脱细胞气管基质材料的免疫原性及生物相容性研究

目的探讨经高氯酸钠（NaClO₄）处理后的脱细胞气管基质材料的免疫原性及其生物相容性。 方法取 2 月龄新西兰兔胫骨骨髓，采用全骨髓贴壁筛选法分离培养 BMSCs。取 10 只 6 月龄成年新西兰兔气管，修剪至每段 1.5 cm，随机分为对照组（A₁ 组，n=5），仅剥离气管外表面疏松结缔组织；实验组（B₁ 组，n=5）采用改良 NaClO₄ 浸泡法脱细胞处理。MTT 法检测两组支架浸提液的细胞毒性；免疫组织化学染色观察支架主要组织相容性复合物（major histocompatibility complex，MHC）类抗原表达。取生长状态良好的第 4 代 BMSCs 接种至两组支架，制备细胞-支架复合物，培养 48 h 时行 Giemsa 染色，倒置显微镜观察材料周围的细胞活性；7、14 d 扫描电镜观察支架上的细胞状态。取 10 只 6 月龄成年新西兰兔，随机分成对照组（A₂ 组，n=5）和实验组（B₂ 组，n=5），分别于颈背部皮下皮囊埋植已制备的新鲜气管和脱细胞气管。术后行大体观察，并于术后 5、10、15、20、25、30 d 分析血清免疫球蛋白 IgM 和 IgG 含量的动态变化，术后 30 d 行 HE 染色观察。 结果MTT 检测示，B₁ 组浸提液的细胞增殖情况与 A₁ 组或纯培养基阴性对照组比较，差异无统计学意义（P>0.05）；免疫组织化学染色观察示，B₁ 组支架经脱细胞处理后可显著降低基质材料的抗原性。细胞-支架复合物培养 48 h Giemsa 染色示，两组材料周围的细胞贴壁生长良好。培养 7、14 d 扫描电镜观察示，细胞在 A₁ 组气管材料外壁上贴附良好，呈扁平的圆形、椭圆形，细胞排列紧密，成簇分布；细胞在 B₁ 组气管材料外壁上呈单片状生长，形态与 A₁ 组相似，生长趋势较好。同种异体动物体内实验显示，B₂ 组材料的排斥反应显著低于 A₂ 组；术后各时间点 A₂ 组的 IgM 和 IgG 含量均显著高于 B₂ 组（P<0.05）；HE 染色示，B₂ 组未见炎性细胞深层渗透或破坏气管结构，未见钙化、排斥等不良反应。 结论经 NaClO₄ 化学脱细胞处理后，兔气管支架材料具有良好的生物相容性，同时其免疫原性降低，适合作为构建组织工程气管的支架材料。     

RKIP regulates CCL5 expression to inhibit breast cancer invasion and metastasis by controlling macrophage infiltration

Accumulating evidence suggests that presence of macrophages in the tumor microenvironment add to the invasive and tumor-promoting hallmarks of cancer cells by secreting angiogenic and growth factors. RKIP is a known metastasis suppressor and interferes with several steps of metastasis. However, the mechanistic underpinnings of its function as a broad metastasis suppressor remain poorly understood. Here, we establish a novel pathway for RKIP regulation of metastasis inhibition through the negative regulation of RANTES/CCL5 thereby limiting tumor macrophage infiltration and inhibition of angiogenesis. Using a combination of loss- and gain-of-function approaches, we show that RKIP hinders breast cancer cell invasion by inhibiting expression of the CC chemokine CCL5 in vitro. We also show that the expression levels of RKIP and CCL5 are inversely correlated among clinical human breast cancer samples. Using a mouse allograft breast cancer transplantation model, we highlight that ectopic expression of RKIP significantly decreases tumor vasculature, macrophage infiltration and lung metastases. Mechanistically, we demonstrate that the inhibition of the CCL5 expression is the cause of the observed effects resulting from RKIP expression. Taken together, our results underscore the significance of RKIP as important negative regulator of tumor microenvironment.     

SH-SY5Y神经细胞中α7神经型尼古丁受体基因沉默对CaM、CaMKⅡ蛋白水平的影响

目的构建稳定的α7 n AChR沉默的神经母细胞瘤细胞(SH-SY5Y)细胞,研究α7神经型尼古丁受体(n AChR)基因沉默对钙调蛋白(Ca M)、钙调素依赖性蛋白激酶Ⅱ(Ca MKⅡ)水平的影响,了解α7 n AChR神经保护作用及其与阿尔茨海默病(AD)发病机制的关系。方法将α7 n AChR shRNA重组质转染到SH-SY5Y,用含嘌呤霉素的培养液筛选,挑选阳性克隆后采用实时荧光定量PCR和蛋白质印迹方法(Western-blot)检测细胞中α7n AChR mRNA及蛋白表达水平的变化;Western-blot方法测定Ca M、Ca MKⅡ蛋白表达水平。结果获得稳定转染α7 n AChR shRNA重组质粒的细胞克隆株,与对照组相比,α7 n AChR mRNA及蛋白表达量分别减少了95%和80%。Ca M、Ca MKⅡ蛋白表达量分别减少了48.5%和35%。结论成功构建了α7 n AChR mRNA沉默的SH-SY5Y细胞细胞株,α7 n AChR沉默降低了Ca M、Ca MKⅡ的蛋白水平,可能影响信号通路转导,这可能与阿尔茨海默病(AD)的发病有一定的关系。     

Prospective DPYD genotyping to reduce the risk of fluoropyrimidine-induced severe toxicity: Ready for prime time

5-Fluorouracil (5-FU) and capecitabine (CAP) are among the most frequently prescribed anticancer drugs. They are inactivated in the liver by the enzyme dihydropyrimidine dehydrogenase (DPD). Up to 5% of the population is DPD deficient and these patients have a significantly increased risk of severe and potentially lethal toxicity when treated with regular doses of 5-FU or CAP. DPD is encoded by the gene DPYD and variants in DPYD can lead to a decreased DPD activity. Although prospective DPYD genotyping is a valuable tool to identify patients with DPD deficiency, and thus those at risk for severe and potential life-threatening toxicity, prospective genotyping has not yet been implemented in daily clinical care. Our goal was to present the available evidence in favour of prospective genotyping, including discussion of unjustified worries on cost-effectiveness, and potential underdosing. We conclude that there is convincing evidence to implement prospective DPYD genotyping with an upfront dose adjustment in DPD deficient patients. Immediate benefit in patient care can be expected through decreasing toxicity, while maintaining efficacy.     

Enrichment of Oct3/4‐positive cells from a human bronchial epithelial cell line

Most adult stem cells are in the G0 phase of the cell cycle, accounting for only a small percentage of the cells in the tissue. Thus, isolation of stem cells from tissues for further study represents a major challenge. The anti‐tumor drug 5‐fluorouracil (5‐FU) selectively kills proliferating cells, sparing cells in the G0 phase. Thus, the objective of this study was to determine whether 5‐FU can be used to enrich stem cells in a human bronchial epithelial (HBE) cell population in vitro. Side population (SP) cells were isolated from untreated HBE cells or HBE cells treated with 5‐FU, and the resulting cells were subjected to colony formation assays, culturing of cell spheres, and tumorigenicity assays. Expression of Oct3/4, Sox2, PCK, and β‐catenin were examined by Western blot analysis and immunofluorescence. Treatment with 5‐FU increased the percentage of SP cells from 0.3% to 1.5%, and the clonogenic ability of 5‐FU‐treated cells was more than twofold higher than that of HBE cells. Cells that survived after 5‐FU treatment exhibited a higher capacity for sphere formation. Furthermore, spheres formed from 5‐FU‐treated cells possessed the capacity to generate differentiated progenies. Cells treated with 5‐FU also exhibited tumorigenic potential, based on tumor formation assays in nude mice, and Oct3/4‐positive cell aggregates were identified in the resulting tumors. In this study, we have shown that 5‐FU treatment enriched the population of cells expressing the putative embryonic markers Oct3/4 and Sox2 and exhibiting nuclear accumulation of β‐catenin. Furthermore, 5‐FU‐treated cells expressed low levels of the epithelial differentiation marker PCK. Analysis of epigenetic modifications suggested that Oct3/4‐positive cells possessed characteristics of stem cells. These results demonstrate that treatment with 5‐FU can enrich the stem cell population present in a human bronchial epithelial cell line, and implicate combined treatment with 5‐FU and serum‐free medium as a new method for isolation of stem‐like cells from the HBE cell line.     

Decreased expression of EphA5 is associated with Fuhrman nuclear grade and pathological tumour stage in ccRCC

The incidence of renal cell carcinoma is increasing all over the world. The molecular mechanisms for tumorigenesis, progression and prognosis are still unknown. The erythropoietin‐producing hepatoma amplified sequence (Eph) receptors have been reported to be expressed aberrantly in many types of human cancers and in particular EphA5 may play a role in certain human cancers. In this study, a set of clear cell renal cell carcinoma (ccRCC) tissues were subjected to immunohistochemistry. The relationship between EphA5 protein expression and clinicopathological parameters was statistically analysed. Our data show that EphA5 protein was negatively (0) or weakly (1+) expressed in 48 of 78 (61.5%), moderately (2+) expressed in 15 of 78 (19.2%) and strongly (3+) expressed in 15 of 78 (19.2%) tumour samples of ccRCC. Decreased expression of EphA5 was detected more often in females than in males (P = 0.017, r_s = ?0.267). Expression of EphA5 was related negatively to Fuhrman grade (P = 0.013, r_s = ?0.279) and pathological tumour stage pT (P = 0.003, r_s = ?0.334). No relation between the expression of EphA5 and age of patients was found (P = 0.107, r_s = 0.184). Fuhrman grade and pT stage are the most important factors used in prognosis of ccRCC. Hence this study may provide a new and useful prognostic marker in the clinical practice of ccRCC.     

CLONAL MONOSOMY 7 AND 5q IN A CHILD WITH MYELODYSPLASTIC SYNDROME

The authors report the case of a 5-year-old boy referred for thrombocytopenia and neutropenia. Bone marrow examination showed a myelodysplasia with clonal monosomy7. The acceleration of the disease was marked by the appearance of an additional cytogenetic abnormality, i.e., the deletion of the long arm of chromosome 5 in the clonal cells. RAS genemutationwas not detected. Chemotherapy was started to achieve complete remission before a bone marrow transplatation. This treatment was complicated by a prolonged a plasia and the patient died of systemic mycotic infection.