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BACKGROUND:Abnormal extracellular matrix accumulation and excessive proliferation of fibroblasts are the main manifestations of pathological scars.Excessive proliferation of fibroblasts leads to the production of large amounts of collagen-based extracellular matrix.Therefore,to investigate the role of fibroblast fibrosis in the formation of pathological scar will provide a new idea for revealing the mechanism of pathological scar and biological therapy. OBJECTIVE:To investigate the effect of RAS-selective lethal small molecule 3(RSL3)on the fibrosis of human pathological scar fibroblasts. METHODS:Then cases of pathological scar tissue and normal skin tissue samples from the same individuals,provided by the Department of Burn Plastic Surgery,General Hospital of Ningxia Medical University,were collected.Fibroblasts of human pathological scar and human normal skin were extracted and used in the following experiments.The general condition of the pathological scar tissue and the normal skin tissue was detected by hematoxylin-eosin staining.The appearance of fibroblasts from pathological scar and normal skin were observed by inverted microscope.The fibroblasts were verified by immunofluorescence assay.The cells were treated with different concentrations of RSL3(1,3,5,7,9,11,13 μmol/L).The inhibitory concentration of RSL3 on fibroblasts was detected by cell counting kit-8.Control group(without treatment)and RSL3 intervention group(treated with 7 μmol/L RSL3 for 24 hours)were set up.The mRNA and protein expressions of glutathione peroxidase 4,type Ⅰ collagen,type Ⅲ collagen and α-smooth muscle actin were detected by Qrt-PCR and western blot,respectively.Level of malondialdehyde in cells was detected.The residual scratch area was measured by cell scratch test after 24 hours to calculate the percentage of residual scratch area. RESULTS AND CONCLUSION:The expression of glutathione peroxidase 4 in the pathological scar group was higher than that in the normal skin group(Mrna:t=3.252,P<0.01;protein:t=5.075,P<0.01).The expression of glutathione peroxidase 4 in the pathological scar fibroblast group was higher than that in the normal skin fibroblast group(Mrna:t=10.32,P<0.01;protein:t=26.22,P<0.01).Compared with the control group,the expression of glutathione peroxidase 4 was decreased(Mrna:t=2.798,P<0.05;protein:t=4.643,P<0.01),the content of malondialdehyde was increased(t=2.917,P<0.05),the expression of type Ⅰ collagen(Mrna:t=15.84,P<0.01;protein:t=4.610,P<0.01),type Ⅲ collagen(Mrna:t=28.86,P<0.01;protein:t=7.713,P<0.01)and α-smooth muscle actin(Mrna:t=2.671,P<0.05;protein:t=7.417,P<0.01)were decreased in the RSL3 intervention group.Compared with the control group,the migration ability was weakened in the RSL3 intervention group(t=14.06,P<0.01).To conclude,RSL3 can inhibit the expression of glutathione peroxidase 4 and then inhibit the ability of fibrosis and migration of pathological scar fibroblasts.
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Objective:To evaluate the efficacy of recombinant humanized type Ⅲ collagen injected by microneedles in facial rejuvenation.Methods:Twenty-five cases recruited from July to December 2021 were included as the research objects in this study. Recombinant humanized type Ⅲ collagen was injected into the face skin using microneedles. Images were collected before the treatment and 1, 2, and 3 months after the end of the treatment. The VISIA image analysis system was used for comparative analysis. Patient′s satisfaction evaluation and the occurrence of complications after treatment were also recorded.Results:According to results of VISIA, the scores of spots were (32.06±6.92) and (27.59±7.69); red areas were (32.79±11.80) and (27.74±9.49); pores were (17.66±9.79) and (14.60±7.07) and brown spots were (43.61±11.93) and (37.59±16.22) after the treatment were lower than those before treatment, and the results were statistically different ( F=3.442, 4.209, 7.473, 7.113, P<0.05). One month after the end of the of treatment. Patient′s self-satisfaction reached 84% (21 cases); three months after the end of the treatment, the patient′s self-satisfaction reached 80% (20 cases). No serious adverse reactions were occurred. Conclusions:The recombinant humanized type Ⅲ collagen injected by microneedles can be effective and durable for facial rejuvenation and get high patient satisfaction. It has high safety and is worthy of clinical promotion.
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Objective:To explore whether chronic fluorosis can cause liver fibrosis in rats by observing expression changes in type Ⅰcollagen (Col-Ⅰ), type Ⅲ collagen (Col-Ⅲ) and alpha smooth actin (α-SMA) in the liver tissue of chronic fluorosis rats.Methods:According to body weight (90 - 100 g), forty-eight SD rats were randomly divided into control group (drinking water fluoride ion concentration < 0.5 mg/L), low, medium and high concentration fluoride groups (drinking water fluoride ion concentration of 5.0, 50.0 and 100.0 mg/L), with 12 rats in each group (half male and half female), and fed for 6 months. Fluoride ion selective electrode method was used to detect bone fluoride and urinary fluoride levels; hematoxylin-eosin staining (HE staining) and Masson staining were used to observe the pathological and morphological changes and the collagen deposition of liver tissue; quantitative real-time polymerase chain reaction and immunohistochemical staining were used to observe Col-Ⅰ, Col-Ⅲ and α-SMA mRNA and protein expressions.Results:There was significant difference in bone fluoride and urine fluoride between the 4 groups [bone fluoride: (92.52 ± 5.64), (112.21 ± 11.86), (142.99 ± 7.87), (235.63 ± 11.55) mg/kg; urinary fluoride: (5.47 ± 0.88), (17.78 ± 1.48), (54.16 ± 5.96), (121.11 ± 6.32) mg/L, P < 0.001]. Under light microscope, with the increase of fluoride concentration, the degree of hepatic cell edema was aggravated, and the deposition of collagen fiber around the central vein and the portal area increased significantly. The mRNA expression level of Col-Ⅰ in low, medium and high concentration fluoride groups (1.20 ± 0.09, 1.80 ± 0.08, 1.58 ± 0.06) was significantly higher than that in control group (1.00 ± 0.00, P < 0.05); Col-Ⅲ and α-SMA mRNA expression levels in medium and high concentration fluoride groups (Col-Ⅲ: 1.15 ± 0.14, 1.64 ± 0.24; α-SMA: 1.69 ± 0.02, 2.34 ± 0.06) were significantly higher than those of low concentration fluoride group (Col-Ⅲ: 0.59 ± 0.17; α-SMA: 0.80 ± 0.13, P < 0.05). With the increase of fluoride concentration, the liver tissue Col-Ⅰ(0.00 ± 0.00, 0.03 ± 0.01, 0.08 ± 0.01, 0.13 ± 0.02), Col-Ⅲ (17 803.05 ± 3 221.16, 47 523.15 ± 3 490.10, 127 786.35 ± 13 008.86, 237 233.03 ± 47 614.63) and α-SMA (516.83 ± 181.18, 2 885.03 ± 864.92, 11 186.94 ± 2 394.08, 37 182.43 ± 12 390.59) protein levels were also increased significantly ( P < 0.05). Conclusion:Long-term excessive intake of fluorine may cause the production of collagen fibers around the central vein and the portal area of the liver in rats to increase, and then lead to the formation of liver fibrosis.
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Chinese herbal medicine ultrafine powder has become a research hotspot for the addition of cosmetic raw materials. Dendrobium candidum is a traditional Chinese herbal medicine. Its extract and stem extract are already cosmetic raw materials and its water extract has the effect of preventing photoaging,but D. candidum ultrafine powder has not been accepted as a raw material for cosmetics,and no relevant research on photoaging prevention has been reported. In this experiment,the ultra-fine powder and fine powder of D. candidum to prevent photoaging were observed and compared,and its mechanism of action was discussed to provide a basis for the prevention of skin photoaging products. Seventy-two female ICR mice were randomly divided into normal group,model group,solvent group,titanium dioxide(Ti O2) group,isooctyl salicylate(2-ES) group,D. candidum ultrafine powder 1(DP1),ultrafine powder 2(DP2) and fine powder(DP3) groups. The photoaging model was established by ultraviolet irradiation for 8 weeks,and the model was intervened while modeling. The skin wrinkle grade,elastic parameters,skin microcirculation blood flow,skin structure and pathological changes(skin thickness,skin collagen fiber,elastic fiber) were observed,the skin transforming growth factor-β1(TGF-β1),Smad3 levels were determined,and the type Ⅰ and type Ⅲ collagen,matrix metalloproteinase-1(MMP-1),activated protein-1(AP-1),VEGF expression were detected. The results showed that ultrafine powder(DP1,DP2) significantly reduced the wrinkle level and skin blood flow of the model mice(P<0. 05,P<0. 01); DP1,DP2 and DP3 could significantly reduce the thickness of the epidermis(P<0. 001),improve collagen fiber,elastic fiber hyperplasia,and distortion and decrease VEGF expression,and DP1 is better than DP2 and DP3; each group could up-regulate type Ⅰ collagen,down-regulate type Ⅲ collagen,AP-1,MMP-1 protein expression,and DP1 improvement optimal. However,it has no obvious effect on TGF-β1 and Smad3. The ultrafine powder and fine powder of D. candidum have certain preventive effect on photoaging,and the effect of ultrafine powder is better than that of fine powder. Ultrafine powder may down-regulate the expression of type Ⅲ collagen,AP-1 and MMP-1 by up-regulating type Ⅰ collagen. Inhibition of collagen degradation plays a role in preventing photoaging.
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Animals , Female , Mice , Dendrobium , Mice, Hairless , Mice, Inbred ICR , Skin , Skin Aging , Ultraviolet RaysABSTRACT
OBJECTIVE:To study the effects of salvianolic acid B(Sal B)on angiotensin Ⅱ(Ang Ⅱ)-induced cardiac fibro-blast proliferation,secretion of type Ⅲ collagen,protein expressions of matrix metalloproteinase 9 (MMP-9),Smad2/3,Smad7, and explore its mechanism of anti-myocardial fibrosis. METHODS:Cells were divided into blank control group(culture medium) Ang Ⅱ model group,Sal B low-dose,medium-dose,high-dose groups (12.5,25,50 μmol/L). After cultured 1 h by blank or drug-containing culture,except for blank control group,cells in other groups were added 1 μmol/L Ang Ⅱ to induce proliferation. for 24 h. MTT method and hematoxylin-eosin staining method were adopted investigate the effect of Sal B on proliferation. Western blot method was adopted to detect the effects of Sal B on protein expressions of type Ⅲ collagen,MMP-9,Smad2/3,Smad7. RE-SULTS:Compared with blank control group,cells in Ang Ⅱ model group were significantly proliferated,protein expressions of type Ⅲ collagen,MMP-9,Smad2/3 were obviously enhanced,protein expression of Smad7 was obviously weakened,with statisti-cal significances(P<0.05). Compared with Ang Ⅱ model group,the cell proliferation in Sal B groups were inhibited,protein ex-pressions of typeⅢcollagen,MMP-9,Smad2/3 were weakened,while protein expression of Smad7 was enhanced. Except the pro-tein expression of type Ⅲ collagen in Sal B low-dose and medium-dose groups,the protein expression of Smad2/3 in Sal B high-dose group did not change significantly,other indexes had statistical significances(P<0.05). CONCLUSIONS:The anti-myo-cardial fibrosis effect of Sal B may be associated with inhibiting the proliferation of cardiac fibroblasts,down-regulating protein ex-pressions of typeⅢcollagen,MMP-9,Smad2/3 and up-regulating protein expression of Smad7.
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OBJECTIVE:To study the effects of salvianolic acid B(Sal B)on angiotensin Ⅱ(Ang Ⅱ)-induced cardiac fibro-blast proliferation,secretion of type Ⅲ collagen,protein expressions of matrix metalloproteinase 9 (MMP-9),Smad2/3,Smad7, and explore its mechanism of anti-myocardial fibrosis. METHODS:Cells were divided into blank control group(culture medium) Ang Ⅱ model group,Sal B low-dose,medium-dose,high-dose groups (12.5,25,50 μmol/L). After cultured 1 h by blank or drug-containing culture,except for blank control group,cells in other groups were added 1 μmol/L Ang Ⅱ to induce proliferation. for 24 h. MTT method and hematoxylin-eosin staining method were adopted investigate the effect of Sal B on proliferation. Western blot method was adopted to detect the effects of Sal B on protein expressions of type Ⅲ collagen,MMP-9,Smad2/3,Smad7. RE-SULTS:Compared with blank control group,cells in Ang Ⅱ model group were significantly proliferated,protein expressions of type Ⅲ collagen,MMP-9,Smad2/3 were obviously enhanced,protein expression of Smad7 was obviously weakened,with statisti-cal significances(P<0.05). Compared with Ang Ⅱ model group,the cell proliferation in Sal B groups were inhibited,protein ex-pressions of typeⅢcollagen,MMP-9,Smad2/3 were weakened,while protein expression of Smad7 was enhanced. Except the pro-tein expression of type Ⅲ collagen in Sal B low-dose and medium-dose groups,the protein expression of Smad2/3 in Sal B high-dose group did not change significantly,other indexes had statistical significances(P<0.05). CONCLUSIONS:The anti-myo-cardial fibrosis effect of Sal B may be associated with inhibiting the proliferation of cardiac fibroblasts,down-regulating protein ex-pressions of typeⅢcollagen,MMP-9,Smad2/3 and up-regulating protein expression of Smad7.
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Background:Activation of hepatic stellate cells( HSCs)plays a pivotal role in development of liver fibrosis. Interleukin-17(IL-17)is the most important effector of T helper 17(Th17)cells that causes inflammatory cell infiltration and tissue damage. Preliminary studies showed that the number of IL-17-positive cells in liver tissue was positively correlated with the severity of liver fibrosis in patients with chronic hepatitis B and autoimmune hepatitis. However,the mechanism of IL-17 in liver fibrosis is not yet clarified. Aims:To investigate the effect of IL-17 on activation of human HSC cell line LX2 and collagen expression. Methods:Human HSC cell line LX2 was treated with different concentrations of IL-17. Viability of LX2 cells was measured by CCK-8 assay. mRNA expressions of α-smooth muscle actin(α-SMA), type Ⅰ collagen(Col-Ⅰ)and Col-Ⅲ were determined by real-time PCR. Protein expressions of α-SMA、Col-Ⅰ and Col-Ⅲ were detected by immunofluorescence. Results: Viability of LX2 cells increased with the increase of IL-17 concentration,but no significant differences were seen between any two groups(P > 0. 05). mRNA expressions of α-SMA, Col-Ⅰ and Col-Ⅲ in IL-17 treatment group(100 ng/ mL)were significantly higher than those in blank control group(P <0. 05). With the increase of IL-17 concentration,protein expressions of α-SMA,Col-Ⅰ and Col-Ⅲ gradually increased. Conclusions:IL-17 can promote the activation of HSCs and expressions of Col-Ⅰ and Col-Ⅲ,thereby contributing to the development of liver fibrosis.
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Objective To investigate the effects of capparis spinosa total alkaloid on the pathological changes and the type Ⅲ collagen(COL?Ⅲ)expression in systemic sclerosis(SSc)mice. Methods Mice models with SSc were established by repeated local injection of bleomycin in BALB/c mice back. After administration of capparis spinosa total alkaloid ,the pathological changes of skin and lung tissue were observed ,and the COL?Ⅲ expression was detected by ELISA. Results Compared with the model group,the inflammation and fibrosis of skin and lung tissue were improved,and the level of COL?Ⅲ was markedly reduced by treatment of high dose capparis spinosa total alkaloid(P<0.05). Conclusion Cap?paris spinosa total alkaloid is effective in treating fibrosis of SSc.
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ObjectiveTo observe the effects of six stagnations elimination therapy on the content of typeⅠ, typeⅢ collagens and matrix metalloproteinase-9 (MMP-9) expression in atherosclerotic mice with vulnerable plaques, to discuss the possible mechanisms of this therapy in stabilizing vulnerable plaques.Methods The ApoE knockout mice were fed on high-fat diets, which built the vulnerable plaques model. Five groups were established, including normal group, model group, high-dose group, low-dose group, and simvastatin group, with 10 mice in each group. Dose groups were given drug intervention, while normal group and model group were given in the same amount of saline. After 12 weeks of drug intervention, the mice were put to death. TypeⅠ and typeⅢcollagens were observed using picric acid-Sirius red staining method. The expression of MMP-9 was detected by immunohistochemical method.ResultsCompared with normal group, vulnerable plaques formed more easily in model group.Compared with model group, typeⅠ collagen increased in high-does and low-dose groups, while typeⅢ collagen, the ratio of typeⅢ andⅠ collagens, and the expression of MMP-9 decreased (P<0.01).ConclusionSix stagnations elimination therapy could stabilize vulnerable plaques. Regulating typeⅠ andⅢ collagens content and inhibiting the expression of MMP-9 may be one of its possible mechanisms.
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BACKGROUND:Shengji Yuhong col agen showed good curative effect of promoting angiogenesis and tissue healing compared with Shengji Yuhong Gao and col agen alone or gelatin alone. OBJECTIVE:To explore the curative effect and mechanism of subcutaneous implantation of Shengji Yuhong col agen in rabbits in promoting angiogenesis and repair. METHODS:Shengji Yuhong col agen as the experimental group and collagen as the control group was implanted inside the rabbit subcutaneous pockets of the back of New Zealand rabbits. The implanted samples and surrounding tissues were obtained at 3, 7, 14, 28 and 56 days fol owing surgery. Pathological sections were made and the repair of surrounding tissue was observed. Hemoglobin levels in col agen were measured. Immunofluorescence and CD34 dyeing marking method were utilized to observe capil ary angiogenesis. Western blot assay was employed to examine vascular endothelial growth factor and angiogenin-1 expression. Immunohistochemistry was used to observe the secretion of typeⅠ and Ⅲ col agen on the surrounding tissues. RESULTS AND CONCLUSION:The experimental group showed increased subcutaneous vascularization. There were reduced inflammatory exudation, granulation tissue hyperplasia, and mature fiber connective tissue at 28 days. Angiogenesis and hemoglobin contents were greater in the experimental group than in the control group (Pidentical between the experimental and control groups. However, the secretion of type Ⅲ col agen was higher in the experimental group than in the control group at 28 and 56 days (Pcol agen was lower in the experimental group than in the control group at 28 and 56 days (Pmechanisms of adjusting the protein expression of vascular endothelial growth factor and angiogenin-1. At the same time, it has the function of regulating col agen formation with better ratio of typeⅠ and type Ⅲ col agen to acquire higher quality of wound healing with reduced scar formation.
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Objective To observe the effects of doxazosin on the expression of type Ⅰ and type Ⅲ collagen fiber in autoantibodies against α1-adrenergic receptors (α1-AA) positive diabetic rats,and to investigate the protective mechanism of doxazosin on cardiomyopathy of diabetic rats.Methods After establishment of diabetes model with streptozocin,diabetic rats were randomly divided into diabetic group (group A,n =10),doxazosin treated group (group B,n =10),α1-AA mediated group (group C,n =8),α1-AA plus doxazosin treated group (group D,n =8).Group C and group D were injected α1-AA (100 μg/100 g) by caudal vein at 0,4,8,12,and 16 weeks.Doxazosin (0.36 mg · kg-1 · d-1) was administered by lavage for 16 weeks in group B and group D,and other groups were given the same volume of saline every day.Expressions of type Ⅰ and type Ⅲ collagen fibers in myocardium of left ventricle were detected by immunohistochemical staining.Pathological changes in the myocardium were observed by both light and electron microscopes.Changes in collagen fiber in myocardium were detected by Van Gieson staining.Results Among various groups,there was no significant difference in blood glucose levels (P ＞ 0.05).After the intervention of doxazosin,body weight in group B and group D was greater than that of group A and group C (P＜0.05 or P＜0.01).Expression of type Ⅰ and type Ⅲ collagen fibers in myocardium in group D was lower than that in group C (P＜0.05).Expression of type Ⅰ and type Ⅲ collagen fibers in group B was lower than that in group A (P＜0.05) as well.Myocardial pathological changes in group C were most serious,showing reduced mitochondrial,vacuolar degeneration,and interstitial collagen hyperplasia.Cardiomyopathy in group D and group B was less marked as compared with that in group C and group A,respectively.Myocardial collagen fiber in group C was significantly increased and showed poor alignment.Compared with group C,myocardial collagen deposition in group D was obviously reduced.Conclusions Doxazosin may suppress type Ⅰ and type Ⅲ collagen expressions in myocardium of α1-AA mediated diabetic rats,resulting in alleviation of myocardial fibrosis and protection of myocardium in diabetic rats.
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Background The tissue-engineered cornea is becoming the hot spot in the ophthalmologic field,while the research of corneal substitute is in the ascendant,because it is more similar to the corneal morpha and easy to survive in vivo.Objective This study was to investigate the biocompatibility of recombinant human type-Ⅲ collagen/poly9 ( 3-( methacryloylamino ) propyl dimethyl ( 3-sulfopropyl ) ammonium hydroxide ) ( PMPDSAH ) interpenetrating polymer network (IPN) (RHC-Ⅲ/PMPDSAH IPN) hydrogel as a tissue-engineered cornea in rabbit eye and its feasibility as the corneal substitute.Methods One hundred and eight rabbits were randomly divided into experimental group( 90 rabbits) and normal control group ( 3 rabbits),and 15 rabbits ( 30 eyes ) used as the donor corneas.RHC-Ⅲ/PMPDSAH IPN,NGF PMPDSAH IPN and corneal grafts were lamellarly transplanted into the right eyes in RHC-Ⅲ/PMPDSAH IPN group,NGF PMPDSAH IPN group and allograft group respectively.The corneal transparency and neovascularization were examined and scored under the slim lamp and compared among three groups using Kraskal-Wallis H test.The corneal epithelization time was observed and compared among these three groups using one way analysis of variance and LSD-t test.The histological examination of corneas was performed at the 3rd day,1st and 2nd week,1 st,3rd and 6th month after the surgery.The immunohistochemistry was used to detect the expression of K3 in cornea at the 6th month.Results The grafts were well attached in RHC-Ⅲ/PMPDSAH IPN group,NGF PMPDSAH IPN group and allograft group,and no rejection reaction was found throughout 6-month following up.Compared with normal control group,no significant differences were found in the scores of corneal opacification and neovescularization in these three groups (x2 =4.34,P =0.23 ;x2 =2.60,P =0.46 ) at the 6th month.NGF PMPDSAH IPN group achieved reepithelialization in (4.97±0.63) days and was obviously shorted than that in RHC-Ⅲ/PMPDSAH IPN group and allograft group ( t =11.97,P =0.00; t =5.80,P =0.00).The re-epithelialization time in RHC-Ⅲ/PMPDSAH IPN was (6.86±0.71) days,and that of allograft group was (5.87±0.43 ) days,showing a significant difference ( t =6.32,P =0.00).Hematoxylin-eosin staining results demonstrated that implanted materials integrated into the host corneal tissue well and support corneal epithelialization.Part of the material degraded at the 2nd week and degraded completely 1 month later.Regular alignment and distribution of collagen fibers were seen in the regenerated cornea and were similar to those of the normal stroma in 6 months.Immunohistochemistry showed the positive expression of keratin-3 in corneal epithelial cells.Conclusions RHC-Ⅲ/PMPDSAH IPN has a good biocompatibility without toxicity to corneal tissue.Furthermore,NGF can promote the corneal wound-healing and re-epithelialization.The material can be used as safe and reliable corneal substitute after improving the mechanical strength.
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Objective To observe the expressions of matrix metalloproteinase 1(MMP1) and tissue inhibitor of matrix metalloproteinase1(TIMP1) and the collagen type Ⅰ,Ⅲ deposition in the liver tissues,and evaluate the possible fibrosis mechanism of patients with chronic hepatitis B(CHB) in the way of degradation of collagen. Methods The specimens of the biopsy liver in 50 cases with CHB were detected for the expressions of type Ⅰ,Ⅲ collagen proteins,MMP1 and TIMP1 by immunohistochemical staining. Results The expressions of type Ⅰ,Ⅲ collagen proteins and TIMP1,were significantly increased along with the advancing of hepatic fibrosis.There was a positive correlation between the expressions of type Ⅰ,Ⅲ collagen proteins and TIMP1. Conclusions Hepatic fibrosis in the patients with CHB may be related to increase of TIMP1 expression that inhibit the degradation of collagen.
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Objective To investigate the effects of different concentrations of glucose on growth and collagen synthesis of rat skin fibroblast cells cultured in vitro. Methods Rat skin fibroblast cells cultured in vitro were treated with glucose of different concentrations (30,35 and 40mmol/L) for 48 h (high glucose group 1,2 and 3). Cell proliferation was detected by MTT method,the hydroxyproline contents in culture media were determined by the commercial kit,and the mRNA expression of typeⅠand Ⅲ procollagen,matrix metalloproteinase-2(MMP-2)and tissue inhibitor of metalloproteinase-2(TIMP-2)were detected by RT-PCR. Cells cultured with 25mmol/L glucose were served as controls. Results With the increase of glucose concentration in culture media,the growth of skin fibroblast cells was significantly inhibited,and the hydroxyproline contents were significantly decreased (P
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Objective To study the mechanism of dermal fibroblasts as a feeder layer to support the growth of human keratinocytes. Methods Human dermis fibroblasts were isolated and cultured and then treated with mitomycin-C. The expression of type Ⅰand type Ⅲ precollagen mRNA and relevant protein in feeder layer were examined by RT-PCR and Immunohistochemistry. KCs were cultured both on FB and NIH3T3 feed layer as control, the adhering numbers and the time of fusion were recorded. Results RT-PCR showed an increase of type Ⅰprecollagen mRNA in FB feeder layer as compared with that of normal fibroblasts (P
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Objective To investigate the changes of connective tissue growth factor(CTGF)gene expression level and the association with pathological staging of liver fibrosis and changes of type Ⅰ,Ⅲ collagen gene expression levels in he- patic fibrogenesis.Methods Hepatic fibrosis rat models were created by subcutaneous injections of CCl_4,and the de- gree of hepatic fibrosis in model rats was determined by HE staining and Sirius red staining.The expressions of CTGF, type Ⅰ and Ⅲ collagen gene of the rat liver tissue specimens were analysed with the Western blot and/or Reverse tran- scription polymerase chain reaction(RT-PCR).Results Grade I fibrosis was observed in rat liver tissue of CCl_4 in- ductiun for 2 weeks,and grade Ⅲ fibrosis in that of CCl_4 induction for 6 weeks.The CTGF mRNA and protein and type Ⅰ and Ⅲ collagen mRNAs in hepatic tissue of grade Ⅰ fibrosis and grad Ⅲ fibrosis were increased by 0.64?0.078、 2.66?0.269、1.83?0.096、2.85?0.096 and 2.81?0.055、6.41?0.224、3.25?0.13、2.69?0.082 fold com- pared with normal control,respectively.Conclusion The changes of CTGF gene expression level significantly correla- ted with the degree of liver fibrosis and type Ⅰ collagen gene expression,but not with type Ⅲ collagen expression in liver fibrogenesis.These findings suggested that the upregulation of CTGF gene expression might play an important role in the process of liver fibrogenesis,and postulating the interference of CTGF expression might be a new strategy.
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Objective To study the expressing status of antisense tissue inhibitor of metalloproteina se-1(TIMP-1) in hepatic stellate cells (HSC) constructed in vitro, and to eval u ate the effects on the production of type Ⅰ and Ⅲ collagens secreted by activated rat HSC. Methods HSC were extracted from normal rat liver by pronase and co llagenase digestion and purified by centrifugal elutriation, and were cultured pla stic until they were activated to a myofibroblastic phenotype after 7-10 days. RT-nest-PCR and gene recombinant techniques were used to construct the rat ant isense TIMP-1 expression plasmid which can express in eukaryotic cells, and seg uenced after being counstructed. The expressing plasmid and the pcDNA3 empty pla smid were transfected into HSC by Effectene reagent separately. The cells were sel ected after growing in DMEM containing 400 ?g/ml G418 for 3 to 4 weeks. Exp ression of TIMP-1 in HSC was d etermined by Northern blot and Western blot. We tested the interstitial collagen ase activity in culture media with FITC-labled type Ⅰ collagen as substrate. U ltimately, we quantified the typeⅠ and type Ⅲ collagen in HSC by Wester n blot. Results The exogenous antisense TIMP-1 recombinant plasmid could block the expression of TIMP-1 greatly, while there were not the same outcome i n pcDNA3 empty plasmid g roup and non-transfecting control group. The ratio of TIMP-1/GAPDH was 0.67, 2 .41 and 2.97 respectively at mRNA level( P
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AIM:To explore the mechanisms of Kangxian Yixin Extract(Radix Codonopsis,Radix Astragali,Poria,Rhizoma Atractylodis macrocephalae,Radix et Rhizoma Salviae miltiorrhizae,Rhizoma Chuanxiong,Flos Carthami,Radix Paeoniae rubra,Herba Lycopi,Herba Leonuri) on ventricular remodeling after dilated cardiomyopathy. METHODS: Ventricular remodeling model was induced by giving Furazolidone in wistar rats for 8 weeks and survival rats were divided into 5 groups(14 rats each group) after another 8 weeks,all the rats were killed.The effects of Kangxian Yixin Extract on typeⅠand type Ⅲ collagen gene expression were determined with SP and RT-PCR methods. RESULTS: Collagen TypeⅠand Ⅲ of model group was significantly higher than that of normal group(P0.05),while higher than that of the low dosage of group(P
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Objective:To investigate the mechanism of hypertrophic scar formation in different age periods.Methods:The expression of type I and type Ⅲ collagen of hypertrophic scar in different age periods was measured with immunohistochemistry which was divided into two groups 1～19 years old and 20～50 years old,and the same measure was adopted in normal skin divided into four groups-fetal,1～19,20～50,and more than 60 years old .The quantities of type I and type Ⅲ collagen and the ratios were analyzed.Results:①The ratio of type Ⅰ/type Ⅲcollagen of normal skin had a guadually increasing tendency.②The ratio of type Ⅰ/type Ⅲ collagen of HTS was increasing compared with normal skin(NS),and relative quantities of type I collagen of HTS was significantly increasing.The ratio of type Ⅰ/type Ⅲcollagen of HTS in group of 1～19 year old was highter than that in the group of 20～50 year old .Conclusion:Normal structure of the skin is decided by quantities and ratio of type I and type Ⅲ collagen.The formation of hypertrophic scar in different age periods relates to maladjustment of the ratio of type I and type Ⅲ collagen.Collagen synthesis and regulation of its constitute are finished after about six months.They keep relatively stable within 6 month to 2～3 years.
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Objective To investigate age-related changes of type Ⅰ and Ⅲ collagen in rat heart. Methods Twenty-one male Wistar rats were divided randomly into infanct,young and old group.Types Ⅰ and Ⅲ collagen expresson of different age hearts were studied by SP immunohistochemical methods. Results 1.Type Ⅰ and Ⅲ collagen all constitute thick fiber and fibril.The fibrils encircled every cardiac muscle and formed collagen net each other.The thick fibers being plaque were located among cardiac muscle groups.The content of type Ⅰ collagen was more than that of type Ⅲ.2.There were distinct expression difference of type Ⅰ and Ⅲ collagen in different aging rat hearts.The collagens distributed densely and evenly in infancy rat heart,and loosely and uniformly in young rat heart,and the contents were increased distinctly with heterogeneous distribution in old rat heart.The cardiac collagen was growing from infanct to old rat,and rapid progress of cardiac collagen was seen in young rat. 3.The content of collagen in right ventricle was more than that of left ventricle of infanct heart.The collagen in all parts of the heart is not of difference both in yound and old rat. Conclusion The contents of type Ⅰ collagen is more than that of type Ⅲ collagen.The two types collagen are increasing with growing.;