INHIBITION OF INVASIVENESS AND METASTATIC POTENTIAL OF HUMAN PE-B LYMPHOME CELLS BY INHIBITING UROKINASE EXPRESSIN

通过抑制尿激酶表达蛋白抑制人PE-B淋巴瘤细胞的侵袭性和转移能力

• 批准号: 08671222
• 负责人: NIIYA Kenji
• 金额: $ 1.47万
• 依托单位: TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08671222/
• 关键词: UROKINASE; ANTISENSE; INVASIVENESS; LYMPHOMA CELLS; ELECTROPOLATION; SUBCLONING; uPA; antisense; metastasis; lymphoma cells; invasion; cell culture; matrix; アンチセンス遺伝子; 遺伝子導入; 浸潤転移能; マトリゲル; cAMP

Although I have moved to Okayama University Medical School from Toyama Medical and Pharmaceutical University January 1998, the above research is continuously but slowly progressing. A new antisense plasmid covering 540 bp from the translation starting site of uPA gene was constructed by inserting an antisense-oriented cDNA fragment of urokinase gene into a PCI vector, The plasmid was transferred with a plasmid containing a Hygromycin-resistant gene into RC-K8 human pre B lymphoma cells by the electroporation. The cells were cultured in the RPMI- 1640 culture medium containing Hygromycin for 2 months and a number of Hygromycin-resistant clones were established. Among them, a couple of clones exerted a reduced uPA production, approximately a half of that of parent RC-K8 cells. The invasiveness examined using a Matrigel chamber was not significantly different from that of parent cells. These results were unfortunately very similar to that seen in the previous experiments performed in 1996 and 1997. Obviously I could not have the conclusion at present, because I do not suceeed yet in construction of the RC-K8 cells whose uPA production was completely bloc ed. Now I am trying to get the urokinase-nonproducing RC-K8 cells using two different antisense plasmid-vectors and proceeding the research with much more careful election and subcloning.

虽然我于1998年1月从富山医科大学转到冈山大学医学部，但上述研究仍在持续但缓慢地进行。将尿激酶基因的反义cDNA片段插入PCI载体，构建了一个新的反义质粒，其长度为540 bp，与潮霉素抗性基因质粒一起电转化人前B淋巴瘤细胞RC-K 8。将细胞在含有潮霉素的RPMI- 1640培养基中培养2个月，并建立了许多潮霉素抗性克隆。其中，一对夫妇的克隆表现出减少uPA的生产，约一半的亲本RC-K8细胞。使用Matrigel小室检查的侵袭性与亲本细胞的侵袭性没有显著差异。不幸的是，这些结果与1996年和1997年进行的先前实验中看到的结果非常相似。显然，我目前还不能得出结论，因为我还没有成功地构建出完全阻断尿激酶产生的RC-K8细胞。现在，我试图用两种不同的反义质粒载体获得不产生尿激酶的RC-K8细胞，并进行更仔细的筛选和亚克隆研究。

项目成果

K.Niiya, et al: "Transcriptional regulation of urokinase-type plasminogen acti-vator receptor by cyclic AMP in PL-21 human myeloid leukemia..." Thrombosis & Haemostasis. 79(3). 574-578 (1998)

K.Niiya 等人：“PL-21 人骨髓性白血病中环 AMP 对尿激酶型纤溶酶原激活剂受体的转录调节......”

N Nomura,K Niiya,et al: "Inhibitory effect of a synthetic prostacyclin analoque, Beraprost, on urokinase-type plasminogen activator expression" Thromb Haemost. 75(6). 928-932 (1996)

N Nomura、K Niiya 等人：“合成前列环素类似物贝前列素对尿激酶型纤溶酶原激活剂表达的抑制作用” Thromb Haemost。

新谷憲治: "RC-K8ヒト悪性リンパ腫細胞株における炎症性サイトカインによるウロキナーゼ(u-PA)の遺伝子発現制御" 臨床病理 2月臨時増刊. 特集(104). 150-158 (1997)

Kenji Shintani：“RC-K8 人恶性淋巴瘤细胞系中炎症细胞因子对尿激酶 (u-PA) 基因表达的调节”临床病理学二月特刊 (104)。