间充质干细胞的旁分泌作用及其在颅颌面骨再生治疗中的应用研究进展

间充质干细胞（mesenchymal stem cells,MSCs）自被发现以来,一直在再生医学中作为理想的种子细胞,利用多向分化的潜能分化为组织细胞,以修复组织缺损。然而近年来人们发现,进入体内的MSCs增殖分化能力有限,其主要是通过旁分泌作用诱导受体自身细胞的增殖分化,从而实现局部组织的修复再生。现对MSCs的旁分泌作用及其在颅颌面骨再生治疗中的应用做一综述。     

尼古丁上调口腔鳞状细胞癌抗氧化蛋白Prx1的表达

目的：探讨口腔鳞状细胞癌（oral squamous cell carcinoma,OSCC）中抗氧化蛋白Prx1与烟草诱导的氧化应激的相关性。方法：采用RT-PCR、Western Blot方法,观察尼古丁作用相同时间（48h）不同浓度（0.1、1、10、100μmol/L）及同一浓度（1μmol/L）不同时间（24、48、72h）对Tca8113细胞Prx1mRNA及蛋白表达的影响。结果：尼古丁可诱导Tca8113细胞Prx1mRNA和蛋白表达增加。结论：Prx1表达增高可能与烟草诱导的氧化应激有相关性,在烟草诱导的OSCC发生发展中可能发挥重要作用。     

转基因间充质干细胞在骨组织工程中的应用

近来，随着基因工程技术的发展，基因治疗在组织工程中得到了广泛地应用。间充质干细胞（mesenchymal stem cells，MSCs）作为骨组织工程的首选种子细胞，自然也成为基因治疗的重要工具和靶目标，利用转基因的MSCs提高骨组织的修复效果也成为了研究热点。     

蜗牛蛋白调控腭部发生发育的机制

蜗牛蛋白（Snail）参与调控腭部融合过程中腭中缝上皮（MES）细胞的上皮间质转化（EMT）、程序性细胞死亡（PCD）或存活以及细胞迁移等生物学行为,Snail基因失去了转化生长因子-β3的抑制而表达水平升高,此时Snail作为MEE/MES细胞的存活因子,导致腭中线上皮（MEE）/MES细胞持续存在并诱发腭裂。Snail基因编码具有锌指结构的Snail1、Snail2和Snail3等转录因子,其家族具有相似的结构,可以结合到E-钙黏蛋白的启动子,抑制其表达,可以在胚胎发生发育和肿瘤转移过程中诱导EMT。Snail基因可防止细胞因为存活因子的减少或PCD因子的积累而死亡,故Snail基因是MEE/MES细胞的存活因子和细胞运动的诱导因子。微小RNA（miRNA）不仅在人类癌细胞中发挥着重要的作用,而且在胚胎健康发育过程中必不可少。miRNA表达模式的变异和miRNA在mRNA的结合位点的结构变异,可能导致颅面发育变异。本文就Snail基因结构与功能、Snail基因在侧腭突发育过程中表达、Snail基因与MEE/MES细胞的EMT、Snail基因与MEE/MES程序性细胞死、Snail基因是MEE/MES细胞的存活因子、微小RNA调控Snail在MEE/MES消失过程中的作用等研究进展作一综述。     

TNF-α刺激下人牙周膜干细胞对CD3+T细胞功能的影响

目的:研究在TNF-α刺激下牙周膜干细胞(periodontal ligament stem cells,PDLSCs)对CD3+T细胞功能的影响.方法:分别获取20-30岁患者的健康离体牙8例,分离培养获得PDLSCs;采用免疫磁珠法分离获取20-30岁健康患者的CD3+T细胞;PDLSCs与CD3+T细胞共培养,PDLSCs加炎症因子(TNFα 10ng/mL)与CD3+T细胞共培养,PDLSCs与CD3+T细胞比例分别为1∶10、1∶20、1∶50、1∶100,共培养时间为48h;共培养48h后分别提取不同组的RNA并反转录为cDNA,检测凋亡、炎症及增殖相关基因的变化.结果:两组细胞生长状态良好;随共培养组PDLSCs比例的降低,正常共培养组CD3+T细胞caspase3/8、IL-6,TNFα mRNA表达水平均降低,在1∶50时趋于稳定;随共培养组PDLSCs比例的降低,炎症因子共培养组CD3+T细胞caspase3/8、IL-6、TNFα mRNA表达水平均降低,在1∶20时趋于稳定.当PDLSCs与CD3+T细胞比例分别为1∶20时H-PDLSCs(Healthy-Periodontal ligament stem cells)、I-PDLSCs(Inflammatory-Periodontal ligament stem cells)对CD3+T细胞CCND-1基因有显著抑制作用,H-PDLSCs对CD3+T细胞CCND-1的抑制作用最显著.结论:在恰当的比例时,正常及炎症PDLSCs对CD3+T细胞的凋亡及炎症因子分泌均有抑制作用,在PDLSCs与CD3+T细胞比例1∶20时,可能是由于炎症因子的存在增强了其抑制作用.     

BmaI1在经典Wnt通路调控间充质干细胞衰老中的作用

目的： 探讨Bmal1与Wnt经典信号通路对间充质干细胞（mesenchymal stem cells,MSCs）的衰老是否具有协同或拮抗作用。方法： 分为转染组和空白对照组,转染组用Bmal1的慢病毒转染NIH-3T3细胞表达增强后,通过实时荧光定量PCR和Western免疫印迹等方法检测转染组和空白组β-catenin、TCF1表达量。采用SPSS 19.0软件包对数据进行统计学分析。结果： 经PCR鉴定,Bmal1基因重组慢病毒载体构建成功,转染Bmal1表达增强后,β-catenin表达也显著增强（P<0.05）,但与TCF1增强比例不同;而TCF1的变化无统计学意义。结论： Bmal1对Wnt 经典信号通路直接或间接调控小鼠 BMSCs 的增龄性改变具有协同作用。     

p75神经营养受体阳性外胚间充质干细胞的体外获取与生物学特性研究

目的 研究鼠胚胎颌面组织中外胚间充质干细胞(ectomesenchymal stem cells,EMSC)的生物学特性,探讨p75+EMSC的体外分化及影响因素,为揭示牙齿发生、发育机制提供实验依据.方法 通过提取12.5 d SD鼠胚胎颌面组织,用免疫荧光染色方法显示EMSC的迁移位置;用p75神经营养因子受体标记EMSC,通过流式细胞技术分选出p75+EMSC,并检测细胞周期和干细胞相关细胞表面抗原.结果 免疫荧光染色结果说明颅神经嵴源性干细胞在鼠胚胎12.5 d迁移到颌突中.p75+EMSC的阳性分选率为6.1％,细胞呈均匀成纤维细胞形态,生长曲线呈“S”型,在传代过程中S期的比例稳定.细胞特异性标志物CD29、CD146、Stro-1标记p75+EMSC,其在p75+EMSC中的表达均较高(＞90％).结论 SD鼠受孕12.5 d时,胚胎的牙齿启动尚未发生;分选后的p75+EMSC在传代过程中具有稳定的增殖能力和干细胞特征,尚未开始分化.     

TNF-α刺激下人牙周膜干细胞对CD3+T细胞功能的影响

目的:研究在TNF-α刺激下牙周膜干细胞(periodontal ligament stem cells,PDLSCs)对CD3+T细胞功能的影响.方法:分别获取20-30岁患者的健康离体牙8例,分离培养获得PDLSCs;采用免疫磁珠法分离获取20-30岁健康患者的CD3+T细胞;PDLSCs与CD3+T细胞共培养,PDLSCs加炎症因子(TNFα 10ng/mL)与CD3+T细胞共培养,PDLSCs与CD3+T细胞比例分别为1∶10、1∶20、1∶50、1∶100,共培养时间为48h;共培养48h后分别提取不同组的RNA并反转录为cDNA,检测凋亡、炎症及增殖相关基因的变化.结果:两组细胞生长状态良好;随共培养组PDLSCs比例的降低,正常共培养组CD3+T细胞caspase3/8、IL-6,TNFα mRNA表达水平均降低,在1∶50时趋于稳定;随共培养组PDLSCs比例的降低,炎症因子共培养组CD3+T细胞caspase3/8、IL-6、TNFα mRNA表达水平均降低,在1∶20时趋于稳定.当PDLSCs与CD3+T细胞比例分别为1∶20时H-PDLSCs(Healthy-Periodontal ligament stem cells)、I-PDLSCs(Inflammatory-Periodontal ligament stem cells)对CD3+T细胞CCND-1基因有显著抑制作用,H-PDLSCs对CD3+T细胞CCND-1的抑制作用最显著.结论:在恰当的比例时,正常及炎症PDLSCs对CD3+T细胞的凋亡及炎症因子分泌均有抑制作用,在PDLSCs与CD3+T细胞比例1∶20时,可能是由于炎症因子的存在增强了其抑制作用.     

同源盒基因Prx1/Prx2在牙齿及骨组织发育中的作用

同源盒基因Prx1/Prx2在调控细胞增殖、骨和牙齿发育等方面具有重要的生物学特性。研究表明,Prx基因在上皮及间充质的相互诱导过程中起关键作用,并且通过调节Shh而间接调控牙齿的发育。本文就其在调控牙齿发育和骨与软骨形成等方面的研究作一综述。     

Gli1阳性细胞在牙周组织发育中的时空分布特点及功能研究

目的 利用转基因小鼠探究牙周膜内Gli 阳性（Gli1 ⁺）细胞的表达分布以及Gli1 ⁺细胞在牙周组织发育过程中的作用。方法 收集3、6和8周龄Gli1 ^lacZ/+小鼠下颌骨,通过β-半乳糖苷酶组织化学染色（X-gal染色）观察牙周膜内Gli1 ⁺细胞的时间和空间分布特点。然后,通过注射他莫昔芬诱导3周龄Gli1-Cre ^ERT2/+;R26R ^tdTomato/+小鼠表达红色荧光蛋白tdTomato,对Gli1 ⁺细胞及其子代细胞（tdTomato ⁺细胞）进行动态追踪。结果 3周龄小鼠牙周膜内分布大量Gli1 ⁺细胞,随着年龄的增长,Gli1在牙周膜内的数量逐渐减少（P<0.05）。tdTomato ⁺细胞随着诱导时间的延长,数量逐渐增加（P<0.05）,且逐渐分化成熟为成纤维细胞、牙骨质细胞和骨细胞。结论 牙周膜内Gli1 ⁺细胞具有多向分化的潜能,是牙周组织发育过程中重要的细胞来源。     

Putative stem cells in regenerating human periodontium

Background and Objective:  Human postnatal stem cells have been identified in periodontal ligament, with the potential to regenerate the periodontium in vivo . However, it is unclear if periodontal ligament stem cells are present in regenerating periodontal tissues. The aim of this study was to identify and localize putative stem cells in block biopsies and explant cultures of regenerating human periodontal tissues.
Material and Methods:  Guided tissue regeneration was carried out on the molars of three human volunteers. After 6 wk, the teeth with the surrounding regenerating tissues and bone were surgically removed and processed for immunohistochemistry. The mesenchymal stem cell-associated markers STRO-1, CD146 and CD44 were used to identify putative stem cells. Cell cultures established from regenerating tissue explants were analysed by flow cytometry to assess the expression of these markers. Mineralization, calcium concentration and adipogenic potential of regenerating tissue cells were assessed and compared with periodontal ligament stem cells, bone marrow stromal stem cells and gingival fibroblasts.
Results:  STRO-1⁺, CD44⁺ and CD146⁺ cells were identified in the regenerating tissues. They were found mainly in the paravascular and extravascular regions. Flow cytometry revealed that cultured regenerating tissue cells expressed all three mesenchymal stem cell associated markers. The regenerating tissue cells were able to form mineral deposits and lipid-containing adipocytes. However, the level of mineralization in these cells was lower than that of periodontal ligament stem cells and bone marrow stromal stem cells.
Conclusion:  Cells with characteristics of putative mesenchymal stem cells were found in regenerating periodontal tissues, implying their involvement in periodontal regeneration.     

Somatic stem cells for regenerative dentistry

Complex human tissues harbour stem cells and/or precursor cells, which are responsible for tissue development or repair. Recently, dental tissues such as periodontal ligament (PDL), dental papilla or dental follicle have been identified as easily accessible sources of undifferentiated cells. The dental stem cell biology might provide meaningful insights into the development of dental tissues and cellular differentiation processes. Dental stem cells could also be feasible tools for dental tissue engineering. Constructing complex structures like a periodontium, which provides the functional connection between a tooth or an implant and the surrounding jaw, could effectively improve modern dentistry. Dental precursor cells are attractive for novel approaches to treat diseases like periodontitis, dental caries or to improve dental pulp healing and the regeneration of craniofacial bone and teeth. These cells are easily accessible and, in contrast to bone-marrow-derived mesenchymal stem cells, are more closely related to dental tissues. This review gives a short overview of stem cells of dental origin.     

TNF-α刺激下牙周膜干细胞与CD3+T细胞共培养对Wnt/β-catenin信号通路的影响

目的:探讨炎症条件下牙周膜干细胞与CD3⁺T细胞共培养对经典Wnt/β-catenin信号通路的影响。方法:拔除健康前磨牙及第三磨牙,获取健康牙周膜干细胞(H-PDLSCs);人外源性TNF-α炎症因子10ng/mL刺激PDLSCs(I-PDLSCs)。免疫荧光染色检测H/I-PDLSCs Wnt通路蛋白β-catenin和LEF-1的表达。采用免疫磁珠法获取人的CD3⁺T细胞。H/I-PDLSCs与CD3⁺T细胞分别以1∶10,1∶20,1∶50,1∶100共培养,共培养48h,提取各组PDLSCs的RNA,反转录为c DNA后,qPCR检测β-catenin、LEF1、TCF4的基因表达;以上述比例共培养后收集各组细胞进行流式细胞仪细胞周期检测。结果:免疫荧光检测I-PDLSCs组β-catenin、LEF-1的表达强于H-PDLSCs组。共培养比例为1∶20和1∶50时,H-PDLSCs组β-catenin、LEF1的mRNA表达水平均降低;1∶20时TCF4 mRNA的表达水平增加,1∶50时降低。1∶20时,I-PDLSCs组β-catenin、LEF1的mRNA表达水平均增加,而在1∶50时基因的表达水平均降低。1∶20及1∶50时TCF4的表达水平均降低。H-PDLSCs与CD3⁺T细胞共培养比例为1∶10时与H-PDLSCs对照组相比其增殖指数显著提高;1∶20、1∶50及1∶100时其PI指数显著降低。I-PDLSCs与CD3⁺T细胞共培养比例为1∶10、1∶50及1∶100时与I-PDLSCs对照组相比其增殖指数显著降低(P<0.05)。结论:炎症条件可激活牙周膜干细胞的Wnt/β-catenin信号通路,H/I-PDLSCs与CD3⁺T细胞以1∶20、1∶50可稳定的下调Wnt/β-catenin经典信号通路,证实Wnt通路参与炎症条件下PDLSCs的免疫调节。     

The use of adult stem cells in rebuilding the human face

BACKGROUND: Stem cells have been isolated from a variety of embryonic and postnatal (adult) tissues, including bone marrow. Bone marrow stromal cells (BMSCs), which are non-blood-forming cells in marrow, contain a subset of skeletal stem cells (SSCs) that are able to regenerate all types of skeletal tissue: bone, cartilage, blood-supportive stromal cells and marrow fat cells. METHODS: Bone marrow suspensions are placed into culture for analysis of their biological character and for expansion of their number. The resulting populations of cells are used in a variety of assays to establish the existence of an adult SSC, and the ability of BMSC populations to regenerate hard tissues in the craniofacial region, in conjunction with appropriate scaffolds. RESULTS: Single-cell analysis established the existence of a true adult SSC in bone marrow. Populations of ex vivo expanded BMSCs (a subset of which are SSCs) are able to regenerate a bone/marrow organ. In conjunction with appropriate scaffolds, these cells can be used to regenerate bone in a variety of applications. CONCLUSIONS: BMSCs have the potential to re-create tissues of the craniofacial region to restore normal structure and function in reconstructing the hard tissues of a face. Ex vivo expanded BMSCs with scaffolds have been used in a limited number of patients to date, but likely will be used more extensively in the near future.     

Prx1调控PINK1/Parkin介导的线粒体自噬在实验性口腔黏膜癌变中的作用

目的检测口腔黏膜癌变过程中线粒体自噬相关蛋白PINK1、Parkin表达变化及抗氧化蛋白Prx1对其调控作用,探讨Prx1在口腔黏膜癌变中的作用机制。方法利用4NQO化学诱导Prx1^+/+和Prx1+/-小鼠舌黏膜癌变模型,采用免疫组织化学染色及Q-PCR方法,检测PINK1、Parkin在小鼠舌正常黏膜、白斑、白斑伴上皮异常增生、鳞癌组织中的表达。结果在Prx1^+/+小鼠舌癌变模型中,PINK1、Parkin蛋白及mRNA表达在舌白斑及白斑伴上皮异常增生组织中显著高于正常黏膜及舌癌组织;与Prx1^+/+小鼠各组相比,Prx1敲除导致小鼠舌白斑、白斑伴上皮异常增生及舌癌中PINK1蛋白及mRNA表达明显降低;小鼠舌白斑及舌癌组织中Parkin mRNA表达显著升高。结论线粒体自噬参与了口腔黏膜癌变过程;Prx1可能通过调控PINK1/Parkin介导的线粒体自噬在其中发挥重要作用。     

Craniofacial tissue engineering by stem cells

Craniofacial tissue engineering promises the regeneration or de novo formation of dental, oral, and craniofacial structures lost to congenital anomalies, trauma, and diseases. Virtually all craniofacial structures are derivatives of mesenchymal cells. Mesenchymal stem cells are the offspring of mesenchymal cells following asymmetrical division, and reside in various craniofacial structures in the adult. Cells with characteristics of adult stem cells have been isolated from the dental pulp, the deciduous tooth, and the periodontium. Several craniofacial structures--such as the mandibular condyle, calvarial bone, cranial suture, and subcutaneous adipose tissue--have been engineered from mesenchymal stem cells, growth factor, and/or gene therapy approaches. As a departure from the reliance of current clinical practice on durable materials such as amalgam, composites, and metallic alloys, biological therapies utilize mesenchymal stem cells, delivered or internally recruited, to generate craniofacial structures in temporary scaffolding biomaterials. Craniofacial tissue engineering is likely to be realized in the foreseeable future, and represents an opportunity that dentistry cannot afford to miss.     

Identification of marker genes distinguishing human periodontal ligament cells from human mesenchymal stem cells and human gingival fibroblasts

BACKGROUND AND OBJECTIVE: Molecular gene markers, which can distinguish human bone marrow mesenchymal stem cells from human fibroblasts, have recently been reported. Messenger RNA levels of tissue factor pathway inhibitor-2, major histocompatibility complex-DR-alpha, major histocompatibility complex-DR-beta, and neuroserpin are higher in human bone marrow mesenchymal stem cells than in human fibroblasts. However, human bone marrow mesenchymal stem cells express less apolipoprotein D mRNA than human fibroblasts. Periodontal ligament cells are a heterogeneous cell population including fibroblasts, mesenchymal stem cells, and progenitor cells of osteoblasts or cementoblasts. The use of molecular markers that distinguish human bone marrow mesenchymal stem cells from human fibroblasts may provide insight into the characteristics of human periodontal ligament cells. In this study, we compared the molecular markers of human periodontal ligament cells with those of human bone marrow mesenchymal stem cells and human gingival fibroblasts. MATERIAL AND METHODS: The mRNA expression of the molecular gene markers was analyzed using real-time polymerase chain reaction. Statistical differences were determined with the two-sided Mann-Whitney U-test. RESULTS: Messenger RNA levels of major histocompatibility complex-DR-alpha and major histocompatibility complex-DR-beta were lower and higher, respectively, in human periodontal ligament cells than in human bone marrow mesenchymal stem cells or human gingival fibroblasts. Human periodontal ligament cells showed the lowest apolipoprotein D mRNA levels among the three types of cells. CONCLUSION: Human periodontal ligament cells may be distinguished from human bone marrow mesenchymal stem cells and human gingival fibroblasts by the genes for apolipoprotein D, major histocompatibility complex-DR-alpha, and major histocompatibility complex-DR-beta.     

Mesenchymal stem cells acquire characteristics of cells in the periodontal ligament in vitro

Mesenchymal stem cells differentiate into multiple types of cells derived from mesenchyme. Periodontal ligament cells are primarily derived from mesenchyme; thus, we expected mesenchymal stem cells to differentiate into periodontal ligament. Using a combination of immunohistochemistry and in situ hybridization on co-cultures of mesenchymal stem cells and periodontal ligament, we observed a significant increase in mesenchymal stem cells' expression of osteocalcin and osteopontin and a significant decrease in expression of bone sialoprotein, characteristics of periodontal ligament in vivo. Increased osteopontin and osteocalcin and decreased bone sialoprotein expression was detected within 7 days and maintained through 21 days of co-culture. We conclude that contact or factors from periodontal ligament induced mesenchymal stem cells to obtain periodontal-ligament-like characteristics. Importantly, analysis of the data suggests the feasibility of utilizing mesenchymal stem cells in clinical applications for repairing and/or regenerating periodontal tissue.     

不同培养条件对CD133+原代口腔鳞癌细胞干性维持的影响

目的: 通过将CD133^+/-细胞从原代口腔鳞癌细胞中分离纯化,探索不同培养条件对CD133⁺原代口腔鳞癌细胞的干性维持及生物学特性的影响。方法: 应用CCK-8法检测CD133^+/-细胞亚群体外增殖能力以及对顺铂抵抗耐受能力,利用Transwell法检测顺铂对CD133^+/-细胞亚群侵袭能力的影响。以无血清培养法（含或不含白血病抑制因子LIF）和含血清培养法分别培养CD133⁺细胞亚群,流式细胞仪分析CD133⁺细胞的比例变化。在动物实验模型中验证CD133⁺和CD133^-细胞致瘤能力的差异,最后将移植瘤取出,行H-E染色和免疫组织化学染色。采用SPSS 25.0软件包对数据进行统计学分析。结果: 与CD133^-细胞亚群相比,CD133⁺细胞具备较强的体外增殖能力（P<0.05）和顺铂化疗耐受能力（P<0.001）。顺铂对CD133^-细胞亚群侵袭能力的影响更强（P<0.01）。无血清培养法更能维持CD133的比例（P<0.05）,无血清培养基是否添加LIF对CD133的比例维持无显著差异（P>0.05）。在裸鼠体内成瘤实验中,CD133⁺细胞亚群以较少的数量级表现出较强的致瘤能力（P<0.05）。结论: 无血清培养法可较好地维持原代口腔鳞癌细胞干细胞特性,添加LIF对原代口腔鳞癌细胞的干性维持无显著影响。