【作者】 施蕊；

【导师】 李淑娟； 董福生；

【作者基本信息】 河北医科大学 ， 口腔临床医学， 2010， 硕士

【摘要】 目的:通过对雄性wistar大鼠去睾丸、牙周结扎建立骨质疏松牙周炎动物模型,比较右归丸方剂和十一酸睾酮对实验性骨质疏松牙周炎及骨代谢的影响,观察右归丸方剂对实验性骨质疏松大鼠牙周炎的治疗作用,探讨男性骨质疏松与牙周炎发生、发展的关系,为临床上治疗男性骨质疏松牙周炎提供实验依据。方法:选用纯种6月龄雄性wistar大鼠60只,随机分为6组:正常组(10只)、假手术牙周炎组(简称牙周炎组) (10只)、去睾丸组(10只)、去睾丸牙周炎组(10只)、去睾丸牙周炎右归丸方剂治疗组(简称中药组) (10只)、去睾丸牙周炎十一酸睾酮治疗组(简称西药组) (10只)。去睾丸组、去睾丸牙周炎组、中药组、西药组大鼠分别切除双侧睾丸,牙周炎组仅做切口,不切除睾丸;手术4周后,牙周炎组、去睾丸牙周炎组、中药组、西药组大鼠用直径0.2mm的正畸不锈钢丝结扎双侧上颌第一磨牙牙颈部。睾丸切除术后3个月(牙周结扎术后2个月),随机抽取去睾丸组及正常组大鼠,处死后,取大鼠股骨及上颌骨,制作股骨及上颌骨组织切片,组织学观察,鉴定骨质疏松模型;随机抽取正常组及牙周炎组大鼠,处死,取上颌骨,制作上颌第一磨牙牙体牙周联合切片,组织学观察,鉴定牙周炎模型。模型鉴定成功后,中药组大鼠用煎好的右归丸方剂,按5mg/kg体重2ml(相当于生药2g)每日灌胃,西药组大鼠用十一酸睾酮,按8 mg/kg体重用2ml生理盐水的比例配成混悬浊液,1ml每日两次灌胃;其余组每日一次2ml生理盐水灌胃;服药后3个月,股动脉放血处死大鼠,取大鼠一侧上颌骨,立即放入10%甲醛缓冲溶液中固定,然后经10%EDTA溶液脱钙,制作牙周组织切片,进行HE染色,光镜观察;抗酒石酸酸性磷酸酶(Tartate resistant acid phosphatase,TRAP)染色,观察破骨细胞的数量、分布,并作统计学分析处理;骨形态发生蛋白-2(Bone morphogenetic protein,BMP-2)免疫组织化学染色,观察BMP-2的表达并作统计学分析处理。取大鼠另一侧上颌第一磨牙和第二磨牙之间的牙龈组织,制作组织匀浆,离心后提取上清液,测定牙龈组织中碱性磷酸酶(Alkaline phosphatase,ALP)的水平,并作统计学分析处理。结果:1正常组股骨及上颌骨骨小梁粗大、密集,顺受力方向规则排列;去睾丸组股骨及上颌骨骨小梁较细小、稀疏,排列紊乱,不规则。说明去睾丸法已成功建立骨质疏松动物模型。2牙周组织学观察,牙龈炎症明显,牙周膜内血管扩张,结合上皮结合不紧密,或已向根方增殖。说明牙周结扎已成功建立牙周炎动物模型。3大鼠牙周组织HE染色组织形态学观察,用药后3个月,正常组:结合上皮位于釉牙骨质界处,牙龈牙周膜纤维排列整齐,牙槽嵴顶无吸收。牙周炎组:牙龈上皮内有炎细胞浸润,结合上皮向根方增殖或移位,形成较深的牙周袋,血管扩张充血,牙槽嵴顶吸收、降低,可见破骨细胞。去睾丸组:结合上皮附着与釉牙骨质界处,与牙体结合紧密、无炎性浸润,牙槽嵴边缘偶见破骨细胞但牙槽骨无明显吸收。去睾丸牙周炎组:可见深牙周袋,有的甚至深达根尖1/3,有的累及根分叉,沟内上皮下方及固有层有大量炎细胞浸润,血管扩张充血,牙槽嵴顶大量吸收,牙槽嵴表面可见大量吸收陷窝,破骨细胞多,成骨细胞较少。中药组:牙周袋存在,但袋较浅,沟内上皮下方及固有层内有少量炎细胞浸润,牙槽嵴及牙骨质的吸收陷窝边缘可见较多成骨细胞,牙槽骨可见有较多新骨形成。西药组:牙周袋存在,沟内上皮下方及固有层有少量炎细胞浸润,牙槽嵴及牙骨质吸收陷窝边缘有成骨细胞,可见牙槽骨有新骨形成。4各组大鼠牙周组织中ALP水平的变化,牙周炎组和去睾丸组明显高于正常组(P<0.01),去睾丸牙周炎组明显高于牙周炎和去睾丸组(P<0.05) ,中药组和西药组明显低于去睾丸牙周炎组(P<0.01),且与正常组无明显差异(P>0.05),中药组明显低于西药组(P<0.01)。5抗酒石酸酸性磷酸酶(TRAP)染色法测定大鼠牙周组织中破骨细胞的分布和表达水平,正常组:牙槽嵴顶区破骨细胞少见,仅在牙槽嵴边缘有个别破骨细胞存在。牙周炎组:牙槽嵴边缘可见较多破骨细胞存在。去睾丸组:牙槽嵴边缘吸收陷窝内可见少量破骨细胞存在。去睾丸牙周炎组:牙槽嵴顶区域可见大量破骨细胞存在。中药组:牙槽嵴边缘破骨细胞少。西药组:牙槽嵴边缘破骨细胞少。牙槽嵴顶区破骨细胞计数情况,牙周炎组明显高于正常组(P<0.01),去睾丸牙周炎组明显高于牙周炎组(P<0.01),去睾丸组与正常组无明显差异(P>0.05),中药组与西药组明显低于去睾丸牙周炎组(P<0.01),且与正常组无明显差异(P>0.05)。6免疫组化测定BMP-2在大鼠牙槽骨中的分布和表达水平结果,BMP-2主要定位于成骨细胞、成纤维细胞及骨细胞中,正常组:阳性细胞数少且在部分成骨细胞及成纤维细胞中呈弱阳性表达,骨细胞中呈阴性表达。牙周炎组、去睾丸组及去睾丸牙周炎组阳性细胞数增多,呈阳性表达。中药组及西药组中阳性细胞数明显增多,且在成骨细胞、成纤维细胞及部分骨细胞中均呈强阳性表达。BMP-2阳性信号强度平均光密度值(OD)测定结果,牙周炎组、去睾丸组、去睾丸牙周炎组、中药组、西药组均明显高于正常组(P<0.01);牙周炎组、去睾丸组及去睾丸牙周炎组无明显差异(P>0.05) ;中药组和西药组明显高于去睾丸牙周炎组(P<0.01),且两治疗组之间无明显差异(P>0.05)。结论:1.切除大鼠双侧睾丸和钢丝结扎上颌第一磨牙成功建立了大鼠骨质疏松牙周炎模型。2.全身骨质疏松的同时颌骨骨质也出现疏松,并可加重牙周组织的病变。3.右归丸显著降低了牙周组织中破骨细胞的表达量。4.右归丸和雄激素均可使牙周组织中BMP-2的表达提高,诱导成骨,促进了牙槽骨新骨的形成,对骨质疏松牙周炎有显著的治疗作用5.右归丸可能通过降低牙龈组织中ALP的水平,减轻牙周局部炎症。更多还原

【Abstract】 Objective: Animal model of osteoporosis and periodontitis was established through the male wistar rat orchidectomized and periodontal ligation. The influence of Youguiwan and Androgen to the experimental osteoporosis periodontitis and the bone metabolism was compared, to observed the therapy effection Youguiwan on experimental periodontitis in rats with osteoporosis. To explored the relations between masculine osteoporosis and the development of periodontitis, and provide experiment basis on clinical treats the masculine osteoporosis periodontitis.Method: Sixty 6-month-old male Wistar rats were randomly divided into six groups: normal group 10(i.e. group Normal), sham-operated periodontitis group 10(i.e. group Sham-PD), orchidectomy group 10(i.e. group ORX), orchidectomized periodontitis group 10(i.e. group ORX-PD), orchidectomized periodontitis with Youguiwan prescription therapy group 10(i.e. group YGT), orchidectomized periodontitis with Testosterone Undecamoate replacement therapy group 10(i.e. group ART). Group ORX, group ORX-PD, group YGW, group ART resection orchidectomy in rats, group Sham-PD only made a incision don’t excise the testicle; 4 weeks after surgery, rats of group Sham-PD, group ORX-PD, group YGT, group ART were ligated bilaterally of their first maxillary molar teeth with 0.2mm steel-wire; after 3 months of orchidectomy (periodontal ligation 2 months), group ORX and group Normal were randomly selected and killed, take the rats’femur and maxillary, Make femur and maxillary tissue sedions and observe under light microscope, to identify osteoporosis model; Group Sham-PD and group Normal were randomly selected,killed,taken on jaw, the Maxillary First Molar tooth periodontal United biopsy was processed for histological observation, to identify periodontitis model. osteoporosis model and periodontitis model was established, group YGT rats were treated with Youguiwan prescription, according to the proportion of 5mg/kg body weight 2ml(equal to crude drugs 2g) daily gavage, group ART rats were treat with Testosterone Undecamoate, according to the proportion of 8mg/kg body weight match suspension with 2ml physiological saline, 1ml two times daily gavage;the remaining four groups was treated with oral administration of normal saline 2ml daily; after 3 months medication and rats were bled to death by femoral arteries bledding, the rats mandible, hemi-maxillary were immediately putted into 10% formaldehyde buffer solution for fixation, then by 10%EDTA decalcification solution, the production of dental Week biopsy were processed for HE staining to observe, light microscope observation; Tartate resistant acid phosphatase(TRAP) staining to observe osteoclast’s quantity and distribution, and make statistics analysis processing; Immunohistochemisty was used to observe bone morphogenetic protein-2(BMP-2), observes BMP-2 the expression and makes statistics analysis processing. The other side of mandible, the check of the periodontal tissue between maxillary first molar and maxillary second molar, the manufacture organization refining, taking the supernatant after centrifugation, determining in gingival tissues the Alkaline phosphatase(ALP) the level, and makes statistics analysis processing.Results:1 Normal femoral and maxillary trabecular thickness, density were evaluated, Shum rules with the force direction; desexualization group manifested smaller femur trabecular bone, sparse, disorder and irregular. The results indicates flat desexualization method has been successfully in setting up animal models of osteoporosis.2 Histological observation, revealed gingival inflammation, periodontal ligament junctional epithelium vasodilator combination is not close, or has the proliferation to the root side.Indicate that periodontal ligation has been successfully set up animal model of periodontitis.3 Rat Periodontal Tissue Morphology HE staining, after 3 months medication, group Normal: junctional epithelium is located in cementum enamel boundary, gingival periodontal ligament fibers were arranged regularly, no absorption of alveolar crest.group Sham-PD: gingival epithelium has inflammatory cell infiltration,combined with epithelial proliferation or aversion to the root side, the formation of deep periodontal pocket,vasodilator hyperemia, alveolar crest was lower, absorption, and osteoclasts. Group ORX: junctional epithelium is located in cementum enamel boundary, with the tooth body union close, infiltrates not inflammatory, the osteoclast occasionally can be seen in the alveolar crest edge, but alveolar has no obvious resorption. Group ORX-PD: deep periodontal pocket was formatted, and some deep to apical 1/3, and some involved furcation, trench beneath the epithelium and lamina propria have a large number of inflammatory cell infiltration, blood vessel expansion of congestive exposed teeth alveolar bone and the surface has cubstantial absorption lacuna, more osteoclasts and less osteoblasts. Group YGT: periodontal pocket exist, but the bag is shallow, some involved furcation, trench beneath the epithelium and lamina propria have few inflammatory cell infiltration, at the alveolar ridge and cementum lacuna absorption edge more osteoblasts can be seen, more new bone formation can also be observed. Group ART: periodontal pocket exist, trench beneath the epithelium and lamina propria have a small amount of inflammatory cell infiltration,alveolar ridge and cementum lacuna absorption edge shows that osteoblasts existed, new bone formation can be seen.4 The change of the periodontal tissues of rat ALP level in every groups, group Sham-PD and group ORX were significantly higher than the group Normal (P<0.01), group ORX-PD was significantly higher than group Sham-PD and group ORX (P<0.05), group YGT and group ART were significantly lower than group ORX-PD (P<0.01), and group Normal had no significant difference (P>0.05), group YGT was significantly lower than group ART (P<0.01).5 Tartate resistant acid phosphatase(TRAP) dyeing determines osteoclast’s distribution and the expression level in the periodontal tissues of rat, normal group: less osteoclast can be seen in the alveolar crest area, only individual osteoclast existence in the alveolar crest edge. Group Sham-PD: many osteoclast existences in the alveolar crest edge. Group ORX: In alveolar crest edge absorption dimple obviously few osteoclast existence. Group ORX-PD: in the alveolar crest area massive osteoclast existence. group YGT: in the alveolar crest edge osteoclast are rare. Group ART: in the alveolar crest edge osteoclast are few. The alveolar crest area osteoclast counting results, group Sham-PD was higher than the normal group significantly (P<0.01), group ORX-PD was higher than group Sham-PD obviously (P<0.01), group ORX and group Normal had no significant difference (P>0.05), group YGT and group ART were significantly lower than group ORX-PD (P<0.01), and group Normal had no significant difference (P>0.05).6 Immuno histochemistry determination of BMP-2 distribution of expression in the periodontal tissues of rat ’s: BMP-2 mainly locates in the osteoblast, fibroblases the bone cell, normal group: The positive cell number are few, and, in the partial osteoblasts and fibroblasts to assume the weak positive expression, in the bone cell assumes the negative expression. Group Sham-PD, group ORX, group ORX-PD positive cell number increased, assumes positive expression. Group YGT and group ART positive cell number increases obviously, and in the osteoblast, fibroblasts and the partial bone cells assumes the strong positive expression. BMP-2 average light density value (OD) determination result, group Sham-PD, group ORX, group ORX-PD, group YGT, group ART obviously to be higher than the normal group (P<0.01); group YGT and group ART were significantly higher than group ORX-PD (P<0.01) And between two treatment groups there was no obvious difference (P>0.05).Conclusion: 1.Orchidectomized rats ligation of maxillary first molar successfully established periodontitis rat model of osteoporosis. 2. Osteoporosis, while the body also appeared jaw bone osteoporosis, and the osteoporosis may increase the severity of periodontal disease. 3.Youguiwan and androgen significantly reduced periodontal tissue osteoclast’s expression quantity. 4.Significantly increased periodontal tissue BMP-2 expression, induces the bone, promoted mandibular bone new bone’s formation that Youguiwan and Androgen have obvious therapeutic effect on periodontitis of osteoporosis. 5.Youguiwan may be reduces periodontal inflammation through down-requlating the periodontal tissue’s ALP level.更多还原