人破骨细胞生成抑制因子编码区基因的克隆及在成肌细胞中的表达

目的：克隆人破骨细胞生成抑制因子（OPG／OCIF）编码区基因并在真核细胞中的表达。方法：以人骨肉瘤细胞系MG63的总RNA为模板，采用RT－PCR法得到OPG／OCIF的编码区cDNA，构建真核表达载体pIRES2-OPG-EGFP，在脂质体介导下转染小鼠成肌细胞系C2C12，经G418压力筛选建立稳定转染人OPG／OCIF的细胞系，共聚焦荧光显微镜及核酸杂交方法检测OPG／OCIF在细胞中的表达，结果：获得的人OPG／OCIF编码区全长cDNA序列与文献报道的核苷酸序列一致，核酸杂交证实稳定转染人OPG／OCIF编码区cDNA的小鼠成肌细胞系中有OPG／OCIF，mRNA的表达。结论：获得人破骨细胞生成抑制因子编码区全长cDNA并证实在真核细胞中稳定表达。     

前列腺特异性膜抗原人源Fab抗体可变区基因的筛选与鉴定

目的:筛选、鉴定抗中国人前列腺特异性膜抗原(PSMA)的人源Fab抗体可变区的编码基因,为PSMA蛋白质生物学功能的研究及前列腺癌的基因治疗研究开辟新途径。方法:采用噬菌体表面展示技术,以PSMA原核表达重组子pET30 a(+)-PSMA诱导表达并纯化后的PSMA为固相抗原,从噬菌体Fab可变区抗体库中经过5轮“吸附-洗脱-扩增”筛选过程,获得抗原结合活性和特异性较强的PSMA人源Fab可变区抗体基因片段的阳性克隆,并对其进行免疫检测及序列测定。结果:筛选得到Fab抗体克隆的Fd段基因核苷酸序列为696 bp,编码由232个氨基酸残基组成的多肽,与人免疫球蛋白γ链恒定区的同源性为98%;轻链κ基因核苷酸序列为630 bp,编码由210个氨基酸残基组成的多肽,与κ链恒定区同源性为93%。结论:利用噬菌体抗体库技术,成功获得了PSMA人Fab抗体可变区编码基因克隆,该克隆抗体基因具有典型的免疫球蛋白轻链和重链可变区的结构特点,基因编码产物具有与PSMA反应的免疫学活性和特异性。     

肾癌G250抗原基因真核表达载体的构建和序列测定

目的构建肾癌G250抗原基因编码区序列的直核表达载体并进行序列测定。方法从肾癌组织中提取总RNA，采用RT-PCR技术扩增肾癌G250抗原的基因编码区序列，将PCR片段定向克隆至真核表达载体pcDNA3．0，并进行序列测定。结果肾癌组织RT-PCR扩增后，均产生特异性条带，位置在1000bp和1600bp之间。与预定相符。pcDNA3．0-G250分别用Hind Ⅲ、Xho Ⅰ双酶切后产生2条带。均与预定位置相符；抗原基因编码区序列与Genbank登录号BC014950文献报道的序列同源性为100％，目的基因与载体正确连接。结论成功克隆了肾癌G250抗原的基因编码区序列，并构建了其真核表达载体pcDNA3．0-G250，为核素标记的G250单克隆抗体在诊断和治疗的心用及G250基因修饰的树突状细胞疫苗的肾癌生物治疗提供了依据。     

人骨形成蛋白2的基因克隆和序列分析

目的 探讨克隆人骨形成蛋白2基因编码区的方法。方法 由人末梢血白细胞中提取细胞总DNA，利用PCR法，从DNA中分别扩增出人骨形成蛋白2外显子编码区基因DNA片段；将获得的基因片段插入PGEM—T—Easy质粒中，转化到大肠杆菌DH5α后挑选阳性克隆，利用限制性内切酶酶切核苷酸序列分析鉴定后，连接获得人骨形成蛋白2基因。结果 通过质粒DNA酶切分析及序列测定，我们获得的基因片段为人骨形成蛋白—2DNA序列。结论 首次由人白细胞DNA中克隆获得人骨形成蛋白2全长基因，为表达、应用骨形成蛋白2提供了前提条件。     

人前列腺干细胞抗原在前列腺组织中的表达

目的：克隆人前列腺干细胞抗原(PSCA)基因，并利用半定量逆转录聚合酶链反应(RT—PCR)对其组织表达谱进行分析。方法：从人前列腺癌组织提取总RNA，利用RT—PCR技术扩增出人PSCA基因编码区序列，并通过半定量RT—PCR方法检测6例人新鲜正常前列腺组织、16例前列腺增生组织及11例前列腺肿瘤组织中PSCA基因表达水平。结果：序列测定表明，克隆获得的369bp片段与文献报道的人PSCA基因编码区cDNA序列一致，PSCA为前列腺组织特异表达，在前列腺癌组织中的表达明显高于前列腺增生及前列腺正常组织。结论：PSCA是一种新的前列腺肿瘤标记物。     

前列腺癌噬菌体抗体库的构建及抗特异性膜抗原抗体的筛选

目的建立国人前列腺癌噬菌体Fab抗体片段库，筛选、鉴定抗前列腺特异性膜抗原（PSMA）的人源Fab抗体可变区的编码基因，为前列腺癌的诊断与基因治疗研究开辟新途径。方法20例前列腺癌患者外周血分离淋巴细胞，提取其总RNA，经RTPCR及半套式扩增得到免疫球蛋白全部轻链和重链Fd段基因，克隆进载体pComb3，并电转化大肠杆菌XLl-Blue，构建前列腺癌噬菌体Fab抗体片段库。采用噬菌体表面展示技术，以PSMA原核表达重组子pET30a（＋）-PSMA诱导表达并纯化后的PSMA为固相抗原，从噬菌体Fab抗体库中经过“吸附洗脱扩增”筛选过程，获得抗原结合活性和特异性较强的PSMA人源Fab可变区抗体基因片段的阳性克隆，并进行免疫检测及序列测定。结果成功构建前列腺癌噬菌体抗体Fab片段库，其轻链及重链Fd段与载体DNA的重组率分别为87％、79％，库容为2．32×10^7，滴度为8．7×10^13pfu／ml。筛选得到Fab抗体克隆的Fd段基因核苷酸序列为696bp，编码由232个氨基酸残基组成的多肽，与人免疫球蛋白γ链恒定区的同源性为98％；轻链κ基因核苷酸序列为630bp，编码由210个氨基酸残基组成的多肽，与κ链恒定区同源性为93％。结论成功构建了前列腺癌噬菌体抗体库，并获得了PSMA人Fab抗体可变区编码基因克隆，该克隆抗体基因具有典型的免疫球蛋白轻链和重链可变区的结构特点，基因编码产物具有与PSMA反应的免疫学活性和特异性。     

胆管癌相关基因FXYD6的克隆及功能区定位

目的从人组织cDNA文库克隆胆管癌相关基因FXYD6并进行功能区定位分析。方法利用人胎肝、脑及脾cDNA文库,通过Goldkey软件进行引物设计,从组织cDNA文库获得FXYD6全长和转录编码区序列,对其进行克隆与纯化;对FXYD6基因重组克隆进行测序鉴定;并应用GenBank核酸数据库及Goldkey软件对其功能区进行定位分析。结果在胎肝和脑cDNA文库中,扩增出FXYD6特异条带,但在脾cDNA文库未见扩增产物。将PCR扩增产物与pGEM-T载体连接,进行转化后确认回收组、阳性对照组均可见单菌落生长。选取阳性克隆进行菌落收获和质粒提取,并行测序分析,该序列包含已测序FXYD6基因大片段和可能的编码区序列。通过序列分析,得到最可能编码区序列为＋1相位编码区序列,进一步分析得到该基因产物作为膜蛋白的可能功能区分布。结论 FXYD6基因克隆、纯化及功能区定位的成功为进一步研究该基因的功能奠定了基础。     

ABCA2基因单核苷酸多态性与胆囊结石病的相关性

目的 检测中国人ABCA2(ATP binding cassette A2)基因编码区单核苷酸多态性(single nucleotide polymorphism,SNP),并探讨其与中国汉族人群胆囊结石病的关系。方法 采用直接测序法检测ABCA2基因编码区及相邻的部分内含子的序列,以确定中国人ABCA2基因SNPs的位置及类型。对位于编码区的SNPs,采用病例对照方法,在胆囊结石病患者和正常对照者中进行关联研究。结果在16 911 bp测序长度中,共发现12个SNP,2个位于编码区,10个位于内含子,其中7个为新发现的SNP。各SNPs在胆石病组和对照组中分布差异无统计学意义。结论SNP在不同种族间存在差异。ABCA2基因编码区SNP在胆石病和对照组间分布差异无统计学意义。     

Msx1编码区突变与非综合性唇腭裂相关性分析

目的：探讨核心家庭中Msx1编码区突变与非综合征性唇腭裂的关系。方法：运用测序技术检测华东地区核心家庭的Msx1基因全部编码区的突变情况，预测其编码氨基酸的突变频率，比较突变位点在患儿与正常儿童中的分布差异并判断是否有统计学意义，比较患者组与父母组的基因型和等位基因频率，并对核心家庭的数据进行单倍型相对风险分析（HRR），对含杂合子父母的核心家庭进行传递不平衡检验（TDT）。结果：Msx1编码区序列共有三个位点突变，分别为编码区1的C101G（A34G）突变，编码区1同义突变C330T（G110G），编码区2的G162A（S278N）突变。其中只有编码区1的同义突变C330T（G110G）在患儿与正常儿童中的分布差异有统计学意义，患者与其父母各位点基因型和等位基因分布差异无统计学意义（P〉0．05），HRR结果和TDT结果无统计学意义，另外两个突变位点各项统计均无统计学意义。结论：编码区1的同义突变C330T（G110G）可能与中国华东人群非综合征性唇腭裂存在相关性，而编码区1的C101G（A34G）突变和编码区2的G162A（S278N）突变与中国华东人群非综合征性唇腭裂不存在相关性。     

雄激素调节的前列腺特异性同源框基因NKX3.1研究进展

同源框基因(Homeobox Genes)最早在果蝇中发现，是一大类编码同源域蛋白(Homeodomain Protein)调控胚胎发育分化的基因。同源域蛋白含有称为同源域(Homeodomai，HOM，在哺乳类称为Hox)的蛋白片断，同源域则是由同源框基因中所含的同源域序列所编码。同源域序列为含有183bp的高度保守序列，所编码的61个氨基酸的同源域蛋白片断进化上高度保守，有识别和结合特异性DNA序列的功能，是真核生物转录因子。     

Current status of non-adjustable gastric banding

Background: The purpose of this study is to review the current status of non-adjustable gastric banding (NGB) and to determine whether this operation is still acceptable in the management of morbid obesity, especially when compared with the adjustable GB (AGB) in long-term results. Materials and Methods: A literature search was conducted of data published on NGB and AGB in Obesity Surgery in the past 12 months or available from other sources, with records of early and late band-related complications, reoperation rate and weight loss in groups reporting ≥100 patients with minimum 3-year postoperative follow-up. Results: 1,812 NGB and 1,968 AGB patients were included. Mean BMI was 42.4 in NGB vs 44.0 in AGB. No statistical difference occurred in the early complication rate (1.4% in NGB vs 1.6% in AGB). A statistical difference was noted in long-term complication rate, (1.9% in NGB vs 6.7% in AGB), and in reoperation rate (3.4% vs 7.2%). There was no difference in excess weight loss at 48 months following both operations (54.2% vs 53.0%). Conclusion: A significant difference in favor of NGB occurred in the long-term reoperation rate. No other differences were identified, other than in band material. NGB is a softer material and therefore, according to computerized images, has greater flexibility in copying gastric peristaltic waves, which may result in less irritation and more physiological behavior by this band.     

脑红蛋白在感染性休克早期大鼠脑组织中的动态变化

目的探讨感染性休克早期大鼠脑组织中脑红蛋白(neuroglobin,NGB)含量的动态变化在感染性休克中可能发挥的作用。方法健康雄性SD大鼠60只,12~15周龄,体重300~350g,随机分为两组,每组30只。M组经尾静脉注射脂多糖(LPS)10mg/kg制备大鼠感染性休克模型,当SBP下降幅度达到基础值的40%时认为感染性休克模型复制成功;C组给予等量生理盐水。分别在给LPS前(T_0)、给LPS后1h(T_1)、2h(T_2)、4h(T_3)、6h(T_4)采用ELISA法检测血清中TNF-α、IL-6浓度和NGB含量,Western blot法检测脑组织中NGB、半胱天冬酶-3(caspase-3)的含量。结果与C组比较,T1~T4时M组血清中的TNF-α、IL-6浓度明显增高(P0.05),脑组织中caspase-3含量明显增加(P0.05);T_3、T_4时血清和脑组织中NGB含量明显增高(P0.05)。结论感染性休克后大鼠脑组织发生了炎症损伤、细胞凋亡病理学改变,脑红蛋白在感染性休克所致的损伤脑组织中含量增加,推测其可能发挥了内源性神经保护功能。     

Report on Bariatric Surgery in the Ukraine

Background: Morbid obesity (MO) is a problem internationally, including in the Ukraine.We present the surgical treatment of MO in the Ukraine over the last 15 years, during which intestinal bypasses and various gastric reduction procedures were performed. Methods: 198 patients with MO underwent: jejunoileal (JI) bypass 64, non-adjustable gastric banding (NGB) 34, Roux-en-Y gastric bypass (RYGBP) 1, horizontal gastroplasty 1, vertical banded gastroplasty (VBG) 2, and abdominal lipectomy 96.The 96 men and 102 women weighed 160-290 kg (mean 210±SD18 kg). Mean body mass index was >60 kg/m2. These patients had a high incidence of hypertension, diabetes, sleep apnea, menstrual disorders, impotency in men and infertility in women. Results: At 1 year, after JI bypass 61 patients lost a mean of 62±17 kg and after NGB 11 kg. After JI bypass, 1 patient died in the early postoperative period from acute respiratory insufficiency and 2 died in the first year from acute liver insufficiency. The JI bypass was reversed in 2 patients due to uncontrollable malabsorption syndrome; 1 year after reversal, the weight of these patients exceeded their preoperative weight. In the early postoperative period, 1 patient died after NGB and 1 after RYGBP, from acute respiratory insufficiency. Postoperative weight loss was associated with decrease in the co-morbidities of MO, but after JIB, there was a high incidence of bypass enteritis, excessive malabsorption, formation of renal stones and gallstones. After NGB, no complications have been identified. Isolated lipectomy was performed in 44 patients, lipectomy combined with a bariatric operation in 31, and lipectomy after loss of the excess body weight in 21. Conclusions: Bariatric surgery was very effective in weight loss, accompanied by reduction or disappear ance of the co-morbidities of MO, with considerable improvement in quality of life.     

Not all neurogenic bladders are the same: a proposal for a new neurogenic bladder classification system

Neurogenic bladder (NGB) has long been defined as a clinical entity that describes a heterogeneous collection of syndromes. The common theme is a bladder disorder concomitant with a neurologic disorder. This definition does not give the clinician much information about the bladder disorder, nor how to treat it, or even what the natural history of the disorder is likely to be. It may be time for a new classification scheme to better define the bladder defect and prognosis, as well as inform treatment. We propose a classification system based on seven categories, each having a neurologic defect in a distinct anatomic location. This is termed SALE (Stratify by Anatomic Location and Etiology). In addition, the presence or absence of bowel dysfunction and autonomic dysreflexia will be reported. In the future, as more definite prognostic information can be gleaned from biomarkers, we anticipate adding urinary nerve growth factor (NGF) and urinary brain-derived neurotrophic factor (BDNF) levels to the definition. We expect the SALE system to efficiently describe a patient suffering from NGB and simultaneously inform the most appropriate treatment, follow-up regimen, and long-term prognosis.     

Medical management of neurogenic bladder with oral therapy

This is a review of the most current literature on medical management of the neurogenic bladder (NGB) to treat detrusor overactivity (DO), improve bladder compliance and treat urinary incontinence. The use of antimuscarinics, alpha blockers, tricyclic antidepressants, desmopressin and mirabegron will be discussed along with combination therapy to improve efficacy. These medical therapies will be the focus of this review with surgical therapy and botulinum toxin injections being the subject of other articles in this series.     

A cross‐sectional study of the catheter management of neurogenic bladder after traumatic spinal cord injury

Aims

This cross‐sectional study describes the catheter management of neurogenic bladder (NGB) in patients with traumatic spinal cord injury (tSCI) with emphasis on the motivations behind transitions between intermittent (IC) and indwelling catheters.

Methods

Patients at the Minneapolis VA with history of tSCI who utilized either intermittent catheterization (IC), urethral (UC) or suprapubic (SP) catheters, participated in a voluntary, anonymous survey regarding their bladder management strategies.

Results

A total of 100 patients participated, 94% were male and 90% Caucasian with median age of 61 years. Patients with current UC or SP were older than those utilizing IC (P = 0.002). The median age at injury and years since SCI were 32 years and 20.5 years, respectively. The median time with current modality was 11 years. A total of 27% of all patients reported at least one transition between catheter type. A total of 14 of 54 patients using IC had prior use of UC or SP, while 12/25 patients using SP and 10/21 patients using UC had prior use of IC. The most common reasons to stop IC included inconvenience, physician recommendation, and dislike of IC. A total of 53% of patients currently using UC or SP reported never using IC. Patients currently using SP were more content with their current catheterization method than those using UC or IC (P = 0.046).

Conclusions

Among patients using catheters for NGB, intermittent catheterization was the most common modality utilized and the transition between intermittent and indwelling catheter was most often influenced by patient preferences and clinician recommendations.     

Human glyceraldehyde 3-phosphate dehydrogenase-2 gene is expressed specifically in spermatogenic cells

Although the process of glycolysis is highly conserved in eukaryotes, several glycolytic enzymes have unique structural or functional features in spermatogenic cells. We previously identified and characterized the mouse complementary DNA (cDNA) and a gene for 1 of these enzymes, glyceraldehyde 3-phosphate dehydrogenase-s (Gapds). This gene is expressed only in spermatids. The enzyme appears to have an essential role in energy production required for fertilization, and it is reported to be susceptible to inhibition by certain environmental chemicals. We have now cloned and sequenced the cDNA for the human homologue of glyceraldehyde 3-phosphate dehydrogenase (GAPD2) and determined the structure of the gene. The messenger RNA (mRNA) was detected in testis, but not in 15 other human tissues analyzed by Northern blot technique. The deduced GAPD2 protein contains 408 amino acids and is 68% identical with somatic cell GAPD. GAPD2 has a 72-amino acid segment at the amino terminal end that is not present in somatic cell GAPD. This segment is proline-rich but contains smaller stretches of polyproline and is 30 amino acids shorter than the comparable segment of mouse GAPDS. The structure of the human GAPD2 gene was determined by polymerase chain reaction (PCR) to identify exon-intron junctions in a genomic clone and in total genomic DNA. The locations of these junctions in the GAPD2 gene corresponded precisely to those of the 11 exon-intron junctions in the mouse Gapds gene. Immunohistochemical studies found that GAPD2 is located in the principal piece of the flagellum of human spermatozoa, as are GAPDS in mouse and rat spermatozoa. GAPD2 extracted from human spermatozoa and analyzed by Western blot technique migrated with an apparent molecular weight of approximately 56,000, although the calculated molecular weight is 44 501. The conserved nature of the mouse, rat, and human enzymes suggests that they serve similar roles in these and other mammalian species.     

Biphasic response of tropoelastin at the poststenotic dilation segment of the rabbit aorta

OBJECTIVE: The purpose of this study was to assess the time course of tropoelastin gene expression in the poststenotic dilatation segment of rabbit aorta with experimental coarctation. METHODS: Midthoracic aortic coarctation was created in rabbits to produce a PSD. The time points of the study after coarctation were 1, 3, and 7 days and 2, 4, and 8 weeks (n = 3 each). Additional animals (n = 6) were subjected to hypercholesterolemia for analysis of tropoelastin expression in intimal lesions. Northern and Western blot analyses were used to quantitate tropoelastin messenger RNA (mRNA) and protein, and immunohistochemistry was used to analyze tropoelastin distribution. RESULTS: Thoracic aortic coarctation produced a moderate stenosis, which resulted in PSD. mRNA levels in the PSD segment decreased at days 1 and 3, followed by an increase at 2 and 4 weeks (P <.05 versus controls). This biphasic change in tropoelastin mRNA was associated with increase in tropoelastin protein levels at 2 and 4 weeks (P <.05 versus controls). PSD diameter reached a maximum at 4 weeks and did not increase significantly thereafter. The number of medial elastic laminae in PSD was reduced slightly, but media thickness was unchanged. Intimal lesions were much smaller in the PSD segment than in the proximal segment in animals with hypertension superimposed with hypercholesterolemia. Moreover, tropoelastin protein distributed not only in the intima but also in the media of the PSD. CONCLUSION: Tropoelastin gene expression is regulated in a biphasic pattern and precedes PSD formation. The differential distribution of tropoelastin in the media suggests a role for tropoelastin in the poststenotic adaptation response, which may provide increased elasticity to the PSD wall.