Objective To compare the influence of different coating materials and cultural conditions on the purification and growth of human epidermal melanocytes. Methods The full-thick foreskin, epidermis and cell suspension obtained from human foreskin were cultured in the plates, which were precoated with matrigel or laminin respectively. When having reached 80%-90%confluence, the cells were treated with 0.05%trypsin-EDTA for 4 minutes and resuspended in M254 medium, which were supplemented with G418 and 5-BrdU, respectively. The purity of melanocytes was observed by an immunofluorescence staining with melanocyte markers. Results During the primary culture, the cell suspension generated more cells at faster speed compared with those of skin explants and epidermal specimen. Moreover, the epidermis released cells earlier and proliferated quickly over skin explants. The melanocytes in the plates coated with laminin other than with matrigel displayed faster and better growth. The unwanted keratinocytes and fibroblasts were removed by using differentiation trypsinition combined with supplement of G418 or 5-BrdU. Conclusion Using a plate coated with laminin to culture cell suspension from human foreskin, and via a differentiation trypsinization combined with supplement of small doses of G418 to subculture the cells, is advantageous to the melanocyte purification, without affecting their growth.

Objective To investigate the location,fine structure of melanocytes in human fetal scalp hair follicles.Methods The scalp with hair follicles was obtained from a dead fetus of 6 months of age,and divided into two parts.One part was embedded in paraffin,tissue sections were prepared with a width of 7 μm and stained with NKI/beteb,monoclonal antibodies to HMB-45,tyrosinase and tyrosinase-related protein 1(TRP1),respectively.The other part with hair follicles was treated with collagenase type Ⅱ 0.1 g/L and trypsin,then,cell suspension was collected and cultured.After 14-day culture,follicle melanocyte cells (FMC)were separated from keratinocytes by differential trypsinization,and fibroblasts were removed with geneticin.Following three times of pure passage,FMC were seeded and fixed on mica for scanning electron microscopy(SEM)and atomic force microscope(AFM)scanning.Results Histopathological examination showed that NKI/beteb positive cells located at the outer root sheath of human hair follicles,and these cells stained negatively for HMB-45,tyrosinase and TRP1 antibodies.However,in the hair bulb,lots of cells expressed HMB-45,tyrosinase and TRP1 antigens.After fibroblasts and keratinocytes were removed,two kinds of melanocytes remained in the culture:one was small in number and showed abundant melanin,which was lost after subsequent passage;the othgr was large in number and had no melanin initially,but proliferated very rapidly.After three passages,almost all the melanocytes were positive for NKI/beteb.As SEM and AFM showed,most cultured melanocytes appeared fusiform with two(rarely three)dendrites,and the cell body was round or oval with a few melanosomes scattered in but no clear secondary branches on the dendrites.Conclusions The melanocytes in outer root sheath of hair follicles from the fetal scalp are presumed as melanocyte stem cells or their progenies.In vitro,these cells proliferate very rapidly during early phases,but the morphology and function of them still remain immature,which is unfavorable for melanosome transport.

Objective To investigate the ultrastructural characteristics of amelanotic melanocytes (AMMCs). Methods Individual hair follicles from normal human scalp were digested with collagenase type V, then washed in phosphate buffer saline. Hair-follicle cell suspensions were prepared by trypsin and cultured in a medium suitable for melanocyte growth. The keratinocytes were removed by differential trypsinization. Geneticin (100?g/mL) was used to eliminate contaminating fibroblasts. After 3 passages the cells were trypsinized, washed in phosphate buffer saline, and finally processed for transmission electron microscopy. Results Under transmission electron microscope, the cultured cells were round or oval-shaped with a single large nucleus and double-layered karyotheca. Abundant euchromosome but sparse heterochromosome was observed within the nucleus. There were various organelles in the cytoplasm, including mitochondria, rough endoplasmic reticulum (RER), ribosomes and abundant melanosomes of nearly uniform size. The electronic density granules distributed in a concentric pattern in most of the melanosomes. Colgi complexes were inconspicuous in the cells. Conclusions Compared to epidermal melanocytes, AMMCs from human hair follicles have different ultrastructural characteristics which implies their functional immaturity. AMMCs may serve as the depot for mature melanocytes.

Objective To investigate the character of aquaporin-1(AQP-1)expression in adrenal gland in diabetes mellitus and evaluate adrenal gland damage and function alterations by DWI with multiple b values. Methods Twenty male Sprague-Dawley rats were randomly selected by computer and randomized into 2 groups:untreated controls(n=10)and diabetes(DM)(n=10). Rats in diabetes group were fed with high-sucrose and high-fat diet, controls were fed with common diet. After fed with high-sucrose and high-fat diet for 4 weeks, rats in diabetes group were injected with streptozotocin(STZ). Forty days after diabetes induction with streptozotocin(STZ), MR imaging was performed in a 7.0 T scanner. Venous blood from the tails was collected before MRI scan to measure blood glucose, blood glucose more than 16.7 mmol/L wasregarded as diabetic status. All the rats underwent DWI with 18 b values(0 to 4500 s/mm2). Maps of pure diffusion coefficients(D), pseudo-diffusion coefficients(D*)and ultra-high ADC(ADCuh)were acquired. Rats were sacrificed after MRI scan for adrenal gland histopathology, AQP-1 immunohistochemistry analysis and AQP-1 optical density(OD)measurements. Student t test was used to compare the difference of D*, D, ADCuh and OD of AQP-1 between two groups. Results Eight diabetic animals developed hyperglycemia(two rats died during the modeling process). MRI scan was performed in all of the 18 rats. Signal intensity of D*map, D map and ADCuh map decreased gradually. ADCuh increased significantly in DM animals(0.24 ± 0.06) × 10-3mm2/s compared with control animals(0.18 ± 0.07) × 10-3 mm2/s(P<0.05), whereas there was no significant difference found between the two groups in their respective D*and D values(P>0.05). There was a noticeable increase in the AQP-1 labeling in the adrenal cell membrane and cytoplasm in DM animals compared with control animals. DM rats showed an increased OD value of AQP-1 in adrenal gland compared with the control animals(P<0.05). Conclusions We found significantly higher AQP-1 expression in adrenal gland in DM animals compared with controls. Ultra-high b-Values DWI may work as a useful way for noninvasive evaluation the change of adrenal function in DM.

Objective To study the effects of learning environment on learning memory and microvessel density in hippocampus of rats after cerebral infarction. Methods 80 Sprague-Dawley rats were coagulated right middle cerebral arteries electrically, and randomly divided into learning envionment (LE) and standard environment (SE) groups. They were tested with Morris Water Maze 7 d and 28 d after operation. The microvessel density in hippocampus was measured with CD34 immunohistochemstry 1, 3, 7, 14 and 28 days after operation. Results The achievement of Morris Water Maze was significantly better in the LE group than in the SE group (P<0.001), while the microvessel density in the hippocampus was more since 7 days after operation (P<0.05). Conclusion The learning environment can promote the angiogenesis and improve the learning and memory function recovery.

Purpose To detect the expression of autophagic genes Beclin 1 and MAP1LC3 in cutaneous malignant melanoma and to ex-plore the relationship between autophagia and malignant melanoma. Methods 85 cases of speicmens including normal skin tissue, in-tradermal nevi, radial growth phase melanomas, vertical growth phase melanomas, and metastatic melanoma were collected, and the protein expression of Beclin 1 and MAP1LC3 were evaluated by immunohistochemistry of SP methods. Results The Beclin 1 and MAP1LC3 expression were pretended to be 100% in normal skin tissue, and they were declined to 85% and 95% in intradermal nevi, 58% and 50% in radial growth phase melanomas, 49. 5% and 44. 4% in vertical growth phase melanomas, both of 17% in melanoma metastases (P<0. 05). Conclusion Beclin 1 and MAP1LC3 autophagic gene expression were significantly decreased with tumor pro-gression, as well as was correlated with conventional histopathologic prognostic factors.

Objective To investigate the characteristics of diet in patients with gestational diabetes mellitus(GDM) and analyze their dietary nutrition.Methods Clinical data of 978 cases of pregnant women in our hospital were collected and divided into GDM group(458 cases) and the healthy control group (520 cases).Dietary questionnaire was designed and diet characteristics were analyzed retrospectively.Daily calories and nutrients intake were calculated by software calculation.Results There were significant differences on the dietary pattern between the two groups:daily coarse grains:(35.12 ±4.87) g vs (46.35 ±5.23) g;seafood:(22.10 ± 8.45) g vs (39.34 ± 12.45) g;grilled:(6.72 ± 1.76) g vs (3.45 ±0.34) g;fried:(25.34 ± 7.84)g vs (10.56 ±4.76) g;sausage:(5.38 ± 1.81) g vs (3.45 ± 0.45) g;dessert type products intake:(50.41 ±20.76) g vs (35.86 ± 10.45) g (t =2.582,10.346,15.452,20.239,18.195,8.472,respectively,P ＜0.05).There was only significant difference between the two groups on calcium supplements in daily intake of nutritional supplements(GDM group 43.89％,control group 72.69％,x2 =83.660,P ＜ 0.001),and there was no significant difference on the rests(all P ＞ 0.05).Vitamin A,B1,B2,C and calcium,iron,zinc,selenium daily intake in GDM patients were less than those in the control group,but there was significant difference only in calcium compared with the control group (GDM group (785.2 ± 194.5) mg,control group (945.5 ± 245.2)mg,t =2.795,P =0.005).Multivariate logistic regression found that excessive intake of fried class (OR =3.357,95％ CI =2.012-11.340),barbecue (OR =2.652,95％ CI =1.834-8.578),sausage type products (OR =2.518,95％ CI =1.743-7.421) etc.were dietary factors of GDM (all P ＜ 0.05).Incidence of GDM was significantly increased if taking more.While the increased intake of coarse grains (OR =0.632 95％ CI =0.134-1.489),seafood class(OR =0.408,95％ CI =0.105-1.189) can reduce the occurrence of GDM(all P ＜ 0.05).Conclusion Many unscientific ways of eating involved in pregnant women with GDM.Dessert,sausage,grilled,fried products intake increased the risk factors for GDM.Coarse grains,seafood intake were the protective factors of GDM.Timely adjustment of maternal diet and adequate calcium supplementation played an important role in the prevention of GDM.

Objective To measure the expressions of zinc finger protein A20 and nuclear factor (NF)-κB in cutaneous squamous cell carcinoma (CSCC) tissue and their correlation with human papillomavirus (HPV) infection.Methods Tissue specimens were obtained from the lesions of 43 patients with CSCC and normal skin of 21 healthy human controls.Gene chips were used to detect the DNA of 21 HPV subtypes in these specimens.The expression levels of zinc finger protein A20 and NF-κB were determined by immunohistochemistry.The correlation between clinicopathological features of CSCC and HPV infection was estimated by Spearman rank correlation analysis.Results No significant difference was found in the expression level of either zinc finger protein A20 or NF-κB among patients of different age groups,gender,and with CSCC of different primary sites or histological grade (all P ＞ 0.05).However,there was a significant difference in the expression levels of zinc finger protein A20 and NF-κB between 39 patients with histological grade Ⅰ or Ⅱ and 4 patients with grade Ⅲ (25.85 ± 3.84 vs.48.34± 7.69 for zinc finger protein A20,46.64 ± 8.93 vs.57.34 ± 10.02 for NF-κB,both P ＜ 0.05),and between 3 patients with nodular metastasis and 40 patients without (35.34 ± 6.02 vs.26.51 ± 4.09 for zinc finger protein A20,57.53 ± 13.32 vs.45.45 ± 9.64 for NF-κB,P ＜ 0.05 or 0.01).The expression rate was 2/8,5/13 and 1/3 for zinc finger protein A20 (P ＞ 0.05),3/8,10/13 and 2/3 for NF-κB (P ＞ 0.05),in CSCC tissue specimens infected with low-risk HPV subtypes,high-risk subtypes,concurrent low-and high-risk HPV subtypes respectively.In HPV-infected CSCC tissue,a significantly positive correlation was found between the staining scores for zinc finger protein A20 and for NF-κB p65(r =3.14,P ＜ 0.05).Conclusion The high expressions of NF-κB and zinc finger protein A20 may play an important role in the oncogenesis of CSCC induced by HPV infection.

Objective: The occurrence of intramyocardial hemorrhage (IMH) and microvascular obstruction (MVO) in myocardial infarction (MI), known as severe ischemia/reperfusion injury (IRI), has been associated with adverse remodeling. APT102, a soluble human recombinant ecto-nucleoside triphosphate diphosphohydrolase-1, can hydrolyze extracellular nucleotides to attenuate their prothrombotic and proinflammatory effects. The purpose of this study was to temporally evaluate the therapeutic effect of APT102 on IRI in rats and to elucidate the evolution of IRI in the acute stage using cardiovascular magnetic resonance imaging (CMRI). Materials and Methods: Fifty-four rats with MI, induced by ligation of the origin of the left anterior descending coronary artery for 60 minutes, were randomly divided into the APT102 (n = 27) or control (n = 27) group. Intravenous infusion of APT102 (0.3 mg/kg) or placebo was administered 15 minutes before reperfusion, and then 24 hours, 48 hours, 72 hours, and on day 4 after reperfusion. CMRI was performed at 24 hours, 48 hours, 72 hours, and on day 5 post-reperfusion using a 7T system and the hearts were collected for histopathological examination. Cardiac function was quantified using cine imaging and IMH/edema using T2 mapping, and infarct/MVO using late gadolinium enhancement. Results: The extent of infarction (p < 0.001), edema (p < 0.001), IMH (p = 0.013), and MVO (p = 0.049) was less severe in the APT102 group than in the control group. IMH size at 48 hours was significantly greater than that at 24 hours, 72 hours, and 5 days after reperfusion (all p < 0.001). The left ventricular ejection fraction (LVEF) was significantly greater in the APT102 group than in the control group (p = 0.006). There was a negative correlation between LVEF and IMH (r = -0.294, p = 0.010) and a positive correlation between IMH and MVO (r = 0.392, p < 0.001). Conclusion APT102 can significantly alleviate damage to the ischemic myocardium and microvasculature. IMH size peaked at 48 hours post reperfusion and IMH is a downstream consequence of MVO. IMH may be a potential therapeutic target to prevent adverse remodeling in MI.

OBJECTIVE: Whether blood-brain barrier (BBB) disruption induced by chronic spontaneous hypertension is associated with beta-amyloid (Aβ) accumulation in the brain remains poorly understood. The purpose of this study was to investigate the relationship between BBB disruption and Aβ influx and accumulation in the brain of aged rats with chronic spontaneous hypertension. MATERIALS AND METHODS: Five aged spontaneously hypertensive rats (SHRs) and five age-matched normotensive Wistar-Kyoto (WKY) rats were studied. The volume transfer constant (Ktrans) obtained from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to evaluate BBB permeability in the hippocampus and cortex in vivo. The BBB tight junctions, immunoglobulin G (IgG), Aβ, and amyloid precursor protein (APP) in the hippocampus and cortex were examined with immunohistochemistry. RESULTS: As compared with WKY rats, the Ktrans values in the hippocampus and cortex of the SHRs increased remarkably (0.316 ± 0.027 min−1 vs. 0.084 ± 0.017 min−1, p < 0.001 for hippocampus; 0.302 ± 0.072 min−1 vs. 0.052 ± 0.047 min−1, p < 0.001 for cortex). Dramatic occludin and zonula occludens-1 losses were detected in the hippocampus and cortex of SHRs, and obvious IgG exudation was found there. Dramatic Aβ accumulation was found and limited to the area surrounding the BBB, without extension to other parenchyma regions in the hippocampus and cortex of aged SHRs. Alternatively, differences in APP expression in the hippocampus and cortex were not significant. CONCLUSION: Blood-brain barrier disruption is associated with Aβ influx and accumulation in the brain of aged rats with chronic spontaneous hypertension. DCE-MRI can be used as an effective method to investigated BBB damage.
Alzheimer Disease ; Amyloid ; Animals ; Blood-Brain Barrier* ; Brain* ; Hippocampus ; Hypertension* ; Immunoglobulin G ; Immunohistochemistry ; Magnetic Resonance Imaging* ; Methods ; Occludin ; Permeability ; Rats* ; Rats, Inbred SHR ; Rats, Inbred WKY ; Tight Junctions