体外培养的人牙胚上皮细胞的成釉分化

人表皮干细胞可以作为牙齿再生中上皮源性的种子细胞,但是其成釉分化的效率低下. 本研究分离培养了人牙胚上皮细胞,利用E13.5的小鼠牙间充质与其重组,构建重组牙胚,对其成釉分化的潜能和机制进行研究. 研究结果发现,体外培养的P1代人牙胚上皮细胞成釉率高达50%. 随着传代次数的增加,成釉率明显下降. 通过对牙上皮发育分化相关基因的表达检测和分析表明,重组牙胚成牙分化能力和成釉潜能的下降与牙上皮发育相关基因的表达状态密切相关. 特别是FGF8表达水平的下调以及PITX2不同亚型在人牙胚细胞中表达量的不均衡,可能是导致人牙胚细胞成釉潜能下降并丧失的主要原因. 本研究结果为理解牙齿再生过程中上皮源性的种子细胞的成釉机制提供了新的实验数据,对进一步提高表皮干细胞在牙齿再生过程中的成釉率有指导意义.     

解离的小鼠牙胚上皮细胞在重聚发育过程仍维持牙齿发育基因的表达

为了解牙胚细胞解离重聚过程的细胞形态和分子机制,将小鼠帽状期牙胚解离细胞重聚,移植到小鼠肾囊膜下培养,组织切片,HE染色,观察再生牙齿的形态发生过程,并用原位杂交的方法进一步检测了与牙上皮发育密切相关的基因在再生牙上皮中的表达情况。结果发现,解离重聚的牙胚细胞在牙齿器官的再生过程重现了正常牙齿的形态发生过程;解离的牙上皮细胞在重聚和再生过程中保持Fgf8、Noggin和Shh等牙上皮发育基因表达。以上结果表明,即便是被解离形成分散状态的牙上皮细胞,在与牙胚间充质细胞重新聚合后,仍保持牙向分化的潜能。该结果为理解牙齿再生的机理提供新的实验数据,对利用干细胞进行牙齿再生的研究有重要的提示意义。     

大鼠胰腺发育阶段Mesothelin的表达

目的:研究Mesothenlin在大鼠胰腺发育阶段的表达和细胞定位。方法:运用RT-PCR和Western Blot技术分别检测Mesothenlin在大鼠胰腺发育阶段的mRNA和蛋白表达水平;运用免疫荧光检测不同时期Mesothenlin在胰腺的组织细胞学定位。结果:RT-PCR结果显示E18.5 Mesothelin mRNA的表达水平显著增高,至P14达到高峰,成年较低。Western Blot结果显示其蛋白表达趋势与mRNA完全相同。免疫荧光结果显示在不同发育时期Mesothenlin与胰岛β细胞和间充质细胞共表达。结论:Mesothenlin在大鼠胚胎胰岛形成及生后结构重塑中出现显著性高表达,并表达于胰岛β细胞和间充质细胞。     

Caveolin一1真核表达载体的构建及其在Hepal-6细胞中的表达

Caveolin-1在不同肿瘤中发挥作用不同,既发挥抑癌基因样作用又发挥癌基因样作用。旨在分析caveolin-l在小鼠肝癌细胞系中的表达情况及建立稳定表达外源caveolin-1的Hepal-6细胞。利用RT-PCR和Western-blot方法检测caveolin-1在小鼠肝癌H22、Hca-F和Hepal-6细胞中的表达;通过分子克隆构建小鼠caveolin-1 cDNA真核表达载体,利用脂质体转染等方法建立稳定表达外源caveolin-1的Hepal-6细胞株;通过RT-PCR、Western-blot、免疫细胞化学等方法鉴定其稳定表达细胞株。结果显示,caveolin-l在Hepal-6细胞中表达呈阴性,在H22和Hca-F中高表达;成功获得小鼠caveolin-1 cDNA真核表达载体pEGFP-N2／Cav-1,筛选并鉴定出高表达外源caveolin-1的Hepal-6稳定细胞株Cl和C4,为进一步分析caveolin-1在肝癌中所发挥的作用奠定了一定的研究基础。     

小鼠胚胎心流出道发育中胰岛因子1的表达及意义

目的观察胰岛因子1(Islet-1,ISL-1)在小鼠胚胎心流出道发育中的表达,探讨ISL-1阳性细胞在第二生心区的时空分布特点及其在心流出道发育中的作用。方法 15只胚龄8.5d~12d小鼠胚胎心连续石蜡切片,选用抗α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、抗心肌肌球蛋白重链(myosinheavy chain,MHC)和抗ISL-1抗体,进行免疫组织化学染色。结果胚龄8.5d~10d,ISL-1阳性细胞相继出现在流出道远端、心包腔背侧脏壁和体壁中胚层、原始咽腹外侧间充质及第2对鳃弓核心间充质,并逐渐分化为心肌细胞添加到流出道远端;胚龄11d,咽腹侧ISL-1阳性细胞形成独特的锥形结构,形成主肺动脉隔的雏形;胚龄12d,ISL-1阳性锥形结构与流出道远端心内膜垫融合形成主肺动脉隔,此时ISL-1阳性细胞开始分化为平滑肌细胞,参与心包腔内升主动脉、肺动脉干和主肺动脉隔的发育。结论第二生心区涵盖了原始咽腹外侧间充质、心包腔背侧壁中胚层及鳃弓核心间充质等多个区域,其中的ISL-1阳性细胞具有多向分化潜能,在心发育的不同阶段可以分化为不同类型的细胞,参与流出道的正常发育。     

Caveolin-1真核表达载体的构建及其Hepa1-6细胞中的表达

Caveolin-1在不同肿瘤中发挥作用不同,既发挥抑癌基因样作用又发挥癌基因样作用.旨在分析caveolin-1 在小鼠肝癌细胞系中的表达情况及建立稳定表达外源caveolin-1的Hepa1-6细胞.利用RT-PCR和Western-blot方法检测caveolin-1在小鼠肝癌H22、Hea-F和Hepa1-6细胞中的表达;通过分子克隆构建小鼠caveolin-1 cDNA真核表达栽体,利用脂质体转染等方法建立稳定表达外源caveolin-1的Hepa1-6细胞株;通过RT-PCR、Western-blot、免疫细胞化学等方法鉴定其稳定表达细胞株.结果显示,caveolin-1在Hepa1-6细胞中表达呈阴性,在H22和Hca-F 中高表达;成功获得小鼠caveolin-1 cDNA真核表达载体pEGFP-N2/Cav-1,筛选并鉴定出高表达外源caveolin-1的Hepa1-6稳定细胞株C1和C4,为进一步分析caveolin-1在肝癌中所发挥的作用奠定了一定的研究基础.     

小鼠骨髓间充质干细胞的培养、鉴定和Survivin的表达研究

为培养及鉴定小鼠来源骨髓间充质干细胞,并测定细胞中Survivin的表达情况,采用全骨髓培养法获取骨髓间充质干细胞,绘制生长曲线,流式细胞仪检测细胞表面标志物,行成骨、成脂检测,RT-PCR测定Survivin表达情况.结果表明培养出的细胞呈长梭状成纤维细胞样,经流式细胞仪检测细胞表面高表达CD29、CD34、CD44、SCA-1,低表达CD117;细胞曲线显示传代细胞培养1～3d生长缓慢,第4d生长加快并于第7d达到高峰;成骨诱导20d经茜素红染色呈红色结节,成脂诱导14d油红O染色显示有大量脂质沉淀;RT-PCR结果显示Survivin mRNA阳性表达.经全骨髓培养法可以培养出大量骨髓间充质干细胞,同时Survivin在小鼠骨髓间充质干细胞中正常表达,提示可能参与骨髓间充质干细胞抗凋亡过程.     

Caveolin-1在围植入期小鼠子宫内膜组织中的动态变化

目的检测caveolin-1在胚胎植入过程中小鼠子宫内膜组织中的表达,探讨其在胚胎植入过程中的作用。方法选择成年雌性昆明小白鼠42只,随机均分为7组(处于动情期的未孕组、妊娠3.5天组、妊娠4.5天组、妊娠5.5天组、妊娠6.5天组、妊娠7.5天组、妊娠9天组),采用免疫组织化学和RT-PCR方法检测子宫内膜组织中caveolin-1蛋白及mRNA水平在围植入期的变化。结果 (1)caveolin-1在胚胎植入前期(0d、3.5d)小鼠子宫内膜组织中的表达高于胚胎植入期(4.5d、5.5d、6.5d),差异有显著性(P<0.05)。(2)caveolin-1在胚胎植入后期(7.5d、9d)小鼠子宫内膜组织中的表达高于胚胎植入期(4.5d、5.5d、6.5d),差异有显著性(P<0.05)。(3)caveolin-1在胚胎植入后期小鼠子宫内膜组织中的表达略高于胚胎植入前期,但差异无显著性(P>0.05)。结论 Caveolin-1在胚胎植入前期和后期均高表达,植入期低表达。这种变化提示caveolin-1是影响胚胎植入的重要因素之一。     

人牙本质涎磷蛋白启动子驱动LacZ报告基因在人牙胚间充质细胞中表达

牙本质涎磷蛋白(DSPP)的表达是细胞向成牙本质细胞分化的标志。试图分析人DSPP启动子及构建人DSPP启动子驱动的Lac Z基因表达的报告体系,从而方便快捷检测细胞是否向成牙本质细胞分化。为了建立能表达DSPP的细胞体系,分离了人牙胚间充质细胞,并用地塞米松诱导培养液进行诱导,结果显示,该诱导培养液能有效地诱导人牙胚间充质细胞DSPP基因的表达。利用双荧光素酶报告系统对4段人DSPP基因5′上游区域(-4 000-+54、-2 500-+54、-1 447-+54和-1 027-+54)进行分析,结果显示-2 500-+54区域的启动子活性最高。5′上游区从?2 500 bp延长到?4 000 bp时,启动子活性下降;5′上游区从-2 500 bp缩短至-1 447 bp时,启动子活性下降;再次将-1 447 bp缩短至-1 027 bp时,启动子活性进一步下降。结果暗示在-4 000 bp至-2 500 bp区域存在转录抑制元件,-2 500 bp至-1 027 bp区域存在转录激活元件。用-2 500-+54启动子区域和Lac Z基因构建ph DSPP-Lac Z慢病毒报告载体,并分别在人牙胚间充质细胞和永生化人牙胚间充质细胞系ih EDMC4上检测ph DSPP-Lac Z报告载体的功能,通过X-Gal染色,结果显示在2种细胞牙向分化过程中均可检测到Lac Z基因的表达。研究构建的ph DSPP-Lac Z慢病毒报告载体可为诱导人源细胞牙向分化、牙齿发育、牙齿再生工程等研究中DSPP的表达检测提供一种更加便捷的手段。     

Glycolytic enzyme Pgk1 is strongly expressed in the developing tooth germ of the mouse lower first molar

This study examined detailed in situ expression patterns and possible functional roles of phosphoglycerate kinase 1 (Pgk1) gene in the developing tooth germ of the mouse lower first molar. The strong expression of Pgk1 mRNA was seen in the odontogenic epithelial cells and surrounding mesenchymal cells of the tooth germ from embryonic day 10.5 (E10.5) to E18.0. Western blotting analysis demonstrated that Pgk1 protein formed 84-kDa protein complex in these embryonic organs. The results of immunoprecipitation-western blotting also suggested this complex to be formed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Moreover, the immunofluorescence expression of those proteins was shown to overlap each other in the tooth germ at E15.0. A strong immunofluorescence expression of both Pgk1 and GAPDH also corresponded to the in situ expression of those mRNAs. These results suggested that Pgk1 plays some functional roles in the development of tooth germ and other embryonic organs by forming protein complex with GAPDH.     

The Drosophila Fry protein interacts with Trc and is highly mobile in vivo

Background

Protogenin (Prtg) has been identified as a gene which is highly expressed in the mouse mandible at embryonic day 10.5 (E10.5) by a cDNA subtraction method between mandibles at E10.5 and E12.0. Prtg is a new member of the deleted in colorectal carcinoma (DCC) family, which is composed of DCC, Neogenin, Punc and Nope. Although these members play an important role in the development of the embryonic central nervous system, recent research has also shed on the non-neuronal organization. However, very little is known regarding the fetal requirement of the non-neuronal organization for Prtg and how this may be associated with the tooth germ development. This study examined the functional implications of Prtg in the developing tooth germ of the mouse lower first molar.

Results

Ptrg is preferentially expressed in the early stage of organogenesis. Prtg mRNA and protein were widely expressed in the mesenchymal cells in the mandible at E10.5. The oral epithelial cells were also positive for Prtg. The expression intensity of Prtg after E12.0 was markedly reduced in the mesenchymal cells of the mandible, and was restricted to the area where the tooth bud was likely to be formed. Signals were also observed in the epithelial cells of the tooth germ. Weak signals were observed in the inner enamel epithelial cells at E16.0 and E18.0. An inhibition assay using a hemagglutinating virus of Japan-liposome containing Prtg antisense-phosphorothioated-oligodeoxynucleotide (AS-S-ODN) in cultured mandibles at E10.5 showed a significant growth inhibition in the tooth germ. The relationship between Prtg and the odontogenesis-related genes was examined in mouse E10.5 mandible, and we verified that the Bmp-4 expression had significantly been decreased in the mouse E10.5 mandible 24 hr after treatment with Prtg AS-S-ODN.

Conclusion

These results indicated that the Prtg might be related to the initial morphogenesis of the tooth germ leading to the differentiation of the inner enamel epithelial cells in the mouse lower first molar. A better understanding of the Prtg function might thus play a critical role in revealing a precious mechanism in tooth germ development.     

Nidogen-1. Expression and ultrastructural localization during the onset of mesoderm formation in the early mouse embryo.

Nidogen-1, a key component of basement membranes, is considered to function as a link between laminin and collagen Type IV networks and is expressed by mesenchymal cells during embryonic and fetal development. It is not clear which cells produce nidogen-1 in early developmental stages when no mesenchyme is present. We therefore localized nidogen-1 and its corresponding mRNA at the light and electron microscopic level in Day 7 mouse embryos during the onset of mesoderm formation by in situ hybridization, light microscopic immunostaining, and immunogold histochemistry. Nidogen-1 mRNA was found not only in the cells of the ectoderm-derived mesoderm but also in the cytoplasm of the endoderm and ectoderm, indicating that all three germ layers express it. Nidogen-1 was localized only in fully developed basement membranes of the ectoderm and was not seen in the developing endodermal basement membrane or in membranes disrupted during mesoderm formation. In contrast, laminin-1 and collagen Type IV were present in all basement membrane types at this developmental stage. The results indicate that, in the early embryo, nidogen-1 may be expressed by epithelial and mesenchymal cells, that both cell types contribute to embryonic basement membrane formation, and that nidogen-1 might serve to stabilize basement membranes in vivo. (J Histochem Cytochem 48:229-237, 2000)     

EMMPRIN (basigin/CD147) is involved in the morphogenesis of tooth germ in mouse molars

The pattern of gene expression for extracellular matrix metalloproteinase inducer (EMMPRIN) was revealed in the tooth germ of mouse mandibular molars using quantitative real-time PCR. In situ hybridization and immunohistochemical study demonstrated the characteristic distribution of EMMPRIN in the different stages of tooth germ development. To investigate the functional role played by EMMPRIN in tooth germ development, EMMPRIN siRNA interference approach was carried out in cultured mouse mandibles at embryonic day 11.0 (E11.0). The results showed that EMMPRIN siRNA-treated explants exhibited a marked growth inhibition of tooth germ compared to the control and scrambled siRNA-treated explants. Meanwhile, a significant increase in MT1-MMP mRNA expression and a reduction in MMP-2, MMP-3, MMP-9, MMP-13 and MT2-MMP mRNA expression were observed in the mouse mandibles following EMMPRIN abrogation. The current results indicate that EMMPRIN could thus be involved in the early stage of tooth germ development and morphogenesis, possibly by regulating the expression of MMP genes.     

Differential expression of type I and type III collagen genes during tooth development

Collagen gene expression during mouse molar tooth development was studied by quantitative in situ hybridization techniques. Different expression patterns of type I and type III collagen mRNAs were observed in the various mesenchymal tissues that constitute the tooth germ. High concentration for pro-alpha 1(I) and pro-alpha 2(I) collagen mRNAs were found within the osteoblasts. We found that the cellular content of type I collagen mRNAs in the odontoblasts varies throughout the tooth formation: whereas mRNA concentration for pro-alpha 1(I) collagen decreases and that of pro-alpha 2(I) increases, during postnatal development. Moreover, different amounts of pro-alpha 1(I) and pro-alpha 2(I) collagen mRNAs were observed in crown and root odontoblasts, respectively. Type III collagen mRNAs were detected in most of the mesenchymal cells, codistributed with type I collagen mRNAs, except in odontoblasts and osteoblasts. Finally, this study reports differential accumulation of collagen mRNAs during mouse tooth development and points out that type I collagen gene expression is regulated by distinct mechanisms during odontoblast differentiation process. These results support the independent expression of the collagen genes under developmental tissue-specific control.     

Functional implication of nucleolin in the mouse first molar development

We examined the functional implication of nucleolin in the mouse first molar development. Both the nucleolin mRNA and protein expressions were demonstrated in the odontogenic epithelial cells in the early stage and in the inner enamel epithelial layer in the late stage. The expression pattern of nucleolin corresponded to the proliferating cells in the tooth germ, thus showing that nucleolin could possibly be related to cell proliferation. No in situ signal of nucleolin was found in the primary enamel knot (PEK). Furthermore, nucleolin protein was demonstrated in the PEK by immunohistochemistry. The existence of nucleolin protein in the PEK may possibly be related to the apoptosis in the PEK cells. An inhibition assay using the hemagglutinating virus of Japan-liposome containing nucleolin antisense phosphorothioated oligonucleotide (AS S-ODN) in cultured mouse mandibles at embryonic day (E) 11.0 showed a marked growth inhibition of tooth germ. Moreover, no developmental arrest was found in the cultured tooth germ at E15.0 treated with nucleolin AS S-ODN. Real time PCR was performed to examine the mRNA expression of nucleolin-related genes, and a significant reduction in the midkine mRNA expression was thus observed in the mouse mandible after being treated with nucleolin AS S-ODN. This inhibition assay indicated that nucleolin could thus be involved in the early stage of tooth germ initiation and morphogenesis, possibly by regulating the midkine expression.     

Developmental regulation and ultrastructure of glycogen deposits during murine tooth morphogenesis

The distribution and ultrastructure of glycogen deposits were investigated in the murine tooth germ by histochemical periodic acid-Schiff (PAS) staining and transmission electron microscopy. Lower and upper first molars were examined in mouse embryos at embryonic days 11.5–17 (E11.5–E17) and in 2-day-old postnatal (P2) mice. The oral and dental epithelia and the mesenchymal cells were generally PAS-positive during tooth morphogenesis. PAS-negative cells were present at E13 in the distal tip of the tooth bud epithelium and in the contacting mesenchyme, and this complete lack of PAS reactivity continued in the dental papilla mesenchyme and inner enamel epithelium during the cap and bell stages. The lack of glycogen deposits in the interacting epithelium and mesenchyme during early morphogenesis may be associated with their demonstrated high signaling activities. Mesenchymal cells in the dental follicle consistently possessed small clusters or large pools of glycogen, which disappeared by P2. Since an intense PAS reaction was seen in mesenchymal cells at future bone sites, the glycogen in the dental follicle cells may be associated with their development into hard-tissue-forming cells. Ultrastructural observation of the enamel organ cells from the cap to early bell stages (E14–E15) revealed the occurrence of glycogen pools, which were associated with the Golgi apparatus and with vesicles having amorphous contents. Glycogen particles were also occasionally present inside vesicles or in the extracellular matrix. These may be associated with the exocytosis of glycosaminoglycan components into extracellular spaces and the formation of the stellate reticulum. Received: 9 November 1998 / Accepted: 17 January 1999