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Fibulin-1 (FBLN1) is involved in the progression of some types of cancer. However, the role of FBLN1 in cutaneous melanoma (CM) has not been examined. The purpose of this study was to understand the molecular mechanisms and clinical significance of FBLN1 inactivation in CM. The expression of FBLN1 mRNA in CM tissues and adjacent normal skin tissues was analyzed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Methylation-specific polymerase chain reaction was performed to examine the methylation status of the FBLN1 gene promoter. Furthermore, the methylation status of FBLN1 was analyzed with the clinicopathological characteristics and overall survival. qRT-PCR showed FBLN1 mRNA levels in cancerous tissues to be significantly decreased compared with that in adjacent normal skin tissues. The rate of FBLN1 promoter methylation was significantly higher in CM tissues than in adjacent normal skin tissues (P

The ventri-yellow pigmentation mouse (temporarily named VYSlac) arose spontaneously in the C57BL/6J inbred mouse strain, found and bred by Shanghai SLAC Laboratory Animal Co., Ltd. VYSlac presented a special phenotype marked by yellow coat on the ventral surface of neck and trunk that was without melanin deposition but maintained a normal structure. The number of melanocytes in epidermis and melanin in hair follicle of the abdominal skin of the mutant mouse were less than that of their background strain, while there was no significant difference between the dorsal skins of the two strains. This mutant phenotype was inherited as single-gene dominant inheritance, confirmed by genetic experiment, and there was no significant difference between VYSlac and B6 for other biological parameters such as weight, anatomic and histological structures of major organs and blood physiology. When the linkage relationship between the genomic DNA samples of F2 48 mice (VYSlacD2F1×D2) and mutant phenotype were evaluated, the mutant gene was confirmed on chromosome 2 near D2Mit229. New microsatellite and SNP markers were selected to amplify genomic DNA samples of 196 F2 mice and the mutant gene was narrowed down to 5.3 Mb region between rs13476833 and rs27310903 on chromosome 2. The preliminary results of our phenotype analysis and gene location provides a solid basis for further identification of this mutant gene.

Kit W-2Bao mice are single-gene autosomal dominant mutation mice with a B6 background that were bred in our laboratory. Heterozygotes had morphological characteristics including albinism of the abdomen, extremities, and tail, whereas the homozygotes had albinism of the body, black eyes, and infertility. The homozygous mutants showed small, structurally abnormal gonads, and lacked germ cells. Heterozygous male mice lacked germ cells in some contorted seminiferous tubules. This mutation has been mapped at 43.8 cM from the centromere in chromosome 5 by linkage analysis and Kit has been identified as the candidate gene. After Kit full-length mRNA amplification, it was found that a G to T conversion at position 1228 in the ORF changed the 410th amino acid from V to F. This amino acid change could affect the protein’s secondary structure. Heterozygous mutant mice were intercrossed and homozygous mutant mice were bred and genotyped. We found that no primordial germ cells (PGCs) appeared in the urogenital ridge area at fetus day 11.5 in the homozygotes. The number of PGCs also significantly decreased in heterozygotes. At fetus day 15.5, the differentiation of the testis tubule structure was unclear; as well, they contained no spermatogonia. Female homozygotes contained no primordial follicles in the ovary. The numbers of PGCs and primordial follicles were significantly decreased in heterozygous mice. W −2Bao is the only mutated site in the extracellular 4th Ig-like domain and this mutant mouse model provides new material for the study of the mechanism of reproductive system development.

The scant hair mutant mouse (locus symbol: snthr1Bao) is a recessive mutation that originated in an ethylnitrosourea chemical carcinogenesis study using the DBA/2J inbred strain. The gene responsible for the mutation was previously determined to be phospholipase C, delta 1 (Plcd1; mutant allele symbol Plcd1snthr1Bao). To map the modifiers of Plcd1, an intercross (DBA/2J-snthr1Bao/snthr1Bao × C57BL/6J+/+) was conducted. The F2 mutant progeny exhibited a variety of alopecia phenotypes; all F2 mutants (n=507) were classified into 3 groups (mild, moderate, and severe alopecia) and genotyped based on 96 microsatellites. A major QTL was identified on mouse chromosome (mChr) 15 at 12 cM with an LOD score greater than 7 (P < 0.0001). Three minor QTLs were detected on mChr 2, 5, and 7 at 40, 84 and 48 cM, respectively. The QTLs on mChr 7 and 15 were associated with minor alopecia while the QTLs on mChr 2 and 5 were associated with moderate to severe alopecia. No antagonistic or synergistic effects among or between the 4 QTLs were found. Integrating the functions of the 4 potential regulatory QTLs and mutant Plcd1snthr1Bao, we found that these QTLs might contribute to variations of scant hair severity by altering the Ca2+ signal pathways in mouse skin.

The direct sequencing of the Kit cDNA obtained from mutant mice was used to reveal the molecular nature of the W(-3Bao) ENU-induced mutation. There was a T to A transversion at the 441st nucleotide in the W(-3Bao) open reading frame (ORF), which introduced a pre-mature termination codon at residue 147. The gross embryonic development, hematopoiesis and spermatogenesis were examined in the mutant mice. There was no visible difference among the W(-3Bao/+), W(-3Bao/3Bao) and wild type embryos before embryonic day 12.5. W(-3Bao/3Bao) embryos appeared pale after E14.5 and dwarf after E16.5. An extremely low level of hematochrome and large red blood cells were found in W(-3Bao/3Bao) 18.5 days old embryos, leading to the stillbirth of the homozygotes. In 18.5 days old embryos the spermatogonia of W(-3Bao/3Bao) embryos did not migrate to the contorted seminiferous tubules properly, but instead were found in the interstitial tissue. The spermatogonia of W(-3Bao/+) or W(+/+) mice were present in both the interstitial tissue and contorted seminiferous tubules. In the adult male hetereozygotes, there are contorted seminiferous tubules with no spermatogonia, suggesting that the migration defect was dominant. In female W(-3Bao/3Bao) ovaries, primordial follicles were absent while primordial follicles appeared clearly in the ovaries of W(-3Bao/+) or W(+/+) mice. With a nonsense mutation in the Kit gene, W(-3Bao/+) mice show white spotting and an abnormal development of the contorted seminiferous tubules and W(-3Bao/3Bao) mice are stillborn due to severe macrocytic anemia, and have abnormal genital glands in both the male and female.

N-ethyl-N-nitrosourea (ENU) mutagenesis in mice can be used to study gene function in vivo and to establish genetic mouse models of human disease. In this study, a white spotted mouse (named KitW-1 Bao) was obtained by ENU-induced mutagenesis. Inheritance testing showed a single-gene dominant mutation and lethality in the KitW-1 Bao homozygous mice. The mutation was mapped to Chromosome 5 between markers D5 Mit356 and D5 Mit308. The region contains the Kit gene, whose mutations are known to lead to pigmentation defects in mice. Sequence analysis of the Kit cDNA from KitW-1 Bao heterozygotes revealed an A to T missense mutation resulting in an amino acid substitution of Asp (D) by Val (V) at amino acid position 849 within a highly conserved tyrosine kinase domain. The combined phenotype displayed by the KitW-1 Bao heterozygous and homozygous mutant mice demonstrates the critical function of the highly conserved aspartic acid residue at position 849 in the Kit gene product.

The W-3Bao mouse, which was obtained in previous ENU mutagenesis project, is a new mutant caused by the nonsense mutation of Kit gene. Mating and gross observing combining with PCR and sequencing were used for determining the genotypes of the W-3Bao mice. Embryonic development, hematological detection and histopathologic section methods were used for their phenotype analysis. The results showed that the W-3Bao/+ mouse was white belly, white limb terminals and white tail tip. However, there was no difference of the external appearance among the W-3Bao/+, W-3Bao/3Bao and wild type mice for the embryo of 12.5 days. The W-3Bao/3Bao embryos looked pale since pregnant 14.5 days and dwarf since pregnant 16.5 days. The extremely low level of haematochrome and big red blood cells of W-3Bao/3Bao 18.5-day-embryos were found in the inspections of blood routine items and blood smear, which resulted in death of W-3Bao/3Bao homozygous mouse around being born, and no live postnatal W-3Bao/3Bao mouse was found in this study. No spermatogonium at different developmental stages was found in some contorted seminiferous tubules in adult W-3Bao/+ mouse. At the age of 18.5-day embryo, the spermatogonium of W-3Bao/3Bao mice only lied in interstitial tissue and no one lied in contorted seminiferous tubules, while the spermatogonium of W-3Bao/+ or W+/+ mice lied in interstitial tissue and in contorted seminiferous tubules in the same time. At the age of 18.5-day embryo, cells arranged irregularly and primordial follicles were not seen in the W-3Bao/3Bao ovaries, while primordial follicles appeared clearly in the ovaries of W-3Bao/+ or W+/+ mice. We concluded that because of the nonsense mutation of Kit gene, the W-3Bao/+ mice show white spot and abnormal development of some contorted seminiferous tubules. The W-3Bao/3Bao mice die around birth resulting from severe macrocytic anemia and show abnormal genital glands of both genders.

“Phenotype-driven” is the name for an approach used to study gene functions through mutagenesis, location and cloning of the mutant gene. In this study, 150 male C57BL/6J(B6) mice were treated with ENU and reproduced a total offspring of 3860. Of these descendants, 210 exhibited mutation phenotypes by screening, and more than 10 of them are hereditable. Four kinds of mutant mice, named Wbct, W−1Bao, W−2Bao, and W−2Bao, showed dominant hereditary white spot mutation with partial albinism on their belly, distal limbs and tail terminal. To map these mutant genes, 39 microsatellites, equally distributed on the mouse genome and with difference between B6 and DBA/2J (D2), were selected to scan the genome after discrimination of the white spots in the F2 mice [(B6xD2)xD2]. It is found that, the log odds score (LODS) between W−1Bao and D5Mitl68 is 0.56, and the LODS of W−1Bao and D5Mit352 is 4.47. With the gradual application of microsatellites D5Mit290, D5Mit312, D5Mit308 and D5Mit356 that are close to the mutant gene, and the number of F2 mice going up to 537, the mutant W−1Bao is located between D5Mit356 and D5Mit308 on chromosome 5, about 42.19 cM from the centromere. In the same way, W−2Bao and W−3Bao are mapped nearby W−1Bao, and Wbct is located on chromosome 1, about 41.6 cM from the centromere. After searching for the mouse genome database (MGD) and performing a one-by-one study of all genes located on chromosome subregion, it is believed that thekit gene is an excellent candidate for the white spot mutations of W−1Bao, W−2Bao and W−3Bao.

In the current study, the alopecia areata gene was introduced into the C57BL/6 (B6) mouse through repeated backcrossing/intercrossing, and the allelic homozygosity of congenic AAtjmice (named B6.KM-AA) was verified using microsatellites. The gross appearance, growth characteristics, pathological changes in skin, and major organs of B6.KM-AA mice were observed. Counts and proportions of CD4+ and CD8+ T lymphocytes in peripheral blood were determined by flow cytometry. Results show that congenic B6.KM-AA mice were obtained after 10 generations of backcrossing/intercrossing. B6.KM-AA mice grew slower than B6 control mice and AA skin lesions were developed by four weeks of age. The number of hair follicles was reduced, but hair structures were normal. Loss of hair during disease progression was associated with CD4+ and CD8+ T lymphocytes infiltration peri- and intra- hair follicles. No pathological changes were found in other organs except for the skin. In the peripheral blood of B6.KM-AA mice, the percentage of CD4+ T cells was lower and percentage of CD8+ T cells higher than in control mice. These findings indicate that B6.KM-AA mice are characterized by a dysfunctional immune system, retarded development and T-cell infiltration mediated hair loss, making them a promising new animal model for human alopecia areata.

Recently, it was reported that soluble MICB (sMICB) may impair tumor immunogenicity by reducing natural killer group 2D ligand densities on malignant cells. The aim of this study was to elucidate the role of sMICB in melanoma patients. In the present study, we determined sMICB serum concentration in 125 melanoma patients of different clinical stages of disease compared with 30 healthy controls using an ELISA. The correlations between sMICB serum concentration and clinicopathologic variables were analyzed. sMICB serum level was significantly elevated (P

Thymidine kinase 1 (TK1) is involved in cancer progression. Metastatic site is a prognostic factor in melanoma. We assessed whether serum levels of TK1 are associated with metastatic sites or prognosis in patients treated for stage IV melanoma. The study included 64 patients, who received dacarbazine or four-drug chemotherapy (dacarbazine, vincristine, bleomycin, and lomustine) both combined with interferon-alfa. Serum samples for TK1 were analyzed by ELISA. The patients (n = 22) with only skin and subcutaneous metastases had significantly lower mean TK1 levels (1,639 pg/ml) than the patients (n = 42) with other distant metastases (2,586 pg/ml, Mann–Whitney, p = 0.031). TK1 levels above the median (1,590 pg/ml) were significantly related to deep lymph node involvement (odds ratios 3.672; 95 % confidence intervals 1.024–12.843, p = 0.036). There were no other significant associations between TK1 levels and tumor burden nor were the levels significantly related to the response to therapy or survival. Those eight patients who had received previous adjuvant IFN-alfa therapy had lower mean TK1 levels (1,735 pg/ml) as compared to those 56 patients without previous IFN-alfa therapy (2,338 pg/ml, analysis of variance, p = 0.026). This is the first study exploring serum TK1 in melanoma. TK1 might be involved in the deep lymphatic dissemination and progression of melanoma metastasis.

The ventri-yellow pigmentation mouse (temporarily named VY(Slac)) arose spontaneously in the C57BL/6J inbred mouse strain, found and bred by Shanghai SLAC Laboratory Animal Co., Ltd. VY(Slac) presented a special phenotype marked by yellow coat on the ventral surface of neck and trunk that was without melanin deposition but maintained a normal structure. The number of melanocytes in epidermis and melanin in hair follicle of the abdominal skin of the mutant mouse were less than that of their background strain, while there was no significant difference between the dorsal skins of the two strains. This mutant phenotype was inherited as single-gene dominant inheritance, confirmed by genetic experiment, and there was no significant difference between VY(Slac) and B(6) for other biological parameters such as weight, anatomic and histological structures of major organs and blood physiology. When the linkage relationship between the genomic DNA samples of F(2) 48 mice (VY(Slac)D(2)F(1)×D(2)) and mutant phenotype were evaluated, the mutant gene was confirmed on chromosome 2 near D2Mit229. New microsatellite and SNP markers were selected to amplify genomic DNA samples of 196 F(2) mice and the mutant gene was narrowed down to 5.3 Mb region between rs13476833 and rs27310903 on chromosome 2. The preliminary results of our phenotype analysis and gene location provides a solid basis for further identification of this mutant gene.

Cyclophosphamide (cy) was used to produce immunosuppressed model mice by intraperitoneal injection. Affusing different-dosage of the female silkworm moth powder, thymus and spleen index, peritoneal macrophage phagocytosis, lymphocyte transformation, serum hemolysin of mice were investigated. The result showed that all the indexes of the tested immunosuppressed mice were improved by the female silkworm moth powder. So the female silkworm moth powder could enhance non-specific immunological function, humoral immunity and cellular immunity in mice.

To investigate the mechanism of sensory nerve regeneration of the reconstructed penis with sensory nerve implantation and to explore a new surgical technique to improve the postoperative sensory function in phallic reconstruction.Adult male New Zealand rabbits were randomly divided into experimental group (n = 20, with sensory nerve implantation) and control group (n = 20, without sensory nerve implantation), which were both performed phalloplasty with a superficial epigastric faciovascular pedicle flap. Postoperatively, the nerve regeneration process of the reconstructed penis was observed histologically.In experimental group, the amount of CGRP positive nerve fibers increased markedly with the time prolonged, whereas merely a few CGRP positive fibers scattered in deep dermis 6 months later in the other group. The cutaneous sensory nerve regeneration of the reconstructed penis in experimental group shows the procedure that the myelinated axon began to exist within 3 months, thereafter the myelinated axon and unmyelinated axon were both observed under the electron microscope.These findings show that the rabbit model of phalloplasty with sensory nerve implantation can acquire well sensory reinnervation, and bring a light to clinical application for restoration of sensory function in reconstructed penis.

To explore the regeneration of mechanical sensory fibers after free nerve transplantation.Neuroelectrophysiological technique (single nerve fiber recording) was used to test the regeneration rate of mechanical sensory fibers, the proportion of rapidly and slowly adapting receptors, the stimulating thresholds of regenerated mechanoreceptors and conduction velocity of regenerated fibers. The regeneration pattern of the mechanoreceptors after free nerve transplantation to the rabbit reconstructed penis was also analyzed.9 months after operation, the number of regenerated mechanical sensory fiber was almost normal. The regenerated rapidly adapting receptors had a higher proportion with higher mature degree than the regenerated slowly adapting receptors. 9 months after nerve transplantation the stimulating thresholds of regenerated mechanoreceptors and conduction velocity of regenerated fibers remained below normal.After free nerve transplantation to the rabbit reconstructed penis, the function of both rapidly and slowly adapting sensory nerve fiber partially recovered, but in different extent.

To explicate the spinal nerve source of the rabbit penis cutaneous sensation.Twelve adult male rabbits were randomly divided into two groups of equal number. While mechanical stimuli were given to the penis by different von Frey hairs, single fiber activities were recorded in vivo in the left (Group A) and right (Group B) S1-S4 spinal nerves, respectively. The mechanical threshold, adaptability and conduction velocity of the fibers were analyzed.When the ipsilateral penis was mechanically stimulated, discharges were detected in S2 and S3 spinal nerve fibers, but not in S1 and S4. The discharge fibers were 39.67 +/- 3.14 (S2) and 21.00 +/- 2.19 (S3) in the left spinal nerve and 40.00 +/- 3.16 (S2) and 19.67 +/- 2.58 (S3) in the right. There was no obvious difference between the numbers of the left spinal nerves and the right ones (P0.05).The rabbit penis cutaneous sensation originates from S2 and S3 spinal nerves.

To trace the segmental distribution of somatic sensory neurons of the skin and dorsal nerve in the rabbitś penis.The experiment was performed on 8 adult male rabbits with the nerve tracing method of retrograde axonal transport of horseradish peroxidase (HRP), which was injected into the dermis around the penis and the dorsal nerve of the penis. The rabbits were sacrificed five days later to harvest the spinal cord segments and the dorsal root ganglia of lumbosacral segments for histological study.The HRP tracing showed that a number of labeled HRP positive neurons appeared in spinal ganglia (S2 - S4) in all the rabbits, and distributed segmentally. The counts of the positive neurons different segments were: S2 (215.0 +/- 10.2) , S3 (242.2 +/- 8.3) and S4 (109.7 +/- 8.4) respectively, with statistically significant difference between the two groups.The rabbit's sensory nerve fibers in both the skin and the dorsal nerve of the penis are rooted in the S2-S4 segments of spinal ganglia, which distribute regularly.

To investigate the effects of transfection of agrin gene on the recovery of muscle function after a free neurovascular muscle transfer.The electrical gene transfection was performed when the gracilis muscle of the SD rat was completed free neurovascular transfer. The experimental group was treated with pCS2+ -agrin, the group with plasmid pCS2+ as the negative control and the group with normal saline as the frank control. The muscle function, expression of neural agrin and the junctional nAChR number was measured after the operation.At 4, 5 and 10 weeks postoperatively, the pCS2+ -agrin group was significantly better than the control groups in muscle function (P0.05 ). The immunohistochemical staining showed an increasing deposition of the agrin protein near the endplate at 1 and 5 weeks after the operation, but decreasing remarkably to the level of control groups at 10 weeks postoperatively. The pCS2+ -agrin group was significantly more than the control groups in junctional nAChR number at every points of the time postoperatively.Transfection of agrin gene in the transferred muscle may increase the early recovery of muscle function.

To investigate the changes of acetylcholine receptor (AChR) distribution at the neuromuscular junction (i.e. motor end-plate) following the free neurovascular muscle transfer.AChR in the gracilis muscle of the Wistar rat following free neurovascular transfer were labeled by fluorescent alpha-bungarotoxin and radioiodinated alpha-bungarotoxin. Then confocal microscope and gamma-counting were estimated to ACHR, qualitatively and quantitatively.The junctional AChR numbers decreased to a minimum at the fourth week postoperatively, whereas the extrajunctional receptor numbers increased. From the fifth week postoperatively, the number of junctional AChR's increased. Even at 30 weeks after transfer, the morphology of the neuromuscular junction failed to return to the preoperative style. The number of acetylcholine receptors at the reinnervated neuromuscular junction also remained lower than the control.The persistent weakness following free neurovascular muscle transfer may be attributed to these qualitative and quantitative changes at the neuromuscular junction.

To study the effect and the key points in the operative procedure of the one-stage reconstruction of postburn whole auricle defect with medpor car scaffold covered with superficial temporoparietal fascia (TPF) flap.Medpor car scaffold was embedded under the superficial temporal (TFP) fascia. Razor-thin skin was grafted onto the surface of the fascia flap.Fifteen patients with postburn whole auricle defect were treated by one-stage reconstruction with Medpor ear scaffold during the last four years. It was successful in all the patients with satisfactory appearance of the reconstructed ears.Medpor possessed friendly biological compatibility. The reconstruction gave satisfactory results, and its advantages consisted of short operational time, easy manipulation, less injury to patients and good auricular contour.

Summary Objective: To establish mouse models for human diseases through N -ethyl- N -nitrosourea (ENU) mutagenesis, and to provide groundwork to clone genes and study their functions after mapping the mutant genes. Methods: 18 male D2 mice (G0) at age of 8–10 weeks old were injected intraperitoneally with ENU (100 mg/kg) once a week for three consecutive weeks. The treated male mice were mated with females of the same strain, and their offspring (G1) were used to screen for dominant and recessive mutation. After breeding the mutant F2 (D2B6 F1 intercrossing) mice, 39 microsatellites that are equally distributed on the mouse genome and are different between B6 and D2 strains were used to scan the genome. According to the log odds score (LODS) we determined whether these microsatellites were linked to the mutant genes and calculated the location of mutant genes based on their recombination ratio. Results: We screened 532 G1 mice, of which 14 exhibited mutation phenotypes. None was dominantly hereditable. Two cases of recessive inheritable scant hair mice were obtained through testing 30 G1 mice with normal phenotype and potential recessive mutant genes. All showed scant coat hair, grew slowly, and hyperkeratoses of epidermis and bollicular horn plug in histological sections. Their visceral organs were not markedly different from normal, and they were named scant hair 1 Baojin (symbol is snthr −1Bao ) and scant hair 2 Baojin (symbol is snthr −2Bao ). Through microsatellite screening we found that the LODS between snthr −1Bao and D9Mit243 was 7.73, and the linkage was determined. After analyzing the recombination ratio between snthr −1Bao and microsatellite D9Mit18 which was near snthr −1Bao based on a total number of 126 F2 mice with the scant hair phenotype, we determined that snthr −1Bao was located at chromosome 9 and was 71 cM from centromere. Using the same technique, snthr −2Bao was mapped to the same position as snthr −1Bao . Conclusion: In our research, two cases of scant hair mice provide good models for the study of dermatology, and the location of mutant genes provides a solid foundation for cloning new mice scant hair genes.