【作者】 马龙；

【导师】 刘洪臣；

【作者基本信息】 中国人民解放军军医进修学院 ， 口腔临床医学， 2009， 博士

【摘要】 糖尿病和骨质疏松症是影响人工种植牙修复的主要的系统性疾病。它们通过引起种植体周围的一系列的病理变化,进而影响骨组织的修复和重建,降低种植牙的成功率。二甲双胍不仅能够降低血糖,而且对种植体周围骨整合有促进作用;抗高脂血症药物普伐他汀,可以有效地治疗骨质疏松症,是目前发现的惟一能够既促进骨形成,又抑制骨吸收的药物。目前,在种植体周围局部给药成为研究的重点,特别是刘洪臣提出的人工种植体给药系统—即通过种植体与骨的接触,使药物转运到局部乃至全身,从而达到更好的治疗效果。为此,我们需要了解骨组织细胞对两种药物的转运能力。本实验通过观察两种药物在成骨细胞中的转运,探讨其可能的转运机制,为人工种植牙给药提供实验依据。第一部分大鼠下颌骨成骨细胞跨膜转运transwell模型的建立目的:建立大鼠下颌骨成骨细胞跨膜转运transwell模型。方法:分离培养大鼠下颌骨成骨细胞,并接种在transwell上室底部的聚碳酸脂膜上,光学显微镜观察其生长情况,扫描和透射电子显微镜观察细胞连接情况,跨膜电阻及荧光素钠通透实验检测细胞的屏障功能。结果:成功培养并接种了大鼠下颌骨成骨细胞于聚碳酸脂膜上;光学显微镜观察,大鼠下颌骨成骨细胞(1×10~5/ml)接种2周后,细胞已完全汇合,连接紧密,并呈复层生长;扫描和透射电子显微镜显示,细胞间呈缝隙连接;细胞屏障功能检测,跨膜电阻随时间的增加而增加,第2周时达到最大(88.65±7.33)Ω/cm~2,并至少持续到接种后3周;荧光素钠通透性检测结果显示,孵育30min后只有0.17%的荧光素钠从transwell上室到达下室。结论:应用transwell小室可以建立体外大鼠下颌骨成骨细胞复层生长的模型,并可用于药物细胞跨膜转运机制的研究。第二部分大鼠下颌骨来源的成骨细胞对二甲双胍的跨膜转运目的:检测成骨细胞对二甲双胍的跨膜转运,研究其可能的转运机制,探讨人工种植牙给药的条件及可行性。方法:高效液相色谱法测定转入及转出成骨细胞二甲双胍的量,同时考察时间、药物浓度、温度、pH值、有机阳离子转运蛋白(Octs)抑制剂西米替丁、二甲双胍类似物苯乙双胍以及P-糖蛋白(P-gp)抑制剂维拉帕米对二甲双胍转运的影响;免疫组化和RT-PCR检测Octs的表达,Western-blot检测二甲双胍对成骨细胞Oct1转运蛋白表达的影响。结果:二甲双胍能够在成骨细胞上进行跨膜转运,且随着转运时间和药物浓度的增加而增加,并可达到饱和;一定范围内提高温度和pH值可以促进二甲双胍的转运;西米替丁和苯乙双胍对二甲双胍转运有明显抑制作用,而维拉帕米则对转运有明显的促进作用;Oct1广泛表达在成骨细胞的胞膜及胞浆中,而Oct2无表达;二甲双胍在不同浓度及不同给药时间均能够上调Oct1的表达,但各组间无明显差别。结论:大鼠下颌骨来源成骨细胞能够对二甲双胍进行跨膜运输,且跨膜运输是通过Oct1为载体介导的协助扩散及P-gp为载体介导的主动转运的过程。第三部分大鼠下颌骨来源的成骨细胞对普伐他汀的跨膜转运目的:检测成骨细胞对普伐他汀的跨膜转运,研究其可能的转运机制,探讨人工种植牙给药的条件及可行性。方法:高效液相色谱法测定转入及转出成骨细胞普伐他汀的量,同时考察时间、药物浓度、温度、pH值、他汀类药物辛伐他汀和洛伐他汀以及P-糖蛋白(P-gp)抑制剂维拉帕米对普伐他汀转运的影响;免疫组化检测Oatpl的表达,Western-blot检测普伐他汀对成骨细胞Oatpl转运蛋白表达的影响。结果:普伐他汀能够在成骨细胞上进行跨膜转运,且随着转运时间和药物浓度的增加而增加,并可达到饱和;一定范围内提高温度和pH值可以促进普伐他汀的转运;辛伐他汀和洛伐他汀对普伐他汀转运有明显抑制作用,而维拉帕米则对转运无明显作用;Oatp1广泛表达在成骨细胞的胞膜及胞浆中;普伐他汀在不同浓度及不同给药时间均能够上调Oatp1的表达,但各组间无明显差别。结论:成骨细胞能够对普伐他汀进行跨膜运输,且跨膜运输是通过Oatp1为载体介导的协助扩散及非P-gp为载体介导的主动转运的过程。更多还原

【Abstract】 Diabetes and osteoporosis are main systemic diseases that affect the dental implant treatment.They impact the restoration and reconstruction on bone tissue by inducing a series of pathological changes around the dental implant and decrease the success ratio of dental implant.Metformin,an anti-hyperglucose drug,can not only lower blood glucose,but also promote osseointegration around the implant;pravastatin,an anti-hyperlipidemic drug,can treat osteoporosis efficiently,and it is the only one that has the ability to promot the bone formation and inhibite the bone resorption.Therefore,topical administration around dental implant become the focus of the study in present.In particular,Liu Hong-chen proposed dental implant drug administration system—through the contact between implant and bone tissue,drugs can be transported to the local and the whole body so as to achieve the better therapeutic effects.So,we need to understand the capacity of transporting drugs by the bone tissue cells.In this study, we explored the possible transport mechanism through observing the transmembrane transport of metformin and pravastatin by osteoblasts from rat’s mandible to provide the experimental basis for drug administration by dental implant.PartⅠThe establishment of the transmembrane transport transwell model of rat mandible osteoblastsObsjective:To built the transmembrane transport transwell model of rat mandible osteoblasts. Methods:Primary osteoblasts were isolated,cultured,identified and inoculated in the filter membrane of polycarbonate at the bottom of the upper well.Light microscope was utilized to observe the cells growth,scanning and transmission electron microscope was applied to observe the cell junction,membrane resistance and Fluorescein Sodium permeation experiment were detected the barrier function of osteoblasts.Results:Osteoblasts were cultured and inoculated in the filter membrane of polycarbonate successfully;cells(1×10~5/ml)converged completely,connected closely and displayed ambi-layer generation 2 weeks after inoculation;tight junction between each cell was observed by scanning and transmission electron microscope;cells barrier function shown that TER of cells was increasing with time adding and reach the maximum(88.65±7.33)Ω/cm~2 which maintained till the 3rd week after inocubation;Fluorescein Sodium permeation experiment presented that after incubated for 30 minutes,only 0.17%of Fluorescein Sodium reached the inferior filter membrane.Conclusions:The transwell can be used to set up the model of osteoblasts with growth of ambi-layer and investigate the mechanic of the transmembrane transport of drugs.PartⅡThe transmembrane transport of metformin by osteoblasts from rat mandibleObjective:To detect the transmembrane transport of metformin by osteoblasts, investigate the possible mechanic of transport and explore the condition and feasibility of dental implant drug delivery.Methods:HPLC(High Performance Liquid Chromatography)was utilized to detect the transmembrane transport of metformin by osteoblasts.The effect of time,drug concentration,temperature,pH,cimetidine(an inhibitor of organic cation transporter),and verapamil(an inhibito of P-protein)on the transport of metformin were studied.Octs(Organic cation transporters)expression were characterized by Immunocytochemistry and RT-PCR.Western blotting was ulilized to detect the effect ofmetformin on the expression of Oct1.Results:Osteoblasts has the ability of transmembrane transport of metformin. The transport of metformin could be saturable with time and drug concentration adding;a certain range of temperature and pH can promote the transport of metformin;The transport of metformin was inhibited by cimetidine and phenformin,but promoted by verapamil;Oct1 but no Oct2 was expressed extensively in cell membrane and cytoplasm of osteoblasts;The protein level of Oct1 can be up-regulated by metformin with different drug concentrations and times,but no obvious differentces from each group.Conclusions:Osteoblasts from rat mandible have the ability of transmembrane transport of metformin.,and the transport of metformin in osteoblasts was facilitated diffusion mediated by Oct1 and an active transportation mediated by P-gp which proves the possibility of the local drug delivery around dental implant in diabetes patients.PartⅢThe transmembrane transport of pravastatin by osteoblasts from rat mandibleObjective:To detect the transmembrane transport of pravastatin by osteoblasts, investigate the possible mechanic of transport and explore the condition and feasibility of dental implant drug delivery.Methods:HPLC(High Performance Liquid Chromatography)was utilized to detect the transmembrane transport of pravastatin by osteoblasts.The effect of time,drug concentration,temperature,pH,simvastain and lovastatin(the other stins)and verapamil on the transport of pravastatin were studied.Oatp1(Organic anion transporting polypeptide 1)expression were characterized by Immunocytochemistry.Western blotting was ulilized to detect the effect of pravastatin on the expression of Oatp1.Results:Osteoblasts has the ability of transmembrane transport of metformin. The transport of metformin could be saturable with time and drug concentration adding;a certain range of temperature and pH can promote the transport of metformin;The transport of metformin was inhibited by simvastain and lovastatin, but not effected by verapamil;Oatp1 was expressed extensively in cell membrane and cytoplasm of osteoblasts;The protein level of Oatp1 can be up-regulated by pravastatin with different drug concentrations,but no obvious differentces from each group.Conclusions:Osteoblasts from rat mandible have the ability of transmembrane transport of pravastatin,and the transport of pravastatin in osteoblasts was facilitated diffusion mediated by Oatp1 and an active transportation which proves the possibility of the loca1 drug delivery around dental implant in osteoporosis patients.更多还原