【作者】 吴银生；

【导师】 林燕萍；

【作者基本信息】 福建中医学院 ， 中医骨伤科学， 2008， 博士

【摘要】 一、目的揭示成骨细胞G1期调节蛋白Cyclin D1、CDK4、p21与实验性绝经后骨质疏松症的关系,并在细胞分子水平研究健骨颗粒对成骨细胞G1期调控的影响,进一步探讨中医药有效防治绝经后骨质疏松症的详细机制。二、方法1、去卵巢骨质疏松模鼠成骨细胞G1期调节蛋白的改变及健骨颗粒干预作用（1）建立实验动物病理模型:切除6月龄SD大鼠卵巢建立绝经后骨质疏松症动物模型。（2）具体分组与药物干预:170只6月龄SD雌性大鼠,随机分成假手术组50只和手术组120只。手术组进行双侧卵巢结扎切除术,假手术组除未行卵巢结扎切除外,其余步骤与手术组相同。手术组在手术后再分成模型组70只,预防组50只。预防组于术后第2天开始喂服健骨颗粒2g·kg^-1·d^-1,每天1次。术后13周模型组再随机分出20只为治疗组。治疗组喂服健骨颗粒2g·kg^-1·d^-1,假手术组和模型组喂服生理盐水2ml·d^-1。健骨颗粒以生理盐水溶解后灌服。治疗组、假手术组及模型组均从术后第13周起灌服。所有大鼠于术后4、8、12、18、24周分批取材,每批每组各处死10只。（3）指标检测:应用双能X线骨密度仪测定全身骨密度;运用放射免疫法检测血清E₂水平;运用免疫组织化学方法检测骨组织中成骨细胞G1期调节蛋白Cyclin D1、CDK4、p21蛋白表达。2、健骨颗粒含药血清对体外培养成骨细胞G1期调节蛋白的影响（1）建立成骨细胞体外培养体系:原代细胞用酶消化法从新生SD大鼠颅盖骨获取,经碱性磷酸酶染色、钙化结节茜素红染色鉴定后,取第三代细胞进行实验。（2）健骨颗粒含药血清制备:取3月龄清洁级雄性SD大鼠20只,随机分为含药血清组和空白血清组,每组10只,含药血清组给予健骨颗粒2g·kg^-1·d^-1,空白血清组灌服生理盐水2ml·d^-1,二组均连续灌胃7天,取血制备成健骨颗粒含药血清和生理盐水空白血清。（3）检测指标:MTT法检测不同含药血清浓度对成骨细胞增殖的影响,得出最佳含药血清浓度;以最佳浓度的含药血清作用于成骨细胞24h、48h和72h,以空白血清干预的细胞为对照,运用流式细胞术进行细胞周期分析、检测Cyclin D1阳性细胞百分含量,运用免疫细胞化学法检测Cyclin D1、CDK4、p21蛋白的表达,运用RT-PCR法检测Cyclin D1、CDK4、p21 mRNA表达。三、结果1、术后12周后,模型组全身BMD明显下降（P＜0.01）,且随时间推移下降趋势明显,预防组、治疗组BMD明显高于同期模型组,但不及假手术组;模型组血清E₂水平下降明显（P＜0.01）,预防组、治疗组E₂水平明显高于同期模型组,预防组高于治疗组,但不及假手术组;Cyclin D1、CDK4蛋白阳性表达主要定位于骨小梁表面的成骨细胞,假手术组有Cyclin D1、CDK4蛋白阳性表达,模型组、预防组、治疗组表达强于假手术组,以预防组表达最高,治疗组次之;p21蛋白阳性表达部位与Cyclin D1相似,假手术组、模型组、预防组和治疗组均有明显阳性表达,假手术组表达最低,模型组的表达维持在较高水平,在各时期均明显高于同期假手术组、预防组和治疗组。2、（1）MTT法检测显示健骨颗粒含药血清浓度为20%时,体外培养成骨细胞增殖速度最快,明显快于同浓度空白血清组（P＜0.05）。（2）流式细胞术检测细胞周期分布结果显示,随着干预时间延长,G0/G1期细胞分数下降,而S期、G2/M期细胞分数上升,增殖指数（PI）升高,以含药血清组变化明显,干预24h、48h、72h,其S期细胞分数、PI均高于空白血清组（P＜0.05）;流式细胞术检测Cyclin D1阳性细胞百分含量,含药血清组明显高于空白血清组（P＜0.01）。（3）免疫细胞化学检测显示,体外成骨细胞Cyclin D1、CDK4蛋白有阳性表达,表达部位主要在细胞核,随着干预时间的延长,含药血清组和空白血清组Cyclin D1、CDK4蛋白表达呈上升趋势,含药血清组明显高于空白血清组（P＜0.05或P＜0.01）;在p21的表达上,二组随时间的推移则呈下降趋势,且含药血清组低于空白血清组,干预48h、72h时二组比较有显著性差异（P＜0.01）。（4）RT-PCR检测结果与免疫细胞化学检测结果基本一致,干预48h、72h,含药血清组Cyclin D1、CDK4 mRNA表达明显高于空白血清组（P＜0.05或P＜0.01）;干预24h,含药血清组p21 mRNA表达明显低于空白血清组（P＜0.05）,干预48h、72h,二者的差距更明显（P＜0.01）。四、结论1去卵巢骨质疏松模型大鼠发病过程中,随着雌激素水平的下降,成骨细胞Cyclin D1、CDK4、p21蛋白出现明显高表达,成骨细胞增殖加快,同时,成骨细胞周期受阻滞亦增多,成骨细胞数量仍显相对不足,骨形成低于骨吸收,最终导致骨质疏松的发生。2健骨颗粒能提高去卵巢骨质疏松模型鼠成骨细胞G1期调节蛋白Cyclin D1、CDK4的表达,抑制负性调节因子p21的表达,促进成骨细胞增殖,这可能是健骨颗粒防治绝经后骨质疏松症的机制之一。3健骨颗粒能提高体外培养成骨细胞G1期调节蛋白Cvclin D1、CDK4的表达,抑制负性调节因子p21的表达,加快细胞从G1期向S期推进,推动成骨细胞增殖周期进程,从而促进成骨细胞增殖,进一步探讨了健骨颗粒通过调节G1期调节蛋白,促进成骨细胞增殖的机理。更多还原

【Abstract】 ObjectiveTo reveal the relationship between Cyclin D1、CDK4、p21 and the experimental postmcnopause osteoporosis,and research the effects of Jiangu granula（JGG） on the cyclins during the G1 phase in osteoblast（OB）,so that to probe into the detailed mechanism of prevention and cure of postmenopause osteoporosis with traditional Chinese medicine.Methods1.To study the change of the cyclins during the G1 phase in OB of the ovariectomized rats and the effect of JGG on the cyclins.（1） Establishing the experimental animal model:SD female（6-month-old） rats were ovariectomized as the postmenopausal osteoporosis animal model.（2） Grouping and treatment:170 SD female rats were randomly divided into 2 groups:sham（50 rats）,ovariectomy（OVX） group（120 rats）.Rats in OVX group were bilaterally ovariectomied and sham group were subjected to sham surgeries.OVX group were divided into two groups then:model group（70 rats） and prevent group（50 rats）. The rats belong to the prevent group perfused with JGG 2g·kg^-1·d^-1 on the second day after opertion.Then after 13 weeks the rats of model group were divided into two group again:momel group and treatment group,20 rats each group.At the beginning of 13 weeks after operation,the rats of treatment group perfused with JGG 2g·kg^-1·d^-1,and the model group and sham group with normal saline 2ml·d^-1.JGG were dissolved in solution of normal saline.10 rats each of the rats remaided belong to the 4 group were sacrificed respectively 4,8,12,18 and 24 weeks after operation. （3） Detective index:Bone mineral density（BMD） were measured by dual-energy X-ray absorptiometry（DXA） for the whole body.Serum concentration of E2 were measured by radioimmunoassay（RIA）.The expression of Cyclin D1,CDK4,p21 proteinum in OB in bone tissue were measured by immunohistochemistry.2.To study the effects of JGG rat serum on the cyclins during the G1 phase in OB which in vitro culture.（1） Establishing the culture in vitro system of OB:Primary cells were isolated from calvaria of newborn SD rats by enzymatic digestion.OB characteristics were identified by alkaline phosphatase（AKP） stain of azo dye method and calcification scobination of alizarin bordeaux dyeing.Cells at the third passage were selected for experiment.（2） Preparation for JGG rat serum:20 male SD rats（3-month-old） were randomly divided into 2 groups:JGG-group and normal saline（NS）group,10 rats each group.The rats of JGG-group perfused with JGG 2g.kg^-1·d^-1,and NS-group with normal saline 2ml·d^-1.The serum containing JGG or NS were extracted after 7 days.（3） Detective index:Cell proliferation of OB cultured in differenent concentration serum was measured by MTT Method,and the optimization concentration of the serum containing JGG would be obtained.Then the serum containing JGG or NS were respectively added and cultured the cells belong to JGG-group or NS-group for 24h, 48h and 72h.The cell cycle were analyzed and Cyclin D1 measured by flow cytometry（FCM）.Immunocytochemical（ICC） method was applyed to measure the expression of Cyclin D1,CDK4,p21,while RT-PCR applyed to measure Cyclin D1、CDK4,p21 mRNA.Results1.12 weeks after operation,BMD for whole body in the model group decreased significantly（P＜0.01）,while BMD in the prevent group and treament group were higher than that in the model group,but than that in the sham group.Serum concentration of E2 in the rats from the model group decreased significantly after opertion（P＜0.01）,and Serum concentration of E₂ from the prevent group and treament group were higher than that in the model group in the same period,while that in the prevent group higher than treament group、but low than sham group.Cyclin D1、CDK4 in OB positive expression were major located at the superficies of bone trabecula.There was Cyclin D1、CDK4 positive expression in the bone tissue from the sham group,but that from the model group,treament group and prevent group were all obviously higher than the sham group.The positive expression of the prevent group was the highest one among the 4 group,and the treament group was the second one.p21 positive expression location was similar to Cyclin D1.Obviously positive expression of p21 appeared in the bone tissue from all four groups,in which that of the sham group was the lowest,while the positive expression of the model group maintained at a high level,and higher than that of the rest 3 groups in different period after operation.2.（1） It showed that cell proliferation rate of OB was the fastest when the concentra-tion of the serum containing JGG was 20%,which was added to the OB of JGG-group.And it was significantly faster than that of NS-group（P＜0.05）.（2） The measure by FCM on cell cycle of OB showed that with the action time lasting,the cell percentage of G0/G1 phase decreased,while that of S and G2/M phases and proliferation index（PI） rising.The change of the JGG-group was more obviously:the cell percentage of S phase and PI were higher than NS-group at the 24h,48h and 72h point（P＜0.05）.The result of Cyclin D1 measured by FCM showed that the positive expression of Cyclin D1 in the JGG-group was significantly higher than that in NS-group.（3） ICC showed that positive expression of Cyclin D1 and CDK4 were locatet at cell nucleus in OB in vitro culture.With the action time lasting,the positive expression of Cyclin D1 and CDK4 increased,and that of JGG-group was more significant（P＜0.05 or P＜0.01）.Positive expression of p21 in OB decreased With the action time lasting,and the expression of JGG-group was lower than the other group,which there were significant difference at 48h and 72h points（P＜0.05）.（4） The results measured by RT-PCR were mainly coincident.Cyclin D1、CDK4 mRNA in OB of JGG-group was significantly higher than that in NS-group（P＜0.05 or P＜0.01） at 48h and 72h points.On the contrary,p21 mRNA of JGG-group was significantly lower than that in NS-group（P＜0.05）.The distinction were even obviously at 48h and 72h.Conclusion1 With the serum concentration of E₂ decreasing,the Cyclin D1、CDK4 and p21 in OB in the process of osteoporosis in OVX model rats are hyper-expressed, speeding up the cell multiplication and bone formation;while the blockage of the cell cycle and the Apoptosis are excessive,so that the relative insufficience of OB and bone formation cause the high transform osteoporosis.2 JGG can increase the expression of Cyclin D1、CDK4 and inhibit p21 expres-sion in OB in OVX osteoporosismodel rats,so that to stimulate cell proliferation of OB. It is,perhaps one of mechanisms that JGG can prevention and cure postmenopausal osteoporosis.3 JGG can increase the Cyclin D1、CDK4 expression and inhibit the p21 expres-sion in OB cultured in vitro,so that to accelerate the proceeding cell generation cycle and stimulate cell proliferation of OB.The thesis deeply approaches the mechanism that JGG stimulate cell proliferation of OB by the effect of the cyclins during the G1 phase.更多还原