LC-MS/MS法测定大鼠血浆中低聚仿刺参糖胺聚糖的浓度及初步药物动力学研究

目的 建立大鼠血浆中低聚仿刺参糖胺聚糖（DAHG-1W）的LC-MS/MS测定方法,研究大鼠单次静脉注射DAHG-1W后的血浆药物动力学。方法 大鼠血浆样品经灰色链霉菌蛋白酶预处理、温和酸水解脱除岩藻糖支链、硫酸软骨素ABC酶降解成主链二糖并用2-氨基吖啶酮衍生后,进行液相色谱-质谱联用（LC-MS/MS）分析。色谱分离采用Cortecs UPLC T3色谱柱（2.1mm×150 mm, 1.6 μm）,流动相为10 mM乙酸铵水溶液-甲醇,梯度洗脱。质谱检测采用加热电喷雾离子源（H-ESI）,以负离子方式检测,AMAC标记的CS-4S6S和CS-2S4S（内标）的多反应监测（MRM）离子对均为m/z 732.0→652.4,保留时间分别为9.0和7.4 min。应用上述LC-MS/MS方法测定大鼠尾静脉注射DAHG-1W（5 mg·kg-1）后的血浆药物浓度,采用WinNonLin 6.0版软件计算药代动力学参数。结果 大鼠血浆中DAHG-1W在3.91-125.00 μg·mL-1浓度范围内线性关系良好;日内和日间精密度均小于15%,准确度为102.60%-111.17%;回收率为89.16%-103.35%;无明显基质效应且血浆样品稳定性好。大鼠静脉注射给药后DAHG-1W的血药峰浓度（Cmax）为（36.73±2.10）μg·mL-1,半衰期（t1/2）为（277.31±56.22）min,清除率（Cl）为（0.82±0.09）mL·min-1·kg-1,0-300 min药时曲线下面积（AUC0-t）为（3638.30±45.84）min·μg·mL-1。结论 本研究建立了大鼠血浆中DAHG-1W的LC-MS/MS分析方法,可应用于DAHG-1W在大鼠体内的药物动力学研究。     

探讨单抗注册生产现场检查中的质量控制要点

目的：探讨单抗生产质量控制中的常见问题,指出单抗注册生产检查中的关注点,统一单抗注册生产现场检查标准。方法：以多年的药品检查工作实践为基础, 结合抗体的生物学特点,从“注册现场检查”的角度分析单抗质量控制和生产中存在的问题。结果与结论：从基于体系的横向检查策略和基于品种的纵向检查策略两个维度,明确单抗生产中的关键质控点和统一检查标准,与业界达成共识,共同提高国内抗体的工艺控制水平和质量水平。     

兔半月板纤维软骨细胞的体外去分化研究

目的:比较研究不同代次间兔半月板纤维软骨细胞生物学特性的改变,为构建组织工程半月板和细胞治疗中的种子细胞优选提供进一步的理论和实践基础。方法:采用机械分离与酶连续消化相结合的方法体外分离兔半月板纤维软骨细胞,单层培养传代至第5代。采用相差倒置显微镜和SEM观察各代细胞的形态变化和超微结构,MTT法检测细胞增殖情况并描绘生长曲线,采用细胞化学染色和免疫组化鉴定细胞分泌的蛋白聚糖和Ⅰ、Ⅱ型胶原蛋白。结果:半月板纤维软骨细胞原代接种后8~12 h开始贴壁,48~72h大部分细胞贴壁,胞质逐渐展开,细胞变大伸长,形成突起,呈多角形,细胞形态规则,轮廓清晰,镜下观察有立体感,7~10 d后,细胞长满传代。传代后细胞贴壁生长能力和增殖速度明显加快,原代至第2代细胞形态较规则,多呈多角形,大小均一。第3代后随着传代次数的增加,细胞中梭形细胞增多,分泌和增殖能力下降,传代周期延长。第5代分裂相少见,密度稀疏,细胞形态不佳,约80%呈长梭形,分泌和增殖能力下降。SEM示第1代半月板细胞形态规则,呈多极性,表面有突起;第5代细胞形态不规则,多呈梭形。生长曲线可见第4代前半月板细胞生长速度及生长周期均相似,第5代后细胞生长速度减慢,增殖减缓。细胞化学和免疫组化染色分析胶原和蛋白聚糖的表达:随传代的进行,Ⅱ型胶原蛋白和蛋白聚糖的表达逐渐减弱而Ⅰ型胶原表达逐渐增强。结论:体外单层培养条件下,随着传代的进行,细胞活力逐渐下降,生长速度减慢,传代周期延长,逐渐变为梭形,形态不规则。体外单层培养系统中半月板纤维软骨细胞表型随传代的进行,Ⅱ型胶原蛋白和蛋白聚糖的表达逐渐减少而Ⅰ型胶原表达逐渐增多,体外单层培养条件下培养的半月板纤维软骨细胞从第3代开始逐渐失去其特异性表型,发生去分化,逐渐变为成纤维细胞的表型。体外单层分离培养的第5代前半月板细胞基本维持纤维软骨细胞的表型和生物学特性,可作为组织工程半月板的种子细胞。     

病变深度对乳腺良恶性病变超声弹性成像诊断效果的影响

目的 探讨占位性病变的深度对乳腺良、恶性病变超声弹性成像诊断效果的影响.方法 选取2010年2月～2014年10月进行超声弹性成像诊断的乳腺占位患者190例,按照能否获得满意弹性图像为标准分为满意组(170例)和不满意组(20例),收集两组患者病灶位置、病灶直径大小等资料,计算不同深度病灶的超声弹性成像的灵敏度、特异度、准确度,并进行统计学分析.结果 深度≤2.0 cm的病灶获得满意弹性成像的数量明显多于＞2.0 cm的病灶,二者图像满意率差异有统计学意义(P＜0.05);直径≤2.0 cm的病灶,图像满意组人数多于图像不满意组(P＜0.05);病灶深度＜0.5 cm时,准确度和特异度最高,＞1.5 cm时灵敏度降低.结论 病灶越浅、脂肪越少、乳房层越薄,越容易得到满意的弹性图像;同等深度时,病灶直径越大,对成像越有利,病灶病变深度会影响成像的准确度、特异度、灵敏度.     

Computed tomography arterial portography for assessment of portal vein injury after blunt hepatic trauma

PURPOSE

Intrahepatic portal vein injuries secondary to blunt abdominal trauma are difficult to diagnose and can result in insidious bleeding. We aimed to compare computed tomography arterial portography (CTAP), reperfusion CTAP (rCTAP), and conventional computed tomography (CT) for diagnosing portal vein injuries after blunt hepatic trauma.

METHODS

Patients with blunt hepatic trauma, who were eligible for nonoperative management, underwent CTAP, rCTAP, and CT. The number and size of perfusion defects observed using the three methods were compared.

RESULTS

A total of 13 patients (seven males/six females) with a mean age of 34.5±14.1 years were included in the study. A total of 36 hepatic segments had perfusion defects on rCTAP and CT, while there were 47 hepatic segments with perfusion defects on CTAP. The size of perfusion defects on CT (239 cm³; interquartile range [IQR]: 129.5, 309.5) and rCTAP (238 cm³; IQR: 129.5, 310.5) were significantly smaller compared with CTAP (291 cm³; IQR: 136, 371) (both, P = 0.002).

CONCLUSION

Perfusion defects measured by CTAP were significantly greater than those determined by either rCTAP or CT in cases of blunt hepatic trauma. This finding suggests that CTAP is superior to rCTAP and CT in evaluating portal vein injuries after blunt liver trauma.The liver is one of the most frequently injured solid abdominal organs in the setting of blunt abdominal trauma (1). Fortunately, most patients with blunt hepatic trauma have relatively stable vital signs and need only supportive treatment or transarterial embolization (TAE) (1–9). Only 15% of patients, who present with hemodynamic instability or fail with nonoperative management, require operative intervention to manage their liver injury.Embolic therapy has been shown to have a high success rate in hemodynamically stable patients with blunt hepatic injury. TAE is associated with decreased abdominal infections, decreased transfusions, and decreased length of hospital stay compared with operative management (2, 3, 7). However, angiography can only detect bleeding from the hepatic artery; it cannot locate bleeding from the hepatic or portal vein. In the literature, portal vein injuries are not commonly described and most are the result of penetrating injuries to the extrahepatic portal veins. Mortality after a portal vein injury due to trauma is primarily due to hypovolemic shock and can be as high as 50% or greater (10, 11).Since the intrahepatic portions of the hepatic and portal veins are low pressure systems, they can bleed insidiously. Nevertheless, this subtle bleeding may require multiple transfusions and result in a prolonged hospital stay. Relative to an extrahepatic portal vein injury, patients with an intrahepatic portal vein injury may have relatively stable vital signs and slowly decreasing hemoglobin levels (10, 11). In addition, traumatic occlusion and/or thrombosis of the portal vein may cause large hepatic parenchymal infarction.Computed tomography arterial portography (CTAP) is a useful method based on portal enhancement of the liver by infusion of contrast material through the superior mesenteric artery for evaluating the portal venous system (12–15) and is widely used in patients with hepatic tumors with portal venous invasion (13, 16, 17). CTAP has a high sensitivity and specificity in the evaluation of portal vein thrombosis due to tumor (90% sensitivity, 99% specificity, 95% positive predictive value, 97% negative predictive value) (14). However, few studies have focused specifically on the utility of CTAP in the evaluation of portal vein injury as a result of trauma.The liver has a dual blood supply and receives between 66% and 75% of its blood supply from the hepatic portal vein with the remainder supplied by the hepatic artery (18). CTAP reflects only portal venous perfusion while reperfusion CTAP (rCTAP) reflects hepatic arterial reperfusion. Both rCTAP and conventional computed tomography (CT) are useful for determining certain liver injuries. However, they do not specifically evaluate the portal vein.The purpose of this study was to compare CTAP, rCTAP, and CT for diagnosing portal vein injuries after blunt hepatic trauma. We hypothesized that CTAP would be superior to rCTAP and CT in assessing portal vein injury after blunt hepatic trauma.     

祛白酊的质量控制方法研究

目的建立祛白酊的质量控制方法。方法采用微乳薄层色谱法,对祛自酊中的枙子、红花进行定性鉴别;以Diamonsil C₁₈（250 mm×4.6 mm,5μm）为固定相,采用HPLC法对栀子中有效成分栀子苷和红花中有效成分羟基红花黄色素A进行含量测定,以（A）乙腈-水（15:85）,（B）甲醇-0.83%磷酸溶液（30:70）为流动相,梯度洗脱,检测波长:0¹0 min,238nm,10²0 min,403 nm,流速:1.0 ml·min^-1,柱温:25℃,进样量:10μl。结果栀子和红花薄层色谱中与对照药材主斑点相应位置斑点清晰,阴性对照无干扰;栀子苷和羟基红花黄色素A分别在9.6¹44.0、2.4³6.0μg·ml^-1范围内线性关系良好,加样回收率分别为100.30%和100.08%。结论本方法简便、准确、重复性好,可作为祛白酊的质量控制方法。     

纳晶微针的促渗透作用及安全性实验研究

目的探讨纳晶针作用于皮肤后,可在皮肤表面形成开放给药通道,从而促进外用药物经皮渗透和吸收的价值及安全性。方法 (1)扫描电镜观察纳晶针作用后在皮肤表面形成针孔的存在和闭合;(2)以0.125%荧光素钠为模型药物,在荧光显微镜下对比观察纳晶针+荧光素钠和单纯外用荧光素钠后,荧光在大鼠皮肤的分布情况。结果 (1)纳晶针可在皮肤表层形成给药通道,该通道在20 min左右即可闭合;(2)经过纳晶针处理过的大鼠皮肤可见耀眼的荧光(+++);而未经纳晶针处理的大鼠皮肤仅在角质层可见明确的线状荧光(+),其他部位可见微弱荧光(-)。结论纳晶针对外用药物有明显的促进渗透作用,且安全简便。