【作者】 吴璇；

【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， 2008， 博士

【摘要】 糖尿病患者骨整合的形成率低,羟基磷灰石涂层是常用的提高骨整合形成率的方法,但它易于感染、溶解、并从种植体表面崩解,长期效果欠佳。1型糖尿病和2型糖尿病晚期均需接受胰岛素治疗,人工种植牙给药系统的提出为胰岛素经颌骨局部植入式给药开拓了新的思路,一经实现可使患者在接受口腔治疗的同时接受系统治疗,并取代涂层发挥促进种植体骨整合的功效。颌骨成骨细胞在骨整合形成中发挥着关键的作用。随着成骨细胞体外培养技术的发展和应用,人们发现成骨细胞是一种对供体来源有较强记忆功能的细胞,但目前尚未见糖尿病供体来源颌骨成骨细胞的研究报导。本文将研究胰岛素对体外培养的糖尿病大鼠下颌骨成骨细胞生物学活性的影响,为胰岛素的局部植入式给药提供理论依据。第一部分糖尿病大鼠下颌骨成骨细胞的原代培养与鉴定目的:探讨四氧嘧啶糖尿病大鼠下颌骨成骨细胞培养的可行性,为进一步研究提供细胞来源。方法:16只6-8周龄SD大鼠,随机分为糖尿病组10只和对照组6只,糖尿病组腹腔注射四氧嘧啶（200mg/kg体重）,对照组注射生理盐水。建模成功10d后,取大鼠两侧下颌骨,用酶消一组织块联合法进行成骨细胞培养。根据建模前后的随意血糖,对照组成骨细胞在含糖量5.5mM、糖尿病组成骨细胞分别在含糖量5.5及25mM的培养基中培养。倒置相差显微镜观察细胞生长情况。取各组第2代细胞用碱性磷酸酶钙-钴染色、Ⅰ型胶原特染、钙结节茜素红染色进行成骨细胞鉴定。透射电镜观察细胞的超微结构。结果:糖尿病组成骨细胞在5.5mM糖浓度培养下,可阳性表达碱性磷酸酶、Ⅰ型胶原,矿化液诱导可形成钙结节,光镜下与对照组的外部形态无明显差别,电镜下具有高尔基体少而溶酶体多的特点。糖尿病组成骨细胞在25mM糖浓度培养下,弱阳性表达碱性磷酸酶、Ⅰ型胶原,传代后形态发生改变、生长缓慢、不能形成钙结节。结论:糖尿病大鼠下颌骨在5.5mM糖浓度下进行成骨细胞培养具有可行性,可为进一步研究提供细胞来源。第二部分糖尿病大鼠下颌骨成骨细胞的生物学活性目的:研究糖尿病大鼠下颌骨成骨细胞的生物学活性。方法:MTT法、PNPP法、考马斯亮蓝法、放射免疫法及钙结节茜素红染色检测对照组及糖尿病组成骨细胞的增殖能力（1-12d）、蛋白校正ALP含量（3、7、14、21d）、骨钙素水平（28d）。RT～PcR检测5d时ALP、ColⅠ、Runx2的mRNA表达。采用RT-PCR、Westernblot、免疫组化染色检测IRα1、GLUT-1的表达。结果:1.糖尿病组2-12d与1d的MTT相对比值高于对照组（P＜0.05）,3d、7d、14d、21d ALP含量低于对照组（P＜0.01）,28d总骨钙素水平低于对照组（P＜0.01）,5d时ALP、ColⅠ的mRNA表达低于对照组（P＜0.05）,Runx2的mRNA表达高于对照组（P＜0.01）。2.糖尿病组IRα1、GLUT-1基因蛋白水平的表达均显著高于对照组。结论:糖尿病大鼠下颌骨成骨细胞分化功能出现了障碍,表现为增殖加快、增殖期延长而基质形成、成熟、矿化能力下降。该细胞保留了对供体来源的高糖、低胰岛素环境做出的适应性变化,这可能是造成其分化功能障碍的原因之一。第三部分胰岛素对糖尿病大鼠下颌骨成骨细胞生物学活性的影响目的:研究胰岛素对糖尿病大鼠下颌骨成骨细胞分化和糖摄取的影响。方法:1.按培养基中胰岛素的浓度(0,10^-5,10^-6,10^-7M)将糖尿病组成骨细胞分为DM组、DM+Inse-5组、DM+Inse-6组及DM+Inse-7四组,设立对照组N。用MTT法、PNPP法、考马斯亮蓝法、放射免疫法、钙结节茜素红染色及RT-PCR检测各组的增殖能力（加药后24-72h）、蛋白校正ALP含量（3、7、14、21d）、骨钙素水平（0-28d）及矿化能力以及2d时Runx2、ColⅠAl的mRNA表达。2.按照培养基中胰岛素的浓度(0,10^-6M),将对照组和糖尿病组分为N、N+Ins、DM、DM+Ins四组,荧光显微镜观察它们对2-NBDG的摄取能力。结果:1.加药后24h,N组的MTT光密度值最高,DM组最低,与其余各组相比均有统计学意义（P＜0.01）;加药后48h及72h,MTT OD值由高到低依次为DM+Inse-6＞DM+Inse-5、DM+Inse-7＞N＞DM,组间差异两两比较均具有统计学意义。2.N组在四个时间点的ALP含量均为最高,DM各组中ALP含量最高者依次分别为3d:DM+Inse-7组、7d及14d:DM+Inse-6组、21d:DM+Inse-5组。3.N组28d骨钙素分泌量高于DM各组,差异具有统计学意义（P＜0.05）。N组0-7d BGP分泌量达峰值,之后逐渐降低,而DM各组的变化趋势与之相反。4.N组可见大面积红染的钙沉积;DM组可见零星钙结节着色点,DM加胰岛素各组钙结节着色点增多、面积加大。5.DM加胰岛素各组的ColⅠA1的mRNA表达较DM组明显增强,而Runx2表达明显减弱。6.2-NBDG的摄取量,20min时由大到小依次为N+Ins＞DM+Ins＞DM＞N;40min时依次为DM＞N+Ins＞N及DM+Ins。结论:胰岛素可以纠正糖尿病大鼠成骨细胞的分化功能障碍,并抑制其过高的糖摄取。更多还原

【Abstract】 Diabetic patients have a low rate of osseointegration,and hydroxyapatite coating is usually used to improve the osseointegration,however,hydroxyapatite coatings are not stable,they are inclined to cause infection and tend to delaminate from the implant surface.Both patients with type 1 and advanced stage of type 2 diabetes mellitus need insulin treatment,and a new design proposed for medicine administration via dental implant may be a possible resolvement for the above problems.If the design be realized,it may take place of coating for osseointegration and patients could get dental therapy as well as system treatment at the same time.Osteoblasts of jaw bone play a key role in the formation of osseointegration.With the development and application of bone cell culture technique,osteoblasts have been found to be able to remember the character of their donor resource.But now there is few report about the osteoblasts obtained from diabetic donor.The present paper is to study the effect of insulin on the biological activity of osteoblasts obtained from diabetic rats’ mandibles,and to provide feasibility evidence for the local application of insulin via jaw bone.Part 1 Primary culture and identification of osteoblasts obtained from diabetic rats’mandiblesObjective:To explore the feasibility of the culture of osteoblasts got from alloxan-induced diabetic rats,and to supply cells for further experiments.Methods:16 SD rats were assigned to two groups:diabetic group（10）induced by alloxan（200mg/kg）peritoneal injection and control group（6）injected physiologic saline.Rats were killed and their mandibles were harvested 10 days after the establishment.And primary bone cells were obtained from association of enzyme digestion and tissue explants in culture media containing 5.5 or 25mM glucose according to postprandial blood sugar examined before and after diabetic model establishment.The cells’ growth situation was observed under inverted phase contrast microscope.The second passage cells were identified by alkaline phosphatase Ca-Co stain,typeⅠcollagen special stain and calcium nodules alizarin bordeaux stain.Cells’ ultrastructure was observed by transmission electron microscope（TEM）.Results:Cells of diabetic group cultured in 5.5mM glucose concentration media had typical osteoblasts’ appearance under light microscope and positive identification stain.TEM results showed that there were few golgiosome but more lysosomes in diabetic osteoblasts’ endochylema,which is different from the control group.Cells in 25mM glucose concentration media,with no obvious osteoblasts’ morphology,had weakly positive identification and could not grow in multilayer to form calcium nodule.Conclusions:A large amount of osteoblasts can be obtained from alloxan-induced diabetic rats’ mandibles when cultured in 5.5mM glucose media,which supply cells for further experiments.Part 2 Biological activity of osteoblasts obtained from diabetic rats’ mandiblesObjective:To study the biological activity of osteoblasts obtained from diabetic rats’ mandibles.Methods:Comparison of cell biological activity was made between the diabetic and control group using MTT,PNPP,BGP content radioimmunoassay and alizarin bordeaux stain of calcified nodules.The expression of ALP,ColⅠ,Runx2 was measured by RT-PCR.The expressions of insulin receptorα（IRα）and glucose transporter-1（GLUT-1）were measured by RT-PCR,Westemblot and immunohistochemistry stain.Results:1.MTT value of diabetic group was significantly higher than the control group （P＜0.05）,yet ALP（3d、7d、14d、21d）and BGP（28d）value were on the contrary （P＜0.01）.The ALP、ColⅠmRNA expression of diabetic group were lower than control group（P＜0.05）,yet Runx2 were on the contrary（P＜0.01）.2.The expressions of IR a and GLUT-1 of diabetic group were higher than control group（P＜0.05）.Conclusion:Osteoblasts obtained from diabetic rats’ mandibles have defects in their differentiation ability,leading to deregulated proliferation and a minimal capacity of these cells to develop into fully differentiated mineralizing osteoblasts. Meanwhile,these cells keep the adaptation changes to hyperglycemia and hypoinsulinemia,which may contribute to their dysfuntion.Part 3 Effect of insulin on the biological activity of osteoblasts obtained from diabetic rats’ mandiblesObjective:To study the effect of insulin on the differentiation and glucose uptake of osteoblasts obtained from diabetic rats’ mandibles.Methods:1.Osteoblasts were divided into 5 groups（N、DM、DM+Inse-5、DM+Inse-6、DM+Inse-7）according to their origin（control group or diabetic group）and insulin concentration(0,10^-5,10^-6,10^-7M insulin)in culture media.Comparison of cell differentiation ability was made among the five groups using MTT,PNPP, BGP content radioimmunoassay and alizarin bordeaux stain of calcified nodules. RT-PCR was used for the measure of ColⅠA1 and Runx2 mRNA expression.2.Osteoblasts were divided into 4 groups（N、N+Ins、DM、DM+Ins）according to their origin（control group or diabetic group）and insulin concentration(0,10^-6M insulin)in culture media.Comparison of glucose uptake was made among the four groups by measuring fluorescence of intracellular 2-NBDGResults:1.N group had the highest MTT OD value after the first 24h（P＜0.01）,DM group the lowest.In the following 48 hours their proliferation rate from the high to low was DM+Inse-6＞DM+Inse-5、DM+Inse-7＞N＞DM in order.2.From 3 to 21 days,the highest ALP value among diabetic groups turned to be DM+Inse-7、DM+Inse-6、DM+Inse-5 by turns.3.N group had the highest general BGP value among the five groups（P＜0.05）. The BGP secretion peak of N group appeared in the first week,the lowest in the fourth week.Diabetic group had the opposite tendency.4.There was large area red stain of calcium deposition in N group.In DM group there was only red stain of calcium nodules odds and ends,and DM plus insulin groups had enlarged red stain area.5.DM plus insulin groups expressed significantly more ColⅠA1 and less Runx2 mRNA than DM group.6.The uptake of 2-NBDG was N+Ins＞DM+Ins＞DM＞N by turn at 20minutes, and DM＞N+Ins＞N and DM+Ins at 40 minutes.Conclusion:Insulin may correct the differentiation defects of osteoblasts obtained from diabetic rats,and inhibit their too high glucose uptake.更多还原