超声微泡联合Ad-EGFP/HIF-1α介导EPCs归巢大鼠缺血心肌

目的 观察超声破坏微泡联合Ad-EGFP/HIF-1α介导EPCs移植归巢大鼠缺血心肌的可行性及有效性。方法 采用Percoll密度梯度离心法分离SD大鼠骨髓人脐血单个核细胞(MNCs),血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)及表皮生长因子(EGF)诱导培养;Ad-EGFP/HIF-1α在HNK293细胞中进行扩增,之后感染体外培养的内皮祖细胞(EPCs)。将30只SD大鼠建立MI模型后随机分为5组:①空白对照组(C),②超声＋微泡组(US+MB),③单纯EPCs组(EPCs),④超声＋EPCs组(US+EPCs),⑤超声＋微泡+EPCs组(US+MB+EPCs)。EPCs移植后48 h处死大鼠,以激光共聚焦显微镜观察大鼠心肌组织内EPCs的分布,Western blot 检测HIF-1α的表达。结果 US+MB+EPCs组绿色荧光强度高于其他各组,Western blot显示,US+MB+EPCs组HIF-1α蛋白表达高于其他组(P<0.05)。结论 超声破坏微泡联合Ad-EGFP/HIF-1α介导EPCs移植可有效促进EPCs归巢,为干细胞移植治疗IHD提供了一种新的途径。     

超声微泡介导血管内皮生长因子基因转染大鼠阴茎组织

目的 探讨超声靶向破坏微泡介导血管内皮生长因子165(VEGF₁₆₅)转染于高脂模型大鼠阴茎海绵体组织的可行性。方法 以含4％胆固醇及1％胆酸饲料饲养36只2月龄雄性SD大鼠3个月,建立大鼠高脂模型,将其随机均分为高脂模型组(对照组)、VEGF₁₆₅组和1.0 W/cm²超声+微泡+VEGF₁₆₅组(US+MB＋VEGF₁₆₅组)。于基因转染后7天处死大鼠,采用荧光定量PCR检测VEGF₁₆₅基因表达水平,以Western Blot检测大鼠阴茎组织VEGF蛋白质的表达水平,用免疫组织化学法(IHC)检测大鼠阴茎组织内皮型一氧化氮合成酶(eNOS)蛋白质表达。结果 转基因7天后,US+MB＋VEGF₁₆₅组VEGF₁₆₅基因水平明显高于VEGF₁₆₅组及对照组(P均<0.05),其阴茎海绵体组织VEGF蛋白质表达较VEGF₁₆₅组及对照组增加(P均<0.05);IHC结果显示,US+MB＋VEGF₁₆₅组大鼠阴茎组织eNOS较其他组高表达(P均<0.05)。结论 超声靶向破坏微泡可介导VEGF₁₆₅基因在大鼠高脂模型阴茎海绵体组织的高效转移,为基因治疗高脂勃起功能障碍提供了实验依据。     

超声靶向破坏微泡介导骨髓间充质干细胞移植促进大鼠血管新生

目的 探讨超声破坏微泡介导骨髓间充质干细胞(MSCs)移植在大鼠缺血骨骼肌中的作用. 方法 24只Wister大鼠离断右侧股动脉7 d后分为4组:①超声+微泡组(US+MB);②干细胞静脉移植组(MSCs-iv);③超声+微泡+干细胞静脉移植组(US+MB+MSCs-iv);④对照组.处理后第7 d取局部缺血骨骼肌行免疫组化检测. 结果 US+MB+MSCs-iv组缺血骨骼肌可见较多移植的MSCs,其新生血管数量较其他组明显增加. 结论 超声靶向破坏微泡有可能成为一种增强MSCs移植和促进血管新生的新方法 .     

益肾汤对糖尿病大鼠阴茎一氧化氮合酶活性影响的实验研究

目的 研究益肾汤治疗糖尿病勃起功能障碍的可能机制.方法 48只Wistar大鼠随机分为3组:正常组、糖尿病组、治疗组,其中糖尿病组予链脲佐菌素60 mg/kg腹腔注射;治疗组予益肾汤17.7g/(kg·d)灌胃.检测各组2个月后的血糖,阴茎勃起潜伏期,酶组化及图像分析观察阴茎海绵体中一氧化氮合酶(NOS)的活性.结果 治疗组的血糖略低于糖尿病组,差异无显著性(P＞0.05).治疗组阴茎勃起潜伏期明显短于糖尿病组,差异具有显著性(P＜0.05).治疗组的勃起潜伏期、NOS阳性反应与正常组接近.结论 益肾汤能明显缩短阴茎的勃起潜伏期,其可能的机制是提高阴茎海绵体肌NOS的活性,而不是依赖降血糖.     

超声破坏微泡对大鼠皮肤创面愈合影响的实验研究

目的 探讨超声破坏微泡(声诺维)对大鼠皮肤创面愈合的影响.方法 96只SD大鼠建立皮肤创面模型后随机分成4组:超声破坏微泡组、单纯微泡组、单纯超声组和对照组.超声破坏微泡组经鼠尾静脉注射微泡造影剂0.5 ml,同时用声强度1 MHz、2.0 W/cm2超声辐照3 min单纯微泡组经鼠尾静脉注射微泡造影剂0.5 ml;单纯超声组经鼠尾静脉注射生理盐水0.5 ml,同样条件超声辐照3 min对照组经鼠尾静脉注射生理盐水0.5 ml.于创伤后第1、3、5、7、14、21 d利用HE染色和免疫组化方法 观察各组创面肉芽组织生长及血管内皮生长因子(VEGF)表达的情况.结果 HE染色:伤后第7 d,超声破坏微泡组肉芽组织明显比其他3组厚,新生毛细血管均垂直于创面生长,其他3组毛细血管排列紊乱.免疫组化观察VEGF表达变化:超声破坏微泡组在第3 d形成表达高峰,其他3组表达高峰在第5～7 d.结论 超声破坏微泡可以提高大鼠背部皮肤的创面愈合质量;新生肉芽组织成熟早,创面愈合加速.超声破坏微泡时产生的高温、高压及某些化学效应可以刺激内源性VEGF分泌,促进血管生成,从而促进创面愈合.

Abstract：

Objective To investigate the effect of microbubble(Sono Vue) destruction with ultrasound on wound healing in rats. Methods Total 96 SD rats were accepted one rounded whole-layer skin incision on back each other and randomly divided into four groups:microbubble destruction with ultrasound(US + MB),microbubble(MB), ultrasound(US) and control group. Rats in US + MB group were injected with 0.5 ml microbubble contrast agent via tail vein,and then ultrasound irradiated for 3 minutes immediately. MB group were injected with 0.5 ml microbubble contrast agent. US group were injected with 0.5 ml physiological saline,and then ultrasound irradiated for 3 minutes immediately under the same condition. Control group were injected with 0.5 ml physiological saline. Feed each rat in single cage. On day 1,3,5,7,14 and 21 after wound creation,the excised wound tissues were analyzed by histology and VEGF expression in wounds by immunohistochemistry. Results HE staining: On day 7, wounds of US + MB group displayed the most accumulation of granulation tissue and all new capillaries were perpendicular to the wound surface, but the new capillaries of other 3 groups were disordered. Immunohistochemical examination of VEGF expression:the peak expression appeared on day 3 in US + MB group, other 3 groups were on day 5 to day 7.Conclusions US + MB treatment could improve the quality of wound healing and granulation tissues were maturated earlier than MB, US treatment and control group, which could accelerate wound healing. High temperature,high pressure and some kind of chemistry effecs induced by microbubble destruction with ultrasound can stimulate the secretion of endogenous VEGF, which may be the mechanism of promoting angiogenesis and wound healing.     

彩色多普勒超声在诊断阴茎勃起功能障碍中的应用

目的 评估彩色多普勒超声在诊断阴茎勃起功能障碍(erectile dysfunction,ED)中的临床应用价值.方法 应用彩色多普勒超声检测116例疑似血管性勃起功能障碍患者,观察海绵体注射血管活性药物前后阴茎海绵体动脉血流动力学变化情况.结果 非血管性勃起功能障碍48例((41.38%),动脉性勃起功能障碍6例((5.17%),静脉性勃起功能障碍26例(22.4100),疑似静脉性勃起功能障碍24例((20.69%),混合性勃起功能障碍12例(10.35%).注射血管活性药物勃起后非血管性ED组的收缩期峰值流速((PSV)、舒张末期流速(EDV)及阻力指数(RD的数值高于动脉性ED组、静脉性ED组、混合性ED组(P<0.05).静脉性ED组阴茎背深静脉血流速度(DDVV)高于非血管性ED组及动脉性ED组,有显著性差异(P<0.05).结论 彩色多普勒超声技术是目前诊断血管性勃起功能障碍的一种微创而可靠的检查方法,其能够定量反应阴茎的血流动力学状态,为合理治疗勃起功能障碍提供有价值的诊断依据.     

内皮祖细胞与骨髓单个核细胞移植治疗糖尿病下肢缺血大鼠的比较研究

目的探讨内皮祖细胞(EPCs)移植与骨髓单个核细胞移植治疗糖尿病(DM)下肢缺血大鼠的有效性和安全性。方法34只DM雄性 SD大鼠,下肢缺血模型造模后分为3组：A组(n=10)：EPCs 移植;B组(n=12)：骨髓单个核细胞移植;C组(n=12)：生理盐水局部肌注。另取20例雄性 SD大鼠进行 DM造模,并提取骨髓单个核细胞分别用于移植及体外培养诱导分化为 EPCs,将 EPCs进行活细胞染色剂(CM-Dil)标记后移植。移植28 d后处死,取大鼠患肢肌肉标本,检测血管内皮生长因子(VEGF)、内皮型一氧化氮合酶(eNOS)及毛细血管密度(经血管性血友病因子vWF检测)。用 CM-Dil 对 EPCs 进行示踪,检测移植后大鼠患肢 CM-Dil 示踪情况。观察急性排斥反应及肿瘤的发生。结果 A组较 B 组及 C 组患肢 VEGF、eNOS 含量及毛细血管密度均明显增高(P<0.05)。EPCs移植后,患肢观察到CM-Dil标记细胞。移植后28 d未观察到急性排斥反应及肿瘤生长。结论 EPCs较骨髓单个核细胞移植能进一步增加DM大鼠下肢缺血局部 VEGF、eNOS含量和毛细血管密度。     

超声辐照微泡促骨髓间充质干细胞归巢于大鼠肾组织实验研究

目的 观察超声辐照微泡对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)归巢于急性肾小管坏死模型肾组织的作用.方法 将制备成功的40只肾小管坏死模型大鼠随机分为5组(8只),即(1)单纯模型组(对照组);(2)0.5 W/cm2超声+微泡组(0.5 US+MB);(3)干细胞组(MSCs); (4) 0.5 W/cm2超声+微泡+干细胞组(0.5 US+ MB+MSCs);(5)1.0 W/cm2超声+微泡+干细胞组(1.0 US+ MB+ MSCs).7d后将各组大鼠处死,取材用于荧光显微镜观察MSCs在肾组织的分布情况,采用荧光定量PCR测定细胞间黏附分子1(ICAM-1)基因表达水平.结果 采用荧光显微镜观察,DAPI标记的MSCs细胞核(细胞核带蓝色荧光),1.0 US+ MB+ MSCs组肾组织骨髓干细胞数量明显多于0.5 US+ MB+ MSCs组及MSCs组(P＜0.05).0.5 US+MB组、0.5 US+ MB+MSCs组及1.0 US+ MB+MSCs组ICAM-1的基因水平明显高于对照组及MSCs组(P＜0.05).结论 1.0 W/cm2超声辐照微泡促进肾组织细胞ICAM-1表达增加,从而促进MSCs归巢于急性肾小管坏死模型的肾组织.     

超声辐照微泡介导肝细胞生长因子基因逆转大鼠肝纤维化

目的 探讨超声辐照载肝细胞生长因子(hepatocyte growth factor,HGF)基因的超声微泡造影剂逆转大鼠肝纤维化的可行性. 方法 将成模后的40只Wistar大鼠随机分为5组,即(1)超声+载基因超声微泡造影剂组(HGF+ US/MB),(2)超声+单纯质粒组(HGF+ US),(3)质粒+超声微泡造影剂组(HGF+MB),(4)单纯质粒组(HGF),(5)单纯模型组(MA).经股静脉注入超声微泡造影剂,同时超声基因转染治疗仪辐照肝区,辐照条件为300 kHz,2 W/cm2,辐照10 s,间隔10 s,共20 min.于超声辐照后14 d行磁共振弥散加权成像(diffusion weighted imaging,DWI)检查;然后处死各组大鼠,取肝组织HE染色,观察肝纤维化恢复情况;Western Blot检测HGF蛋白在大鼠肝脏中的表达. 结果 HGF+ US/MB组的表观弥散系数(apparent diffusion coefficient,ADC)值明显高于其他各组;相反地,指数表观弥散系数(exponential apparent diffusion coefficient,EADC)低于其他各组;病理组织片可见HGF+ US/MB组汇管区纤维结缔组织增生,但肝小叶结构完整,其余各组纤维组织增生程度强于HGF+US/MB组.同时,HGF+US/MB组的HGF蛋白的表达均高于其他各组(P＜0.05). 结论 超声靶向破坏微泡能够介导HGF基因在肝组织内高效表达,并产生抗纤维化效应,为肝纤维化的基因治疗提供一种新的基因转移途径.     

彩色多普勒超声在诊断阴茎勃起功能障碍中的应用价值

目的 评估彩色多普勒超声在诊断阴茎勃起功能障碍(erectile dysfunction,ED)中的临床应用价值.方法 应用彩色多普勒超声检测79例疑似为血管性勃起功能障碍患者,观察海绵体注射血管活性药物前后阴茎海绵体动脉血流动力学变化情况,测量并加以分析阴茎探动脉收缩期最大血流速度(PSV)、舒张末期最小流速(EDV)、海绵体动脉血流阻力指数(RI)以及阴茎背深静脉的血流状态.结果 非血管性ED 23例(29.1％),动脉性ED 35例(44.3％),静脉性ED 21例(26.6％).注射血管活性药物后非血管性ED组的收缩期峰值流速(PSV),舒张期流速(EDV)及阻力指数(RI)的数值高于动脉性ED组,差异有统计学意义(P＜0.05).结论 彩色多普勒超声技术是目前诊断血管性勃起功能障碍的一种微创而可靠地检查方法,其能够定量反应阴茎的血流动力学状态,为合理治疗勃起功能障碍提供有价值的诊断依据.     

超声破坏微泡联合粒细胞集落刺激因子促进大鼠心肌血管新生的实验研究

目的 探讨超声破坏微泡联合粒细胞集落刺激因子(G-CSF)促进大鼠心肌血管新生的有效性.方法 40只健康成年Wistar大鼠随机分为4组.A组在超声微泡治疗前一天行G-CSF皮下注射预处理;B组仅行超声微泡治疗;C组仅行G-CSF皮下注射;D组为对照.于治疗后2周取材,HE染色光镜观察心肌结构变化,免疫组化法检测心肌组织中VEGF及CD34表达,评定心肌血管新生疗效.结果 A组心肌中有大量VEGF和CD34表达,新生血管较多;B组心肌中有部分VEGF和CD34表达,新生血管相对较少;C组及D组仅有极少量VEGF和CD34表达,未见明显新生血管.结论 超声微泡可刺激心肌内源性VEGF分泌,促进心肌血管新生,G-CSF能明显增强其血管新生作用.     

Antitumor Effect of Docetaxel‐Loaded Lipid Microbubbles Combined With Ultrasound‐Targeted Microbubble Activation on VX2 Rabbit Liver Tumors

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.     

Experimental research on therapeutic angiogenesis induced by hepatocyte growth factor directed by ultrasound-targeted microbubble destruction in rats.

OBJECTIVE: The purpose of this study was to explore the feasibility of therapeutic angiogenesis in myocardial infarction induced by hepatocyte growth factor (HGF) mediated by ultrasound-targeted microbubble destruction. METHODS: Forty Wistar rats were divided into 4 groups after the models of myocardial infarction were prepared: (1) HGF, ultrasound, and microbubbles (HGF+US/MB), (2) HGF and ultrasound, (3) HGF and microbubbles, and (4) surgery alone. Destruction of ultrasound-targeted microbubbles loaded with the HGF gene with an electrocardiographic trigger mode was performed in the HGF+US/MB group. All the rats were killed after being transfected for 14 days. Enhanced green fluorescent protein expression was examined in the myocardium, liver, and kidney in all groups by fluorescence microscopy; CD34 expression was detected by immunohistochemistry, and microvessel density (MVD) was counted in the high-power field on microscopy. Hepatocyte growth factor expression in the myocardium was detected by western blotting and an enzyme-linked immunosorbent assay. RESULTS: Enhanced green fluorescent protein expression was detected in the myocardium of the HGF+US/MB group, but a few areas of HGF expression were detected only in small vessels and the capillary endothelium, and no expression was found in the surgery-alone and HGF and microbubbles groups. The results of MVD counting by microscopy showed that the MVD in the myocardium of the HGF+US/MB group was the highest among all the groups. The results of western blotting and the enzyme-linked immunosorbent assay showed that the amount of HGF in the myocardium was highest in the HGF+US/MB group. CONCLUSIONS: Ultrasound-targeted microbubble destruction could deliver HGF into the infracted myocardium and produce an angiogenesis effect, which could provide a novel strategy for gene therapy of myocardial infarction.     

Ultrasound-Mediated Microbubble Cavitation Transiently Reverses Acute Hindlimb Tissue Ischemia through Augmentation of Microcirculation Perfusion via the eNOS/NO Pathway

Ultrasound-mediated microbubble cavitation improves perfusion in chronic limb and myocardial ischemia. The purpose of this study was to determine the effects of ultrasound-mediated microbubble cavitation in acute limb ischemia and investigate the mechanism of action. The animal with acute hindlimb ischemia was established using male Sprague-Dawley rats. The rats were randomly divided into three groups: intermittent high-mechanical-index ultrasound pulses combined with microbubbles (ultrasound [US] + MB group), US alone (US group) and MB alone (MB group). Both hindlimbs were treated for 10 min. Contrast ultrasound perfusion imaging of both hindlimbs was performed immediately and 5, 10, 15, 20 and 25 min after treatment. The role of the nitric oxide (NO) pathway in increasing blood flow in acutely ischemic tissue was evaluated by inhibiting endothelial nitric oxide synthase (eNOS) with N^ω-nitro-L-arginine methyl ester hydrochloride (L-NAME). In the US + MB group, microvascular blood volume and microvascular blood flow of the ischemic hindlimb were significantly increased after treatment (both p values <0.05), while the microvascular flux rate (β) increased, but not significantly (p > 0.05). The increases were observed immediately after treatment, and had dissipated by 25 min. Changes in the US and MB groups were minimal. Inhibitory studies indicated cavitation increased phospho-eNOS concentration in ischemic hindlimb muscle tissue, and the increase was significantly inhibited by L-NAME (p < 0.05). Ultrasound-mediated microbubble cavitation transiently increases local perfusion in acutely ischemic tissue, mainly by improving microcirculatory perfusion. The eNOS/NO signaling pathway appears to be an important mediator of the effect.     

Normal erectile function

A complex interaction of neural, vascular, and biomolecular activity results in normal erectile function. Two chambers in the penis, the corpus cavernosa, contain sinusoids, which fill with blood and expand to become rigid when smooth muscle is relaxed. Blood flow out of the penis is temporarily stopped (veno-occlusion) to maintain the erection. Neural control is complex but, in general, the sympathetic nervous system inhibits, and the parasympathetic system excites erectile function. The central nervous system can induce erections without peripheral stimuli, e.g. in nocturnal erections. In general, there are 5 recognized phases in erectile response: latent, tumescent, full erection, rigid erection, and detumescent. The key biochemical event in erectile function is an increase in levels of cyclic guanosine monophosphate, which causes smooth-muscle relaxation and allows an erection to occur. This process is mediated by nitric oxide, produced by neurons and elsewhere, which acts as a gaseous messenger molecule.     

NOSTRIN在糖尿病性勃起功能障碍大鼠阴茎海绵体组织中的表达

目的 检测糖尿病性勃起功能障碍（ED）大鼠阴茎海绵体组织中内皮型一氧化氮合成酶运输转导物（NOSTRIN）的表达.方法 将12周龄雄性SD大鼠20只随机分为2组,对照组8只,糖尿病组12只,链脲佐菌素诱导糖尿病性ED模型,8周后检测两组大鼠阴茎海绵体内压,并取阴茎海绵体组织行real time RT-PCR、Western blot、免疫组化染色测定NOSTRIN、eNOS的表达.结果 糖尿病组大鼠阴茎海绵体内压比造模前明显降低（P＜0.01）,而对照组无明显差异.与对照组比较,糖尿病组NOSTRIN表达升高,eNOS表达降低（P＜0.01）.结论 NOSTRIN在糖尿病性ED大鼠阴茎海绵体中表达升高,eNOS表达降低.     

Effect of combined ultrasound and microbubbles treatment in an experimental model of cerebral ischemia

Combined 2-MHz ultrasound (US) and second-generation, sulfur hexafluoride microbubbles (MB) treatment (US+MB) was performed in a permanent middle cerebral artery (MCA) occlusion model in rats to evaluate possible effects on the ischemic cascade. We used 16 Wistar rats and the MCA occlusion model for stroke induction. Glutamate, pyruvate, lactate and glycerol levels were measured by intracerebral microdialysis before and after stroke induction and after US+MB application (n = 8) for 20 h. After 24 h, brain infarct volume, apoptosis and IL-6 and TNF-alpha levels were evaluated. The infarct volume was significantly reduced (p < 0.05) in the US+MB-treated group compared with control animals. In additional, glutamate levels were significantly lower in US+MB-treated animals, and these animals showed a higher rate of apoptotic cell death in the infarcted area. The levels of IL-6 and TNF-alpha concentrations were not different in both groups, and there was no apoptotic cell death outside the infarction in animals treated with US+MB. The results demonstrate that US+MB with second generation microbubbles does not have a harmful effect on ischemic stroke in an MCA occlusion model of the rat. (E-mail: fatar@neuro.ma.uni-heidelberg.de).