β1 were used.CONCLUSION: TGF β1 may down regulate the expression of SMAD3,but up regulate that of SMAD7 .展开更多
Objective: To explore the effects of connective tissue growth factor (CTGF) on the pathogenesis of human keloid. Methods: CTGF antisense oligonucleotides (ASODN) conjugated with isothiocyananate fluorescence was encap...Objective: To explore the effects of connective tissue growth factor (CTGF) on the pathogenesis of human keloid. Methods: CTGF antisense oligonucleotides (ASODN) conjugated with isothiocyananate fluorescence was encapsulated by liposome, and then added into the human keloid fibroblasts (HKFs) culture media. The intracellular distribution of CTGF ASODN was observed by fluorescence microscopy in the fixed HKFs. The proliferation of HKFs was measured by MTT test. The collagen synthesis of HKFs was measured by 3H-proline incorporation method. Results: Compared with control group, the CTGF ASODN can inhibit the proliferation and collagen synthesis of the HKFs (P<0.01). Conclusion: CTGF ASODN has anti-fibrotic effects on keloid in vitro, and CTGF play an important role in promoting the fibrosis of keloid.展开更多
Objective:The aim of this study was to investigate the effect of different dose of pentoxifylline(PTX)on intedeukin-6(IL-6)and platelet-derived growth factor(PDGF)expression in human normal skin fibroblast(NF)and kelo...Objective:The aim of this study was to investigate the effect of different dose of pentoxifylline(PTX)on intedeukin-6(IL-6)and platelet-derived growth factor(PDGF)expression in human normal skin fibroblast(NF)and keloid fibroblast(KF),to explore the pathogenesis of PTX on prevention and treatment of KF.Methods:Human NF and KF were passaged till the 5 th to 7 th generations by tissue block adhering wall method,and then were cultured in vitro with different dose of PTX.Levels of IL-6 and PDGF were detected by ELISA at various periods.Results:(1)Compared with control group,the expressions of IL-6 in NF were significantly inhibited with 0.5 mg/ml PTX for 72 h,1.0 mg/ml and 2.0 mg/ml PTX for 24 h,48 h and 72 h separately(p<.01);The lower expressions of IL-6 were also showed in KF with 0.25 mg/ml PTX for 48 h and 72 h,0.5 mg/ml,1.0 mg/ml and 2.0 mg/ml PTX for 24 h,48 h and 72 h separately(p<.01).(2)After cultured with 0.25 mg/ml PTX for 72 h,0.5 mg/ml,1.0 mg/ml and 2.0 mg/ml PTX for 24 h,48 h and 72 h separately,the expressions of PDGF in NF were decreased significantly compared to the controls(p<.01);The reduced PDGF expressions were also found in KF after cultured with 0.25 mg/ml,0.5 mg/ml,1.0 mg/ml and 2.0 mg/ml PTX for 24 h,48 h and 72 h separately(p<.05).Repeated measures analysis of variance indicated the difference source of cells and doses with statistical significance.Conclusions:The expressions of IL-6 and PDGF in KF were higher than those in NF.PTX plays a notable inhibitory role in the expression of IL-6 and PDGF,which could be used as a promising medicine for Keloid treatment.展开更多
Objective: To define the mechanisms underlying the keloid formation. Methods: The gap junctional intercellu- lar commumication (GJIC) of fibroblasts derived from pathological scars and noed skin was investigated. Six ...Objective: To define the mechanisms underlying the keloid formation. Methods: The gap junctional intercellu- lar commumication (GJIC) of fibroblasts derived from pathological scars and noed skin was investigated. Six Samples of hyperplastic scars, keloids and normal skin were obtained. Fibroblasts from these samples were cultured in ho and vended by type Ⅰ collagen, and the GJIC among the fibroblasts was investigated by adherent cell analysis with soning interactive laser coytometer(ACAS 570) for fibroblasts culturing. Results: The fibroblasts from normal skin showed nounal GJIC, which are depressed between fibroblasts from hyperplastic scare and was completely blocked in fibroblasts from the keloids. Conclusion: The blockade of interoellular communication may play an important role in the pathogenesis of excessive and invasive growth of fibroblasts derived from the keloids.展开更多
The levels of hsa circular RNA_0002198(hsa_circ_0002198)have been found to be significantly upregulated in keloid dermal fibroblasts.However,the functional role of hsa_circ_0002198 in keloid fibroblasts and the underl...The levels of hsa circular RNA_0002198(hsa_circ_0002198)have been found to be significantly upregulated in keloid dermal fibroblasts.However,the functional role of hsa_circ_0002198 in keloid fibroblasts and the underlying molecular mechanism for its effects have not been reported.In this study,the levels of hsa_circ_0002198 and nucleotide-binding and oligomerization domain,leucine rich repeat and pyrin domain containing 3(NLRP3)expression in keloid scar tissues and adjacent normal skin tissues were determined by quantitative real-time PCR and western blotting,respectively.In vitro models of keloid tissue were created by culturing primary keloid fibroblasts obtained from patients.A series of functional experiments,including CCK-8 assays,Transwell assays,and ELISA assays were performed to analyze the functional role of hsa_circ_0002198/NLRP3.Our data showed that hsa_circ_0002198 and NLRP3 were upregulated in keloid scar tissues when compared with adjacent normal tissues.Knockdown of hsa_circ_0002198 expression significantly suppressed cell proliferation,migration,and invasion,and those effects could be partially reversed by forced NLRP3 overexpression in keloid fibroblasts.At the molecular level,knockdown of hsa_circ_0002198 downregulated the levels of Col I,α-SMA,and NLRP3 proteins,as well as the levels of TGF-β,IL-1ß,and IL-33,but upregulated caspase 3 expression in keloid fibroblasts.All those effects were partially reversed after NLRP3 overexpression.In conclusion,our results suggest hsa_circ_0002198 as a potential target for treating keloid lesions.展开更多
The various fibroproliferative disorders affecting humans have in common excess fibroblast activity and persistent overexpression or dysregulated activity of transforming growth factor beta (TGF-β). Cancer has many s...The various fibroproliferative disorders affecting humans have in common excess fibroblast activity and persistent overexpression or dysregulated activity of transforming growth factor beta (TGF-β). Cancer has many similar characteristics. Antineoplastic drugs can downregulate fibroblast activity and cytokine growth factors. This study evaluates the effect of six antineoplastic drugs on keloid and Dupuytren’s disease fibroblasts. Keloid, normal scar, Dupuytren’s affected palmar fascia, and normal palmar fascia fibroblasts were grown and seeded into Fibroblast Populated Collagen Lattices (FPCLs). The FPCLs were treated with one of six antineoplastic drugs or left untreated as controls. At 7 days, supernatants were extracted from all FPCLs and assayed for expression of Transforming Growth Factor beta (TGF)-β<sub>1</sub> and TGF-β<sub>2</sub>. All six antineoplastic drugs significantly inhibited FPCL contraction in both fibroproliferative conditions compared with the untreated controls (p β<sub>1</sub> and TGF-β<sub>2</sub> expression was downregulated in the supernatants of all FPCLs by the drug exposure. Cytotoxicity did not occur in these studies and was not the reason for the results. Although antineoplastic drugs can have significant side effects when given systemically, these results may be minimized when given to small areas involved in fibroproliferative scarring or when given topically or intralesionally. These in vitro results suggest that antineoplastic drugs may have a utility for treating various fibroproliferative disorders and warrant further investigation.展开更多
文摘Objective To investigate the effects of quercetin and X-ray on collagen synthesis of cultured human keloid-derived fibroblast and the mechanism.Methods Collagen synthesis of cultured human keloid and normal fibroblasts were detected by hydroxyproline colorimetric analysis. Immunocytochemical staining was used to investigate collagen I and III expression. mRNA expression of collagen I and III, and transforming growth factor (TGF)-β1 were assayed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Results Quercetin inhibited the collagen synthesis of both keloid and normal fibroblasts in a dose-dependent manner. Immunocytochemical staining indicated that collagen I and III were down-regulated by quercetin and X-ray (P<0.05), particularly collagen I (P<0.05). mRNA expression of both collagen I and III in quercetin groups significantly decreased compared with that in control group (P<0.05), especially in the group treated with both quercetin and X-ray (P<0.01). mRNA level of TGF-β1 gene was down-regulated by quercertin (P<0.05). Conclusions Quercetin will probably be one of the new medicines which could effectively treat keloid. Quercetin combined with X-ray could reduce the dose of radiation.
文摘AIM: To study the expression of SMAD3 and SMAD7 of transforming growth factor β1 (TGF β1)on keloid derived fibroblasts. METHODS: The expression of SMAD3 (at 1,2,4,24,48 h)and SMAD7(at 0.5 1,1.5, 2,4,24 h) were detected by using methods of Western blot after 500 pmol/L TGF β1 were added to the monolayer culture system.RESULTS: The level of SMAD3 were down regulated at 24 h and the SMAD7 were maximum up regulated at 4 h when TGF β1 were used.CONCLUSION: TGF β1 may down regulate the expression of SMAD3,but up regulate that of SMAD7 .
文摘Objective: To explore the effects of connective tissue growth factor (CTGF) on the pathogenesis of human keloid. Methods: CTGF antisense oligonucleotides (ASODN) conjugated with isothiocyananate fluorescence was encapsulated by liposome, and then added into the human keloid fibroblasts (HKFs) culture media. The intracellular distribution of CTGF ASODN was observed by fluorescence microscopy in the fixed HKFs. The proliferation of HKFs was measured by MTT test. The collagen synthesis of HKFs was measured by 3H-proline incorporation method. Results: Compared with control group, the CTGF ASODN can inhibit the proliferation and collagen synthesis of the HKFs (P<0.01). Conclusion: CTGF ASODN has anti-fibrotic effects on keloid in vitro, and CTGF play an important role in promoting the fibrosis of keloid.
文摘Objective:The aim of this study was to investigate the effect of different dose of pentoxifylline(PTX)on intedeukin-6(IL-6)and platelet-derived growth factor(PDGF)expression in human normal skin fibroblast(NF)and keloid fibroblast(KF),to explore the pathogenesis of PTX on prevention and treatment of KF.Methods:Human NF and KF were passaged till the 5 th to 7 th generations by tissue block adhering wall method,and then were cultured in vitro with different dose of PTX.Levels of IL-6 and PDGF were detected by ELISA at various periods.Results:(1)Compared with control group,the expressions of IL-6 in NF were significantly inhibited with 0.5 mg/ml PTX for 72 h,1.0 mg/ml and 2.0 mg/ml PTX for 24 h,48 h and 72 h separately(p<.01);The lower expressions of IL-6 were also showed in KF with 0.25 mg/ml PTX for 48 h and 72 h,0.5 mg/ml,1.0 mg/ml and 2.0 mg/ml PTX for 24 h,48 h and 72 h separately(p<.01).(2)After cultured with 0.25 mg/ml PTX for 72 h,0.5 mg/ml,1.0 mg/ml and 2.0 mg/ml PTX for 24 h,48 h and 72 h separately,the expressions of PDGF in NF were decreased significantly compared to the controls(p<.01);The reduced PDGF expressions were also found in KF after cultured with 0.25 mg/ml,0.5 mg/ml,1.0 mg/ml and 2.0 mg/ml PTX for 24 h,48 h and 72 h separately(p<.05).Repeated measures analysis of variance indicated the difference source of cells and doses with statistical significance.Conclusions:The expressions of IL-6 and PDGF in KF were higher than those in NF.PTX plays a notable inhibitory role in the expression of IL-6 and PDGF,which could be used as a promising medicine for Keloid treatment.
文摘Objective: To define the mechanisms underlying the keloid formation. Methods: The gap junctional intercellu- lar commumication (GJIC) of fibroblasts derived from pathological scars and noed skin was investigated. Six Samples of hyperplastic scars, keloids and normal skin were obtained. Fibroblasts from these samples were cultured in ho and vended by type Ⅰ collagen, and the GJIC among the fibroblasts was investigated by adherent cell analysis with soning interactive laser coytometer(ACAS 570) for fibroblasts culturing. Results: The fibroblasts from normal skin showed nounal GJIC, which are depressed between fibroblasts from hyperplastic scare and was completely blocked in fibroblasts from the keloids. Conclusion: The blockade of interoellular communication may play an important role in the pathogenesis of excessive and invasive growth of fibroblasts derived from the keloids.
基金This study was supported by The Natural Science Foundation of Hainan Province(Project No.817338)Hainan Province in 2019–2021 Provincial Key Subject Construction Project of Traditional Chinese Medicine(The Oncology Department)(No.S100111.401).
文摘The levels of hsa circular RNA_0002198(hsa_circ_0002198)have been found to be significantly upregulated in keloid dermal fibroblasts.However,the functional role of hsa_circ_0002198 in keloid fibroblasts and the underlying molecular mechanism for its effects have not been reported.In this study,the levels of hsa_circ_0002198 and nucleotide-binding and oligomerization domain,leucine rich repeat and pyrin domain containing 3(NLRP3)expression in keloid scar tissues and adjacent normal skin tissues were determined by quantitative real-time PCR and western blotting,respectively.In vitro models of keloid tissue were created by culturing primary keloid fibroblasts obtained from patients.A series of functional experiments,including CCK-8 assays,Transwell assays,and ELISA assays were performed to analyze the functional role of hsa_circ_0002198/NLRP3.Our data showed that hsa_circ_0002198 and NLRP3 were upregulated in keloid scar tissues when compared with adjacent normal tissues.Knockdown of hsa_circ_0002198 expression significantly suppressed cell proliferation,migration,and invasion,and those effects could be partially reversed by forced NLRP3 overexpression in keloid fibroblasts.At the molecular level,knockdown of hsa_circ_0002198 downregulated the levels of Col I,α-SMA,and NLRP3 proteins,as well as the levels of TGF-β,IL-1ß,and IL-33,but upregulated caspase 3 expression in keloid fibroblasts.All those effects were partially reversed after NLRP3 overexpression.In conclusion,our results suggest hsa_circ_0002198 as a potential target for treating keloid lesions.
文摘The various fibroproliferative disorders affecting humans have in common excess fibroblast activity and persistent overexpression or dysregulated activity of transforming growth factor beta (TGF-β). Cancer has many similar characteristics. Antineoplastic drugs can downregulate fibroblast activity and cytokine growth factors. This study evaluates the effect of six antineoplastic drugs on keloid and Dupuytren’s disease fibroblasts. Keloid, normal scar, Dupuytren’s affected palmar fascia, and normal palmar fascia fibroblasts were grown and seeded into Fibroblast Populated Collagen Lattices (FPCLs). The FPCLs were treated with one of six antineoplastic drugs or left untreated as controls. At 7 days, supernatants were extracted from all FPCLs and assayed for expression of Transforming Growth Factor beta (TGF)-β<sub>1</sub> and TGF-β<sub>2</sub>. All six antineoplastic drugs significantly inhibited FPCL contraction in both fibroproliferative conditions compared with the untreated controls (p β<sub>1</sub> and TGF-β<sub>2</sub> expression was downregulated in the supernatants of all FPCLs by the drug exposure. Cytotoxicity did not occur in these studies and was not the reason for the results. Although antineoplastic drugs can have significant side effects when given systemically, these results may be minimized when given to small areas involved in fibroproliferative scarring or when given topically or intralesionally. These in vitro results suggest that antineoplastic drugs may have a utility for treating various fibroproliferative disorders and warrant further investigation.
文摘目的:探讨长链非编码RNA 8975-1(Long non-coding RNA 8975-1,LNCRNA8975-1)通过微小RNA-203(microRNA-203,miR-203)/血管内皮生长因子-A(Vascular endothelial growth factor-A,VEGF-A)信号轴对人瘢痕疙瘩成纤维细胞(Human keloid fibroblast,HKF)的细胞增殖周期和迁移的影响及其机制。方法:培养人皮肤成纤维细胞(Human skin fibroblasts,HSF)和HKF,采用定量PCR(quantitative polymerase chain reaction,qPCR)及western blot法检测LNCRNA8975-1、miR-203和I型胶原蛋白(Type I collagen,Col-I)的表达量。将HKF细胞分为Control组、小干扰RNA载体的阴性对照(the negative control for small interfering RNA vector,siNC)组、沉默LNCRNA8975-1的小干扰RNA载体(the small interfering RNA vector for silence of LNCRNA8975-1,siLNCRNA8975-1)组、pcDNA3.1空载体(the empty pcDNA3.1 vector,pcDNA)组、过表达LNCRNA8975-1的pcDNA3.1载体(overexpression of LNCRNA8975-1 in the pcDNA3.1 vector,pcDNA-LNCRNA8975-1)组、miR-203拟似物的阴性对照(the negative control of miR-203 mimic,NC mimic)组、miR-203的拟似物(miR-203 mimic)组、miR-203抑制剂的阴性对照(negative control of miR-203 inhibitor,NC inhibitor)组、miR-203的抑制剂(miR-203 inhibitor)组、siLNCRNA8975-1+miR-203 inhibitor组、VEGF-A+miR-203 mimic组。CCK-8法检测细胞增殖活性,免疫荧光检测细胞周期标志物细胞周期蛋白D1(cyclin D1),伤口愈合实验和Transwell实验检测细胞迁移功能。荧光素酶基因报告实验检测HKF细胞中LNCRNA8975-1和miR-203、miR-203和VEGF-A的直接作用。结果:与HSF相比,HKF中LNCRNA8975-1和Col-I表达量上调(P<0.05),而miR-203降低(P<0.05)。与siNC相比,siLNCRNA8975-1抑制了HKF细胞周期蛋白cyclin D1表达和细胞的迁移(P<0.05)。与NC mimic相比,miR-203 mimic抑制了HKF中cyclin D1蛋白表达和细胞的迁移(P<0.05)。LNCRNA8975-1充当miR-203的“分子海绵”靶向抑制miR-203,且VEGF-A是miR-203的靶基因。VEGF-A表达被miR-203 mimic抑制(P<0.05)。miR-203 inhibitor充分逆转了siLNCRNA8975-1对HKF中cyclin D1表达和迁移的抑制作用(P<0.05),而且VEGF-A则逆转了miR-203 mimic对HKF中cyclin D1表达和迁移的抑制功能(P<0.05)。结论:LNCRNA8975-1通过抑制miR-203上调VEGF-A调控HKF的周期和迁移。
