【作者】 张颖；

【导师】 苗登顺；

【作者基本信息】 南京医科大学 ， 人体解剖与组织胚胎学， 2015， 硕士

【摘要】 我们前期利用基因工程方法在小鼠甲状旁腺素相关肽基因第84位氨基酸序列后插入一个终止密码子TGA,使其只表达1-84位而缺乏NLS和C-末端,建立了PTHrP NLS和C-末端缺失的PTHrP Knock-In（PTHrP KI）小鼠模型。PTHrP KI小鼠表现为严重的生长阻滞和成熟前衰老以及骨质疏松表型。然而,PTHrP NLS和C-末端在调节骨骼发育和成骨细胞骨形成中的作用及机制尚不明确。为明确PTHrP核定位序列和C末端是否通过调节氧化应激和DNA损伤反应介导骨骼生长发育,我们利用流式细胞术、Real-time RT-PCR和Western blot检测PTHrP KI小鼠骨组织中氧化应激和DNA损伤反应相关指标的变化。结果发现,PTHrP KI小鼠骨组织ROS水平升高,DNA损伤及其反应相关蛋白γ-H2AX、p-Chk2和p53蛋白表达水平明显上调,而抗氧化酶SOD1、SOD2、过氧化氢酶（CAT）、谷胱甘肽还原酶（GSR）和谷胱甘肽过氧化物酶4（GPX4）mRNA表达水平均明显下调。因此,我们建立了Chk2和PTHrP NLS及C-末端双基因缺失的(Chk2^-/-KI)小鼠模型,运用影像学、组织化学、免疫组织化学、Real time RT-PCR和Western Blot等方法,比较分析其与同窝WT、Chk2^-/-和PTHrP KI小鼠的表型差异。为明确Chk2敲除是否能够纠正PTHrP NLS和C末端缺失引起的生长阻滞和成熟前衰老,我们比较分析了Chk2^-/-KI与同窝WT、Chk2^-/-和PTHrP KI小鼠的体型、体重和生存期的差异。结果发现:PTHrP KI小鼠的平均生存期仅为2周（5-18天）,而Chk2^-/-KI小鼠平均生存期延至3周（12-24天）。与WT小鼠相比,2周龄Chk2^-/-小鼠体型有所增大,体重显著增加;PTHrP KI和Chk2^-/-KI小鼠体型较小,体重明显降低。然而,与PTHrP KI小鼠相比,Chk2^-/-KI小鼠体型有所增大,体重明显增加。这些结果表明Chk2敲除能够延长PTHrP KI小鼠的生存期,并能改善其生长发育。为明确Chk2敲除是否能够纠正PTHrP NLS和C末端缺失引起的骨骼生长阻滞,我们利用X线摄影和组织学的方法,比较分析了2周龄同窝WT、Chk2^-/-、PTHrP KI和Chk2^-/-KI四种基因型小鼠骨骼生长发育相关指标的变化。结果发现:与WT小鼠相比,长骨大小和长度,生长板和增殖带宽度在Chk2^-/-小鼠明显增加,在PTHrP KI和Chk2^-/-KI小鼠明显下降。然而,与PTHrP KI小鼠相比,这些指标在Chk2^-/-KI小鼠明显增加。这些结果表明Chk2敲除能够改善PTHrP KI引起的骨骼生长阻滞。为明确Chk2敲除是否能够纠正PTHrP NLS和C末端缺失引起骨密度和骨容量降低,我们利用微CT扫描和三维重建以及总胶原组织化学染色的方法,比较分析了同窝四种基因型小鼠骨密度和骨容量相关指标的变化。结果发现:与WT小鼠相比,骨密度、小梁骨骨容量/组织容量（BV/TV,%）百分率、骨骺和皮质骨骨容量以及骨小梁数目在Chk2^-/-小鼠明显增加,在PTHrP KI和Chk2^-/-KI小鼠明显下降。然而,与PTHrP KI小鼠相比,这些指标在Chk2^-/-KI小鼠明显增加。此外,与WT小鼠相比,小梁骨分离度在Chk2^-/-小鼠明显下降,在PTHrP KI和Chk2^-/-KI小鼠明显增加。然而,与PTHrP KI小鼠相比,小梁骨分离度在Chk2^-/-KI小鼠明显降低。这些结果表明Chk2敲除能够改善PTHrP KI引起的骨密度和骨容量降低。为明确Chk2敲除改善PTHrP KI小鼠的骨密度和骨容量是否与成骨细胞骨形成有关,我们利用组织学、ALP组织化学染色、I型胶原（COL-I）免疫组织化学染色和Real-time PCR等方法,比较分析了四种基因型小鼠成骨细胞骨形成相关指标的变化。结果显示:与WT小鼠相比,成骨细胞数、ALP和COL-I阳性面积百分比以及成骨相关基因包括Runx2、ALP、OCN和COL-I表达水平在Chk2^-/-小鼠均明显增加,在PTHrP KI和Chk2^-/-KI小鼠均明显下降。然而,与PTHrP KI小鼠相比,这些参数在Chk2^-/-KI小鼠明显增加。这些结果表明Chk2敲除能够通过促进成骨细胞骨形成而改善PTHrP KI引起骨容量减少。为明确Chk2敲除改善PTHrP KI引起的成骨细胞骨形成减少是否与骨髓间充质干细胞（BM-MSC）增殖和分化的改变有关,我们体外培养了四种基因型小鼠来源的BM-MSC,进行甲基蓝和ALP细胞化学染色,利用图像分析方法检测了甲基蓝染色阳性成纤维细胞集落形成单位（CFU-f）和ALP阳性CFU-f（CFU-fap）面积的变化。结果显示:与WT小鼠相比,Chk2^-/-小鼠骨髓细胞甲基蓝染色阳性CFU-f面积和ALP阳性CFU-f面积百分率明显增加,在PTHrP KI和Chk2^-/-KI小鼠明显降低。然而,与PTHrP KI小鼠相比,这些指标在Chk2^-/-KI小鼠明显增加。这些结果表明Chk2敲除通过促进BM-MSC增殖和向成骨细胞分化纠正PTHrP KI引起的成骨细胞骨形成减少。为明确Chk2敲除改善PTHrP KI引起的骨密度和骨容量降低是否与破骨细胞骨吸收改变有关,我们利用TRAP组织化学染色和Real-time RT-PCR的方法,比较分析了四种基因型小鼠破骨细胞骨吸收相关指标的变化。结果显示:与WT小鼠相比,TRAP阳性破骨细胞面（Oc.S/B.S,%）、RANKL与OPG比值以及破骨相关基因TRAP和RANKL表达水平在Chk2^-/-小鼠明显减少,在PTHrP KI和Chk2^-/-KI小鼠明显增加。然而,与PTHrP KI小鼠相比,Chk2^-/-KI小鼠的这些指标均明显降低。这些结果表明Chk2敲除通过抑制破骨细胞骨吸收改善PTHrP KI引起的骨密度和骨容量的降低。为明确Chk2敲除改善PTHrP KI引起的成熟前衰老和骨质疏松表型是否与氧化应激有关,我们利用Real time RT-PCR和Western blot方法比较分析了四种基因型小鼠骨组织中氧化应激相关基因和蛋白的表达水平变化。结果显示:与WT小鼠相比,抗氧化酶基因包括SOD1/2、过氧化物酶（CAT）、谷胱甘肽还原酶（GSR）和谷胱甘肽过氧化物酶4（GPX4）表达水平以及SOD1和Bmi1蛋白表达水平在Chk2^-/-小鼠明显上调,在PTHrP KI和Chk2^-/-KI小鼠明显下调。然而,与PTHrP KI小鼠相比,这些指标在Chk2^-/-KI小鼠均明显上调。这些结果表明Chk2敲除能够通过抑制氧化应激纠正PTHrP KI引起的成熟前衰老和骨质疏松表型。为明确Chk2敲除改善PTHrP KI引起的成熟前衰老和骨质疏松表型是否与DNA损伤及其反应有关,我们通过Real-time RT-PCR和Western blot方法比较分析了四种基因型小鼠骨组织中DNA损伤及其反应相关指标的变化。结果显示:与WT小鼠相比,DNA损伤指标γ-H2AX、凋亡相关指标Caspase-3和DNA损伤反应蛋白p53的蛋白表达水平,以及细胞周期依赖性激酶抑制因子（CDKI）包括p16、p19、p53、p21和p27的mRNA表达水平在Chk2^-/-小鼠明显下调,在PTHrP KI和Chk2^-/-KI小鼠明显上调。然而,与PTHrP KI小鼠相比,这些指标在Chk2^-/-KI小鼠均明显下调。这些结果说明Chk2敲除能够通过抑制DNA损伤及其反应,下调细胞周期依赖性激酶抑制因子,促进细胞增殖,抑制细胞凋亡,从而纠正PTHrP KI引起的成熟前衰老和骨质疏松表型。以上结果表明PTHrP核定位序列和C末端缺失能够导致氧化应激增加和DNA损伤及其反应增强。敲除DNA损伤反应关键基因Chk2能够抑制氧化应激和DNA损伤及其反应,从而促进软骨内成骨、BM-MSC增殖和向成骨细胞分化以及成骨细胞骨形成,同时抑制破骨细胞骨吸收,部分纠正PTHrP KI引起的骨骼生长阻滞和骨质疏松。我们的结果证明DNA损伤检查点通路作为PTHrP胞内分泌作用的下游靶标,在PTHrP调节骨骼生长发育过程中起重要作用。本研究进一步阐述了PTHrP核定位序列和C末端在调节骨骼生长发育中的作用机制,为临床应用PTHrP核定位序列和C末端或抗氧化剂防治骨质疏松提供了理论和实验依据。更多还原

【Abstract】 We previously generated a parathyroid hormone related peptide（PTHrP）knock-In（PTHrP KI）mouse model by introducing a premature termination codon TGA in PTHrP in ES cells and that expresses PTHrP（1–84）,a truncated form of the protein that is missing NLS（the nuclear localization sequence）and the C-terminal region of the protein.PTHrP KI mice displayed early senescence,skeletal growth retardation and defective osteoblastic bone formation.However,the role of PTHrP NLS and C-terminus in regulating skeletal development still remains unclear.To determine whether oxidative stress and DNA damage response pathways are involved in the regulation of skeletal growth and development by PTHrP,we examined the alterations of oxidative stress and DNA damage response related molecules in PTHrP KI skeletal tissues in vivo.These results revealed that compared with wild-type controls,the levels of ROS and the DNA damage-related molecules includingγ-H2AX,p-Chk2,and p53 were significantly increased,while the expression of mRNA in anti-oxidative enzymes including SOD1 and 2,catalase,glutathione reductase（GSR）,and glutathione peroxidase 4（GPX4）was decreased in PTHrP KI skeletal tissues.Furthermore,we disrupted the DNA damage response pathway by Chk2 deletion in PTHrP KI mice(Chk2^-/-KI).Their phenotypes were compared with WT,Chk2^-/-,and PTHrP KI littermates using imageology,histochemistry,immunohistochemistry,Real time RT-PCR,and Western blot.To assess whether the deletion of Chk2 can rescue the growth retardation and premature senescence caused by PTHrP KI,we examined the effect of the deletion of Chk2 on lifespan,habitus,and body weight in PTHrP KI mice.These results showed that the mean lifespan of Chk2^-/-KI mice was extended to 3 weeks（12-24days）from 2 weeks（5-18 days）in PTHrP KI mice.The habitus and body weight were significantly increased in Chk2^-/-KI mice compared with PTHrP KI mice,However,they were both reduced in PTHrP KI and Chk2^-/-KI mice compared with wildtype mice.These results demonstrated that the deletion of Chk2 prolong the lifespan and increase the body size and weight in PTHrP KI mice.To determine whether the deletion of Chk2 can rescue skeletal growth retardation caused by PTHrP KI,we analyzed the bone phenotypes of the 2-week-old four genotype mice using X-ray and histology.Our results showed that the length of long bones,the width of cartilage growth plate and proliferation area were increased in Chk2^-/-mice,which,however,were significantly reduced in both PTHrP KI and Chk2^-/-KI mice compared to WT mice.Notably,these parameters were increased significantly in Chk2^-/-KI mice compared to PTHrP KI mice.These results indicated that the deletion of Chk2 improve skeleton growth defect displayed in PTHrP KI mice.To determine whether the deletion of Chk2 can rescue the loss of bone mineral density and the decrease in bone volume caused by PTHrP KI,we examined the effect of deletion of Chk2 on bone mineral density and bone volume of PTHrP KI mice using Micro-CT,3D reconstruction,and total collagen histochemistry.Results showed that the number of BMD,BV/TV,trabecular and cortical bone volume and trabecular bone were significantly higher in Chk2^-/-mice than in both PTHrP KI and Chk2^-/-KI mice.However,these parameters were significantly increased in Chk2^-/-KI mice compared to PTHrP KI mice.These results indicated that deletion of Chk2 can partly improve the loss of bone mineral density and bone volume in PTHrP KI mice.To test whether the improvement in bone mineral density and bone volume in PTHrP KI mice by deletion of Chk2 was related to osteoblastic bone formation,we examined the effect of deletion of Chk2 on osteoblastic bone formation of PTHrP KI mice using histology,immunohistochemistry,and molecular biology.Results showed that the number of osteoblasts,ALP and type I collagen（COL-I）-positive bone areas,the mRNA expression of osteoblast related genes including Runx2,ALP,OCN,and COL-I were significantly increased in Chk2^-/-mice,which,however,were significantly reduced in both PTHrP KI and Chk2^-/-KI mice compared to WT mice,Moreover,these parameters were markedly increased in Chk2^-/-KI mice compared to PTHrP KI mice.These results indicated that deletion of Chk2 can improve the decline in bone mineral density and bone volume by enhancing osteoblastic bone formation in PTHrP KI mice.To assess whether the effects of deletion of Chk2 on osteoblastic bone formation of PTHrP KI mice was associated with the elevation of proliferation and differentiation in bone marrow mesenchyme stem cells（BM-MSC）,we performed colony forming units-fibroblast（CFU-f）assay and cytochemical staining for ALP.Results showed that compared to PTHrP KI mice,CFU-f and ALP-positive area were significantly increased in Chk2^-/-KI mice.These results indicated that deletion of Chk2 in PTHrP KI mice can improve osteoblastic bone formation by promoting the proliferation and differentiation of BM-MSCs into osteoblasts.To explore whether the improvement in bone mineral density and bone volume caused by deletion of Chk2 in PTHrP KI mice was associated with osteoclastic bone absorption,we examined the effects of deletion of Chk2 on osteoclastic bone absorption of PTHrP KI mice using histochemistry and molecular biology.Results showed that Oc.S/B.S,RANKL/OPG,and the mRNA expression of osteoclast-related genes TRAP and RANKL were significantly decreased in Chk2^-/-mice,which,however,were markedly increased in both PTHrP KI and Chk2^-/-KI mice compared to WT controls.Additionally,these parameters were significantly lower in Chk2^-/-KI mice than in PTHrP KI mice.These results indicated that deletion of Chk2 can improve premature osteoporosis by inhibiting osteoclastic bone absorption in PTHrP KI mice.To determine whether the deletion of Chk2 improved the premature osteoporosis phenotype of the PTHrP KI mice through inhibiting their oxidative stress,Real-time RT-PCR and Western blot were performed to examine the expression of antioxidant enzyme genes in bony tissue of grouped mice.Results showed that the expression of antioxidant enzyme genes including SOD1/2,CAT,GSR and GPX4 mRNA and SOD1,Bmi1 protein was remarkably increased in bony tissues of Chk2^-/-KI mice compared with PTHrP KI littermates.These results indicated that the deletion of Chk2 can rescue the premature osteoporosis exhibited in PTHrP KI mice by inhibiting the oxidative stress.To evaluate whether the improvement in the premature osteoporosis caused by deletion of Chk2 in the PTHrP KI mice was associated with the decline of DNA damage,we examined the DNA damage and response-related parameters in bony tissues of indicated group of mice using Real-time RT-PCR and Western blot.These results showed that the expression ofγ-H2AX,Caspase-3,p53 and cyclin dependent kinase inhibitors including p16,p19,p53,p21,and p27 was dramatically decreased in bony tissues of Chk2^-/-KI mice compared to PTHrP KI littermates.These results indicated that deletion of Chk2 can rescue the premature osteoporosis of PTHrP KI mice by inhibiting the DNA damage and response.These results indicated that deletion of PTHrP nuclear localization sequence and C terminal result in an increase in oxidative stress and DNA damage,whereas deletion of Chk2 can rescue the early senescence and growth retardation caused by PTHrP KI.Furthermore,deletion of Chk2 can improve osteoblastic bone formation,promote the proliferation and differentiation of BM-MSC into osteoblasts,and suppress osteoclastic bone absorption by inhibiting the oxidative stress and DNA damage response pathway.Our data demonstrated that the DNA damage checkpoint pathway may be involved in the role of PTHrP in regulating skeletal growth and development.This study might provide experimental and theoretical evidence for the application of PTHrP NLS and the C-terminus in the prevention and treatment of osteoporosis.更多还原