鼠口腔黏膜上皮干细胞的分离培养与鉴定

目的 探索和建立鼠口腔黏膜上皮干细胞体外分离与培养的技术方法。方法 利用纤维结合素-胶原包被的培养瓶快速黏附法分离鼠口腔黏膜上皮干细胞，以鼠成纤维细胞条件培养基和无钙EMEM培养基配．制表皮干细胞培养基进行培养。通过β1整合素和角蛋白15（K15）免疫组化染色对培养细胞进行鉴定。结果 鼠口腔黏膜上皮干细胞呈明显克隆性生长，β1整合素和角蛋白15（K15）免疫组化染色呈强阳性。结论 利用纤维结合素-胶原包被的培养瓶快速黏附法分离鼠口腔黏膜上皮干细胞，用鼠成纤维细胞条件培养基和无钙EMEM培养基配制的表皮干细胞培养基进行培养，可以初步实现对鼠口腔黏膜上皮干细胞体外分离与培养。     

Ⅳ型胶原粘附法纯化富含干细胞的鼠切牙颈环上皮细胞

目的:探讨Ⅳ型胶原对实验室培养条件下富含干细胞的鼠切牙颈环上皮细胞纯化的影响.方法:以Ⅳ型胶原包被培养瓶,再接种颈环上皮混杂细胞,以未包被胶原的培养瓶做对照,观察贴擘细胞的生长形态;用β1整合素和CK角蛋白对贴壁细胞进行免疫组织化学染色鉴定.结果:与对照组相比,Ⅳ型胶原包被组纤维样细胞被完全抑制,贴壁生长的细胞全部为上皮细胞,形成大的克隆,β1整合索和CK角蚩白呈阳性表达.结论:Ⅳ型胶原粘附法可以获取较纯的鼠切牙颈环上皮细胞.     

富含干细胞的鼠切牙颈环上皮细胞的分离和原代培养

目的:获取较纯的鼠切牙颈环上皮细胞.方法:分离出生2 d的Wistar大乳鼠下颌切牙完整的牙胚,机械剥离颈环组织,酶消化法与组织块法结合培养原代混合细胞.采用Ⅳ型胶原包被培养法纯化上皮干细胞.结果:原代培养的细胞为混杂细胞,经2～3次纯化可完全去除纤维样细胞.颈环上皮细胞呈多角形,克隆状生长.结论:Ⅳ型胶原包被培养法可以获取纯化的鼠颈环上皮细胞.     

人牙周韧带细胞膜片的体外实验研究

目的 观察体外培养人牙周韧带细胞(hPDLCs)的成骨、成脂能力;构建hPDLCs膜片并鉴定膜片细胞外基质的主要结构蛋白.方法 通过酶联组织块法分离并纯化培养hPDLCs;免疫细胞化学法鉴定hPDLCs来源及干细胞标志物STRO-1表达情况;取第2～4代细胞分别用成骨诱导培养基和成脂诱导培养基培养,茜素红及油红O染色检测hPDLCs的成骨、成脂能力;制备成熟的hPDLCs膜片,苏木素-伊红(HE)及免疫组织化学染色法鉴定膜片细胞外基质主要结构蛋白Ⅰ型胶原、层黏蛋白及纤连蛋白的表达情况.结果 经原代培养的hPDLCs抗波形蛋白染色阳性,干细胞标志物STRO-1染色阳性,证实hPDLCs来源于间充质并具有干细胞潜在分化能力;hPDLCs在诱导成骨、成脂分化培养14～16 d后,茜素红及油红O染色结果阳性提示hPDLCs具有良好的成骨及成脂分化能力;HE染色及免疫化学染色发现hPDLCs膜片高表达细胞外基质主要结构蛋白Ⅰ型胶原、层黏蛋白及纤连蛋白.结论 本研究从细胞分离培养,来源鉴定、多向分化潜能诱导及细胞膜片技术等方面证实PDLCs中存在成体干细胞,提示hPDLCs膜片能为牙周组织再生种子细胞提供新来源,可进一步深入研究.     

Beagle犬脂肪基质干细胞的培养及分化研究

目的：体外培养Beagle犬脂肪基质干细胞（dog adipose-derived stromal cells，dADSCs），定向诱导分化为脂肪、成骨细胞，为口腔组织工程提供种子细胞。方法：取Beagle犬皮下脂肪组织，剪碎，经Ⅰ型胶原酶消化培养犬脂肪基质干细胞，取第3代细胞鉴定其来源及干细胞标记并向脂肪、成骨细胞诱导分化。结果：该细胞波形丝蛋白表达阳性，角蛋白表达阴性，CD44表达阳性；成脂诱导后，经油红O染色可见细胞内脂滴的积累；成骨诱导后，ALP、钙结节、骨钙素、Ⅰ型胶原均阳性表达。结论：Beagle犬脂肪组织中存在着具有能分化为脂肪、成骨细胞的脂肪基质干细胞，此脂肪基质干细胞可作为口腔组织工程种子细胞来源。     

人CD146＋牙周膜干细胞的分选及其干细胞特性的鉴定

目的：通过酶消化法及流式细胞分选技术分选CD146＋牙周膜干细胞（Periodontal ligament cells,PDLCs）,探讨其干细胞特性。方法：采用流式细胞技术从人牙周膜细胞中分选CD146＋PDLCs,并对其进行免疫组化染色（角蛋白、波形丝蛋白及Stro-1）,成骨、成脂诱导分化及克隆形成实验鉴定其干细胞特性。结果：CD146＋PDLCs表达波形丝蛋白及Stro-1,具有成骨及成脂分化能力,体外培养能形成细胞克隆。结论：以CD146为分选指标的流式细胞分选技术可作为牙周膜干细胞筛选的方法。     

犬牙周膜干细胞体外分离培养和鉴定的实验研究

目的体外分离培养犬牙周膜干细胞,并对其进行生物学鉴定。方法采用有限稀释法进行犬牙周膜细胞克隆筛选,获得单细胞克隆来源细胞,检测其克隆形成率,并采用免疫组织化学染色、碱性磷酸酶染色、苏木精-伊红染色以及生长因子分化诱导等方法观察其生物学特性。结果1）克隆化分离培养的犬牙周膜干细胞呈集落状生长,增殖快,克隆形成率为0.95％。2）苏木精-伊红染色见细胞呈成纤维样,体积小、核大,有较多的双核细胞。3）碱性磷酸酶染色阳性。4）细胞表型组化鉴定波形丝蛋白、Ⅰ型胶原、Ⅲ型胶原阳性,间充质干细胞表面标志STRO-1染色阳性,角蛋白染色阴性。5）在含β-磷酸甘油钠、维生素C、地塞米松的矿化液中,细胞被诱导分化为成骨细胞,碱性磷酸酶活性增强,Ⅲ型胶原染色阴性,能形成矿化结节,细胞表达成骨细胞特异的骨涎蛋白,在含β-羟基乙醇的培养液中,被诱导分化为神经细胞样细胞。结论克隆化分离培养的犬牙周膜干细胞具有很强的克隆形成能力,具有间充质干细胞表型和多向分化潜能。     

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。     

大鼠脂肪干细胞体外培养及成骨诱导分化研究

目的:观察SD大鼠脂肪干细胞(adipose-derived stem cells,ASCs)体外培养的基本生物学特性、鉴定其诱导成骨分化潜能。方法:无菌条件下取6周龄SD大鼠腹股沟处脂肪组织,0.1%I型胶原酶消化,分离培养。显微镜下观察细胞形态;采用MTT比色法绘制细胞生长曲线;用流式细胞仪检测细胞表面标志物;取第3代细胞行成骨和成脂诱导培养,分别用碱性磷酸酶(ALP)、茜素红染色和油红O染色鉴定成骨诱导效果。结果:从SD大鼠脂肪组织中分离获得的ASCs呈长梭形或多角形,呈集落生长;细胞生长曲线呈"S"型,具有较强的增殖能力;流式细胞仪分析示:CD29、CD90高表达,CD34、CD45低表达;成骨诱导后ALP染色及茜素红染色阳性,成脂诱导后油红O染色阳性。结论:分离培养的细胞为ASCs,具有成骨潜能。     

鼠尾胶原在培养鼠下切牙颈环上皮细胞中的应用

目的: 建立用自制鼠尾胶原为贴附底物的大鼠颈环上皮细胞的体外培养模式.方法: 制作鼠尾胶原,观察用自制的鼠尾胶原培养出大鼠颈环上皮细胞的效果,并对纯化的上皮细胞进行免疫组织化学染色鉴定.结果: 自制的鼠尾胶原能正常地培养出大鼠颈环上皮细胞,呈多角形,克隆状生长,β1整合素和角蛋白免疫组织化学染色阳性.结论: 鼠尾胶原可作大鼠颈环上皮细胞的体外培养贴附底物,为今后研究牙齿发育机制提供简便的方法和途径.     

Isolation of oral epithelial progenitors using collagen IV

Objective:  Although oral mucosal epithelial stem cells are thought to reside in the basal layer, such cells have not yet been isolated. We isolated a population of rabbit oral epithelial progenitor cells containing putative stem cells.
Materials and methods:  Epithelial cells harvested from rabbit buccal mucosa were allowed to adhere to dishes coated with collagen IV for periods ranging from 10 min to 16 h. The properties of individual cell populations were evaluated using BrdU, Ki-67, integrin β 1, integrin α 6 and keratin 13 using colony forming efficiency (CFE).
Results:  Cells that adhered to collagen IV-coated dishes within 10 min were enriched about sixfold in terms of BrdU incorporation, Ki-67, integrin α 6 and integrin β 1 were strongly expressed. Interestingly, keratin 13 was faintly expressed. The CFE of rapidly adherent cells among oral epithelial cells was significant compared with other cell populations.
Conclusions:  These results suggested that rabbit oral epithelial cells could be isolated by depending on adhesiveness to collagen IV, especially when segregated according to progenitor cell properties. Putative progenitor cells with stem cell properties were most effectively harvested within 10 min. Our separation procedure should be a useful tool with which to isolate epithelial stem cells for regenerative medicine.     

The culture of human buccal and vaginal epithelial cells for permeability studies.

The objective of the present study was to develop a single improved technique to culture human vaginal and buccal epithelial cells, whereby the cultured cells can be used in drug permeability studies. Cells were obtained from healthy human vaginal and buccal mucosa following vaginal hysterectomies and various oral surgical procedures. Tissue obtained was washed extensively in phosphate buffered saline (PBS, pH 7.3) containing antibiotics and amphotericin-B. Tissue specimens were cut into small pieces and plated out in 24-well plates. After drying, the full medium was added. Cell growth occurred within 4-6 days from primary explants and confluency was reached within 2-3 weeks. Primary explants yielded epithelial cells with minimal fibroblast contamination. After trypsinization, cells were seeded into collagen-coated wells and onto Transwell membranes. Trypsinized cells grew best on collagen-coated surfaces yielding more than one layer. The average steady state flux for the collagen-coated membranes (ccm's) containing either buccal or vaginal epithelial layers towards water was 6-10 times lower than that found for the cell-free ccm's. Fluxes for cultured cells on ccm's were 3x higher than those obtained for intact buccal and vaginal mucosa. Growth and permeability to water of the vaginal and buccal epithelial cells were comparable, confirming the similarity of these two tissues and the suitability of using the former as a model for the latter in permeability to tritiated water.     

成体人牙髓干细胞的分离与鉴定

目的从成体人牙髓组织中分离培养牙髓干细胞，初步探讨其分化潜能。方法选取年轻患者因正畸或阻生拔除的健康第三磨牙，取出牙髓，采用酶消化及过滤法得到单细胞悬液，有限稀释法原代培养。扩大培养细胞克隆，检测STRO-1的表达。体外诱导分化后对各克隆从碱性磷酸酶(ALP)活性、矿化结节形成、牙本质涎蛋白(DSP)表达、Oil Red—O染色、PPARr2基因表达等方面进行检测。结果克隆来源细胞STRO-1表达阳性。在矿化液诱导下，克隆细胞呈现明显高的ALP活性；能够形成矿化结节；可以分泌表达DSP，已向成牙本质细胞方向发生了分化。成脂肪诱导后，Oil Red—O染色阳性，可检测到PPARr2基因表达。结论从成体人牙髓组织中可以分离培养出干细胞，在体外能有效增殖并保持低分化状态。     

The fibroblast population in oral submucous fibrosis

The purpose of the investigation was to compare the morphology of fibroblasts cultured from healthy oral mucosa and mucosa of patients with oral submucous fibrosis (OSF) and to collate the occurrence of cell types of similar morphology. Cells cultured from biopsy specimens from the buccal mucosa of six subjects who did not chew the areca nut and six patients with OSF who chewed areca nut were grown according to standard techniques. Ninety cells per cell line were recorded daily for 8 days, classified into types F1, F2 and F3 according to their morphology, and the results statistically analyzed. We found that there was a relative increase of F3 cells in relation to Fl cells in OSF resulting in the ratio of F3 to F1 cells being significantly larger in OSF than the ratio in the controls. As it has been reported that F3 cells m rat connective tissues produce significantly more collagen types I and III than F1 cells, we concluded that a change of fibroblast population has occurred in OSF and that this relative increase of F3 cells in humans, which could be committed to the production of large quantities of collagen, can be an explanation for the excessive collagen formation in OSF.     

牙囊干细胞诱导成骨的体外研究

目的:体外研究牙囊干细胞(DFSC)的成骨能力,为其在牙周组织工程中的应用提供理论基础。方法:结合组织块法和酶消化法分离培养DFSC,用有限稀释法纯化细胞,经细胞形态、免疫组化和流式细胞技术鉴定DFSC后,然后进行成骨诱导,并通过碱性磷酸酶和茜素红染色、Real-timePCR、Western-blot检测成骨/牙骨质细胞表面标记物ALP、OCN、BMP2、RUNX2的表达。结果:DFSC具有较强的增殖能力,免疫组化示波形丝蛋白表达阳性,角蛋白表达阴性;DFSC高表达间充质干细胞表面标记物CD44、CD90、CD105、CD146;随着诱导时间的增加,其ALP、OCN、BMP2、RUNX2的表达明显上调(P＜0.05)。结论:DFSC是牙周组织再生良好的种子细胞,必将为牙周组织再生治疗开辟新的途径。     

大鼠颊黏膜鳞状细胞癌单克隆细胞系Rca-B的建立及其生物学特性研究

目的：建立大鼠颊黏膜鳞癌单克隆细胞系，并观察其生物学特性，为口腔癌的研究提供大鼠来源的恶性肿瘤细胞系、移植瘤和转移动物模型。方法：将4-硝基喹啉-1-氧化物诱发的SD大鼠颊黏膜鳞癌组织标本进行体外传代培养，利用有限稀释法获得单克隆细胞。光学和电子显微镜观察该细胞的形态变化，细胞计数和四唑盐比色法观察细胞的增殖能力，染色体分析确定细胞核型特点，流式细胞仪检测其细胞周期；动物实验观察细胞裸鼠皮下接种成瘤率及实验性肺转移能力。结果：成功得到单克隆细胞系，细胞形态和生物学观察结果表明，该细胞系符合鳞癌细胞的基本特征，65代细胞群体倍增时间为25．44h，平板克隆形成率为56．30％。细胞周期分布S期占20．13％；染色体为四倍体核型。角质蛋白和波形蛋白免疫组织化学染色均为阳性。细胞系裸鼠皮下接种成瘤为16／16；细胞系行裸鼠尾静脉注射后，其实验性肺转移为10／10；该单克隆细胞系SD大鼠同种异体皮下接种不能成瘤。结论：成功建立SD大鼠颊黏膜鳞癌细胞系Rca—B，细胞系具有高转移鳞癌细胞的典型生物学特征；该细胞系是研究口腔鳞癌分子发生机制和防治策略的又一理想模型。     

毛囊bulge干细胞培养方法的比较研究

目的通过对限定性角质形成细胞无血清培养基（DK-SFM）和3T3滋养层培养毛囊bulge干细胞的比较,寻求既能方便获得大量毛囊bulge干细胞,又能减少其分化的理想培养条件。方法采用DK-SFM和3T3滋养层分别培 养毛囊bulge干细胞,通过倒置显微镜观察毛囊bulge干细胞的形态,免疫荧光染色检测毛囊bulge干细胞的特异性标 记细胞角蛋白19（CK19）和分化相关抗体群34（CD34）的表达鉴定毛囊bulge干细胞。通过比较2种方法培养的毛囊bulge干细胞的克隆形成率和CD34阳性率的差异,分别比较毛囊bulge干细胞的增殖能力和干细胞的数量,综合评估 2种培养方法的优劣。结果倒置显微镜观察2种培养方法所得毛囊bulge干细胞均为铺路石状。免疫荧光染色检测 2种培养方法培养的毛囊bulge干细胞CK19和CD34均呈阳性。DK-SFM和3T3滋养层培养的毛囊bulge干细胞克隆形成 率分别为69.4%和62.2%。流式细胞仪检测DK-SFM培养的毛囊bulge干细胞中CD34阳性率为72.3%;3T3滋养层培养 的毛囊bulge干细胞中CD34阳性率为34.7％。结论DK-SFM和3T3滋养层培养作为毛囊bulge干细胞的2种培养方法, 均可以获得未分化的毛囊bulge干细胞。用3T3滋养层培养毛囊bulge干细胞的方法比较复杂,且所获的细胞混杂有 3T3细胞,但用此种方法可获得大量的毛囊bulge干细胞。用DK-SFM培养毛囊bulge干细胞方法简单,但细胞不易贴 壁,且数量较少,但能有效分离出较纯的毛囊bulge干细胞,保持较好的细胞活性。     

Mucosal keratinocyte isolation: a short comparative study on thermolysin and dispase

New techniques for reconstructing large defects of the floor of the mouth include the use of cultured mucosal substitutes. The purpose of this study was to compare dispase and thermolysin for keratinocyte isolation. Keratinocyte yield per surface area of rabbit buccal mucosa was assessed by histology, cytokeratin 13 (CK13) staining, seeding efficiency analysis and cell diameter quantification. Surface areas of cultured mucosa were calculated. Histology showed that treatment by thermolysin resulted in incomplete separation of epidermis from dermis. Also, the absolute number of keratinocytes/cm(2) isolated mucosa, cell yield, cell size and seeding efficiencies was higher in the dispase group. A 3.45-fold larger graft could be reconstituted using dispase. The use of dispase, rather than thermolysin, to isolate cells from buccal mucosa is concluded to be favourable.     

人血清白蛋白对人牙龈上皮细胞粘附作用的影响

目的：探讨人血清白蛋白（human serum allbumin,HSA）对人牙龈上皮细胞（human gingival epithelial cels,HGE)粘附作用的影响。方法：使用角化细胞无血清培养基分离酶原代培养HGE，并用广谱细胞角蛋白单抗做细胞鉴定，细胞接种24h内用MTT法连续观测HSA聚苯乙烯表面预孵育组及培养基含HSA组HGE的粘附，在含HSA培养基中观测HGE的生长曲线。结果HGE免疫组化染色阳性；HSA预孵育组8h以内粘附的细胞数量显著少于培养基含HSA组及对照组，10－24h两实验组与对照间差异无显著性；生长曲线各时间点细胞的数量， 实验组与对照组相比差异无显著性。结论：HSA预孵育于聚苯乙烯表面，对早期HGE的粘附产生抑制作用。     

槟榔提取物抑制人类口腔粘膜角朊细胞生长的实验研究

目的：建立人类口腔粘膜上皮角朊细胞体外原代培养方法：探讨口腔粘膜下纤维性变患者上皮厚度改变的机理;方法;先对人类口腔粘膜上皮角朊细胞进行分离,培养和鉴定,然后用四唑盐比色试验检测ＯＳＦ患者和正常人口腔粘膜上皮ＫＣ增殖状况,并且观察槟榔提取物对ＫＣ增殖的影响。