Rotator cuff tear is a prevalent musculoskeletal condition. The complex soft-to-hard transition at the tendon-bone interface (TBI) makes the healing process more challenging than in homogeneous tissues. In this study, low-intensity pulsed ultrasound (LIPUS), a non-invasive modality for bone repair, was employed as an external stimulus in combination with tissue engineering. A composite scaffold, designated as BMP-2/bFGF@GM-PLA, was developed by incorporating a gelatin-methacryloyl (GM) hydrogel loaded with bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (bFGF) into polylactic acid (PLA) electrospun fibers. Under LIPUS stimulation, the BMP-2/bFGF@GM-PLA scaffold effectively promotes migration, osteogenic and tenogenic differentiation of the bone marrow mesenchymal stem cells through synergistic mechanical and chemical cues. In vivo, the combined system of BMP-2/bFGF@GM-PLA implantation and post-operative LIPUS treatments significantly enhances healing in rat models of rotator cuff tear, as evidenced by enhanced biomechanical properties, accelerated bone defect repair and improved histological structure of the TBI. This study proposes a novel approach that combines LIPUS stimulus with acoustic-responsive biomaterial scaffolds, thereby coordinating mechanical and chemical cues to facilitate rotator cuff healing.

Thanks to containing tumor‐related molecules, small extracellular vesicles (sEVs) are emerging as biomarkers in tumor liquid biopsy and commonly employed to diagnose a specific cancer. However, a pan‐cancer screening method for pre‐symptomatic patients is crucial to achieving the early cancer detection. Herein, a lipid‐protein capture system on herringbone (HB) microfluidic chip ( HB EV‐Chips) to isolate multiple tumor‐derived sEVs is constructed. Phosphatidylserine (PS) is abnormally surface‐supposed on tumor‐derived sEVs and binds T‐cell immunoglobulin domain and mucin domain‐containing protein 4 (Tim4) in calcium‐dependent manner. PS + sEVs isolated by the HB EV‐Chips is perform on liquid chromatography electrospray ionization tandem mass spectrometry and it is found that PS + sEVs are highly correlated with tumor‐related pathway ( P < 0.05), such as Cdc42 protein signal transduction, neuropilin signaling pathway, and Wnt signaling pathway. And it is further validated in clinical sample and found that PS + sEV number shows statistical difference between cancer patients and healthy donors in plasma ( P < 0.01) and has efficient diagnostic power (area under curve = 0.86), suggesting PS + sEV as potential biomarker for pan‐cancer screening. The Tim‐4 functionalized device facilitates the early multiple cancer detection, providing the potential to be used as a rapid‐screening tool in clinical setting.

外泌体是一类参与多种生理病理过程的细胞间信息交流载体,其脂质双分子层结构能有效维护所携带的生物信息分子的稳定性.因此,外泌体相对于血清、血浆等更加适合于组学研究,如蛋白质组学、转录组学及代谢组学等.随着质谱技术的不断发展,蛋白质作为诸多生物功能的直接执行者受到越来越多的关注,尤其是外泌体内的蛋白质及其生物功能.外泌体用于蛋白质组学的研究有助于阐明肿瘤的发生和发展机制,进而寻找特异性的肿瘤标志物和可用于精准治疗的靶点.

Exosomes, which are lipid membrane-enclosed nanovesicles (30-150 nm in diameter), contain abundant proteomic and genetic information. Specifically, tumor-derived exosomes have been recognized as ideal biomarkers for cancer diagnosis. However, effective methods for isolating as well as detecting exosomes are still challenging. Herein, we have developed an immunoaffinity-based microfluidic device HBEXO-Chip which can directly separate exosomes from plasma. This separation technology enables capturing the exosomes through tumor specific surface markers, such as the pancreatic cancer target Glypican-1 employed in this study, to prevent the interference of non-specific exosomes. Compared with the traditional exosomes separation workflow, (EXO)-E-HB-Chip shows excellent specific exosomes separation performance. We demonstrated that GPC1+ exosomes can distinguish pancreatic cancer from Pancreatitis group and healthy control group. Additionally, we identificate a miRNA signature for pancreatic cancer through RNA sequencing. Finally, we found 2 miRNAs with diagnostic value for pancreatic cancer in these differentially expressed miRNAs, namely miR-125b-5p and miR-214-3p.

Due to extensive metastasis, poor blood supply, and drug‐resistant, there is still no effective clinical means to treat peritoneal dissemination of gastric cancer. Here, an aptamer‐siRNA chimera (Chim)/polyethyleneimine (PEI)/5‐fluorouracil (5‐FU)/carbon nanotube (CNT)/collagen membrane is constructed, which could be divided into 15 layers with a thickness of 70–100 µm. Sustained release experiments show that the collagen membranes can control 5‐FU release for more than 2 weeks. Aptamer‐siRNA chimera can specifically bind to gastric cancer cells, enabling targeted delivery of 5‐FU and silencing drug‐resistant gene. In vitro experiments demonstrated that Chim/PEI/5‐FU/CNT nanoparticles promoted the apoptosis of 5‐FU‐resistant gastric cancer cells, inhibited their invasion and proliferation. Animal experiments show that Chim/PEI/5‐FU/CNT/collagen membrane significantly inhibits the expression of mitogen‐activated protein kinase (MAPK), and effectively treats peritoneal dissemination of 5‐FU‐resistant gastric cancer. Compared with siRNA/PEI/5‐FU/CNT group, ki‐67 proliferation index, and matrix metallopeptidase 9 (MMP9) expression are significantly decreased in the Chim/PEI/5‐FU/CNT group, while the proportion of apoptotic cells is markly increased. In conclusion, a chimera/PEI/5‐FU/CNT/collagen membrane is constructed, which can effectively treat peritoneal dissemination of drug‐resistant gastric cancer. The study provides a new therapeutic approach for relevant clinical treatment.

Borna disease virus (BDV) is a neurotropic and non-cytolytic virus, which causes behavioral disorders in a wide range of warm-blooded species. It is well established that BDV induces neurodegeneration by impairing neurogenesis and interfering with neuronal functioning in the limbic system. In the present study, the potential role of BDV infection in SH-SY5Y cells was identified, and comparisons of two original BDV strains (the human Hu-H1 and the laboratory Strain V) were performed to further elucidate the phenotypes of BDV pathogenesis with strain differences. Cell Counting Kit-8 and flow cytometric analyses revealed that the two BDV strain-infected groups exhibited marked anti-proliferation and cell cycle arrest compared with the control group, and the Hu-H1 strain caused more evident effects. However, the Hu-H1 strain did not exert effects on the apoptosis of SH-SH5Y cells, while Strain V led to a marked increase in apoptosis upon initial infection. Western blot analysis confirmed the upregulation of apoptosis regulator BAX protein and the downregulation of apoptosis regulator Bcl-2 protein caused by the two BDV strains. The results of the present study provided evidence that infection with BDV suppressed SH-SY5Y cellular functioning and exhibited divergent antiproliferative and apoptotic roles in cells between the two strains. The present study provided an insight for future investigation of strain differences and underlying pathomechanisms.

Nonviral gene transfer by ultrasound-targeted microbubble destruction (UTMD) is an promising technique for RNA interference (RNAi) therapy. Targeting silence survivin gene may provide an important therapeutic option for patients with ovarian cancer. However, UTMD mediated RNAi therapy typically uses nontargeted microbubbles with suboptimal gene transfection efficiency. In this work, a LHRHa targeted microbubble agent and recombinant expression plasmid of shRNA targeting survivin gene (pshRNA survivin) were constructed for UTMD mediated pshRNA survivin therapy in ovarian cancer A2780/DDP cells that express LHRH receptors. The targeted microbubbles (TMBs) mixed with the pshRNA survivin were added to cultured ovarian cancer cells followed by ultrasound exposure (1 MHz, 0.5 W/cm(2)) for 30 s. After transfection for 48 h, the expression of survivin mRNA and protein were (0.36 ± 0.036) and (0.05 ± 0.02), respectively. The cell proliferation inhibitory rates at 24, 48, and 72 h after treatment are (42.08 ± 3.20)%, (54.60 ± 1.02)%, and (74.25 ± 2.14)%, respectively, and the apoptosis rate was (28.99 ± 2.70)%. The expression of apoptosis related protein caspase-9 and caspase-3 were (0.95 ± 0.09) and (2.6 ± 0.21). In comparison with the other treatment groups, ultrasound mediation of targeted microbubbles yielded higher RNAi efficiency and higher cell apoptosis rate and cell proliferation inhibitory rate (p < 0.05). Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles will enhance RNAi efficiency in ovarian cancer cells. This novel method for RNA interference represents a powerful, promising no viral technology that can be used in the tumor gene therapy and research.

Pericardiocentesis is the life-saving procedure and accurate needle tip location is one of the important considerations to lower the procedure-related complications [ [1] Loukas M. Walters A. Boon J.M. Welch T.P. Meiring J.H. Abrahams P.H. Pericardiocentesis: a clinical anatomy review. Clin Anat. 2012; 25: 872-881 Crossref PubMed Scopus (47) Google Scholar ]. Although, based on strategies, like ECG, fluoroscopy or echocardiography, the perforation of myocardium and coronary artery are largely reduced in comparison to the blind manipulation, it is difficult to confirm the needle tip in some cases [ [2] Ainsworth C.D. Salehian O. Echo-guided pericardiocentesis: let the bubbles show the way. Circulation. 2011; 123: e210-e211 Crossref PubMed Scopus (30) Google Scholar ].

Objective The objective was to explore the feasibility of ultrasound-microbubble-mediated hepatocyte growth factor (HGF) gene transfer for treating rat hepatic fibrosis induced by CCl4. Methods Forty-eight male SD rats were divided into ultrasound-microbubble-HGF group (U-M-HGF group), ultrasound-HGF group (U-HGF group), microbubble-HGF group (M-HGF group), HGF group (HGF group), CCl4 group (control group), and normal group. The serum levels of alanine transaminase (ALT), aspartate transaminase (AST), total protein, albumin (ALB), and globulin (GLB) and the ratio of ALB/GLB were determined after treatment. The degree of hepatic fibrosis was evaluated by histopathological numerical scores. The protein expressions of HGF, collagen I, collagen III, and α-smooth muscle antibody (α-SMA) were detected by immunohistochemistry. Results Ultrasound-microbubble-mediated HGF therapy significantly reduced the serum level of ALT and AST to 59.88% and 49.18% of the control group, respectively. Ultrasound-microbubble-mediated HGF therapy prevented liver fibrosis, with an obvious decrease in fibrosis areas and extracellular matrix production of collagen I, collagen III, and α-SMA. The gene therapy could induce HGF delivery into the fibrotic liver effectively. Conclusions Ultrasound-microbubble-mediated HGF gene therapy can reduce liver fibrosis, which provides a novel strategy for gene therapy of chronic liver disease.

Objective To explore the anti-tumor effects of lipid microbubbles loaded hematoporphyrin(LMLH) on rabbit liver VX2 tumor using ultrasound mediated target drug delivery.Methods Thirty-five New-Zealand rabbits were induced into liver VX2 tumor and randomly divided into 7 groups(each n=5),i.e.Ultrasound+LMLH group(US+LMLH),Hematoporphyrin(HP) +ultrasound group(US+HP),ultrasound + lipid microbubbles group(US+MB),LMLH alone group(LMLH),HP alone group(HP),US alone group(US) and normal saline(NS) alone as control group(C).Ultrasonography,including 2D,CDFI and CEUS were applied to detect the size,echo and blood of tumor,and the tumor growth inhibiting rate was calculated.The tumors were harvested to observe in transmission electron microscope,while the intrahepatic metastasis and distant metastasis were observed.Results The tumors in US+LMLH group had mixing echo.CDFI and CEUS showed that the blood vessels obviously decreased,and the tumor growth inhibiting rate was higher in US+LMLH than in other groups.There were less metastases in US+LMLH group than in other groups.Ultramicrostructure showed cellular membrane was broken,and mitochondria were obviously swell.Conclusion HP can be efficiently activated by ultrasound.This technique can inhibit tumor growth significantly in vivo.

Objective To explore the safety and effectiveness of diagnostic ultrasound associated with microbubbles to open the blood brain barrier(BBB).Methods Microbubbles were injected through caudal vein,the rat head was radiated by GE Vivid 7 diagnostic ultrasound immediately.The radiated depth was located in the basal ganglia assisted by magnetic resonance imaging(MRI)scanning.The degree of BBB opening was evaluated by enhanced MRI and Evans blue dyeing.The safety was inspected by observation of cell morphology under hematoxylin eosin(HE)staining.Results After the rat head radiated by diagnostic ultrasound with microbubbles,signal enhancement of the radiated area was observed on post contrast T1-weighted images.Red fluorescence of Evans blue was detected by fluorescence microscope in the same area.Normal cellular morphology and structural integrity were showed by HE staining.Conclusions The BBB of rat could be opened targetedly and noninvasively by diagnostic ultrasound associated with microbubbles.This may provide a new strategy for the drugs and stem cells treatment in the central nervous system diseases. Key words: Ultrasonography; Microbubbles; Blood-brain barrier

Objective To observe the inhibition effect of docetaxel-loaded lipid microbubbles (DLLM) combined with ultrasound targeted microbubbles destruction (UTMD) on microvessel in rabbit VX2 liver tumor models.Methods Sixty rabbits were randomly divided into 6 groups (n= 10),i.e.Doc group (used docetaxel only),DLLM group (used docetaxel-loaded lipid microbubbles),Doc+US group (used docetaxel combined with ultrasound positioning irradiation),PLM+US group (used microbubbles combined with ultrasound positioning irradiation),DLLM+US group (used docetaxel-loaded lipid microbubbles combined with ultrasound positioning irradiation) and control group.The expression of CD34 and VEGF and microvessel density (MVD) were compared among different groups.Results After treatment,the expression of CD34 in DLLM+US group was lower,the MVD of DLLM+US group was markedly lower than that of the other groups (P＜0.01),while the expression of VEGF in this group was the lowest among all 6 groups (P＜ 0.01).Conclusion DLLM combined with UTMD can inhibit the generation of microvessels in rabbit VX2 liver tumor,thus inhibit the growth of the tumor.

OBJECTIVE:The purpose of the study was to explore the antitumor effect of docetaxel-loaded lipid microbubbles combined with ultrasound-targeted microbubble activation (UTMA) on VX2 rabbit liver tumors.METHODS:Docetaxel-loaded lipid microbubbles were made by a mechanical vibration technique. VX2 liver tumor models were established in 90 rabbits, which were randomly divided into 6 groups, including control, docetaxal-loaded lipid microbubbles alone, docetaxal alone, docetaxal combined with ultrasound, pure lipid microbubbles combined with ultrasound, and docetaxel-loaded lipid microbubbles combined with ultrasound (DOC+MB/US). The tumor volume and inhibition rate (IR) of tumor growth were calculated and compared. Apoptosis was detected by terminal deoxyuridine nick end labeling. Proliferating cell nuclear antigen and matrix metalloproteinase 2 (MMP2) protein expression was detected by immunohistochemistry. Caspase 3 and MMP2 messenger RNA (mRNA) expression was detected by in situ hybridization histochemistry. The tumor metastasis rate and survival time of the animals were compared.RESULTS:The IR and apoptotic index of the DOC+MB/US group were the highest among all groups, and the proliferating labeling index was the lowest. Matrix metalloproteinase 2 protein and mRNA expression in the DOC+MB/US group was the lowest among all groups, and caspase 3 mRNA expression in the DOC+MB/US group was the highest. The extensive metastasis rate in the DOC+MB/US group was the lowest, and the survival time of the animals in the DOC+MB/US group was the longest.CONCLUSIONS:Docetaxel-loaded lipid microbubbles combined with UTMA could inhibit the growth of VX2 rabbit liver tumors by deferring proliferation and promoting apoptosis, which may provide a novel targeted strategy for chemotherapy of liver carcinoma.

Objective To assess the application of contrast-enhanced ultrasound(CEUS) in evaluation of the renal cortical and medullary perfusion changes before and after acute renal failure(ARF) in rabbits.Methods Rabbit ARF models were established with intramuscular 50% glycerin(12—15 ml/kg) injection into rabbits' thighs.One day before and after ARF models establishment,CEUS was performed on each rabbit.The renal perfusion time-intensity curve(TIC) was analyzed,including parameters like AT(arrival time),TTP(time to peak intensity),A(amplitude of peak intensity),AUC(area under the curve),β(slope rate of TIC) of renal cortex and medulla.Results The value of A and β before model establishment was(17.36±13.73) dB and(5.38±2.08) dB/s,respectively,both was significantly higher than those after glycerin injection([6.59±4.25] dB and [1.58±1.41] dB/s,respectively)(P0.05).The value of TTP and AUC before model establishment was(2.46±1.76) s and(329.31±171.70) dBs,both was significantly lower than those after injection([5.93±4.80] s and [722.28±354.14] dBs,respectively)(P0.05).No significant difference was found in AT of renal cortex and AT,A,TTP,AUC,β of renal medulla,nor in SCr or BUN.Conclusion CEUS can display the changes of perfusion image of the renal cortex of the ARF earlier than SCr and BUN.

Rationale and Objectives. Ultrasound-targeted microbubble destruction is a promising technology for the targeted gene delivery. The purpose of the present Study is to prepare it novel lipid ultrasound microbubble-carrying gene and transactivating transcriptional activator (Tat) peptide and to investigate its transfection effect in vivo.Methods and Materials. Lipid Ultrasound microbubbles were prepared using mechanical vibration. and the appearance, distribution, concentration, diameter, and zeta potential of the lipid ultrasound microbubbles were measured. The efficiencies of the microbubble carrying gene and Tat peptide were investigated using the fluorospectrophotometer. Contrast-enhanced Ultrasonography was performed oil normal rabbits to observe the duration and intensity of enhancement in myocardium. Quantitative analysis wits detected using the DFY Ultrasound Image Analyzer. Transfection in vivo was performed using the CGZZ ultrasound gene transfection instrument. The expression of enhanced green fluorescent protein in the organs was observed using confocal laser scanning microscope.Results. The diameter of the lipid microbubbles carrying gene and Tat wits (2.3 +/- 0.4) mu m, the concentration was (3.1 +/- 0.4) x 10(9)/mL, and Zeta potential wits (2.0 +/- 0.1) mV. The gene encapsulation efficiency of the lipid ultrasound microbubbles was 32%, and the Tat encapsulation efficiency was 35%. In vivo experiment showed that lipid ultrasound microbubbles Could enhance the echo intensity and transfection efficiency.Conclusion. Lipid microbubbles containing gene and Tat peptide can be used as a new vehicle for gene transfection.

OBJECTIVE:The purpose of this study was to explore the feasibility of using ultrasound-targeted microbubble destruction to treat liver fibrosis induced by hepatocyte growth factor (HGF).METHODS:Forty Wistar rats were divided into five groups after the models of liver fibrosis were prepared: (1) HGF, ultrasound, and microbubbles (HGF+US/MB); (2) HGF and ultrasound (HGF+US); (3) HGF and microbubbles (HGF+MB); (4) HGF (HGF); and (5) model alone (MA). All rats were killed after being transfected for 14 days. Recovery of the liver was detect by diffusion-weighted imaging (DWI) and pathological methods. Collagen I expression was detected by immunohistochemistry. Hepatocyte growth factor expression in the liver was detect by western blotting.RESULTS:The results of DWI and pathological examination showed the recovery of liver in HGF+US/MB group were better than those of other groups. In HGF+US/MB group, collagen I expression was less, and HGF protein was the highest among all the groups.CONCLUSIONS:Ultrasound-targeted microbubble destruction could deliver HGF into the fibrotic liver and produce an antifibrosis effect, which could provide a novel strategy for gene therapy of liver fibrosis.

目的以固相合成法合成穿膜肽TAT,并对合成产物进行活性评价。方法采用Nа-芴甲氧羰基(fluorenyl-methyloxyloxycarbonyl,FMOC)作为α-氨基的保护基,以逐个延伸的固相合成法合成穿膜肽TAT。应用高效液相色谱和质谱仪测定其纯度及相对分子质量;荧光显微镜观察穿膜肽TAT介导增强型绿色荧光蛋白(enhanced green fluorescent protein,pEGFP)质粒在体外培养人脐静脉内皮细胞中的转染效果以评价穿膜肽TAT的生物活性;MTT法检测穿膜肽TAT与质粒DNA复合物对人脐静脉内皮细胞生长活性的影响。结果高效液相色谱和质谱鉴定所制备穿膜肽TAT的纯度为96.6%,相对分子质量1880。它能够携带质粒DNA穿过细胞膜进行基因转染,并对细胞活性无明显影响。结论采用Fmoc固相肽合成法可以成功地合成有生物活性的穿膜肽TAT。

The aim of the present study was to explore the gene transfection efficiency of Tat peptide/plasmid DNA/ liposome (TDL) compound combined with ultrasound-targeted microbubble destruction (UTMD) in human umbilical vein endothelial cell (HUVEC). Tat peptide, plasmid DNA (pIRES2-EGFP-HGF) and Lipofectamine™ 2000 were used to prepare the TDL compound. Microbubbles were prepared using mechanic vibration. The expression of the report gene enhanced green fluorescent protein (EGFP) was observed using fluorescent microscopy and flow cytometry. The viability of HUVEC was measured by MTT assay. mRNA and protein of HGF was analyzed by reverse transcription–polymerase chain reaction and Western Blot. The intensity of green fluorescence and the gene transfection efficiency of TDL compound + microbubbles + ultrasound group were higher than those of other groups, and no significantly different viability was found between TDL compound + microbubbles + ultrasound group and the other groups. The HGF mRNA and HGF protein of TDL compound + microbubbles + ultrasound group were higher than those of other groups. Our finding demonstrated that UTMD could enhance the transfection efficiency of TDL compound without obvious effects on the cell viability of HUVEC, suggesting that the combination of UTMD and TDL compound might be a useful tool for the gene therapy of ischemic heart disease. (E-mail: xcshan@163.com)

RATIONALE AND OBJECTIVES:The aim of this study was to explore the antitumor effects on mice xenografted ovarian carcinoma using the technique of ultrasound-mediated drug release from paclitaxel-loaded lipid microbubbles (PLMs).MATERIALS AND METHODS:Twenty-five ovarian cancer-bearing nude mice were randomly divided into five groups of five mice each. Each group received a unique kind of treatment once a day. These treatments were PLMs combined with ultrasound, intravenous paclitaxel administration, non-drug-loaded microbubbles combined with ultrasound, intravenous PLM administration, and normal saline administration (the control group). After 7 days of consecutive treatment, all mice were sacrificed, and their tumors were harvested to measure volumes and weights. The tumor inhibition rate was calculated by weight. Expressions of vascular endothelial growth factor (VEGF) and p53 in tumor tissues were detected by immunohistochemical staining.RESULTS:Mean tumor volume and weight were the lowest in the first group (PLMs combined with ultrasound), so this group's tumor inhibition rate was the highest (P < .05). On immunohistology, VEGF and p53 expression levels were lowest (P < .05) in the first group.CONCLUSION:Ultrasound irradiation mediates PLM destruction so that the drug is released from the vehicles at the same time. It helps achieve targeted chemotherapy in tumor tissues. This technique has potential to be adopted as a novel tool for ovarian cancer chemotherapy.

目的研究HIV-1Tat蛋白转导域/质粒DNA/Liposome(TDL)复合物介导增强型绿色荧光蛋白(enhanced green fluorescent protein,pEGFP)在体外培养人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)的转染效率。方法将质粒DNA与Tat肽以不同的电荷比混匀,再加入2μl Lipofectamine2000制成TDL复合物。琼脂糖凝胶电泳分析质粒DNA与Tat肽的结合力;荧光显微镜和流式细胞仪观察TDL复合物与lipofectamine2000介导基因在体外培养人脐静脉内皮细胞中的转染效果;MTT法检测TDL复合物对人脐静脉内皮细胞生长活性的影响。结果TDL复合物组琼脂糖凝胶电泳未发现明显DNA条带。TDL复合物的转染率优于单用Lipofectamine2000(P<0.05)。Tat/DNA电荷比为8∶1时,TDL复合物的转染效率最高。TDL复合物组对细胞活性均无明显影响。结论TDL复合物可明显提高基因在体外培养人脐静脉内皮细胞的转染效率,该方法可作为提高基因转染效率的有效手段之一。