Objective To promote in vitro tissue-plasminogen activator(t-PA)thrombolysis by using microbubbles augmented low frequency ultrasound(US) insonication.Two kinds of microbubbles,albumin-coated vs.lipid-coated were compared.Methods Human blood clot weighted 200～300 mg was made from of 0.8 ml fresh blood.Twenty kHz ultrasound,lipid and albumin-coated microbubbles were applied to separate clots groups with or without t-PA administration.The clots before and after processing were weighed and then the clot dissolution ratios were calculated.Results The simple ultrasound group and the basic t-PA group dissolved((24.72)?(4.83))% and((35.66)?(3.34))% of the clots,respectively.The clot dissolution ratio rised to((42.06)?(4.20))% when coordinated US and t-PA(P(0.05)).Conclusions The in vitro US augmentation of t-PA thrombolysis can be significantly promoted by introduction of microbubbles.But there is no significant thrombolysis difference between albumin and lipid-coated microbubbles.

Objective To investigate the effect of microbubbles mediated ultrasound insonation on proliferation and apoptosis of vascular smooth muscle cells (VSMCs) in different phase of cell cycle. Methods Rat thoracic aortic VSMCs were cultured in vitro by the method of tissue adherence. The cells were synchronized by the methods of serum starvation and double thymidine block. The synchronization results were detected by flow eytometer. VSMCs in different phases of cell cycle were exposed to 1 MHz continuous waves ultrasound for 120 s at intensity 0.3 W/cm2 in the presence of lipid-coated microbubbles (1 ml/L). Apoptosis of VSMCs was analyzed by AnnexinV/PI staining using flow eytometry. The proliferation and the proliferating cell nuclear antigen(PCNA) protein expression of VSMCs were detected by MTT assay and immunoeytochemistry, respectively. Results The synchronized G0/G1 and S phase VSMCs were achieved, with synchronized rates to 89.53 % and 66.87 %, respectively. Ultrasound sonication for 120 s with microbubbles could significantly inhibit the proliferation and downregulate the PCNA expression of S phase VSMCs,but the proliferation and PCNA expression of G0/G1 phase VSMCs were not affected. After treatment of ultrasound with microbubbles, the apoptotic ratio were found to reach (7.05 ± 2.04)% in G0/G1 phase VSMCs and (27.01 ±3.87)% in S phase VSMCs. Conclusions Microbubbles mediated ultrasound insonation can significantly inhabit the proliferation and induce apoptosis in VSMCs at proliferation stage.

Objective To investigate the effect of ultrasound irradiation combined with liposome membrane microbubbles on the reorgnization of cytoskeleton in vascular smooth muscle cells (VSMCs). Methods Rat thoracic aortic VSMCs were cultured in vitro. VSMCs were exposed to 1 MHz continuous waves ultrasound radiation for 120 s at intensity 0.3 W/cm2in the presence of liposome membrane microbubbles (1 μl/ml) after treated with platelet derived growth factor-BB (PDGF-BB). The reorganizations of microfilaments, microtubules and intermediate filaments were examined by using immunofluorescence and fluorocytochemistry techniques. Results There was a substantial increase in the expression of F-actin and assembly of long bundles of stress fibers in the transversed cell body when treated with PDGF-BB. Neither alterations of β-tubulin nor of vimentin cytoskeletal protein organization were observed in PDGF-BB treated cells as compared to those of the contol group. After ultrasound irradiation combined with liposome membrane microbubbles, the expression of F-actin, β-tubulin and vimentin were reduced along with the simultaneous changes in microfilaments, microtubles and intermediate filaments array. Conclusions Ultrasound irradiation combined with liposome membrane microbubbles can induce significant changes in cytoskeleton structure of VSMCs cultured in vitro.

Objective To investigate the value of ultrasound guided autobiopsy technology in interventional diagnosis of pulmo-nary peripheral disease .Methods 117 cases of pulmonary peripheral disease proved by X-ray or CT ,confirmed by ultrasound ,but could not be identified the nature by the examination above ,were biopsied under the guidance of ultrasound .Results All the 117 ca-ses were biopsed successfully .115 cases of them had definite pathological results ,the pathological positivity rate was 98 .3% ,in which contained 20 cases of chronic inflammation ,14 cases of tuberculosis ,30 cases of squamous cell carcinoma ,45 cases of adeno-carcinoma ,6 cases small cell undifferentiated carcinoma .No complication ,such as pneumatothorax ,hemoptysis ,bleeding occurred during the operation .Conclusion The ultrasound guided autobiopsy technology in interventional diagnosis of pulmonary peripheral disease is a safe and effective technique ,the tissues obtained from ultrasound guided autobiopsy were adequate to pathological slide which can provide the basis of the clinical diagnosis .

Objective To explore the targeting homing capacity of bone-marrow derived mesenchymal stem eells(MSCs) on rabbit myocardial ischemia by intravenous injection of MSCs under the mediation of diagnostic ultrasound and microbubble. Methods Density gradient centrifugation and adherent culture method were used in the isolation and cultivation of MSCs. MSCs were labeled with DAPI. Rabbit myocardial infarction(MI) models were builded by totally ligation of left anterior descending branch of coronary artery. DAPI labeled MSCs were implanted by intravenous injection with or without the mediation of diagnostic ultrasound and microbubbles. Forty-eight hours after cell transplantation, the hearts of MI rabbits were made of frozen section and observed under fluorescent microscope. The DAPI positive cells were counted in the MI and border area of rabbit heart and compared between two groups. Pathological changes of MI area were observed with HE staining under light microscope and transmission electronic microscope. Results The number of DAPI positive cells in MI and border area of rabbit in both groups were counted under fluorescent microscope. There were more DAPI positive cells in the MI area in ultrasound + microbubble + MSCs group (213.2±26.5) than that in the intravenous injection group (146.8±18.78, P<0.01). There were erythrocytes leaking out of the vessels in MI area in HE staining section under light microscope in ultrasound + microbubble + MSCs group while there were nearly none in the intravenous injection MSCs group. The intercellular space of endothelial cells of the vessels wall was increased and serum component leaked out of the vessel wall in ultrasound + microbubble + MSCs under transmission electronic microscope. Conclusions The targeted homing capacity of BM-MSCs in the MI area of rabbit heart can be enhanced under the mediation of diagnostic ultrasound and microbubbles.

Objective To investigate the mechanism of the increased permeability of the blood-brain barrier during transcranial ultrasound contrast imaging. Methods Sprague-Dawhy (SD) rats were performed transcranial ultrasound contrast imaging, the lanthanum nitrate and the evans-blue were used as tracers,the distribution of the tracers were observed and the transports mechanism were also investigated. Results The opening of the tight junction and increased permeability of the cellular membrane were observed after the transcranial ultrasound contrast imaging. Conclusions The main mechanism of the increased permeability of the blood-brain barrier was the opening of the tight junction and increased permeability of the cellular membrane.

Objective To explore the value of diagnostic ultrasound mediated microbubble destruction in improving the myocardial perfusion and left ventricular systolic function when cooperated with the mecsenchymal stem cells(MSCs) transplantation in rabbit myocardial ischemia. Methods One week after myocardial ischemia (MI) modeling,36 rabbits were divided into 3 groups,the control group(group Ⅰ) ,intravenous injection of MSCs group(group Ⅱ) and ultrasound + microbubble + MSCs group (group Ⅲ). Myocardial contrast enhancement (MCE) was performed and quantification analysis of anterior wall was assessed with Photoshop. Left ventrieular systolic function was assessed with M-mode echocardiography and bi-plane Simpson's method. CD34 expression in heart was detected with immunohistochemisty(IHC). Western blotting was applied to detect the level of VEGF in three groups. Results The differences of gray scale analyzed with histogram of Photoshop in anterior wall of ischemia myocardium between the group Ⅰ and group Ⅱ or group Ⅲ were significant,and P value was 0. 032 and 0. 000 , respectively. There were significant differences of FS between group Ⅲ (30. 43±4.09)% and group Ⅱ (26.29±2.93)%, P<0.01, and similar to group Ⅰ (19.28 ± 2.84)%. The difference of EF(%) between group Ⅲ and group Ⅱ was significant [(61.5±5.8 vs 53.6±4. 71), P<0. 05] ,or markedly significant between group Ⅲ and group Ⅰ [(61.5±5.8 vs 42.6± 5.0), P <0.01]. EF(%) assessed with bi-plane Simpson's method was significantly increased from (34.64 ± 4.59) in group Ⅰ to (41.78 ± 4.21) in group Ⅱ and (48.6±3.96) in group Ⅲ. The expression of CD34 assessed with immunohistochemistry was the highest in group Ⅲ. The level of VEGF with western blotting in group Ⅲ was significantly higher than other two groups. Conclusions It is an efficacious transplantation means of MSCs infusion under the ultrasound mediated microbubles destruction in improving the myocardial perfusion and cardiac systolic function.

Objective To compare two effective preparation methods for paclitaxel-loaded lipid microbubbles, and to evaluate the physiochemical properties as for acoustic activated drug delivery. Methods Paclitaxel-loaded lipid microbubbles were prepared with two methods: one was mixed with phospholipids directly (Ⅰ); the other was added in triacetin, then mixed with phospholipids (Ⅱ). Concent ration, size, pH, drug entrapment efficiency, drug-loading amounts of these two kinds of paclitaxel-loaded lipid microbubbles were studied, while drug release with ultrasound and tumor imaging enhanced on rabbit breast tumor were observed. Results There was no significant difference in tumor imaging between two kinds of microbubbles which could be ruptured by low energy ultrasound. Compared with Ⅰ, the mean diameter of Ⅱ decreased significantly ([ 1.07±0.38] μm vs [2.79± 0.41] μm, P<0.01), the surface potencial was higher ([19.10±0.32] mV vs [-5.90±0.21] mV, P<0.01), whereas entrapment efficiency and drug-loading amounts increased markedly ([ 95.00±1.22]% vs [36.10±4.74]%, P<0.01; [5.60±0.11]% vs [0.50±0.04]%, P<0.01). Conclusion The Ⅱ paclitaxel-loaded lipid microbubbles added triacetin have an important clinical value.

OBJECTIVETo investigate the effect of cyclopamine on metastatic ability of human esophageal cancer EC109 cells and explore the possible mechanism.

METHODSTranswell chamber assay and angiogenesis assay were used to examine the metastatic ability, invasiveness and angiogenesis of EC109 cells treated with cyclopamine for 48 h. The expression of Gli-1 mRNA was detected using RT-PCR, and Western blotting was used to examine the protein expressions of Gli-1, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF).

RESULTSInhibition of the hedgehog signaling pathway by cyclopamine suppressed the migration, invasion, and angiogenesis of EC109 cells. Cyclopamine treatment significantly lowered the expression of Gli-1 mRNA (P<0.05) and the protein expressions of Gli-1, MMP-9 and VEGF (P<0.05).

CONCLUSIONCyclopamine can significantly inhibit the metastatic capacity of EC109 cells possibly by down-regulating MMP-9 and VEGF expression as a result of Gli-1 inhibition.

Cell Line, Tumor ; Esophageal Neoplasms ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Metastasis ; RNA, Messenger ; genetics ; Signal Transduction ; drug effects ; Transcription Factors ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism ; Veratrum Alkaloids ; pharmacology ; Zinc Finger Protein GLI1

BACKGROUND: A series of basic researches have confirmed that,the olfactory ensheathing cell transplantation can promote spinal cord regeneration and recover some neurological functions of spinal cord in animal models of spinal cord injury.Some clinical trials also prove that transplantation of olfactory ensheathing cells can indeed improve neurological function in patients with spinal cord injury,and then improve their quality of life.OBJECTIVE: To verify the effectiveness and safety of olfactory ensheathing cell transplantation in repair of neurological function of spinal cord injury patients.METHODS: The aborted embryonic olfactory bulb was collected and digested into single olfactory ensheathing cells.After they were cultured and purified 2 weeks,olfactory ensheathing cell suspension was prepared.A total of 213 cases of spinal cord injury were selected.Under general anesthesia,the prepared olfactory ensheathing cell suspension was injected through several target sites surrounding the injured spinal cord.ASIA scale was used to assay the patients before transplantation,3 weeks to 2 months after transplantation,so as to evaluate spinal cord recovery.RESULTS AND CONCLUSION: The spinal cord nerve function in all patients altered to different degrees at 3 weeks postoperation.Spinal cord function score,the sensory and motor functions were significantly increased compared with preoperation(P < 0.001),and showed a trend of continuous improvement with time; the patients were visited as follow-up for no more than 5 years,and no impairment of the restored nervous function or transplant adverse reactions were observed.It is confirmed that olfactory ensheathing cell transplantation can promote the recovery of nerve function in patients with spinal cord injury,it can restore and improve some spinal cord functions,and the treatment is safe.