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標題: To Investigate the Roles of Four VEGF Isoforms in Non-small Cell Lung Cancer Angiogenesis
作者: 施富元
Shih, Fu-Yuan
關鍵字: 血管新生;Angiogenesis;血管內皮新生因子;非小細胞肺癌;女性肺腺癌細胞株 (CL1-0);人類臍帶血管內皮細胞;Vascular endothelial growth factor;VEGF;Non-small cell lung cancer;Femal lung adenocarcinoma cell line;CL1-0;Human umbilical vein endothelial cells;HUVECs
出版社: 分子生物學研究所
血管新生是指由已經存在的血管中形成新的血管,並且在許多生理和病理上的事件中扮演著決定性的機制。血管內皮新生因子 (VEGF),也被稱為血管滲透因子 (VPF),是血管新生、管脈新生和血管滲透性最首要的調控者。現在已經被證實單一VEGF基因藉由選擇性接合作用產生至少六種亞型的單體,分別由121、165、189、206、145和183個胺基酸所組成。為了探討不同的VEGF亞型在非小細胞肺癌腫瘤發生與血管新生的角色,四種主要的亞型基因 (VEGF121、VEGF165、VEGF189或VEGF206) 被大量表現在女性肺腺癌細胞株 (CL1-0)。我們把受到Tet-Off系統調控的VEGF cDNA質體轉染到CL1-0,而該系統是藉由pTet-Off所產生的tTA轉錄活化因子,與TRE操作子結合後啟動VEGF基因的表現。轉染成功的細胞株先以即時定量RT-PCR偵測VEGF mRNA的表現量,再藉由ELISA定量釋放到細胞培養液的VEGF蛋白質。VEGF可能的自體分泌作用被排除,是依據CL1-0在活體外的增生能力並沒有因為大量表現四種VEGF亞型而產生顯著的差異。以大量表現VEGF165的CL1-0細胞培養液刺激人類臍帶血管內皮細胞 (HUVECs),其MAPK磷酸化疑似比其他VEGF亞型更為顯著。為了探討VEGF在活體內對於腫瘤生長的影響,將大量表現VEGF的CL1-0注射到免疫缺陷小鼠的皮下,結果顯示大量表現VEGF165的腫瘤會形成密度較高的血管網路。因此,我們推測VEGF165促進腫瘤血管新生的能力比其他VEGF亞型更為顯著。

Angiogenesis is a term that describes the formation of new capillaries from a pre-existing vasculature and is a crucial mechanism required for a number of physiological and pathological events. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a prime regulator of angiogenesis, vasculogenesis, and vascular permeability. It is now well established that alternative exon splicing of a single VEGF gene results in at least six isoforms, having respectively 121, 165, 189, 206, 145 and 183 amino acids. To investigate the roles of different VEGF isoforms in non-small cell lung cancer tumorigenesis and angiogenesis, the four major isoforms (VEGF121, VEGF165, VEGF189 or VEGF206) were overexpressed in a female lung adenocarcinoma cell line (CL1-0). CL1-0 cells were stably transfected with constructs encoding VEGF cDNA under the control of the Tet-Off system. In the system, the tetracycline-controlled transactivator (tTA), is encoded by the pTet-Off regulator plasmid, binds the tetracycline-response element (TRE) and turn on the VEGF gene expression. Successfully transfected clones were identified using real-time quantitative RT-PCR to detect VEGF mRNA. Secretion of VEGF protein into the conditioned medium was assayed using a VEGF-specific ELISA. The possible autocrine effects of VEGF were ruled out by observations that overexpression of four VEGF isoforms did not have a significant effect on the in vitro growth rates of CL1-0 cells in monolayer culture. The condition medium derived from VEGF165-overexpressing CL1-0 cells could effectively stimulate mitogen activated phosphorylated kinase (MAPK) phosphorylation in human umbilical vein endothelial cells (HUVECs) than other VEGF isoforms. To examine the effects of VEGF on tumor growth in vivo, VEGF-overexpressing CL1-0 cells were injected subdermally into SCID mice. The results indicated tumor overexpressing VEGF165 generated dense vessel networks. Therefore, we suggest that VEGF165 is a more potent tumor angiogenesis activator than other VEGF isoforms.
Appears in Collections:分子生物學研究所

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