發(fā)布時(shí)間：2018-04-04 14:45

  本文選題：人骨髓間充質(zhì)干細(xì)胞　切入點(diǎn)：梯度密度離心　出處：《廣西醫(yī)科大學(xué)》2015年博士論文

【摘要】：第一部分人骨髓間充質(zhì)干細(xì)胞體外培養(yǎng)與鑒定[目的]運(yùn)用實(shí)驗(yàn)室已經(jīng)成功創(chuàng)建的人骨髓間充質(zhì)干細(xì)胞培養(yǎng)方案,分離、培養(yǎng)和鑒定hMSCs,為后續(xù)研究提供充足的干細(xì)胞來(lái)源。[方法]采用密度梯度離心法分離出單個(gè)核細(xì)胞,細(xì)胞貼壁法在α-MEM培養(yǎng)基中獲得原代hMSCs,隨后進(jìn)行傳代培養(yǎng)。對(duì)成骨細(xì)胞分化采用堿性磷酸酶活性檢測(cè)和Alizarin Red S染色進(jìn)行鑒定；對(duì)脂肪細(xì)胞分化應(yīng)用Oil Red-O染色方法進(jìn)行鑒定；應(yīng)用Alcian blue染色對(duì)軟骨細(xì)胞分化進(jìn)行鑒定。[結(jié)果]骨髓細(xì)胞培養(yǎng)6-24小時(shí),胞體形狀由圓形逐步變?yōu)槿切突虿灰?guī)則形狀。培養(yǎng)第2-4天間充質(zhì)細(xì)胞形態(tài)發(fā)生改變,細(xì)胞形態(tài)逐漸變?yōu)槌衫w維細(xì)胞樣的梭型,并聚集成集落；培養(yǎng)7-14天,細(xì)胞長(zhǎng)滿培養(yǎng)瓶底面積的70%-80%,細(xì)胞排列更加緊密,此時(shí)的骨髓間充質(zhì)干細(xì)胞為單層細(xì)胞。對(duì)細(xì)胞進(jìn)行成脂誘導(dǎo)分化,可檢測(cè)不同形狀的Oil Red-O陽(yáng)性染色細(xì)胞形成；成骨細(xì)胞誘導(dǎo)分化后,堿性磷酸酶活性檢測(cè),以及Alizarin Red S染色的陽(yáng)性細(xì)胞形成；誘導(dǎo)成軟骨細(xì)胞分化后可見Alcian blue染色陽(yáng)性細(xì)胞形成。[結(jié)論]本試驗(yàn)采用的方法可以成功分離、培養(yǎng)及擴(kuò)增人骨髓間充質(zhì)干細(xì)胞,并鑒定其具有成骨、成脂、成軟骨分化能力。第二部分DHEA上調(diào)IGF-I基因表達(dá)并促進(jìn)hMSCs向成骨分化[目的]本研究驗(yàn)證DHEA是否通過(guò)調(diào)節(jié)IGF-I從而促進(jìn)骨髓間充質(zhì)干細(xì)胞向成骨細(xì)胞轉(zhuǎn)化。[方法]采用不同時(shí)間點(diǎn)、不同濃度DHEA藥物刺激人骨髓間充質(zhì)干細(xì)胞,進(jìn)行時(shí)間和濃度依賴試驗(yàn),并采用生物化學(xué)和小分子酶抑制劑去闡明DHEA在人骨髓間充質(zhì)干細(xì)胞中調(diào)節(jié)IGF-I基因表達(dá)并促進(jìn)其向成骨分化的機(jī)制。在本實(shí)驗(yàn)研究,應(yīng)用PKA信號(hào)通路抑制劑H-89, PKC信號(hào)通路抑制劑CHE,JNK信號(hào)通路抑制劑SP600125分別阻斷PKA信號(hào)通路,PKC信號(hào)通路,JNK信號(hào)通路。應(yīng)用P13K信號(hào)通路抑制劑LY294002,P38 MAPK信號(hào)通路阻斷劑SB203580,P42/44信號(hào)通路阻斷劑PD098059, IGF-IR受體阻斷劑AG1024分別阻斷PI3K、P38 MAPK、P42/44 MAPK、IGF-IR信號(hào)通路。[結(jié)果]在骨髓間充質(zhì)干細(xì)胞中,DHEA可上調(diào)IGF-I基因表達(dá),并且存在時(shí)間和濃度依賴。IGF-I基因表達(dá)高峰期是在DHEA刺激后4-6小時(shí)；在刺激時(shí)間為6小時(shí)情況下,最佳藥物濃度為10nM。阻斷PI3K,P38 MAPK, P42/44 MAPK信號(hào)通路,DHEA上調(diào)IGF-1基因表達(dá)的作用被抑制。隨后樣本經(jīng)特殊酶抑制劑作用后,對(duì)成骨分化相關(guān)基因和堿性磷酸酶檢測(cè)均出現(xiàn)表達(dá)降低。[結(jié)論]DHEA可能經(jīng)過(guò)IGF-I信號(hào)通路,包括PI3K, P38 MAPK, P42/44 MAPK信號(hào)通路來(lái)上調(diào)人骨髓間充質(zhì)干細(xì)胞中IGF-I基因表達(dá)并促進(jìn)其向成骨方向分化。第三部分[目的]研究老齡化與1 α-羥化酶關(guān)系,并闡明DHEA在維生素D代謝中的作用機(jī)制。[方法]運(yùn)用RT-PCR方法研究在人骨髓間充質(zhì)干細(xì)胞中維生素D代謝關(guān)鍵酶1α-羥化酶的表達(dá),并切分析年齡對(duì)其表達(dá)的影響。應(yīng)用IGF-IR受體阻斷劑AG1024, CERB阻滯劑KG501分別阻斷IGF-1, CERB信號(hào)通路,從蛋白水平研究DHEA在維生素D轉(zhuǎn)化代謝中的作用機(jī)制。[結(jié)果]一組包括19例患者的樣本中,年齡從40到87歲,平均年齡56±11歲,研究發(fā)現(xiàn)隨著年齡的增長(zhǎng),CYP27B1/1α-羥化酶的表達(dá)逐漸下降。老年組(年齡55歲,n=12)CYP27B1/1α-羥化酶表達(dá)是年輕組CYP27B1/1α-羥化酶表達(dá)(年齡50歲,n=7)的64.4%。在老年組和年輕組中,我們通過(guò)RT-PCR分別對(duì)250HD3或1,25(OH)2D3±DHEA對(duì)人骨髓間充質(zhì)干細(xì)胞成骨分化的影響進(jìn)行研究。在年輕組中,成骨誘導(dǎo)培養(yǎng)3d后,我們發(fā)現(xiàn)25OHD3,1,25(OH)2D3, DHEA均可以促進(jìn)成成骨相關(guān)標(biāo)志基因的表達(dá),如堿性磷酸酶(alkaline phosphatase, ALP)和骨涎蛋白(bone sialoprotein, BSP)。但在老年組中,250HD3對(duì)相關(guān)成骨標(biāo)志基因刺激作用較弱。在老年組中,經(jīng)DHEA預(yù)處理后,250HD3和1,25(OH)2D3均可以誘導(dǎo)人骨髓基質(zhì)干細(xì)胞向成骨方向分化。在人骨髓基質(zhì)干細(xì)胞中,通過(guò)不同的小分子抑制劑的應(yīng)用,發(fā)現(xiàn)cAMP反應(yīng)元件結(jié)合蛋白(cAMP response element binding protein, CREB)和[GF-I分別介導(dǎo)了脫氫表雄酮調(diào)節(jié)CYP27B1的作用。通過(guò)應(yīng)用小分子抑制劑AG-1024(一種IGF-IR的抑制劑),發(fā)現(xiàn)脫氫表雄酮上調(diào)CYP27B1的表達(dá)都是通過(guò)IGF-I的激活。而通過(guò)應(yīng)用小分子抑制劑KG-501(特殊抑CREB的下游基因),發(fā)現(xiàn)脫氫表雄酮上調(diào)CYP27B1的表達(dá)同樣需要通過(guò)CREB的激活。由此推測(cè)CYP27B1上調(diào)則是由于DHEA激活的IGF-I信號(hào)系統(tǒng),繼發(fā)激活CREB所介導(dǎo)的。[結(jié)論]1α-羥化酶/CYP27B1的表達(dá)及活性隨著人年齡的增長(zhǎng)而下降。脫氫表雄酮可能上調(diào)IGF-I,從而激活CREB,最終上調(diào)CYP27B1,增強(qiáng)人骨髓間充質(zhì)干細(xì)胞對(duì)于25OHD3的反應(yīng)能力,從而增強(qiáng)其成骨分化能力。
[Abstract]:The first part of human bone marrow mesenchymal stem cells in vitro culture and identification of [Objective] by laboratory has successfully created human bone marrow mesenchymal stem cell culture solution, separation, culture and identification of hMSCs, provide sufficient dry by density gradient centrifugation to isolate mononuclear cells. Methods for the follow-up study source, cell the adherent method in a -MEM medium primary hMSCs medium, then were cultured on the differentiation of osteoblasts. Alkaline phosphatase activity assay and Alizarin Red S staining; on adipocyte differentiation using Oil Red-O staining method was used to identify the application of Alcian; blue staining of chondrocytes were identified. Results: bone marrow cells after 6-24 hours, the cell body shape gradually into triangular or irregular shape. The culture of 2-4 days between the morphology of mesenchymal cells changed, the cells gradually turned into fiber The spindle cell type, and gathered into the colony; 7-14 days in culture, cells covered with 70%-80% culture bottle bottom area, cells arranged more closely, the bone marrow mesenchymal stem cells into single cells. The cells were adipogenicdifferentiation, can detect the different shapes of Oil Red-O positive staining cell formation; osteoblast differentiation, detection of alkaline phosphatase activity, and Alizarin Red S staining positive cells formation; the chondrogenic differentiation was seen after Alcian blue staining positive cells. Conclusion] forming method used in this experiment can be successfully isolated, cultured and amplified in human bone marrow mesenchymal stem cells, and identify its osteogenic adipogenic, chondrogenic differentiation ability, part DHEA. Up regulation of IGF-I gene second expression and promotes the osteoblast differentiation of hMSCs [Objective] this study tested whether DHEA by adjusting IGF-I to promote bone marrow mesenchymal stem cells into Bone cell transformation. By using different time points, DHEA concentrations stimulate human bone marrow mesenchymal stem cells, the time and concentration dependent tests, and the biochemical and molecular enzyme inhibitors to elucidate DHEA in human bone marrow mesenchymal stem cells in regulating IGF-I gene expression and its mechanism to promote osteogenesis differentiation. In this study, the application of PKA signaling pathway inhibitor H-89, PKC inhibitor of CHE signaling pathway, JNK signaling pathway inhibitor SP600125 were used to block PKA signaling pathway, PKC signaling pathway, JNK signaling pathway. P13K signaling pathway inhibitor LY294002, P38 MAPK signal pathway inhibitor SB203580 P42/44 signal pathway inhibitor PD098059, IGF-IR blocking PI3K receptor antagonist AG1024, P38 MAPK, P42/44 MAPK, IGF-IR signaling pathway. Results in bone marrow mesenchymal stem cells, DHEA can upregulate the expression of IGF-I gene, and the presence of time And concentration dependent expression of.IGF-I gene in DHEA is the peak 4-6 hours after stimulation; the stimulation time was 6 hours, the best concentration was 10nM. PI3K P38 MAPK, P42/44 block, MAPK signaling pathway, the expression of DHEA gene suppressed the upregulation of IGF-1. Then the samples by special enzyme inhibitors after the action of bone differentiation related gene and alkaline phosphatase assay showed decreased expression. Conclusion]DHEA may be through the IGF-I pathway, including PI3K, P38, MAPK, P42/44, MAPK signaling pathway to increase human bone marrow mesenchymal stem cells in IGF-I gene expression and promote osteogenic differentiation. The third part to study the aging and the relationship between the 1 alpha hydroxylase the expression, and clarify mechanism. Methods DHEA in vitamin D metabolism in the use of RT-PCR method in human bone marrow mesenchymal stem cells in vitamin D metabolism key enzyme 1 alpha hydroxylase, and Analysis of the effect of age on its expression. The application of IGF-IR receptor antagonist AG1024, CERB inhibitor KG501 were used to block IGF-1, CERB signaling pathway, from the study of DHEA protein level conversion mechanism. Results in the metabolism of vitamin D in a group of samples including 19 patients, aged from 40 to 87 years old, the average age of 56. At the age of 11, the study found that with the increase of age, the expression of CYP27B1/1 alpha hydroxylase decreased gradually. The elderly group (age 55, n=12 CYP27B1/1) alpha hydroxylase expression is CYP27B1/1 hydroxylase expression in young group (aged 50, n=7 64.4%.) in the elderly group and the young group, we separately by RT-PCR on 250HD3 or 1,25 (OH) 2D3 + DHEA to study the effect of human bone marrow mesenchymal stem cell into osteogenic differentiation. In young group, osteoblast induced 3D, we found that 25OHD3,1,25 (OH) 2D3, DHEA can promote osteoblast related gene expression signature, Such as alkaline phosphatase (alkaline phosphatase, ALP) and bone sialoprotein (bone sialoprotein, BSP). But in the old group, 250HD3 of osteoblast marker gene stimulation is weak. In the older group, treated with DHEA, 250HD3 and 1,25 (OH) 2D3 can stem cells to differentiate into osteoblasts induction of human bone marrow stromal. In human bone marrow stromal stem cells, through the application of small molecule inhibitors of different cAMP response element binding protein (cAMP response element binding protein, CREB and [GF-I respectively) mediated by dehydroepiandrosterone regulates CYP27B1 function. Through the application of small molecule inhibitors of AG-1024 (an inhibitor of IGF-IR), found that the expression of dehydroepiandrosterone up-regulated the expression of CYP27B1 is through the activation of IGF-I. Through the application of small molecule inhibitors of KG-501 (gene special inhibition of CREB), found that the expression of dehydroepiandrosterone up-regulated CYP27B1 also need to pass After the activation of CREB. The results indicated that CYP27B1 increase is due to the activation of DHEA IGF-I signaling system, the expression and activity of secondary activation mediated by CREB. Conclusion]1 hydroxylase /CYP27B1 decreased with age. DHEA may upregulate IGF-I, which activates CREB, the upregulation of CYP27B1, enhancement of human bone marrow mesenchymal stem cells for the reaction ability of 25OHD3, so as to enhance the capability of osteogenic differentiation.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R68

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉莉治;保健食品中DHEA含量的測(cè)定[J];中國(guó)衛(wèi)生檢驗(yàn)雜志;2005年01期

2 沈敏;向平;沈保華;王萌燁;嚴(yán)慧;;外源性DHEA的體內(nèi)代謝研究[J];中國(guó)司法鑒定;2007年03期

3 葉雅萍;歐奇志;覃愛(ài)平;;育齡女性血清硫酸脫氫表雄酮(DHEA-S)檢測(cè)分析及其臨床意義[J];生殖與避孕;2013年11期

4 恒力;;DHEA,幫你找回年輕[J];金秋科苑;1998年08期

5 張衛(wèi)東;去氫表雄甾酮(DHEA)對(duì)老年病的預(yù)防效果[J];日本醫(yī)學(xué)介紹;2005年03期

6 杜昕;;脫氫表雄甾酮(DHEA)不能預(yù)防衰老[J];英國(guó)醫(yī)學(xué)雜志(中文版);2007年01期

7 陳新娜;;DHEA和卵巢功能低下1例及文獻(xiàn)復(fù)習(xí)[J];現(xiàn)代婦產(chǎn)科進(jìn)展;2010年09期

8 李莉,倉(cāng)公敖,江夕夫;保健食品中DHEA的高效液相色譜分析方法研究[J];中國(guó)衛(wèi)生檢驗(yàn)雜志;2000年03期

9 吳賽珠,王子?xùn)|,周忠江,周可祥,吳炎賢,孫飛,容志毅,馬瑞;The Effect of Dehydroepiandrosterone on the Expression of AT_1 receptor and TNF-induced ICAM-1 in Vascular Smooth Muscle Cells[J];South China Journal of Cardiology;2005年01期

10 嚴(yán)衛(wèi)星,賈群山;DHEA的安全性與功能(綜述)[J];中國(guó)食品衛(wèi)生雜志;2000年01期

相關(guān)會(huì)議論文 前10條

1 P.C.Ho;;Role of DHEA in Ovarian Stimulation-is it useful[A];中國(guó)中西醫(yī)結(jié)合學(xué)會(huì)生殖醫(yī)學(xué)分會(huì)首屆學(xué)術(shù)年會(huì)暨生殖醫(yī)學(xué)專業(yè)委員會(huì)成立大會(huì)論文匯編[C];2014年

2 殷復(fù)建;劉琳;丁逍;周英俏;沈?qū)W懷;馬海田;;DHEA調(diào)節(jié)自然衰老大鼠脂代謝中的研究[A];全國(guó)動(dòng)物生理生化第十二次學(xué)術(shù)交流會(huì)論文摘要匯編[C];2012年

3 ;The expression of serum steroid sex hormones and steroidogenic enzymes following intraperitoneal administration of dehydroepiandrosterone(DHEA)in male rats[A];全國(guó)動(dòng)物生理生化第十一次學(xué)術(shù)交流會(huì)論文摘要匯編[C];2010年

4 ;Comparative Phosphoproteomic Analysis of the Effects and Regulation of DHEA on Hepaticin Lipids Metabolism in Broiler Chickens[A];全國(guó)動(dòng)物生理生化第十一次學(xué)術(shù)交流會(huì)論文摘要匯編[C];2010年

5 殷復(fù)建;劉琳;沈?qū)W懷;周英俏;丁逍;馬海田;;DHEA對(duì)自然衰老大鼠抗氧化功能的影響[A];全國(guó)動(dòng)物生理生化第十二次學(xué)術(shù)交流會(huì)論文摘要匯編[C];2012年

6 沈?qū)W懷;謝正露;殷復(fù)建;劉琳;丁逍;周英俏;馬海田;;DHEA對(duì)TM-3細(xì)胞周期的影響[A];全國(guó)動(dòng)物生理生化第十二次學(xué)術(shù)交流會(huì)論文摘要匯編[C];2012年

7 沈?qū)W懷;殷復(fù)建;謝正露;劉琳;周英俏;丁逍;馬海田;;DHEA對(duì)TM-3細(xì)胞線粒體超微結(jié)構(gòu)和功能的影響[A];全國(guó)動(dòng)物生理生化第十二次學(xué)術(shù)交流會(huì)論文摘要匯編[C];2012年

8 ;Regulation of DHEA on hepatic lipid metabolism in broil chicken and its cellular molecular mechanisms involved[A];膳食變遷對(duì)民眾健康的影響：挑戰(zhàn)與應(yīng)對(duì)——第二屆兩岸四地營(yíng)養(yǎng)改善學(xué)術(shù)會(huì)議學(xué)術(shù)報(bào)告及論文摘要匯編[C];2010年

9 周征;呂明莊;;耳針對(duì)衰老大鼠血清DHEA-S、E2、T及腺體影響的實(shí)驗(yàn)研究[A];全國(guó)第16屆針灸臨床學(xué)術(shù)研討會(huì)、全國(guó)第11屆耳穴診治學(xué)術(shù)研討會(huì)、當(dāng)代臨床治驗(yàn)論壇暨中西部十省區(qū)學(xué)術(shù)研討會(huì)論文集[C];2008年

10 沈?qū)W懷;馬海田;;脫氫表雄酮(DHEA)在TM-3細(xì)胞中的生物轉(zhuǎn)化及其對(duì)類固醇代謝相關(guān)酶表達(dá)的影響[A];中國(guó)生理學(xué)會(huì)消化內(nèi)分泌生殖代謝生理專業(yè)委員會(huì)2011年消化內(nèi)分泌生殖學(xué)術(shù)會(huì)議論文摘要匯編[C];2011年

相關(guān)重要報(bào)紙文章 前7條

1 記者 盧蘇燕;服用DHEA不是時(shí)尚是賭命[N];新華每日電訊;2001年

2 楊駿;DHEA可治肺動(dòng)脈高壓[N];中國(guó)醫(yī)藥報(bào);2004年

3 楊駿;DHEA可治肺動(dòng)脈高壓？[N];醫(yī)藥經(jīng)濟(jì)報(bào);2004年

4 束梅英 張驍;脫氫表雄酮不只是激素之母[N];醫(yī)藥經(jīng)濟(jì)報(bào);2001年

5 科文;法將抗老丸DHEA列入藥品管制[N];中國(guó)醫(yī)藥報(bào);2001年

6 ;法專家提醒慎用雄激素[N];中國(guó)高新技術(shù)產(chǎn)業(yè)導(dǎo)報(bào);2001年

7 曹海;性愛(ài)也是一種體育鍛煉[N];北京科技報(bào);2004年

相關(guān)博士學(xué)位論文 前10條

1 譚曉冬;脫氫表雄酮對(duì)實(shí)驗(yàn)性自身免疫性神經(jīng)炎保護(hù)作用的研究[D];山東大學(xué);2009年

2 劉雁勇;慢性輕度應(yīng)激致?lián)p傷及7-oxo-DHEA干預(yù)機(jī)制研究[D];中國(guó)協(xié)和醫(yī)科大學(xué);2003年

3 梁曉南;脫氫表雄酮（DHEA）在維生素D轉(zhuǎn)化代謝中的作用機(jī)制研究[D];廣西醫(yī)科大學(xué);2015年

4 盧佳宏;針刺對(duì)治療患者面部痤瘡、血清IGF-1、DHEA及改善其抑郁焦慮狀態(tài)的研究[D];中國(guó)中醫(yī)科學(xué)院;2013年

5 郝群;補(bǔ)腎寧心方合并DHEA對(duì)去勢(shì)雌兔動(dòng)脈粥樣硬化的防治作用[D];復(fù)旦大學(xué);2004年

6 姜艷芳;去氫表雄酮抑制腫瘤細(xì)胞增殖及其分子機(jī)制的研究[D];吉林大學(xué);2005年

7 楊筍;去氫表雄酮（Dehydroepiandrosterone）的腫瘤化學(xué)預(yù)防和抗骨質(zhì)疏松作用及其機(jī)制研究[D];中國(guó)協(xié)和醫(yī)科大學(xué);2001年

8 黃建珍;胚胎期肉雞肝臟脂類代謝關(guān)鍵因子的篩選及DHEA的調(diào)控研究[D];南京農(nóng)業(yè)大學(xué);2008年

9 趙素梅;兩種肉雞胚胎期脂肪代謝及DHEA調(diào)控研究[D];南京農(nóng)業(yè)大學(xué);2007年

10 王凌;脫氫表雄酮及補(bǔ)腎寧心方對(duì)成骨細(xì)胞—免疫網(wǎng)絡(luò)的調(diào)控作用[D];復(fù)旦大學(xué);2006年

相關(guān)碩士學(xué)位論文 前10條

1 程恒輝;DHEA的抗AS作用以及細(xì)胞色素P450芳香酶對(duì)DHEA抗AS作用的影響[D];華中科技大學(xué);2007年

2 王江偉;膽甾醇和DHEA的化學(xué)修飾及抗菌活性初步研究[D];湖南科技大學(xué);2012年

3 劉琳;DHEA對(duì)原代大鼠睪丸間質(zhì)細(xì)胞生物學(xué)特性的影響及其調(diào)節(jié)睪酮合成機(jī)制的研究[D];南京農(nóng)業(yè)大學(xué);2013年

4 王萌燁;體內(nèi)DHEA及其相關(guān)類固醇激素的檢測(cè)與評(píng)價(jià)[D];復(fù)旦大學(xué);2008年

5 王小岑;以AD/DHEA為原料甾體7位衍生物的合成研究[D];天津大學(xué);2010年

6 孫紹鳳;以DHEA為原料甾體類重要中間體的合成研究[D];天津大學(xué);2005年

7 劉奔;DHEA對(duì)小鼠脾淋巴細(xì)胞活化的調(diào)節(jié)[D];天津醫(yī)科大學(xué);2005年

8 沈?qū)W懷;DHEA對(duì)TM-3細(xì)胞生物學(xué)特性的影響及其生物轉(zhuǎn)化規(guī)律研究[D];南京農(nóng)業(yè)大學(xué);2011年

9 杜計(jì)燕;7α—羥基 DHEA、5α—雄甾—16—烯—3—酮及4—羥基雄甾化合物合成研究[D];華東師范大學(xué);2005年

10 單威;唾液DHEA-S、T及血清IGFBP-3在游泳運(yùn)動(dòng)員機(jī)能監(jiān)控中的應(yīng)用可行性[D];北京體育大學(xué);2007年

，

本文編號(hào)：1710412