Extraction and Isolation of Alkaloids of Sophora Alopecuroides and Their Anti-Tumor Effects in H22 Tumor-Bearing Mice

Background

Alkaloids of Sophora alopecuroides have good biological activity, and are widely used in clinical settings, which not only have pharmacological activities of anti-cancer, cancer suppression, as well as the inhibition, and killing of various microorganisms; but also possess extensive pharmacological effects on immune system, nervous system and cardiovascular system. The objective of this paper was to extract and isolate total alkaloids of Sophora alopecuroides (TASA), and to study their anti-tumor effects in H22 tumor-bearing mice.

Materials and Methods

TASA were extracted and isolated using thin-layer chromatography, and column chromatography; and the isolated compounds were analyzed using nuclear magnetic resonance. The inhibitory effects of TASA on tumor in H22-bearing mice were determined by MTT assay.

Results

Three compounds were isolated from Sophora alopecuroides L., which were matrine, oxymatrine and sophoridine, respectively. Meanwhile, mouse H22 sarcoma model was established and different doses of TASA apparently inhibited solid H22-tumor in mice; it inhibited the thymus, and spleen to some extent; the degree of inhibition was more obvious for the spleen.

Conclusion

TASA has an anti-tumor effect in H22 tumor-bearing mice.     

骨髓基质干细胞复合煅烧骨的皮下成骨

背景：研究证明骨髓基质干细胞与煅烧骨支架材料结合后可形成组织工程化骨,但在动物体内的生物相容性及皮下诱导成骨的能力国内报道较少。 目的：观察骨髓基质细胞复合异种煅烧骨植入BALB/c裸鼠背部皮下的成骨性能及煅烧骨材料作为组织工程骨支架材料的可行性。 方法：选用经脱脂及脱蛋白处理后高温煅烧形成的骨支架材料与梯度密度离心法分离培养至第3代的羊骨髓基质干细胞构建细胞-煅烧骨复合物植入BALB/c裸鼠背部皮下,选同期对侧背部皮下植入单纯煅烧骨为对照组。 结果与结论：煅烧后的松质骨块为白垩色,表面呈蜂窝状多孔结构,保留了天然松质骨的多孔状空间结构。骨小梁结构完整,孔隙相互连通。骨髓基质干细胞接种到煅烧骨后24 h可见大量细胞黏附于支架上,7 d后细胞分泌大量细胞外基质,细胞与基质分界不清,细胞能在材料上良好地黏附、增殖与生长,细胞活性未受到支架材料的影响。植入4周后,两组均可见煅烧骨边缘出现少量残片,细胞-煅烧骨复合物组煅烧骨孔隙周边可发现骨细胞,对照组煅烧骨表面可见纤维结缔组织包绕。植入后8周,两组均可见到煅烧骨部分降解为片状类骨质,周围有成纤维细胞包绕,排列紧密,形态多样,细胞-煅烧骨复合物组煅烧骨孔隙内可见煅烧骨表面有排列成行的成骨细胞,孔隙间有散在淋巴细胞浸润。对照组标本可见孔隙内有大量结缔组织长入,未见明显成骨迹象。结果说明,经高温煅烧后的松质骨材料,具有良好的生物相容性和生物安全性,可作为骨髓基质干细胞的良好载体,复合后植入体内能够诱导新生骨组织形成,可作为骨缺损组织工程修复的支架材料。     

Systemic Lupus Erythematosus and Related Diseases

NZB (H-2^d) mice are well known for the production of IgM autoantibodies to ssDNA. However, an FI cross between NZB and either NZW or SWR mice is required to produce IgG nephritogenic antibodies to dsDNA and glomerulonephritis. The contribution of parental class II loci in the hybrid mice is clearly important to the development of anti-dsDNA antibodies, In contrast, NZB mice congenic with the la^bm12 mutation develop IgG autoantibodies to dsDNA despite being homozygous for Ia. As a part of our effort to examine the mechanisms of disease development in NZB.H-2^bm12 mice, we have generated a panel of monoclonal antibodies against nucleic acids. A subgroup of these antibodies exhibited strong electrostatic interaction with nucleic acids as evidenced by inhibition of their binding by a moderate increase in ionic strength. Interestingly, the effect of salt was either all or none; e.g., antibodies were either markedly inhibited or virtually unaffected. The importance of this ionic interaction was highlighted by analysis of DNA binding of antibodies from serum and nephritic kidneys of NZB.H-2^bm12 mice. Antibodies specific for ssDNA, which are common in NZB mice and not associated with nephritic lupus, are largely unaffected by salt. However, serum and kidney eluted IgG antibodies specific for dsDNA were markedly inhibited by salt. We postulate that B cell clones whose antibodies exhibit electrostatic interaction with DNA are preferentially expanded during the course of lupus in NZB.H-2^bm12 mice and that such antibodies contribute significantly to glomerulonephritis.     

Hyperreactivity of Neutrophils and Abnormal T Cell Homeostasis: a New Insight for Pathogenesis of Behçet's Disease

Neutrophil hyperfunction of Behçet's disease is in part regulated by genetical factors, especially related to HLA-B51 genes and by immunological abnormalities. Regarding the latter points, Behçet's disease worsens with abnormal regulation by γδ T cells and αβ T cells. Indeed, our own studies and those of other laboratories suggest that human heat shock protein may be one of the triggering factors for activation of both the γδ T cells and αβ T cells. These activated T cells may produce proinflammatory and/or inflammatory cytokines, and lead to tissue injury possibly via delayed-type hypersensitivity reaction, macrophage activation, and activation and/or recruitment of neutrophils. Here we discuss the crosstalk between the immune system and neutrophils in this disease.     

Flexibility of the Thyroiditogenic T Cell Repertoire for Murine Autoimmune Thyroiditis in CD8-Deficient (B2m -/-) and T Cell Receptor VBC Congenic Mice

In murine experimental autoimmune thyroiditis (EAT), previous studies have revealed a highly adaptable thyroiditogenic T cell repertoire which involves both CD4⁺ and CD8⁺ T cells in the susceptible H2k strain. To further test this flexibility, congenic B10.K mice lacking CD8⁺ T cells (B2m -/-) or harboring 70% T cell receptor (TCR) V6 gene deletions (V6^C) were immunized with mouse thyroglobulin (MTg) and evaluated for EAT 28 days later. All 62m -/- mice developed moderate antibodies to MTg, and thyroidal inflammation was comparable to B10.K mice, averaging 35-40%. Spleen cells (SC) from MTg-immunized mice were then injected into syngeneic recipients after stimulation in vitro with MTg or with conserved, thyroxine (T4)- or thyronine (TO)- containing 12mer peptides, hT4(5), hT0(2553), or hT4(2553), derived from the primary hormonogenic sites at position 5 or 2553 of human Tg. As previously shown in another H2^k strain (CBA/J), all three peptides activated MTg-primed SC to transfer EAT in B10.K mice. hT4(5) and hT4(2553) were further tested in B10.K-V6^C and ß2m^- B10.K mice. Both peptides expanded thyroiditogenic T cells in either strain, resulting in severe thyroiditis in syngeneic recipients. That EAT can develop in the absence of CD8⁺ T cells or in the presence of a severely restricted TCR repertoire underscores the remarkable flexibility of the thyroiditogenic T cell profile in the susceptible k haplotype.     

Neurotrophin system activation in pleural effusions

Neurotrophins (NTs) expression was assessed in malignant and non-malignant pleural effusions (inflammatory exudates and transudates). Enzyme-linked immunosorbent assay, in malignant exudates from small and non-small cell lung cancer (SCLC and NSCLC), detected nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), and their levels are higher as compared with inflammatory and transudative effusions. By immunoblots, in cultured cancer cells coming from malignant pleural effusions, NTs and low- and high-affinity NT receptors were detected in a percentage of SCLC and NSCLC. Proliferation assay demonstrated that BDNF significantly increased cancer cell proliferation in vitro, on the contrary, NT-3 reduced cancer cell growth rate and NGF did not modify cell growth. Moreover, NGF protects cells from death during starvation. These effects are reverted by the addition of NT receptor antagonists. Cultured cancer cells injected into the lung of immunodeficient mice generate lung tumors expressing NTs and NT receptors. These findings suggest that NTs may be able to modulate cancer cell behavior and their growth.     

反义锁核酸在转基因小鼠体内阻断乙肝病毒C基因表达

目的 探讨针对乙肝病毒C基因反义锁核酸在转基因小鼠体内阻断病毒基因表达的效果.方法 各实验组经尾静脉给药后,采用real-time PCR、时间分辨免疫荧光技术、免疫组织化学法等技术分别检测血清HBV DNA、HBeAg含量及肝细胞内HBcAg的表达情况.结果 注射锁核酸后,对乙肝病毒核酸的复制和乙肝病毒e抗原的合成均有较强的抑制作用,注射后1、3 和5 d,HBV DNA的平均抑制率分别19.24％、39.20％和44.47％;HBeAg的平均抑制分别为30.71％、51.14％和63.46％;小鼠肝细胞的HBcAg阳性细胞数也较对照组明显减少.结论 针对乙肝病毒C基因的反义锁核酸在转基因小鼠体内能有效抑制病毒基因的复制和表达,提示C基因可作核酸药物治疗的有效靶位.     

SPARC在db/db小鼠肾脏组织中高表达

目的 检测db/db鼠肾脏组织中的富含半胱氨酸的酸性分泌蛋白(SPARC)的表达情况.方法 RT-PCR、Western blot及免疫荧光方法检测db/db鼠肾脏组织中的SPARC mRNA及蛋白的表达.结果 SPARC在db/db鼠肾脏组织中呈现高表达.结论 db/db鼠肾脏组织中的SPARC呈现高表达(P＜0.05),可能与糖尿病肾病的发生与发展有关.     

急性髓系白血病干细胞NOD/SCID小鼠白血病模型的建立

目的探讨用非肥胖型糖尿病/严重联合免疫缺陷（NOD/SCID）小鼠和经流式细胞仪分选出的KG1a中CD34＋CD38-的干细胞亚群建立白血病干细胞（LSCs）动物模型,为靶向治疗LSCs的药物筛选奠定体内实验基础。方法 18只雌性NOD/SCID小鼠,体质量18～20 g,鼠龄6～8周龄。实验组12只,应用流式细胞仪分选KG1a细胞中具有LSCs特性的CD34＋CD38-亚群,以2×106个/只尾静脉注射经全身X射线照射2Gy的NOD/SCID小鼠;正常对照组6只,只注射磷酸盐缓冲溶液。观察两组小鼠的一般情况和白血病发生情况,应用形态学、组织病理检查、流式细胞术、骨髓染色体检查等检测实验组小鼠的外周血、骨髓、肝脏、脾脏的白血病细胞标志。结果接种2周后实验组小鼠外周血可见白血病细胞,接种30 d实验组小鼠的白血病发病率为100%,无自发缓解;外周血、骨髓、肝、脾中均可发现大量的白血病细胞浸润;实验组小鼠骨髓细胞中CD13抗原阳性率15.47%～23.66%,并可见KG1a细胞的核型特征。结论尾静脉接种流式细胞仪分选后的CD34＋CD38-KG1a细胞于全身亚致死量X射线照射后的NOD/SCID小鼠,能成功建成全身播散的白血病模型,为进一步研究LSCs的靶向治疗药物奠定实验基础。     

Diabetic Basement Membrane Thickening Does Not Occur in Myocardial Capillaries of Transgenic Mice When Metallothionein is Overexpressed in Cardiac Myocytes

Diabetic cardiomyopathy is a clinically distinct disease characterized by impaired cardiac function as a result of reduced contractility and hypertension‐induced athero‐ or arteriosclerosis. This may be due either to generalized vascular disease, tissue‐based injury such as focal cardiomyocyte dysmorphia, or microvascular damage manifested by myocardial capillary basement membrane (CBM) thickening. Hyperglycemia‐driven increases in reactive oxygen species (ROS) have been proposed to contribute to such damage. To address this hypothesis, we utilized light (LM) and transmission electron microscopy (TEM) to demonstrate cardiomyocyte morphology and myocardial CBM thickness in the left ventricles of four mouse genotypes: FVB (background Friend virus B controls), OVE (transgenic diabetics), Mt [transgenics with targeted overexpression of the antioxidant protein metallothionein (MT) in cardiomyocytes], and OVEMt (bi‐transgenic cross of OVE and Mt) animals. Mice were prepared for morphometric analysis by vascular perfusion. Focal myocardial disorganization was identified in OVE mice but not in the remaining genotypes. Not unexpectedly, myocardial CBM thickness was increased significantly in OVE relative to FVB (P < 0.05) and Mt (P < 0.05) animals (+28% and +39.5%, respectively). Remarkably, however, OVEMt myocardial CBMs showed no increase in width; rather they were ～3% thinner than FVB controls. Although the molecular mechanisms regulating CBM width remain elusive, it seems possible that despite a significant hyperglycemic environment, MT antioxidant activity may mitigate local oxidative stress and reduce downstream excess microvascular extracellular matrix (ECM) formation. In addition, the reduction of intra‐ and perivascular ROS may protect against incipient endothelial damage and the CBM thickening that results from such injury. Anat Rec, 296:480–487, 2013. © 2013 Wiley Periodicals, Inc.