【作者】 葛保健；

【导师】 夏仁云；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 目的:观察不同剂量的人工合成的植物雌激素—依普拉芬对去卵巢大鼠骨质疏松的防治作用;体外培养大鼠成骨细胞,观察依普拉芬对成骨细胞增殖和成骨分化的影响;检测依普拉芬对体外培养的大鼠成骨细胞和去卵巢大鼠一氧化氮(NO)以及一氧化氮合酶(NOS)生成的影响;探讨依普拉芬防治绝经后骨质疏松的作用以及NO调控机制,为其更好的在临床应用提供理论依据。方法:(1) 60只SD雌性大鼠,随机分成六组:设一假手术组;腹腔手术切除大鼠双侧卵巢,分为阴性对照组、依普拉芬低、中、高剂量组和雌激素对照组,分别给予基础饲料和不同剂量受试物,12周后进行骨密度、骨组织形态计量、生物力学以及骨代谢指标测定,与雌激素对照,观察给与依普拉芬对绝经后骨质疏松的防治作用。(2)体外分离、培养、纯化新生大鼠颅盖骨成骨细胞,碱性磷酸酶染色及矿化结节染色鉴定,将不同浓度的依普拉芬加入培养液,测定细胞增殖情况、碱性磷酸酶(ALP)活性及钙化结节。与雌激素对照,了解依普拉芬对成骨细胞增殖和分化的影响。(3)体外分离培养新生SD大鼠颅盖骨成骨细胞,取第二代细胞,培养液中分别加入不同浓度的依普拉芬,72小时后,测定各组细胞培养基中的NO浓度,RT-PCR方法检测各组细胞中内皮型一氧化氮合酶(eNOS)mRNA的表达。(4)按第一部分方法建立绝经后骨质疏松模型及分组,给与受试物12周后测定大鼠血清一氧化氮(NO)以及一氧化氮合酶(NOS)含量。用免疫组织化学方法检测骨组织中eNOS以及iNOS的表达。结果:(1)大鼠去卵巢后骨密度显著下降,股骨的力学性能以及骨代谢指标有较大变化,弯曲强度和弯曲弹性模量明显降低。给予依普拉芬后,可使骨密度显著提高(P<0.01),存在一定的剂量-效应关系,同时弯曲强度和弯曲弹性模量明显增加,但均低于雌激素对照组。(2)与阴性对照组相比,各浓度组的依普拉芬均可增加成骨细胞数量(P<0.01),提高成骨细胞的ALP活性和促进钙化结节形成(P<0.01)。其中10-8～10-5mol/L作用更为显著。(3)与阴性对照组相比,各浓度组的依普拉芬均可增加成骨细胞培养液中的NO浓度并促进eNOS mRNA的表达(P<0.01)。其中10-8～10-5mol/L之间作用更为显著。(4)与假手术组相比,去卵巢大鼠阴性对照组血清NO、NOS浓度以及股骨eNOS表达均明显降低,依普拉芬各剂量组高于阴性对照组,与假手术组差异无显著意义。同时低于雌激素组。结论:依普拉芬对去卵巢大鼠具有显著的骨保护效应,同时可以促进大鼠成骨细胞的增殖和成骨分化,并存在一定的剂量效应关系,其作用弱与雌激素。依普拉芬防治绝经后骨质疏松作用可能是通过NO-NOS系统进行调控的。更多还原

【Abstract】 Objective: To investigate the effect of Ipriflavone(7-isopropoxyisoflavone, IP), a synthetic phytoestrogen,on prevention and treatment of osteoporosis in ovariectomized rats;Isolation, expansion, and culture the new born rat calvarid osteoblastic cell in vitro,then evaluate the effect of Ipriflavone on the proliferation and osteogenic differentiation of cultured rat osteoblast in vitro;Investigate the effect of Ipriflavone on nitric oxide (NO ) synsthesis and expression of endothelium nitric oxide synathase (eNOS) mRNA in cultured rat osteoblast in vitro. Observe the effect of Ipriflavone on synthesis of nitric oxide (NO) and nitric oxide synathase (NOS) inovariectomized rats. Thereby ascertain the effects of Ipriflavone on osteoporosis in ovariectomized rats and its possible mechanisms through NO pathway,provides the theory basis to clinical practice much better.Methods: (1) 60 six-month-old Sprague-Dawley female rats were randomly assigned to four groups: sham,ovx, ovx+IP(50 mg/kg/d),ovx+IP(100 mg/kg/d), ovx+IP(200 mg/kg/d),and ovx+17β-estradiol (E2) (10ug/kg/d). After 12 weeks, bone mineral density, bone histomorphometry, biomechanics of bone and the markers of bone metabolism were measured. Observe the prevention and treatment effect of Ipriflavone on postmenopausal osteoporosis. (2) Isolation, expansion, and culture the new born rat calvarid osteoblastic cell in vitro, identified by alkaline phosphatase staining and mineralized nodules. The osteoblasts were cultured with the medium containing Ipriflavone of different concentration.The proliferation,the activity of alkaline phosphatase(ALP) and calcium node were determined and compared with control.Observe the effect of Ipriflavone on the proliferation and differentiation of cultured rat osteoblast in vitro. (3) New born rat calvarid osteoblastic cell were isolated and cultured with the medium containing Ipriflavone of different concentration.After 72hours, the levels of NO in the media were detectetd and the mRNA expression of eNOS were examined by reverse-transcriptase polymerase chain reaction (RT-PCR). (4) The model of postmenopausal osteoporosis was established and divided into different groups use the same methodsof the first part. After 12 weeks, the the level of serum NO and NOS were detected in grouped rats. nitric oxide (NO) and nitric oxide synthase (NOS). expression of eNOS in the bone tissue was detected by immunohistochemical staining.Results: (1) The bone mineral density (BMD) of OVX in ovariectomized rats decreased significantly . The mechanical properties and biomechanics markers of femur changed obviously. Ipriflavone and estrogen can increase the BMD(P<0.01)and demonstrate a dose-dependent manner. Meanwhile flexural strengh and flexural elastic module of femur increase, but lower than the estrogen group. (2)Compared with the negative control, Ipriflavone could increase the quantity of osteoblast(p<0.01). Ipriflavone could elevate the activity of ALP and ability of calcification(p<0.01). 10-8～10-5mol/L ipriflavone had more significant effect than other concentration. (3) In comparison to the negative control, Ipriflavone could increase the concentration of NO in the media and the expression levels of eNOS mRNA (P<0.01)。10-8～10-5mol/L ipriflavone had more significant effect than other concentration . (4) In comparison to sham group ,The level of NO and NOS in ovariectomized(OVX) rats decreased significantly. Ipriflavone could significantly increase the level of NO and NOS comparison to OVX . There was no statistical significance of the difference between Ipriflavone and sham group. Estrogen groups obtained higher level of NO and NOS than other groups.Conclusions : Ipriflavone demonstrated significant protective effect on bone in ovariectomized rats,also can promote the proliferation and osteogenic differentiation of rat osteoblast, and there is a definite dose-response relationship. It’s effect was weaker than estrogen. The effect of Ipriflavone on prevention and treatment of postmenopausal osteoporosis(PMOP) probably regulated through NO-NOS system.更多还原