A系小鼠胚腭突细胞培养及生物学特性的研究

目的:研究A系小鼠胚愕突上皮与间充质细胞在DMEM/F-12培养基中进行原代和传代联合培养及其生物学特性。方法:采用机械分离及胰蛋白酶消化法，简便、准确、快速地获得高成活率的A系小鼠胚愕突细胞，并通过流式细胞仪、相差显微镜、光镜等观察其DNA及蛋白质合成、细胞骨架及其结构。结果:联合培养的A系小鼠胚鳄突细胞中，愕突上皮及间充质细胞均能较好地生长，光镜下观察见上皮型细胞呈多角型，单层铺路石状排列，细胞骨架为栅栏型或巢穴型;间充质细胞呈梭型，单层漩涡状排列，细胞骨架呈梭型、多平行排列的纤维。流式细胞仪检测见细胞DNA及蛋白质含量较丰富，细胞增殖活跃。结论:A系小鼠胚愕突上皮与间充质细胞联合培养模型能较好地保持体内胚愕突的上皮细胞及间充质细胞细胞成分的基本特征。     

超声造影时间-强度曲线评价裸鼠食管癌移植瘤血管生成情况的研究

目的 探讨超声造影(CEUS)时间-强度曲线(TIC)对裸鼠食管癌移植瘤血管生成情况的评价效果。方法 选取18只裸鼠并于背部皮下注射人食管癌细胞株建立食管癌移植瘤模型,分别于移植瘤移植后4周、6周、8周时,选取6只裸鼠,使用CEUS检查选择感兴趣区行TIC分析,比较移植后不同时间点食管癌移植瘤CEUS TIC定量参数和影像学特点,苏木素－伊红(HE)染色观察移植瘤病理变化,免疫组化分析移植瘤组织血管内皮生长因子(VEGF)蛋白表达和微血管密度(MVD),Pearson分析CEUS TIC定量参数与移植瘤MVD、VEGF相关性。结果 与移植后4周时比较,移植后6周、8周的峰值强度(PI)、曲线下流入面积(AWI)和曲线下流出面积(AWO)均升高(P<0.05);与移植后6周时比较,移植后8周的PI和AWO均升高(P<0.05)。移植后4周时食管癌移植瘤的常规二维灰阶超声表现为类椭圆形低回声肿块、边界清晰、内部回声不均匀,且多普勒血流显像显示裸鼠移植瘤体内和周边见少许血流信号;移植后6周时食管癌移植瘤内部显示灌注不均匀增强,移植后8周时食管癌移植瘤内部出现灌注缺损。HE染色结果显示,在移植4周时移植瘤有角化珠且细胞间有细胞间桥,随着移植后时间延长角化珠和细胞间桥逐渐减少。与移植后4周时比较,移植后6周、8周的MVD均升高(P<0.05),移植后8周的VEGF蛋白表达升高(P<0.05)。Pearson分析显示裸鼠人食管癌移植瘤VEGF蛋白表达、MVD均与PI、AWI、AWO呈正相关(P<0.001)。结论 CEUS技术TIC分析可以有效评价裸鼠人食管癌移植瘤血管生成情况。     

Shh在鼠磨牙牙胚发育晚期的基因表达

目的：观察信号分子Sonic hedgehog(Shh)在小鼠下颌第一磨牙牙胚发育晚期的基因表达，探讨其在成釉细胞、成牙本质细胞分化中的作用。方法：制备昆明小鼠磨牙牙胚发育晚期(E16．5—P1．5)标本，用原位杂交法分析Shh mRNA在牙胚中的表达和分布。结果：Shh mRNA在牙胚发育晚期的前成釉细胞和中间层细胞呈阳性表达，在成牙本质细胞层表达较弱。结论：在牙胚发育晚期，Shh可能通过自分泌途径和旁分泌途径，参与了成釉细胞和成牙本质细胞分化。     

Smo基因在小鼠胚胎颌面部的表达

目的:探讨Smoothened(Smo)基因在小鼠胚胎颌面部正常发育中的表达。方法: 应用免疫组织化学ABC法和图像分析系统研究Smo基因在小鼠胚胎11.5、13.5及17.5d颌面部的表达情况。结果:Smo基因在胚胎11.5、13.5及17.5d的颌面上下颌突均有表达,且较对照组有显著性差异(P<0.05),但在上下颌突之间表达没有显著性差异,在上皮和结缔组织之间表达基本无差异。结论: Smo基因在小鼠胚胎颌面部正常发育有明显表达,提示Smo基因可能参与颌面部生长发育。     

Virulence of a polymicrobic complex,Treponema denticola and Porphyromonas gingivalis,in a murine model

The effect of a polymierobic infection employing Treponema denticola and Porphyromonas gingivalis in the murine lesion model was used to determine the synergistic virulence of these two periodontopathic bacteria. At high doses of P. gingivalis W50, addition of T. denticola in the infection mixture had no effect on the formation and size of the spreading lesion caused by this microorganism. However, at low P. gingivalis challenge doses, T. denticola significantly enhanced the virulence of P. gingivalis compared with monoinfection of this microorganism. A potential role of the trypsin-like protease enzyme activity of P. gingivalis in this synergistic virulence was tested using P. gingivalis mutants deficient (i.e., BEI) or devoid (i.e., NG4B19) of this protease activity. These findings demonstrated that T. denticola—P. gingivalis complexes exhibit enhanced virulence in this model and that even using a polymicrobic challenge infection, the trypsin-like protease activity was important to P. gingivalis virulence expression.     

Effect of Myd88 deficiency on gene expression profiling in salivary glands of female non-obese diabetic (NOD) mice

ObjectivesSjögren's syndrome (SS) is a chronic autoimmune disease characterized by inflammatory lesions in the salivary and lacrimal glands, which are caused by distinct lymphocytic infiltrates. Female non-obese diabetic (NOD) mice spontaneously develop inflammatory lesions of the salivary glands with SS-like pathological features. Previous studies have shown that MyD88, a crucial adaptor protein that activates innate immune signaling, affects lymphocytic infiltration, but its detailed role remains unclear. In this study, we investigated the role of MyD88 through gene expression profiling in the early phase of pathogenesis in the salivary glands of female NOD mice.MethodsSubmandibular glands collected from 10-week-old female wild-type and Myd88-deficient NOD mice were used for RNA preparation, followed by microarray analysis. The microarray dataset was analyzed to identify Myd88-dependent differentially expressed genes (DEGs). Data generated were used for GO enrichment, KEGG pathway, STRING database, and INTERFEROME database analyses.ResultsMyd88 deficiency was found to affect 230 DEGs, including SS-associated genes, such as Cxcl9 and Bpifa2. Most of the DEGs were identified as being involved in immunological processes. KEGG pathway analysis indicated that the DEGs were putatively involved in autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Furthermore, the DEGs included 149 interferon (IFN)-regulated genes.ConclusionsMyD88 is involved in the expression of specific genes associated with IFN-associated immunopathological processes in the salivary glands of NOD mice. Our findings are important for understanding the role of MyD88-dependent innate immune signaling in SS manifestation.     

In vivo and in vitro study of the effects of chlorpromazine on tooth mineralization in rats and mice

The effects of chlorpromazine (CPZ) on tooth mineralization were examined using incisor dentine in adult rats and cultured tooth germs of mandibular first molars dissected from mouse embryos. CPZ (10, 50 and 250 mg/kg, s.c.) substantially inhibited dentine mineralization as evaluated by contact microradiographs. Plasma calcium and phosphorus concentrations were not decreased by CPZ (10 and 50 mg/kg). Physicochemical effects were not involved in the action of CPZ on the mineralization. In vitro experiments showed that CPZ (1 and 10 μM) inhibited mineralization and alkaline phosphatase (ALP) activity in the tooth germs. As CPZ has the properties of a calmodulin antagonist, the calmodulin antagonists W-7 and W-5 were also examined. Both inhibited mineralization and ALP activity in tooth germs; W-5 had less effect than W-7. These in vivo and in vitro findings suggest that CPZ inhibited cell-mediated mineralization in dentine without affecting the calciumdashregulating system and physicochemical mineral deposition. In addition, calmodulin could be involved in cell-mediated mineralization.     

Potential function of TGF-β isoforms in maturation-stage ameloblasts

Objectives

To investigate potential functions of transforming growth factor-beta (TGF-β) isoforms in maturation-stage ameloblasts during amelogenesis.

Differences in mouse strain influence leukocyte and immunoglobulin phenotype response to Porphyromonas gingivalis

This study examined the nature of the infiltrating cells in Porphyromonas gingivalis-induced lesions and immunoglobulins in the serum samples of BALB/c (H-2^d), C57BL6 (H-2^b), DBA/2J (H-2^d) and CBA/CaH (H-2^k) mice. Mice were immunized intraperitoneally with P. gingivalis outer membrane antigens or sham-immunized with phosphate-buffered saline followed by subcutaneous challenge with live organisms 1 week after the final immunization. The resulting skin abscesses were excised 7 days later, cryostat sections cut and an immunoperoxidase method used to detect the presence of CD4⁺ and CD8⁺ T-cell subsets, CD14⁺ macrophages and CD19⁺ B cells. Peroxidase positive neutrophils and IgG1- and IgG2a-producing plasma cells were also identified. Anti P. gingivalis IgG1 and IgG2a subclass antibodies were determined in serum obtained by cardiac puncture. Very few CD8⁺ T cells and CD19⁺ B cells were found in any of the lesions. The percentages of CD4⁺ cells, CD14⁺ cells and neutrophils were similar in lesions of immunized BALB/c and C57BL6 mice, with a trend towards a higher percentage of CD14⁺ cells in sham-immunized mice. The percentage of CD14⁺ cells was higher than that of CD4⁺ cells in immunized compared with sham-immunized DBA/2J mice. The percentages of CD4⁺ and CD14⁺ cells predominated in immunized CBA/CaH mice and CD4⁺ cells in sham-immunized CBA/CaH mice. The percentage of neutrophils in immunized CBA/CaH mice was significantly lower than that of CD14⁺ cells and CD4⁺ cells in sham-immunized mice. IgG1⁺ plasma cells were more dominant than IgG2a⁺ cells in immunized BALB/c, C57BL6 and DBA/2J mice, whereas IgG2a⁺ plasma cells were more obvious in sham-immunized mice. IgG2a⁺ plasma cells were predominant in immunized and sham-immunized CBA/CaH mice. In the serum, specific anti-P. gingivalis IgG2a antibody levels (Th1 response) were higher than IgG1 levels (Th2 response) in sham-immunized CBA/CaH and DBA/2J mice. In immunized BALB/c mice, IgG2a levels were lower than IgG1 levels, while IgG2a levels were higher in immunized C57BL6 mice. In conclusion, this study has shown differences in the proportion of infiltrating leukocytes and in the subclasses of immunoglobulin produced locally and systemically in response to P. gingivalis in different strains of mice, suggesting a degree of genetic control over the response to P. gingivalis.     

Modulation of the antibody response by Porphyromonas gingivalis and Fusobacterium nucleatum in a mouse model

Successive immunization of mice with Fusobacterium nucleatum and Porphyromonas gingivalis has been shown to modulate the specific serum IgG responses to these organisms. The aim of this study was to investigate these antibody responses further by examining the IgG subclasses induced as well as the opsonizing properties of the specific antibodies. Serum samples from BALB/c mice immunized with F. nucleatum (gp1-F), P. gingivalis (gp2-P), P. gingivalis followed by F. nucleatum (gp3-PF) F. nucleatum followed by P. gingivalis (gp4-FP) or saline alone (gp5-S) were examined for specific IgG1 (Th2) and IgG2a (Th1) antibody levels using an ELISA and the opsonizing properties measured using a neutrophil chemiluminescence assay. While IgG1 and IgG2a subclasses were induced in all immunized groups, there was a tendency towards an IgG1 response in mice immunized with P. gingivalis alone, while immunization with F. nucleatum followed by P. gingivalis induced significantly higher anti-P. gingivalis IgG2a levels than IgG1. The maximum light output due to neutrophil phagocytosis of P. gingivalis occurred at 10 min using nonopsonized bacteria. Chemiluminescence was reduced using serum-opsonized P. gingivalis and, in particular, sera from P. gingivalis-immunized mice (gp2-P), with maximum responses occurring at 40 min. In contrast, phagocytosis of immune serum-opsonized F. nucleatum demonstrated peak light output at 10 min, while that of F. nucleatum opsonized with sera from saline injected mice (gp5-S) and control nonopsonized bacteria showed peak responses at 40 min. The lowest phagocytic response occurred using gp4-FP serum-opsonized F. nucleatum. In conclusion, the results of the present study have demonstrated a systemic Th1/Th2 response in mice immunized with P. gingivalis and/or F. nucleatum with a trend towards a Th2 response in P. gingivalis-immunized mice and a significantly increased anti-P. gingivalis IgG2a (Th1) response in mice immunized with F. nucleatum prior to P. gingivalis. Further, the inhibition of neutrophil phagocytosis of immune serum-opsonized P. gingivalis was modulated by the presence of anti-F. nucleatum antibodies, while anti-P. gingivalis antibodies induced an inhibitory effect on the phagocytic response to F. nucleatum.