【作者】 毛永利；

【导师】 刘鲁川；

【作者基本信息】 第三军医大学 ， 口腔临床医学， 2007， 硕士

【摘要】 背景目的:牙周病是一波及广、危害大的口腔感染性疾病,该疾病严重影响人们的咀嚼消化功能,导致牙齿脱落,还可以成为某些全身疾病的危险因素,所以牙周病一直是国内外学者关注的焦点。由于牙周病慢性、迁延的特点,加之耐药性的产生,目前治疗效果尚不理想,寻找新的药物和新的用药方法受到关注。前期我们针对牙周病的病因及病理特点,筛选以复方奥硝唑甲磺酸培氟沙星合理配伍,在体外抑菌实验研究上取得了满意效果;并依牙周生理外形特点,采用新的缓释材料,设计出适用于不同牙位、不同深度、符合前后牙周袋形态的牙栓,克服了液体制剂易流失,凝胶制剂易污染,固体制剂需修剪等缺点。为了进一步的开发应用,本研究对该缓释牙栓进行了系列动物毒理学试验研究;牙栓在牙周袋中抑菌消炎的同时必然也会作用于牙周组织,而人牙周膜成纤维细胞(HPDLF)是牙周组织的主要功能细胞,在创伤修复和组织再生中发挥着重要作用,进行相关分析探讨缓释牙栓对HPDLF的生物学活性的影响,为进一步动物、临床试验提供试验依据。方法:1.采用冷压法自制复方奥硝唑甲磺酸培氟沙星缓释牙栓,自然凉干,紫外照射30min,消毒备用,每枚重10mg,含原药量1mg。2.以1 L蒸馏水缓释牙栓的最大浓度剂量即210 g/L,制成混悬液,按0.4ml/10g最大允许容积一次给予昆明小鼠灌胃,连续观察14 d,观察小鼠接受过量受试药物所产生的急性中毒反应,为多次反复给药的毒性试验设计剂量、分析人体过量时可能出现的毒性反应。3.将40只SD雌雄大鼠,平均分到4个组中;①小剂量组:按每日5g/kg给药;②中剂量组:按每日10g/kg给药;③大剂量组:按每日20g/kg给药;④空白对照组:按等容量的生理盐水灌胃。观察动物反复给予受试药物后,对机体产生的毒性反应及其严重程度,主要的毒性靶器官及其损害的可逆性,提供无毒性反应剂量及临床上主要的监测指标,为制定人用剂量提供参考。4. 40只豚鼠平均分为4组进行皮肤过敏试验:①空白对照组;②缓释牙栓组(210g/L);③辅料基质组;④阳性对照组。试验前24 h在豚鼠背部两侧,剪出3cm×3cm的去毛区,观察缓释牙栓对皮肤的影响。5.将20只SD大鼠下唇缝成一盲袋,然后随机分为2组,分别用缓释牙栓(210g/L)及等体积基质注入袋内,了解药物对牙周粘膜毒性及刺激的影响。6.选择经2%荧光素钠溶液检查证实,无角膜损伤的健康新西兰兔6只,将缓释牙栓(210g/L)滴入右眼结膜囊内,观察药物对眼部刺激的影响。7.利用免疫组化ABC法对原代培养的HPDLF,进行波形丝蛋白和角蛋白单克隆抗体染色,作细胞来源鉴定。8.取生长良好的第5代HPDLF,制备成浓度为4×10~7 /L细胞悬液,每孔加入浓度分别为0、0.1、0.5、2.5、10、50、200 g/L经DMEM缓释的牙栓,单纯加培养液作空白对照,于不同时间用酶标仪检测各个浓度缓释牙栓OD值,评定药物对细胞增殖的影响。9.取在标准环境下培养24 h的96孔培养板HPDLF,加入浓度分别为0、0.1、0.5、2.5、10、50、200 g/L经DMEM缓释的牙栓,每孔加入1μCi3H-TdR,在液闪记数仪上测量每分钟闪烁记数(cpm),评定药物对HPDLF DNA合成的影响。10.将加有不同浓度缓释牙栓的96孔板HPDLF,每孔加入100μl 0.2% TritonX-100,考马斯亮兰染,在波长570处检测OD值,探讨缓释牙栓对HPDLF蛋白含量的影响。结果:1.经一次性给予最高浓度(210g/L),及小鼠最大灌胃容量(0.4ml/10g)缓释牙栓后,小鼠无一中毒死亡,小鼠对缓释剂的MTD为8.4g/kg。按公斤体重计算,相当于50kg成人每日用量(假设32颗牙齿均用药,每颗牙放置10mg牙栓一枚)的1300倍以上。2.缓释牙栓组与对照组大鼠,在60 d试验期内均活动正常、行为活泼、毛发光泽,无一死亡;体重增长基本一致;血常规、肝肾功检验未见显著性差异;5个脏器的比重量与对照组相比无统计学差异;经病理切片镜下检查,心肌纤维横纹清晰,细胞分化好;肝小叶结构清晰,肝细胞分化良好;未见肾小管变性坏死,间质无出血或炎症;脾无水肿;肺无充血、水肿和炎症反应。3.口腔粘膜完整,缓释牙栓组与基质组无差异,粘膜病理切片未见改变。4.缓释牙栓组动物自给药72 h内未出现红斑及水肿,与空白对照组无差异;基质组无致敏现象;阳性对照组动物皮肤受试区自激发给药6 h后,有明显轻度红斑出现,无水肿,致敏率100%。5.新西兰兔眼部刺激试验出现结膜充血,轻微水肿,少量分泌物,评分≤3分。在用药48 h后全部消失,为可逆变化,由于大多数人认为兔眼较人眼敏感,因此可以推测牙栓缓释剂对人眼基本无刺激性。6.原代HPDLF呈长梭形,胞质均匀,核呈圆形或椭圆形,核仁清晰,免疫组化染色表明,波形丝蛋白染色阳性,角蛋白染色阴性。7.经单因素方差分析的结果显示缓释牙栓在0.1、0.5、2.5、10、50、200 g/L时OD值与对照组相比没有显著性差异(P>0.05)。8.缓释牙栓在0.1、0.5、2.5、10、50、200 g/L浓度时,与对照组比,蛋白质OD值、DNA合成均无显著性差异(P>0.05)。结论:1.自制复方奥硝唑甲磺酸培氟沙星缓释牙栓每一批次制备规范,粒差小,体外缓释稳定,符合2005年版《中国药典》对栓剂的规定。2.急、慢性毒性试验结果表明缓释牙栓无明显毒性作用,大鼠的一般状态、行为活动、饮食、尿便、体重增长、血液学、血液生化学及主要脏器系数与对照组比较均未见明显差异,各脏器病理学检查均未见明显药物损害性病理改变,无局部刺激作用及过敏反应,不同动物对该缓释牙栓有很大的耐受性,提示该药的使用安全性高。3.免疫组化染色表明,证明所培养的细胞为来自中胚层的成纤维细胞。4.在200 g/L缓释浓度范围内,该缓释牙栓有较佳的生物相容性,未显示有HPDLF抑制作用。更多还原

【Abstract】 Backgrounds and ObjectivePeriodontitis is one of prevalent, deleterious oral infection diseases. This disease has serious effect on the function of mastication and digestion and results in the defluvium of the teeth. It is a dangerous factor which will induce the diseases of the whole body. so periodontitis is the focus of scholars both domestic and international in the long run. Periodontitis is a chronic, proof-chemical disease and it has close relation with other apparatus, so up to now, the therapeutics is not so ideal. As a result, we pay more attention to the research of new method and medicine. In the prophases, according to the periodontal pathogeny and its pathologic characteristics, we reasonably mix Compound Ornidazole and pefloxacinmesylat. According to the outer characteristics of the periodontal physiology, we work out periodontal suppository which is fit to periodontal pocket with different teeth, different depth and fit to different periodontal configuration. By adopting new sustained-release material, we made great contribution to the bacteriostasis in vitro, overcoming many disadvantages such as the easily loss of the liquor medicine, the easily contamination of the gel medicine and trimming of solid medicine and so on.In order to make further development and application, we did a series of animal toxicological research in the spirit of the related new medical regulation. When Periodontal Suppository bacteriostatic and anti-inflammatory effect exists in the periodontal pocket, in the meanwhile, it will impact the periodontal tissue. The HPDLF is the main functional cell of periodontal tissue, which plays great role in the renovation of the wound and the rebirth of the tissue. We did related analysis on the biological active influence that Periodontal Suppository to HPDLF, which can supply foundation to further animal and clinical trial.Material and method1. Take use of cold compression to manufacture Periodontal Suppository, cooled naturally, using ultraviolet radiation to irradiate 30min, sterilizing befor use, 10mg each piece, contained raw medicine 1 mg.2. Using the highest concentration dosage (210g/L) in 1L distilled water sustained-release periodontal suppository to produce blending suspending solution. Pouring the highest dimension (0.4ml/10g) solution into stomach of mouse at one time. Obserived in the following 14 days. Using the same capability NS to administrate for control mouse. Observing the acute intoxication reflection of the mouse after taking up overdose reagent, Choose the appropriate dose the periodontal suppository repeated administration toxic experiment, analyzing the primary target organs of the toxic effect and the potential toxic reaction when human body are in overdose.3. 20 male and 20 female SD rats weigh 135±10 g are divided into 4 groups according to the experiment design.①low dosage group(5g/kg per day).②middle-dose group(10g/kg per day).③high-dose group(20g/kg).④control group (same capability NS). Observing the toxic reaction and primary target organs and reversibility damage of rats after repeated administration of the reagent. Offering nontoxic reaction dose and primary inspected index and referenced dose for human being.4. Using 40 guinea pigs dividing into 4 groups evenly to carry out skin scratch test:①control group.②sustained-release Periodontal Suppository group (10g/kg).③complement substrate group.④positive control group. Shearing 3cm×3cm hairless area on both sides back of guinea pig 24 hours before test. Then observing the effect of medicine on skin.5. The ectolabium of 40 SD rats being sewed into blind bags. Then the rats divided into 2 groups randomly. Pouring separately sustained-release Periodontal Suppository (210g/L) and the same volume substrate into bags and daubing the lower Gingival mucous membrane. Finding out the toxicity and stimulating effect of medicine to periodontal mucous membrane.6. Choosing six healthy and no cornea injury proved by 2% fluorescence New Zealand rabbits. Dripping 0.2ml sustained-release Periodontal Suppository (210g/L) into conjunctival sac of right eye, dripping the same quantity NS into the left eye for comparison. Observing the stimulate effect of medicine for eyes.7. Taking use of immunohistochemistry ABC method to human periodontal ligament fibroblast to colour the undee silk protein and cell keratin monoclone antibody coloration and identify the origin of cells. 8. Choosing the fourth generation well growth ligament fibroblast and then confect hang cell solution at 4×10~7 /L concentration. Putting DMEM which contained and sustained-released periodontal suppository 0.5 percent FBS into each hole. The cultivation of sustained-release Periodontal Suppository is divided into seven concentration groups as follows 0, 0.1, 0.5, 2.5, 10, 50, 200 g/L,in which the former acts as control group. Each group will be cultivated for different time, putting 5 g/L MTT 20ul into each hole in one culture board. Taking use of enzyme mark apparatus to test the OD value at 470 nm wavelength ,which is to evaluate effects of the drug on multiplication of cells .9. Chosing a culture board with 96 holes cultivated for 24 hours under standard condition, and putting 0、0.1、0.5、2.5、10、50、200 g/L concentration DMEM sustained-release periodontal suppository into holes,and putting 1μCi3H-TdR into each holes. Testing glittering count in one minute by glitter counting apparatus to evaluate effects of drug on HPDLF DNA synthesis.10. Taking use of 96 holes full of different concentration sustained-release periodontal suppository. Putting 100ul 0.2% TritonX-100 into each hole. Putting over 20ul solution from each hole into 200ul dying fluid, keeping on surging 10 minutes, testing the OD value at 570 wavelength.Results1. None of the Jimpy mice died from poisoning,even at maximum concentration (210g/L) and the maximum intragastric administration volume (0.4ml/10g)of sustained-release periodontal suppository reagent,the MTD of retarder is 8.4g/kg for Jimpy mice.This is more than 1,300 times of the adult’s(50kg) dose per day( provided each of the thirty two teeth is placed a suppositorium).2. The rat of Medication’s group act normally, behavior lively and the pelage is gloss, and none of them died during experimental stage of the sixty days, which is coincident with those of control group; There is no significant difference as for hemo-rout, test of Hepatic and renal function, partly remarkably heighten than control group, though these changes are within normal limits, without statistical difference,To check the pathological section by microscope,the transverse striation of cardiac muscle fiber is clear, cellular differentiation is well; the structure of hepatic lobules is clear; cellular differentiation of liver is well; there is no denaturation , and atosis for the nephric tubule, renal interstitium is non-haemorrhage or inflammatory; spleen didn’t hydrops, there are no congestion, hydroncus and inflammatory reaction for pulmo.3. The mucous membrane of mouth is integrity, there is no difference between each group.4. The animal of sustained-release periodontal suppository group didn’t appear erythema and dropsy, which has no difference with the control group; substrate group has no sensibilization; the skin of animal ,received test ,blonging to positive control group, obviously emerge erythema slightly, with no dropsy, sensitize rate for 100%.5. It’s reported that there are conjunctival congestion, light hydroncus and little secreta by stimulation test of ocular region, with scoring no more than three points. These symptoms are reversible change and vanish totally after medication for 48 hours. Majority of people think that hare’s eye is more sensitive than human’s, so we can presume that periodontal suppository has no stimulation to human’s eyes.6. The primary cell is long fusiform shape, and it’s cytoplam is uniform, with nucleus round or ellipse, chromatospherite clear. Immunohistochemistry coloretur indicated that the undee silk protein is masculine, keratin negative.7. By analysis of variance of single factor, It’s showed that the OD value (0.1、0.5、2.5 10、50、200 g/L) of sustained-release Periodontal Suppository has no significant difference compared with control group(P>0.05).8. When sustained-release Periodontal Suppository is at the concentration of 0、0.1、0.5、2.5 10、50、200 g/L, contrast to the control group, both OD value of protein and DNA’s synthesis has no significant difference. (P>0.05)Conclusions1. The preparation artwork is simple for Compound Ornidazole and pefloxacinmesylat sustained-release Periodontal Suppository, nodule equation is little, the extraorgan sustained-release stable, which is consistent with the regulation for suppositories in the book《dispensatory of China》in 2005.2. The result of acute and chronic toxicity test manifests that sustained-release Periodontal Suppository has no overt toxicity, the general condition of rat, behavior activity, drink and food, urine and stool, growing of body weight, hematology and blood biochemics, coefficient of major organ as well, have no obviously difference with the control group, the check of pathology for each organ has no manifest medicinal impairment patho-alter, no part stimulation and allergic response, diversity of animals can tolerance this sustained release Periodontal Suppository greatly, which means using this medicine is safe.3. Immunohistochemistry proved that cultured cytes come from collagenoblast of mesoderm.4. When the concentration of sustained-release is within 200 g/L, the sustained-release Periodontal Suppository has better biocompatibility, without cyto-inhibition of HPDLF.更多还原