Objective: To investigate the expression and function of collagen type ⅩⅦ α1 (COL17α1) in aging mouse skin and its effect on the stemness and proliferation of human epidermal stem cells (ESCs), and to explore the mechanism of related microRNA (miR) in intervening the expression of COL17α1 of human ESC. Methods: The method of experimental research was used. Twelve 2-month-old (young) and twelve 24-month-old (aged) male C57BL/6J mice were selected, and full-thickness skin samples from their upper back were taken for follow-up detection. After hematoxylin-eosin staining of the full-thickness skin samples of young mice and aged mice, the structure of the epidermis was observed and the thickness of the epidermis was measured; the morphology of epidermal basement membrane and hemidesmosomes were observed by transmission electron microscopy, and the hemidesmosomes were counted; the mRNA and protein expressions of COL17α1 were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively, and the protein expression and distribution of COL17α1 was observed and detected by immunofluorescence method. The fresh foreskin tissue discarded after surgery was obtained from 3 healthy men aged 20-30 years who underwent circumcision at the Fourth Medical Center of PLA General Hospital, ESCs were extracted and well-grown cells were wsed for follow-up experiments. According to the random number table (the same grouping method below), ESCs were divided into blank control group, transfection reagent control group, empty vector plasmid group, and COL17α1 knockdown plasmid group with corresponding treatment. After 48 hours of culture, the mRNA expression of COL17α1 was detected by real-time fluorescent quantitative RT-PCR, the protein expressions of COL17α1 and cytokeratin 14 (CK14) were detected by Western blotting, and the cell proliferation level was detected by cell counting kit 8. miRs that might act on the 3' non-coding region of COL17α1 mRNA were screened through DIANA, miRTarBase, miRNAMap, TargetScan, and microRNA databases. The ESCs were divided into negative control group transfected with miR mimic negative control and each miR mimic group transfected with each of the previously screened miR mimics. Forty-eight hours after transfection, the protein expression of COL17α1 was detected by Western blotting. Based on the sequencing data set GSE114006 in Gene Expression Omnibus (GEO), the GEO2R tool was used to statistically analyze the expression of the previously screened miRs that could cause the reduction of COL17α1 protein expression in the skin of 30 young (18-25 years old) and 30 elderly (>70 years old) human skins. The full-thickness skin samples of young mice and aged mice were taken, and the expressions of increased miRs in the aforementioned aged human skin were detected by real-time fluorescent quantitative RT-PCR. Two batches of human ESCs were taken, the first batch was divided into COL17α1 wild type+miR-203b-3p negative control group and COL17α1 wild type+miR-203b-3p mimic group, and the second batch was divided into COL17α1 mutant+miR-203b-3p negative control group and COL17α1 mutant+miR-203b-3p mimic group. Each group of ESC was transfected with corresponding sequences respectively. Forty-eight hours later, the luciferase reporter gene detection kit was used to detect the gene expression level of COL17α1. The number of samples in the tissue experiment was 6, and the number of samples in the cell experiment was 3. Data were statistically analyzed with independent sample t test, one-way analysis of variance, least significant difference test or Dunnett's test, Mann-Whitney U test or Kruskal-Wallis H test. Results: Compared with those of young mice, the boundary between the epidermis and the dermis of the aged mice skin was blurred and the cell layers were less, and the thickness of epidermis was significantly thinner (Z=-2.88, P<0.01); the morphology of basement membrane was discontinuous, with less unevenly distributed hemidesmosomes at the epidermis-dermis junction, and the number of hemidesmosomes was significantly reduced (Z=-2.91, P<0.01); the mRNA and protein expression levels of COL17α1 in the skin of aged mice were significantly decreased (with t values of 10.61 and 6.85, respectively, P<0.01). Compared with those of young mice, the protein expression of COL17α1 in the basal layer of epidermis and the bulb of hair follicle in the skin of aged mice was significantly decreased (Z=-2.24, P<0.05). After 48 hours of culture, the protein expression levels of COL17α1 in ESCs of blank control group, transfection reagent control group, empty vector plasmid group, and COL17α1 knockdown plasmid group were 1.00±0.27, 1.12±0.21, 1.13±0.23, and 0.42±0.18, respectively. Compared with those of blank control group, the mRNA and protein expression levels of COL17α1, the protein expression level of CK14, and the proliferation level of ESCs in transfection reagent control group and empty vector plasmid group did not change significantly (P>0.05), while these indexes in COL17α1 knockdown plasmid group were significantly decreased (P<0.05 or P<0.01). miR-203a-3p, miR-203b-3p, miR-512-5p, miR-124-3p, miR-28-5p, miR-590-3p, and miR-329-5p might bind to the 3' non-coding region of COL17α1 mRNA. Forty-eight hours after transfection, compared with 1.000±0.224 in negative control group, the protein expression level of COL17α1 in ESCs of miR-329-5p mimic group, miR-203b-3p mimic group, and miR-203a-3p mimic group decreased significantly (0.516±0.188, 0.170±0.025, and 0.235±0.025, with t values of 3.17, 5.43, and 5.07, respectively, P<0.05 or P<0.01). Only the expression level of miR-203b-3p in the skin of the elderly was significantly higher than that of the young (t=3.27, P<0.01). The expression level of miR-203b-3p in the skin of aged mice was significantly higher than that of young mice (Z=-2.88, P<0.01). Forty-eight hours after transfection, the gene expression level of COL17α1 in ESCs of COL17α1 wild type+miR-203b-3p mimic group was significantly lower than that of COL17α1 wild type+miR-203b-3p negative control group (t=7.66, P<0.01). The gene expression level of COL17α1 in ESCs of COL17α1 mutant+miR-203b-3p mimic group was similar to that of COL17α1 mutant+miR-203b-3p negative control group (P>0.05). Conclusions: The mRNA and protein expression levels of COL17α1 decrease with age increasing in mice, which may lead to the detachment of mouse ESC from the epidermal basement membrane. Decreased expression of COL17α1 can inhibit the expression of CK14 and ESC proliferation, which may be responsible for the thinning of the epidermis and slower wound healing in aged human skin. The increased expression of miR-203b-3p in aged mouse skin can target and bind to the 3' non-coding region of COL17α1 mRNA, hindering the post-transcriptional translation process, thus resulting in decreased COL17α1 protein expression.
Adolescent ; Adult ; Aged ; Animals ; Autoantigens ; Humans ; Keratin-14 ; Male ; Mice ; Mice, Inbred C57BL ; MicroRNAs/genetics* ; Non-Fibrillar Collagens/pharmacology* ; Polyesters ; RNA, Messenger ; Skin Aging ; Stem Cells ; Young Adult

Bullous pemphigoid is a chronic autoimmune blistering disease characterized clinically by tense bullae that develop on normal or erythematous skin. Bullous pemphigoid is associated with autoantibodies to two hemidesmosomal proteins, BPAG1 (230 kD) and BPAG2 (180 kD). The localized form of BP is an unusual variant that occurs in 5~30% of the patients. A 58-year-old man who had been suffering from right hemiplegia since 2006, presented with multiple tense bullae localized on both arms and hands. Direct immunofluorescence test showed linear deposition of IgG and C3 along the basement membrane zone. The antibodies against the recombinant NC16a-domain of BP180 were positive by ELISA and immunoblotting using epidermal extract of normal human foreskin demonstrated that the patient's serum reacted with only BP180 antigen. Here, we report a case of localized bullous pemphigoid on both upper extremities in a hemiplegic patient predominantly on the opposite side to the hemiplegia.
Antibodies ; Arm ; Autoantibodies ; Autoantigens ; Basement Membrane ; Blister ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique, Direct ; Foreskin ; Hand ; Hemiplegia ; Humans ; Immunoblotting ; Immunoglobulin G ; Middle Aged ; Non-Fibrillar Collagens ; Pemphigoid, Bullous ; Proteins ; Skin ; Stress, Psychological ; Transcutaneous Electric Nerve Stimulation ; Upper Extremity

We performed photorefractive keratectomy(PRK) on 10 rabbit eyes and determined the distribution of collagen type III, IV VI, VII at postoperative 2, 4 and 6 months to examine immunohistochemical changes after PRK. Type III collagen was not found in the normal cornea but strongly detected in the regenerated corneal stroma at all intervals. It was most prominent at 2 months after surgery and then decreased. Type IV collagen was detected in basement membrane in both normal and ablated corneas at all intervals and the staining was more intense in ablatd corneas than in normal cornea. There was no difference of staining intensity among the groups of different intervals. Type IV collagen was found in both normal and healed corneal stroma at all intervals and there was no difference of staining intensity between normal and ablated corneas and among the groups of different intervals. Type VII collagen was observed as a linear continuous band along the basal surface of epithelium in normal cornea. At 2 months after surgery, type VII collagen staining in basement membrane zone became denser than normal cornea, but segmented. At 4 months after surgery, continuous band of collagen type VII staining was observed, but it was less intense than in normal cornea. At 6 months after surgery, the intensity of continuous band of collagen type VII was the same as in normal cornea. This results suggest that the presence of type III collagen in the regenerated cornea may be related to the development of postoperative subepithelial opacity after PRK and the normalization of collagen type IV and VII at postoperative 6 months may mean the complete reestablished of the adhesion of regenerated epithelium and stroma.
Basement Membrane ; Collagen Type III* ; Collagen Type IV ; Collagen Type VII ; Collagen* ; Cornea* ; Corneal Stroma ; Epithelium ; Immunohistochemistry ; Lasers, Excimer* ; Photorefractive Keratectomy

OBJECTIVETo observe the histological characteristics of constructed basement membrane in tissue-engineered skin.

METHODSForskins from circumcision in normal children were obtained with informed consent of the parents, and then the epidermal keratinocytes (KC) and dermal fibroblasts (Fb) were isolated with trypsin and collagenase D digestion in sequence. Tissue engineered skin with composite chitosan was maintained in a submerged state for 3 days, and then at the air-liquid interface. The tissue-engineered skins were fixed in neutral formalin and then embedded in paraffin after culture for 7, 10 and 15 days, respectively for immunohistological examination of the basement membrane component,including the condition of collagen type IV (COL-IV), collagen type VII (COL-VII), and laminin (LN).

RESULTSHE staining showed that the keratinocytes formed a fine stratified squamous epithelium with the presence of basal, spinous, granular and corneous cell layers, and there was various amount of cells in flat and fusiform shape in each layer. It was found that a regular red staining strip situated at the dermal epidermal junction. Positive staining of collagen IV, collagen VII as well as LN was observed by immunohistological examination.

CONCLUSIONThe results suggest that the composite chitosan tissue engineered skin has a good prospect for clinical use because it presents a perfect reconstruction of basement membrane.

Basement Membrane ; cytology ; Cells, Cultured ; Child ; Chitosan ; metabolism ; Collagen Type IV ; metabolism ; Collagen Type VII ; metabolism ; Humans ; Laminin ; metabolism ; Organ Culture Techniques ; Skin, Artificial ; Tissue Engineering ; methods

OBJECTIVETo study the effects of Si-Wu-Tang on serum protein of blood deficient mice b y proteomicstechnique and study the enriching and regulating blood mechanism of Si-Wu-Tang on mocular level.

METHODThe blood deficient mice was induced by using a single dose of 3.5 Gy radiation from a 60Cogamma source, and high resolution two-dimensional polyacryamide gel electrophoresis (2-DE), computer-assisted image analysis, and mass spectrometry were used to detect regulated protein by Si-Wu-Tang.

RESULT12 lower and 4 higher protein in sera could be recovered by Si-Wu-Tang, 4 protein might be DNA-dependent protein kinase catalytic subunit, Dystrophin, KIF13A, dystonin. They play a part in DNA double-stranded break repair, recombination and modulation of transcription, transportation of mannose-6-phosphate receptor, etc.

CONCLUSIONSi-Wu-Tang can regulate serum protein in blood deficient mice, resulting in improving hematopoiesis and lessening irradiated injury.

Animals ; Autoantigens ; blood ; Carrier Proteins ; Cytoskeletal Proteins ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Dystonin ; Dystrophin ; blood ; Female ; Kinesin ; blood ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins ; Non-Fibrillar Collagens ; blood ; Plants, Medicinal ; chemistry ; Radiation Injuries, Experimental ; blood ; Radiation-Protective Agents ; pharmacology ; Whole-Body Irradiation

INTRODUCTIONAnti-BP180 IgG titres were observed to parallel disease activity in case series of bullous pemphigoid (BP). This study aimed to examine whether anti-BP180 titres are an indicator of disease severity, clinical course and outcome in Asian patients with BP.

MATERIALS AND METHODSThis was a prospective observational study conducted between March 2005 and March 2008 in the Immunodermatology Clinic at the National Skin Centre, Singapore. Disease activity and anti-BP180 IgG titres were measured 4-weekly for 12 weeks and during disease flares and clinical remission. Associations between anti-BP180 titres and disease activity, disease flare, clinical remission and cumulative prednisolone dose were examined.

RESULTSThirty-four patients with newly diagnosed BP were recruited. Median follow-up duration was 3 years. Notable correlations between disease activity and anti-BP180 titres were at baseline (r = 0.51, P = 0.002), and disease flare (r = 0.85, P <0.001). Lower titres at Week 12 were associated with greater likelihood of clinical remission (P = 0.036). Post hoc, patients with anti-BP180 titres above 87.5 U/mL at time of diagnosis who reached remission within 2 years of diagnosis received significantly higher cumulative doses (mg/kg) of prednisolone (median, 72.8; range, 56.5 to 127.1) than those with titres <87.5 U/mL (median, 44.6; range, 32.5 to 80.8); P = 0.025).

CONCLUSIONAnti-BP180 titres may be a useful indicator of disease activity at time of diagnosis and at disease flare. Lower titres at Week 12 may predict greater likelihood of clinical remission. Titres above 87.5 U/mL at time of diagnosis may suggest the need for higher cumulative doses of prednisolone to achieve remission within 2 years.

Adult ; Aged ; Aged, 80 and over ; Antibodies, Anti-Idiotypic ; blood ; Asian Continental Ancestry Group ; Autoantibodies ; blood ; Autoantigens ; blood ; Disease Progression ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Male ; Middle Aged ; Non-Fibrillar Collagens ; blood ; Outcome Assessment (Health Care) ; Pemphigoid, Bullous ; diagnosis ; ethnology ; immunology ; Predictive Value of Tests ; Prospective Studies ; Singapore

The preeclamptic nephropathy is characterized by swelling of endothelial cells, interposition of mesangial cells and matrix, subendothelial deposits of incompletely defined material, and thickening of the capillary walls. To determine the distribution of extracellular matrix (ECM) components in preeclamptic nephropathy, the immunohistochemical study was performed in ten renal biopsy cases using antisera to human type I, III, IV, and VI collagens, fibronectin, and laminin. In preeclamptic nephropathy, the accumulation of type IV and VI collagens, fibronectin was observed in moderate amount in the mesangium and, to some extent, in the thickened capillary walls, particularly in the subendothelial layer. In segmentally sclerotic lesions seen in six cases, the amount of type IV collagen was partly decreased, whereas those of type VI collagen and fibronectin were slightly increased. Type I collagen was expressed to a mild degree in the expanded mesangium and segmentally sclerotic lesions. The results suggest that the expression of ECM in the mesangium is increased in preeclamptic nephropathy, and the deposition of ECM components may be involved in the development and the reparative process of the characteristic glomerular lesions. The formation of sclerotic lesions may be linked to the alternative accumulation of ECM components.
Biopsy ; Capillaries ; Collagen ; Collagen Type I ; Collagen Type IV ; Collagen Type VI ; Endothelial Cells ; Extracellular Matrix* ; Fibronectins ; Humans ; Immune Sera ; Laminin ; Mesangial Cells

PURPOSE: Alport syndrome is clinically characterized by hereditary progressive nephritis causing ESRD with irregular thickening of the GBM and sensory neural hearing loss. The mutations of type IV collagen gene(COL4A5) located on the long arm of X chromosome is considered responsible for most of the structural abnormalities in the GBM of Alport patients. Since no definite clinical prognostic predictor has been reported in the disease yet, we designed this study to evaluate the significance of genetic polymorphism of the angiotensin converting enzyme in children with Alport syndrome as a prognostic factor for disease progression. METHODS: ACE I/D genotype were examined by PCR amplification of the genomic DNA in 12 patients with Alport syndrome and 12 of their family members. Alport patients were divided into two groups; the conservative group, those who had preserved renal function for more than 10 years of age, the early CRF group, those who had progressed to CRF within 10 years of age. RESULTS: The mean age of onset was 3.45+/-2.4 years in the conservative group, 4.4+/-1.2 years in the early CRF group. Sex ratios were 5:3 and 2:1 in each group. Among 12 cases of patients, 4 cases were in early CRF group and their mean duration of onset to CRF was 4.5 years(8.9 years of age). Eight patients(67%) were in the conservative group and they had normal renal function for more than 10 years of age(mean duration of renal preservation was 10.6 years). The incidence of II type ACE gene were in 25.0%(3 cases), ID type in 41.7 %(5 cases), DD type in 33.3%(4 cases). There was no significant difference between Alport patient and normal control(II type 44.3%, ID type 40.9%, DD type 14.8%). The incidence of DD type of early CRF group were higher than that of the conservative group(75% vs 12.5 %)(p<0.05). There was no difference in ACE gene polymorphism between normal Alport family members and control group. CONCLUSION: Even though there was no significant difference of ACE polymorphism between Alport patients and the normal control group, the incidence of DD type is significantly increased in early CRF group which means DD type of ACE polymorphism has a possibility of being a predictor for early progression to CRF in Alport patients.
Age of Onset ; Angiotensins* ; Arm ; Child ; Collagen Type IV ; Disease Progression ; DNA ; Genotype ; Hearing Loss ; Humans ; Incidence ; Kidney Failure, Chronic ; Nephritis ; Nephritis, Hereditary* ; Peptidyl-Dipeptidase A* ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Sex Ratio ; X Chromosome

OBJECTIVEAlport syndrome (AS) is a progressive renal disease characterized by hematuria and progressive renal failure. X-linked dominance is the major inheritance form of the syndrome, accounting for almost 80% of the cases, caused by mutations in COL4A5 genes. There is currently no effective treatment that has been shown to favorably affect the outcome of AS, so early diagnosis and even prenatal diagnosis is very important.

METHODSIn this study mutation of COL4A5 was detected by amplifying the entire coding sequence mRNA of peripheral blood lymphocytes using nested PCR in two Chinese X-linked dominant Alport syndrome (XLAS) families, then the first prenatal diagnosis of XLAS in China was performed. Mutation analysis of the fetus was performed on both cDNA-based level and DNA-based level of amniocytes. Fetus sex was determined by PCR amplification of SRY as well as karyotypes analysis. Maternal cells contamination was excluded by linkage analysis.

RESULTSThere was a deletion mutation in the proband of the first family, 2696 - 2705 del gtatgatggg in the 32 exon of COL4A5, but the mother did not carry the mutation (de novo). There was a G to A substitution at position 4271 in exon 46 of COL4A5 gene (c.G4271A) in the second family, the mother also carried this mutation. After genetic counselling, only the second family accepted prenatal diagnosis. Both amniocytes cDNA level and amniocytes genomic DNA level based prenatal diagnosis showed that the fetus did not carry the same mutation as the mother. PCR amplification of SRY and karyotypes analysis showed a male fetus. Linkage analysis of X chromosome polymorphic microsatellite markers showed that there was no MCC in amniocytes.

CONCLUSIONBoth cDNA level and DNA level analysis could enhance the accuracy and reliability of prenatal diagnosis. PCR amplification of SRY was faster than karyotypes analysis in the fetal sex determination. Linkage analysis was useful in the detection of maternal cells contamination in amniocytes.

China ; Chromosomes, Human, X ; Collagen Type IV ; genetics ; DNA ; analysis ; DNA Mutational Analysis ; trends ; DNA, Complementary ; analysis ; Exons ; physiology ; Female ; Genetic Counseling ; Genetic Linkage ; Genetic Testing ; Humans ; Mutation ; Nephritis, Hereditary ; diagnosis ; genetics ; Pedigree ; Pregnancy ; Prenatal Diagnosis ; methods ; RNA, Messenger

OBJECTIVETo identify the pathogenic mutation in a Chinese family with Alport syndrome.

METHODSBlood samples were collected from the members of the family. Direct DNA sequence analysis of the entire coding region and exon-intron boundaries of the COL4A5 gene was performed, and restriction fragment length polymorphism (RFLP) analysis was used to confirm the sequencing results and to test the mutation in all the family members and 200 controls.

RESULTSA novel splicing mutation of c.1517-1G to T in the COL4A5 gene was identified in all patients in the family. RFLP analysis did not detect this mutation in all the unaffected family members and the 200 controls.

CONCLUSIONThis data revealed a novel splicing mutation of c.1517-1G to T in the COL4A5 gene causing Alport syndrome in a Chinese family. Author's study enriched the spectrum of COL4A5 mutation associated with Alport syndrome.

Adult ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Case-Control Studies ; Chromosomes, Human, X ; genetics ; Collagen Type IV ; genetics ; DNA Mutational Analysis ; Female ; Genetic Linkage ; Humans ; Male ; Mutation ; Nephritis, Hereditary ; genetics ; Pedigree ; Polymorphism, Restriction Fragment Length ; RNA Splicing ; genetics