成骨生长肽羧基端5肽OGP_（10-14）及其类似物G48A对大鼠骨代谢的影响
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摘要
目的 通过大鼠血清骨转换生化标志物检测、离体骨密度测定、骨组织形态计量学和生物力学试验综合评价了OGP_(10-14)及其类似物G48A对去势SD大鼠骨代谢的影响，并初步探讨了各项参数间的相关关系。方法 雌性SD大鼠86只，按体重随机分层分组：OP预防组和OP治疗组。其中OP预防组再分为以下7组：Ⅰ．假手术组(SHAM)，Ⅱ．去势组(OVX)，Ⅲ．阿伦膦酸钠组(ALEN)，Ⅳ．G36G低剂量组(G36GL)，V．G36G高剂量组(G36GH)，Ⅵ．G48A低剂量组(G48AL)，Ⅶ．G48A高剂量组(G48AH)，每组8只，计56只。术后次日起，以上各组大鼠每日皮下给予各种处理因素：Ⅰ，Ⅱ组每日皮下注射磷酸盐缓冲液(PBS)，Ⅲ，Ⅳ，Ⅴ，Ⅵ，Ⅶ组按体重每日皮下给予阿伦膦酸钠、低剂量G36G、高剂量G36G、低剂量G48A、高剂量G48A PBS溶液，干预9周后，处死大鼠；OP治疗组，再分为以下6组：Ⅷ．假手术组(SHAM-T)，Ⅸ．去势组(OVX-T)，X．利塞膦酸钠组(RISE-T)，Ⅺ．G36G高剂量+利塞膦酸钠组(36GRI-T)，Ⅻ．G36G高剂量组(G36G-T)，ⅩⅢ．G48A高剂量组(G48A-T)，均于造模术后15周起，每日皮下给予各种处理因素：Ⅷ，Ⅸ组每日皮下注射PBS，Ⅹ，Ⅺ，Ⅻ，ⅩⅢ组按体重每日皮下给予利塞膦酸钠、利塞瞵酸钠+高剂量G36G、高剂量G36G、高剂量G48A PBS溶液，治疗9周后处死大鼠。分离
天津医别一火学疼士学位论文
股骨、胫骨和腰椎Ll一。。双能X线骨密度仪(DExA)小动物软件测定各组大鼠
腰椎L卜4和股骨远端干髓端、股骨全段骨密度。股骨与腰椎L，分别行三点弯
曲试验及腰椎压缩试验检测其生物力学性质。近端胫骨干髓端制备不脱钙骨
切片，行骨计量学测定。所得资料以SPSS10.O软件进行统计。结果(l)骨转
换生化标志物:G36GH，G48AH组IGF一2水平显著高于SHAM组，ALEN，OVX
组BGP显著低于G48AH组。G36G一T，G48A一T，RISE一T组BGP，IGF一2显著低
于36GRI一T组。(2)股骨干重、灰重及骨密度:OVX，OVX一T组股骨全段、远端
干箭端、腰椎L卜4三个部位的骨密度(BMD)及股骨灰重/干重比值(AW/DW)
分别显著低于SHAM，S以M一T组;ALEN组上述各项指标均显著高于OVX组;
G36GH，G36GL，G48AH，G48AL组AW/DW显著高于OVX组，三个部位骨密度均
较OVx组有增高趋势，但无显著性差异。OVX一T组三个部位BMD值显著低于
36GRI一T和SHAM一T组:G36G一T，G48A一T组显著低于SHAM一T组。(3)生物力学:
股骨三点弯曲试验OVX，OVX一T组最大应变(￡b)，弯曲能量(U)，弹性载荷
(Fp)与SHAM，SHAM一T组无显著差异。腰椎压缩试验OVx组强度极限(ob)
显著低于SHAM组，ALEN组显著高于OVX组、G48AH和G48AL组;OP
各测试指标间无显著性差异。(4)骨计量学:
OVX，OVX一T
治疗组
组骨小
粱体积与全部骨组织体积之比(TBv/TTv)、骨小粱板厚度(MTPT)和密度(MT即)
分别较S以M，SHAM一T组显著降低，骨小粱板间距(MTPS)较SHAM，SHAM一T
组显著增加。G36GH，G48AH，G48AL组TBV/TTV，骨小粱体积与海绵骨体积
之比(TBV/SBV)，价PT均较OVX组显著增加，MTPS和骨小粱表面与体积之
比(S/V)较OVX组下降。G36G一T，G48A一T，36GRI一T组TBV/TTv，TBv/SBV
较OVX组显著增加，但显著低于SHAM一T组。动力学参数OVx组四环素双标
表面{Sfract(d)}较SH胡组显著增加，类骨质表面占骨小粱表面百分比(TOS)
低于SHAM组。ALEN组Sfraet(s)，Sfra。t(d)均较OVX;组下降。G36GH，G36GL，
GSAL组Sfraet(d)低于OVX组，TOS高于OVX和ALEN组。G48A一T组Sfraet(s)
一3·
及津医刊一大学唯士学位沦文
显著高于OVX一T组。RISE一T，36GRI一组几乎无双标记。(5)相关分析:预防
组股骨远端干髓端、股骨整体和腰椎L，一;BMD值与骨计量学参数TBV/TTV，MTPD
之间呈显著正相关，与MTPS呈显著负相关。生物力学部分指标虽然与骨密度、
骨计量参数有显著相关性，但从决定系数评价密切程度低，相关效率也较低。
结论OGP(:0一14，及其类似物G48A可有效防治大鼠去势后的骨丢失，改善干髓
端骨小梁的连接性，短期治疗对骨生物力学性质无明显影响。作为评价抗骨
质疏松药物作用的指标，骨计量学与骨密度参数之间存在一定的关联，生物
力学试验方法尚待进一步改进。
objective: To investigate the effects of OGP(10-14) and its analogue G48A on biochemical markers of bone turnover, bone mineral density(BMD), histomorphometry and biomechanical properties in ovariectomized rats while the relationships between BMD, biomechanics and histomorphometric parameters were explored. Methods: Eighty-six female SD rats, four months old, were divided into following groups by body weight randomly: OP-prevention group and OP-therapy group. The ovariectomized rats were used as a model for osteoporosis. The prevention group was further subdivided into seven groups(n= 8/group): shamed-operated group(SHAM), Ovariectomized group(OVX), Alendronate-therapy group(ALEN), G36G high dosage-therapy group (G36GH), G36G low dosage-therapy group (G36GL), G48A high dosage -therapy group (G48AH), G48A low dosage -therapy group (G48AL). The rats of prevention groups were injected subcutaneously with phosphate buffered saline (PBS) on SHAM and OVX group or G36G, G48A , alendronate on other groups once per day for 9 weeks one day after the ovariectomy
separately, then sacrificed. The therapy group was further subdivided into six groups(n= 5/group) :shamed-operated group(SHAM-T), Ovariectomized group(OVX-T), Risedronate-therapy group(RISE-T), Risedronate with G36G therapeutic alliance group(36GRI-T), G36G high dosage-therapy group (G36G-T), G48A high dosage-therapy group (G48A-T). The rats of therapy groups were injected subcutaneously with PBS on SHAM and OVX group or G36G, G48A, Risedronate on other groups once per day for periods of 9 weeks initiated 15 weeks after ovariectomy separately, then sacrificed. Lumbar spine,the bilateral tibia and femur were dissected out. Serum BGP and IGF-2 levels were determined by radioimmunoassay , while The morphological and dynamic parameters for bone histomorphometry , biochemical markers for bone formation and other indices of bone metabolism were measured. Biomechanical property parameters of femora and lumbar spine (L5) were measured with the three point bending tests and compressive test. The BMD of total femur .distal femur metaphysis and L1-4 were measured by dual energy X-ray absorptiomtry (DEXA) using DMS equipment while histomorphometry of proximal tibia undecalcif ied specimen sections were performed which include trabecular volume, trabecular thickness, dynamic data , et al. The relationship between BMD, biomechanic characters and histormorphometry in OP-prevention group were investigated. These data were analyzed by SPSS 10. 0 software with analysis of variance and correlation. Results: Akaline phosphatase activity levels were not statistically significantly different in the prevention and therapy groups. An elevation of BGP serum level were observed in OVX and ALEN
group as compared with G48AH group however the descent of IGF-2 serum level were determined in G36GH, G48AH group as compared with SHAM group. The results showed that both G36G and G48A had significant stimulation on bone formation as the tetracycline labeling on the trabecular bone surface and the rate of bone formation while no significant effect on the biomechanics, but both of them could not completely prevent bone loss in OVX rats. The ratio of femora ash weight to dry weight(AW/DW) and BMD of total femur, distal metaphysis , L1-4 in OVX and OVX-T group were significantly lower than in SHAM group, while the above-mentioned indices were remarkably increased in ALEN group than others. There was only an increased trend of BMD on the three sites of bone in G36GH, G36GL, G48AH, G48AL group as compared with OVX group. Bone histomorphometry in the OVX group was showed significant decrease on trabecular bone volume%total tissue (TBV/TTV), mean trabecular plate thickness(MTPT), mean trabecular plate density( MTPD) while mean trabecular plate spacing( MTPS) seemed to be higher than other groups. In the G36GH and G48AH groups, parameters of the TBV/TTV, trabecular bone volume%total sponge( TBV/SBV), MTPT were statistically increased and parameters of MTPS, trabecular surface%volume
天津医别一火学疼士学位论文
股骨、胫骨和腰椎Ll一。。双能X线骨密度仪(DExA)小动物软件测定各组大鼠
腰椎L卜4和股骨远端干髓端、股骨全段骨密度。股骨与腰椎L，分别行三点弯
曲试验及腰椎压缩试验检测其生物力学性质。近端胫骨干髓端制备不脱钙骨
切片，行骨计量学测定。所得资料以SPSS10.O软件进行统计。结果(l)骨转
换生化标志物:G36GH，G48AH组IGF一2水平显著高于SHAM组，ALEN，OVX
组BGP显著低于G48AH组。G36G一T，G48A一T，RISE一T组BGP，IGF一2显著低
于36GRI一T组。(2)股骨干重、灰重及骨密度:OVX，OVX一T组股骨全段、远端
干箭端、腰椎L卜4三个部位的骨密度(BMD)及股骨灰重/干重比值(AW/DW)
分别显著低于SHAM，S以M一T组;ALEN组上述各项指标均显著高于OVX组;
G36GH，G36GL，G48AH，G48AL组AW/DW显著高于OVX组，三个部位骨密度均
较OVx组有增高趋势，但无显著性差异。OVX一T组三个部位BMD值显著低于
36GRI一T和SHAM一T组:G36G一T，G48A一T组显著低于SHAM一T组。(3)生物力学:
股骨三点弯曲试验OVX，OVX一T组最大应变(￡b)，弯曲能量(U)，弹性载荷
(Fp)与SHAM，SHAM一T组无显著差异。腰椎压缩试验OVx组强度极限(ob)
显著低于SHAM组，ALEN组显著高于OVX组、G48AH和G48AL组;OP
各测试指标间无显著性差异。(4)骨计量学:
OVX，OVX一T
治疗组
组骨小
粱体积与全部骨组织体积之比(TBv/TTv)、骨小粱板厚度(MTPT)和密度(MT即)
分别较S以M，SHAM一T组显著降低，骨小粱板间距(MTPS)较SHAM，SHAM一T
组显著增加。G36GH，G48AH，G48AL组TBV/TTV，骨小粱体积与海绵骨体积
之比(TBV/SBV)，价PT均较OVX组显著增加，MTPS和骨小粱表面与体积之
比(S/V)较OVX组下降。G36G一T，G48A一T，36GRI一T组TBV/TTv，TBv/SBV
较OVX组显著增加，但显著低于SHAM一T组。动力学参数OVx组四环素双标
表面{Sfract(d)}较SH胡组显著增加，类骨质表面占骨小粱表面百分比(TOS)
低于SHAM组。ALEN组Sfraet(s)，Sfra。t(d)均较OVX;组下降。G36GH，G36GL，
GSAL组Sfraet(d)低于OVX组，TOS高于OVX和ALEN组。G48A一T组Sfraet(s)
一3·
及津医刊一大学唯士学位沦文
显著高于OVX一T组。RISE一T，36GRI一组几乎无双标记。(5)相关分析:预防
组股骨远端干髓端、股骨整体和腰椎L，一;BMD值与骨计量学参数TBV/TTV，MTPD
之间呈显著正相关，与MTPS呈显著负相关。生物力学部分指标虽然与骨密度、
骨计量参数有显著相关性，但从决定系数评价密切程度低，相关效率也较低。
结论OGP(:0一14，及其类似物G48A可有效防治大鼠去势后的骨丢失，改善干髓
端骨小梁的连接性，短期治疗对骨生物力学性质无明显影响。作为评价抗骨
质疏松药物作用的指标，骨计量学与骨密度参数之间存在一定的关联，生物
力学试验方法尚待进一步改进。
objective: To investigate the effects of OGP(10-14) and its analogue G48A on biochemical markers of bone turnover, bone mineral density(BMD), histomorphometry and biomechanical properties in ovariectomized rats while the relationships between BMD, biomechanics and histomorphometric parameters were explored. Methods: Eighty-six female SD rats, four months old, were divided into following groups by body weight randomly: OP-prevention group and OP-therapy group. The ovariectomized rats were used as a model for osteoporosis. The prevention group was further subdivided into seven groups(n= 8/group): shamed-operated group(SHAM), Ovariectomized group(OVX), Alendronate-therapy group(ALEN), G36G high dosage-therapy group (G36GH), G36G low dosage-therapy group (G36GL), G48A high dosage -therapy group (G48AH), G48A low dosage -therapy group (G48AL). The rats of prevention groups were injected subcutaneously with phosphate buffered saline (PBS) on SHAM and OVX group or G36G, G48A , alendronate on other groups once per day for 9 weeks one day after the ovariectomy
separately, then sacrificed. The therapy group was further subdivided into six groups(n= 5/group) :shamed-operated group(SHAM-T), Ovariectomized group(OVX-T), Risedronate-therapy group(RISE-T), Risedronate with G36G therapeutic alliance group(36GRI-T), G36G high dosage-therapy group (G36G-T), G48A high dosage-therapy group (G48A-T). The rats of therapy groups were injected subcutaneously with PBS on SHAM and OVX group or G36G, G48A, Risedronate on other groups once per day for periods of 9 weeks initiated 15 weeks after ovariectomy separately, then sacrificed. Lumbar spine,the bilateral tibia and femur were dissected out. Serum BGP and IGF-2 levels were determined by radioimmunoassay , while The morphological and dynamic parameters for bone histomorphometry , biochemical markers for bone formation and other indices of bone metabolism were measured. Biomechanical property parameters of femora and lumbar spine (L5) were measured with the three point bending tests and compressive test. The BMD of total femur .distal femur metaphysis and L1-4 were measured by dual energy X-ray absorptiomtry (DEXA) using DMS equipment while histomorphometry of proximal tibia undecalcif ied specimen sections were performed which include trabecular volume, trabecular thickness, dynamic data , et al. The relationship between BMD, biomechanic characters and histormorphometry in OP-prevention group were investigated. These data were analyzed by SPSS 10. 0 software with analysis of variance and correlation. Results: Akaline phosphatase activity levels were not statistically significantly different in the prevention and therapy groups. An elevation of BGP serum level were observed in OVX and ALEN
group as compared with G48AH group however the descent of IGF-2 serum level were determined in G36GH, G48AH group as compared with SHAM group. The results showed that both G36G and G48A had significant stimulation on bone formation as the tetracycline labeling on the trabecular bone surface and the rate of bone formation while no significant effect on the biomechanics, but both of them could not completely prevent bone loss in OVX rats. The ratio of femora ash weight to dry weight(AW/DW) and BMD of total femur, distal metaphysis , L1-4 in OVX and OVX-T group were significantly lower than in SHAM group, while the above-mentioned indices were remarkably increased in ALEN group than others. There was only an increased trend of BMD on the three sites of bone in G36GH, G36GL, G48AH, G48AL group as compared with OVX group. Bone histomorphometry in the OVX group was showed significant decrease on trabecular bone volume%total tissue (TBV/TTV), mean trabecular plate thickness(MTPT), mean trabecular plate density( MTPD) while mean trabecular plate spacing( MTPS) seemed to be higher than other groups. In the G36GH and G48AH groups, parameters of the TBV/TTV, trabecular bone volume%total sponge( TBV/SBV), MTPT were statistically increased and parameters of MTPS, trabecular surface%volume
引文
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