亮肽素、TLCK和乳铁蛋白对人结肠肥大细胞类胰蛋白酶释放的影响

目的：探讨亮肽素、N-甲苯磺酰-L-赖氨酸氯甲基化酮（TLCK）和乳铁蛋白对人结肠肥大细胞释放类胰蛋白酶的影响。 方法： 人结肠组织经酶消化后，细胞成份用全HBSS重新悬浮。激发过程在LP4试管中、37 ℃ 条件下完成。类胰蛋白酶水平用酶联免疫吸附试验（ELISA）方法测定。 结果： 亮肽素、TLCK和乳铁蛋白无明显刺激人结肠肥大细胞释放类胰蛋白酶的作用。但可以剂量依赖性的方式抑制抗IgE抗体诱导的类胰蛋白酶释放，抑制范围在36.6%-47.6%。在37 ℃条件下同结肠细胞预培养20 min与无预培养相比，亮肽素和TLCK对抗IgE抗体诱导的类胰蛋白酶释放的抑制作用无明显改变。亮肽素、TLCK和乳铁蛋白均可以抑制CI诱导的类胰蛋白酶释放，抑制范围在27.1%-44.1%。在37 ℃条件下同结肠细胞预培养20 min与无预培养相比，亮肽素对CI诱导的类胰蛋白酶释放的抑制作用明显增强，TLCK则无此特点。 结论： 我们首次发现类胰蛋白酶抑制剂可抑制人结肠肥大细胞IgE依赖性和非依赖性类胰蛋白酶释放，提示类胰蛋白酶抑制剂可治疗炎症性肠病或其它肥大细胞相关疾病。     

Activation of Intestinal Mucosal Immunity in Tumor-bearing Mice by Lactoferrin

We have previously demonstrated that oral administration of bovine lactoferrin (bLF) markedly increases CD4⁺ and CD8⁺ T cells and NK (asialoGM1⁺) cells in the blood of tumor-bearing mice and enhances anti-metastatic activity. In this paper, we document that oral administration of bLF and bLF-hydrolysate (bLFH) is associated with strong increases in CD4⁺ and CD8⁺ T, as well as asialoGM1⁺ cells in lymphoid tissues and lamina propria of the small intestine in mice, especially in tumor-bearing animals in which Co26Lu cells were implanted subcutaneously. Moreover, IgM⁺ and IgA⁺ B cells in lamina propria of the small intestine were also significantly increased by bLF and bLFH. Bovine apo-transferrin (bTF) did not exhibit such activity. In the colon, only CD8⁺ cells were significantly increased by treatment with bLF, while asialoGM1⁺ cells were significantly decreased. bLF and bLFH induced cytokines to activate T, B and asialoGM1⁺ cells. Administration of bLF and bLFH, but not bTF, increased production of interleukin-18 (IL-18), interferon-gamma (IFN-γ) and caspase-1 in the mucosa of the small intestine. Particularly high levels of IL-18 were found in the epithelial cells of the small intestine. Moreover, administration of bLF and bLFH, but not bTF, induced IFN-γ presenting cells in the small intestine. Caspase-1, which processes proIL-18 to mature IL-18, was also induced in the epithelial cells of the small intestine following treatment with bLF and bLFH, but not with bTF. These results suggest that enhanced production of IL-18 and IFN-γ and caspase-1 induction by treatment with bLF may be important for elevation of intestinal mucosal immunity.     

Inhibition of poliovirus type 1 infection by iron-, manganese- and zinc-saturated lactoferrin

In this study we investigated the inhibitory activity of different milk proteins on poliovirus infection in Vero cells. Proteins analyzed were mucin, α-lactalbumin, β-lactoglobulin, and bovine and human lactoferrin. Viral cytopathic effect was not prevented by mucin, α-lactalbumin or β-lactoglobulin, whereas the lactoferrins tested were able to inhibit the replication of poliovirus in a dose-dependent manner. Further experiments were carried out in which apo- and native lactoferrin or lactoferrin fully saturated with ferric, manganese or zinc ions were added to the cells during different phases of viral infection. Results obtained demonstrated that all lactoferrins were able to prevent viral replication when present during the entire cycle of poliovirus infection or during the viral adsorption step. Only zinc lactoferrin strongly inhibited viral infection when incubated with the cells after the viral attachment, being the inhibition directly correlated with the degree of zinc saturation. Our results demonstrated that all lactoferrins interfered with an early phase of poliovirus infection; zinc lactoferrin was the sole compound capable of inhibiting a phase of infection subsequent to virus internalization into the host cells. Received: 30 November 1998     

干眼症患者泪液乳铁蛋白检测的意义

目的探讨泪液乳铁蛋白（LF）与干眼症的发病关系及其意义。方法用放射免疫分析法测定40例（40眼）干眼症患者与35例（35眼）正常人的泪液LF值，并将干眼症患者泪液LF与SchirmerⅠ试验、BUT时间以及年龄的关系进行分析。结果干眼症组泪液LF含量与正常组对比，明显下降，差异有显著性（P〈0．0005）。干眼症泪液LF与SehirmerⅠ试验之间呈正相关性（r=0．48，〈0．005），与BUT时间之间呈正相关性（r=0．78，P〈0．005），与年龄之间呈负相关性（r=-0．74，P〈0．006），说明泪液LF随年龄每年下降约0．02mg／ml。结论泪液LF的含量下降是干眼症发病因素之一，非干燥综合征（Siogren，s syndrome，SS）患者的治疗除补充人工泪液外，补充具有生物活性的泪液蛋白也是必要的措施之一。     

Decreased lactoferrin levels in peritoneal fluid of women with minimal endometriosis

OBJECTIVE: The aim of the study was to evaluate for the presence of lactoferrin (LTF) in peritoneal fluid (PF) of women with and without endometriosis. PATIENTS: Seventy-eight women were studied, including 49 women with endometriosis and, as a reference group, 29 patients with functional follicle ovarian cysts. RESULTS: Lactoferrin levels were detectable in all peritoneal fluid samples. Women with minimal endometriosis had lower PF lactoferrin concentrations compared to both patients with high revised American Fertility Society classification scores and women with follicle ovarian cysts. No significant difference in the peritoneal LTF levels was found between patients with stage II endometriosis, stage III or IV endometriotic disease and women with functional cysts of ovaries. CONCLUSIONS: Owing to its antibacterial properties lactoferrin is probably an important defense factor in the peritoneal cavity, however its role in the pathogenesis of endometriosis remains enigmatic.     

良性前列腺增生的前列腺液中低相对分子质量前列腺特异抗原及乳铁蛋白的表达

目的:检测良性前列腺增生患者和正常男性的前列腺液中低相对分子质量前列腺特异抗原(lw-PSA)及乳铁蛋白的表达水平.方法:采用蛋白双向电泳的方法,对20例良性前列腺增生患者和20例正常男性前列腺液的蛋白表达进行检测,并通过质谱分析方法确定蛋白点的性质.随后采用Western blotting的方法来验证蛋白的表达水平.结果:在良性前列腺增生患者的前列腺液中发现了相对分子质量为10×103、等电点(pI)为8.5～9.3的蛋白点A和相对分子质量为35×103、pI为7～7.5的蛋白点B.进行质谱分析后确定、蛋白点A为lw-PSA,蛋白点B为乳铁蛋白(lactoferrin).Western blotting检测进一步确定良性前列腺增生患者的前列腺液中有lw-PSA表达,而前列腺癌组织中和正常男性的前列腺液中未见lw-PSA的表达.另外,Western blotting检测还发现,良性前列腺增生患者前列腺液的乳铁蛋白表达水平高于正常男性,而前列腺癌组织中无乳铁蛋白的表达.结论:良性前列腺增生患者的前列腺液中有lw-PSA表达,而正常男性的前列腺液中未见lw-PSA表达,lw-PSA可能与良性前列腺增生的病理变化存在一定关联,或许可以作为良性前列腺增生的蛋白标志物.另外,良性前列腺增生患者前列腺液的乳铁蛋白表达水平增高,正常男性前列腺液的乳铁蛋白表达水平较低,而前列腺癌的乳铁蛋白表达缺失.     

乳铁蛋白对骨关节炎软骨细胞增殖活力及细胞外调节蛋白激酶表达的影响

目的 观察人乳铁蛋白(hLF)对人骨关节炎软骨细胞(HACs)增殖活力及细胞外调节蛋白激酶(ERK)基因表达的影响. 方法 噻唑蓝(MTT)比色法检测HACs的增殖力,并选择hLF最佳工作浓度;LIVE/DEAD染色检测HACs的活力;实时定量聚合酶链反应(Real-time PCR)和免疫细胞化学染色检测磷酸化细胞外调节蛋白激酶( p-ERK )/ERK的表达.结果 MTT检测显示hLF对HACs的增殖促进作用呈剂量-效应关系(P＜0.05),以200 mg/L为最佳药物浓度.LIVE/DEAD染色显示hLF可以显著促进HACs的活力,与空白对照组和无血清培养组比较,差异有统计学意义[(98.53±2.76)％比(89.12 ±6.98)％比(65.37 ±2.08)％,P＜0.05].Real-time PCR检测显示,hLF作用48 h后ERK mRNA相对表达量为(1.589±0.113),与对照组(1.024±0.026)比较差异有统计学意义(P＜0.05).IN Cell Analyzer 2000定量检测,hLF能够显著促进p-ERK/ERK蛋白的表达,较对照组明显增高,差异有统计学意义[(0.887±0.003)比(0.637±0.002),P＜0.05].结论 hLF可明显促进HACs增殖力和活力,其作用机制可能与上调增殖基因p-ERK/ERK表达有关.     

Lactoferrin inhibits infection-related osteoclastogenesis without interrupting compressive force-related osteoclastogenesis

BackgroundControl of periodontal tissue inflammation during orthodontic treatment is very important in achieving a favourable therapeutic goal. We previously demonstrated that orally applied bovine lactoferrin (bLF) inhibited LPS-induced bone resorption but not orthodontic force-induced tooth movement in vivo. This study is designed to examine the underlying mechanism of it.MethodsWe examined the inhibitory effects of bLF on the expression of RANKL, OPG, TNF-α and COX-2 in osteoblasts loaded with compressive stress (CS) in comparison with LPS stimulated osteoblasts. Formation of osteoclasts was evaluated by co-culture system.ResultsBoth CS- and LPS-applications upregulated COX-2 and RANKL but downregulated OPG. TNF-α was upregulated in LPS-stimulated osteoblasts but downregulated in CS-loaded osteoblasts. NS398 (a specific inhibitor of COX-2) significantly inhibited CS-induced RANKL-upregulation but not LPS-induced RANKL upregulation, indicating a critical role of COX-2/PGE₂ pathway in CS-induced osteoclastogenesis. bLF significantly downregulated LPS-induced upregulation of RANKL and eliminated OPG suppression but not affected in CS-induced changes. Moreover, bLF significantly decreased LPS-induced osteoclast formation, whereas bLF had no effect on PGE₂-induced osteoclast formation.ConclusionsbLF can effectively suppress harmful bone destruction associated with periodontitis without inhibiting bone remodelling by CS-loading. Therefore, oral administration of bLF may be highly beneficial for control of periodontitis in orthodontic patients.     

Regulation of PKB/Akt-pathway in the chemopreventive effect of lactoferrin against diethylnitrosamine-induced hepatocarcinogenesis in rats

BackgroundAbnormal activation of protein kinase B (PKB) is associated with many cancers. This makes inhibition of PKB signaling pathway a promising strategy for cancer therapy. Lactoferrin (Lf) has been reported for its inhibition of tumor growth and metastasis, however, the mechanism is not completely understood. Its anti-hepatocarcinogenic activity has not taken the deserved recognition despite the additional advantages of Lf as an antiviral against hepatitis C virus, the main cause of hepatocellular carcinoma (HCC), and as a targeting ligand for delivering chemotherapeutics to hepatoma cells.MethodsThis study evaluated the anti-hepatocarcinogenic effect of Lf, and the role of PKB in this effect using diethylnitrosamine (DENA)-induced HCC rat model, and a primary cell culture prepared from the induced hepatic lesions (DENA?HCC cell culture).ResultsUp-regulation of activated PKB in the hepatocytes of rats with DENA-induced HCC was observed, as measured biochemically in the liver homogenate, and localized immunohistochemically. This was accompanied by increment of hepatocytes proliferation, and expression of vascular endothelial growth factor and endothelial nitric oxide synthase. Involvement of PKB in DENA-induced HCC was confirmed by the observed decrease in cell proliferation in DENA?HCC cell culture that was treated with PKB inhibitor. In Lf-treated rats, a dose-dependent chemopreventive effect was observed, with decreased expression and activation of PKB, amelioration of the other DENA-induced alterations, and stimulation of apoptosis. In vitro, Lf blocked PKB activator-induced cell proliferation.ConclusionThese findings support the chemopreventive activity of Lf against HCC, and suggest regulation of PKB-pathway as a potential mechanism underlying this effect.