大豆生理活性肽的研究(Ⅱ)--抗氧化性和ACE抑制活性的初步研究

采用AS1.398和Alcalase两种蛋白酶,制备了水解度为10%～24%的大豆多肽,对其抗氧化性、ACE抑制活性和相对分子质量的分布进行了研究,结果表明采用AS1.398酶水解的DH为12%的产品抗氧化活性最高,添加量为6 mg/mL时使亚油酸的氧化诱导期延长2.92倍,其相对分子质量分布在1 000以上的组分较多;采用Alcalase酶水解的DH为14%的大豆多肽产品,ACE抑制活性最高,IC50为0.144 mg/mL,其相对分子质量分布大多在200～600之间.     

合成角质细胞生长因子活性短肽促进表皮细胞增殖的实验研究

目的 应用噬菌体随机7肽库筛选角质细胞生长因子(Keratinocyte growth factor,KGF)的关键序列,进行KGF活性短肽的调整和合成,并研究其对表皮细胞增殖的作用。方法 以KGF单克隆抗体为靶,筛选噬菌体随机7肽库,获得KGF的特异性序列,并进行序列调整。用免疫荧光法检测KGF活性短肽的表皮细胞亲和力。用CCK-8法检测KGF活性短肽对表皮细胞增殖的影响。用RT-PCR法和Western-blot法检测表皮细胞上特异性受体KGFR的表达水平。结果筛选噬菌体随机7肽库,获得2个与KGF相似的特异性序列,合成获得2个KGF活性短肽,并纯化至98%纯度,短肽的C端进行氨基化封闭,N端进行罗丹明激光染料标记。免疫荧光检测结果显示,2个KGF活性短肽均能够与表皮细胞相结合。CCK-8检测结果显示,与阴性对照组相比,2个KGF活性短肽能够促进表皮细胞增殖,并呈浓度依赖性。RT-PCR和Western-blot检测结果显示,2个KGF活性短肽组中表皮细胞表达KGFR增强。结论 从噬菌体随机7肽库中筛选到2个KGF的关键序列,合成的2个KGF活性短肽能够与KGFR相结合,并增强KGFR的表达,从而促进表皮细胞增殖,有望应用于促进创面愈合。     

人结肠癌血管特异结合短肽的噬菌体肽库筛选及鉴定

目的 建立小鼠结肠癌模型,筛选并鉴定能够与人结肠癌血管内皮细胞特异结合的噬菌体呈现短肽.方法 建立小鼠荷瘤模型.鉴定阳性噬菌体的体内归巢能力及内皮细胞结合能力.人工合成噬菌体呈现短肽,通过竞争结合实验观察短肽对其呈现噬菌体的竞争结合抑制作用.并检测短肽与共培养内皮细胞及结肠癌血管的特异性结合能力.结果 鉴定出两种外源肽噬菌体,称为pCV1及pCV2.体内实验显示,pCV1、pCV2均能特异性归巢于人结肠癌移植瘤组织,滴度比值pCV1/pCont、pCV2/pCont分别为15.9、20.1倍,明显高于对照组织.体外细胞酶联免疫吸附试验(ELISA)结果显示pCV2在Co-HUVECs上的结合显著高于在HUVECs上的结合,其比值达2.61.人工合成短肽CV2能特异性竞争抑制pCV2向结肠癌移植瘤内的归巢及与Co-HUVECs的特异性结合.免疫荧光染色结果显示,FITC-CV2特异性结合于Co-HUVECs的胞膜与核周胞质,以及结肠癌移植瘤的血管组织.结论 得到两个能特异性结合于结肠癌移植瘤的噬菌体单克隆pCV1及pCV2.pCV2能够特异性结合于Co-HUVECs,pCV2所呈现的环状九肽CV2介导了它与Co-HUVECs的特异结合.短肽CV2具有与Co-HUVECs及结肠癌移植瘤血管特异性结合的能力,有可能用于结肠癌的血管靶向治疗.

Abstract：

Objective To select, identify and analyze a peptide binding specifically to blood vessels of human colon cancer by phage displayed peptide library in vivo. Methods Animal models were established using sub-renal capsular assay (SRCA) in immunosuppressive mice implanted with human colon cancer xenografts. The phage displayed peptide library was injected intravenously into mice. After 4 rounds of selection, 20 clones were picked up randomly and sequenced individually. The homing ability to human colon cancer xenografts and co-cultured human umbilical vein endothelial cells ( Co-HUVECs with human colon cancer Lovo cells) of the positive phage clones were determined by in vivo binding assay and in vitro cell enzyme linked immunosorbent assay ( ELISA ). The binding ability to Co-HUVECs of peptide-displayed phage clone was identified by immunocytochemical stain. Peptide displayed on the phage was synthesized and competitive binding assays were performed to observe the competitive inhibition effect of the peptide with their phage counterpart. Immunofluorescence microscopy was used to study the binding of synthesized peptides to Co-HUVECs and vascular vessels in colon cancer. Results Two peptide sequences were obtained finally and named CV1 and CV2. In vivo binding assay showed that the homing ability to human colon cancer xenografts of peptides of CV1 or CV2 was higher than that of control organs. In vitro cell ELISA suggested that CV2 phage preferably binded to Co-HUVECs rather than control HUVECs. Then CV2 phage clone was identified further. Immunocytochemical staining revealed that CV2 phage preferably binded to Co-HUVECs rather than the control. Competition binding assays demonstrated a significant competition between the synthesized peptide CV2 and the phage displaying CV2 while binding to Co-HUVECs or human colon cancer xenografts. Under the immunofluorescence microscopy, fluorescence-labeled CV2 peptide was seen on the membrane and in the perinuclear cytoplasm of Co-HUVECs, and bound to colon cancer xenografts rather than control organs. Conclusion Two phage clones displaying CV1 and CV2 peptide could target to human colon cancer xenografts. The peptide CV2 and its displayed phage were identified binding preferably with Co-HUVECs. And the cyclic nonapeptide (CV2) was binding site of the CV2 phage with Co-HUVECs. Synthesized nonapeptide CV2 had specificity to Co-HUVECs and colon cancer vascular endothelial cells. The peptide CV2 could be used in target therapy of tumor angiogenesis.     

聚乳酸聚乙醇酸／RNAⅢ抑制肽缓释微球的制备及评价

目的探讨聚乳酸聚乙醇酸（PLGA）／RNAⅢ抑制肽（RIP）缓释微球的合理制备方法,测定其载药率和体外释药特征。方法采用改良复乳－溶剂挥发法制备PLGA／RIP缓释微球,观察其表征,测定缓释微球的载药率和包封率。采用高效液相色谱法测定不同时点PLGA／RIP的释放速度,观察PLGA／RIP微球的体外释药特点。结果采用改良复乳－溶剂挥发法制备的PLGA／RIP缓释微球粒径均匀、表面光滑,包封率约为（68．22±6．20）％,载药率为（15．35±3．26）％。PLGA／RIP释药动力学方程为y=31．016x＋59．611,符合Huguchi方程。结论采用改良复乳-溶剂挥发法制备的PLGA／RIP缓释微球粒径均匀、表面光滑,包封率和载药率较高,体外释放动力学符合Huguchi方程,是理想的缓释载药体系。     

血管活性肠肽及其受体与胆囊胆固醇结石患者胆囊排空的相关研究

目的　探索胆囊胆固醇结石患者胆囊排空与血浆和胆汁血管活性肠肽 (VIP)的浓度及其胆囊壁VIP受体 (VIP R)表达的关系以及在胆囊结石形成中的意义。方法　采用B超测定胆囊排空功能 ,同时抽空腹静脉血及胆囊结石手术病人胆汁放免测定VIP浓度 ,免疫组化测定VIP R的表达。结果　胆囊结石病人胆囊排空障碍 ;血浆VIP浓度正常人为 ( 8.2 8± 0 .98)ng/L ,胆囊结石病人为 ( 15 .64± 2 .5 1)ng/L ,显著升高 ( P <0 .0 1) ;胆汁VIP高于血浆VIP ;空腹容积越大 ,血浆VIP(P <0 .0 5 )和胆汁VIP(P <0 .0 1)浓度越高 ,VIP R表达越强。结论　胆囊排空功能在胆囊结石形成中起重要作用 ,胆囊结石病人胆囊排空障碍与VIP明显相关 ,空腹容积与VIP及其受体明显正相关     

血管活性肠肽瘤：1例报道及文献资料分析

目的探讨胰腺血管活性肠肽瘤（VIPoma）的临床、实验窜和影像学特点,以及诊断办法和治疗手段方法报道1例胰腺VIPoma,同时检索国内文献得到49例胰腺VIPoma患者临床资料,许对此50例病例进行分析结果分泌性腹泻、低血钾和代谢性酸中毒是胰腺VIPoma的主要临床表现,InL浆血管活性肠肽（VIP）水平增高具有诊断价值。经手术治疗,腹泻症状可减轻或消失;对于远处转移患者,生长抑素、化疗、干扰素等治疗均有效。结论VIPoma早期诊断闲难,确诊依赖下典捌临床症状、血浆VIP水平、影像学检仓以及免疫组化检查。手术切除可改善颅后,生长抑素等治疗可缓解症状,发生转移者也应积极治疗。     

胰腺血管活性肠肽瘤的诊治

胰腺血管活性肠肽瘤的诊治北京丰台铁路中心医院外科（１０００７１）何永清１９５８年Ｖｅｒｎｅｒ－Ｍｏｒｒｉｓｏｎ［１］首次报告２例因腹泻、低血钾酸中毒而死亡，经尸解及病理证实为胰岛非β恶性细胞瘤的病例，此后称之Ｖ－Ｍ综合征。此征又称ＷＤＨＡ或ＷＤＨＨ综...     

Correlates of ACE activity in macroalbuminuric type 2 diabetic patients treated with chronic ACE inhibition.

The activity of the renin-angiotensin-aldosterone system (RAAS) plays an important role in the development and progression of diabetic nephropathy. However, the effect of angiotensin-converting enzyme (ACE) inhibition on the RAAS appears to be modulated by a number of factors including the I/D polymorphism of the ACE genotype. In this study, we attempted to find independent correlates of ACE activity in 121 macroalbuminuric type 2 diabetic Iranian patients under chronic ACE inhibition. Both univariate and multivariate analyses were used. The presence of the D allele was independently associated with significantly higher levels of ACE activity (with the II genotype as reference, P < 0.001, B = 27.3, 95% CI = 17.6-37.1), and this association was not eliminated by potentially confounding variables. In conclusion, the D allele is a significant independent correlate of ACE activity in macroalbuminuric type 2 diabetic Iranian patients under long-term ACE inhibition.     

Stunting growth: association of the blood pressure levels and ACE activity in early childhood

Angiotensin converting enzyme (ACE) converts angiotensin I to angiotensin II and inactive bradykinin. Several studies carried out in our laboratory have consistently identified three isoforms of ACE, at 65, 90 and 190 kDa, with the 90-kDa isoform being a possible genetic marker of hypertension. Based on these observations and the fact that nutritional stunting can be associated with hypertension, we have investigated the expression and activity of ACE in stunted children and its association with blood pressure (BP) levels and nutritional state. Sixty children aged 2–7 years were selected for this study. A urine sample was collected from each child. Angiotensin converting enzyme activity was evaluated using two different substrates, and ACE expression was detected by Western blotting. Our results show that nutritional stunting is associated with high ACE activity in childhood and that adjustment by gender does not modify the strength of this association. A greater percentage of stunted children had increased BP levels, and this clinical parameter was inversely correlated with anthropometric indicators. A greater urinary protein expression of the three ACE isoforms was observed in the group of children with growth stunting. Our findings suggest that the reported high risk of hypertension in stunted adolescents and adults are, at least partly, associated with abnormalities in the renin–angiotensin system.     

Activity of testis angiotensin converting enzyme (ACE) in ejaculated human spermatozoa

Testicular angiotensin converting enzyme (ACE) isozyme is likely to play important functional roles in male reproduction. Several studies have shown that ACE is released from human spermatozoa during capacitation and that ACE is associated with reduced sperm motility. Recently, we established an assay to detect testicular ACE activity in human spermatozoa. The purpose of this study was to determine if testicular ACE activity is related to sperm motility in human ejaculates. Semen samples were collected from 80 infertile patients. According to the semen characteristics, they were divided into four (WHO) categories. Enzyme activities of ACE in spermatozoa (testicular ACE) and seminal plasma (somatic ACE) were spectrophotometrically determined. Total testicular ACE activity in spermatozoa was measured by solubilization of spermatozoa with Triton X-100. Membrane testicular ACE activity was measured in a sperm : PBS suspension. Sperm concentration and sperm motility were 136.6 +/- 154.1 x 10(6)/mL and 58.6 +/- 23.4%, respectively (mean +/- SD). Enzyme activities of membrane testicular ACE, total testicular ACE and somatic ACE were 0.273 +/- 1.219 microU/10(6) spermatozoa, 0.35 +/- 1.34 microU/10(6) spermatozoa and 684.7 +/- 226.6 mU/mL, respectively. A negative correlation was observed between sperm motility and membrane testicular ACE activity (p < 0.05). Membrane testicular ACE activity in 44 normal semen samples was 0.04 +/- 0.02 microU/10(6) spermatozoa, whilst that in 36 abnormal semen samples was 0.24 +/- 0.42 microU/10(6) spermatozoa. There was a significant difference between these two groups (p < 0.01). Membrane testicular ACE in sperm samples from normozoospermic men was significantly lower than that from oligoasthenozoospermic men (p < 0.05). These findings suggest that testicular ACE is released from normal functional spermatozoa for them to have fertilizing ability.     

Inhibition of angiotensin-converting enzyme reduces rat liver reperfusion injury via bradykinin-2-receptor

BACKGROUND: Bradykinin is both a potent vasodilatator and a central inflammatory mediator. Similar to findings in myocardial reperfusion injury, bradykinin might mediate the protective effects of angiotensin-converting enzyme (ACE) inhibition after liver ischemia via increased bradykinin-2-receptor (B-2) stimulation. On the other hand, B-2-inhibition has been shown to reduce liver reperfusion injury. This study was designed to investigate the role of Bradykinin in hepatic reperfusion injury. MATERIALS AND METHODS: Twenty eight rats were allocated randomly to Sham procedure (Sham), 30-min normothermic ischemia (ischemia), ischemia with Ramiprilat (ACE-I), or ischemia with Ramiprilat and B-2-inhibitor HOE 140 (ACE-I+B-2-I). Liver microcirculation and leukocyte adherence were investigated using intravital microscopy 30 min after reperfusion (n = 7 per group). In addition, serum activities of AST and ALT were measured for 7 days (n = 28). RESULTS: Ischemia was associated with a loss of perfused sinusoids, sinusoidal vasoconstriction, and a reduction in microvascular blood flow. Permanent leukocyte adherence increased both in sinusoids and in postsinusoidal venoles. ACE-I restored sinusoidal perfusion, normalized vasoregulation, maintained sinusoidal blood flow, and inhibited leukocyte adhesion. ACE-I+B-2-I abolished the protective effects linked to ACE-I. Ischemia-induced liver cell injury after 5 h of reperfusion was ameliorated by ACE-I. In the ACE-I+B-2-I group, reduction in liver cell injury was reversed. CONCLUSION: After hepatic ischemia, ACE-I reduced reperfusion injury in a B-2-dependent manner. These results suggest a pivotal role for bradykinin in the treatment of reperfusion injury by Ramiprilat, mediating sinusoidal dilation and blunting hepatic inflammation.     

Effect of chronic ACE inhibition on the diagnostic value of renography for renovascular hypertension: a preliminary report

We compared the effects of acute and chronic ACE inhibition(ACEi) on the ¹²³I-hippurate in the stenotic kidney of two 2-kidney,1-clip hypertensive dogs. In the period after clip implantationpoststenotic renograms without ACEi of both dogs were normal.Acute ACEi always resulted in delayed hippurate handling. ChronicACEi, however, induced abnormal poststenotic renograms in only36% of the cases. Withdrawal of chronic ACEi restored the phenomenonof acute ACE-induced delayed hippurate handling within 5 monthsin both dogs. These data indicate that chronic ACEi or recent ACEi medicationreduces the effectiveness of ACEi renography in diagnosing hypertensiondue to a moderate renal-artery stenosis. This phenomenon mayexplain why the sensitivity of ACEi renography in human studiesvaries more than in animal studies.     

影响芦荟制品非酶褐变的因素及其控制方法

利用分光光度法测定芦荟制品加工和贮存过程中褐变指数的变化 ,结果表明 ,影响芦荟制品非酶褐变的主要因素有 :多酚类物质的质量浓度、pH、环境温度、Mg2 +、Fe3+、Cu2 +等金属离子以及包装材料的质量 ;控制这些因素可有效抑制芦荟制品的非酶褐变 .     

丹参对马兜铃酸肾损害大鼠肾组织病理学改变及ACE、ACE2表达的影响

目的：观察丹参对马兜铃酸诱导的大鼠肾组织病理改变、血管紧张素转化酶（ACE）7L其同源物AC1、2以及细胞因子表达的影响。方法：建立马兜铃酸肾损害大鼠模型,并灌服不同剂量丹参水溶液（5、10、15g·kg^-1·d^-1）,于实验第12、16、20、24周留取肾组织标本,HE、P／kS、Masson观察肾组织病理改变,免疫印迹检测肾组织ACE、ACE2的蛋白表达量,RT—PCR检测AngⅡ、TGF—β1、PAI-1mRNA水平。结果：第12周开始,模型大鼠出现肾小管结构损伤,表现为刷状缘消失、细胞空泡变性、脱落,并出现裸膜,伴炎细胞浸润,实验后期出现局部动脉管壁增厚及轻度纤维化表现。模型大鼠肾组织ACE的表达升高,并随实验进展而明显;第16周开始艘的表达降低。AngⅡ、TGF—β1、PAI-1mRNA表达上调,以AngⅡ mRNA上调明显。丹参干预后肾组织病变有所减轻,未出现纤维化表现;丹参可降低肾组织ACE的表达,并下调AngⅡ mRNA水平。结论：丹参可能通过降低ACE的表达、促进ACE2的合成,抑制了AngⅡ的活性,并下调TGF-β1、PAI-1的过高表达,从而减轻马兜铃酸诱导的肾小管间质病变的发生发展。     

氧化石墨烯对大鼠骨髓间充质干细胞生物活性的影响

目的：探讨氧化石墨烯对体外培养的大鼠骨髓间充质干细胞生物学活性的影响。方法采用改良Hummers法制备氧化石墨烯，将不同浓度的氧化石墨烯溶液与大鼠骨髓间充质干细胞共培养，检测其对细胞增殖和形态的影响，相关结果采用SAS V8软件进行统计学分析。结果经表征分析，改良Hummers法可以成功制备出高纯度的氧化石墨烯。在细胞密度较低时，氧化石墨烯对大鼠骨髓间充质细胞的分裂增殖有抑制作用；而在细胞密度较高，达到生长接触抑制的浓度时，氧化石墨烯具有促进细胞死亡，降低活性细胞数量的作用。连续镜下观察后发现，氧化石墨烯对细胞形态有显著影响，可使细胞伸展性下降，折光率降低。这些细胞形态的变化与氧化石墨烯的浓度呈正相关。结论氧化石墨烯可抑制大鼠骨髓间充质干细胞的生物学活性，其作用呈浓度依赖性。     

Change in Body Mass Index After Total Knee Arthroplasty and Its Influence on Functional Outcome

Background

There is often an assumption by patients that weight loss will occur once their knee pain is relieved by total knee arthroplasty (TKA). This study aims to evaluate (1) the change in patients' body mass index (BMI) after TKA; (2) if postoperative change in BMI influences functional outcome and survival rate of TKA; and (3) the predictive factors associated with change in BMI.