目的：建立L6565白血病克隆细胞株，为白血病的生物学、病毒学和实验治疗学提供体外研究模型。方法：有限稀释法建成克隆细胞，从生长情况、染色体、形态、超微结构、XOC检测、癌基因表达及分化标志等方面了解其生物学特性。结果：L6565细胞生长旺盛，形态大小不一，染色体条数从38到144条不等，众数为42条，有标记染色体A、B、M。超微结构细胞内核仁清楚，细胞器丰富，核占3/4左右，细胞内有A型和C型病毒颗粒，XOC检测阳性，c2myc、c2fos癌基因高表达，分化标志CD4和CD8阴性。结论：我们成功地建立了含有致白血病病毒的L6565克隆细胞株，该细胞性质属于淋巴干细胞性白血病细胞。

目的：研究胞嘧啶脱氨酶/5-氟胞嘧啶系统对前列腺癌细胞株的作用。方法：细胞培养，细胞转染，药物敏感性实验，观察旁观者效应，动物实验。结果：腺病毒载体可以转染所有已建立的前列腺癌细胞株，但是每种细胞株转染所要求的载体浓度以及暴露时间不同。转染的胞嘧啶脱氨酶基因在细胞内的表达高峰出现在不同的时间，但一直持续到11天以后。腺病毒载体介导的胞嘧啶脱氨酶基因转染可以使前列腺癌细胞株提高对5-氟胞嘧啶的敏感性。在LNCap和RM-1细胞株中，只有5%的细胞转染了胞嘧啶脱氨酶基因就可以引起100%的细胞杀伤效应。在动物实验中，用400MOI转染过的肿瘤细胞建立小鼠皮下肿瘤并同时腹腔注射5-氟胞嘧啶，可以有效的抑制肿瘤生长。结论：腺病毒载体介导的胞嘧啶脱氨酶基因转染可以提高前列腺癌细胞株对5-氟胞嘧啶的敏感性，胞嘧啶脱氨酶/5-氟胞嘧啶的联合应用能显著抑制小鼠肿瘤生长。

目的：研究c-myc基因在L6565小鼠白血病发生中的作用以及抑制其表达所起的治疗作用。方法：用逆转录病毒载体pGNC构建一个含有小鼠反义c-myc基因的逆转录病毒表达系统pGNCas。用磷酸钙沉淀法将pGNCas导入包装细胞PA317，筛选获得病毒生产细胞。用pGNCas病毒感染L6565白血病克隆细胞，筛选后，获得抗G418阳性克隆细胞L6565as。PCR方法检测反义c-myc序列是否稳定整合入L6565as细胞中。最后从细胞生长、形态、周期、软琼脂集落形成能力以及c-myc基因表达方面观察其恶性和表型的改变。结果：L6565as细胞形态变圆，大小较一致。生长曲线显示：L6565as细胞生长被抑制;流式细胞仪检测细胞周期：L6565as阻滞于G0/G1期，S期细胞减少；软琼脂集落形成试验发现L6565as细胞形成克隆数明显减少。免疫组化检测：L6565as细胞c-myc蛋白表达呈弱阳性，而对照组细胞呈强阳性。结论：c-myc基因在L6565小鼠白血病的发生中起着重要的作用；抑制c-myc的表达，可部分逆转L6565白血病的恶性表型和恶性行为。

目的：研究整合素（integrins）αvβ5对造血细胞功能的影响。方法：用逆转录病毒载体系统将αv、β5基因转染K562细胞,观察αv、β5亚基的表达;用去除血清的方法,观察造血细胞凋亡和分化时αvβ5和αvβ3的变化；用抗αvβ5单抗屏蔽造血细胞上αvβ5时对凋亡和增殖的影响。结果：将含有β5基因的逆转录病毒导入K562细胞,β5亚基没有得到表达；诱导造血细胞分化和凋亡时,αvβ5表达升高,而αvβ3表达下降;用单抗封闭细胞表面的αvβ5，可抑制去血清诱导的细胞凋亡。结论：造血细胞αvβ5的过度表达可抑制细胞增殖并与细胞凋亡有关。

Background: Protease activated receptor-2 is cleaved and activated by trypsin or mast cell tryptase and may play an important role in inflammation. However, it is unknown whetehr PAR-2 can mediate tryptase-induced inflammatory reaction. This study was conduct to investigate wheter PAR-2 could be the activated by mast cell tryptase and medicated the tryptase induced interleukin-8 expression in endothelial cells. Methods: Protease activated receptor-2 expression was found in endothelial cell lines ECV304 cell by reverse transcription-polymerase chain reaction（RT-PCR）and Western blotting. Interleukin-8 stimulated by purified human mast cell tryptase was determined by RT-PCR and enzyme linked immunosorbent assay（ELISA）. Data were analysed by the S-N-K one-way ANOVA test. Results: The present study shows that mRNA and protein of protease activated receptor-2 could be expressed in ECV304 cells, and tryptase upregulated the expression levels of both interleukin-8 mRNA and protein. The increased expression of interleukin-8 was inhibited by an antiprotease activated receptor-2 monoclonal antibody, SAM11. An additional band was observed by Western blotting after the incubation of ECV304 cells with tryptase for 2 hours, which suggested that protease activated receptor-2 was activated. Conclusion: Protease activated receptor-2 can mediate the mast cell tryptase stimulated expression of interleukin-8 in ECV304 cell.

Objective To inve stigate the function of the αvβ5 integrin in hematopoietic cells1 Met h o ds Tissue culture, integrin expre ssion vectors, gene transfer, polymera se chain reaction ( PCR), apopto sis analyse s and cytometic analysis were made on hematopoietic cells1 Re s ult s Theβ5 integrin cDNA wa s not expre ssed in hematopoietic cells following expo sure to the β5 integrin retrovirus vector p Gβ5CHT1 Unbalanced expre ssion of the αvβ3 and αvβ5 integrins occurred during apopto sis induced by serum depletion and upon differentiation1 The treatment of hematopoietic cells with anti2αvβ5 monoclonal antibody inhibited apopto sis induced by serum depletion1 Inducible expre ssion of the β5 integrin cDNA in the hematopoietic cell line K562 caused cellular proliferation inhibition1 Co nclusion The αvβ5 integrin cDNA in hematopoietic cells can inhibit the proliferation of the hematopoietic cell, cause the differentiation of the hematopoietic cells and induce the apopto sis of the hematopoietic cells.

Tryptase is involved in proteinase-activated receptor-2 (PAR-2) mediated up-regulation of IL-8 expression. The present report showed the effects of tryptase on gene expression and activation, including up-regulation IL-8 expression. The expression of mRNA for NF-κB first increased at 1h after tryptase-treatment (1ng/ml) and reached the plateau after 4 h. The NF-κB mRNA increased by 3-fold (n=3, P<0.05), AP-1 by 2-fold (n=3, P<0.05), and PKB by 10-fold (n=3, P<0.05). However, tryptase-treatment did not affect the expression of JNK and p38 MAPK when compared with control cells at mRNA level. Furthermore, in addition to increasing phosphorylation of p38 MAPK, tryptase-treatment also increased phosphorylation of PKB by 2-fold at 15 min following the treatment. The up-regulation and phosphorylation of PKB by tryptase could be abolished by either phosphoinositol-3-kinase (PI3K) inhibitor (LY294002) at 10 μM or antisense PKB cDNA transfection. The up-regulation of NF-κB expression could be inhibited by LY294002 and antisense PKB cDNA. These results indicate that tryptase can activate PI3K-PKB pathway and enhance IL-8 expression.

The steroidogenic acute regulatory (StAR) protein promotes intramitochondrial delivery of cholesterol to the cholesterol side-chain cleavage system. In this experiment, we first demonstrated that StAR expressed in endothelial cells as well. Immunochemistry showed positive staining of StAR in endothelial cells. To investigate whether steroids and oxysterols regulate StAR expression in endothelial cells, mouse brain microvascular endothelial cell line (bEnd.3) was treated with various steroids and oxysterols, including free cholesterol (CHO), low density lipoprotein (LDL), and 25-hydroxycholesterol (25-OH). All these three compounds increased StAR mRNA and protein expression in a time- and dose-dependent manner. When treated with CHO and LDL, the StAR mRNA change was prior to the protein change, suggesting that transcription may be one of the mechanisms of CHO and LDL regulation. In contrast to CHO and LDL, 25-OH increased StAR protein levels independently of mRNA amount. It suggested that 25-OH might regulate StAR activity at post-transcriptional level.

目的：建立C57BL6近交系小鼠前列腺癌原位移植瘤模型并探讨其应用价值。方法：用微量注射器在C57BL6近交系小鼠的前列腺左右背外侧叶包膜下接种小鼠前列腺癌RM21细胞2×106个建立原位移植瘤模型，以皮下移植瘤模型作为对照比较两者的肿瘤转移发生情况及生存期。结果：原位模型小鼠全部发生肿瘤转移，其中盆腔淋巴结和肺的转移发生率分别为（10/10）、（8/10），对照组仅有肿瘤的局部生长和浸润，无转移发生；原位模型的小鼠平均生存期为（11.0±2.4）d较对照组（23.0±4.7）d明显缩短，差异有非常显著性（P<0.01）。结论：小鼠的前列腺癌原位移植瘤模型较好地模拟了人癌体内的自然生长状况，是观察转移性前列腺癌较为适宜的模型。

目的：探讨原位CD基因转导配合全身应用5-FC对于前列腺癌原位移植瘤模型肿瘤和转移的抑制作用。方法：通过向C57BL/6小鼠的前列腺包膜下原位接种前列腺癌RM21细胞建立前列腺癌原位移植瘤模型。两天后进行原位CD基因注射，腹腔注射300mg/kg/天的5-FC溶液共14天。结果：与应用Adβ-gal/52FC，52FC和PBS的三组比较，接受原位CD基因转导配合全身应用52FC治疗的荷瘤鼠存活时间明显延长，局部肿瘤的生长受到明显抑制。更重要的是，病理学观察发现原位模型中的PBS、52FC和Adβ-gal三组小鼠全部发生肿瘤转移，包括髂淋巴结的转移（10/10，10/10，10/10）和肺转移（8/10，7/10，7/10），而AdCD/52FC治疗组中肿瘤转移的发生机率仅为2/10，且均为髂淋巴结的转移，而各主要脏器及腹主动脉旁淋巴结均无转移发生，结果提示这一治疗方法具有很强的抗肿瘤转移作用。结论：原位CD基因转导配合全身应用5-FC对于前列腺癌原位移植瘤模型的肿瘤和转移具有抑制作用。未来对于各种复发性或转移性的前列腺癌通过局部注射法作基因转导可能是一种新型的治疗方法。