两种黑色素瘤B16细胞肺转移小鼠模型的构建和比较

目的采用尾静脉接种法建立C57BL/6小鼠和KM小鼠两种黑色素瘤B16细胞肺转移动物模型,观察小鼠肺部成瘤过程,并对两种模型进行比较。制作肿瘤切片,通过HE染色分析两种肿瘤模型的组织病理学特征。方法培养B16黑色素瘤细胞至对数生长期时收集细胞,用生理盐水调整浓度为5×106/ml备用。将备好的B16细胞通过尾静脉接种小鼠,每只注射0.2 ml。接种后,两种小鼠每隔一定时间随机各取一只小鼠处死,解剖取肺,观察成瘤情况,计数肺表面的瘤结节数,测量瘤结节大小。以出现瘤结节开始计时,记录小鼠荷瘤时间。形成稳定的肿瘤模型后,制作肿瘤组织切片,HE染色进行病理学观察分析。结果 B16细胞尾静脉接种C57BL/6小鼠和KM小鼠,均能形成肺转移模型,但两种小鼠成瘤情况差异较大。KM小鼠的荷瘤时间比C57BL/6小鼠荷瘤时间长。HE染色结果显示黑色素瘤B16细胞在两种小鼠的肺组织中均形成巢团状的转移瘤,且两种模型肺转移瘤均多分布在支气管周围。结论通过对C57BL/6小鼠和KM小鼠两种黑色素瘤B16细胞肺转移小鼠模型的成瘤特点的分析比较,可以看出KM小鼠更适合用于研究新的肺肿瘤治疗药物,为研究黑色素瘤的发生发展提供依据。     

重组人内皮抑制素抑制肺黑色素瘤转移

目的 研究rhEndostatin的抗肿瘤活性。 方法：鸡胚绒毛尿囊膜实验观察rhEndostatin的体内抗血管生成活性；将B16-BL6黑色素瘤细胞尾静脉注射入C57BL/6小鼠体内构建肺转移瘤模型，评价rhEndostatin的抗肿瘤活性。结果：rhEndostatin可以明显抑制血管的生成。与盐水对照组相比，高、中、低剂量rhEndostatin明显减少肺转移瘤数目，并对由于黑色素瘤体积的增长而引起的肺重的增加也有抑制趋势。结论：rhEndostatin通过抑制血管生成可以抑制肺黑色素瘤的转移。     

内毒素休克猕猴早期肺损伤选择素的改变及意义

目的 探讨L-选择素和P-选择素在内毒素休克肺损伤中的作用。方法11只猕猴随机分为两组，脂多糖（LPS）组（n-6），制备LPS休克模型，分别在LPS攻击前（0min）、攻击60min和120min时采血，检测L-选择素和P进择素；空白对照组（n=5），也相应时间点采血。于两组均于实验120min时处死动物，取肺组织进行电镜及选择素免疫组化观察。结果LPS攻击120min时电镜下可见猕猴肺组织出现Ⅰ、Ⅱ型上皮损伤及血管内皮、基底膜损伤，少量红细胞渗出。两组在LPS攻击0、60和120min时L-选择素浓度均有所下降，其中LPS攻击120min时两组L-选择素均较LPS攻击前明显下降（P均〈0．05），两组间比较差异均无显著性（P均〉0．05）。两组P-选择素在各时间点均无明显变化（P均〉0．05），组间比较差异也无显著性（P均〉0．05）。LPS组动物肺泡内可见P进择素表达，血管内皮可见L-选择素，而空白对照组肺组织未见选择素表达。结论休克早期出现肺损伤，肺组织P-选择素及L-选择素的表达早于血浆中的变化；选择素在猕猴肺损伤中起重要作用。     

原发性高血压患者血浆活化血小板表达的研究

目的：探讨高血压病患者血小板活性因子指标CD62P（P-选择素）、CD63的变化。方法：取158例原发性高血压患者血浆，用流式细胞仪进行检测CD62P（P-选择素）、CD63，自动生化仪测定肾功能、放射免疫法测定血、尿微球蛋白。结果：高血压组CD62P（P-选择素）、CD63均明显高于正常对照组，且随高血压病患者血压分级水平的增高而增高。结论：高血压病患者分级水平的增高与血小板活化指标CD62P（P-选择素）和CD63过度表达有一定的关系，表明其可能参与高血压的发生、发展。     

急性胰腺炎患者血浆内皮素和P-选择素的变化及意义

目的 观察急性胰腺炎（AP）患者血浆内皮素（ET）和P-选择素的变化特点，探讨其临床意义。方法 患者分两组：急性轻型胰腺炎（MAP）组24例，急性重型胰腺炎（SAP）20例。以健康体检者10例作对照组，采用放射免疫法测定血浆ET和P-选择素。结果 AP患者血浆ET和P-选择素增高，增高程度与急性胰腺炎病情程度有关。结论 血浆内皮素和P-选择素参与了急性胰腺炎的发病过程，可作为判断病情轻重和预后的指标。     

P-选择素和胰岛素抵抗对糖尿病肾病的影响

目的 研究P-选择素、胰岛素抵抗（IR）与糖尿病肾病（DN）的关系。方法 对109例2型糖尿病（T2DM）患者（有肾病者为DN组、无肾病者为DM组）和正常对照组（NC）,进行空腹血糖（FPG）、胰岛素、P-选择素测定,计算HOMA法胰岛素抵抗指数（HOMA-IR）。结果 DN和DM组血中的FPG、胰岛素、P-选择素及HOMA-IR均高于NC组（P〈0.01）,且DN组上述结果都高于DM组（P〈0.01）,相关分析表明IR与P-选择素呈正相关,多元回归显示DN发生与高FPG、高胰岛素、IR及P-选择素相关。结论P-选择素参与了IR,且P-选择素与IR一起参与了他T2DM患者DN的发生、发展过程。     

急性脑梗死患者外周血血小板表面P-选择素阳性表达率变化及药物干预影响

目的 探讨血小板活化因子P-选择素在急性脑血管疾病患者中的改变及应用奥扎格雷钠治疗后的变化。方法采用流式细胞仪测定急性脑梗死患者及正常对照组外周血血小板表面P-选择素阳性表达情况，对比观察治疗前后P-选择素的变化。结果急性脑梗死患者外周血血小板表面P-选择素阳性表达率显著高于正常对照组（P〈0．01）；应用奥扎格雷钠治疗后的外周血血小板表面P-选择素阳性表达率明显低于正常对照组（P〈0．01）。结论血小板活化在急性脑梗死的发生、发展中起重要作用；P-选择素可作为选择药物治疗的一项可靠指标，奥扎格雷钠可减少血小板活化。     

P-选择素在早期糖尿病肾病中的诊断意义

目的：探讨P-选择素在早期糖尿病肾病中的诊断意义.方法：应用酶联免疫吸附法（ELISA）测定143例2型糖尿病患者[2型糖尿病组根据有无蛋白尿分为三个亚组：2型糖尿病无蛋白尿组（A组）52例、2型糖尿病微量白蛋白尿组（B组）41例和2型糖尿病临床蛋白尿组（C组）50例]及34例健康体检者（对照组）血清P-选择素水平.并采用全自动生化分析仪葡萄糖氧化酶比色法检测空腹血糖的水平;采用电泳仪琼脂糖凝胶电泳法检测糖化血红蛋白的水平;采用特种蛋白分析仪免疫乳胶增强散射比浊法测定高敏C反应蛋白及尿微量白蛋白的水平.结果：2型糖尿病组血清P-选择素的水平显著高于对照组（P＜0.01）,糖尿病临床蛋白尿组血清P-选择素水平高于糖尿病微量白蛋白尿组（P＜0.01）;而糖尿病微量白蛋白尿组血清P-选择素水平高于糖尿病无蛋白尿组（P＜0.01）.2型糖尿病的P-选择素与空腹血糖、糖化血红蛋白、高敏C反应蛋白均呈正相关（r分别0.40、0.84、0.87,P均＜0.01）.结论：血清P-选择素参与了糖尿病微血管病变的发病过程,是糖尿病肾病的早期指标之一.     

肝癌患者血清可溶性P-选择素和细胞内粘附分子-1的检测及意义

目的探讨肝癌患者血清可溶性P-选择素和细胞内粘附分子-1的检测意义。方法应用ELISA法测定原发性肝癌患者血清可溶性P-选择素和细胞内粘附分子-1的含量，并分析其与临床病理特征的关系。结果肝癌患者血清可溶性P-选择素和细胞内粘附分子-1的含量分别高于正常人血清含量，差异均有统计学意义（均P〈0．01）；肝癌患者血清可溶性P-选择素和细胞内粘附分子-1的含量在肿瘤直径≥5cm组、有肝外脏器转移组和临床分期Ⅲ和Ⅳ期组分别高于肿瘤直径〈5cm组、无肝外脏器转移组和临床分期Ⅰ和Ⅱ期组，差异均有统计学意义（P〈0．01或0．05）。结论原发性肝癌患者血清可溶性P-选择素和细胞内粘附分子-1的含量与肿瘤的发展、转移和临床分期密切相关。     

中国广西人群P-选择素基因rs6131 C/T多态性的研究

目的研究P-选择素基因单核苷酸多态性rs6131C/T等位基因及其基因型在中国广西地区人群中的分布频率,并比较其与不同种族间分布的差异。方法采用单碱基延伸PCR扩增技术和DNA测序检测199例中国广西人群的P-选择素基因rs6131C/T多态性,并与人类基因组计划（HapMap）公布的欧洲人、非洲人、日本人和中国北京人的SNP分型数据比较,分析这5个人群的基因型及等位基因的分布频率。结果 P-选择素基因rs6131C/T存在多态性,其基因型及等位基因频率男女组间比较无统计学意义（P〉0.05）,但与非洲人、日本人比较存在显著性差异（P〈0.05）,与欧洲人和北京人比较差异无统计学意义（P〉0.05）。结论在中国广西人群中P-选择素基因rs6131C/T存在多态性,与其他种族人群比较存在差异,这种差异对于人类学的研究可能起重要的作用。     

Increased metastatic potential of tumor cells in von Willebrand factor-deficient mice

BACKGROUND: The key role played by von Willebrand factor (VWF) in platelet adhesion suggests a potential implication in various pathologies, where this process is involved. In cancer metastasis development, tumor cells interact with platelets and the vessel wall to extravasate from the circulation. As a potential mediator of platelet-tumor cell interactions, VWF could influence this early step of tumor spread and therefore play a role in cancer metastasis. OBJECTIVES: To investigate whether VWF is involved in metastasis development. METHODS: In a first step, we characterized the interaction between murine melanoma cells B16-BL6 and VWF in vitro. In a second step, an experimental metastasis model was used to compare the formation of pulmonary metastatic foci in C57BL/6 wild-type and VWF-null mice following the injection of B16-BL6 cells or Lewis lung carcinoma cells. RESULTS: In vitro adhesion assays revealed that VWF is able to promote a dose-dependent adhesion of B16-BL6 cells via its Arg-Gly-Asp (RGD) sequence. In the experimental metastasis model, we found a significant increase in the number of pulmonary metastatic foci in VWF-null mice compared with the wild-type mice, a phenotype that could be corrected by restoring VWF plasma levels. We also showed that increased survival of the tumor cells in the lungs during the first 24 h in the absence of VWF was the cause of this increased metastasis. CONCLUSION: These findings suggest that VWF plays a protective role against tumor cell dissemination in vivo. Underlying mechanisms remain to be investigated.     

Contrast-aided diagnostic ultrasound does not enhance lung metastasis in a mouse melanoma tumor model.

OBJECTIVE: The purpose of this research was to test the hypothesis that contrast-aided diagnostic ultrasound (CADUS) could exacerbate the metastatic spread of mouse melanoma tumor cells to the lungs. METHODS: The melanoma cell lines B16 and B16-D5 (metastatic specifically to lung) were implanted on a hind leg of female C57/bl6 mice. Growing tumors were scanned by 1.5-MHz diagnostic ultrasound in a 37 degrees C water bath. Four hundred image frames were triggered at a 1-Hz rate with 4 retro-orbital injections of an ultrasonographic contrast agent at dosage of 10 microL/kg at 100-second intervals. Sham-treated mice received 400 frames of ultrasonography followed by the contrast agent with the ultrasound off. The primary tumor was surgically removed 1 day after ultrasound administration. Lungs were removed and evaluated blind after 2 weeks of bleaching in Fekete solution. RESULTS: Three experiments were performed. The first experiment involved scanning sham and CADUS groups of 20 mice each with B16 tumors; B16 metastasis was not enhanced. The second experiment repeated this test with the D5 cell line; the metastasis enhancement was marginally significant for average number (0.3 and 3.2; P = .06) and incidence (3 and 9 of 19; P = .08) in mice without tumor recurrence. Finally, a third experiment was performed to clarify ambiguous results in the second experiment and consisted of 2 groups of 40 mice each. In this larger experiment, the results were essentially equal for the sham and CADUS groups. CONCLUSIONS: Overall, the results do not support the hypothesis of CADUS-enhanced metastasis.     

The effect of storage on the metastatic potential of tumor cells collected in autologous blood. An animal model

Malignancy in a potential autologous blood donor is considered by some as a relative contraindication for the collection of autologous blood. There is, however little experimental information regarding the safety of transfusion of autologous blood that may contain viable tumor cells. The purpose of this study was to determine if tumor cells seeded into autologous blood retain their metastatic potential and, if so, for how long. A well-established model of the pulmonary metastasis of B16/F10 (F10) melanoma in C57BL/6 mice was used. Equal numbers of F10 cells were seeded into CPDA-1 blood from the mice or into saline, and the number of lung tumors was evaluated 14 days after infusion of blood or saline. The CPDA-1 blood seeded with F10 cells was stored for up to 21 days in transfer packs at 4 to 6 degrees C, and the metastatic potential of F10 cells in the stored blood was ascertained as above. F10 cells seeded into autologous blood that was then transfused without storage gave rise to the same number of metastatic foci (404 +/- 32 metastases) as did those cells transfused in saline (374 +/- 38, p = 0.5). In contrast, the number of metastatic foci resulting from the transfusion of blood containing F10 cells decreased progressively after storage of the blood (7 days = 124 +/- 14 metastases; 14 days = 7 +/- 1; 21 days = 2 +/- 1; all p values less than 0.001 vs fresh blood). Also, the predilection of F10 cells to metastasize to the lung was unchanged by storage in blood.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blocking of lung endothelial cell adhesion molecule-1 (Lu-ECAM-1) inhibits murine melanoma lung metastasis.

The 90-kD lung endothelial cell adhesion molecule-1 (Lu-ECAM-1) selectively promotes Ca(2+)-dependent adhesion of lung-metastatic B16 melanoma cells. Corresponding with their metastatic performance, high lung-metastatic B16-F10 melanoma cells bind in significantly higher numbers to Lu-ECAM-1 than their intermediate and low lung-metastatic counterparts B16-L8-F10 and B16-F0, respectively. Maximum attachment is observed at a density of approximately 2.4 x 10(2) Lu-ECAM-1 sites/microns2 of plastic surface. B16 melanoma cell binding to Lu-ECAM-1 is blocked by mAb 6D3 and is competitively inhibited by soluble Lu-ECAM-1. C57B1/6 mice passively immunized with anti-Lu-ECAM-1 mAb 6D3 or actively immunized with purified Lu-ECAM-1 exhibit an anti-Lu-ECAM-1 antibody titer-dependent reduction in the number of B16 experimental metastases. Lu-ECAM-1 promotes neither binding nor metastasis of other lung-metastatic tumor cells (e.g., KLN205). Our data indicate that an "antiadhesion" therapy directed at interfering with the adherence of blood-borne tumor cells to organ-specific vascular endothelium is efficient in the control of metastasis formation in selective organ sites.     

来氟米特对糖尿病大鼠肾组织中P-选择素表达的影响

目的探讨P-选择素在糖尿病大鼠肾组织中的表达及来氟米特对其影响。方法高糖高脂饮食联合小剂量腹腔注射链脲佐菌素(streptozotocin,STZ)建立2型糖尿病动物模型。随机分为糖尿病组(B组)和来氟米特干预组(C组),另设鼠龄匹配的正常对照组(A组)。第8、9、12、16周,监测各组血糖(FBG)、糖化血红蛋白A1(HbA1c)、血脂、血肌酐(Scr)、尿素氮(BUN)、胰岛素(Insulin)及24小时尿蛋白定量。第9、16周各组随机处死6只大鼠,测量肾重指数。另外观察肾脏病理情况。用半定量RT-PCR方法检测肾皮质P-选择素(P-selectin)mRNA表达水平。结果大鼠肾脏病理符合2型糖尿病肾病的改变。与A组相比,第16周时B组血肌酐、尿素氮明显升高(P0.01),24小时尿蛋白定量自糖尿病成模后即开始高于对照组(P0.05),并于12、16周时仍持续增加(P0.01,P0.01)。与B组相比,C组大鼠BUN和Scr第16周时降低,差异有统计学意义(P0.05,P0.01),24小时尿蛋白定量12、16周时较B组降低(P0.05,P0.01)。糖尿病大鼠肾组织中P-selectin表达明显高于对照组(P0.01),来氟米特干预后其表达降低(P0.05)。结论来氟米特可能通过抑制糖尿病大鼠肾组织中P-选择素的表达发挥对肾脏的保护作用。     

蛋白酶活化受体1在B16F10瘤细胞体内外迁移中的作用

目的:研究蛋白酶活化受体 1(PAR1)在肿瘤细胞转移过程中的作用,评估凝血系统与肿瘤转移的关系。方法:首先通过逆转录聚合酶链反应(RT-PCR)和 Western-blot 方法检测了 siRNA 对 B16F10 黑色素瘤细胞中 PAR1的内源表达的影响,然后在 PAR1 受 siRNA 干扰的情况下检测 B16F10细胞在体内外迁移能力,其结果与未受siRNA 干扰的试验结果相对照,来确定 PAR1 在 B16F10 瘤细胞体内外迁移中的作用。结果:75nmol/L 的 siRNA 干扰48h 后可明显抑制 B16F10中 PAR1 的表达,其转录水平抑制率是60%,蛋白水平的抑制率为85%;体外迁移实验表明,PAR1 表达量减少后与对照组相比细胞迁移能力减弱(P<0.01);动物实验表明.干扰 B16F10细胞中的 PAR1后,肿瘤细胞经血管迁移能力减弱,在肺部形成的转移瘤数目减少(P<0.01)。结论:PAR1 具有促进肿瘤细胞迁移作用。开发 PAR1 封闭剂有望抑制肿瘤转移,可作为研制抗肿瘤转移药物的新靶点。     

血清P-选择素联合尿微量蛋白检测对过敏性紫癜患儿肾损害的早期诊断价值

目的探讨血清P-选择素联合尿微量蛋白检测对过敏性紫癜（HSN）患儿肾损害的早期诊断意义。方法选择76例HSP患儿作为HSP组,并选取同期65例健康儿童作为对照组。采用常规放免法检测p2微球蛋白（陉一MG）、白蛋白（ALB）及转铁蛋白（TRF）含量,采用双抗夹心酶联免疫法测N乙酰-β-氨基葡萄糖苷酶（NAG）含量,血清肌酐（Scr）和尿素氮（NJN）测定运用终点法,血清P-选择素定量测定采用酶联免疫吸附法（ELlSA）。比较2组尿ALB、TRF、NAG、β2-MG及血清P-选择素的水平,并分析血清P-选择素水平与尿微量蛋白间的相关性。结果HSP组患儿BUN及Scr水平均在正常范围内,且尿ALB、TRF、NAG、β2-MG及血清P-选择素水平均显著高于对照组患儿,差异有统计学意义;血清P_选择素水平与尿微量蛋白系列呈正相关;HSP组血清P-选择素及尿微量蛋白4项指标中的1项或〉1项的异常率显著高于传统以尿常规阳性来判定早期肾损害的尿常规异常率,差异有统计学意义。结论联合检测血清P-选择素及尿微量蛋白对HsP患儿的肾损害具有早期诊断价值。     

平喘固本合剂对哮喘小鼠气道急性炎症的影响

目的 探讨平喘固本合剂对哮喘小鼠气道急性炎症的影响及可能的机制。方法 健康雌性昆明小鼠40只,随机分为空白对照组（A组）、哮喘模型组（B组）、平喘固本合剂治疗组（C组）。用卵清清蛋白致敏建立哮喘小鼠气道急性炎症模型,各组分别给予相应药物治疗10 d。对各组小鼠支气管肺泡灌洗液（BALF）中细胞进行分类与计数,苏木精-伊红（HE）染色观察肺组织的病理变化,同时应用ELISA法测定BALF中白细胞介素4（IL-4）、干扰素γ（IFN-γ）水平的变化。结果 与A组比较,B组BALF中细胞总数、嗜酸性粒细胞、淋巴细胞、巨噬细胞明显增多（F=85.70-127.69,P＜0.05）,哮喘模型制作成功;C组细胞总数、嗜酸性粒细胞、淋巴细胞、巨噬细胞较B组均明显减少,但较A组升高,差异有显著性（q=2.15-10.08,P＜0.05）。与A组比较,B组中IL-4含量、IL-4/IFN-γ明显升高,IFN-γ明显降低,差异有统计学意义（F=186.17-378.06,q=17.02-21.46,P＜0.05）;与B组比较,C组IL-4、IL-4/IFN-γ降低,IFN-γ升高,差异有显著性（q=4.80-7.18,P＜0.05）。HE染色显示,C组炎症细胞浸润、平滑肌肥厚及肺组织黏膜水肿等炎症表现较B组明显减轻。 结论 平喘固本合剂对急性哮喘小鼠气道炎症有明显的抑制作用,其作用机制可能与其抑制IL-4的表达、抑制炎性细胞聚积及促进IFN-γ的表达有关。