用PGA支架体外构建人口腔黏膜固有层的实验研究

目的:探讨利用可吸收生物材料聚羟基乙酸(polyglycolicacids,PGA)和口腔黏膜成纤维细胞(oralfibroblast,OFC)在体外构建组织工程化口腔黏膜固有层的可能性。方法:取细胞经体外培养,扩增至第3代,与PGA混合培养,形成细胞—生物材料复合物,每2d换液一次。动态观察细胞形态和粘附生长状况,于体外培养1周左右,取组织作电镜观察、组织学和RT-PCR检测。结果:复合物体外培养6d,OFC粘附在生物支架上,沿PGA纤维纵轴方向向两极伸展,细胞分泌基质并形成拉网状结构。HE染色和Masson染色均提示胶原纤维形成,RT-PCR显示胶原成分主要为Ⅰ型胶原。结论:应用PGA支架可以在体外成功构建口腔黏膜固有层组织。     

槟榔碱对口腔黏膜肌成纤维细胞分化的影响

目的探讨口腔黏膜下纤维性变组织中肌成纤维细胞的来源。方法通过体外口腔黏膜角质形成细胞和成纤维细胞共培养，用免疫组化、反转录聚合酶链反应等方法观察α-平滑肌肌动蛋白的表达。结果体外培养的口腔黏膜下纤维性变成纤维细胞中α-平滑肌肌动蛋白的阳性率为（85.80±3.56）％，正常口腔黏膜成纤维细胞中阳性率为（3.82±0.76）％。槟榔碱直接干预成纤维细胞后α-平滑肌肌动蛋白的表达与对照组相比差异无统计学意义（P〉0.05）；角质形成细胞与成纤维细胞共同培养组成纤维细胞中α-平滑肌肌动蛋白的表达增强；角质形成细胞经槟榔碱预处理后与成纤维细胞共同培养组α-平滑肌肌动蛋白的表达强于无干预共同培养组。结论口腔黏膜下纤维性变组织中成纤维细胞向肌成纤维细胞分化可能是槟榔成分与角质形成细胞共同作用的结果。     

口腔癌相关成纤维细胞的特殊体外行为研究

目的 探讨口腔颊癌中癌相关成纤维细胞(Carcinoma Associated Fibroblasts,CAFs)的特殊体外生物学行为。方法 组织块贴壁法培养CAFs及正常颊黏膜成纤维细胞(Normal Fibrolasts,NFs);通过体外生长形态观察,增殖、粘附行为差异的区别,比较两种细胞的生物学行为差别。结果 与NFs相比,CAFs均具有更高的增殖活性、贴壁率及迁移能力(P<0.05)。结论口腔癌CAFs的体外增殖、粘附能力均高于NFs,这或许提示其在口腔癌的侵袭、转移中发挥了重要的作用。     

人口腔黏膜角化上皮细胞的体外培养及生物学特性研究

目的:探讨人口腔黏膜角化上皮细胞的体外培养扩增方法及其生物学特性。方法:取牙周手术中切取的正常角化牙龈组织,经0.25％Dispase分离上皮层后,0.05％的胰酶分离为单个细胞,接种在无血清培养基中,进行原代培养及传代培养,并进行形态学观察、角蛋白免疫组化染色及其生长曲线、传代特点、各代生长特点的观察。结果:口腔黏膜角化上皮细胞能够在Epilife无血清培养基中稳定增殖传代,可连续传4-6代,成活45-60d,细胞生长呈铺路石状,角蛋白免疫组化染色阳性。结论:应用Epilife无血清培养基,可在短期内获得大量具有增殖能力的口腔黏膜角化上皮细胞。     

口腔黏膜上皮细胞体外癌变模型的建立

目的诱导永生化口腔上皮细胞系（HIOEC）细胞恶性转化，建立口腔黏膜上皮细胞体外癌变模型。方法通过化学致癌剂苯并芘[B（a）P]体外诱导HIOEC细胞，逐步筛选到鳞状细胞癌细胞系HIOEC-B（a）P。通过微分干涉显微镜和HE染色观察细胞形态学改变；用软琼脂集落形成及裸小鼠异体皮下成瘤的实验鉴定HIOEC-B（a）P细胞的恶性表型。结果①HIOEC细胞在B（a）P诱导培养后可以在正常生理钙离子浓度、含胎牛血清培养液中生长；②细胞在诱导过程中形态多变，最后稳定为多角形铺路石样上皮细胞；③第93代HIOEC-B（a）P细胞开始具备软琼脂集落形成能力；④HIOEC—B（a）P第55代细胞在裸小鼠皮下首次形成角化团块，第69代细胞形成分化较好的典型鳞状细胞癌，第74代、第96代细胞均形成与临床病理相似的Ⅰ～Ⅱ级鳞状细胞癌。结论生物因素合并化学致癌因素B（a）P可以诱导永生化口腔黏膜上皮细胞恶性转化，为进一步研究口腔黏膜上皮细胞多因素、多步骤癌变机制提供实验模型。     

3种义齿粘附剂的体外细胞毒性研究

目的:体外研究3种商品化义齿粘附剂的细胞毒性。方法:采用MTT法检测不同浓度义齿粘附剂浸提液对人原代口腔黏膜角质细胞、成纤维细胞和小鼠成纤维细胞系L929的细胞毒性。结果:3种义齿粘附剂对人原代口腔黏膜角质细胞有1~2级细胞毒性,对人原代口腔黏膜成纤维细胞有1级细胞毒性,对L929细胞却无毒性。高浓度义齿粘附剂浸提液一般比低浓度浸提液具有更强的细胞生长抑制作用。结论:义齿粘附剂对人原代口腔黏膜角质细胞和成纤维细胞有细胞毒性,对L929细胞却无细胞毒性,提示在义齿粘附剂的细胞毒性测试方面,人原代口腔黏膜细胞可能会提供更有价值的信息。     

白细胞介素-1β（IL-1β）和地塞米松对成纤维细胞增殖的影响

目的：检测白细胞介素-1β（IL-1β）和地塞米松（DEX）对体外培养的口腔正常黏膜与口腔扁平苔藓（OLP）黏膜成纤维细胞增殖的影响。方法：应用MTT法检测3种浓度梯度的IL-1β和DEX对体外培养的14例OLP黏膜成纤维细胞和11例正常口腔黏膜成纤维细胞增殖活性的影响。结果：MTT测定结果显示,IL-1β对OLP黏膜成纤维细胞和正常口腔黏膜成纤维细胞的增殖均有促进作用,且随浓度增加促进作用增强;相反,DEX却抑制两种成纤维细胞的增殖,浓度越高抑制作用越强。结论：白细胞介素-1β可促进成纤维细胞的增殖,地塞米松则抑制成纤维细胞的增殖。     

以胶原膜为支架构建组织工程化人口腔黏膜研究

目的：应用组织工程技术,以胶原膜为支架构建组织工程化人口腔黏膜。方法：体外培养、扩增人口腔黏膜角化细胞,通过细胞形态学及角蛋白免疫组化染色等对细胞进行定性研究,角化细胞接种于胶原膜上液面下培养1周后,升至液-气平面培养1周,光镜、电镜下观察大体组织形态及超微结构。结果：口腔黏膜上皮细胞可在生物支架材料胶原膜上粘附生长,并可形成上皮样组织,但无成熟的桥粒及基底膜样结构。胶原膜降解快,14d左右难以操作。结论：胶原膜与口腔黏膜角化细胞具有良好的生物相容性;由于降解速度和不易操作等特点,胶原膜作为支架材料构建组织工程化口腔黏膜有待深入研究。     

人口腔黏膜成纤维细胞的原代培养和在聚乳酸羟基乙酸膜上的种植实验

目的探索人口腔黏膜成纤维细胞体外培养,观察其在聚乳酸羟基乙酸(PLGA)膜上生长情况。方法体外分离培养人口腔黏膜成纤维细胞,苏木精-伊红染色,光镜、倒置相差显微镜、扫描电镜(SEM)观察其形态结构、免疫组化Vimentin,鉴定其间叶来源。结果光镜、倒置相差显微镜、SEM观察显示培养的细胞形态结构符合成纤维细胞特征,免疫组化结果显示Vimentin染色阳性。结论成功培养人口腔黏膜成纤维细胞,且其在PLGA膜上生长良好。     

口腔组织病理学

口腔黏膜上皮细胞及成纤维细胞与PGA的生物相容性研究，槟榔碱诱导体外培养的血管内皮细胞凋亡研究，表皮生长因子对MDPC-23细胞增殖和ALP活性的影响，bFGF、TGF-β对人牙髓干细胞增殖和分化能力的影响，体外三维培养下诱导人胚胎面突外胚间充质干细胞向成牙本质样细胞分化，牙本质涎磷蛋白反义核酸对体外培养牙胚发育、矿化的影响．     

口腔黏膜上皮细胞和成纤维细胞的复合培养

目的 为癌前病变临床病理研究建立一种简便、迅速和有效的口腔黏膜上皮细胞和成纤维细胞复合培养的方法,实现体外模拟组织工程化口腔黏膜的发生发展。方法 用DispaseⅡ分离上皮和皮下组织,用KGM培养口腔黏膜上皮细胞。用细胞培养法和组织块培养法获取验用的口腔黏膜上皮细胞和成纤维细胞,并采用复合培养法对两种细胞进行共同培养。结果 用DispaseⅡ可成分地分离上皮和皮下组织。KGM可明显促进口腔黏膜上皮细胞的分裂繁殖。复合培养的HE染色切片显示薄层的结缔组织之上有方形的基底细胞、颗粒细胞和角化层。结论 KGM可明显促进口腔黏膜上皮细胞的分裂和成熟。采用组织培养法获取原代成纤维细胞是适合口腔黏膜取材等特点的有效方法。气液相培养的口腔黏膜下结缔组织可促进上皮细胞的分层和分化。     

Smokeless tobacco extracts modulate keratinocyte and fibroblast growth in organotypic culture

Smokeless tobacco is associated with pathologic alterations of the oral mucosa, yet its direct effects on human keratinocytes and fibroblasts in stratified squamous epithelium are not well-understood. We hypothesized that smokeless tobacco could modulate the growth of keratinocytes and fibroblasts in an in vivo-like, organotypic tissue model. To test this, we exposed organotypic cultures for 3 days to smokeless tobacco aqueous extracts and determined the changes in morphology and proliferation of human keratinocytes and fibroblasts. All smokeless tobaccos stimulated keratinocyte proliferation at low doses (0.25% w/v) and suppressed growth at higher doses (> 0.5% w/v). In contrast, smokeless tobacco extracts promoted fibroblast growth at all concentrations without inducing fibroblast turnover. Fibroblasts and keratinocytes, therefore, were differentially affected by smokeless tobacco extracts in an organotypic tissue model, suggesting incipient changes that may occur in vivo.     

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。     

Nifedipine induces gingival epithelial hyperplasia in rats through inhibition of apoptosis

BACKGROUND: Nifedipine is used as a long-acting vasodilator; one of its side effects is gingival overgrowth, characterized by an accumulation of collagenous components within the gingival connective tissue and epithelial hyperplasia with elongated, branched rete pegs penetrating into the connective tissue. We investigated the effect of nifedipine on apoptosis of gingival keratinocytes of rats to elucidate the mechanism of nifedipine-induced gingival epithelial hyperplasia. METHODS: Twenty-day-old rats were fed a powdered diet containing or lacking nifedipine for 8 to 30 days. The mandibular gingiva and palatal mucosa were removed on days 8, 15, or 30, and epithelial thickness was examined by light microscopy. In situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was used to examine apoptosis of keratinocytes in the epithelium. In addition, we examined the effects of nifedipine on proliferation of keratinocytes and epithelial cell life on day 8 by 5-bromo-2'-deoxyuridine (BrdU) staining. RESULTS: Microscopic examination showed gingival epithelial hyperplasia in nifedipine-treated rats after day 15. Apoptosis of gingival keratinocytes was seen to be inhibited in nifedipine-treated rats on day 8 and 15. Also, nifedipine did not induce an increase of keratinocyte proliferation activity in terms of the number of cells showing positive staining with BrdU. Prolongation of cell life by nifedipine was observed on day 8 in gingival epithelium through a delay of upward cell movement compared to controls. However, epithelial hyperplasia was not detected in palatal mucosa, and there were no significant differences in apoptotic rates of keratinocytes and cell life between nifedipine-treated rats and control rats. CONCLUSIONS: These results suggest that nifedipine induces epithelial hyperplasia in gingival overgrowth not by an increase in keratinocyte proliferation, but by prolongation of cell life through reduction of apoptosis before epithelial hyperplasia is detectable.     

槟榔提取物刺激口腔黏膜角朊细胞培养上清对成纤维细胞增殖活性的影响

目的：探讨槟榔提取物刺激口腔黏膜角朊细胞在黏膜下纤维性变发病中的作用。方法：采用不同浓度槟榔提取液刺激体外培养的角朊细胞,取细胞培养上清,MTT法观察细胞培养上清对成纤维细胞增殖的影响。结果：一定浓度槟榔提取液刺激的角朊细胞培养上清能促进成纤维细胞增殖;细胞培养上清对成纤维细胞增殖的促进作用存在个体差异。结论：槟榔成分可能通过改变口腔黏膜角朊细胞的活性而导致口腔黏膜下纤维性变的发生。     

Oral mucosa model based on a collagen-elastin matrix

Golinski PA, Gröger S, Herrmann JM, Bernd A, Meyle J. Oral mucosa model based on a collagen–elastin matrix. J Periodont Res 2011; 46: 704–711. © 2011 John Wiley & Sons A/S Background and Objective: The collagen‐elastin matrix (Matriderm^®) is used to treat deep and full‐thickness burns and was recently described as a suitable scaffold for tissue engineering. The aim of the present study was to investigate the biocompatibility of Matriderm^® for gingival use through creation of an oral mucosa model ex vivo. Material and Methods: Gingival fibroblasts and keratinocytes were cultured. A dermal area on the base of the collagen–elastin matrix was repopulated with fibroblasts. After 14 days, keratinocytes were seeded on this dermal area to engineer a multilayered mucosa. Analysis of the architecture was performed using light and electron microscopy. Immunohistochemical detection of collagen IV and cytokeratin was carried out. Results: Based on this scaffold we generated a multilayered oral mucosa‐like structure. Histological, immunohistochemical and electron microscopic analysis of the dermal/epidermal junction showed a typical basement membrane and hemidesmosomal structures. Neighboring keratinocytes formed desmosomes in the epidermal sections. Cytokeratin was detectable in all epidermal layers. These experiments revealed that the collagen–elastin matrix was highly biocompatible with gingival cells under ex vivo conditions. Conclusion: Employing tissue‐engineering techniques with dermal and epidermal cells from the gingiva, a multilayered oral mucosa was generated and characterized with respect to biocompatibility for Matriderm^®. The results indicate that Matriderm^® is suitable for the ex vivo growth of gingival tissue cells and is a useful scaffold with possible applications in periodontal therapy.     

The immunohistology of CD40 in human oral epithelium in health and disease

BACKGROUND: CD40 has a role in the regulation of immune responses, cell proliferation and migration, and apoptosis. Little is known of its distribution in oral mucosal pathology. METHODS: Oral keratinocyte lines were tested for CD40 protein by Western blotting. Immunohistochemistry was used to stain paraffin sections of oral mucosa in health and in inflammatory, reactive, dysplastic and malignant disease. RESULTS: Western blotting confirmed the presence of CD40 in oral keratinocytes. CD40 was generally expressed by keratinocytes in the basal layer, with variable parabasal expression. Langerhans cells also stained positively. Expression was lost in nine of 33 (27%) epithelial dysplasias, seven of which were severe. Eighty-one percent of well, 69% of moderately and 50% of poorly differentiated oral squamous cell carcinomas (OSCC) expressed CD40. Overall, 45 of 65 (69%) OSCC were positive. The pattern of expression was unrelated to tumour differentiation. CONCLUSION: CD40 expression by basal and parabasal oral keratinocytes is physiological. Expression is lost in approximately one-third of oral epithelial dysplasias and OSCC. The significance of such loss remains unknown, but may be related to immunological or other abnormalities of keratinocyte homeostasis.     

Culturing primary human gingival epithelial cells: comparison of two isolation techniques.

BACKGROUND: Cultured epithelial cells offer many potential clinical applications. There have generally been two techniques that have been used to cultivate oral keratinocytes, which include the direct explant technique and the enzymatic method. Little work has been done comparing these two techniques and their capacity to isolate and cultivate oral keratinocytes. Objectives: The objectives of this study were to (1) investigate the difference in the percentage of keratinocyte isolation between the direct explant technique and the enzymatic method of human gingival epithelial cell culture and (2) to examine the effect of age and sex of the subjects providing the tissue samples on (a) the success in cultivation and (b) the growth patterns of gingival keratinocytes. MATERIAL AND METHODS: Gingival tissue was obtained from healthy human subjects and was used for keratinocyte isolation using the direct explant technique or the enzymatic method. Epithelial cell cultures from each of the two culture techniques were selected randomly for flow cytometry analysis for cell expression of vimentin and cytokeratin. Growth rate assays were also conducted. RESULTS: The success rate for cultivation from the direct explant technique was higher (82%) than in the enzymatic method (57.9%). The success rate of both methods was not significantly associated with either age or sex of the subjects providing the tissue. From flow cytometry, the average percentage of cells that was positive to anti-pan cytokeratin was nearly the same for both methods at about 97%. It was noted that the cells from the enzymatic method gave significantly higher percentages of cells that were positive to anti-pan cytokeratin only. CONCLUSION: Both the direct explant technique and the enzymatic method can be used for isolating and culturing human oral keratinocytes. The direct explant technique appeared to be more successful in culturing human oral keratinocytes than the enzymatic method, although there were limitations found with both methods. The age and sex of the subjects providing the gingival samples did not appear to be a factor influencing the success rate in culturing the keratinocytes. However, contamination by oral microbiological flora from the gingival tissue samples remained an ever present problem. Further studies are needed in the investigation of clinical applications of these two epithelial cell isolation methods.     

Reduced expression of nuclear factor kB in oral mucosa undergoing preoperative chemoradiotherapy

Radiotherapy as well as chemotherapy in head and neck cancer induces severe oral mucositis. Even after healing of the mucositis, however, the oral mucosa looking atrophic is known to be susceptible to injury and infection. In order to investigate such vulnerability of mucosa, we immunohistochemically studied the expressions of Ki-67, proliferating cell nuclear antigen (PCNA), cyclin D₁, nuclear factor (NF)-kB, and keratinocyte growth factor (KGF) receptor in the oral mucosal keratinocytes undergoing preoperative concurrent chemoradiotherapy for oral cancer, compared with those of the oral mucosa without such therapy. As a result, the expressions of Ki-67, PCNA, and cyclin D₁ were decreased in the chemoradiotherapy-treated oral keratinocytes. Interestingly, NF-kB expression, which is known to be enhanced in oral mucositis, was reduced after chemoradiotherapy. The chemoradiotherapy had no effect on the expression of KGF receptor in oral keratinocytes. In conclusion, the vulnerability of oral mucosa undergoing chemoradiation may be associated with reduced NF-kB expression and impaired growth activity.