基于星形聚赖氨酸的基因载体研究

目的：研究合成的以聚乙烯亚胺（PEI）为内核、聚赖氨酸（PLL）为外围的星形嵌段共聚物PEI-g-PLL的基因载体性能。方法：将PEI-g-PLL与pDNA复合形成PEI-g-PLL/pDNA复合物,并用琼脂糖凝胶电泳和zeta电位粒径分析仪表征;CCK-8法检测聚合物的HEK293细胞毒性;检测复合物在HEK293转染后Luciferase表达量以评价其转染效率。结果：PEI-g-PLL能复合pDNA形成尺寸约为200 nm的复合物,在HEK293中有较高的转染效率和较低的细胞毒性。结论：合成的PEI-g-PLL可用作潜在的基因载体。     

还原性多肽共载基因和化学治疗药载体的构建与体外评价

目的 制备一种应用于共载基因和化疗药的硫辛酸（LA）修饰的非内吞机制进入细胞的固有无序正常蛋白-细胞质定位的内化肽6（CL）的纳米复合物(LA-CL),并考察其对HEK293细胞的转染效率、胞摄取情况,以及体外释放规律。方法 以不同比例（2.5%、5%、10%、20%）半胱氨酸作为交联剂,合成四种不同交联度的LA-CLss (LA-CLss1, LA-CLss2,LA-CLss3, LA-CLss4),利用1H-NMR和凝胶色谱鉴定合成的LA-CLss。取增强绿色荧光蛋白质粒(plasmid Enhanced Green Fluorescent Protein, pEGFP) 和LA-CLss以不同氮磷比（N/P）（2.5, 5, 10, 20, 40, 80）自组装形成纳米复合物,粒度测定仪测定复合物的粒径和zeta电位,琼脂糖凝胶电泳测定载体LA-CLss对pEGFP的包裹能力以及保护作用。用超声乳化法制备载多西他赛的载药胶束,并用芘荧光探针法测定其临界胶束浓度。用LA-CLss/ pEGFP纳米复合物与人胚肾HEK293细胞共同培养,考察不同交联度复合物的细胞转染情况。结果 通过核磁结果确定LA-CLss合成成功;在N/P=40时,HEK293细胞对LA-CLss3/ pEGFP的转染效率高于其他四种 复合物(LA-CLss, LA-CLss1,LA-CLss2, LA-CLss4)。超声乳化法制备的载药胶束包封率为85.25 ± 0.04%,载药量为8.81 ± 0.02%。细胞摄取结果表明该载体可以有效地将基因递送进细胞内。体外释放实验结果表明该多肽胶束具有还原性条件敏感释药行为。结论 制备的LA-CLss纳米复合物有望成为一种高效的共载基因和化疗药的载体。     

聚氨基酯载基因纳米粒的研究

目的合成一种新型阳离子聚合物聚氨基酯(PBAE),并以此作为基因载体进行研究。方法通过迈克尔加成反应合成PBAE,以自主装法制备PBAE/pDNA纳米复合物,并考察PBAE/pDNA纳米复合物在HEK293细胞中的转染效率。结果当PBAE/pDNA的质量比为50∶1时,pDNA被完全包裹;在HEK293细胞转染实验中,当PBAE/pDNA质量比为200∶1时,其转染效率高于阳性对照组PEI/pDNA的转染效率(43.3%±3.7%vs 30.3%±2.1%,P<0.05)。结论带正电荷的PBAE能够通过静电作用浓缩包裹pDNA,在体外转染实验中具有较高的转染效率。     

壳聚糖修饰对细胞摄入和细胞毒性的影响

目的研究精氨酸或十六烷基修饰壳聚糖对其细胞摄入和细胞毒性的影响及作用机制。方法用α-32P-dATP标记质粒DNA,分别与十六烷基化壳聚糖和精氨酸修饰的壳聚糖通过复凝聚方法形成壳聚糖DNA纳米复合物。采用血管平滑肌A10细胞进行摄入测试,并用β液闪计数仪检测结果。用MTT方法对壳聚糖DNA纳米复合物的细胞毒性进行评价。结果经32P标记的DNA与不同修饰的壳聚糖复合所形成的纳米复合物粒径约为55·9~174·9nm,zeta电位约为1·8~10·8mV。细胞摄入实验显示通过精氨酸或十六烷基修饰的壳聚糖与DNA形成的纳米复合物更易于进入细胞。其中十六烷基化壳聚糖DNA纳米复合物(HCNC)在N/P为1∶1时细胞摄入量最高,精氨酸修饰的壳聚糖DNA纳米复合物(ACNC)细胞摄入次之,与未经修饰的壳聚糖DNA纳米复合物(UCNC)相比,差异具有显著性(HCNC、ACNC比UCNC高1·3倍,P<0·05)。细胞毒性实验显示,修饰后的壳聚糖纳米复合物的毒性远远小于商品化的细胞转染试剂Lipofectamine2000。结论两种修饰对壳聚糖DNA纳米复合物的血管平滑肌细胞摄入有明显促进作用,其作用机制各不相同;同时其细胞毒性远小于商品化的细胞转染试剂Lipofectamine2000。     

新型基因载体巯基烷基化壳聚糖的制备及体外性质的初步研究

目的 制备巯基烷基化壳聚糖(TACS)载基因纳米粒,并对其体外相关性质进行初步研究.方法 复凝聚法制备TACS/pDNA复合纳米粒子,并用透射电镜对其的形态和粒径进行观察和表征;凝胶阻滞分析观察其对基因的保护情况;以Lipofectamine 2000转染试剂作为阳性对照,检测其对人胚胎肾细胞(HEK293)的转染活性;四嚷氮唑盐(MTT)比色法测定其对细胞活性的影响.结果 TACS/pDNA复合纳米粒子形态不很均一,粒径在390 nm左右;凝胶阻滞分析证明了载体能有效地包裹和保护基因不受DNase Ⅰ酶的消化;体外基因转染实验证实了TACS纳米粒子能够有效地转染HEK293,其转染效率远高于壳聚糖纳米粒子,略低于脂质体;MTT测定其对细胞活力影响甚微,对细胞的毒性明显低于脂质体.结论 TACS有望成为一种有价值的新型基因载体.     

聚-羟丙基-天冬氨酸-谷氨酸-聚乙烯亚胺共聚物作为非病毒载体的研究

目的:选用聚-羟丙基-天冬氨酸-谷氨酸材料作为骨架偶联低分子量聚乙烯亚胺,以构建低毒、高效的新型非病毒性基因载体.方法:用聚-羟丙基-天冬氨酸-谷氨酸(PHPAG)为基本骨架,偶联低分子量的聚乙烯亚胺(PEI 1.8 kDa)形成聚-羟丙基-天冬氨酸-谷氨酸-聚乙烯亚胺(PHPAG-PEI 1.8 kDa)的载体材料.通过核磁共振氢谱(1H-NMR)、粒径测定、凝胶体积排除色谱法(GPC)等化学物理方法,凝胶电泳阻滞实验、MTT细胞毒性实验、细胞转染等生物学实验,对聚合物的结构及性能进行研究.结果:成功合成载体材料PHPAG-PEI 1.8 kDa.通过1H-NMR证实材料PHPAG-PEI 1.8 kDa在5或6个氨基酸上能偶合1个PEI 1.8 kDa.GPC结果表明PHPAG、PHPAG-PEI 1.8 kDa 2种材料的分子量约为1.2×104.粒径检测结果显示,PHPAG-PEI/pDNA复合物的平均粒径为200 nm左右.凝胶电泳阻滞实验表明,PHPAG-PEI/pDNA复合物在N/P为3.5∶1时可以完全阻滞DNA.细胞毒性实验表明,在COS-7和A293 2种不同的细胞中,载体材料显示出较低的毒性,与对照组PEI 1.8 kDa相近.在B16细胞、Hela细胞上的转染实验表明,PHPAG-PEI/pCAG-Luc3的复合物在N/P为25∶1时的转染效率最高,高于对照组PEI 25 kDa.结论:PHPAG-PEI聚合物载体材料是一种有潜在用途的非病毒基因药物载体.     

尿刊酸修饰壳聚糖载基因纳米复合物的制备及体外性质

制备新型壳聚糖衍生物——尿刊酸修饰壳聚糖（UAC)/质粒DNA（pDNA）纳米复合物,采用凝胶电泳阻滞实验评价UAC包裹pDNA的能力;通过对各种影响因素（包括pH、渗透压、脱氧核糖核酸酶及肝素）的考察,对该纳米复合物的稳定性进行评价;利用激光粒度仪测定该纳米复合物的粒径和Zeta电位,透射电镜对其进行形态学观察。研究结果表明,高取代度（50.0%）的载体UAC在氮磷比（N/P）≥3时将pDNA完全包裹形成纳米复合物;N/P越高,该纳米复合物对碱性环境、高渗透压、DNaseⅠ、肝素的稳定性越好;低相对分子质量壳聚糖（20 kD）的UAC能够更有效地保护pDNA免受DNaseⅠ的降解,且形成的纳米复合物较容易被肝素解聚;N/P为30的纳米复合物的粒径为125.2 nm,Zeta电位为+37.9 mV,呈类球形结构。因此,高取代度、低相对分子质量壳聚糖的载体UAC与pDNA形成高N/P的纳米复合物能够具有良好的特性,适合进行细胞转染实验,有望成为一种新型载基因纳米复合物。     

聚乙烯亚胺修饰纳米金基因载体制备及体外实验研究

目的 制备聚乙烯亚胺修饰纳米金基因载体并研究其理化性质的表征参数和体外转染效率.方法 通过化学还原法制备聚乙烯亚胺修饰的纳米金基因载体,用绿色荧光蛋白质粒(pAcGFP-N1)做报告基因,纳米基因载体可通过静电吸附的方式结合质粒DNA.用紫外分光光度计检测其吸收光谱,用透射电镜观察其形态特征,激光粒度分析仪测定其粒度分布、表面电位(Zeta电位),1％琼脂糖凝胶电泳检测该基因载体与质粒DNA的结合稳定性,CCK-8实验检测聚乙烯亚胺修饰纳米金基因载体及DNA-纳米金复合物对HEK293细胞的细胞毒性作用,通过荧光显微镜观察聚乙烯亚胺纳米基因载体介导pAcGFP-N1在体外培养的HEK293细胞中的表达,并分析其转染效率.结果 聚乙烯亚胺还原氯金酸可以得到带正电荷的纳米颗粒,呈单分散球形分布,其粒径为(12.3 ±3.3)nm.在pH =7.2时,Zeta电位为+(29.7±5.1)mV.1％琼脂糖凝胶电泳结果表明,当纳米金/质粒DNA≥0.5时,质粒DNA可完全结合到纳米金表面.体外转染实验表明,聚乙烯亚胺修饰纳米金基因载体能介导pAcGFP-N1转染HEK293细胞并在细胞中表达绿色荧光蛋白,其转染效率可达25％.结论 聚乙烯亚胺修饰纳米金是一种新型非病毒基因载体,具有转染效率高、对细胞毒性小等优势.     

寡肽阳离子脂质体作为基因载体的体外研究

采用阳离子寡肽脂质材料赖氨酰化谷氨酸双十四醇酯(T2GL)制备阳离子脂质体(CL),与增强型绿色荧光蛋白质粒(pEGFP)静电结合形成复合物(CL/pDNA),CL/pDNA粒径和表面电位分别为(132.3±1.0)nm和+(31.35±2.99)mV。考察CL/pDNA对肝素及脱氧核糖核酸酶Ⅰ(DNase I)的稳定性,结果表明,CL/pDNA可被适宜浓度的肝素竞争性解组装释放pDNA,其对DNase I的稳定性明显优于市售制剂Lipofectamine 2000。研究CL与pDNA的比例(N/P)及质粒用量对转染效率的影响,结果显示当N/P为3,质粒用量4μg时,CL/pDNA的转染效率最佳。采用CL/pDNA对3种不同的细胞进行转染,发现CL/pDNA在人源非小细胞肺癌A549细胞中转染效果最好,与Lipofectamine 2000相比,转染效率相当但细胞毒性更低,因此为进一步研究其体内行为提供了良好的基础。     

可降解非病毒基因载体PEG-b-(PG-g-PEI)的合成与表征

目的结合重组低分子量PEI 和PEG结构修饰两种手段合成出新型可生物降解的非病毒基因载体聚乙二醇-b-(聚谷氨
酸-g-聚乙烯亚胺)。方法用相对分子质量600 的聚乙烯亚胺氨解嵌段聚合物PEG-b-PBLG,合成非病毒基因载体聚乙二
醇-b-(聚谷氨酸-g-聚乙烯亚胺);利用核磁共振氢谱、凝胶渗透色谱、激光粒度分析仪、zeta电位仪和凝胶电泳对载体及其与DNA
复合物进行了表征,并通过体外细胞实验考察了载体的细胞毒性与转染效率。结果我们成功合成出窄分布的非病毒基因载体
聚乙二醇-b-(聚谷氨酸-g-聚乙烯亚胺);凝胶电泳测定结果表明当N/P比大于5时载体能很好地包裹DNA;载体与DNA形成的
复合物粒径为120 nm,zeta电位25 mV;通过MTT实验和体外质粒转染实验显示出载体在测量范围内具有极低的细胞毒性和
很高的转染效率。结论聚合物聚乙二醇-b-(聚谷氨酸-g-聚乙烯亚胺)有望作为非病毒基因传递载体。
     

Value of D-Dimers in patients with acute aortic dissection

Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P ＜ 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (＞0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P ＜0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P ＜ 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.     

Research of combined liver-kidney transplantation model in rats

Objective： To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods： SD rats served as both donors and recipients. 4℃ sodium lactate Ringer＇s was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava （IVC） inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results： Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion： This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.     

APPLICATION OF LORNOXICAM TO PATIENT-CONTROLLED ANALGESIA IN PATIENTS UNDERGOING ABDOMINAL SURGERIES

FOR anesthesiologis s ,treatingpostoperativepainhas alwaysbeen a problem.Althoughopioidshave been provedtobe effective,theirsideeffectscouldnotbeignored.With thedevelopmentofscienceand pharmacology,many drugs with aspectsof satisfactoryanalgesicefficacyand couldbe welltoleratedby patientshave been developed.And lornoxicamisone of them, which isa non-steroidalanti-inflammatorydrug (NSAID ), with analgesic, anti-infl-ammatory,andantipyreticproperties.Itseliminationhalf-time(3 to 5 hours) isle…     

BLOOD PRESSURE CHANGE WITH AGE IN SALT-SENSITIVE TEENAGERS

Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P＜ 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P＜ 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.     

Development on Adjustable Calibration Marker for Shock Wave Focus

Shock wave lithotripsy （SWL） is a treatment of choice for upper urinary stones. However, this procedure is inappropriate for obese patients because the focus is often unable to reach the target owing to the limited focal distance in shock wave source. Although treating such patients in a blast path may increase the application length of shock wave source, it＇s difficult to find this path on the lithotripter monitor. For this reason, we invented an adjustable calibration marker in order to set an effective focus in the shock wave hath.     

Study on the TCM Pathogenesis of Angiopathic Complications of Type II Diabetes

Excess production of reactive oxygen species(ROS)of mitochondrion mediated by hyperglycemia is the common pathogenesis of angiopathic complications of diabetes.TCM holds that the damp from the dysfunction of spleen.kidney and liver is the causative factor of complications of diabetes.This is similar to the mechanism of Ros resulting in angiopathic complications of diabetes.When the angiopathic complications of type II diabetes mellitus(T2DM)are difierentiated as caused by turbid damp in TCM can be explained as ROS.Since the obstruction of pathogenic damp in channels and collaterals is said to be the main pathogenesis,the treating principle should be dissolving the damp to remove the obstruction.     

Leptin, Ghrelin and TNF-α before and after Hypo-caloric Traditional Chinese Diet and Auricular Acupuncture

INTRODUCTION Obesity is a complex emergent problem, which can be possibly solved not only by the diet but also by the life style and promotion of a constant physical exercise. 1, 2 No doubt careful attentions must be given to the nutritional condition of obese people, the dietary habits, the somatic build (i.e. distribution of fat mass) and the organic functions linked to formation of the fat mass. All the parameters should be constantly monitored before, during and after a diet treatment. 3, 4, 5     

Relationship between Dysglycemia and Carotid Atherosclerosis in Tibetan Population

People with dysglycemia are at high risk for atherosclerotic diseases. This study aims at investigating the atherosclerotic vascular damage in dysglycemia and its metabolic origin in Tibetan population.     

Acupuncture Combined with Spinal Tui Na for Treatment of Primary Dysmenorrhea in 30 Cases

Objective: To observe the therapeutic effects in acupunture treatment of primary dysmenorrhea combined with spinal Tui Na, and study its mechanism. Methods: Thirty cases of the treatment group were treated by acupuncture combined with spinal Tui Na, and thirty cases in the control group were treated by routine acupuncture. Results: The total effective rate was 93.3% in the treatment group, and 73.3% in the control group, with a significant difference between the two groups (P<0.05). Conclusions: Acupuncture combined with spinal Tui Na has good prospects for treatment of primary dysmenorrhea.     

Luo Lingjie's Experience in Treating Chronic Nephropathy

In treating chronic nephropathy,Luo Lingjie,a chief physician,pays attention to regulating the balance between yin and yang,treating infection if present,and removing pathogenic factors.He prescribes gentle drugs and uses carefully strongly warming-tonifying ones,emphasizes the importance of persuading the patient to persist in treatment with medication and nurse one's health for recuperation,and is good at combined use of TCM and western medicine therapy and brings the merits of various therapies into full play,with obvious theraoeutic effects.