新型的病毒-基因治疗系统CNHK500-hγ的构建

目的研制出一种新的基因-病毒治疗系统。方法通过基因操作技术将主要晚期启动子(MLP)调控的人干扰素-γ基因插入腺病毒E1A基因受hTERT启动子调控、E1B基因受HRE启动子调控的增殖病毒载体质粒PXC70-HRE-TP的E1A上游,得到腺病毒质粒pSG500-hγ。通过pSG500-hγ与质粒pBHGE3在293细胞中同源重组得到重组病毒CNHK500-hγ。用TCID50方法测定病毒滴度。通过增殖实验观察重组病毒的选择性增殖能力。利用ELISA法检测人干扰素-γ抗癌基因的表达。结果CNHK500-hγ的病毒滴度为4×109pfu/ml,增殖实验结果证实CNHK500-hγ可以选择性地在端粒酶阳性的肝癌细胞中增殖,CNHK500-hγ所携带的人干扰素-γ基因在肝癌细胞株中的表达量(442μg/L)明显高于携带该基因的非增殖型腺病毒载体(120μg/L)Ad-hγ(P<0.005)。结论CNHK500-hγ是一种具备治疗肝癌潜力的新型基因-病毒治疗系统。     

双重调控选择增殖型腺病毒CNHK500的构建及初步研究

目的　研制出一种双重调控的选择增殖型腺病毒载体系统。方法　先后经 2次定点突变重叠聚合酶链反应 (PCR)技术使腺病毒E1A及E1B启动子缺失 ,并将人工合成的人端粒酶逆转录酶启动子和缺氧反应启动子分别插入E1A及E1B基因的上游 ,得到腺病毒载体质粒pSG5 0 0。通过 pSG5 0 0与质粒 pBHGE3在 2 93细胞中同源重组得到重组病毒CNHK5 0 0。扩增、纯化病毒 ,用TCID5 0方法测病毒滴度。通过病毒增殖实验观察重组病毒的选择性增殖能力。结果　成功构建了由双重调控选择增殖型腺病毒CNHK5 0 0 ,病毒滴度为 1.9× 10 10 pfu/ml ,增殖实验结果证实CNHK5 0 0可以选择性地在端粒酶阳性肿瘤中增殖。结论　CNHK5 0 0为肿瘤的生物治疗提供了一种新的策略。     

携带治疗基因的增殖型腺病毒CNHK300-m干扰素-γ的构建

目的 研制出一种携带抗癌基因的选择增殖型腺病毒载体系统.方法 克隆鼠干扰素(IFN)-γ基因序列,利用分子克隆技术将其插入肿瘤特异性增殖病毒的病毒基因组中,得到腺病毒载体质粒pSG300-m IFN-γ.通过pSG300-m IFN-γ与质粒pBHGE3在293细胞中同源重组得到重组病毒CNHK300-mIFN-γ.扩增、纯化病毒,用TCID50方法测病毒滴度.通过病毒增殖实验观察重组病毒的选择性增殖能力,通过Western blot检测腺病毒蛋白的表达,通过双抗体夹心法酶联免疫吸附试验(ELISA)检测重组病毒在不同细胞及不同时相的mIFN-γ表达量.结果 成功构建了携带治疗基因的增殖型腺病毒CNHK300-mIFN-γ,病毒滴度为1.0×109 pfu/ml,增殖实验证实CNHK300-mIFN-γ可以选择性地在端粒酶阳性肿瘤中增殖,Western blot分析结果显示腺病毒的E1A蛋白选择性在端粒酶阳性的肿瘤细胞中表达,ELISA显示CNHK300-mIFN-γ感染端粒酶阳性肿瘤后有大量mIFN-γ的表达,并随着感染的时间表达量也相应上升.结论 CNHK300-mIFN-γ为肿瘤的生物治疗提供了一种新的策略.     

多重调控病毒.基因载体的构建及其体外抗肿瘤活性

目的 构建一种新型的病毒.基因治疗载体.方法 通过克隆技术将人肿瘤坏死因子相关凋亡诱导配体(TRAIL)基因插入质粒载体pBHGE3的E3区,得到由主要晚期启动子(MLP)调控的腺病毒质粒pPE3-hTRAIL,再通过与质粒pSGS00在293细胞中进行位点特异性重组得到E1A、E1B基因分别受hTERT启动子与HRE启动子双重调控的重组增殖型腺病毒,用TCID50方法测定病毒滴度.通过增殖实验观察重组病毒的选择性增殖能力.利用酶联免疫吸附试验(ELISA)检测人TRAIL基因的表达.并进行噻唑蓝(MTT)比色法实验检测其杀伤肿瘤细胞的能力.结果 CNHK500-hTRAIL的病毒滴度为2.39×1010pfu/ml,增殖实验结果证实CNHK500-hTRAIL可以选择性地在端粒酶阳性的人肺癌细胞A549中增殖,其所携带的人TRAIL基因在A549中的表达量(183.12μg/L)明显高于携带该基因的非增殖型腺病毒载体Ad-hTRAIL(24.53μg/L,P<0.01).MTr显示CNHK500-hTRAIL对A549的杀伤能力明显高于携带该基因的非增殖型腺病毒载体Ad-hTRAIL,其半数抑制MOI值分别为17.825、0.197(P<0.01).结论 CNHKS00一hTRAIL是一种具备治疗肺癌潜力的新型病毒-基因治疗系统.     

基因-病毒治疗系统CNHK300-murine endostatin的构建及体外研究

目的构建一种新型的肿瘤基因．病毒治疗系统CNHK300-murine endostatin（CNHK300-mE），以肿瘤增殖病毒为载体，携带抗肿瘤新生血管生成的基因。方法利用基因重组技术将人端粒酶逆转录酶（hTERT）启动子插入腺病毒E1A基因上游，将小鼠内皮抑素基因表达盒插入到腺病毒包装信号和hTERT启动子之间，通过病毒增殖试验、电镜技术、Western blot分析、酶联免疫吸附实验（ELISA）、鸡胚绒毛尿囊膜（CAM）试验，观察CNHK300-mE的复制情况、mE的表达情况及对鸡胚绒毛尿囊膜新生血管的抑制情况。结果成功构建了基因-病毒治疗系统CNHK300-mE，该系统为hTERT启动子调控腺病毒E1A基因表达并携带小鼠内皮抑素基因的重组腺病毒。该病毒可以在端粒酶阳性的胃癌细胞株SGC-7901和肝癌细胞株HepGⅡ中大量增殖及复制，而在端粒酶阴性的正常纤维细胞中不增殖。电镜证实该重组病毒在胃癌细胞株SGC-7901中复制及增殖。ELISA检测表明SGC-7901感染CNHK300-mE后7d，小鼠内皮抑素的表达量为1000μg／L。CAM实验证实该病毒感染胃癌细胞后的培养液上清具有抗新生血管生成的生物活性。结论基因-病毒治疗系统CNHK300-mE能在端粒酶阳性的肿瘤细胞中特异性增殖复制，并大量表达具有抗新生血管生成生物活性的小鼠内皮抑素。     

双靶向溶瘤腺病毒携带内皮抑素基因对肝癌的抑制作用

目的 构建双调控溶瘤腺病毒,携带小鼠内皮抑素基因(mE),研究其对裸鼠肝癌移植瘤的抗瘤活性.方法 以人端粒酶逆转录酶启动子(hTERT)和缺氧调控元件序列(HRE)调控腺病毒E1a和E1b基因,基因组插入mE基因,构建双调控溶瘤腺病毒CNHK500-mE;在裸鼠模型中观察CNHK500-mE对肝癌移植瘤模型的疗效.结果 CNHK500能够在hTERT阳性的肝癌细胞中增殖[24 h:(16.67±4.04)％;48 h:(65.33 ±7.02)％;P＜0.01],并介导mE高效表达;与空白对照组( 1895.80±323.37) mm3比较,CNHK500-mE和Ad-mE的抑瘤率分别为50.95％[(929.80±211.10) mm3,P＜0.01]和29.99％[(1327.23 ±319.36) mm3,p＜0.05];CNHK500-mE对癌组织间质血管的抑制作用明显强于Ad -mE(P ＜0.05).结论 将mE与溶瘤腺病毒结合,发挥病毒增殖的溶瘤作用和基因产物的血管抑制作用,表现出明显的协同抗瘤作用.     

新型增殖性腺病毒的构建及对肝癌细胞的抑制

目的 构建携带p53基因新型增殖性腺病毒CNHK600-p53,研究其对肝癌细胞株抑制效应是否优于Ad-p53.方法 PCR扩增p53基因,利用酶切连接方法 将其插入CNHK600载体,PCR鉴定.经293细胞包装成病毒,抽提病毒DNA,PCR鉴定.氯化铯密度梯度离心法纯化病毒,TCID50 方法 测病毒滴度.病毒增殖实验检测病毒在不同细胞增殖能力.四甲基偶氮唑盐(methyl-thiazolyl tetrazolium assay,MTT)法观察CNHK600-p53、Ad-p53两种病毒分别对肝癌细胞株的抑制率.结果 成功构建新型增殖性腺病毒载体CNHK600-p53;293细胞包装成病毒,PCR方法 鉴定无野生型病毒存在;病毒滴度为1.99×10~（10）pfu/ml;CNHK600-p53在肝癌细胞HepG2、SMMC-7721内复制能力明显高于正常肝细胞HEL-1和L02.对6种肝癌细胞(PLC/PRF5、SMMC7721、HepaG2、BEL-7402、BEL-7404、QGY-7703)而言,随着MOI值的不断增高.其对肝癌细胞的抑制作用也不断增强;在相同MOI情况下,CNHK600-p53组较Ad-p53组的细胞抑制率高(P＜0.05).当细胞抑制率达到80%以上时.两组之间差异无统计学意义(P＞0.05).结论 利用新型增殖性腺病毒CNHK600一p53较 Ad-p53能更有效的抑制肝癌细胞,可能成为肝癌的基因治疗更有效的一种基因治疗手段.     

增殖性腺病毒CNHK600-p53可增强肝癌细胞株SMMC7721对化疗药物的敏感性

目的构建携带p53基因新型增殖性腺病毒CNHK600-p53,研究其是否增加肝癌细胞株对化疗药物的敏感性。方法采用四甲基偶氮唑盐(m ethylth iazolyl tetrazolium assay,MTT),观察化疗药物5-氟尿嘧啶(F luorourac il,5-Fu)、丝裂霉素(M itomyc in,MMC)和表阿霉素(Ep irub ic in,EPI)组与携带p53基因的重组腺病毒CNHK600-p53联合上述化疗药物组对肝癌细胞株SMMC7721的杀伤效应,同时观察其是否优于Ad-p53。结果单用CNHK600-p53或Ad-p53对肝癌细胞SMMC7721的杀伤效果无明显差别;肝癌细胞株SMMC7721在5-Fu浓度为100 ug/m l时细胞抑制率为58%,MMC浓度为2.5 ug/m l时细胞抑制率为50%,EPI浓度为1.25ug/m l时细胞抑制率为57%,但转入CNHK600-p53(MO I=10)后再使用上述浓度的化疗药物,细胞抑制率分别为76%、60%和62%。结论单用CNHK600-p53或Ad-p53对肝癌细胞SMMC7721的杀伤效果无明显差别,携带p53基因的CNHK600-p53能提高肝癌细胞株SMMC7721对化疗药物的敏感性,CNHK600-p53联合化疗药物治疗可望开辟克服肝癌耐药的新途径。     

非增殖型和靶向增殖型腺病毒携带TRAIL基因治疗肝癌的实验研究

目的比较携带人TRAIL基因的增殖型腺病毒（CNHK500-hTRAIL）和非增殖型腺病毒（Ad-hTRAIL）对TRAIL基因的表达以及对SMMC-7721肝癌细胞的杀伤能力。方法通过病毒增殖实验评估增殖型腺病毒CNHK500-hTRAIL的选择性增殖能力。通过MTT实验,评估增殖型腺病毒CNHK500-hTRAIL以及非增殖型腺病毒Ad-hTRAIL对人正常肝细胞株L02、人肝癌细胞株SMMC-7721的杀伤能力。采用ELISA法检测CNHK500-hTRAIL和Ad-hTRAIL感染SMMC-7721肝癌细胞后TRAIL基因的表达情况。以及通过流式细胞术（FCM）检测其对细胞早期凋亡的影响。结果CNHK500-hTRAIL能选择性地在SMMC-7721细胞内大量增殖,感染96 h后增殖达225137倍,在极低的MOI值（MOI=0.1）即可大量杀伤SMMC-7721细胞,明显强于Ad-hTRAIL,而对L02细胞无明显杀伤。CNHK500-hTRAIL和Ad-hTRAIL感染SMMC-7721细胞后,其TRAIL基因表达量,前者是后者的近10倍;CNHK500-hTRAIL可选择性地诱导SMMC-7721细胞早期凋亡,其能力显著高于Ad-hTRAIL。结论靶向增殖型腺病毒载体携带TRAIL基因对肿瘤细胞的杀伤能力和目的基因的表达,明显优于传统的非增殖型腺病毒载体,应用前景广阔。     

Hyperbaric Oxygen and N-Acetylcysteine Treatment in L-Arginine-Induced Acute Pancreatitis in Rats

ABSTRACT

Background: This study was designed to evaluate the combined effects of hyperbaric oxygen (HBO) and N-acetylcysteine (NAC) on acute necrotizing pancreatitis in rats. Methods: Experiments were performed in 50 male Wistar rats, which were divided into five groups (N = 10 for each group). The first group received normal saline (0.9% NaCl) intraperitoneal and served as the control group. In the second group, acute pancreatitis was induced by 3.2-g/kg body weight L-arginine intraperitoneal twice at an interval of 1 hr, which has been shown previously to produce severe necrotizing acute pancreatitis. In the third group, NAC treatment (1000 mg/kg) was given after 1 hr of the induction of acute pancreatitis twice 24 hr apart. In the fourth group, animals received HBO, 6 hr after the induction of pancreatitis twice 12 hr apart. In the fifth group, animals received together NAC as in Group 3 and HBO treatment as in Group 4. Groups 1, 2, and 3 were left under normal atmospheric pressures. Twelve hours after last treatment, the animals were killed by exsanguinations. Blood samples were studied for amylase, calcium, and lactate dehydrogenase (LDH), pancreatic histology, pancreatic tissue malondialdehyde, superoxide dismutase, and glutathione levels. Results: Acute pancreatitis is reduced by the treatment of NAC, HBO, NAC + HBO. HBO + NAC groups performed statistically the best in preventing L-arginine-induced acute necrotising pancreatitis. Conclusions: NAC especially combined with HBO, decreases oxidative stress parameters, serum amylase, calcium, and LDH levels, as well as histopathologic score.     

硝普钠阴茎海绵体内注射治疗阳萎的临床研究

本研究选择４２例阳萎患者，采用硝普钠进行阴茎海绵体注射（ＩＣＩ），并选择罂粟碱／酚妥拉明进行对照，结果表明，硝普钠ＩＣＩ后：（１）阴茎外形性状（长度、周径等）明显改变。（２）Ｖｉｒａｇ硬度计点表明硝普钠与罂粟碱／酚妥拉明效果之间无明显差别。（３）所有测试患者无一例出现低血压或局部不适等副反应，与罂粟碱／酚妥拉明相比各有优劣，但总体差异不大，这充分表明，硝普钠作为一种ＮＯ供体可导致阴茎平滑肌松弛，血窦充盈阴茎勃起，其副反应较小，有其临床应用之价值。     

糖皮质激素对肿瘤坏死因子引起的大鼠中性粒细胞粘附的影响

采用中性粒细胞（ＰＭＮ）与玻璃珠粘附和ＰＭＮ与血管内皮细胞（ＥＣ）粘附两种模型，以肿瘤坏死因子（ＴＮＦ），作为ＰＭＮ的刺激因子，研究糖皮质激素（ＧＣ）对ＴＮＦ引起的大鼠ＰＭＮ粘附的影响，同时给予糖皮质激素受体（ＧＲ）阻断剂ＲＵ３８４８６观察ＧＲ在粘附中的作用。结果发现，ＴＮＦ能明显增强大鼠ＰＭＮ的粘附（Ｐ＜０．０１）；Ｄｅｘ不能抑制经ＴＮＦ预处理的ＰＭＮ的粘附（Ｐ＞０．０５），但有一定的预防作用；经ＴＮＦ预处理再同时给予Ｄｅｘ和ＲＵ３８４８６的ＰＭＮ粘附同样明显增强（Ｐ＜０．０１）。     

Kelfiprim,a new sulpha-trimethoprim combination,versus cotrimoxazole,in the treatment of urinary tract infections: A multicentre,double-blind trial

Summary A new combination of trimethoprim with a sulphonamide, named Kelfiprim, differs from cotrimoxazole in that: a) the sulpha drug is sulphamethopyrazine instead of sulphamethoxazole; b) the trimethoprim to sulpha ratio is 5:4 instead of 1:5;c) the presence of a long-acting sulphonamide allows the administration of a daily dose of one capsule, following an initial loading dose of two capsules; d) a reduced amount of trimethoprim is given, as compared to cotrimoxazole, without any decrease of efficacy. Kelfiprim [KP] was compared to contrimoxazole [Co] in a multicentre double blind trial. Sixty four patients suffering from acute and chronic infections of the upper and lower urinary tract entered the study. Urine sterilisation and clinical improvement without relapses showed no differences from the two treatment groups. Tolerance was excellent except in two patients, one treated with KP and the other treated with Co, who showed a transient exanthema.     

Time-dependent hexaminolaevulinate induced protoporphyrin IX distribution after topical application in patients with cervical intraepithelial neoplasia: A fluorescence microscopy study

BACKGROUND AND OBJECTIVES: Compared to the conventional management of cervical intraepithelial neoplasia (CIN) the potential advantage of photodynamic therapy (PDT) for the treatment of cervical human papilloma virus (HPV)-related disease encompasses a minimal invasive procedure with reduced risk of profuse bleeding as a consequence of conization, and possibly more favorable long-term results avoiding cervical stenosis. At present little is known about the precise time-dependent distribution and histological localization of hexaminolaevulinate (HAL) induced protoporphyrin IX (PPIX) fluorescence in healthy tissue and in CIN. The aim of this study was to use ex vivo fluorescence microscopy to determine whether PPIX is selectively induced by neoplastic cells of the cervical epithelium at various times after topical application. STUDY DESIGN/MATERIALS AND METHODS: Cold cream containing 0.5% HAL was applied by means of cervical cap over various periods of time. We analyzed 52 healthy cervical mucosa and 84 CINs. RESULTS: At time delay 100 (+/-10) minutes, high epithelial fluorescence and a significant selectivity between epithelium and underlying lamina propria was found. By contrast, no significant difference between healthy and neoplastic tissues, or between low and high-grade epithelial dysplasia (P > or = 0.05), was observed at any time point. CONCLUSIONS: Application of HAL 0.5% cream to the cervix induced selective fluorescence in epithelial cells. The optimal ratio with a homogeneous PPIX distribution was obtained after 100 ( +/- 10) minutes cream application, which should be evaluated further for PDT.     

An updated review on the principles of intraoperative neurophysiological monitoring and the anaesthetic considerations

It is known that any surgery to the nervous system poses risks to neural structures and their surrounding structures. These mechanisms of injury are the result of mechanical manipulations, haemodynamic alterations, chemical or thermal injuries. Intraoperative neurophysiological monitoring (IONM), using various modalities, is employed to facilitate the assessment of the functional integrity of neural structures, and it is used to provide a real-time alerting system when changes caused by surgically induced insults are detected. The primary goal of IONM is reducing the risk of postoperative neurological deficits during these surgical procedures. It is used to provide information that allows the surgeon to correct any surgical interventions that may have compromised these systems and this also in turn provides guidance on what neurological deficits to anticipate postoperatively. Apart from being utilized as an alerting system to avoid catastrophic outcomes, IONM also assists as a guidance system using stimulation techniques to map out eloquent areas within the cortex, allowing identification of specific neuronal structures, particularly when landmarks cannot be easily recognized due to infiltration by pathological lesions.In this article, we focus on updating our previous paper published in 2019 and again, to provide attention to the various neurophysiological modalities that are employed in IONM. We will look at the basic underlying physiological principles and their individual indications for use clinically. We will explain the information that each modality provides. Importantly, and the primary reason for this article, we look at the various anaesthetic agents, their effects on each neurophysiological modality and other anaesthetic considerations such as haemodynamic and temperature effects. We will also recommend the use of an alert checklist for the multidisciplinary team should an intraoperative alert be issued during surgical procedures.