【作者】 徐丽丽；

【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:通过建立雄性大鼠骨质疏松模型,观察右归丸方剂对实验性骨质疏松大鼠颌骨骨代谢的影响,探讨右归丸方剂抑制骨质疏松大鼠颌骨吸收的机制。为临床上治疗骨质疏松患者颌骨的吸收,特别是老年男性骨质疏松患者颌骨的吸收提供理论依据。方法:选用纯种6月龄雄性Wistar大鼠50只,随机分为5组:正常组、激素组(模型组1)、去势组(模型组2)、激素右归丸治疗组(治疗组1)、去势右归丸治疗组(治疗组2),每组10只。去势组及去势右归丸治疗组大鼠在10%水合氯醛腹腔注射全身麻醉下,取仰卧位,纵隔两侧约1cm处各开一纵行切口,分别将双侧睾丸与附睾分离、结扎,切除睾丸,缝合切口,腹腔注射庆大霉素1ml。睾丸摘除术后一月激素组及激素右归丸治疗组大鼠灌服氢化可的松注射液,按3.6mg/kg给药,前30天每周灌胃6次,后30天改为每周给药2次,共2月。去势术后3个月(激素灌胃后2个月)随机抽取动物,制作上颌骨和股骨HE染色组织学切片,光镜观察确定骨质疏松模型成功。模型鉴定成功后,两治疗组给予右归丸方剂,每只大鼠每天2ml(5mg/kg)灌胃,其他三组灌服2ml生理盐水。灌药后3个月,各组大鼠在10%水合氯醛腹腔注射全身麻醉下测定股骨骨密度,股动脉放血处死大鼠,留取全血,放射免疫法检测血清中睾酮(T)和雌二醇(E2)的含量;取大鼠上颌骨和股骨,立即放入10%甲醛缓冲液固定,24h后,15%EDTA脱钙液脱钙2个月,常规制作组织切片,HE染色,光镜观察上颌骨及股骨骨组织形态学的变化;制作免疫组织化学切片,免疫组织化学染色,观察转化生长因子β1(TGF-β1)在颌骨骨组织中的表达。结果:1造模一周后,大鼠出现消瘦、毛发稀疏、倦怠少动、畏寒喜扎堆、少食少饮和小便次数及小便量增多等“肾阳虚”症状。去势术后3个月(激素灌胃2个月),激素组体重增长较慢(P<0.01),而去势组体重增加较快(P<0.01)。随机抽取模型组及正常组大鼠,取上颌骨和股骨并处死动物,制作组织切片,正常组骨小梁粗大、密集,顺受力方向规则排列;模型组上颌骨及股骨骨小梁细小疏松、排列紊乱不规则。说明激素法及去势法已成功建立骨质疏松动物模型。右归丸治疗后3个月,激素组大鼠体重明显低于正常组(P<0.01),去势组大鼠的体重明显高于正常组(P<0.01);激素右归丸治疗组体重较激素组升高(P<0.05),去势右归丸治疗组较去势组体重降低(P<0.01),右归丸治疗两组大鼠的体重与正常组比较无明显变化(P>0.05),且两治疗组之间没有显著性差异(P>0.05)。2灌服右归丸方剂后3个月,各组大鼠股骨骨密度比较,两模型组较正常组低,且有显著性差异(P<0.01),但两组之间无明显差异(P>0.05),治疗组明显高于模型组(P<0.01),而低于正常组,有显著性差异(P<0.01),两治疗组之间无明显差异(P>0.05)。3放射免疫法测定大鼠血清中E2和T的结果,各组大鼠E2浓度无显著差异(P>0.05)。两模型组T含量较正常组显著降低(P<0.01),且两组之间无明显差异(P>0.05);激素右归丸治疗组明显高于激素组(P<0.01),且高于正常组,有统计学意义(P<0.05);去势右归丸治疗组与去势组之间无显著性差异(P>0.05),较正常组低(P<0.01);激素右归丸治疗组明显高于去势右归丸治疗组,有显著性差异(P<0.01)。4大鼠上颌骨及股骨HE染色骨组织形态学的观察及骨组织形态计量学参数的测量灌服右归丸后3个月,骨组织形态学观察:正常组股骨骨小梁排列紧密,有序,数目多,面积大;颌骨未见吸收,松质骨间充满红骨髓,骨小梁呈片状,较粗大。两模型组股骨骨小梁排列疏松,数目少,破坏严重部位出现骨小梁的明显缺失,导致骨髓腔扩大;颌骨可见吸收,骨小梁变细,间隙增大,牙槽骨边缘可见破骨细胞及吸收陷窝。两治疗组股骨的骨小梁数量较多,排列较紧密,与正常对照组相近,无明显扩大的骨髓腔;颌骨松质骨内骨小梁变粗大,间隙减小,其间充满红骨髓,吸收陷窝内可见成骨细胞。骨组织计量学参数的测量:两模型组颌骨及股骨骨小梁面积比、骨小梁平均厚度均明显低于正常组(P<0.01) ,骨小梁平均间隔宽度较正常组显著升高(P<0.01),但两组之间无显著性差异(P>0.05);治疗组骨小梁面积比、骨小梁平均宽度均较模型组升高(P<0.01),骨小梁平均间隔宽度较模型组显著降低(P<0.01),两治疗组之间无明显差异(P>0.05)。5 TGF-β1在上颌骨中的表达,TGF-β1在正常组的骨髓腔、成骨细胞胞浆中呈阳性显色,部分骨基质及骨细胞胞浆内可见表达;在模型组骨髓腔基质细胞内呈弱阳性表达,成骨细胞胞浆内未见明显表达;治疗组TGF-β1在颌骨骨髓腔基质细胞及成骨细胞内呈强阳性表达,部分血管内皮细胞、骨细胞及骨基质中也可见阳性表达。6 TGFβ-1免疫组化平均光密度值(OD)测定,激素组及去势组明显低于正常组(P<0.01),两组之间无差异性(P>0.05);治疗组明显高于模型组(P<0.01),且两治疗组间无显著性差异(P>0.05),治疗组与正常组之间无明显差异(P>0.05)。结论:1氢化可的松灌胃和去睾丸均能成功建立雄性大鼠骨质疏松模型。2全身骨质疏松的同时颌骨也疏松,但颌骨骨吸收小于股骨骨吸收。3右归丸治疗减缓了因睾酮水平降低引起的骨质疏松症的发生,减缓了颌骨的骨质吸收,促进新骨形成,改善了颌骨的组织形态结构,对颌骨骨质疏松有一定的治疗作用。4右归丸方剂可改变未完全受损的性器官的病理状态,增加性激素的分泌;并不能改善去睾丸大鼠激素水平,但具有雄性激素样作用。5右归丸可使颌骨骨组织中TGF-β1的表达升高,诱导成骨,促进新骨形成,使颌骨组织形态结构的改变得以恢复。更多还原

【Abstract】 Objective: To establish animal model of osteoporosis of the male rats, to observed the effect of Yougui pill on bone metabolism in experimental rat with osteoporosis, and to explore Yougui pills’effect of inhibiting rat alveolar bone absorption. For further understand the role of traditional Chinese medicine for prevention and treatment alveolar bone absorption in patients with osteoporosis, especially gerontism male to provide an experimental reference.Methods: Fifty 6-month-old male Wistar rats were randomly divided into five groups: normal group, hormone group(group model 1), orchidectomy group(group model 2), hormone with Yougui pill therapy group(group THER 1), orchidectomy with Yougui pill therapy group(group THER 2) and each group 10. Group ORX, group ORX-YGT to get dorsal position removal didymus under general anaesthesia with 10% KOD. Suture nick, peritoneal injection GM 1ml. 4 weeks after orchidectomized, rats of group H, group H-YGT drench Hydrocortisone injection, 3.6mg/kg, 6 times per week for the first 30 days, 2 times per week for the later 30 days, 2 months in all. After 3 months of orchidectomy, draw-off animal randomly, the maxilla and femur tissue sections were stained with HE for histological examination. Osteoporosis model identification is proved to be successful, two therapy groups rats were treated with Yougui pills, each rat gavage daily from 2ml(5mg/kg), the remaining three groups were treated with oral administration of normal saline 2ml water daily. After 3 months, determine femoral bone BMD, then all rats were bled to death by femoral arteries bledding, collecting whole blood for serum T and E2 with radioimmunity; collected maxillae and femoral bone, and was immediately put into 10% formaldehyde buffer solution for fixation, after 24h, changed to 10%EDTA decalcification solution 2 months, the tissue sections were stained with HE for histological examination; and the TGF-β1 expression were detected by immunohistochemical method.Results:1 A week after modeling, the rats exhibited weight loss, hair thinning, fatigue less dynamic, chills and crowding together, eat and drink less, and the urination frequency and volume of urine increased "deficiency of kidney-YANG" symptoms. 3 months after orchidectomy, body weight slower growth in hormone group (P<0.01), while the ORX group gained weight faster (P<0.01). Model and normal group rats were randomly sampled, take the maxillae and femur and the animals were killed, making biopsy section, trabecular bone thick, dense in the normal group; model group exhibited femoral trabecular bone loose, arrangement of disorder. Indicates that the two methods has been successfully in establishing animal models of osteoporosis. After 3 months Yougui Pill treatment, the hormone group body weight was significantly lower than normal group (P<0.01), body weight of orchidectomized rats was significantly higher than the normal group (P<0.01); group H-YGT weight higher than the hormone group (P<0.05), group ORX-YGT reduced body weight compared with ORX group (P<0.01), the weight of two treatment groups rats compared with group normal were no significant change (P>0.05), and between the two treatment groups there was no significant difference (P> 0.05).2 After 3 months Yougui Pill treatment, the rats femur bone mineral density among five groups were compared. Two model group was significantly lower than group normal(P<0.01), and no significant difference between the two groups (P>0.05); two treatment group was significantly higher than the model group(P<0.01) and lower than group normal(P<0.01), between the two treatment groups was no significant difference (P>0.05).3 Radioimmunoassay determination of serum E2 and T, the E2 concentration in rats was no significant difference (P>0.05). The T content of two model group was significantly lower than group normal (P<0.01), and no significant difference between the two groups (P>0.05); group H-YGT was significantly higher than the group hormone (P<0.01), and higher than the group normal (P<0.05); group ORX-YGT and group ORX no significant difference (P>0.05), lower than the group normal (P<0.01); group H-YGT was significantly higher than group ORX-YGT , there was significant difference (P<0.01).4 Rat maxillae and femur tissue HE staining and bone histomorphometry parameters were measured.Three months after medication, morphology observation: normal femoral trabecular was thick, orderly, cis-arranged by the rules of force direction; maxillae has no absorption, which is fraught with bone marrow, trabecular bone was flaky, enlargement. Model group femoral trabecular was smaller and trabecular spaces scale-up, resulting the bone marrow cavity expansion; maxillae resorption, trabecular thinning, alveolar bone surface has absorption lacuna and osteoclasts. Two treatment groups has larger number of femoral trabecular, arranged more closely, close to the group normal, no significant expansion of the bone marrow cavity; maxillae trabecular bone thicker and trabecular spaces decreases, which is fraught with red bone marrow , absorption lacuna more osteoblasts can be seen.Bone histomorphometry parameters were measured: two model group, maxillae and femur Tb.Ar(%),Tb.Th(μm) were significantly lower than the group normal(P<0.01), the Tb.Sp(μm)was significantly higher than group normal (P<0.01), but between the two groups no significant difference (P>0.05); two treatment group Tb.Ar(%),Tb.Th(μm) were significantly higher than the model group(P<0.01), the Tb.Sp(μm)was significantly lower than model group(P<0.01), the two treatment groups has no significant difference (P>0.05).5 TGF-β1 expression in maxillae, in group normal, cavitas medullaris, osteoblasts showed positive expression, some bone matrix and bone cells can also be stainned; in model group, cavitas medullaris revealed weakly positive expression, osteogenic cells has no obvious expression; in treatment group, cavitas medullaris, osteoblasts showed strong positive expression, in some vascular endothelial cells, bone cells and bone matrix can also be found positive expression.6 TGF-β1 immunohistochemistry mean optical density (OD) measured, group hormone and group ORX was significantly lower than group normal(P <0.01), no difference between the two groups (P> 0.05); treatment group was significantly higher than the model group (P <0.01), and the two treatment groups no significant difference (P> 0.05).Conclusion: 1 Hydrocortisone and orchidectomized successfully established rat model of osteoporosis. 2 When all over the body occurrence osteoporosis, the jaw bones also can be occurrenced osteoporosis at the same time, but the jaw bone resorption is less than the femur bone resorption. 3 Yougui pill slowed down osteoporosis occurrence due to lower testosterone levels, slowed down the jaw bone absorption, and promoted new bone formation, improved the alveolar bone structure, indicates it has certain therapeutic effect on jaw bone absorb caused by osteoporosis. 4 Yougui pill can increased sex hormones level; has androgen-like effect. 5 Yougui pill can increased TGF-β1 expression in jaw bone tissue, induction bone formation improve the jaw bone structure.更多还原