人牙周膜干细胞的初步鉴定及体外成骨诱导

目的：体外培养、鉴定人牙周膜干细胞（periodontal ligament stem cells，PDLSCs）并定向诱导分化为成骨细胞，探讨人PDLSCs的多向分化潜能：方法：体外分离、培养人牙周膜细胞，待细胞达一定量后用有限稀释法进行克隆化培养，筛选鉴定牙周膜干细胞（PDLSC），矿化诱导培养21d后检测钙结节形成情况、ALP活性，免疫细胞化学检测骨涎蛋白（BSP）、Ⅰ型胶原表达，RT—PCR检测ALP、BSP mRNA表达。结果：人PDLSCs体外诱导培养21d后可见钙结节形成，成骨细胞相关蛋白及mRNA均阳性表达。结论：人PDLSCs在体外诱导条件下具有成骨潜能。     

颌骨骨髓基质细胞与牙周膜细胞的生物学特性比较

目的:比较颌骨骨髓基质细胞与牙周膜细胞的基本生物学特性。方法:分离颌骨来源骨髓基质细胞和牙周膜细胞,进行改良体外原代培养。倒置显微镜观察细胞生长及克隆形成情况;CCK-8检测细胞生长曲线;免疫荧光染色检测STR0-1表达;成脂诱导后检测脂滴形成,矿化诱导后检测碱性磷酸酶的变化并用RT-PCR检测7、14 d成骨相关基因OCN的表达水平。结果:两种细胞体外培养均呈成纤维样细胞外形,能克隆生长,均具有活跃的增殖能力;两种细胞STR0-1表达阳性;成脂诱导后可见脂滴形成;矿化诱导后骨髓基质细胞的碱性磷酸酶活性较强,而且OCN基因表达较早且较强。结论:颌骨来源骨髓基质细胞具有较强的增殖及成骨分化能力,具有干细胞特性,可能是有较大临床应用潜力的组织工程种子细胞。     

Bone Morphogenic Protein‐2 Induces Apoptosis and Cytotoxicity in Periodontal Ligament Cells

Background: Periodontal ligament (PDL) expresses endogenous growth factors, such as bone morphogenic proteins (BMPs), which facilitate maintenance of tissue homeostasis. Inflammatory conditions, such as chronic periodontitis, could disrupt this homeostasis, and physiologic levels of growth factors may be insufficient to maintain tissue homeostasis. BMPs facilitate periodontal bone regeneration but also are implicated in causing tooth ankylosis and root resorption. The underlying mechanism of tooth ankylosis is unclear. However, there is evidence that BMPs induce apoptosis in progenitor cells. Little is known about BMP‐induced cytotoxicity in PDL cells, which contain a population of progenitor cells. The aim of this study is to determine BMP2‐induced osteogenic mediators and cytotoxic effects in PDL cells and compare these cells to osteoblasts. Methods: Human PDL cells and primary osteoblasts were stimulated with doses of 1 to 200 ng/mL BMP2. Expression of alkaline phosphatase (ALP), in vitro mineralization along with osteonectin expression, induction of apoptosis, and cytotoxicity assays were performed. Results: PDL cells and osteoblasts upregulated ALP and in vitro mineralization in a dose‐dependent manner with BMP2 stimulation. However, at BMP2 concentrations >10 ng/mL, ALP, in vitro mineralization, and osteonectin were downregulated in PDL cells. Relative to osteoblasts, PDL cells were susceptible to apoptosis and cytotoxicity with 10 times lower concentration of BMP2. Conclusions: Relative to osteoblasts, PDL cells are susceptible to BMP2‐induced cytotoxicity. BMP‐induced tooth ankylosis is controversial and is poorly understood. Disruption of PDL homeostasis by BMP‐induced apoptosis could play a role in tooth ankylosis.     

改良法分离培养人牙周膜干细胞

目的 改良法体外分离培养人牙周膜干细胞(PDLSC),并进行鉴定.方法采用酶消化组织块法获得人牙周膜细胞,通过有限稀释法克隆化培养、分离得到PDLSC,用含10%FBS的α-MEM培养液培养并传代:测定克隆形成率:免疫组织化学检测角蛋白及波形蛋白表达:流式细胞术分析细胞周期及表面标志物STRO-1、CD146的表达;并...     

干细胞表面标志物在牙髓干细胞中的表达

目的:探讨磁珠分选后培养的人牙髓干细胞的表面标记抗原随培养代数增加的变化情况。方法:改良组织块法培养的人牙髓细胞,至第2代（P2）时,用磁珠方法分选出STRO-1阳性细胞,用流式细胞术分别检测P2、P3、P4、P5、P6、P7、P8代的干细胞表面标志物CD73、CD90、CD105、 CD166、STRO-1。取P3代细胞,分别进行成骨诱导和成脂诱导。21 d后,分别行茜素红染色和油红O染色,观察矿化物形成情况和脂滴形成情况,同时以未诱导细胞为对照。结果:STRO-1在牙髓干细胞表面随代数增加而下降,CD73、CD90、CD105、 CD166的表达比较稳定,茜素红染色可见矿化结节形成,油红O染色显示形成大量脂滴。结论:STRO-1在牙髓干细胞表面随代数增加而下降,其他干细胞标志物比较稳定。     

富血小板纤维蛋白通过Wnt/β-catenin信号通路促进BMSCs成骨分化的研究

目的 :研究富血小板纤维蛋白(PRF)通过Wnt/β-catenin信号通路对骨髓基质干细胞(BMSCs)分化的影响。方法:分离、培养原代兔BMSCs,取第三代细胞进行干细胞鉴定,收集同一只兔耳缘静脉血离心制备PRF,将PRF与BMSCs置于Transwell小室中共培养并进行成骨分化。实验分组:A组为单纯BMSCs对照组;B组为PRF与BMSCs共培养组;C组为加入DKK1的PRF与BMSCs共培养组;D组为加入Wnt3a的PRF与BMSCs共培养组。茜素红染色观察各组钙结节形成情况,碱性磷酸酶(ALP)试剂盒检测各组ALP活性,定量聚合酶链反应(q RT-PCR)检测成骨成脂相关基因Runx2、OCN、PPARγ2及LPL的m RNA表达,同时检测Wnt/β-catenin信号通路关键因子Cyclin D1及β-catenin的m RNA表达。结果:流式细术检测结果显示,原代培养的BMSCs符合间充质干细胞鉴定标准。茜素红染色结果显示,B组和D组的钙结节数量明显多于A组和C组,A组和C组之间无明显差异。碱性磷酸酶(ALP)活性检测结果显示,B组与D组的ALP活性较A组和C组明显增加(P<0.05),且两组之间差异无明显统计学意义。q RTPCR结果显示B组与D组中的成骨分化标志基因Runx2、OCN的m RNA表达,较A组和C组显著增加(P<0.05),而成脂分化标志基因PPARγ2、LPL的m RNA表达显著降低(P<0.05)。此外,B组和D组中Wnt/β-catenin信号通路关键因子Cyclin D1及β-catenin的m RNA表达较A组和C组显著增加(P<0.05)。结论:PRF通过激活Wnt/β-catenin信号通路促进BMSCs成骨分化而抑制其成脂分化。     

人牙髓细胞与牙周韧带细胞多向分化能力的比较

目的 比较人牙髓细胞(dental pulp cells,DPC)和牙周韧带细胞(periodontal ligamentcells,PDLC)的多向分化能力,揭示其干细胞成分特征,为开展干细胞介导的生物牙根再生奠定实验基础.方法 酶消化法分离培养人DPC和PDLC,流式细胞术检测STRO-1的表达.诱导细胞成牙本质及成骨分化、成脂分化和成软骨分化,von Kossa染色、抗骨钙素(osteocalcin,OCN)和牙本质涎蛋白(dentin sialoprotein,DSP)免疫组化染色、油红O染色、阿新蓝染色、抗Ⅱ型胶原免疫组化染色以及实时荧光定量反转录聚合酶链反应(RT-PCR)等检测DPC和PDLC的多向分化.结果 DPC和PDLC体外呈克隆样生长,STRO-1阳性率分别是(16.5%±4.2%)和(11.6%±1.1%).100%的DPC和83.3%的PDLC样本可多向分化.细胞诱导分化后,OCN、牙本质涎磷蛋白(dentinsialophosphoprotein,DSPP)、过氧化物酶体激活物增生受体2(peroxisomal proliferator activated receptorgamma 2,PPARγ2)、脂蛋白脂酶(lipoprotein lipase,LPL)和Ⅱ型胶原mRNA表达上调,与诱导前相比差异均有统计学意义(P<0.001),DPC和PDLC间OCN和PPARγ2基因上调倍数的差异有统计学意义(P<0.001).结论 人DPC和PDLC的间充质干细胞比例和多向分化能力相似.     

CKIP-1对骨髓间充质干细胞体外增殖和分化能力的影响

目的：探讨CKIP-1基因对小鼠骨髓间充质干细胞（BMSCs）的增殖和分化能力的调控。方法：选择CKIP-1基因敲除型小鼠（KO）和野生型C57小鼠（WT）,采用全骨髓贴壁法培养BMSCs,取第3代BMSCs,分为KO组和WT组,分别采用成骨及成脂诱导液对细胞进行诱导培养,MTT法检测细胞增殖,流式细胞仪检测目的细胞表面标记分子,用ALP染色、茜素红染色、油红O染色分别对细胞成骨及成脂能力做定量分析。结果：2种细胞增殖和干细胞分子表达相似;成骨诱导后ALP染色显示,KO组的细胞染色的阳性率要大于WT组。茜素红染色观察显示KO组的矿化结节要多于WT组;油红O染色显示KO组细胞的脂质沉淀量大于WT组。结论：CKIP-1基因缺失可以使BMSCs成骨及成脂分化能力增强,对其增殖影响不明显。     

BCOR基因敲除促进骨髓间充质干细胞成骨分化的研究

目的研究BCL6共抑制因子-BCOR对骨髓间充质干细胞成骨定向分化能力的影响。方法利用慢病毒载体的BCORshRNA基因敲除BCOR,进行丧失性功能研究。通过检测ALP活性、茜素红染色、钙离子定量分析、成骨分化相关基因的表达,观察骨髓间充质干细胞体外成骨分化能力。结果BCOR在牙源性间充质干细胞和骨髓间充质干细胞的表达无明显差异。基因敲除BCOR促进了骨髓间充质干细胞ALP活性、体外矿化能力及成骨分化相关基因骨涎蛋白、骨钙素的表达。结论BCOR基因表达降低能促进骨髓间充质干细胞成骨分化,证实BCOR是骨髓间充质干细胞成骨分化的抑制基因。     

人脂肪组织来源血管周干细胞成骨、成脂、成软骨分化能力研究

目的    研究从人脂肪组织中提取的血管周干细胞（human perivascular stem cells,hPSCs）的特点,并探讨其成骨、成脂及成软骨分化的能力。方法    采用流式荧光细胞分选技术（FACS）在12例行吸脂手术患者的脂肪标本中分选出人基质血管成分（human stromal vascular fraction,hSVF）和由周皮细胞（CD34-、CD146+、CD45-）与外膜细胞（CD34+、CD146-、CD45-）组成的hPSCs进行培养,比较其克隆增殖能力,然后将2种细胞进行成骨、成脂和成软骨诱导,诱导结束后分别进行茜素红染色、油红O染色和阿尔新蓝染色,并检测成骨诱导后的成骨相关基因mRNA表达。结果    hSVF和hPSCs均以纺锤形成纤维样细胞生长,hPSCs呈现出更快的融合趋势,且细胞形态更均一。hPSCs细胞相比于hSVF具有更强的克隆增殖能力（P < 0.05）。hPSCs细胞在成骨及成软骨方向比hSVF具有更强优势,而hSVF在成脂方向比hPSCs具有更强优势。成骨诱导后,hPSCs的成骨基因碱性磷酸酶（alkaline phosphatase,ALP）和骨钙素（osteocalcin,OCN）的相对表达量要明显高于hSVF（P < 0.05）。结论    hPSCs细胞具有干细胞的多项分化潜能,且其在成骨方向具有很强优势,可成为骨组织工程学中理想的种子细胞来源。     

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??Objective    To extract human perivascular stem cells??hPSCs??from fat tissue??and then to detect its abilities of osteogenic differentiation??adipogenic differentiation??and chondrogenic differentiation. Methods     Human stromal vascular fraction??hSVF??and hPSCs were purified from 12 samples of human lipoaspirate by FACS. After cell culture?? the proliferation abilities of hSVF and hPSCs were compared. Then two kinds of cells were induced into osteogenic differentiation??adipogenic differentiation??and chondrogenic differentiation. Results were obtained by alizarin red staining??oil red staining??alcian blue staining??and qRT-PCR. The mRNA expression of osteogenesis-related genes were detected. Results    hSVF and hPSCs grew like spindle fibroblast. hPSCs fused faster than hSVFs??and had homogeneous morphology. hPSCs had stronger cloning and proliferation ability than hSVF??P < 0.05??. hPSCs had stronger osteogenic differentiation ability and chondrogenic differentiation ability than hSVF??P < 0.05????while hSVF had stronger adipogenic differentiation ability than hPSCs. Alkaline phosphatase??ALP??and osteocalcin??OCN??had higher expression in hPSCs than in hSVF after osteogenesis??P < 0.05??. Conclusion    hPSCs have the multipotential differentiation ability as stem cells??and have strong advantages in the direction of osteogenesis. hPSCs can be an ideal source of seed cells in bone tissue engineering.     

Stem cell properties of human periodontal ligament cells

BACKGROUND AND OBJECTIVE: Stem cells have been used for regenerative therapies in various fields. The proportion of cells that possess stem cell properties in human periodontal ligament (PDL) cells is not yet well understood. In this study, we quantitatively characterized human PDL cells to clarify their stem cell properties, including self-renewal, multipotency, and stem cell marker expression. MATERIAL AND METHODS: PDL cells were obtained from extracted premolar or wisdom teeth, following which a proliferation assay for self-renewal, a differentiation assay for multipotency, immunostaining for STRO-1, and fluorescence-activated cell sorter (FACS) analysis for stem cell markers (including CD105, CD166, and STRO-1) were performed. RESULTS: Approximately 30% of 400 PDL cells were found to possess replicative potential and formed single-cell colonies, and 30% of these colonies displayed positive staining for STRO-1, 20% differentiated into adipocytes and 30% differentiated into osteoblasts. FACS analysis revealed that PDL cells, including cell populations, expressed the stem cell markers CD105, CD166, and STRO-1. CONCLUSION: The findings of this study indicated that PDL cells possess crucial stem cell properties, such as self-renewal and multipotency, and express the mesenchymal stem cell markers CD105, CD166, and STRO-1 on their cell surface, although there were some variations. Thus, PDL cells can be used for periodontal regenerative procedures.     

FGF-2 induces proliferation of human periodontal ligament cells and maintains differentiation potentials of STRO-1(+)/CD146(+) periodontal ligament cells

The presence of human STRO-1⁺/CD146⁺ periodontal ligament (PDL) cells has been reported, but obtaining a large amount of these cells is difficult. The purpose of this study was to evaluate the percentages of STRO-1⁺/CD146⁺ cells in PDL cells and determine the effects of FGF-2 on the proliferation and multilineage differentiation potency of these cells. Human PDL (HPDL) cells were individually prepared from 15 extracted teeth. HPDL cells were cultured with or without FGF-2, and the percentages of STRO-1⁺/CD146⁺ cells in each HPDL cell culture was examined using FACSAria?. The STRO-1⁺/CD146⁺ cells were sorted with FACSAria?, and the mRNA expression and differentiation potency of the sorted cells were subsequently examined. The numbers of the STRO-1⁺/CD146⁺ cells in the FGF-2 cultures were significantly higher than those cultured in the absence of FGF-2. The sorted STRO-1⁺/CD146⁺ cells expressed mRNA of PDL markers and differentiated into adipocytes and osteoblast-like cells. The present study shows that FGF-2 augmented the proliferation of the STRO-1⁺/CD146⁺ cells in the HPDL cultures whilst retaining adipogenic and osteogenic differentiation potentials. Thus, it may be useful to culture HPDL cells with FGF-2 for the application of the human STRO-1⁺/CD146⁺ PDL cells in periodontal tissue regeneration.     

年龄因素对人牙周膜干细胞生物学行为影响的研究

目的　研究年龄因素对人牙周膜干细胞（periodontal ligament stem cells,PDLSC）生物学行为的影响。方法  选取2017年10月至2018年10月于青岛市口腔医院口腔外科就诊的因阻生齿或正畸需要拔除牙齿的患者18例,根据不同年龄段分为3组,分别为A组（18 ~ 20 岁）、B组（30 ~ 35岁）、C组（50 ~ 55岁）,每组6例。原代培养人PDLSC,采用衰老相关β-半乳糖苷酶染色、MTT法检测各组细胞衰老程度和增殖能力。成骨诱导人PDLSC,采用碱性磷酸酶（ALP）染色和茜素红染色检测各组细胞的成骨分化能力。采用实时荧光定量PCR（qRT-PCR）检测各组多能性相关转录因子Nanog和Sox2的表达水平。结果　各组均成功原代培养出人PDLSC,经衰老相关β-半乳糖苷酶染色发现,A组阳性染色细胞率最低、B组次之、C组最高,组间差异均有统计学意义（均P < 0.05）。通过MTT法检测发现,不同年龄组OD值比较,差异有统计学意义（F = 22.354,P = 0.009）,且各组OD值均随着培养时间的延长而增大（F = 28.367,P = 0.018）,年龄与时间之间有明显的交互作用（F = 26.431,P = 0.015）。ALP染色和茜素红染色均发现,A组染色面积百分比大于B组和C组,B组染色面积百分比大于C组,差异均有统计学意义（均P < 0.05）。qRT-PCR结果显示,A组Nanog和Sox2的相对表达量均高于B组和C组,B组Nanog和Sox2的相对表达量高于C组,差异均有统计学意义（均P < 0.05）。结论　随着年龄增加,衰老的人PDLSC增多,其增殖能力、成骨分化能力以及多能性相关转录因子的表达水平也显著下降。     

小肠黏膜下层对牙周膜干细胞体外增殖、骨向分化的影响

目的 观察小肠黏膜下层 (SIS)与人牙周膜干细胞(PDLSCs)的生物相容性及其作为支架材料对PDLSCs的骨向分化诱导作用。方法 体外分离培养 PDLSCs,分别用四唑盐比色法（MTS）、茜素红染色法、碱性磷酸酶(ALP)和逆转录聚合酶链式反应（RT-PCR）方法观察 SIS 生物材料对PDLSCs增殖活性和骨向分化能力的影响。结果 SIS 显著地刺激体外培养的PDLSCs增殖,诱导了细胞的矿化和提高细胞ALP活性。RT-PCR结果显示SIS诱导后细胞 mRNA 水平表达骨涎蛋白(BSP)和骨钙素（OCN)。结论 体外培养条件下,SIS与PDLSCs有良好的生物相容性,能够诱导PDLSCs骨向分化。     

Physiologic Levels of Endogenous Hydrogen Sulfide Maintain the Proliferation and Differentiation Capacity of Periodontal Ligament Stem Cells

Background: Many invading oral bacteria are known to produce considerable amounts of hydrogen sulfide (H₂S). The toxic activity of exogenous H₂S in periodontal tissue has been demonstrated, but the role of endogenous H₂S in the physiologic function of periodontal tissue remains poorly understood. The purpose of the present study is to investigate the biologic functions of H₂S in the proliferation and differentiation of human periodontal ligament stem cells (PDLSCs). Methods: PDLSCs were isolated from periodontal ligament tissues of periodontally healthy volunteers or patients with periodontitis. Immunocytochemical staining, flow cytometry, and Western blot analysis were used to examine the expression of H₂S‐synthesizing enzymes cystathionine‐β‐synthase (CBS) and cystathionine‐γ‐lyase (CSE). The proliferation capacity of PDLSCs was determined by cell counting kit‐8 assay, carboxyfluorescein succinimidyl ester analysis, and 5‐ethynyl‐2′‐deoxyuridine assay. The osteogenic potential of PDLSCs was tested using alkaline phosphatase staining, Alizarin Red staining, and in vivo transplantation experiments. Oil Red O staining was used to analyze adipogenic ability. Results: The results show that human PDLSCs express both CBS and CSE and produce H₂S. Blocking the generation of endogenous H₂S with CBS inhibitor hydroxylamine significantly attenuated PDLSC proliferation and reduced the osteogenic and adipogenic differentiation capacity of PDLSCs. In contrast, CSE inhibitor dl ‐propargylglycine had no effect on PDLSC function. Exogenous H₂S could inhibit the production of endogenous H₂S and impair PDLSC function in a dose‐dependent manner. Conclusion: Physiologic levels of endogenous H₂S maintain the proliferation and differentiation capacity of PDLSCs, and CBS may be the main source of endogenous H₂S in PDLSCs.     

组蛋白去甲基化酶FBXL11抑制牙髓干细胞成骨和成牙本质分化

目的 研究组蛋白去甲基化酶FBXL11对牙髓干细胞定向分化能力的影响.方法 成骨分化诱导培养基诱导牙髓干细胞体外成骨/成牙本质分化.逆转录病毒转染构建过表达FBXL11的牙髓干细胞稳定转染细胞,进行FBXL11获得性功能研究.碱性磷酸酶活性实验及碱性磷酸酶染色检测成骨/成牙本质分化早期分化指标-碱性磷酸酶活性.茜素红染色及钙离子定量分析检测牙髓干细胞体外成骨/成牙本质分化能力.实时定量RT-PCR检测FBXL11及成骨/成牙本质分化相关基因-骨涎蛋白、骨桥蛋白和骨钙素的表达.结果 成骨诱导牙髓干细胞抑制FBXL11的表达.过表达FBXL11明显抑制牙髓干细胞的碱性磷酸酶活性、牙髓干细胞体外矿化能力以及骨涎蛋白和骨桥蛋白的表达.结论 组蛋白去甲基化酶FBXL11具有抑制牙髓干细胞成骨和成牙本质分化的潜能.     

Mechanisms of the mechanically activated ion channel Piezo1 protein in mediating osteogenic differentiation of periodontal ligament stem cells via the Notch signaling pathway

目的 探究机械激活性离子通道压电蛋白Piezo1通过Notch信号通路介导人牙周膜干细胞（hPDLSC）成骨分化作用机制。方法 选取自2016年1月1日—2018年1月1日就诊于北京儿童医院正畸科的8~14岁儿童因阻生而拔出的年轻恒牙的牙周膜组织为细胞来源,采用酶消化法对hPDLSC进行提取。采用免疫组织化学染色法检测角蛋白、波形蛋白的表达和流式细胞术对hPDLSC的标志物CD146和STRO-1进行鉴定。构建和筛选siRNA-Piezo1基因干扰载体和Piezo1基因过表达质粒。应用Flexcell 4000T机械牵张应力仪器构建体外牵张力学hPDLSC细胞模型。实验分成5组：siRNA干扰组、过表达组、空白对照组、牵张应力组和阴性对照组。荧光定量聚合酶链反应（RT-qPCR）检测Piezo1、Notch1和成骨基因碱性磷酸酶（ALP）、Runt相关基因2（Runx2）、骨钙素（OCN）和骨涎蛋白（BSP）的表达。Western blot检测成骨标志蛋白ALP和Runx2的表达。Fluo-3 AM探针检测细胞内钙离子含量。结果 hPDLSC波形蛋白染色阳性,角蛋白染色阴性。流式细胞仪检测hPDLSC标志物STRO-1表达阳性,CD146表达阳性。空病毒载体、siRNA-Piezo1干扰序列和Piezo1过表达载体序列均可以通过慢病毒转染hPDLSC,而且转染效率较高,均在90%。逆转录聚合酶链反应结果显示,siRNA干扰组、过表达组、空白对照组、牵张应力组和阴性对照组Piezo1 mRNA的表达水平差异有统计学意义（F=9.573,P<0.05）;过表达组Piezo1 mRNA的表达水平明显高于siRNA干扰组,差异有统计学意义（q=3.893,P<0.05）;牵张应力组Piezo1 mRNA的表达水平明显高于空白对照组,差异有统计学意义（q=2.006,P<0.05）。Notch1和成骨基因ALP、Runx2、OCN和BSP的mRNA表达具有同样的趋势。Western blot检测结果显示siRNA干扰组、过表达组、空白对照组、牵张应力组和阴性对照组成骨标志蛋白ALP蛋白表达差异有统计学意义（F=11.207,P<0.001）;过表达组ALP蛋白的表达水平明显高于siRNA干扰组,差异有统计学意义（q=2.991,P<0.05）;牵张应力组ALP蛋白的表达水平明显高于空白对照组,差异有统计学意义（q=3.007,P<0.05）。Runx2蛋白表达具有同样趋势。细胞内钙离子检测结果显示,过表达组和牵张应力组细胞内钙离子荧光强度明显高于siRNA干扰组。结论 机械牵张应力可以促进Piezo1蛋白的表达,以Ca²⁺为第二信使,激活Notch1信号通路,激活ALP、Runx2、OCN和BSP的表达,促进hPDLSC成骨分化。而siRNA-Piezo1干扰质粒可以阻断这一进程,反之,Piezo1的过表达质粒可以促进hPDLSC成骨分化进程。     

年龄因素对人牙周膜干细胞生物学行为影响的研究

 目的　研究年龄因素对人牙周膜干细胞（periodontal ligament stem cells，PDLSC）生物学行为的影响。方法  选取2017年10月至2018年10月于青岛市口腔医院口腔外科就诊的因阻生齿或正畸需要拔除牙齿的患者18例，根据不同年龄段分为3组，分别为A组（18 ~ 20 岁）、B组（30 ~ 35岁）、C组（50 ~ 55岁），每组6例。原代培养人PDLSC，采用衰老相关β-半乳糖苷酶染色、MTT法检测各组细胞衰老程度和增殖能力。成骨诱导人PDLSC，采用碱性磷酸酶（ALP）染色和茜素红染色检测各组细胞的成骨分化能力。采用实时荧光定量PCR（qRT-PCR）检测各组多能性相关转录因子Nanog和Sox2的表达水平。结果　各组均成功原代培养出人PDLSC，经衰老相关β-半乳糖苷酶染色发现，A组阳性染色细胞率最低、B组次之、C组最高，组间差异均有统计学意义（均P < 0.05）。通过MTT法检测发现，不同年龄组OD值比较，差异有统计学意义（F = 22.354，P = 0.009），且各组OD值均随着培养时间的延长而增大（F = 28.367，P = 0.018），年龄与时间之间有明显的交互作用（F = 26.431，P = 0.015）。ALP染色和茜素红染色均发现，A组染色面积百分比大于B组和C组，B组染色面积百分比大于C组，差异均有统计学意义（均P < 0.05）。qRT-PCR结果显示，A组Nanog和Sox2的相对表达量均高于B组和C组，B组Nanog和Sox2的相对表达量高于C组，差异均有统计学意义（均P < 0.05）。结论　随着年龄增加，衰老的人PDLSC增多，其增殖能力、成骨分化能力以及多能性相关转录因子的表达水平也显著下降。     

牙周膜成纤维细胞-富血小板胶的合成及其成骨活性表达的研究

目的：体外合成牙周膜成纤维细胞-富血小板胶（Periodontal ligament fibroblasts-Platelet—Rich Plasma gel，PDLFs／PRP—gel）并探讨其在临床应用中最佳植入时间。方法：体外合成PDLFs／PRP—gel；并测定其碱性磷酸酶（Alkaline phosphatase，ALP）含量；免疫组化法检测基质中骨涎蛋白（Bone sialoprotein，BSP）、骨桥蛋白（Osteopontin，OPN）含量；Von Kossa染色法观察沉积钙盐。结果：PDLFs／PRP—gel基质内第6d时ALP含量明显增高，第8-10d达到峰值，11d后迅速下降，与对照组比较差异有统计学意义。基质内骨桥蛋白在15—16d时表达明显，与其他时间的实验组比较有统计学差异；骨涎蛋白在4～6d和15～18d时表达明显，与其他时间的实验组比较有统计学差异。PRP—gel缓释液培养的人牙周膜成纤维细胞约在第15d时出现钙化结节。结论：临床牙周组织再生术中宜选用在体外培养8—15d的PDLFs／PRP—gel进行植入。