40,896 research outputs found
The feasibility of establishing immortal caprine mammary epithelial cell line by transfection of telomerase
No full text自中大型產乳經濟動物乳腺產製醫療用重組生物製劑，是科學農業
未來的重點方向之ㄧ。但是，產製基因轉殖動物現今仍普遍存在低效
率、成本高及耗時長等問題。造成效率低的原因，主要有質體架構不當、
外源基因來自不同物種或組織，導致質體所表現的蛋白不具生物活性。
因此，若能先於體外培養之乳腺上皮細胞內，測試質體架構與表現的能
力，則可解決產製基因轉殖動物所遭遇的困難。惟體外初級培養之乳腺
上皮細胞有無法長期繼代培養之問題。利用大量表現致癌基因或γ 放射
線照射等方式，能夠造成細胞的不朽化，但是轉型後的細胞伴隨不正常
分化，甚至是癌化。穩定表現外源性端粒酶反轉錄酶，亦能誘導初級培
養之細胞成為不朽化，不論外形或分化功能上，皆與正常細胞較為相似。
本研究主要目的乃建立一株穩定表現外源性端粒酶之山羊乳腺上
皮細胞株 (CMEC)，做為產製基因轉殖動物質體架構之測試平台。初級
乳腺上皮細胞取自性成熟之雌性撒能山羊，利用cytokeratin 18 抗體進行
免疫螢光染色分析，證實CMEC 表現大量cytokeratin 18 蛋白質；同時，
當CMEC 長滿後，於接觸性誘導下，形成與活體乳腺發育中，乳腺泡內
腔類似的構造。進一步將CMEC 培養於MATRIGEL 上，更會被誘導形
成立體中空乳泡構造。經由上述定性分析，證實CMEC 保有乳腺上皮細
胞最基本而重要的功能。利用表現外源性人類端粒酶反轉錄酶於CMEC
中，經抗生素篩選後，已得到一株能夠穩定表現外源性端粒酶之山羊乳
腺上皮細胞株。
此細胞株之建立，不僅能做為產製基因轉殖動物質體架構的測試平
台，更能應用於研究乳腺發育調控、細胞衰老及癌症生成機制等領域。
於癌症應用上，已知Caveolin-1 基因扮演癌症生成的抑制角色 (如：乳
癌、結腸癌)；因此，為研究Caveolin-1 與癌症生成關係，本研究製備
Caveolin-1 胜肽多株抗體。此抗體以西方吸漬及免疫螢光染色法，證實
能夠辨識來自小鼠、山羊及人類等不同物種之內源性與外源性
Caveolin-1 蛋白質。此抗體進一步以免疫組織化學染色法，進行人類乳
癌、肺癌及直腸癌切片之測試比較，結果顯示，在乳癌及直腸癌切片試
驗中，與相同部位組織之正常切片做比較，此抗體可明顯區辨癌化與正
常切片中，Caveolin-1 表現的差異。證實此Caveolin-1 多株抗體，可應
用於人類乳癌及結腸癌之臨床診斷試驗。
結合穩定表現外源性端粒酶之山羊乳腺上皮細胞株與Caveolin-1 胜
肽多株抗體，將成為我們未來研究乳腺發育調控、基因轉殖動物質體架
構測試及細胞老化與癌化的重要利器。Establishment of a stable telomerase-expressed caprine mammary epithelial cell line
Abstract
Production of recombinant biopharmaceuticals from the mammary gland of domestic animals through transgenic nuclear transfer technology is a dominant system in scientific agriculture. Low efficiency, high costs and time-consuming, however, are common problems in producing transgenic domestic animals. Furthermore, improper plasmid constructs and gene source from different species or tissues are more serious problems making protein lose its biological activity. In order to solve the above problems, the culture of primary mammary epithelial cell for pioneer testing the expressed recombinant protein biological activity and characteristics is recommended, but the finite life span of cultured primary mammary epithelial cell is more concerned. SV40 large T-antigen transformed cells are also immortal but they accompany with carcinogenesis and lose of normal cellular functions. Stable expression of telomerase reverse transcriptase (TERT) in primary culture cells can induce cells immortalized and maintain cells with both normal cellular morphology and functions.
The aim in this study is to establish a telomerase-immortalized caprine mammary epithelial cell line (CMEC) as a pioneer testing platform to examine the biological activity of interesting gene expressed in transgenic animal. In the beginning, the characteristics of the cultured CMEC were examined and verified it specifically expressed cytokeratin 18 and formed alveolar structure on MATRIGEL matrix, showing the CMEC still possess the normal function of mammary epithelial cell. Further, we transfected CMEC with hTERT and selected with proper antibiotics for establishing a stable hTERT-expressed caprine mammary epithelial cell line.
As the immortal CMEC cell line is established, it can not only be used for mammary gland development study but also for cellular senescence and breast cancer research. Using mRNA subtractive hybridization and differential display techniques, caveolin-1 was seem to be lost or down-regulated in human mammary adenocarcinoma-derived cells comparing to normal mammary epithelial cell. For further researches, a rabbit polyclonal peptide antibody specially recognizing different species’ caveolin-1 protein was developed. This antibody could against 22-24 kDa caveolin-1 protein extracted from human epithelial cell line A431, CMEC, mice fibroblast cell line NIH3T3 and recombinant caveolin-1 transiently-expressed rat pituitary epithelial cell line GH3 by western blotting and immunostain techniques. This result had proved this antibody can be used for examining caveolin-1 protein expressed in mouse, rat, goat and human species. To explore whether this antibody can be applied for breast cancer diagnosis, we performed immunohistochemistry on breast tumor biopsy and showed significant difference in caveolin-1 expression between normal with tumor was observed.
To the conclusion, once the immortal CMEC being established, we can study the development of mammary gland and the production of recombinant protein biopharmaceuticals. Further, combining with caveolin-1 antibody, the study of cellular senescence and signal transduction through caveolin-1 can also be achieved.壹、摘要 ………………………………………………………………………………1
貳、前言 ………………………………………………………………………………3
參、文獻檢討 …………………………………………………………………………6
一、活體中乳腺發育與構造 ……………………………………………………6
(一) 乳腺構造 ………………………………………………………………6
(二) 影響活體乳腺發育因素 ………………………………………………7
1. 胜肽與固醇類內泌素 ………………………………………………7
2. 胞外基質 ……………………………………………………………8
二、體外培養下乳腺上皮細胞之發育 …………………………………………8
(一) 胞外基質對乳腺泡之發育調控 ………………………………………9
(二) 胜肽與固醇類內泌素對培養中乳腺細胞發育及功能之影響 ………9
1. 型態上的分化 ………………………………………………………10
2. 乳蛋白分泌調控 ……………………………………………………10
3. β-酪蛋白啟動子序列調控β-酪蛋白表現 ………………………11
三、乳腺上皮細胞的老化與不朽 ………………………………………………11
(一) 細胞衰老 ………………………………………………………………11
(二) 哺乳類動物細胞體外培養的衰老現象 ………………………………12
(三) 衰老細胞的生物性標記 ………………………………………………13
(四) 染色體端粒、端粒酶與細胞衰老之關係 ……………………………13
(五) 誘導乳腺上皮細胞不朽化的方法 ……………………………………14
(六) 經SV40 large T-antigen 轉型與端粒酶不朽化人類乳腺上皮細胞株之
比較 ……………………………………………………………………14
肆、材料與方法 ………………………………………………………………………15
一、山羊乳腺之取樣 ……………………………………………………………15
二、山羊乳腺上皮細胞之分離與初級培養 ……………………………………15
三、乳腺上皮細胞豐富化 ………………………………………………………16
四、細胞繼代培養及生長曲線之計算 …………………………………………17
五、細胞冷凍儲存 ………………………………………………………………18
六、細胞解凍活化 ………………………………………………………………18
七、萃取及純化大量質體DNA ………………………………………………18
八、細胞轉型感染 ………………………………………………………………19
九、細胞免疫染色 ………………………………………………………………20
十、抗生素篩選建立穩定表現外源性蛋白質之細胞株 ………………………21
十一、 不同胞外基質上之細胞培養 …………………………………………21
十二、 CMEC細胞total RNA之萃取 ………………………………………22
十三、 反轉錄 …………………………………………………………………22
十四、 聚合酶鏈鎖反應 ………………………………………………………23
十五、 Senescent associated β-Gal activity (SA β-Gal) stain …………………24
十六、 誘導CMEC 酪蛋白之表現 …………………………………………25
伍、結果 ………………………………………………………………………………26
一、山羊乳腺上皮細胞之分離 …………………………………………………26
二、乳腺上皮細胞之enrichment …………………………………………………26
三、山羊乳腺上皮細胞表現cytokeratin 18 蛋白質之檢測 …………………26
四、接觸性CMEC 培養促進類內腔構造形成 ……………………………………27
五、山羊乳腺上皮細胞體外培養之生長曲線 …………………………………27
六、山羊乳腺上皮細胞於不同生長代數之外觀變化 …………………………28
七、不同代數山羊乳腺上皮細胞SA β-Gal 活性測試 …………………………28
八、比較selection前後不同代數之山羊乳腺上皮細胞p16 gene 表現情
形………………………………………………………………………………29
九、乳泡狀構造之形成 …………………………………………………………29
十、hTERT 質體表現外源性端粒酶於山羊乳腺上皮細胞內 …………………30
十一、 抗生素篩選轉形感染後之山羊乳腺上皮細 …………………………30
十二、 山羊乳腺上皮細胞穩定表現外源性端粒酶 …………………………31
陸、討論 ………………………………………………………………………………32
一、 山羊乳腺上皮細胞之外觀與體外培養相關特性 ………………………32
二、 山羊乳腺上皮細胞體外培養之衰老現象 ………………………………33
三、 體外培養山羊乳腺上皮細胞p16 基因的表現變化 ……………………35
四、 表現外源性端粒酶於山羊乳腺上皮細胞 ………………………………35
五、 建立穩定表現外源性端粒酶之山羊乳腺上皮細胞株 …………………37
柒、結論 ………………………………………………………………………………38
捌、參考文獻 …………………………………………………………………………39
玖、英文摘要 …………………………………………………………………………44
壹拾、 附圖 …………………………………………………………………………45
圖一、山羊乳腺上皮細胞之分離 ……………………………………………………45
圖二、山羊乳腺上皮細胞之enrichment ……………………………………………46
圖三、Enrichment 前後，初級培養之山羊乳腺細胞表現cytokeratin 18 蛋白
質 ………………………………………………………………………………47
圖四、山羊乳腺上皮細胞形成類內腔構造 …………………………………………48
圖五、山羊乳腺上皮細胞體外培養之生長曲線 ……………………………………49
圖六、山羊乳腺上皮細胞於不同生長代數之外觀 …………………………………50
圖七、不同代數山羊乳腺上皮細胞SA β-Gal 活性測試 ……………………………51
圖八、比較selection 前後不同代數之山羊乳腺上皮細胞 p16 gene 表現情形 ……52
圖九、乳泡狀構造之形成 ……………………………………………………………53
圖十、pLPC-hTERT 表現外源性端粒酶於山羊乳腺上皮細胞 ……………………54
圖十一、pIRES2-hTERT 表現外緣性端粒酶於山羊乳腺上皮細胞 …………………55
圖十二、抗生素篩選轉形感染後之山羊乳腺上皮細胞 ……………………………56
圖十三、穩定表現外源性端粒酶之山羊乳腺上皮細胞株 …………………………57
附錄一、pLPC-hTERT mammary cell expressed plasmid ……………………………58
附錄二、pIRES2-hTERT mammary cell expressed plasmid …………………………59
附錄三、Development and Characterization of an Anti-caveolin-1 Peptide antiserum for
Diagnosing Human Breast and Colon Cancer ……………………………60
附錄四、Bromocriptine Sensitizes Rat Pituitary GH3 Cells to Caveolin-1 Induced
apoptosis ……………………………………………………………………8
The political role of the people's liberation army 1949-1973
Full text linkThis thesis is to study the political role of the People's Liberation Army from the approach of structure and function. The framework of the thesis consists of three major parts, first, the influence of Chinese traditional political culture on, and the formation of, the political role of the PL A; second, the influence of domestic political struggles and external military conflicts on the development of the political role of the PLA; and the third, the analysis of the transition of the PLA's political role from the structure and personnel arrangements of the CCPCC Within the above-mentioned three scopes, this thesis make a thorough discussion on the following: (1) The relationship between the structure of the PRC and the formation of the PLA's political role; (2) How has ideology influenced the army's political role; (3) What is Mao's viewpoint and his influence on the development of the army's political role; (4) What is the link between the army and the party, and how has this developed; (6) What accounts for the expansion of the PLA's political functions; (7) What is the influence of political factional struggles on the PLA's political role; (8) Is it political institution or military institution that controls the recruitment of the military elite; (9) What are the disparities between the military elite in handling international conflicts and what are their political considerations; (10) What is the Party's position in the army; (11) How have the Party’s important meetings and personnel arrangements influenced the rise and fall of the PLA's political role
Beginning Android Application Development
No full textCreate must-have applications for the latest Android OSThe Android OS is a popular and flexible platform for many of today's most in-demand mobile devices. This full-color guide offers you a hands-on introduction to creating Android applications for the latest mobile devices. Veteran author Wei Meng Lee accompanies each lesson with real-world examples to drive home the content he covers. Beginning with an overview of core Android features and tools, he moves at a steady pace while teaching everything you need to know to successfully develop your own Android applications.Explains what an activ
Wai ke shi fa
No full text程國彭撰.綫裝.框16.3x10.5公分, 11行22字, 小字雙行同. 白口, 左右雙邊, 單黑魚尾. 版心上鐫題名, 中鐫卷次, 下鐫葉次.書名頁刻"醫學心悟, 歙程山齡原本, 附外科十法, 經綸堂梓".目錄首葉版心下刻"經綸堂". 卷六為"外科十法".前有乾隆五十六年[1791]王文治序, 言汪氏重刻此書.《中國中醫古籍總目》05661著錄乾隆五十六年[1791]汪氏書粟軒刻本.鈐"莊兆祥印"朱, 白文各一方.Xian zhuang.Kuang 16.3 x 10.5 gong fen, 11 hang 22 zi, xiao zi shuang hang tong. Bai kou, zuo you shuang bian, dan hei yu wei. Ban xin shang juan ti ming, zhong juan juan ci, xia juan ye ci.Detailed notes in vernacular field only.Mu lu shou ye ban xin xia ke "Jing lun tang". Juan liu wei "Wai ke shi fa"Detailed notes in vernacular field only.Detailed notes in vernacular field only.Cheng Guopeng zhuan.Qian "Zhuang Zhaoxiang yin" zhu, bai wen ge yi fang
sj-pdf-1-ajs-10.1177_03635465221090605 – Supplemental material for H-loop Knotless Double-Row Repair Versus Knotted Suture Bridge for Rotator Cuff Tears: A Biomechanical and Histological Study in an Animal Model
No full textSupplemental material, sj-pdf-1-ajs-10.1177_03635465221090605 for H-loop Knotless Double-Row Repair Versus Knotted Suture Bridge for Rotator Cuff Tears: A Biomechanical and Histological Study in an Animal Model by Jiang Guo, Yi Long, Min Zhou, Zhang-Hai He, Wei-Ke Zeng, Meng-Lei Yu, Alike Yamuhanmode, Yi-Yong Tang, Fang-Qi Li, Ke Meng, Jing-Yi Hou and Rui Yang in The American Journal of Sports Medicine</p
Role of functional domains of disabled-2 gene in hTERT-immortalized caprine mammary epithelial cells
No full text初級培養之山羊乳腺上皮細胞 (CMECs)具有近似活體乳腺上皮之功能性，受到泌乳激素與胞外基質的刺激，可歷經形態及功能之分化，適合應用於乳腺發育、泌乳機制調節、乳癌發生等領域之研究，亦可做為測試重組基因表現效率之平台，提升產製轉基因動物之成功率。惟初級培養CMECs之應用受限於有限的繼代次數，主要肇因為染色體末端之端粒，逐漸耗損於每一回細胞分裂，終至喪失保護染色體的功用而引發細胞生長休止。據此，可長期繼代培養且保有乳腺上皮特性之不朽化山羊乳腺上皮細胞株 (CMCs)，乃本研究之主要目標。
藉由表現外源性人類端粒酶基因 (hTERT)，業已成功建立五株CMCs，以特定細胞系骨架蛋白 (cell lineage specific cytoskeleton)抗體，經西方印漬術 (western blotting)證實分別為二株肌上皮細胞 (myoepithelial cell lineage)，與三株腔上皮細胞系 (luminal epithelial cell lineage)。Telomeric repeat amplification protocol (TRAP)顯示CMCs與初級培養CMECs相較，具有較高端粒酶活性。核型分析試驗發現二株肌上皮細胞與一株腔上皮細胞，保有完整染色體數目 (2n = 60)與結構，另二株腔上皮細胞之染色體數明顯缺失。染色體異常多與細胞癌化相關，檢測抑癌因子p53蛋白質於DNA受損狀態下的表現，顯示二株染色體缺失之腔上皮細胞，缺乏p53蛋白質表現，同時具有癌化細胞anchorage-independent之生長特性，說明其潛在性之癌化傾向。關於CMCs功能性分化之能力，三株腔上皮細胞皆可在Matrigel胞外基質形成類乳泡構造，受到泌乳激素誘導，其中一株腔上皮細胞更可測得乳蛋白基因 (αs1- and β-casein genes)之表現。此結果證實，表現外源性人類端粒酶基因可有效不朽化初級培養之山羊乳腺上皮細胞，惟具備正常染色體數目與結構，對於保有乳腺上皮細胞之重要分化特性甚為重要。
為探討乳腺上皮細胞分化過程之分子調節機制，研究中選定細胞溶質之承接蛋白質 (cytosolic adaptor protein) Dab2，進行其功能性探討。比較不同發展階段之山羊乳腺組織，顯示Dab2蛋白質含量在懷孕末期與離乳期較高於泌乳階段，且表現位置主要位在乳泡構造。將初級培養CMECs誘導生成類乳泡構造，模擬乳腺上皮細胞之分化，顯示Dab2蛋白質表現量隨類乳泡構造之形成而有增加的趨勢。據此，推測Dab2之功能與乳泡構造的形成有關。結構上，Dab2蛋白質具有二個功能領域 (functional domains)，分別名為phosphotyrosine-binding domain (PTB)及proline-rich domain (PRD)，主宰Dab2的功能。因此，進一步將PTB domain與PRD domain架接於慢病毒表現載體 (lentivector)，並產製具感染能力之慢病毒粒子 (lentivirus particles)。經由慢病毒感染，選殖具穩定PTB domain或PRD domain表現之山羊乳腺上皮細胞株 (CMC-PTB or CMC-PRD)。已知Dab2會抑制Grb2/Sos/MAPK訊號途徑，血清饑餓試驗證實在CMC-PTB與CMC-PRD細胞內，Erk分子的磷酸化明顯受到干擾。因乳泡形成過程涉及細胞移動，以創傷癒合試驗得知CMC-PTB與CMC-PRD細胞之移動能力顯著高於未表現外源基因之控制組別。免疫細胞化學法顯示CMC-PTB與CMC-PRD細胞缺乏磷酸化focal adhesion kinase (FAK)分布於focal adhesion構造，推測此結果與其較高之細胞移動能力有關。比較PTB domain與PRD domain對山羊乳腺上皮細胞形成類乳泡構造的影響，不同於CMC-PTB細胞呈現具完整空腔之類乳泡構造，CMC-PRD細胞所形成之類乳泡構造周圍具有明顯凸起結構。此結果表明PTB domain與PRD domain對於乳腺上皮細胞之形態分化具有不同作用，異常表現PRD domain會影響類乳泡構造的完整性。
綜合上述研究結果，本論文主要利用人類端粒酶基因建構穩定且可完整定性之不朽化山羊乳腺上皮細胞株，可廣泛應用於乳腺發育、乳癌生成與產製重組蛋白質之體外測試平台等研究領域。另外，以不朽化山羊乳腺上皮細胞作為研究模式，發現Dab2蛋白質之PTB domain與PRD domain，對於乳腺上皮細胞形態分化成完整之乳泡構造，扮演重要的調節作用。Finite lifespan limited the application of primary cultured mammary epithelial cells (MECs), whereas immortal cell lines retaining major characteristics of primary cultured MECs were more desirable. For the purpose of obtaining immortal caprine mammary epithelial cells (CMCs), human telomerase reverse transcriptase (hTERT) gene was introduced into primary cultured caprine-MECs (CMECs). Both luminal and myoepithelial cells were successfully immortalized and expressed their cell-lineage specific cytoskeleton markers. Activated telomerase in obtained immortal CMCs was confirmed by telomeric repeat amplification protocol (TRAP). The integrity of chromosomal structure and Capra hircus origin of CMCs was examined by karyotypic analysis. For morphologic differentiation, CMCs of luminal group, but not myoepithelial group, formed well-organized alveolar structures (acinus) when grown in Matrigel extracellular matrix (ECM). Furthermore, one luminal CMC clone expressed αs1- or β-casein gene in response to lactohormone stimulation. These results demonstrated that hTERT-immortalized CMCs do reserve important functional characteristics of primary cultured CMECs.
As well-organized acinus is essential to full differentiation of MECs, the function of a cytosolic adaptor protein disabled-2 (Dab2), which was measured with a moderate level in pregnancy, followed by evident decrease during lactation and peaked in wean of normal caprine mammary epithelium, was explored. Immunohistochemistry (IHC) further showed Dab2 majorly distributed in alveolar structures. Thus, Dab2 was hypothesized to engage in process of alveologenesis, which was supported by evidence of elevated level of Dab2 during in vitro acinus formation of primary cultured CMECs. Dab2 molecule hold two functional domains, the amino-terminus phosphotyrosine-binding domain (PTB) and the carboxyl-terminus proline-rich domain (PRD), CMC clones stably expressed each domain were established for exploring their contributions on alveologenesis. The effect of PTB or PRD domain in CMC clones was confirmed by significant reduction of Erk phosphorylation after growth factor stimulation. As alveologenesis was associated with cell migration, wound healing assay showed enhanced cell motility of both PTB and PRD CMC clones. To examine the effect of PTB or PRD domain on alveolar structure formation, in vitro acinus culture exhibited CMC clone bearing PRD domain, but not PTB domain, represented obvious protrusions of the acinus. These data indicated both PTB and PRD domains of Dab2 were involved in regulating alveologenesis of MECs, but different mechanisms might be employed by different domains.
In this study, the establishment and elaborate characterization of hTERT-immortalized CMC cell lines was demonstrated. These immortal CMC cell lines were valuable models for exploring the molecular mechanisms regulating the development of caprine mammary gland and provided a platform to examine the expression of recombined plasmids used to generate transgenic livestock. In addition, the findings of PTB and PRD domains in mediating alveologenesis of immortal CMC cells, which might claim the importance of Dab2 during the development of caprine mammary gland
Fig. 1 in Iridoid glycosides and lignans from the fruits of Gardenia jasminoides Eills
No full textFig. 1. Compounds isolated from the fruits of G. jasminoides.Published as part of Cao, Yan-Gang, Ren, Ying-Jie, Liu, Yan-Ling, Wang, Meng-Na, He, Chen, Chen, Xu, Fan, Xi-Ling, Zhang, Yan-Li, Hao, Zhi-You, Li, Hong-Wei, Zheng, Xiao-Ke & Feng, Wei-Sheng, 2021, Iridoid glycosides and lignans from the fruits of Gardenia jasminoides Eills, pp. 1-8 in Phytochemistry (112893) 190 on page 3, DOI: 10.1016/j.phytochem.2021.112893, http://zenodo.org/record/825772
Ng Wei Sheng dinobat Anugerah Kecemerlangan Heitech
Full text linkPAYA BESAR, 6 November 2022 – “Sentiasa berpijak di bumi nyata dan hidup di masa kini. Jangan membiarkan diri dikurung oleh apa yang telah berlaku pada masa lalu dan terjebak oleh mimpi masa depan”, begitulah kata-kata yang sentiasa dipegang oleh Ng Wei Sheng, 24 graduan Universiti Malaysia Pahang (UMP) yang meraih Anugerah Kecemerlangan Heitech pada Majlis Konvokesyen UMP Ke-17 baru-baru ini
LIPIcs, Volume 186, ICDT 2021, Complete Volume
No full textLIPIcs, Volume 186, ICDT 2021, Complete Volum
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