人乳腺癌上皮细胞miR-217对DNMT1调控作用的研究

目的 验证在乳腺癌上皮细胞系中,miR-217通过直接作用于DNMT1 3′UTR调控DNMT1表达,为后期动物实验和临床试验提供实验基础.方法 利用瞬时转染技术,在对应细胞系中,过表达、抑制miR-217,Western blot检测各实验组中DNMT1蛋白表达,并用含有miR-217野生型及突变型识别位点的DNMT1 3′UTR载体质粒分别转染293T细胞,检测相对荧光素酶活性改变.结果 过表达miR-217,DNMT1表达下降;抑制miR-217,DNMT1表达增高;双荧光素酶检测实验,DNMT1-UTR-WT的相对荧光素酶活性显著低于DNMT1-UTR-MUT,差异有统计学意义（P〈0.05）.结论 在乳腺癌上皮细胞系中,miR-217对DNMT1表达具有调控作用,且miR-217通过直接作用于DNMT1 3′UTR调控DNMT1表达.     

Morin inhibits cell proliferation and fibronectin accumulation in rat glomerular mesangial cells cultured under high glucose condition

Morin, is a natural bioflavonoid isolated from Chinese herbs of the Moraceae family, has been reported to possess antidiabetic activity. However, the role of morin on glomerular mesangial cells (MCs) proliferation and extracellular matrix (ECM) accumulation in diabetic condition is still unclear. Therefore, in this study, we investigated the role of morin on cell proliferation and ECM accumulation in rat glomerular MCs cultured under high glucose (HG) condition. Our results showed that morin inhibited HG-induced MC proliferation, arrested HG-induced cell-cycle progression, reversed HG-inhibited expression of p21^Waf1/Cip1 and p27^Kip1. It also inhibited HG-induced ECM expression, ROS generation and NOX4 expression in MCs. Furthermore, morin suppressed HG-induced phosphorylation of p38 MAPK and JNK1/2 in MCs. These data suggest that morin inhibits HG-induced MC proliferation and ECM expression through suppressing the activation of p38 MAPK and JNK signaling pathways. Thus, morin may be useful for the prevention or treatment of diabetic nephropathy.     

冠心病患者血清基质金属蛋白酶-2的检测及其临床意义

目的 探讨冠心病患者血清基质金属蛋白酶（MMP）-2水平与冠状动脉病变的关系。方法 采用双抗体夹心ELISA法测定74例冠脉造影证实有至少1支以上冠状动脉狭窄≥50％的冠心病患者的血清MMP-2水平。结果 血清MMP-2水平在临床诊断急性冠脉综合征的患者中明显增高，与正常组和稳定型心绞痛组比较差异有非常显著性（P〈0．01）；而稳定型心绞痛组与正常对照组比较差异没有显著性（P〉0．05）；双支和三支血管病变患者血清MMP-2水平高于单支病变组。结论 冠心病患者血清MMP-2水平增高，且增高程度与冠脉病变程度、病变活动度有关；冠状动脉粥样硬化性心脏痛的发生发展与炎症反应密切相关。     

Decellularization of bovine small intestinal submucosa and its use for the healing of a critical‐sized full‐thickness skin defect,alone and in combination with stem cells,in a small rodent model

In this study, we initially described an efficient decellularization protocol for bovine‐derived small intestinal submucosa (bSIS), involving freeze–thaw cycles, acid/base treatment and alcohol and buffer systems. We compared the efficacy of our protocol to some previously established ones, based on DNA content and SEM and histochemical analyses. DNA content was reduced by ~89.4%, significantly higher than compared protocols. The sulphated GAG content of the remaining interconnected fibrous structure was 5.738 ± 0.207 µg/mg (55% retained). An in vitro study was performed to evaluate whether rat bone marrow mesenchymal stem cells (MSCs) could attach and survive on bSIS membranes. Our findings revealed that MSCs can preserve their viability and proliferate on bSIS for > 2 weeks in culture. We conducted in vivo applications for the treatment of an experimental rat model of critical sized (7 cm²) full‐thickness skin defect. The wound models treated with either MSCs‐seeded (1.5 × 10⁶ cells/cm²) or non‐seeded bSIS membranes were completely closed by week 7 without significant differences in closure time; on the other hand, the open wound control was closed at ~47% at this time point. Immunohistopathology results revealed that the group which received MSCs‐seeded bSIS had less scarring at the end of the healing process and was in further stages of appendage formation in comparison with the non‐seeded bSIS group. Copyright © 2015 John Wiley & Sons, Ltd.     

B淋巴细胞刺激因子及其受体在多发性骨髓瘤中的表达及其意义

目的 探讨B淋巴细胞刺激因子(BLyS)及其受体在多发性骨髓瘤(MM)中的表达及其意义.方法 采用流式细胞术(FCM)分析人MM肿瘤细胞系KM3及CZ-1细胞表面BLyS及其受体的表达;RT-PCR及Western blot进一步验证Blys及其受体的表达;采用荧光免疫细胞化学法和激光共聚焦技术鉴定KM3细胞中Blys蛋白的表达与定位;WST细胞增殖实验检测Blys对MM肿瘤细胞生长与存活的影响.ELISA及荧光定量.聚合酶链反应(RTQ-PCR)测定MM患者BLyS蛋白与mRNA的表达水平.同时分析乳酸脱氢酶(LDH)、β2微球蛋白浓度与BLyS蛋白及mRNA表达水平的关系.结果 ①MM细胞能够表达BLyS及其受体;②BLyS定位表达于KM3细胞的浆膜上;③Blys促进MM细胞的生长与存活;④MM患者BLyS蛋白或mRNA表达水平显著高于正常对照组(P值均<0.01);⑤直线相关分析显示LDH、β2微球蛋白浓度与BLyS蛋白及mRNA表达水平呈显著相关性.结论 BLyS及其受体对于MM发生、发展可能起着重要的作用.     

Artificial extracellular matrices of collagen and sulphated hyaluronan enhance the differentiation of human mesenchymal stem cells in the presence of dexamethasone

In this study we investigated the potential of artificial extracellular matrix (aECM) coatings containing collagen II and two types of glycosaminoglycan (GAGs) with different degrees of sulphation to promote human bone formation in biomedical applications. To this end their impact on growth and osteogenic differentiation of human mesenchymal stem cells (hMSCs) was assessed. The cell proliferation was found to be significantly retarded in the first 14 days of culture on surfaces coated with collagen II and GAGs (coll‐II/GAG) as compared to tissue culture polystyrol (TCPS) and those coated with collagen II. At later time points it only tended to be retarded on coll‐II/sHya3.1. Heat‐inactivation of the serum significantly reduced cell numbers on collagen II and coll‐II/sHya3.1. Alkaline phosphatase (ALP) activity and calcium deposition, on the other hand, were higher for coatings containing sHya3.1 and were not significantly changed by heat‐inactivation of the serum. Expression levels of the bone matrix proteins bone sialoprotein (BSP‐II) and osteopontin (OP) were also increased on aECM coatings as compared to TCPS, which further validated the differentiation of hMSCs towards the osteogenic lineage. These observations reveal that aECM coatings, in particular those containing sHya3.1, are suitable to promote the osteogenic differentiation of hMSCs. Copyright © 2012 John Wiley & Sons, Ltd.     

Ultrasonographic evaluation of cervical lymph node metastases in esophageal cancer with special reference to the relationship between the short to long axis ratio (S/L) and the cancer content

Cervical lymph node metastasis was evaluated sonographically in 58 esophageal cancer patients. The short to long axis ratio (S/L) is a useful way to detect lymph node metastasis as opposed to the long axis alone. In other words, the lymph node exceeding 10 mm in long axis and with S/L over 0.5 showed a much higher incidence of metastasis than S/L under 0.5 in the analysis of the 126 detected lymph nodes. The cancer content was calculated with a microcomputer in each of the total 77 metastatic lymph nodes by enlarging the microscopic specimen 8 or 16 times using a magnifying apparatus. The average cancer content in the metastatic lymph nodes with S/L under 0.5 and over 0.5 was 26.0% and 59.1%, respectively, revealing a statistically significant difference (p less than 0.01). Thus, cancer proliferation in the metastatic lymph nodes of esophageal cancer is closely related to the increase in S/L.     

A recombinant protein TmSm(T34A) can inhibit proliferation and proapoptosis to breast cancer stem cells(BCSCs) by down-regulating the expression of Cyclin D1

Cancer stem cells (CSCs), a small fraction of cancer cells lines proved with stem cell characteristics, were regarded as “bad seeds” related to recurrence, metastasis and chemotherapy resistance of breast carcinoma in recent years. So inhibiting the growth or inducing the differentiation and apoptosis of CSCs were considered as one of the effective pathways to fight against breast cancer. Based on the recombinant protein TmSm(T34A) that was designed and prepared in our previous experiments for targeting survivin, an inhibitor of apoptosis protein(IAP), in this study, we explored the effects of TmSm(T34A) on BCSCs obtained by enriching in serum-free suspension, sorting and characterizing of MCF-7/ADM. The results showed that TmSm(T34A) could not only inhibit the proliferation and growth of BCSCs by decreasing CD44⁺CD24⁻ proportion and down-regulating the expression of Cyclin D1 significantly, but also induce BCSCs apoptosis evidently. Furthermore, in BCSCs xenograft nude mice administrated TmSm(T34A), the tumor growth was slower than that of the control obviously. Thus it can be seen TmSm(T34A) would be a promising potential protein for treatment of breast cancer by effecting on BCSCs.     

Influence of in vitro biomimicked stem cell ‘niche’ for regulation of proliferation and differentiation of human bone marrow‐derived mesenchymal stem cells to myocardial phenotypes: serum starvation without aid of chemical agents and prevention of spontaneous stem cell transformation enhanced by the matrix environment

Niche appears important for preventing the spontaneous differentiation or senescence that cells undergo during in vitro expansion. In the present study, it was revealed that human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) undergo senescence‐related differentiation into the myocardial lineage in vitro without any induction treatment. This phenomenon occurred over the whole population of MCSs, much different from conventional differentiation with limited frequency of occurrence, and was accompanied by a change of morphology into large, flat cells with impeded proliferation, which are the representative indications of MSC senescence. By culturing MSCs under several culture conditions, it was determined that induction treatment with 5‐azacytidine was not associated with the phenomenon, but the serum‐starvation condition, under which proliferation is severely hampered, caused senescence progression and upregulation of cardiac markers. Nevertheless, MSCs gradually developed a myocardial phenotype under normal culture conditions over a prolonged culture period and heterogeneous populations were formed. In perspectives of clinical applications, this must be prevented for fair and consistent outcomes. Hence, the biomimetic 'niche' was constituted for hBM‐MSCs by cultivating on a conventionally available extracellular matrix (ECM). Consequently, cells on ECM regained a spindle‐shape morphology, increased in proliferation rate by two‐fold and showed decreased expression of cardiac markers at both the mRNA and protein levels. In conclusion, the outcome indicates that progression of MSC senescence may occur via myocardial differentiation during in vitro polystyrene culture, and this can be overcome by employing appropriate ECM culture techniques. Copyright © 2013 John Wiley & Sons, Ltd.