類別:http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/1332024-03-28T11:08:49Z2024-03-28T11:08:49ZT細胞第四因子(Tcf4)與細胞死亡相關蛋白(Daxx)結合作用之探討Shu-Ling Tzeng曾淑玲http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/319382021-06-13T03:25:11Z2006-01-01T00:00:00Z標題: T細胞第四因子(Tcf4)與細胞死亡相關蛋白(Daxx)結合作用之探討; Studies on the interaction between T cell factor 4 (Tcf4)
and cell death-associated protein (Daxx)
作者: Shu-Ling Tzeng; 曾淑玲
摘要: 近年來的研究報導指出,在人類大腸癌細胞株細胞中,因為抑癌基因APC的突變或是乙型卡特寧本身的突變,導致細胞核內的人類T細胞因子與乙型卡特寧蛋白複合物活性的持續活化,因而造成人類T細胞因子所調節的下游基因失控是造成大腸上皮細胞的轉型以及息肉生成的主要原因。事實上,人類T細胞因子與乙型卡特寧蛋白複合物對轉譯活性的調節,不管是在胚胎發育或是癌症生成的過程中都存在著相當的意義。在嘗試探討T細胞因子在癌症生成過程中所可能扮演的角色之前,必須先釐清T細胞因子的調控網絡如何運作。雖然T細胞因子可以透過HMG直接與去氧核醣核酸結合,然而這樣的結合在缺乏與乙型卡特寧結合形成蛋白複合體時,T細胞因子並不足以獨立啟動其下游基因的轉譯,除此之外,更多的實驗結果顯示乙型卡特寧不只有如上所述與T細胞因子形成蛋白質複合體以啟動下游基因的轉譯活性,同時也可以將T細胞因子對下游基因的調控功能,由原本的轉譯抑制轉換為轉譯活化,鑑於對相同下游基因的不同作用,科學家們深信必定存在有一嚴謹的開關,得以嚴密調控並且決定細胞發育生長過程中不同的命運。
在T細胞因子轉譯活性的調控中,可能存在與T細胞因子結合的介質蛋白以及其彼此之間交互作用的機轉激發我們相當的研究興趣。由於在人類大腸上皮細胞中存在大量的T細胞第四因子蛋白,因此實驗設計以T細胞第四因子為餌,透過酵母菌雜交的方式順利分離出可能與T細胞第四因子結合的候選蛋白-人類細胞死亡相關蛋白:Daxx。
Daxx蛋白最早被發現是在細胞質中扮演Fas/JNK相關訊息傳遞的媒介蛋白,不過近年來更多的證據指出,其實Daxx蛋白主要是存在細胞核中,扮演基因轉譯活性的調節者。藉由在人類293T細胞中的共同免疫沈澱法以及在酵母菌Y190菌種中的雜交法,證實了Daxx蛋白與T細胞第四因子蛋白之間的結合作用,並且定出T細胞第四因子與Daxx蛋白的結合區間。在細胞核內,Daxx蛋白藉由減低T細胞第四因子與去氧核醣核酸結合的能力而達到抑制T細胞第四因子轉譯活性。因此,在人類大腸癌細胞中大量表現Daxx蛋白時,T細胞第四因子下游基因的調控也跟著改變,包括有cyclinD1和Hath-1基因,同時造成人類大腸癌細胞生長週期G1時期的停頓。除此之外,在人類大腸腺癌病人的檢體中,比較該病人癌化的與正常的大腸配對組織,Daxx蛋白的表現則有明顯減少的現象。綜合以上的發現推論Daxx蛋白可能透過與T細胞第四因子的結合,繼而調控人類細胞的生長週期,以達到細胞增生與細胞分化的生理目的。
另外,當共同表現Daxx蛋白時,可以透過SDS-PAGE的分離,觀察到另一個較小型態的T細胞第四因子蛋白的存在,並且隨著Daxx蛋白的表現劑量或時間有專一的相關聯性。T細胞第四因子的轉譯活性也可以被SUMO-1以及PIAS3加強,不過這樣的轉譯加強效果仍然可以被Daxx蛋白所抑制。以上的研究發現與推論,除了可以進一步勾勒出T細胞第四因子與Daxx蛋白結合時對人類生理功能的影響,同時也可以提供創新的思考模式,達到癌症治療的目的。; Recently, it has been reported that the nuclei of colon carcinoma cell lines contain constitutively active Tcf4 / b-catenin complexes as a direct consequence of either loss of function of the tumor suppressor gene APC or gain of function mutations in b-catenin itself. This is believed to result in the uncontrolled transcription of TCF target genes, leading to transformation of colon epithelial cells and initiation of polyp formation. Regulation of the transcriptional activity of b-catenin / Tcf4 has important implications for embryonic development as well as for carcinogenesis in the intestinal epithelium. Prior to discuss the potential role for LEF / TCF transcription factors in cancer, it is important to outline the mechanism by which they have been proposed to operate. Although LEF / TCF transcription factors bind directly to DNA through their HMG domains, they are incapable of independently activating gene transcription. On the other hand, most experimental data support the view that TCF is a repressor when Wnt does not convert it into an activator. TCF can repress target genes as well as activate those same target genes in cells instructed to change developmental fate, there are several mechanisms by which this switch is achieved.
We are interested in the possibility of the bridging protein that interacts with TCF during the regulation of transcriptional activity. Because of its predominant expression in the human colonic epithelium, the full-length Tcf4 is used as bait in yeast two-hybridization, and Daxx is isolated. Daxx, a human cell death associated protein, has been reported to mediate the Fas / JNK-dependent signals in the cytoplasm. However, several lines of evidence have suggested that Daxx is mainly located in the nucleus and functions as a transcriptional regulator. Co-immunoprecipitation in HEK-293T cells and yeast two-hybrid screen in Y190 cells are performed to identify the interaction between Tcf4 and Daxx, and co-immunoprecipitation is also used to map the binding regions of Tcf4. In the nucleus, Daxx reduces DNA binding activity of Tcf4 and represses Tcf4 transcriptional activity. Overexpression of Daxx alteres expression of genes downstream of Tcf4, including cyclin D1 and Hath-1, and induces G1 phase arrest in colon cancer cells. Besides, a reduction in Daxx protein expression is observed in colon adenocarcinoma tissue when compared with normal colon tissue. These findings suggest a possible physiological function of Daxx, via interaction with Tcf4, to regulate cell cycle progression, and hence cell proliferation and differentiation. Furthermore, a small form of Tcf4 is observed specifically in SDS-PAGE when Daxx is co-expressed in a dose- and time-dependent manner. Moreover, transcriptional activity of Tcf4 is enhanced by SUMO-1 and PIAS3, but this transactivation still can be repressed by Daxx. Taken together, these findings not only outline the functional link between Tcf4 and Daxx, but also provide a new idea to develop novel cancer therapies.2006-01-01T00:00:00Z黃麴毒素B1對斑馬魚胚胎發育的影響Ya-Chih Cheng鄭雅之http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/684212021-06-17T02:20:31Z2017-01-01T00:00:00Z標題: 黃麴毒素B1對斑馬魚胚胎發育的影響; Effects of Aflatoxin B1 on the Development of Zebrafish Embryos
作者: Ya-Chih Cheng; 鄭雅之
摘要: 黃麴毒素B1(Aflatoxins B1, AFB1)為黃麴黴 (Aspergillus flavus) 等黴菌產生之二級代謝產物,已知其對人類及動物具有肝毒性及致肝癌性。目前對於AFB1在胚胎發育上的影響及毒性機制尚不明瞭,因此本研究將利用斑馬魚胚胎作為動物模式來探討AFB1對於胚胎發育的影響。實驗結果發現,AFB1處理斑馬魚胚胎達120 hpf (hours post fertilization) 後,雖然導致胚胎的高死亡率,卻不會對胚胎造成明顯的不良型態變化。以0.25及0.5 μM的AFB1處理轉殖肝螢光魚胚胎Tg(lfabp:eGFP)以觀察AFB1對肝臟發育的影響 (6-96 hpf),結果顯示肝臟螢光強度隨毒素劑量上升而顯著下降,組織切片分析也顯示肝臟有縮小現象。末端脫氧核苷酸轉移酶脫氧尿苷三磷酸切口末端標記螢光染色 (Terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL)的結果指出AFB1會促進肝臟細胞凋亡,磷酸化組蛋白H3免疫染色 (phospho-histone H3 immunostaining) 的結果則顯示AFB1可能可以抑制肝臟細胞增生。以prox1和hhex探針進行全胚體原位雜交法 (Whole-mount in situ hybridization) ,發現胚胎肝臟在特化 (specification) 階段(24hpf)就會受到AFB1的傷害,進一步在肝臟發育不同的不同時期以AFB1處理轉殖肝螢光魚胚胎Tg(lfabp:eGFP)觀察,得知肝臟在出芽/分化 (budding/ differentiation) 及增長 (outgrowth) 階段都有可能受AFB1影響而造成肝臟縮小的情形。AFB1的暴露亦會導致肝臟特異性的miR-122表現下降,但不會影響胚胎中凝血因子基因(f7、f3a、f3a)和肝臟特異性基因(vtna、cp)的表現。此外AFB1會誘發斑馬魚肝細胞(ZFL)中的氧化壓力上升,而抗氧化劑N-乙醯半胱氨酸(N-acetylcysteine , NAC)和維生素C (Vitamin C, Vit C) 的使用可以降低AFB1造成的細胞傷害、胚胎死亡率,而維生素C更可能可以改善肝臟縮小的情形。
過去研究指出AFB1也會造成中樞神經的損傷。我們由行為測試觀察到AFB1的暴露造成斑馬魚胚胎異常的游泳模式,同時也使胚胎移動的距離下降,因此推測AFB1可能會影響胚胎發育階段的神經生長。早期神經元的標記(HuC)和神經毒性的生物指標(gfap)的mRNA變化證實了AFB1會影響胚胎神經發育而產生神經毒性。乙醯化微管蛋白免疫染色 (Acetyl-alpha Tubulin staining, AcTub staining) 的結果也顯示,0.25及0.5 μM的AFB1明顯干擾了24 hpf胚胎神經元的發育。將 6 hpf 斑馬魚胚胎以 0.5 μM AFB1 處理至 48 hpf 後進行微陣列技術分析(microarray),發現多個和神經發育相關基因的表現皆受到AFB1的影響。以半定量及定量聚合酶鏈鎖反應 (PCR) 確認在處理AFB1達24h及48h的胚胎中,ngfa 及atp1b1b mRNA的表現量顯著下降prtga mRNA的表現量則上升,上述結果表示AFB1可能可以干擾神經元的能量來源並抑制斑馬魚胚胎神經元的早期分化。
本篇研究證實AFB1會干擾早期胚胎肝臟和神經的發育,而相關的發育毒性的機制仍需要進一步的實驗來確認。; Aflatoxins B1 (AFB1) is a naturally occurring mycotoxin produced by the Aspergillus flavus group of fungi. It is also a well-known hepatocarcinogen that contributes significantly to the high incidence of hepatocellular carcinoma. Since there are few studies examining the developmental toxicity of AFB1, we used the zebrafish embryo as a vertebrate model herein. Treatment of zebrafish embryos from 6 hours post-fertilization (hpf) to 120 hpf resulted in a high mortality rate, but the morphology of embryos remained unchanged. To observe the effect of AFB1 on liver development, the embryos of transgenic zebrafish Tg(lfabp:eGFP) were treated with AFB1 ranging from 0.25 to 0.5 μM. We found that the liver fluorescence was decreased in a dose-dependent manner and histological analysis also indicated a shrunk liver size. TUNEL assay revealed that AFB1 promoted liver apoptosis and pH3 immunostaining implied that AFB1 might also inhibited liver proliferation. Whole-mount in situ hybridization with prox1 and hhex probes and observation of Tg(lfabp:eGFP) showed that the embryonic liver was affected by AFB1 at the specification and the budding/ differentiation stages (24-72hpf). Also, AFB1 treatment led to the down-regulation of liver-specific miR-122. However, AFB1 did not affect the gene expression of coagulation factors (f7, f3a, and f3a) and liver-specific genes (vtna and cp) in 6-72 hpf embryos. As AFB1 was found to increase the oxidative stress in zebrafish liver cell line (ZFL), ZFL cell death as well as embryo mortality mediated by AFB1 was restored in the presence of antioxidants N-acetylcysteine (NAC) and Vitamin C (Vit C). In addition, Vit C is likely to rescue the liver shrinkage that caused by AFB1.
Previous studies indicate that AFB1 also leads to brain damage. From our results of behavior test, AFB1 exposure decreased the total distance moved of embryos which swam in an abnormal shaking patterns. Therefore, we hypothesized that AFB1 might affect the neurodevelopment during developmental stage of embryos. The marker of early born neurons (HuC) and the biomarker of neurotoxicity (gfap) were both modulated by AFB1. Additionally, acetylated alpha-tubulin (AcTub) staining indicated that AFB1 disrupted the development of embryonic neurons. Microarray profiles of embryos after 48 hr AFB1 treatment showed an alteration of several genes which were related to neurodevelopment, including nerve growth factor a (ngfa), atp1b1b, and protogenin homolog a (prtga). PCR analysis further confirmed that AFB1 significantly decreased the expression of both ngfa and atp1b1b, and increased that of prtga gene in 6-48 hpf embryos, which suggest that AFB1 might have the ability to suppress neuronal differentiation and reduce the source of energy demand in neurons.
In conclusion, AFB1 might interfere with the embryonic liver and neural development. Further experiments are required to confirm the mechanism of developmental hepatotoxicity and neurotoxicity associated with AFB1.2017-01-01T00:00:00Z高血糖狀態調控間質幹細胞分化成脂肪細胞及造骨細胞之機制探討Chia-Chi Chuang莊佳琪http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/382042021-06-13T16:27:55Z2005-01-01T00:00:00Z標題: 高血糖狀態調控間質幹細胞分化成脂肪細胞及造骨細胞之機制探討; Studies on the Mechanisms of Adipocyte and Osteoblast Differentiation from Mesenchymal Stem Cell under Hyperglycemia
作者: Chia-Chi Chuang; 莊佳琪
摘要: 糖尿病是指人體內的胰臟不能製造足夠的胰島素或是胰島素失去功能,導致葡萄糖無法充分進入細胞內,使血糖濃度升高,因而形成糖尿病。雖然糖尿病是一種多樣性的症候群,但其基本表現都以高血糖為主。長期處於高血糖狀態會產生一些糖尿病慢性併發症,包括視網膜病變、神經病變、腎臟病變及糖尿病足等等。而糖尿病患者也常出現骨折後骨頭癒合不良的情況,但是糖尿病造成骨質疏鬆的原因尚未釐清。基於骨頭內的間質幹細胞可以分化成造骨細胞及脂肪細胞等等,而且在臨床上也發現隨著年齡的增加,骨頭內的造骨細胞減少,取而代之的是脂肪細胞的增加,進而造成骨質疏鬆症。因此,本篇主要是探討在高血糖狀態下影響間質幹細胞的分化及其相關的分子機制。我們使用25.5mM的高葡萄糖溶液處理間質幹細胞來模擬糖尿病患者的高血糖狀態。
首先,我們證明高葡萄糖會經由phosphoinositide 3-kinase (PI3K)/Akt而促進間質幹細胞分化成脂肪細胞。取6~8週大的FVB/N雄性小鼠的股骨及脛骨,沖出骨髓後培養七天,先單獨處理10-8 M Dexamethasone (DEX) 及 5 μg/ml胰島素,觀察間質幹細胞分化成脂肪細胞的情況。我們發現在第12天脂肪細胞數目明顯的增加,因此在10-8 M DEX 及 5 μg/ml 胰島素下合併處理25.5 mM的高葡萄糖及一個已知會促進脂肪細胞分化的內生性peroxisome proliferators-activated receptor gamma (PPARγ)受質- 15-Deoxy-△12,14-prostaglandin J2 (15d-PGJ2)。12天後以流式細胞儀分析,發現在有25.5 mM的高葡萄糖情況下,脂肪細胞分化的數目比只有處理10-8 M DEX 及 5 μg/ml胰島素的這組增加了1倍,而處理1 μM 15d-PGJ2這組增加了4倍的脂肪細胞。接著,我們探討高葡萄糖促進脂肪細胞分化的機制。在1999年,Wu等人就發現PPARγ這個轉錄因子會調控脂肪細胞的分化。因此,我們合併處理高葡萄糖溶液及15d-PGJ2 12天,發現高葡萄糖溶液及15d-PGJ2會增加PPARγ表現量,而且會被PPARγ拮抗劑- GW 9662 (20 μM) 所抑制。另外,合併處理高葡萄糖溶液及15d-PGJ2也會增加Akt的磷酸化,然而處理PI3K的抑制劑- LY 294002 (7.5 μM) 後,除了會抑制Akt的磷酸化外,也會抑制高葡萄糖溶液及15d-PGJ2促進的PPARγ表現。而且主要是抑制PPARγ的isoform-PPARγ2。由以上的實驗,我們認為高葡萄糖溶液及15d-PGJ2皆會藉由PI3K及Akt這個路徑來促進PPARγ的表現,進而促進脂肪細胞的分化。為了加以確認,我們以轉染作用給予隱性變異質體DN-p85 和 DN-Akt,皆可以減少高葡萄糖溶液及15d-PGJ2促進的PPARγ表現。
由以上實驗,我們認為PI3K/Akt路徑在調控間質幹細胞的分化可能扮演一個很重要的角色。所以,我們更進一步探討PI3K/Akt是否會調控間質幹細胞分化成造骨細胞及其相關的分子機制。我們把沖出的骨髓培養7天後,單獨處理10-8 M DEX,5 μg/ml維他命C及10 mM β-甘油磷酸觀察間質幹細胞分化成造骨細胞的情況。1990年,Owen等人就發現從大鼠的間質幹細胞分化成造骨細胞到造骨細胞死亡的過程需要28天,而在這整個時期,鹼性磷酸酶的活性是屬於早期造骨細胞分化的指標,而骨鈣素的表現及骨礦物質化是屬於晚期的造骨細胞分化指標。而我們的實驗也發現在造骨細胞的分化過程中,第12天的鹼性磷酸酶活性最高,同時合併處理1 μM 15d-PGJ2及25.5 mM 高葡萄糖溶液後,會抑制鹼性磷酸酶的活性,但處理GW 9662 (20 μM) 及LY 294002 (7.5 μM) 後會增加被15d-PGJ2所抑制的鹼性磷酸酶活性,而LY 294002也會增加被高葡萄糖溶液所抑制的鹼性磷酸酶活性。另外我們也觀察造骨細胞分化的晚期指標,發現在第22天骨鈣素的mRNA表現量最大,同時合併處理15d-PGJ2及高葡萄糖後,15d-PGJ2會抑制骨鈣素mRNA的表現,而此現象也會被GW 9662及LY 294002所阻斷,但高葡萄糖溶液則沒有作用。為了加以確認,我們以轉染作用給予隱性變異質體DN-p85 和 DN-Akt,在造骨細胞分化的第22天,發現會抑制15d-PGJ2的作用,進而增加骨礦物質化,但高葡萄糖溶液仍沒有影響。因此,第二部分,我們證明高葡萄糖會經由PI3K/Akt而抑制早期的間質幹細胞分化成造骨細胞。
最後,我們以低劑量streptozotocin (STZ)連續腹腔注射5天,誘導6~8週大的FVB/N雄性小鼠成第一型糖尿病,經過3週後,檢查其血糖是否高於400 mg/dl,沖出控制組及糖尿病組小鼠骨頭內骨髓培養,經過7天後,處理DEX、胰島素及合併高葡萄糖溶液或15d-PGJ2,發現糖尿病組小鼠的間質幹細胞分化成脂肪細胞的比例較控制組高。另外,我們也取控制組及糖尿病組小鼠的脛骨和股骨的骨幹端,大約在生長板的位置,利用液態氮磨碎後,加入溶解緩衝液,測其三酸甘油酯的含量及鹼性磷酸酶的活性,發現糖尿病組小鼠骨頭內的三酸甘油酯含量比控制組小鼠增加1倍,而糖尿病組小鼠的骨頭鹼性磷酸酶活性比控制組小鼠稍低。最後,再利用南方點墨法分析,也發現糖尿病組小鼠的骨頭內PPARγ表現量比控制組高。因此,第三部分,我們證明高血糖的糖尿病小鼠,其骨頭內脂肪組織比控制組多。
綜合以上實驗,我們認為PI3K/Akt在調控間質幹細胞分化上扮演很重要的角色,包括活化PI3K/Akt會增加PPARγ表現進而促進間質幹細胞分化成脂肪細胞,而相反地活化PI3K/Akt會減少鹼性磷酸酶、骨鈣素表現及骨礦物質化進而抑制間質幹細胞分化成造骨細胞。; Diabetes is characterized by mild to moderate hyperglycemia, glucosuria, polyphagia, hypoinsulinemia, hyperlipidemia, and weight loss. All forms of diabetes are characterized by chronic hyperglycemia and the development of many serious complications, for example, heart disease (cardiovascular disease), blindness (retinopathy), nerve damage (neuropathy), and kidney damage (nephropathy). Diabetes has also been reported with a net loss of bone. However, the effect of hyperglycemia on bone loss remains unclear. Bone loss in aging and osteoporosis are associated with a decrease in the number and activity of osteoblasts and a parallel increase in the number of adipocytes. Here we have demonstrated that hyperglycemia affected the mesenchymal stem cells (MSCs) differentiation by enhancing adipocyte differentiation (adipogenesis). We used high glucose (HG, 25.5 mM) to mimic the hyperglycemia condition.
To determine the effects of hyperglycemia on adipogenesis, we cultured mouse MSCs in an adipogenic hormonal cocktail, and adipogenesis was strong enhancement by supplementation of HG and 15-deoxy-∆12,14-PGJ2 (15d-PGJ2), which has been identified as an endogenous ligand for peroxisome proliferator-activated receptor gamma (PPARγ), inducing adipogenesis in vitro. This ligand improves insulin sensitivity through the activation of the transcription factor, PPARγ. In addition to sensitizing cells to insulin, the PPARγ2 isoform appears to be critical for the regulation of osteoblast differentiation (osteoblastogenesis) and adipocyte differentiation (adipogenesis) of MSCs in bone marrow. In this HG cultures, the expression of PPARγ2 was up-regulated even prior to adipogenic induction. Moreover, treatment with PPARγ agonists, GW 9662 (20 μM) or inhibitors of phosphatidylinositol 3-kinase (PI3K), LY 294002 (7.5 μM), leads to the complete blockade of HG-enhanced adipogenesis of MSCs by inhibited the PPARγ expression. HG-activated Akt on adipogenesis of MSCs was also inhibited by LY 294002. Likewise, blocking the PI3K or Akt activity with the dominant-negative vectors DN-p85 or DN-Akt, respectively, also greatly inhibited the HG-enhanced the expression of PPARγ. These suggesting that HG enhanced adipogenesis of MSCs in the adipogenic medium may through a PI3K/Akt regulated PPARγ pathway.
Another intriguing finding was that 15d-PGJ2 (1 μM) enhanced adipogenesis by increasing the PPARγ expression which was inhibited by LY 294002, DN-p85 and DN-Akt. Collectively, these data provide a new insight into the PI3K/Akt pathway on MSCs differentiation. So, next we examine the mechanisms of osteoblastogenesis of MSCs. First, we found that 15d-PGJ2 and HG decreased alkaline phosphatase activity, which was used as early differentiation markers of osteoblastogenesis from MSCs in osteoblastogenic medium, and treatment with GW 9662 or LY294002 was significantly increasing the alkaline phosphatase activity. Second, the 15d-PGJ2 inhibited the expression of the osteocalcin, which gene marked the late stages of osteoblastogenesis, and treatment with GW 9662 or LY294002 was significantly increasing the osteocalcin gene expression. But HG was no effect on osteocalcin expression. Third, mineralization was reduced by treatment of MSCs with 15d-PGJ2 in osteoblastogenic medium and dominant-negative vectors DN-p85 or DN-Akt greatly increased mineralization. HG was also no effect on mineralization. Mineralization correlated closely with osteocalcin gene expression. Taken together, these results indicate that activation of PI3K/Akt pathway in MSCs may increase adipogenesis and decrease osteoblastogenesis of MSCs.
On the other hand, in in vivo study, mice were made diabetic by multiple low-dose streptozotocin (STZ) treatment, and controls were treated with vehicle alone. After 3 weeks, chose the diabetic mice which had ≧400 mg/dl blood sugar. We cultured diabetic MSCs and control MSCs in adipogenic medium combined with HG or 15d-PGJ2 and compared their adipogenesis of MSCs. We found that the level of diabetic adipogenesis of MSCs was higher than the level of control mice. Moreover, the tibial bone from the proximal metaphysis to the tibiofibular junction was snap frozen in liquid nitrogen, pulverized, and we found that the expression of PPARγ and triglyceride amounts were significantly increased in the diabetic bone whereas alkaline phosphatase activity was reduced in the diabetic bone.
These findings support a reciprocal relationship between the development of bone and fat under hyperglycemia, and may prompt further exploration of the PI3K/Akt regulated MSCs differentiation as a potential target for intervention in diabetic osteoporosis.2005-01-01T00:00:00Z高血糖狀態調控細胞增殖及死亡訊息機制之探討Meei-Ling Sheu許美鈴http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/392812021-06-13T17:25:20Z2005-01-01T00:00:00Z標題: 高血糖狀態調控細胞增殖及死亡訊息機制之探討; Studies on the cellular signaling pathways in hyperglycemia-induced cell proliferation and apoptosis
作者: Meei-Ling Sheu; 許美鈴
摘要: 糖尿病為一常見的內分泌代謝疾病,目前已經成為公共衛生問題的一部份。在世界各地隨著肥胖人數日驅增加,糖尿病的人數也隨之顯著提高。而糖尿病對身體造成最大的問題是對實質性組織器官的傷害;尤其是心血管系統以及腎臟的影響。也由於在世界各地糖尿病的病人愈來愈多,其導致的複雜變化以及合併病發症更值得我們去重視。
糖尿病病人體內血糖過高的訊息是不可逆的並且最後導致組織器官的功能障礙或損傷。由過去的文獻報導以及臨床病人報告中已知:糖尿病的病理過程可引起血管內皮細胞功能缺失,造成的血管功能障礙、加速冠狀動脈硬化並對預後不佳。然而進一步地導致血管內皮細胞凋亡的機制至今仍不十分清楚。探討此一異常訊息是如何造成人類血管內皮細胞凋亡?其機制為何?進而如何避免糖尿病在人類心血管系統功能障礙及合併症的產生,以及病程的惡化是十分重要的課題。於本論文第一部分的研究主要為探討高血糖的狀態下誘導人類血管內皮細胞凋亡之機制,以及PI3K所調控之環氧化蛋白酶表現在其中的角色。本實驗發現暴露於高血糖的環境中皆可測得早期血管內皮細胞凋亡訊號。在細胞凋亡的形態學上以Hoechst染色觀察細胞形態以及以Annexin V/Propidium Iodide偵測早期細胞凋亡訊號。高血糖狀態也會誘導環氧化蛋白酶的表現增加,同時也誘導環氧化蛋白酶的下游產物PGE2生成,進而促使凋亡酶-3 (caspase-3)活性增強而導致細胞走向凋亡。出乎意外地,給予PI3K抑制劑皆可有效地抑制高血糖狀態之環氧化蛋白酶蛋白表現、環氧化蛋白酶的下游產物PGE2生成、凋亡酶-3活性和細胞凋亡。高血糖誘導PI3K活化也依序促使Akt的磷酸化。更進一步地,高血糖引發氧化自由基的生成和NF; Diabetes has become a public health crisis. With the incidence of obesity rising in the world, the number of diabetics will grow considerably. Of greatest concern is the impact this trend will have on damage to the kidneys and cardiovascular disease. The incidence of diabetes is increasing worldwide, with subsequent increase in the incidence of diabetic complication. The hyperglycemic signal exchange occurs ubiquitously and irreversibly in patients with diabetes mellitus, and its consequences are especially relevant to organ dysfunctions. In type-2 diabetes, a greater proportion of patients have overt nephropathy at shortly and vascular complications after diagnosis of diabetes. In this thesis, the studies are divided into two parts for description. Diabetes has been demonstrated to accelerate vascular dysfunction, coronary atherosclerosis, and the prognosis were worse following cardiac events. Therefore, the part I will investigate the regulation of abnormal signalings that promote human umbilical vein endothelial cells apoptosis under high glucose condition and relevant in understanding the intracellular signaling associated with vascular disease and preventing the development of vascular complication in diabetics, principally the evolution of this practice from its beginning until cell death. Diabetes mellitus causes endothelial dysfunction. The precise molecular mechanisms by which hyperglycemia causes apoptosis in endothelial cells are not yet well understood. The aim of this study was to explore the role of cyclooxygenases-2 (COX-2) and the possible involvement of phosphoinositide 3-kinase (PI3K) signaling in high glucose (HG)-induced apoptosis in human umbilical vein endothelial cells (HUVECs). For detection of apoptosis, the morphological Hoechst staining and Annexin V/Propidium Iodide staining were used. Glucose up-regulated COX-2 protein expression, which was associated with the induction of prostaglandin E2 (PGE2), caspase-3 activity and apoptosis. Unexpectedly, we found that PI3K inhibitors could suppress COX-2 expression, PGE2 production, caspase-3 activity, and the subsequent apoptosis under HG condition. Glucose-induced activation of PI3K resulted in the down stream effector Akt phosphorylation. PI3K inhibitors effectively attenuated the intracellular reactive oxygen species (ROS) generation and NF-2005-01-01T00:00:00Z