前列腺特异性膜抗原基因复制缺陷型腺病毒载体的构建及表达

目的 构建携带前列腺特异性膜抗原(PSMA)基因的复制缺陷型腺病毒载体,并检测其在真核细胞中的表达.方法 利用质粒pET-30a-PSMA行PCR扩增、酶切获得PSMA cDNA片段,并插入腺病毒穿梭质粒pAdTrack-CMV的启动子下游,构建重组穿梭载体pAdTrack-CMV-PSMA,线性化后与骨架质粒pAdEasy-1在细菌BJ5183内同源重组得到腺病毒质粒pAd-PSMA,经293A细胞包装成复制缺陷型腺病毒Ad-PSMA.将Ad-PSMA体外感染DU145细胞,蛋白质印迹法检测目的基因表达.结果 构建了携带PSMA基因的复制缺陷型腺病毒,病毒滴度1.4×109 pfu/ml,能有效感染DU145细胞,转染率约95%,并经蛋白质印迹法检测到相对分子质量约100 000的目的蛋白PSMA在DU145中表达.结论 成功构建了表达PSMA基因的复制缺陷型腺病毒载体,为开展靶向PSMA的基因免疫治疗提供了实验基础.     

后腹腔镜下根治性肾切除术及其应用解剖学研究

目的 总结后腹腔镜下根治性肾切除术,并对镜下肾周区域相关解剖结构进行研究.方法 2006年1月至2008年3月行后腹腔镜下根治性肾切除术85例.肿瘤位于左肾38例,右肾47例;肿瘤直径2.5～10.5 cm,平均(5.5±1.7)cm;T1N0Mo 74例,T2:N0M0 11例.根据肾筋膜外切除原则,在"两个间隙、上下两极间"分离切除患肾,腹侧为肾旁前间隙(后腹膜与肾前筋膜之间),背侧为腰肌前间隙(肾后筋膜与腰肌筋膜之间),上极达膈下,下方到髂窝.术中辨认重要的解剖结构:后腹膜及其返折、肾前筋膜、侧锥筋膜、肾后筋膜、腰大肌及重要血管. 结果 除1例因与周围粘连严重中转开放手术外,其余均获得成功.手术时间50～165 min,中位数65 min.估计失血量25～600ml,中位数58 ml.术中腹膜破裂5例,肾蒂周围小血管损伤出血6例,无重要脏器及大血管损伤.术后平均随访10(2～25)个月,均无瘤生存,无局部复发或发生穿刺通道种植转移. 结论 后腹腔镜下根治性肾切除术时,熟悉重要的镜下解剖结构有利于提高手术安全性、减少副损伤.     

睾丸支持细胞的基本生物学特性

目的通过了解睾丸支持细胞(Sertoli cells,SC)的基本生物学特性,探讨SC鉴定的良好方法。方法联合应用复合胶原酶及差速贴壁法从睾丸组织中分离、培养SC,光镜和电镜下观察细胞的形态、MTT法测定细胞的生长曲线,观察其在体外培养条件下的增殖特性;利用免疫细胞染色及免疫荧光染色的方法,检测Fas配体(FasL)的表达,观察其免疫功能;应用吖啶橙荧光染色进行细胞鉴定。结果联合应用复合胶原酶及差速贴壁法分离、培养的细胞在光镜下呈长柱状及三角形,增殖能力强,电镜下胞核中可见特异性卫星小体,胞质中细胞器丰富,免疫细胞染色及免疫荧光染色证实其高表达FasL,吖啶橙荧光染色可见胞核中含有大量异染色质,核仁明显,证实其为SC。结论复合胶原酶及差速贴壁法分离的SC具有良好的增殖能力及免疫功能,电镜、吖啶橙荧光染色是鉴定SC方便、有效的方法。     

HIV感染者血β2微球蛋白(β2MG)的临床意义

目的 分析HIV感染者血β2微球蛋白(β2MG)的临床意义.方法 检测HIV感染者血β2MG、蛋白电泳、T淋巴细胞亚群计数、肌酐,分析血β2MG与蛋白电泳、T淋巴细胞亚群计数的相关性.结果 HIV感染者血β2MG升高者占86.5%(64/74),其中无症状期、AIDS期患者中血β2MG升高者分别占90.9%(20/22)、84.6%(44/52).AIDS期患者血β2MG水平显著高于无症状期感染者(P＜0.01),且CD4＜200/ml的HIV感染者血β2MG明显高于CD4＞200/ml的感染者(P＜0.05);78.8%(67/85)HIV感染者血清γ球蛋白百分比升高,其中无症状期和AIDS期感染者分别为75%(27/36)和81.6%(40/49).未观察到无症状期和AIDS期感染者γ球蛋白百分比有统计学差异(P＞0.05),但是无症状期感染者血清白蛋白百分比明显高于AIDS期感染者(P＜0.05).HIV感染者血β2MG与血清γ球蛋白百分比呈正相关:r=0.455(P＜0.01),而与血清白蛋白百分比呈负相关:r=-0.586(P＜0.01).HIV感染者血β2MG与CD4计数呈负相关:r=-0.279(P＜0.05),也与CD4/CD8比值呈负相关:r=-0.399(P＜0.01).对血β2MG＞3.0 mg/L的HIV感染者,其血β2MG与CD4计数、CD4/CD8比值呈负相关:r=-0.434、r=-0.427 (P＜0.01).几乎所有的HIV感染者肾功能指标都正常.结论 绝大多数HIV感染者血β2MG升高,且随着疾病进展而升高,可能与免疫系统被激活和淋巴细胞被破坏有关.HIV感染者血β2MG升高是因机体产生了更多的β2MG,而非因HIV感染者肾小球滤过率降低,滤过功能受损所引起.     

细胞周期素依赖性蛋白激酶-5参与疼痛、阿片耐受

疼痛及其治疗是临床上的一大难题,主要由于机制不明.Cdk5是一种特殊的细胞周期素依赖性激酶家族,几乎存在于整个中枢和外周神经系统的神经元内.Cdk5作为丝氨酸/苏氨酸激酶,可调节众多底物和蛋白,目前发现在疼痛信号通路及阿片耐受中具有重要作用,可能作为治疗疼痛的新方法,对其在疼痛通路及阿片耐受中作用的深入了解,将有助于解决这一难题.     

Association of bone morphogenetic protein-2 gene polymorphisms with susceptibility to ossification of the posterior longitudinal ligament of the spine and its severity in Chinese patients

A case–control study was conducted to examine the association between two single nucleotide polymorphisms (SNPs) in exon 2 of the bone morphogenetic protein-2 gene (BMP-2) and ossification of the posterior longitudinal ligament (OPLL), and to investigate whether SNPs of the Ser37Ala (T/G) and the Ser87Ser (A/G) in the BMP-2 gene are associated with genetic susceptibility to OPLL and its severity in Chinese subjects. The Ser87Ser (A/G) SNP has been implicated in bone mineral density (BMD) and increases the risk of OA in women. The Ser37Ala (T/G) SNP is associated with BMD and the rate of bone loss in osteoporosis and osteoporosis fractures. A total of 57 OPLL patients and 135 non-OPLL controls were studied. Radiographs of the cervical spine were analyzed to determine the presence and the severity of OPLL. The association of two SNPs with the occurrence and the extent of OPLL were statistically evaluated. There was a significant association between the Ser37Ala (T/G) polymorphism and the occurrence of OPLL in the cervical spine. However, no significant association was found between the Ser37Ala (T/G) polymorphism and the more number of ossified cervical vertebrae in OPLL patients. There was a significant association between the Ser87Ser (A/G) polymorphism and the more number of ossified cervical vertebrae in OPLL patients. However, there was no statistical difference between the Ser87Ser (A/G) SNP and the occurrence of OPLL in the cervical spine. In addition, the Ser87Ser (A/G) polymorphism in male patients and in female patients showed no statistical difference between cases and controls. The present results demonstrate that BMP-2 Gene is not only a factor associated with the occurrence of OPLL, but also a factor related to more extensive OPLL. The “G” allele in the Ser37Ala (T/G) polymorphism is associated with the occurrence of OPLL, but not more extensive OPLL in the cervical spine. The “G” allele in the Ser87Ser (A/G) polymorphism promotes the extent of OPLL, whereas the “A” allele in the Ser87Ser (A/G) polymorphism restricts ectopic ossification in the cervical spine at least in Chinese subjects.     

Solitary Lymph Node Metastasis in Gastric Cancer

The feasibility and diagnostic reliability of sentinel node (SN) biopsy for gastric cancer are still controversial. We studied the clinicopathological features and localization of solitary lymph node metastasis (SLM) in gastric cancer to provide useful information for use of the SN concept in gastric cancer. From 2000 to 2004, 3,267 patients with gastric cancer underwent D2 radical gastrectomy. The clinicopathological features of 195 patients with histologically proven SLM and the distribution of metastasized nodes were assessed. The incidence of SLM was 6.0% in all cases. Compared with the node-negative patients, significant differences were observed in age, tumor size, depth of invasion, and surgical type. The cumulative 5-year survival rate of patients with SLM was 80.5%, which was significantly lower than 90.2% for node-negative patients (P < 0.001). Of patients with SLM, 82.6% had it in the perigastric node area (N1), and the other 17.4% patients had skip metastasis in the N2-N3 nodes. Perigastric nodes were the most common first sites of drainage from the tumor, making them the main targets of the operative SN mapping procedure. Due to the higher than expected incidence of skip metastasis in gastric cancer, D2 lymphadenectomy should be performed until the reliability of SN navigation surgery is validated in multicenter prospective clinical trials.     

足跟皮肤软组织缺损的皮瓣修复

目的介绍应用多种皮瓣修复足跟皮肤软组织缺损的经验。方法1993年8月-2007年4月治疗242例足跟部皮肤软组织缺损患者。男157例，女85例；年龄27～76岁。病变类型：不稳定瘢痕35例，慢性溃疡46例，鳞状细胞癌57例，恶性黑色素瘤72例，创伤引起的组织缺损32例。创面均位于足跟部，51例伴轻度感染，皮肤软组织缺损范围2cm&#215;2cm～14cm&#215;8cm。患者病程1h～5年。根据创面的部位、皮肤软组织缺损的范围分别选用岛状或游离（肌）皮瓣修复足跟创面，其中足跟外侧皮瓣34例，足背岛状皮瓣15例，足底内侧岛状皮瓣108例，腓肠神经营养血管皮瓣36例，隐神经营养血管皮瓣26例，游离（肌）皮瓣23例。行腓肠神经营养血管皮瓣和隐神经营养血管皮瓣感觉重建13例。切取（肌）皮瓣范围3．0cm&#215;2．5cm～15．0cm&#215;9．0cm。供区采用直接缝合或皮片移植修复。结果术后235例皮瓣全部成活，创面I期愈合；5例皮瓣远端部分表皮坏死，经局部换药后成活；2例切口轻度感染，经局部换药处理后创面II期愈合。供区创面均I期愈合，移植皮片完全成活。217例患者获随访，随访时间1～60个月，皮瓣色泽正常，质地较正常足跟组织柔软、可耐磨、外形轮廓满意；经训练术后6个月基本可正常负重行走，步态正常。118例肿瘤患者获随访，局部均无肿瘤复发，其中22例恶性黑色素瘤患者术后12～26个月发现有远处转移病灶，随访至60个月的32例恶性黑色素瘤患者未发现远处转移病灶。结论应用不同岛状或游离（肌）皮瓣修复足跟部创面，可达到较理想的足跟功能重建和外形重塑效果。     

糖尿病对大鼠自体移植静脉内膜增生的影响

目的:探讨AGE-RAGE系统对糖尿病大鼠自体移植静脉内膜增生的作用。方法:雄性SD大鼠40只，随机分为糖尿病组及正常对照组。两组均建立自体静脉移植模型，制模后的7 d和14 d测定血清AGE含量，取自体静脉移植标本行组织形态学观察，半定量RT-PCR检测RAGE，NF-κB p65及VCAM-1mRNA的表达，Western蛋白印迹和免疫组化方法检测RAGE，NF-κB p65及VCAM-1蛋白表达,并用免疫组化方法检测PCNA的表达。结果:术后7 d及14 d，与对照组比较，糖尿病组自体移植静脉内膜增生明显为重，PCNA，RAGE，NF-κB p65及VCAM-1mRNA和蛋白表达均增强，血清AGE含量增加，差异均有统计学意义（均P<0.05）。 结论:AGE-RAGE系统激活NF-κB，增强黏附分子的表达，可能是糖尿病大鼠自体移植静脉内膜增生加重的原因。