【作者】 李娜；

【导师】 许彦枝；

【作者基本信息】 河北医科大学 ， 口腔临床医学， 2011， 硕士

【摘要】 目的:牙周病是一种慢性破坏性疾病,可造成牙周支持组织的破坏和附着丧失,最终导致牙齿丧失。牙周病治疗的最终目的是获得牙周支持组织的再生,即牙周膜、牙槽骨和牙骨质的再生。随着组织工程学的发展,应用组织工程支架材料修复牙周组织的缺损,成为目前研究牙周病治疗的热点。玉米醇溶蛋白是从玉米中萃取而成,具有良好的成膜性、黏接性和防水、防湿性能,并且还具有耐酸、耐油等特性。根据玉米醇溶蛋白的特性,其被广泛应用于食品医药方面,而将其作为支架材料应用于牙周组织工程的研究却很少。本研究将中药双黄补作为一种生长因子与玉米醇溶蛋白支架结合,探讨双黄补对牙周膜细胞在玉米醇溶蛋白支架材料上黏附、生长及增殖的影响,研究双黄补-玉米醇溶蛋白作为一种复合材料应用于牙周组织工程的可行性。方法:1牙周膜细胞原代培养选择健康青少年因正畸拔除的健康前磨牙,无菌条件下用PBS或DMEM冲洗3次,刮取根中1/3牙周膜组织,采用组织块法培养牙周膜细胞。2免疫组化ABC法进行来源鉴定取生长良好的第2代细胞进行爬片培养72h,PBS清洗3次,4%的多聚甲醛室温固定20分钟;PBS清洗3次,0.1%Triton X 100孵育10min;3%的过氧化氢室温阻断20min;PBS清洗3次,加入稀释度为1:80的鼠抗人波形丝蛋白(Vimentin)和1:120的兔抗人角蛋白(Cytokeratin,CK)一抗室温1h;PBS清洗3次,加入二抗室温孵育20min;PBS清洗3次,加入DAB显色剂,苏木素复染;同时用PBS代替一抗作阴性对照,显微镜下观察照相。3双黄补煎液的制备将黄连、黄芩、骨碎补按一定比例混合,煎前用过滤水或蒸馏水浸泡30分钟,分别加8倍体积及6倍体积水煎2次,第1次2h,第2次1.5h,水提醇沉法制备成1 g/mL生药的双黄补煎液,加1%活性炭脱色3次,调pH至7.0～7.1, 4℃保存备用。4玉米醇溶蛋白支架材料的制备与预处理按一定比例称取氯化钠,将其加入玉米醇溶蛋白酒精溶液中,边加热边搅拌至均匀分散,采用溶剂浇铸/粒子沥滤法制备玉米醇溶蛋白支架。玉米醇溶蛋白支架紫外线消毒2h后备用。5 MTT法检测牙周膜细胞的增殖活性将玉米醇溶蛋白支架按1 cm~2/mL的比例置含10% FBS的DMEM培养液中,37℃浸提72 h,收集浸提液并配制50μg/mL、100μg/mL、150μg/mL、200μg/mL、500μg/mL、1000μg/mL质量浓度的双黄补培养液,备用。取第5代培养细胞,调整细胞浓度为1×10~4 /ml接种于96孔板,每孔100μl,37℃5% CO2培养24h,弃孔内培养液。随机分为玉米醇溶蛋白支架浸提液配制的不同浓度双黄补组(双黄补-Zein支架浸提液组)、玉米醇溶蛋白支架浸提液组及细胞对照组,MTT法检测牙周膜细胞的增殖活性。6倒置相差显微镜及扫描电镜观察将预先处理好的玉米醇溶蛋白支架放于24孔板中,用含10% FBS的DMEM培养液37℃浸泡24h。取第5代培养细胞,调整细胞浓度为1.5×10~5 /ml接种于24孔板中支架上,每孔200μl,随后加入800μl含10% FBS的DMEM培养液,37℃5%CO2培养24h。弃孔内培养液,随机分为实验组和对照组,三周后倒置相差显微镜及扫描电镜进行观察。7统计学处理采用SPSS 13.0统计软件进行数据处理,数据以ˉx±s表示,进行单因素方差分析, P<0.05具有统计学意义。结果:1细胞培养及来源鉴定牙周膜细胞于培养的第5～7天从组织块游出,呈放射状排列生长,细胞为梭形或星形,胞体丰满,胞浆均匀,核圆形或卵圆形。来源鉴定结果显示,波形丝蛋白阳性,胞浆棕黄色着色,细胞角蛋白阴性,证实其为中胚层来源的成纤维细胞,可用于实验。2双黄补对牙周膜细胞在玉米醇溶蛋白支架材料上增殖活性的影响MTT结果显示,与细胞对照组及玉米醇溶蛋白支架浸提液组相比,玉米醇溶蛋白支架浸提液配制的不同浓度双黄补均能促进牙周膜细胞的增殖,具有剂量依赖性,且以100μg/mL质量浓度的双黄补促增殖作用最明显(P <0.01),统计学意义显著。玉米醇溶蛋白支架浸提液组与细胞对照组相比,两者之间无明显差异(P >0.05),说明玉米醇溶蛋白支架浸提液不影响牙周膜细胞的生长。3双黄补对牙周膜细胞在玉米醇溶蛋白支架材料上黏附生长影响的形态学观察倒置相差显微镜观察,在双黄补作用下玉米醇溶蛋白支架上黏附生长的牙周膜细胞数目较多,细胞呈长梭形,横跨支架孔隙交织成网状,而无双黄补作用的玉米醇溶蛋白支架上黏附生长的细胞数目较少。扫描电镜观察,在双黄补作用下玉米醇溶蛋白支架上生长的牙周膜细胞数目较多,主要黏附生长在支架孔隙的周边,呈长梭形或星形,伸出伪足并横跨孔隙交织成网状,在支架表面及孔隙陷窝内也有生长。而无双黄补作用的玉米醇溶蛋白支架上生长的细胞数目较少,多呈星形,主要黏附生长在孔隙陷窝内。在细胞形态上,双黄补作用下在玉米醇溶蛋白支架上黏附生长的牙周膜细胞多为梭形,胞体丰满,胞浆均匀,立体感强。结论:双黄补能促进牙周膜细胞在玉米醇溶蛋白支架上的黏附、生长与增殖。双黄补作为一种生长因子与玉米醇溶蛋白支架结合,作为一种复合支架材料应用于牙周组织工程具有可行性。更多还原

【Abstract】 Objective: Periodontal disease is an inflammatory destructive chronic disease, which can cause destruction and loss of attachment of periodontal tissue and eventually leading to tooth loss. The ultimate goal of periodontal therapy is to achieve regeneration of periodontal tissue, including periodontal ligament, alveolar bone and cementum. With the development of tissue engineering, application of scaffolds to repair periodontal tissue defects has become the focus of periodontal therapy. Zein is extracted from corn, it has good properties in film forming, bonding, waterproofing and moisture resistance, and also tolerance with acid and oils. Zein is widely used in food and medicine because of the characteristic, however it is less used in tissue engineering as scaffolds. In this research, Shuanghuangbu as a growth factor was combined with zein scaffolds, the aims were to study the effects of Shuanghuangbu on the adhesion, growth and proliferation of human periodontal ligament cells (HPDLCs) in zein scaffolds, and to evaluate the feasibility of shuanghuangbu-zein compound scaffold materials in periodontal tissue engineering.Methods:1 HPDLCs primary cultureHealthy premolars removed for orthodontic reasons from healthy adolescents were selected. The study was performed with the approval of the hospital’s Human Investigations Committee. After washing with PBS or DMEM for 3 times, the periodontal membranes at 1/3 of roots were aseptically collected and used for primary cultures with the explant culture method.2 Source identification with immunocytochemistry and ABC staining method The second passage cells were cultured on slide for 72 h, and then it was washed with PBS 3 times. After immobilization of the cultured cells with 4% paraformaldehyde, the cell membranes were permeabilized for 10 min with 0.1% Triton X 100 in PBS and then blocked for 20 min with 3% H2O2, then it was washed with PBS three times. Mouse anti-Vimentin and rabbit anti-Cytokeratin (Zhongshan, Beijing, China) were diluted to their final concentration of 1:80 and 1:120 respectively. Both antibodies were incubated with the cells at room temperature for 1 h. Following a wash-out step with PBS, the second antibody was added and incubated with the cells at room temperature for 20 min, and then with DAB colouration and hematoxylin afterstain. Meanwhile, for negative controls, the specific primary antibody was replaced with PBS. That was observed under light microscope3 Shuanghuangbu preparationCoptidis rhizoma, scutellariae radix and rhizoma drynariae were weighed according to a certain proportion and mixed well, The mixture was soaked in distilled water for 30 min and then boiled in 8 volumes of water (v/w) for 2 h and 6 volumes of water (v/w) for 1.5 h, Shuanghuangbu decoction was prepared to concentration of 1 g/ml with water extraction and alcohol precipitation method. The decoction was discolored with 1% active carbon for three times, pH adjusted to 7.0 ~ 7.1, and then stored at 4°C.4 Preparation and pretreatment of zein scaffoldsSodium chloride according to a certain proportion was weighed and added to alcoholic solution of zein, the solution was then heated with stirring until evenly distributed, zein scaffolds were prepared by solvent casting/particle leaching, UV irradiated for 2 h and stored for use.5 MTT assay of periodontal ligament cell proliferationAt 1 cm~2/mL (v/v), the zein scaffold slices were soaked in DMEM containing 10% FBS at 37°C for 72 h. The zein scaffold extract were collected and then Shuanghuangbu was diluted to final concentrations of 50, 100, 150, 200, 500 and 1000μg/ml with the zein scaffold extract. The fifth passage cells were trypsinized, collected and made into cell suspensions at 1× 10~4/ml. Then the suspensions were seeded at 1×10~3 cells per well in 96-well plates. After culture at 37°C in 5% CO2 for 24 h. The wells were randomly divided into different concentrations of Shuanghuangbu group, zein scaffold extraction group and control group, MTT assay of periodontal ligament cell proliferati.6 Inverted phase contrast microscopy and scanning electron microscopy The zein scaffold slices were soaked in DMEM medium containing 10% FBS at 37°C for 24 h in a 24-well plate. The fifth passage cells were trypsinized,collected and made into cell suspensions at 1.5×105/ml. Then the suspensions were seeded at 3×104 cells per well in 24-well plates with zein scaffold slices, then 800μl of DMEM containing 10% FBS was added. Afterculture at 37°C in 5% CO2 for 24 h, the cells were divided into the experimental group and the control group in random. Cells were cultured for 3 weeks and then observed under inverted phase contrast microscope and scanning electron microscope.7 Statistical analysisAll data were presented as means±standard deviation (S.D.) and analyzed with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Comparisons of group means were assessed with One-Way ANOVA. P < 0.05 was considered significant difference.Results:1 Cell culture and identificationPeriodontal ligament cells migrated from the tissue after 5 to 7 days in culture, oriented radially with growth; the cells were spindle or stellate in shape, with round cell body and even cytoplasm, round or oval nuclei. Identification result showed that the cells were vimentin-positive with brown staining in the cytoplasm and cytokeratin negative, confirming that the cells were fibroblasts of mesoderm origin and suitable for the experiments.2 The effect of Shuanghuangbu on HPDLCs proliferation in zein scaffoldsMTT assay result showed that compared with the control group and zein scaffold extract group, the different concentrations of Shuanghuangbu prepared with zein scaffold extract showed promotion of HPDLCs proliferation, and the most significant effect was achieved at 100μg/mL of Shuanghuangbu (P < 0.01), indicating that different concentrations of Shuanghuangbu significantly promoted the proliferation of HPDLCs in a dose-dependent manner. There was no significant difference between Zein scaffold extract group and control group (P > 0.05), which showed that zein scaffolds did not affect the proliferation of HPDLCs.3 Morphological observations of HPDLCs in zein scaffolds after Shuanghuangbu treatmentUnder inverted phase contrast microscope, Shuanghuangbu treatment increased the number of periodontal ligament cells in zein scaffolds, the cells were long spindle in shape, forming a net across the pores. Without Shuanghuangbu, a small number of cells were present in the zein scaffolds. Under scanning electron microscope, Shuanghuangbu treatment increased the number of periodontal ligament cells in zein scaffolds, the cells were mostly attached to the peripheral of the pores of the scaffold, spindle or star in shape; extending pseudopodia across the pores and forming a mesh; the cells were also on the scaffold surface and lacunae. Without Shuanghuangbu, a small number of cells were present in the zein scaffolds, mostly star in shape and mainly on lacunae. The cells in zein scaffolds under Shuanghuangbu treatment were mostly spindle shaped with stout cell body, uniform plasma and stereoscopic.Conclusion: Shuanghuangbu could promote the adhesion, growth and proliferation of human periodontal ligament cells in zein scaffolds, this indicates that Shuanghuangbu as a growth factor and shuanghuangbu-zein as composite scaffold materials used in periodontal tissue engineering were feasible.更多还原