非PLC依赖PKC通路特异性甲状旁腺素模拟肽的建立及其对骨代谢作用的初步研究

发布时间：2018-06-05 16:47

本文选题：甲状旁腺素 + 信号转导通路　；参考：《南方医科大学》2015年硕士论文

【摘要】：背景：甲状旁腺素(PTH)是目前应用于临床的唯一促骨形成药物,它可以有效增加骨量,治疗骨质疏松及骨质疏松性骨折、关节假体松动及长期使用二磷酸盐引起的非典型骨折等。2002年美国FDA批准低剂量PTH间断性皮下注射用以治疗骨质疏松,2010年PTH被批准进入中国,治疗绝经后的重度骨质疏松。随着中国人口老龄化问题的日益严重,以及人们对于预防骨质疏松的意识尚浅薄等原因,骨质疏松症愈来愈成为困扰中老年人的疾病之一。骨质疏松症是一种以骨量减少及骨组织显微结构退化为特征,导致骨脆性增加及骨折危险性增加的一种全身代谢性骨病,目前骨质疏松症的治疗药物种类很多,其适用范围和作用机制也各不相同。国内的治疗主要以抗骨质吸收类药物为主,包括二膦酸盐类、降钙素、雌激素及选择性雌激素受体调节剂等,辅以促进骨矿化的钙剂、维生素D及其活性代谢物等,其对于骨质疏松症的治疗效果有限。PTH作为一种骨形成促进剂,研究证明其对骨密度提高的程度优于既往抗骨吸收类药物,对减少骨质疏松性骨折的作用也更为突出。但是,PTH在临床上的应用也受到诸多限制,如价格高,疗程长(2年),高剂量、持续应用会促进骨吸收(不能复合到内植入物以促进骨融合),可能存在致癌的风险,长期使用还存在疗效降低的可能性等,因此优化PTH,促进疗效,缩短使用时间,避免其副作用,是目前PTH领域研究的重要方向之一。人体中天然的PTH由84个氨基酸构成[PTH(1-84)],与Ⅰ型PTH受体(PTHR1)结合后激活PTHR1及其下游的多个信号途径,参与骨组织代谢的过程。主要的信号通路有：(1) Gs/cAMP/PKA信号转导通路,目前认为是PTH作用于骨组织的主要机制。(2) PLC/PKC通路。(3)非PLC依赖PKC激活途径(PTH/nonPLC/PKC)。(4) β-arrestin通路等。目前认为,PTHR1受体至少通过两种途径激活PKC,其一为激活质膜上的PLC,进而激活PKC；另一途径为不依赖PLC的PKC激活途径(nonPLC/PKC),即不激活PLC而通过其他分子介导机制激活PKC,具体信号特征并不清楚,其信号转导机制及调节骨代谢功能的研究是时下PTH研究领域的热点之一。PTH激活不同的信号通路与其自身的多肽结构密不可分,任何氨基酸的突变及构象的改变都有可能导致某些信号通路激活能力的缺失。研究证实,改变PTH的氨基酸序列能改变PTH的信号转导特征,如PTH(1-34)片段中,1-3位的氨基酸决定PTH的cAMP/PKA和PLC信号通路的激活功能；Ser1变成Gly1可使得PTH(1-34)失去激活PLC的能力；第19位氨基酸Glu19变成Arg19能够补偿29-34氨基酸残基缺失引起的受体结合力减弱；Leu24,Leu28,va131突变成Glu后,PTH(5-34)不与受体结合Aib1,3,Nle8,21,Gln10,Har11,Ala12,Trp14,Arg19构成的组合突变亦称M突变,M突变可使PTH(1-34)的cAMP合成能力提高约40倍,PLC的激活能力提高66倍,与PTHR的结合力提高约10倍；在PTH(1-34)中,Ile5,Glul9,Val21等位点的突变严重干扰PTH(1-34)与受体的结合力：PTH(1-34)第1、3位氨基酸突变成氨基异丁酸(Aib)后促进与PTHR的结合和cAMP的形成。根据以上模拟肽结构的研究基础,我们拟通过氨基酸突变的方法来达到基本不影响PTH肽与受体结合能力,屏蔽cAMP/PKA和PLC激活能力的目的,设计nonPLC/PKC通路特异性PTH模拟肽的序列。我们的前期研究也表明,Gly1Arg19hPTH(1-28) (GR(1-28)和Gly1Arg19hPTH(1-34) (GR(1-34))均失去了激活PLC的能力,但GR(1-34)仍可激活PKC,而GR(1-28)却没有这样的作用,即PTH可以通过nonPLC/PKC通路激活PKC,且功能区域是PTH(29-34)片段。通过C57BL小鼠皮下注射观察发现,GR(1-34)显著增加骨量,尤其是受力的松质骨区域,显著强于GR(1-28)的作用,尽管后者具有与前者相同的cAMP激活特性,提示PTH作用下nonPLC/PKC通路的激活能够促进受力区松质骨合成,改善骨小梁微结构,促进新骨的形成。近期我们发现GR(1-34)具有比PTH(1-34)和GR(1-28)更强的促进脊柱融合的作用,具有一定通路选择性的PTH模拟肽具有较PTH更好的促骨形成作用和改善骨微结构的功能。但是nonPLC/PKC通路的具体信号介导分子仍不清楚,其对于骨代谢的作用机制仍需要深入研究。因此我们拟设计和筛选nonPLC/PKC信号通路选择性PTH模拟肽,并分析该通路特有的下游效应分子,进一步探讨PTH通过nonPLC/PKC信号通路促进骨形成的作用机理。目的：利用原代成骨细胞,通过相关信号通路屏蔽和基因表达分析,筛选与核实甲状旁腺激素29-34位蛋白结构域(PTH(29-34))的效应基因,分析其对骨代谢的影响。以PTH结构为模板,通过改变其氨基酸序列,构建nonPLC/PKC信号通路选择性PTH模拟肽,并在相关通路阻滞剂与激活剂的干扰下利用FRET技术和ELISA法对其信号特征进行验证核实。并研究新肽对成骨相关基因的影响,探究PTH/nonPLC/PKC通路的骨代谢功能。方法：2-3日龄C57BL乳鼠10只,取颅盖骨分离培养成骨细胞,经成骨诱导液培养14d和28d时分别进行ALP染色和茜素红染色,鉴定原代成骨细胞。取贴壁生长的第1代细胞,分别接受100 nmol/L GR(1-28),10 nmol/L GR(1-34),10 nmol/L PTH(1-34)及空白对照作用4h,提取总RNA,行小鼠全基因组表达谱芯片分析,进行相关通路分析,并筛选出可能与nonPLC/PKC信号转导通路相关的差异表达基因。RT-PCR筛选及验证上述差异表达基因。培养MC3T3-E1细胞,使用cAMP通路抑制剂(RP-cAMP)阻断PTH诱发的cAMP/PKA信号通路,比较GR(1-28)和GR(1-34)引起的基因变化情况。构建PTHR稳定转染的HEK293细胞,转染CKAR报告分子后,利用PKC激活的荧光能量共振转移(FRET)技术,检测PTHR稳转细胞在cAMP通路抑制剂(RP-cAMP)干扰下分别接受GR(1-28)和GR(1-34)刺激下PKC的激活情况,确定PTH的nonPLC/PKC通路相关区域。以PTH氨基酸为基础,用氨基酸突变的方法增强nonPLC/PKC通路相关区域作用,合成PKA"PLCPKC+肽(MY1肽)。用FRET技术检测MYl肽对转染CKAR的PTHR稳转HEK293细胞中PKC的激活能力,并观察加入PKC抑制剂(Go6983)后PKC激活的变化情况,检测MY1肽作用下转染CKAR的HEK293细胞(无PTHR)中PKC的激活情况,探究其与PTHR的关系。用ELISA法检测MY1肽对MC3T3-E1细胞中cAMP、PLC激活能力。以验证核实MYl肽为PTH的nonPLC/PKC通路特异性模拟肽。MC3T3-E1细胞分别接受10umol/LMY1肽、10umol/L PTH(3-34、100nmol/LPTH(1-34)及空白对照组作用4h,提取总RNA, RT-PCR检测成骨相关基因的表达。结果：原代培养成骨细胞细胞形态较均一,呈梭形或多边形。待细胞长满后,见细胞排列紧密,呈铺路石状。成骨诱导培养14d,细胞排列紧密,呈复层生长,行ALP染色,镜下显示胞质中出现蓝色颗粒,成骨诱导培养至28d行茜素红染色,镜下显示出现红染的矿化结节。行小鼠全基因组芯片检测,通过相关通路分析,得到了最可能与PTH的nonPLC/PKC信号转导途径相关的14条信号通路。根据芯片结果,经过进一步分析,我们挑选出了与PTH的nonPLC/PKC信号通路相关性最高的56个基因,作为RT-PCR筛选验证的基因对象。筛选的56个基因中,我们发现CITED1的表达量PTH(1-34)组明显高于其他各组,且GR(1-34)组显著高于GR(1-28)组,PTH(1-34)与GR(1-34)组均显著高于空白对照组。MC3T3-E1细胞经各组模拟肽刺激后,提取总RNA,进行RT-PCR检测,CITED1的表达量GR(1-34)组仍显著高于GR(1-28)组,与原代成骨细胞实验结果一致,且在加入PKC抑制剂后,CITED1表达量明显下降,证实CITED1表达量的升高由nonPLC/PKC通道激活引起,不依赖PLC及PKA的激活。PTHR稳转细胞经PKC特异性激活剂TPA刺激后,PKC被激活,C/Y值显著增高,表明我们检测PKC激活的FRET技术检测平台构建成功,其可以作为后续试验中检测PKC激活的有效手段。PTHR稳转细胞在PTH(1-34)的作用下,C/Y值显著增高,即检测到PKC被激活,表明我们成功地构建了PTHR稳转细胞。在转染CKAR的PTHR稳转细胞细胞中,阻断cAMP通路后,GR(1-28)无法激活PKC,而GR(1-34)仍可激活PKC,nonPLC/PKC通路的激活与PTH的29至34氨基酸片段有关。在MYl肽作用下,FRET检测显示C/Y值明显升高,PKC被激活,而PTH(3-34)及空白对照中,PKC未被激活。在MYl肽刺激使C/Y值明显升高后,加入PKC阻滞剂后,C/Y值明显下降,并逐渐降至起始水平。转染CKAR的HEK293细胞(无PTHR)在PTH(3-34)或是MYl作用下,C/Y值均未发生变化,而在加入TPA (PKC特异性激活剂)后,C/Y值明显升高,PKC被激活。ELISA法检测MC3T3-E1经各模拟肽作用下cAMP、PLC的含量,实验结果显示,PTH(1-34)组中cAMP、PLC含量明显高于其它各组,而其它三组之间cAMP含量无统计学差异,即MYl肽没有激活cAMP、PLC的能力。MC3T3-E1细胞经各PTH模拟肽作用后行RT-PCR检测,结果显示MYl组CITED1的表达量明显高于空白对照组和PTH(3-34)组,在加入PKC抑制剂Go6983后,CITED1的表达量明显下降,与原代成骨细胞实验中结果相一致。在成骨基因检测中,ALP的表达量在MYl组明显高于空白对照组和PTH(3-34)组,且在加入PKC抑制剂后,其表达量明显下降。结论：1.原代成骨细胞培养成功,经基因芯片分析,发现与PTH的nonPLC/PKC信号转导途径相关的14条信号通路。经RT-PCR筛选,我们发现PTH(29-34)蛋白机构域可通过PKC信号途径促进CITED1的表达,介导PTH对成骨代谢的作用。该途径不依赖PLC和PKA信号的激活。2.经FRET分析,PTHR稳定转染细胞成功建立,我们构建的MY1肽具有通过PTHR激活PKC的特性,且不依赖PKA及PLC信号转导。表明MY1为PTH/nonPLC/PKC通路特异性模拟肽。MYl肽的建立为PTH/nonPLC/PKC的进一步研究奠定了基础,据我们所知,单纯具有nonPLC/PKC的信号特征的PTH模拟肽国内外未有报道。3.MYl肽促进转录因子CITED1和成骨基因ALP的表达,表明PTH的nonPLC/PKC通路至少通过调控转录因子CITED1或成骨基因ALP的表达量参与骨代谢调节过程。但其对骨代谢的影响和机理需要进一步的研究。
[Abstract]:Background: parathyroid hormone (PTH) is the only bone forming drug used in clinical practice. It can effectively increase bone mass, treat osteoporosis and osteoporotic fractures, joint prosthesis loosening and atypical fractures caused by long-term use of two phosphate and other.2002 years, FDA batch low dose PTH intermittent hypodermic injection for the treatment of bone Loosely, in 2010, PTH was approved to enter China for the treatment of postmenopausal severe osteoporosis. Osteoporosis is becoming one of the diseases that plagued middle and old people as the problem of aging population in China is increasingly serious and the awareness of osteoporosis is still shallow. Osteoporosis is a kind of bone loss and bone. A kind of systemic metabolic bone disease characterized by the degeneration of microstructure and the increase of bone fragility and increased risk of fracture. There are many kinds of drugs for the treatment of osteoporosis, and the scope and mechanism of its application are different. The main treatment in China is anti bone absorption drugs, including two phosphonic acid salts, calcitonin, and female irritable disease. Hormone and selective estrogen receptor modulators, supplemented with calcium, vitamin D and its active metabolites that promote bone mineralization, and its therapeutic effect on osteoporosis is limited.PTH as a bone formation promoter. Studies have shown that its increase in bone density is better than previous anti bone resorption drugs for reducing osteoporotic fractures. However, the clinical application of PTH is also limited, such as high price, long course (2 years), high dose, continuous application will promote bone absorption, which can not be combined with internal implant to promote bone fusion. It may have the risk of carcinogenesis, and the long-term use still has the possibility of reducing the curative effect, so optimizing the PTH and promoting the curative effect, Shortening the use time and avoiding its side effects are one of the most important directions in the field of PTH. The natural PTH in the human body consists of 84 amino acids [PTH (1-84)], which is combined with the type I PTH receptor (PTHR1) to activate PTHR1 and its downstream signal pathway to participate in the metabolic process of bone tissue. The main signal pathways are: (1) Gs/cAMP/PKA signal Transduction pathway is considered to be the main mechanism that PTH acts on bone tissue. (2) PLC/PKC pathway. (3) non PLC dependent PKC activation pathway (PTH/nonPLC/PKC). (4) beta -arrestin pathway, etc. at present, the PTHR1 receptor activates at least two ways to activate PKC, one is activating the PLC on the plasma membrane and activating PKC; the other is not dependent on PLC PKC excitation. The active pathway (nonPLC/PKC), which is not activated by PLC, activates PKC through other molecular mediated mechanisms. The specific signal characteristics are not clear. The signal transduction mechanism and the regulation of bone metabolism are one of the hotspots in the current field of PTH research..PTH activation of different signaling pathways is inseparable from its own peptide structure and any amino acid mutation. Changes in conformation and conformation may lead to loss of activation ability of certain signal pathways. Studies have shown that changing the amino acid sequence of PTH can change the signal transduction characteristics of PTH, such as PTH (1-34) fragments, the 1-3 amino acids determine the activation function of the cAMP/PKA and PLC signaling pathways of PTH; Ser1 becomes Gly1 can cause PTH (1-34) to lose activation of PLC. Nineteenth bit amino acid Glu19 Arg19 can compensate for the weakening of the receptor binding force caused by the deletion of the 29-34 amino acid residues; after Leu24, Leu28, and va131 process into Glu, PTH (5-34) does not combine Aib1,3 with the receptor, Nle8,21, Gln10, Har11, Ala12, and is also called the mutation. 40 times, the activation ability of PLC increased by 66 times and the binding power of PTHR increased by about 10 times; in PTH (1-34), the mutation of Ile5, Glul9, Val21 and other sites seriously interfered with the binding force of PTH (1-34) with the receptor: PTH (1-34) 1,3 amino acid suddenly became the combination of amino isobutyric acid (Aib) and PTHR binding and cAMP formation. On the basis of this, we intend to design the sequence of the nonPLC/PKC pathway specific PTH analog peptide by means of amino acid mutation, which basically does not affect the binding ability of PTH peptide to the receptor, shielding the activation ability of cAMP/PKA and PLC. Our previous study also showed that Gly1Arg19hPTH (1-28) (GR (1-28) and Gly1Arg19hPTH (1-34) (GR (1-34)) were lost. The ability to activate PLC, but GR (1-34) still activates PKC, while GR (1-28) does not function, that is, PTH can activate PKC through the nonPLC/PKC pathway, and the functional region is PTH (29-34). By subcutaneous injection of the C57BL mice, it is found that GR (1-34) significantly increases the bone mass, especially the region of the stressed cancellous bone, which is significantly stronger than GR (1-28). Although the latter has the same cAMP activation characteristics as the former, it is suggested that activation of the nonPLC/PKC pathway under the action of PTH can promote the synthesis of cancellous bone in the stressed area, improve the microstructure of the trabecular bone and promote the formation of new bone. In the near future, we found that GR (1-34) has a stronger role in promoting spinal fusion than PTH (1-34) and GR (1-28), and has a certain pathway selectivity. The PTH mimic peptide has a better function of promoting bone formation and improving bone microstructure than PTH. However, the specific signaling molecules of the nonPLC/PKC pathway are still unclear, and the mechanism of its action on bone metabolism still needs to be studied. Therefore, we intend to design and screen the selective PTH analog peptide of the nonPLC/PKC signaling pathway and analyze the pathway of this pathway. Some downstream effectors further explore the mechanism of PTH to promote bone formation through the nonPLC/PKC signaling pathway. Objective: to screen and verify the effect genes of parathyroid hormone 29-34 protein domain (PTH (29-34)) by using primary osteoblasts, screening and verifying the gene expression domain (PTH (29-34)) of parathyroid hormone, and analyzing its effect on bone metabolism. With the PTH structure as a template, the nonPLC/PKC signaling pathway selective PTH mimic peptide was constructed by changing the amino acid sequence, and the signal characteristics were verified by FRET and ELISA under the interference of the related pathway blockers and activators, and the effect of the new peptide on the osteogenic phase gene was investigated and the PTH/nonPLC/PKC pathway was explored. Methods: bone metabolism function. Methods: 10 C57BL mice aged 2-3 days old were isolated and cultured for osteoblasts. ALP staining and alizarin red staining were carried out when 14d and 28d were cultured by osteogenic induction solution to identify the primary osteoblasts. The first generation cells attached to the wall were taken to receive 100 nmol/L GR (1-28), 10 nmol/L GR (1-34), 10 nmol/L PTH (1-34) and empty, respectively. The total RNA was extracted from the white control 4h, the whole genome expression spectrum chip was analyzed, the related pathway was analyzed, and the differential expression gene.RT-PCR related to the nonPLC/PKC signal transduction pathway was screened and tested to verify the differential expression genes. MC3T3-E1 cells were cultured and cAMP pathway inhibitor (RP-cAMP) was used to block cAMP induced cAMP. /PKA signaling pathway, comparing the gene changes caused by GR (1-28) and GR (1-34). Construction of PTHR stable transfected HEK293 cells, after transfection of CKAR reporter molecules, using PKC activated fluorescence energy resonance transfer (FRET) technology to detect the PTHR metastable cells under cAMP pathway inhibitor (RP-cAMP) and receive GR (1-28) and stimulus (1-34) stimulation respectively. Activation and determination of the nonPLC/PKC pathway related regions of PTH. Based on PTH amino acids, the function of nonPLC/PKC pathway related regions was enhanced by amino acid mutation, and PKA "PLCPKC+ peptide (MY1 peptide) was synthesized. The activation ability of MYl peptide to PTHR stabilized HEK293 cells transfected with CKAR was detected by FRET technology. The activation of PKC was detected by detecting the activation of PKC in HEK293 cells transfected with CKAR under the action of MY1 peptide (PTHR), and the relationship between them and PTHR was explored. ELISA assay was used to detect cAMP and PLC activation of MY1 peptides in MC3T3-E1 cells. 1 0umol/L PTH (3-34100nmol/LPTH (1-34) and blank control group acted 4h, extracted total RNA and RT-PCR to detect the expression of bone related genes. Results: the cells of primary cultured osteoblasts were homogenous, shuttle or polygon. After the cell was full, the cells were arranged closely and showed a paved stone. The osteogenesis was induced and cultured for 14d, the cells were arranged close and complex. The layer was grown and stained with ALP. The blue particles appeared in the cytoplasm under the microscope. The osteogenesis was induced to 28d with alizarin red staining. The red stained mineralized nodules were displayed under the microscope. All the 14 signal pathways related to the nonPLC/PKC signal transduction pathway of PTH were obtained by the whole genome chip detection. As a result, after further analysis, we selected 56 genes with the highest correlation with the nonPLC/PKC signaling pathway of PTH, which were selected as RT-PCR screening tests. Among the 56 selected genes, we found that the PTH (1-34) group of CITED1 was significantly higher than the other groups, and GR (1-34) group was significantly higher than the GR (1-28) group, PTH (1-34) and GR (1-34). The group of.MC3T3-E1 cells was significantly higher than that of the blank control group. After the stimulation of the analog peptide, the total RNA was extracted and the RT-PCR was detected. The expression of CITED1 was still significantly higher than that in the GR (1-28) group, which was consistent with the experimental results of the original osteoblast, and the expression of CITED1 was obviously decreased after adding the PKC inhibitor. The increase of the CITED1 expression was confirmed by nonPLC. The increase of the CITED1 expression was confirmed by nonPLC. The activation of /PKC channel, which does not depend on the activation of PLC and PKA, is activated by PKC specific activator TPA, PKC is activated and C/Y value increases significantly. It indicates that our detection platform for FRET is successful. It can be used as an effective means to detect PKC excitation in the follow-up test. At the same time, the C/Y value increased significantly, that is, the detection of PKC was activated, indicating that we successfully constructed the PTHR stable cells. In the PTHR stable transfected cell cells transfected with CKAR, GR (1-28) can not activate PKC, and GR (1-34) still activates PKC. The activation of the nonPLC/PKC pathway is related to the 29 to 34 amino acid fragments of PTH. The test showed that the C/Y value was obviously increased and PKC was activated, while PKC was not activated in PTH (3-34) and blank control. After the MYl peptide stimulated the C/Y value, the C/Y value decreased obviously, and gradually decreased to the beginning level. The HEK293 cells transfected with CKAR were not changed in PTH (3-34) or under the action. After TPA (PKC specific activator), the C/Y value increased obviously. PKC was activated by.ELISA method to detect the content of cAMP and PLC under the action of each analogue peptide of MC3T3-E1. The experimental results showed that the PLC content in PTH (1-34) group was obviously higher than that of the other groups, but the cAMP content of the other three groups was not statistically different. The expression of CITED1 in MYl group was significantly higher than that of the blank control group and PTH (3-34) group. The expression of CITED1 in the MYl group was significantly higher than that in the blank control group and the PTH (3-34) group. The expression of ALP was significantly higher in the MYl group than in the original osteoblast test. In the blank control group and the PTH (3-34) group, and after the addition of PKC inhibitor, the expression of the 1. primary osteoblasts was successfully cultured. The 14 signal pathways associated with the nonPLC/PKC signal transduction pathway of PTH were detected by gene chip analysis. We found that the PTH (29-34) protein mechanism domain could be promoted by the PKC signal pathway through the RT-PCR screening. The expression of CITED1 mediates the effect of PTH on osteoblastic metabolism. This pathway does not depend on the activation of PLC and PKA signals by FRET analysis, and PTHR stable transfection cells are successfully established. The MY1 peptide we constructed has the characteristics of activating PKC through PTHR, and does not depend on PKA and PLC signal transduction. The foundation is established for further study of PTH/nonPLC/PKC. As we know, the PTH analog peptide with nonPLC/PKC signal characteristics has not reported the expression of.3.MYl peptide promoting transcription factor CITED1 and osteogenic gene ALP, indicating that the nonPLC/PKC pathway of PTH is at least by regulating the expression of the transcription factor CITED1 or the expression of ALP of the osteogenic gene. Volume is involved in the process of bone metabolism regulation, but its effects on bone metabolism and mechanism need further study.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R580

【共引文献】