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目的 观察微泡造影剂联合超声对胶质瘤大鼠血脑屏障通透性及化疗药物疗效的影响。方法 通过透射电镜观察超微结构、Western blotting检测紧密连接蛋白claudin-5的表达、脑组织伊文蓝染色检测并确认影响Wistar大鼠血脑屏障通透性的最佳造影剂浓度及最佳超声模式。采用大鼠胶质瘤细胞系9L脑纹状体注射建立Wistar大鼠胶质瘤模型,予以替莫唑胺化疗（50mg/kg,连用5d）后,计算瘤体大小,ELISA检测血清中肿瘤标志物胶质纤维酸性蛋白（GFAP）的浓度。结果 微泡造影剂联合超声可使血脑屏障内皮细胞间的连接松弛,最佳造影剂浓度为1ml/kg,最佳超声模式为间隙触发（间隙400ms）10min;声空效应组大鼠中伊文蓝通过血脑屏障的含量明显多于对照组（P〈0.05）。替莫唑胺化疗后,声空效应组肿瘤体积明显低于对照组,而血清GFAP浓度明显高于对照组（P〈0.05）。结论 微泡造影剂联合超声可提高胶质瘤大鼠血脑屏障通透性及化疗药物的疗效。

目的探讨炎症激活条件下间充质干细胞分泌的微泡（LPS-exosomes）对糖尿病慢性创面的治疗作用。方法用终浓度为100ng/ml的脂多糖预处理间充质干细胞2d,收集上清液,利用超速梯度离心法分离提取微泡,并对其进行鉴定。建立糖尿病大鼠皮肤缺损模型,将实验大鼠随机分为3组：未治疗组、间充质干细胞源微泡治疗组、炎症激活间充质干细胞源微泡治疗组,分次多点注射微泡60μg于伤口周边,1次/d,连续10d。于治疗后第3、7、14天进行创面大体观察,q RT-PCR法检测创面炎症相关因子表达水平及巨噬细胞亚型表面标记物的表达改变。结果与未治疗组相比,炎症激活间充质干细胞源微泡治疗组创面愈合速度明显加快,治疗后第7、14天促炎因子IL-1、IL-12及M1型巨噬细胞表面标记物i NOS表达水平明显降低,同时抗炎因子IL-10、TGF-β及M2型巨噬细胞表面标记物CD163表达明显增加,差异均有统计学意义（P〈0.05）。结论炎症激活间充质干细胞源微泡可能通过调节巨噬细胞表型改变,抑制创面局部慢性炎症反应,进而加速糖尿病皮肤创面的愈合。

目的探讨超声、超声造影剂微泡介导基因转染的可行性及所需超声照射时间。方法选择超声能量在MI=1.5、超声频率f=8MHz,向人脐静脉内皮细胞(HUVEC)悬液加入微泡后分别超声辐照20s、1、5、10、15、30min,继续培养24h。然后用台盼蓝拒染法计数活细胞,各组与对照组(无辐照)行统计学分析,选择超声辐照的最佳时长。然后,微泡与增强型绿色荧光蛋白(EGFP)质粒充分孵育后,采用该时长进行超声微泡介导基因转染试验,细胞继续培养48h后,通过荧光显微镜观察EGFP的表达。结果超声辐照10、15、30min组细胞计数比对照组降低,差异有统计学差异(P<0.05)。利用超声能量在MI=1.5、超声频率f=8MHz条件下辐照5min,继续培养48h后荧光显微镜观察,有EGFP表达,细胞生长良好。结论超声微泡能够促进质粒进入细胞并转染基因。超声能量MI=1.5、超声频率f=8MHz时,超声微泡介导基因转染的最佳辐照时长为5min。

Aim： Microvesicles （MVs） are nanoscale membrane fragments released from virtually all cell types upon activation or apoptosis, and may contribute to the beneficial effects of stem cell therapy. In this study, we investigated the therapeutic effects of mesenchymal stem cell （MSC） derived MVs （MSC-MVs） on pulmonary artery hypertension （PAH） in rats. Methods： MSC-MVs were isolated from rat bone marrow MSCs that were cultured in a serum-free conditioned medium. Transmission electron microscopy （TEM）, flow cytometry and nanoparticle tracking analysis （NTA） were used to characterize the MVs. Adult SD rats were injected with monocrotaline （50 mg/kg, sc） to induce PAH. Three weeks later, the rats were intravenously injected with MSCs, MSC-MVs or saline for 2 weeks. At the end of treatments, the hemodynamic parameters and pathological right ventricular and pulmonary arterial remodeling were analyzed in each group. Results： The MSC-MVs showed general morphologic characteristics of MVs and expressed annexin V and CD29 markers under TEM, and their size ranged from 40 to 300 nm. Intravenous injection of MSC-MVs or MSCs significantly ameliorated the mean pulmonary artery pressure （mPAP） and mean right ventricle pressure （mRVP） in PAH rats. Furthermore, intravenous injection of MSC-MVs or MSCs significantly decreased the right ventricle （RV） hypertrophy and pulmonary arteriole area index （AI） and thickness index （TI） in PAH rats. Conclusion： Intravenous injection of MSC-MVs or MSCs produces similar beneficial effects for treating PAH, and our results provide a basis for cell-free approach in stem cell therapy.

Aim： To investigate whether human multiple myeloma （MM） cells secrete microvesicles （MVs） and whether the MVs secreted from MM cells （MM-MVs） promote angiogenesis. Methods： RPMI8226 human MM cells and EA.hy926 human umbilical vein cells were used. MVs isolated from RPMI 8226 cells were characterized under laser confocal microscopy, electron microscopy and with flow cytometry. The fusion of MM-MVs and EA.hy926 cells was studied under confocal microscopy, and the transfer of CD138 to EA.hy926 cells was demonstrated with flow cytometry. The proliferation, invasion and tube formation of EA.hy926 cells in vitro were evaluated using M]-r, transwell migration and tube formation assays, respectively. The vasculization of EA.hy926 cells in vivo was studied using Matrigel plug assay. The expression of IL-6 and VEGF was analyzed with PCR and ELISA. Results： MM-MVs from the RPMI 8226 cells had the characteristic cup-shape with diameter of 100-1000 nm. Most of the MM-MVs expressed phosphatidylserine and the myeloma cell marker CD138, confirming that they were derived from myeloma cells. After added to EA.hy926 cells, the MM-MVs transferred CD138 to the endothelial cells and significantly stimulated the endothelial cells to proliferate, invade, secrete IL-6 and VEGF, two key angiogenic factors of myeloma, and form tubes in vitro and in vivo. Conclusion： Our results confirm the presence of MVs in MM cells and support the idea that MM-MVs are newfound mediators for myeloma angiogenesis and may serve as a therapeutic target to treat MM.

Microvesicles （MVs）, also known as microparticles, are small membrane vesicles released from different cell types under different conditions. MVs have been detected in the circulation and in organs/tissues in various diseases, including diabetes. Patients with different types of diabetes and complications have different cellular MV patterns. Studies have shown that MVs may mediate vascular thrombosis, vascular inflammation, angiogenesis, and other pathological processes of the disease through their procoagulant, pro-inflammatory, pro-angiogenic, proteolytic, and other properties. Therefore, MVs contribute to the development of diabetic macrovascular and microvascular complications. In addition, clinical studies have indicated that changes in MV number and composition may reflect the pathophysiological conditions of disease, and therefore, may serve as potential biomarkers for diagnostic and prognostic use. Understanding MVs＇ involvement in the pathophysiological conditions may provide insight into disease mechanisms and would also be helpful for the development of novel therapeutic strategies in the future. Here, we review the latest publications from our group and other groups and focus on the involvement of MVs in diabetic complications.

Aim： To investigate the effects of serum deprivation （SD） on microvesicles （MVs） secreted from human myeloma cells and the implications for disease progression. Methods： RPMI 8226, U266, and KM3 human myeloma cells were incubated in medium containing 10% （non-SD） or 1% fetal bovine serum （SD） and MVs were isolated. The levels and size distribution of MVs were analyzed with flow cytometry. The protein profiles of MVs were studied using 2D SDS-PAGE, MALDI-TOF.MS, and Western blotting. NF-KB activation was analyzed using EMSA. Angiogenesis was examined in Eahy926 endothelial cells. Results： Exposure of RPMI 8226 ceils to SD for 24 h did not alter the number of apoptotic cells. However, SD increased the number of MVs from RPMI 8226, U266, and KM3 cells to 2.5-, 4.3-, and 3.8-fold, respectively. The size distribution of SD MVs was also significantly different from that of non-SD MVs. Three proteins ZNF224, SARM, and COBL in SD MVs were found to be up-regulated, which were involved in cell cycle regulation, signal transduotion and metabolism, respectively. Co-culture of SD MVs and RPMI 8226 cells increased NF-KB activation in the target RPMI 8226 cells. Furthermore, SD MVs from RPMI 8226 cells significantly increased the microtubule formation capacity of Eahy926 endottlelial cells compared with non-SD MVs. Conclusion： SD elevates the levels of microvesicles with different size distribution and selectively enriched proteins in human myeloma cells in vitro. The selectively enriched proteins, especially ZNF224, may play key roles in regulation of myeloma cells, allowing better adaptation to SD.

The cyclonic-static micro-bubble flotation column （FCSMC） is a highly efficient mineral processing equipment. In this study, a cell-column （FCSMC） integration process was investigated for the separation of bauxite and its feasibility was analyzed on a theoretical basis The properties of low-grade bauxite ore from Henan Province, China were analyzed. Parameters such as reagent dosage, scraping bubble time, and pressure of the circulating pump during the sorting process were investigated and optimized to improve the flotation efficiency. On the basis of these parameters, continuous separation experiments were conducted. Bauxite concentrate with an aluminum-to-silicon （A/S） mass ratio of 6.37 and a 77.63wt% recovery rate were achieved via a flow sheet consisting of ＂fast flotation using a flotation cell, one roughing flotation and one cleaning flotation using flotation columns＂. Compared with the full-flotation-cells process, the cell-column integration process resulted in an increase of the A/S ratio by 0.41 and the recovery rate by 17.58wt%. Cell-coltmm integration separation technology represents a new approach for the separation of middle-to-low-grade bauxite ore.

In the present study, ultrasound-mediated microbubble destruction （UMMD） alone and combined with liposome technology was used as a novel nonviral technique to transfect a Nogo receptor （Nogo-R） shRNA plasmid （pNogo-R shRNA） into neural stem cells （NSCs）. Using green fluorescent protein as a reporter gene, transfection efficiency of NSCs was significantly higher in the group transfected with UMMD combined with liposomes compared with that of the group transfected with UMMD or liposomes alone, and did not affect cell vitality. In addition, Nogo-R mRNA and protein expression was dramatically decreased in the UMMD combined with liposome-mediated group compared with that of other groups after 24 hours of transfection. The UMMD technique combined with liposomes is a noninvasive gene transfer method, which showed minimal effects on cell viability and effectively increased transfer of Nogo-R shRNA into NSCs.