p21WAF1与p27KIP1真核双表达载体的构建鉴定及在胃癌细胞中的表达

目的 构建p21WAF1与p27KIP1真核双表达载体,体外转染胃癌7901细胞,观察其在胃癌细胞中的表达及相关特性.方法 提取人全血总RNA,利用逆转录-聚合酶链反应(RT-PCR)扩增p21WAF1和p27WAF1基因并将其分别克隆到真核双表达载体pIRES中构建重组质粒pIPES-p27KIP1-p21WAF1,经酶切和测序鉴定重组质粒.脂质体介导重组质粒plRES-p27KIP1.p21WAF1"转染胃癌7901细胞,收集并提取转染后12、24、48、72 h和5 d各细胞的总RNA,逆转录成cDNA,荧光定量聚合酶链反应(FQ-PCR)检测各细胞中p21WAF1和p27KIP1在RNA水平的表达.带有绿色荧光蛋白的pIRES-EGFP作为对照.结果 RT-PCR扩增分别获得约500和600 bp大小的产物,重组质粒pIRES-p27KIP1.p21WAF1经酶切和测序鉴定证实为p21WAF1和p27KIP1基因.转染胃癌7901细胞48 h时转染率为68%.与空质粒对照组比较,重组质粒转染胃癌7901细胞12、24、48、72 h和5 d后FQ-PCR证实p27KIP1的Ct值(cycle threshold,Ct)分别减少8.2、10、10、7.4和6.7,p21WAF1的Ct值分别减少7.4、8.2、8.2、6.3和5.70其中24 h和48 h的Ct值减少最大.结论 真核双表达载体pIRES-p27KIP1.p21WAF1构建成功并能在胃癌7901细胞内高效表达.     

Differential Regulation of RGS-2 by Constant and Oscillating PTH Concentrations

PTH has diverse effects on bone metabolism: anabolic when given intermittently, catabolic when given continuously. The cellular mechanisms underlying the varying target cell response are not clear yet. PTH induces RGS-2, a member of the Regulator of G-protein Signaling protein family, via cAMP/PKA, and inactivates PKC-mediated signaling. To investigate intracellular signaling pathways with different PTH concentration-time patterns, we treated UMR 106-01 osteoblast-like cells in a perfusion system. PTH was administered intermittently (4 min/h, 10⁻⁷ M) or continuously at an equivalent cumulative dose (6.6 × 10⁻⁹ M). cAMP was measured using radioimmunoassay, mRNA levels using real-time rtPCR and ribonuclease protection assay, and protein levels using Western immunoblotting. A single PTH pulse transiently increased cAMP levels by 2000% ± 1200%. In contrast to continuous PTH exposure, cAMP induction remained unchanged with intermittent PTH, ruling out desensitization of the PTH receptor. In continuously perfused cells, RGS-2 abundance was three to five times higher than in cells intermittently exposed to PTH for up to 12 h. MKP-1 and -3 were significantly less induced with pulsatile PTH; exposure-mode-dependent differences in MMP-13 and IGFBP-5 were small. Pulsatile but not continuous PTH administration prevents PTHrP receptor desensitization and accumulation of RGS-2 in osteoblasts, which should preserve PKC-dependent signaling.     

Gene expression profiling of human lymph node metastases and matched primary breast carcinomas: Clinical implications

The genetic program that drives tumor metastasis and the mode and timing of its initiation are of great practical significance to clinical management. Modern technical advances open new opportunities for gaining useful relevant information. Gene expression profiles of histologically‐verified viable tissue from lymph node metastases were compared with those of matched primary breast cancers from 10 different patients, among samples from over 400 cases, using high‐throughput oligonucleotide arrays comprising probes for 22,000 genes. It was observed that metastases have very similar expression signatures to their parent tumors. However, detailed computational analysis revealed that a small number of genes were consistently differentially expressed between 100% of tumors and metastases, suggesting that these are mechanistically important. Lists of such candidate genes, of potential clinical interest, are provided. We interpret these results in the framework of a meta‐analysis of previous investigations by others and ourselves and of existing clinical knowledge on the behavior of human tumors. The collective data show that metastases resemble their primary tumors but the signatures obtained in different studies are not sufficiently reproducible or informative to be prognostically useful, although they do give valuable insights into the pathogenesis and biology of human tumor metastasis. The findings indicate that the genetic program encoding metastasis is implemented progressively over time although, occasionally, this evolution can occur rapidly, early in the life of the neoplasm. The important clinical significance of this deduction is that, in most patients, early detection provides time for appropriate therapeutic intervention to be effective in obstructing metastasis.     

肝癌多药耐药产生与低糖环境的关系

目的探讨局部微环境低糖与肝细胞癌多药耐药性(MDR)产生的关系及影响机制。方法低糖培养HepG2细胞,应用流式细胞术Annexin V/PI法检测低糖培养的细胞在化疗药物5-氟脲嘧啶(5-Fu)作用后的凋亡情况,分别应用荧光定量聚合酶链反应(PCR)技术和Western blot技术检测低糖培养后HepG2细胞内多药耐药相关基因mdr1、MRP1、LRP的mRNA和蛋白水平的表达。结果在低糖环境下生长时间越长的HepG2细胞对5-Fu的抵抗越强,而且随着低糖培养时间的增加,5-Fu诱导的HepG2细胞的凋亡高峰延迟。低糖培养的HepG2细胞内多药耐药相关基因mdr1、MRP1、LRP在mRNA和蛋白水平的表达随低糖培养时间的延长而升高,以LRP的改变最为显著。结论肝癌生长微环境葡萄糖耐量不足也是肝癌产生MDR的原因之一。低糖可通过上调一组多药耐药相关基因的表达而诱导肝癌的多药耐药性。     

睾丸旁促结缔组织增生性小圆细胞肿瘤1例报告及文献复习

目的:探讨睾丸旁促结缔组织增生性小圆细胞肿瘤的临床、病理特征、诊断及治疗。方法:回顾分析1例睾丸旁促结缔组织增生性小圆细胞肿瘤的诊治及随访资料,并复习相关文献。患者,男,27岁,因阴囊内无痛性肿块4个月就诊。体检于左侧阴囊内可扪及囊性肿块,同侧睾丸不能扪及。行左侧睾丸根治性切除术,术后辅以化疗。结果:术中见左侧睾丸旁多发结节状隆起肿块,质偏硬;术后病理检查肿瘤细胞呈巢状或梁索状结构并埋没在增生的纤维结缔组织中;免疫组化显示瘤细胞具有上皮源性、间质性和神经源性等多向分化的特点。术后已随访3年无瘤生存,预后良好。结论:促结缔组织增生性小圆细胞肿瘤有特异的临床病理特征,好发于年轻男性,手术切除肿瘤联合化疗是治疗的主要方法,睾丸旁促结缔组织增生性小圆细胞肿瘤比腹腔型预后相对好。     

Association of the Platelet Glycoprotein Ⅰa C807T Gene Polymorphism With Risk of Myocardial Infarction in Chinese

Objectives To investigate the relationship of the GPIa C807T dimorphism to the risk of myocardial infarction (MI) in Chinese. Methods We did a case-control study including 100 patients and 110 controls with same race. An allele-specific polymerase chain reaction (PCR) was used for genotyping of C807T polymorphism. Results An apparent association was found between the T807 allele and MI among individuals younger than the mean age of 60 years (odds ratio,2.49; 95% confidence interval, 1.08 ～ 6.22 ). The T807 allele remained an independent risk factor for MI when age, sex, smoking, hypertension, diabetes, bodymass index, LDL-cholesterol and HDL-cholesterol were adjusted by logistic regression. Conclusions GPⅠa T807 appears to be an independent risk factor for MI.     

CT的分叶征表现在肺内孤立结节影像诊断中的价值

目的探讨CT的分叶征表现在肺内孤立结节影像诊断的价值以及CT多平面重组（MPR）对此征检出的意义。方法观察病理证实的周围型小肺癌137例及良性肺内结节45例，将结节的边缘分为4型。对28例肺癌、22例良性结节行MPR，考察其显示分叶征的作用。引入可能性比值（likelihood ratios，LR）以量化良恶性病变在形态学上存在重叠的表现。结果Ⅰ型边缘结节对良性病变的特异性为83．3％（20／24），敏感性为44．4％（20／45）。Ⅳ型边缘结节对周围型小肺癌的特异性为97．6％（83／85），敏感性为60．7％（83／137）。表明Ⅰ型边缘是良性病变的特点；而Ⅳ型边缘是恶性病变的特征。Ⅳ型边缘的结节恶性病变在MPR重组图像上的检出率为64．3％（18／28），与横断面扫描差异有统计学意义（P〈0．01）。结论分叶征对肺内孤立结节的诊断有价值，MPR配合横断面薄层图像有助于提高该征的检出率。     

9 Survivin反义寡核苷酸对人肝癌细胞的抑制作用

目的:探讨survivin反义寡核苷酸（ASODN）对人肝癌细胞HepG2的抑制作用。方法:采用免疫组织化学法检测肝细胞癌（HCC）组织中survivin的表达。采用脂质体介导survivin ASODN体外转染人肝癌细胞株HepG2，Western blot检测细胞survivin蛋白的表达，FCM检测细胞的凋亡率，观察细胞在软琼脂中的集落形成能力。建立人肝癌裸鼠皮下移植瘤模型，观察survivin ASODN的体内抑癌作用。 结果:（1）肝癌组织中survivin表达阳性率为75.8％(25/33)，明显高于癌旁组织和正常肝组织(P<0.01);（2）survivin ASODN体外转染可明显下调HepG2细胞survivin蛋白表达，ASODN组HepG2细胞凋亡率明显高于空白对照组和SODN组(P<0.01)，ASODN组HepG2细胞在软琼脂中形成的集落数目明显少于空白对照组和SODN组(P<0.01);（3）ASODN组瘤体生长速度较空白对照组和SODN组明显减慢(P<0.01)，ASODN组瘤体重量较空白对照组和SODN组明显减轻(P<0.05)。结论:Survivin在HCC组织中高表达;survivin ASODN可以诱导HepG2细胞凋亡，在体外实验和动物实验中对HepG2细胞生长都有抑制作用。     

重组人骨形态发生蛋白4基因腺相关病毒载体对兔骨髓基质干细胞生物学行为的影响

目的应用重组人骨形态发生蛋白4基因腺相关病毒载体(AAV-hBMP4)转染兔骨髓基质干细胞(BMSCs),观察其对BMSCs生物学行为的影响,从而为骨组织工程寻找理想的病毒载体及种子细胞。方法全骨髓法培养兔BMSCs,按感染复数(MOI)值不同设定为四组,分别转染兔BMSCs,观察病毒量对细胞形态的影响。选取影响最小的MOI值,进行后续实验。转染兔BMSCs,MTT法描记细胞生长曲线,观察AAV对细胞增殖活性的影响。以重组增强型绿色荧光蛋白基因的腺相关病毒载体(AAV-EGFP)为参照,行流式细胞仪检测,计算转染效率。AAV-hBMP4与对照病毒AAV-EGFP分别转染细胞,观察细胞形态,行碱性磷酸酶(ALP)染色、Von Kossa染色及ALP含量测定,观察成骨活性。兔肌袋实验观察异位成骨情况。结果MOI值为5×10~4 vg/cell时,AAV对细胞形态影响最小,以此值进行后续实验。AAV转染后,细胞增殖活性良好,转染效率为55％～65％。AAV-hBMP4转染后,细胞形态呈现典型的成骨改变,ALP染色及Von Kossa染色均出现成骨的特征性改变,而AAV-EGFP组无上述改变。细胞上清ALP含量测定显示,实验组ALP含量显著增高,与对照组比较差异有统计学意义(t=218．65,P＜0．01)。兔肌袋实验术后4周组织学检测可见大量钙盐沉积,矿化结节形成。结论AAV-hBMP4转染效率高,对BMSCs的增殖活性影响小,AAV-hBMP4转染的BMSCs可望成为组织工程化骨的理想种子细胞。     

低位直肠癌的保肛手术

目的 探讨如何选择合适的保肛手术方法治疗低位直肠癌。方法 44例肿块下缘距肛缘5～7cm的低位直肠癌患者,对21例采用改良的结肠肛管吻合术(A组)、23例应用吻合器技术的前切除术(B组)进行治疗;并对两组疗效予以对比。结果 A组患者无远端直肠残端肿瘤残留,B组有2例。肿瘤远端直肠切除距离A组平均为(2.81±0.35)cm,B组(1.73±0.42)cm。两组比较,差异有显著性意义(t=9.083,P<0.001)。A组术后两年均无吻合口复发,B组有4例,B组吻合口复发率明显高于A组(x~2=4.234,P=0.04)。A组术后早期排便功能较差,但均能在半年内改善;B组术后排便功能良好。结论 低位直肠癌患者肿瘤下缘距肛缘5～7cm、肿瘤“T”分期为Ⅱ、Ⅲ期、术前指诊肿瘤可推动、肿瘤侵犯肠壁范围不到1周可行保肛手术。应根据患者体型、骨盆宽窄、肿瘤分化程度及其侵犯肠管的周径合理选择改良的结肠肛管吻合术或吻合器技术的前切除术。