细胞外基质与肾小球硬化

细胞外基质(ECM)位于细胞的周围,主要由胶原蛋白、糖蛋白及蛋白多糖三大类物质聚合组成,每类物质又包括多种成分。不同器官组织的ECM中各种成分的比例及其排列不尽相同,从而适应不同的功能。ECM对细胞的生理功能起着非常重要的作用,它不仅是细胞的粘附及支持所必需     

细胞外基质与下肢静脉曲张关系研究

静脉壁的细胞外基质维持了静脉壁正常的结构和功能。曲张静脉壁的细胞外基质中各种成分的分布和含量发生了不同程度的改变，且对静脉曲张的形成具有重要的意义。本文就曲张静脉壁的细胞外基质的改变作一综述。     

甲状旁腺素对人肾小管上皮细胞细胞外基质合成与降解的影响

目的 研究甲状旁腺素（PTH）对人肾小管上皮细胞（HK-2）合成分泌Ⅲ型胶原、纤连蛋白以及对纤溶酶原激活物抑制物1（PAI-1）、基质金属蛋白酶1（MMP-1）、金属蛋白酶1组织抑制剂（TIMP-1）基因表达的影响,以探讨PTH在肾间质纤维化发病机制中的作用。 方法 HK-2细胞培养于含5％ FBS的DMEM-F12培养液中,加入终浓度为0、10-12、10-11、10-10、10-9、10-8 mol/L PTH的培养液刺激HK-2细胞48 h,或10-8 mol/L PTH刺激HK-2细胞不同时间（0、12、24、48、72 h）。应用半定量RT-PCR法检测细胞中Ⅲ型胶原、纤连蛋白、PAI-1、MMP-1、TIMP-1基因的表达;Western印迹法检测HK-2细胞中Ⅲ型胶原的蛋白表达;ELISA法检测细胞培养上清液中纤连蛋白的含量。 结果 PTH 呈剂量和时间依赖性促进HK-2细胞中Ⅲ型胶原、纤连蛋白、PAI-1、TIMP-1 mRNA的表达,抑制MMP-1 mRNA的表达,MMP-1/TIMP-1 比值降低。PTH呈剂量和时间依赖性增加HK-2细胞中Ⅲ型胶原的蛋白表达及细胞培养上清液中纤连蛋白的含量。 结论 PTH通过促进HK-2细胞中PAI-1、TIMP-1的基因表达,抑制MMP-1的基因表达,导致细胞外基质合成增加而降解减少。     

肝素对肾小球系膜细胞和细胞外基质的影响

肝素对肾小球系膜细胞和细胞外基质的影响王宗谦周希静近年来，单纯肝素或肝素并用激素治疗肾病综合征获良好疗效。Ｐｕｒｋｅｒｓｏｎ等报道，Ｎ－Ｄｅｓｕｌｆａｔｅｄ／ＡｃｅｔｙｌａｔｅｄＨｅｐａｒｉｎ可能延缓肾衰进程，但机理尚未明。我们通过观察肝素对体外培养...     

人转化生长因子β1重组腺病毒转染传代髓核细胞对细胞外基质合成的影响

目的探讨人转化生长因子融（transforming growthfactorβ，TGF-β1）对传代羊髓核细胞的细胞外基质（extracellular matrix，ECM）和DNA合成调节因子的作用。方法取1岁龄成年山羊腰椎间盘，体外分离培养羊髓核细胞，传至第3代后以携人TGF-β1（humanTGF-β1，hTGF-β1）或lacZ基因的复制缺陷型腺病毒（Ad／hTGF—β1及Ad／lacZ）感染，分别为实验组和阴性对照组；未加病毒液的细胞为空白对照组；原代髓核细胞为原代组。然后继续单层或藻酸钙凝胶三维（3-D）培养10d。对两种系统培养的细胞分别行DNA荧光定量、Westernblot分析和蛋白多糖（glycosaminoglycan，GAG）定量检测。结果DNA荧光定量显示，单层培养时实验组细胞的DNA合成显著高于两对照组（P〈0．05），藻酸钙凝胶3-D培养各组间比较差异无统计学意义（P〉0．05）。Western blot检测hTGF—β1、Ⅱ型胶原、Ⅰ型胶原和Aggrecan的表达显示，两种培养系统中，实验组hTGF—β1、Ⅱ型胶原和Aggrecan的表达均显著高于两对照组（P〈0．05），Ⅰ型胶原的表达显著低于两对照组（P〈0．05），实验组Ⅱ型胶原／Ⅰ型胶原比值较两对照组显著增高（P〈0．05）。GAG定量结果显示，两种培养系统中实验组细胞的GAG合成均显著高于两对照组（P〈0．05）。结论hTGF-β1在很大程度上可起到维持髓核细胞表型，并在细胞传代后仍发挥表型的调节作用。通过基因工程方法使髓核细胞表达hTGF—β1，有望遏制、甚至逆转椎间盘退变；而以Ad／hTGF—β1感染过的髓核细胞，在藻酸钙凝胶3-D培养系统中培养则表现出原始表型。     

降糖通脉片对人肾小球系膜细胞及细胞外基质影响的实验研究

目的：探讨降糖通脉片对人肾小球系膜细胞（MC）及其细胞外基质（ECM）的影响。方法;采用血清药理学方法和体外人肾小球系膜细胞培养技术,观察具有清热化瘀,滋阴补气作用的中药降糖通脉片对MC增殖及其ECM成分的影响。结果：降糖通脉片具有抑制MC增殖及ECM成分中纤维连接蛋白（FN）,层粘连蛋白（LN）和IV型胶原（ColIV)的作用,该抑制作用具有一定的量效关系。结论：MC是降糖通脉片发挥治疗作用的 重要靶细胞,抑制MC增殖和ECM的产生可能是该方延缓肾小球硬化的重要机制之一。     

增塑剂对人腹膜间皮质细胞细胞外基质合成和分泌的影响

目的：探讨增塑剂邻苯二甲酸二辛酯（DEHP）与腹膜硬化的关系和可能机制。方法：分离人腹膜间皮细胞（HPMC）作体外培养，选择3种不同浓度的DEHP连续刺激5d，同时设阴性对照，分别在第1、5天进行检测。以^3H－脯氨酸掺入法测定细胞总胶原的合成和分泌；ELISA法检测培养液中纤连蛋白（FN）的含量；FN－mRNA、I型胶原mRNA(Co I-mRNA)、Ⅲ型胶原mRNA(Co Ⅲ-mRNA）和转化生长因子（TGF）－β1mRNA的表达采用半定量逆转录聚合酶链反应（RT－PCR）方法测定。结果：（1）HPMC表达FN、层连蛋白（LN）、Co Ⅲ和TGF－β1；（2）HPMC在不同浓度DEHP刺激下第5天时培养液上清FN及胶原的分泌量明显增加，其中高浓度组与对照组相比差异有显著性意义（P＜0．05）；（3）不同浓度DEHP刺激HPMC下在第5天时FN－mRNA、CoI-mRNA和CoⅢ-mRNA的表达与对照组相比均明显增加，差异有显著性意义，但各浓度之间差异无显著性意义；（4）不同浓度的DEHP刺激HPMC24h均引起TGF－β1mRNA表达增加，第5天时表达消失。结论：体外研究证实DEHP可促进人腹膜间皮细胞合成和分泌细胞外基质，且有一定的时间和剂量依赖性。早期可能通过TGF-β1介导。其在长期腹膜透析患者腹膜硬化的发生中可能起了一定作用。     

微量元素与细胞外基质

目的 论述微量元素对细胞外基质代谢的影响,探讨创伤愈合的生理和相关病理机制。方法 以基础研究结合相关临床资料,分析微量元素与细胞外基质代谢的关系。探讨创伤修复机制。结果 在创伤愈合过程中,微量元素通过参与酶的构成与激活、构成体内重要的载体及蛋白质成份、参与激素和维生素的合成以及调控自由基水平等功能,广泛参与到胶原蛋白和其它细胞外基质的合成、分解、沉积与重建过程中。结论 微量元素对细胞外基质的代谢起着一定的调控作用,在创伤愈合过程中发挥着重要作用。     

复方积雪草对肾小球系膜细胞及细胞外基质的影响

目的 :探讨复方积雪草在体外及体内对大鼠肾小球系膜细胞及细胞外基质增生的影响。方法 :体外实验在培养的大鼠肾小球系膜细胞中进行 :应用氮蓝四唑盐 (MTT)比色法、流式细胞术对DNA倍体进行分析 ,分别检测复方积雪草对系膜细胞增殖的影响。体内实验在单侧肾切除术合并阿霉素静脉双次注射的大鼠肾小球硬化模型中进行 ,用定量病理分析观察复方积雪草对系膜细胞增殖及系膜基质增殖的影响。结果 :体外实验提示 ,复方积雪草明显抑制血清加血管紧张素Ⅱ刺激的系膜细胞增殖 (与对照组比较P <0 .0 1) ,G0 /G1期细胞比例增加 ,S G2 /M期细胞比例逐渐下降 ,且呈量效和时效依赖关系。体内实验中 ,造模大鼠经复方积雪草治疗后第 8周 ,系膜细胞增殖、细胞外基质沉积均明显减少 (与对照组比较P <0 .0 1)。结论 :复方积雪草可以抑制系膜细胞增生 ,阻止系膜细胞由G1期进入S期 ,减少细胞外基质沉积     

甲状旁腺素对人肾小管上皮细胞细胞外基质合成与降解的影响

Objective To investigate the effects of parathyroid hormone (PTH) on the synthesis and secretion of collagen Ⅲ and fibronectin (FN), and the expressions of plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA in cultured human renal tubular epithelial cells (HK-2).Methods HK-2 cells were cultured in DMEM-F12 medium supplemented with 5% FBS. Cells were exposed to different concentrations of PTH (0, 10-12, 10-11, 10-10, 10-9, 10-8 mol/L) for 48 h, or 10-8 mol/L PTH at different time (0, 12, 24, 48, 72 h). The gene expressions of collagen Ⅲ,FN, PAI-1, MMP-1, and TIMP-1 were detected by semi-quantitative RT-PCR. The protein expression of collagen Ⅲ was detected by Western blotting. The level of FN in the supernatant was assayed by enzyme linked immunosorbent assay (ELISA). Results PTH increased gene expressions of collagen Ⅲ, FN, PAI-1 and TIMP-1 in a dose- and time-dependent manner, but decreased MMP-1 gene expression. Then the ratio of MMP-1/TIMP-1 was decreased. PTH increased the collagen Ⅲ protein expression in cultured HK-2 cells and the level of FN in the supernatant of cultured HK-2 cells in a dose- and time-dependent manner. Conclusion PTH can up-regulate PAI-1, TIMP-1 gene expressions, and down-regulate MMP-1 gene expression,resulting in elevation of extracellular matrix (ECM) synthesis and reductim of degradation.     

Matrix extracellular phosphoglycoprotein (MEPE) is highly expressed in osteocytes in human bone

The matrix extracellular phosphoglycoprotein (MEPE) gene is highly expressed in tumors that cause oncogenic hypophosphatemic osteomalacia (OHO). MEPE is also known as one of the bone-tooth matrix proteins and is associated with bone mineralization. We developed a rabbit polyclonal antibody directed against recombinant human MEPE (rhMEPE) after cloning its cDNA from the cDNA library of a nasal tumor tissue causing OHO. Using this antibody, we analyzed the distribution of MEPE in human bones by immunohistochemistry. In bone specimens from normal subjects, MEPE was predominantly expressed by osteocytes and not by osteoblasts. In bone specimens from patients with osteomalacia, however, MEPE was focally expressed by deeply located osteocytes. We also compared the MEPE positivity of osteocytes in mineralized bone and non-mineralized osteoid obtained from patients with osteomalacia and osteoporosis. Among osteomalacia patients, MEPE positivity was seen in 87.5 ± 8.6% of the osteocytes from mineralized bone compared with 7.8 ± 6.4% of those from osteoid. Among osteoporosis patients, MEPE positivity was found in 95.3 ± 0.5% of the osteocytes from mineralized bone compared with 4.9 ± 5.7% of those from osteoid. MEPE was mainly expressed by osteocytes embedded in the matrix of mineralized bone from patients with osteomalacia or osteoporosis. Our data provide the first histological evidence that MEPE is predominantly expressed by osteocytes in human bone, with significant expression by osteocytes within mineralized bone.     

辛伐他汀对人牙髓干细胞成骨活性的影响

目的：研究辛伐他汀对人牙髓干细胞成骨活性的影响.方法：将体外分离培养的人牙髓干细胞分别接种于含有不同浓度辛伐他汀的矿化培养液中诱导培养,测定碱性磷酸酶活性及骨唾液酸蛋白基因的表达情况.结果：与对照组比较,适宜浓度辛伐他汀（1×10^-6mol/L、1×10-7mol/L、1×10-8mol/L）促进人牙髓干细胞的成骨活性作用更为明显,差异具有统计学意义（P＜0.05）,当辛伐他汀浓度为1×10-7mol/L时,促进作用最显著.结论：适宜浓度的辛伐他汀可以有效促进人牙髓干细胞的成骨活性.     

辛伐他汀对人牙髓干细胞增殖和分化的影响

目的:研究辛伐他汀对人牙髓干细胞(dental pulpstem cells,DPSCs)增殖和成骨分化的影响。方法:将第3代人DPSCs在矿化培养液中诱导培养,同时加入不同浓度的辛伐他汀(1×10-5mol/L、1×10-6mol/L、1×10-7mol/L、1×10-8mol/L),噻唑蓝(methyl thiazolyl tetrazolium,MTT)法检测细胞增殖情况,碱性磷酸酶试剂盒检测碱性磷酸酶(alkaline phosphatase,ALP)活性,茜素红染色鉴定成骨分化。结果:各浓度辛伐他汀均抑制人DPSCs增值,辛伐他汀浓度为1×10-5mol/L时,抑制作用最明显。适宜浓度辛伐他汀(1×10-6mol/L、1×10-7mol/L、1×10-8mol/L)促进人DPSCs向成骨细胞分化,其中,1×10-7mol/L的辛伐他汀促进ALP活性的作用最明显。结论:辛伐他汀抑制人DPSCs的增值,适宜浓度的辛伐他汀可有效促进人DPSCs的成骨分化。     

瘦素对人牙髓干细胞骨向分化作用的实验研究

目的：探讨瘦素（leptin）对人牙髓干细胞（Human dental pulp stem cells,hDPSCs）增殖和骨向分化的影响。方法：采用酶消化法体外培养人牙髓干细胞,分别加阶梯浓度leptin处理,通过四唑盐（MTT）比色试验和碱性磷酸酶（Alkalinephosphatase,ALP）活性测试来检测瘦素对牙髓干细胞体外增殖分化的影响;再将人牙髓干细胞与羟基磷灰石/磷酸三钙材料制作为复合体植入免疫缺陷裸鼠,12周后采用组织学和免疫组化方法检测体内生成物。结果：瘦素对人牙髓干细胞细胞增殖无明显促进作用,但leptin能促进人牙髓干细胞碱性磷酸酶的活性表达;体内试验证明leptin可提高牙髓干细胞向成成牙本质细胞分化及生成类牙本质的能力。结论：瘦素对人牙髓干细胞的增殖活性无明显作用,但人牙髓干细胞与羟基磷灰石磷酸三钙制作为复合体,可明显促进人牙髓干细胞骨向分化。     

大鼠牙髓干细胞与骨髓间充质干细胞分化成骨样细胞能力对比研究

目的：以骨髓间充质干细胞作为参考,讨论牙髓干细胞作为骨组织修复种子细胞的可行性。方法：通过酶消化法获得大鼠牙髓干细胞,离心法获得骨髓间充质干细胞,贴壁培养,通过倒置光学显微镜观察二者形态差异;流式细胞技术鉴定骨髓间充质干细胞的间质细胞表面标志物表达;MMT法检测细胞生长曲线;特定的成骨诱导液诱导干细胞成骨分化,免疫荧光法检测成骨细胞表面标志物表达。结果：分离的大鼠骨髓间充质干细胞与牙髓干细胞形态学以及生长特性具有相似性,且符合间充质干细胞特性,牙髓干细胞第4天进入对数生长期,第7天进入平台期,骨髓间充质干细胞第4天进入对数生长期,第8天进入平台期;经过成骨诱导后二者都具备成骨样细胞分化能力,牙髓干细胞在成骨诱导后的骨钙素表达上与骨髓间充质细胞有着相似的能力;牙本质泌涎蛋白表达阳性。结论：牙髓干细胞具有与骨髓间充质干细胞都具有成骨分化能力,但牙髓干细胞细胞成分相对复杂,增殖能力较强。     

In vitro isolation of stem cells derived from human dental pulp

Agha‐Hosseini F, Jahani M‐A, Jahani M, Mirzaii‐Dizgah I, Ali‐Moghaddam K. In vitro isolation of stem cells derived from human dental pulp.
Clin Transplant 2009: DOI: 10.1111/j.1399‐0012.2009.01137.x.
© 2009 John Wiley & Sons A/S. Abstract: Stem cells are characterized by the ability to differentiate and to self‐renew. Stem cells derived from human dental pulp have been shown to differentiate into osteoblasts serving as a potential source of autologous bone produced in vitro. The purpose of the present study was to isolate mesenchymal stem cells from dental pulp. Dental pulp was gently extracted from 27 intact human permanent third molars of patients aged 18–25. Cow horn forceps were used to isolate intact dental pulp in sterilized condition. The pulps were cultured in a medium containing Dulbecco’s modified Eagle’s medium‐low glucose (DMEM)‐LG and Amphotericin 1%. The cells were subsequently expanded by passages, two passages were performed before they were stored in liquid nitrogen for further examination. DMEM + fetal bovine serum (FBS) 10% L‐Glutamin 0.1% + Trypsin 2.5% + ethylene diamine tetraacetic acid (EDTA) were used for passage. Light microscope and flow cytometry were used to study the cells. The isolated dental pulp cells expressed mesenchymal stem cell markers. The cells were negative for CD34 and CD31 and CD45 but were positive for CD13, CD44, CD90, CD166, and CD105. These results indicate that dental pulp can be use as a source of stem cells that we can isolate and culture.     

Histologic analysis of the effect on dental pulp of a 9.6-microm CO(2) laser

BACKGROUND AND OBJECTIVE: Both patients and dentists would like a replacement of the dental drill. During the last decade, lasers have been investigated as a possible replacement. For lasers to be accepted, studies must show that their effect on the dental pulpal tissues is equal to or less noxious than those effects caused by the dental handpiece (drill). STUDY DESIGN/MATERIALS AND METHODS: In this study, two laser systems were used; the first was a breadboard CO(2) laser and the second a prototype clinical CO(2) laser system both emitted 60-micros-long pulses of 9.6-microm radiation. On the delivery system of both lasers, a scanner moved the focussed beam in a circular pattern and a water spray system served to cool the ablation site. Both lasers were used to create holes of similar dimensions in canine teeth. The treated teeth were then restored and harvested at either 4 days or 4 weeks. The teeth were decalcified, sectioned, and stained for examination via light microscopy. RESULTS: The histologic examination revealed normal pulpal tissues in the canine teeth treated with both CO(2) lasers. Some histologic sections showed an increase in the predentin layer, 28 days after laser treatment. While many histologic sections showed normal pulpal architecture following handpiece treatment, some sections showed total disruption of the normal pulpal histology. CONCLUSIONS: Histologic evaluation revealed that the lasers produced no noticeable damage to the dental pulpal tissue and appear to be a safe method for removing dental hard tissues. From this study, it appears that 9.6 microm CO(2) laser does not cause damage to the dental pulpal tissues in dogs.     

Localization of alkaline phosphatase in developing bovine dental pulp

The distribution of alkaline phosphatase in dentinogenically active bovine dental pulp tissues was investigated histochemically and biochemically. Histochemical observation showed a high enzyme activity in the subodontoblastic layer, especially in coronal pulp and also in the core of radicular pulp. Biochemical results were well consistent with histochemical findings. Alkaline phosphatase activity in the coronal pulp was twice that in radicular pulp. Most of the enzyme activity in coronal pulp was in the subodontoblastic layer (91%), whereas the activity in the radicular pulp was evenly distributed. Since undifferentiated mesenchymal cells were observed in the areas showing relatively high ALPase activity, it seemed that there were some relations between the inducion of ALPase and the undifferentiated mesenchymal cells.     

Comparison of human dental pulp and bone marrow stromal stem cells by cDNA microarray analysis

We compared the gene expression profiles of human dental pulp stem cells (DPSCs) and bone marrow stromal stem cells (BMSSCs) as representative populations of odontoprogenitor and osteoprogenitor cells, respectively. Total RNA from primary cultures was reverse-transcribed to generate cDNA probes and then hybridized with the Research Genetics human gene microarray filter GF211. The microarrays were analyzed using the P software package. Human DPSCs and BMSSCs were found to have a similar level of gene expression for more than 4000 known human genes. A few differentially expressed genes, including collagen type XVIII 1, insulin-like growth factor-2 (IGF-2), discordin domain tyrosine kinase 2, NAD(P)H menadione oxidoreductase, homolog 2 of Drosophila large disk, and cyclin-dependent kinase 6 were highly expressed in DPSCs, whereas insulin-like growth factor binding protein-7 (IGFBP-7), and collagen type I 2 were more highly expressed in BMSSCs. Furthermore, we confirmed the differential expression of these genes by semiquantitative polymerase chain reaction (PCR) and northern blot hybridization. The protein expression patterns for both IGF-2 and IGFBP-7 correlated with the differential mRNA levels seen between DPSCs and BMSSCs. This report describes the gene expression patterns of two distinct precursor populations associated with mineralized tissue, and provides a basis for further characterization of the functional roles for many of these genes in the development of dentin and bone.     

牙髓干细胞移植治疗大鼠脊髓损伤的实验研究

目的:探讨牙髓干细胞移植治疗脊髓损伤的神经修复及运动功能恢复情况.方法:培养取材第2代来源于第三磨牙的人牙髓于细胞,鉴定其表面标记物,应用B27、碱性呈纤维细胞生长因子和胰岛素转铁蛋白硒进行神经诱导,并行免疫荧光染色检测.改良Allen's法制作SD大鼠脊髓损伤模型,3d后随机分为实验组及对照组,每组各20只大鼠.分别于脊髓损伤处注入人牙髓干细胞及生理盐水,于造模后1d、治疗前1d、治疗后3d、治疗后7、14、28d进行动物后肢运动功能检测.28d时脊髓取材进行HE染色,观察脊髓空洞形成,计算空洞面积;Tunnel法检测两组细胞凋亡情况;免疫荧光双标法标记HuNu-NeuN和HuNu-GFAP,观察人牙髓干细胞体内生长分化情况.结果:细胞培养传代后呈长梭形,细胞形态均匀,胞浆丰富、胞核增大,细胞平行排列呈漩涡状或螺旋状.流式细胞仪分析人牙髓干细胞诱导分化后高表达CD44、CD90和CD146,低表达STRO-1,CD34、CD45表达阴性.人牙髓干细胞神经诱导14d,免疫荧光标记GFAP、NeuN呈阳性.细胞移植3d、7d,两组间BBB评分无统计学差异;细胞移植14d及28d实验组BBB评分分别为3.8±0.8、7.2±1.6,对照组BBB评分为2.2±0.8、3.6±1.1,两组间比较差异有显著性(P＜0.05).28d时HE染色两组均可见脊髓出血、炎性细胞浸润、小血管增生及空洞形成.实验组脊髓空洞面积百分比(26.75±2.50)％,对照组为(49.50±6.25)％,两组差异有统计学意义(P＜0.05);Tunnel法检测实验组神经细胞凋亡百分比(32.33±1.54)％,对照组为(46.33±1.53)％,实验组显著减少神经细胞的凋亡(P＜0.05).免疫荧光双标法检测到部分细胞为带有HuNu-NeuN和HuNu-GFAP双抗体细胞.结论:人牙髓干细胞能够在体外特定条件下及移植入脊髓损伤大鼠体内可分化为神经细胞,用于治疗脊髓损伤时可减少神经细胞的凋亡,促进后肢运动功能的恢复.