牙本质非胶原蛋白诱导颅神经嵴干细胞向成牙本质细胞表型分化

目的探讨颅神经嵴干细胞(CNCSC)用于牙再生研究的可行性,观察牙本质基质非胶原蛋白(DMNCP)对CNCSC的成牙本质样细胞表型分化的影响。方法小鼠神经管组织块无血清条件培养法获取颅神经嵴干细胞,以DMNCP体外诱导CNCSC的分化,形态学、免疫细胞化学、茜素红特染等方法,分别从Ⅰ型胶原表达、牙本质涎磷蛋白(DSPP)表达、碱性磷酸酶(ALP)活性及钙结节形成情况,鉴定CNCSC的成牙本质样细胞表型。结果诱导后的CNCSC呈现I型胶原、DSPP阳性表达,ALP活性增高,钙结节形成,具有成牙本质细胞表型特征。结论DMNCP具有诱导CNCSC向成牙本质细胞表型分化的作用,利用CNCSC开展牙再生研究是可行的。     

Streptococcus mutans binding to solid phase dextran mediated by the glucan-binding protein C

Streptococcus mutans GbpC is a wall-anchored surface protein which is involved in dextran-dependent aggregation. The GbpC phenotype is observed only in cells grown under stress conditions. In order to detect the GbpC protein of S. mutans, we isolated the wall fraction following digestion of the cell wall of this organism by N-acetylmuramidase, and detected the GbpC protein from S. mutans cells by western analysis with anti-GbpC serum. Interestingly, S. mutans cells exhibiting the negative dextran(alpha-1,6 glucan)-dependent aggregation (ddag) phenotype expressed the protein and could bind to immobilized dextran.     

A comparative study of sialic acid-rich proteins in rat bone and dentin

Four sialic acid-rich (SA-rich) proteins found in bone and dentin, osteopontin (OPN), bone sialoprotein (BSP), bone acidic glycoprotein-75 (BAG-75), and dentin matrix protein 1 (DMP1), share some common features. We used SDS-PAGE and Western immunoblots to analyze and compare SA-rich proteins in bone and dentin extracts from rats with a single chromatographic procedure. OPN was detected in dentin extracts, with a relative level less than one-seventieth of that in bone. Both bone and dentin BSP demonstrated an extremely broad distribution pattern, probably due to a high degree of heterogeneity in post-translational modifications. BAG-75 in both bone and dentin was detected as an 83 kDa band, dramatically distinct from that of DMPI. Using a polyclonal antibody raised against a purified bone 57 kDa protein (a portion of DMPI), we detected 150 kDa protein bands in bone fraction; the same bands were recognized by antirecombinant rat DMPI antibody. Bands from dentin migrating at about 150 kDa in earlier fractions and progressing to 200 kDa in later fractions showed a clear immunoreactivity to the anti-57 kDa antibody. We conclude that the majority of DMPI in rat bone is processed into fragments, whereas that in dentin remains intact.     

人牙周膜细胞中内源性骨形成蛋白的流式细胞仪分析

目的：定量检测和分析人牙周膜细胞（ＰＤＬＣ）表达内源性骨形成蛋白（ＢＭＰ）的情况。方法：应用ＢＭＰ单克隆抗体，通过流式细胞仪和免疫组化ＡＢＣ的方法双重判定。结果：在离体培养的人ＰＤＬＣ中有一半左右的细胞能表达ＢＭＰ。结论：牙周膜细胞具有一定的合成和分泌ＢＭＰ的能力；可以进一步认为人的牙周膜细胞是具有成骨潜能的，在牙周组织再生中有积极作用。     

Immunochemical detection of CD14 on human gingival fibroblasts in vitro

The activation of monocytes and macrophages induced by lipopolysaccharide has been shown to contribute to the binding of lipopolysaccharide and lipopolysaccharide-binding protein complex to the cell surface CD14 molecule. To clarify the mechanism of the lipopolysaccharide-induced modulation of the function of gingival fibroblasts, we investigated the effect of anti-CD14 on interleukin 6 (IL-6) production on human gingival fibroblasts in vitro. Immunochemical staining revealed weak positivity for CD14 on fibroblasts from healthy gingiva, while strong positivity for CD14 was found on fibroblasts from inflamed gingiva. Western blot profiles of the fibroblasts and monocytes showed a CD14-positive reaction at 55 kDa. Fluorescein isothiocyanate-conjugated Escherichia coli lipopolysaccharide bound to fibroblasts more strongly in the presence of 10% fetal bovine serum than without serum. This binding, as well as IL-6 production, was blocked by anti-CD14 monoclonal antibody. The results showed that CD14 was present on human gingival fibroblasts, which suggests that lipopolysaccharide modulation of gingival fibroblast function depends on CD14.     

Aggregatibacter actinomycetemcomitans leukotoxin is post-translationally modified by addition of either saturated or hydroxylated fatty acyl chains

Aggregatibacter actinomycetemcomitans, a common inhabitant of the human upper aerodigestive tract, produces a repeat in toxin (RTX), leukotoxin (LtxA). The LtxA is transcribed as a 114-kDa inactive protoxin with activation being achieved by attachment of short chain fatty acyl groups to internal lysine residues. Methyl esters of LtxA that were isolated from A. actinomycetemcomitans strains JP2 and HK1651 and subjected to gas chromatography/mass spectrometry contained palmitoyl (C16:0, 27-29%) and palmitolyl (C16:1 cis Δ9, 43-44%) fatty acyl groups with smaller quantities of myristic (C14:0, 14%) and stearic (C18:0, 12-14%) fatty acids. Liquid chromatography/mass spectrometry of tryptic peptides from acylated and unacylated recombinant LtxA confirmed that Lys(562) and Lys(687) are the sites of acyl group attachment. During analysis of recombinant LtxA peptides, we observed peptide spectra that were not observed as part of the RTX acylation schemes of either Escherichia coliα-hemolysin or Bordetella pertussis cyclolysin. Mass calculations of these spectra suggested that LtxA was also modified by the addition of monohydroxylated forms of C14 and C16 acyl groups. Multiple reaction monitoring mass spectrometry identified hydroxymyristic and hydroxypalmitic acids in wild-type LtxA methyl esters. Single or tandem replacement of Lys(562) and Lys(687) with Arg blocks acylation, resulting in a >75% decrease in cytotoxicity when compared with wild-type toxin, suggesting that these post-translational modifications are playing a critical role in LtxA-mediated target cell cytotoxicity.     

几丁质/rhBMP2/胶原复合物修复骨缺损的实验研究

目的：研究几丁质作为骨缺损充填材料和rhBMP2载体的可行性。方法：制备多孔几丁质及几丁质/rhBMP2/胶原复合物，进行了几丁质，几丁质/rhBMP2/胶原复合物修复兔颅骨缺损的实验研究。通过X线片，组织学检查评价其骨缺损修复能力。结果：实验结果表明几丁质具有一定的骨引导活性，与rhBMP2复合后，成为既具有骨引导活性，又具有骨诱导活性的复合植骨材料，具有较强的骨缺损修复能力。结论：几丁质适于作为骨替代材料及BMP的载体，但在材料的强度和表面活性上还需要改进。     

小鼠Notch1胞外段高抗原区编码基因的克隆与表达

目的 :克隆小鼠Notch1胞外段高抗原区编码基因 ,并进行原核表达及蛋白纯化 ,为制备mNotch1的单克隆抗体做准备。方法 :计算机分析小鼠Notch1全长 ,PCR扩增其胞外段高抗原区编码基因 ,克隆入载体T -vector进行序列测定 ,然后将测定正确的片段连入GST融合表达载体pGEX - 4T - 1,得到pG -NEF ,转化宿主菌DH5α ,经IPTG诱导 ,SDS -PAGE分析 ,以及新生条带表达形式分析以后 ,用GST亲和层析柱纯化。结果 :小鼠Notch1分子胞外段近膜处约 170个氨基酸区域抗原性较高 ,PCR扩增得到大小约 5 0 0bp的特异性片段 ,序列测定结果与文献报导的完全一致 ;原核表达产物大小约Mr 43× 10 3 ,以包涵体形式存在 ,约占总蛋白的2 5 % ,纯化后目的蛋白含量 >95 %。结论 :成功地进行了小鼠Notch1胞外段高抗原区编码基因的基因克隆、原核表达及蛋白纯化。     

Ten-year results following treatment of intra-bony defects with enamel matrix proteins and guided tissue regeneration

Background: Surgery utilizing an enamel matrix protein derivative (EMD) or guided tissue regeneration (GTR) has been shown to promote periodontal regeneration.
Aim: To evaluate the 10-year results following treatment with EMD, GTR, EMD+GTR, and open flap debridement (OFD).
Material and Methods: Thirty-eight patients out of an initial group of 56 participants were treated with one of the four modalities. Results were evaluated before surgery, at 1 year, and at 10 years. Primary outcome variable was CAL change.
Results: Treatment with EMD yielded a mean CAL gain of 3.4±1.0 mm ( p <0.001) and 2.9±1.4 mm ( p <0.001) at 1 and 10 years, respectively. GTR resulted in a mean CAL gain of 3.2±1.4 ( p <0.001) at 1 year and 2.8±1.2 mm ( p <0.001) at 10 years. Mean CAL gain in the EMD+GTR group was of 3.3±1.1 mm ( p <0.001) and 2.9±1.2 mm ( p <0.001) at 1 and 10 years, respectively. Treatment with OFD demonstrated a mean CAL gain of 2.0±1.2 mm ( p <0.01) at 1 year and 1.8±1.1 mm ( p <0.01) at 10 years. Compared with OFD, the three regenerative treatments resulted in statistically significant ( p <0.05) higher CAL gain, at both 1 and 10 years. The CAL change between 1 and 10 years did not present statistically significant differences in any of the four groups.
Conclusion: The present results indicate that the clinical outcomes obtained with all four approaches can be maintained over a period of 10 years.     

变形链球菌母子传播与其对牙面初始粘附关系的研究

目的:探索变形链球菌母子传播与其对牙面初始粘附能力的关系。方法:首先以AP-PCR检测Mutans streptococci(MS)在20对3~4岁儿童和母亲之间的传播,并从母亲口腔中筛选出传播株和非传播株;再采用同位素标记法,检测传播株和非传播株对SHA的粘附差异,同时对其表面蛋白粘附功能区spap-a和spap-pv分别用限制性内切酶haeⅢ和AluⅠ进行RFLP分析。结果:传播株较非传播株具有较弱的粘附力(P<0.01),spap-a和spap-pv经酶切后分别呈现的3种和2种基因型在传播和非传播人群的MS中分布不同(P<0.01)。结论:初始粘附能力弱的菌株被传播的可能性较大,不同MS株传播能力的差异可能与其表面蛋白A区,PV区的基因多态性有关。