单核细胞趋化因子1发卡环RNA真核表达载体的构建

目的：构建单核细胞趋化因子1发卡环RNA真核表达载体。 方法：实验于2002—10／2003-05在解放军第二军医大学中心实验室完成。①单核细胞趋化因子1发卡小分子干扰RNA片段合成。（2）将发卡小分子干扰RNA克隆人质粒pSilencer产生重组质粒SiRNA—pSilencer-单核细胞趋化因子1。③分别用BamHⅠ和HindⅢ酶切鉴定，随机分为1，2样本，每份样本进行10次电泳，并且采用460bpi标准参照样。④将经酶切鉴定后的重组质粒作测序分析。 结果：①单核细胞趋化因子1发卡小分子干扰RNA片段被成功克隆进pSilencer-U6质粒中。②BamHⅠ和HindⅢ酶切鉴定可切出450bpi大小的片段，结果表明样本1，2均与参照以及marker相一致，从片段大小的角度初步确定酶切的准确。③DNA测序分析显示，重组质粒小分子干扰RNA编码序列与设计片段的序列完全一致。 结论：针对单核细胞趋化因子1发卡小分子干扰RNA片段的真核表达载体SiRNA—pSilencer-单核细胞趋化因子1的成功构建，为进一步研究其基因功能打下了基础。     

Octreotide decreased liver metabolic activity in patients with hepatitis B surface antigen-positive cirrhosis.

The influence of octreotide and somatostatin on liver metabolic activity were studied in 16 patients with cirrhosis that was positive for hepatitis B surface antigen (HBsAg). In patients receiving a 50 micrograms bolus and a 50 micrograms/hr infusion of octreotide, the hepatic blood flow, hepatic clearance, and the maximum velocity/metabolic elimination rate constant (Vmax/km) were significantly reduced after octreotide infusion compared with basal values. Similarly, the hepatic blood flow, hepatic clearance, and Vmax/km were significantly decreased in patients receiving a 250 micrograms bolus and a 250 micrograms/hr infusion of somatostatin. The extraction ratio and the systemic hemodynamic values, including cardiac index, heart rate, mean arterial pressure, and systemic vascular resistance, showed no significant changes in patients receiving either octreotide or somatostatin. These findings suggest that, as with somatostatin, octreotide reduced hepatic blood flow and impaired liver metabolic activity in patients with HBsAg-positive cirrhosis. These effects may have important clinical implications in the management of bleeding esophageal varices in patients with cirrhosis.     

Adenovirus-mediated GM-CSF gene and cytosine deaminase gene transfer followed by 5-fluorocytosine administration elicit more potent antitumor response in tumor-bearing mice.

Antitumor effects of combined transfer of suicide and cytokine genes were investigated in this study. Adenovirus harboring E. coli cytosine deaminase gene (AdCD) and adenovirus harboring murine granulocyte-macrophage colony-stimulating factor gene (AdGMCSF) were used simultaneously for in vivo gene transfer in melanoma-bearing mice. Growth inhibition of established tumors and prolongation of survival period were observed more significantly in tumor-bearing mice after transfection with AdGMCSF and AdCD followed by continuous injection of prodrug 5-fluorocytosine (5FC) when compared with mice treated with control adenovirus AdlacZ/5FC, AdCD/5FC or AdGMCSF alone (P < 0.01). After combined therapy the expression of MHC-I (H-2Db) and B7-1 molecules on freshly isolated tumor cells increased greatly and more dendritic cells and CD8+ T cells infiltrated into the tumor mass. The activity of specific cytotoxic T lymphocytes was also found to be induced more significantly after the combined therapy. Further experiments showed that apoptosis of tumor cells and induction of antitumor immune response might be involved in the mechanisms of the tumor cell killing by the combined therapy. Our results demonstrated that combined transfer of the GM-CSF and CD suicide genes, being able to inhibit the growth of melanoma synergistically and induce specific antitumor immune response efficiently, thus addressing the drawbacks of suicide gene therapy or cytokine gene therapy which were proved to be not satisfactory when used alone, might be of therapeutic potential for gene therapy of cancer.     

骨髓干细胞移植治疗运动神经元病的近期效果:72例疗效分析

目的:观察自体骨髓干细胞移植治疗运动神经元病的近期疗效,探讨对其功能改善作用。方法:2004-02/10对经解放军第四六三医院神经内科临床确诊为运动神经元病的72例患者实施自体骨髓干细胞移植,围手术期给予营养神经等药物治疗。疗效评估以肌张力是否下降,言语不清、吞咽困难是否改善,抬头困难是否改善,肌束震颤次数是否减少,肌力是否增高为标准。如以上各项任何一种症状缓解即为有效。结果:5种症状均减轻者2例,任意4种症状得到缓解者6例,任意3种症状得到缓解者20例,任意2种症状得到缓解者28例,任意1种症状得到缓解者10例,所有上述5种症状均未得到缓解者6例。以上5种症状中的任何一种症状得到缓解均视为有效,有效率为92%(66/72)。无任何并发症出现。结论:自体骨髓干细胞移植具有改善运动神经元病功能障碍的可行性,近期疗效确切。     

新辅助化疗在老年乳腺癌治疗中的意义

目的：探讨新辅助化疗在老年乳腺癌患者治疗中的意义。方法：35例老年乳腺癌患者术前用CTF方案化疗1疗程，化疗后1周手术。结果：无完全缓解病例，部分缓解4例（占11．43％），轻度缓解20例（占57．14％），无变化11例（占31．43％），无临床进展病例。病理组织学观察20例镜下见肿瘤细胞有小点片状坏死。1例因白细胞降低推迟手术，其余患者按计划手术。全组随访1～6年，在随访期间发现5例局部复发，2例骨转移，2例有肺和脑转移。6例在随访期间因本病死亡，3例因其它合并症死亡，余均健在。结论：新辅助化疗对老年乳腺癌可缩小肿瘤，增加手术机会，提高治疗效果，并可能减少术中血行转移。     

Experimental study of bone marrow-derived mesenchymal stem cells combined with hepatocyte growth factor transplantation via noninfarct-relative artery in acute myocardial infarction

We investigated the impact of bone marrow-derived mesenchymal stem cells (BM-MSCs) alone or in combination with hepatocyte growth factor (HGF) transplantation via noninfarct-relative artery in a swine myocardial infarction (MI) model. Donor BM-MSCs were derived in vitro from swine auto-bone marrow cultures labeled by bromodeoxyuridine (BrdU) incorporation. Host MI swine model was created by ligating the distal left anterior descending artery. After 4 weeks, age-matched male MI swines were used for the transplantation. Male MI swines were transfused via noninfarct-relative artery with vehicle (control, n=6) or BrdU-labeled BM-MSCs (5 x 10(6)) alone (MSCs, n=6) or BrdU-labeled BM-MSCs (5 x 10(6)) combined with HGF (4 x 10(9) PFU) (MSCs+HGF, n=6). To evaluate the collateral artery growth (Rentrop) and cardiac perfusion in these animals, gate cardiac perfusion imaging and coronary angiography were performed before and 4 weeks after transplantation, respectively. To assess the contribution of donor-originated cells in stimulation of cardiomyocyte regeneration and angiogenesis, immunohistochemistry for BrdU and alpha-smooth muscle actin (alpha-SMA) and quantitative image analysis were performed at 4 weeks after transplantation. The results are as follows: (1) BrdU-positive cells were detected in host myocardium in both MSCs and MSCs+HGF groups, but not in the vehicle group. Most BrdU-positive cells expressed myosin heavy chain beta. (2) alpha-SMA(-)positive arteriole densities in the infarcted border area and infarcted area were increased significantly in both transplantation groups compared with the vehicle group. (3) Gate cardiac perfusion imaging demonstrated that the cardiac perfusion was significantly improved in transplantation groups compared with the vehicle group. (4) Ejection fraction and alpha-SMA-positive arteriole densities were increased significantly in both transplantation groups compared with the vehicle group. However, there was no difference in ejection fraction and alpha-SMA-positive arteriole densities between the MSCs group and the MSCs+HGF group. Growth of collateral arteries was not detected by coronary angiography in all three groups. In conclusion, the current study indicates that BM-MSCs transplantation via noninfarct-relative artery stimulates cardiomyocyte regeneration and angiogenesis and improves cardiac function, but does not stimulate collateral artery growth. BM-MSCs transplantation combined with HGF therapy is not superior to BM-MSCs alone transplantation. BM-MSCs transplantation via noninfarct-relative artery may be an alternative for those patients who cannot be transplanted via infarct-relative artery in clinical practice.     

Hypertension promotes phosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 in rats: implication for the pathogenesis of hypertensive vascular disease

Atherosclerosis is initiated by adhesion and infiltration of inflammatory leukocytes into the intima, where non-receptor protein tyrosine kinases, such as focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2), play important roles as intracellular messengers of mechanical and biochemical signals. In the present study, we examined whether FAK and PYK2 are up-regulated by elevated blood pressure or circulating humoral factors in hypertension. We used a rat model of abdominal aortic banding that allows separate evaluation of elevated blood pressure (upper body) and circulating humoral factors (lower body). We obtained the proximal and distal aortas of the banding site, 6 hours, 3 days, and 1 and 4 weeks after the banding procedure, for evaluation of phosphorylation of FAK and PYK2 by Western blotting. Arterial pressure was significantly elevated only in the upper body throughout the experimental period. The expression of FAK and the FAK phosphorylation were significantly increased at 1 and 4 weeks only in the proximal aorta. This was also the case for the expression of total PYK2 and the PYK2 phosphorylation. In contrast, there was no significant change in FAK or PYK2 phosphorylation in the distal aorta, whereas plasma levels of angiotensin II were systemically elevated. In sham-operated rats, no change in FAK or PYK2 phoshorylation was noted in the proximal and distal aortas. These results indicate that phosphorylation of FAK and PYK2 is upregulated by elevated blood pressure but not by humoral factors in the rat aorta, demonstrating novel aspects of atherogenesis in hypertension.     

TWEAK induces liver progenitor cell proliferation

Progenitor ("oval") cell expansion accompanies many forms of liver injury, including alcohol toxicity and submassive parenchymal necrosis as well as experimental injury models featuring blocked hepatocyte replication. Oval cells can potentially become either hepatocytes or biliary epithelial cells and may be critical to liver regeneration, particularly when hepatocyte replication is impaired. The regulation of oval cell proliferation is incompletely understood. Herein we present evidence that a TNF family member called TWEAK (TNF-like weak inducer of apoptosis) stimulates oval cell proliferation in mouse liver through its receptor Fn14. TWEAK has no effect on mature hepatocytes and thus appears to be selective for oval cells. Transgenic mice overexpressing TWEAK in hepatocytes exhibit periportal oval cell hyperplasia. A similar phenotype was obtained in adult wild-type mice, but not Fn14-null mice, by administering TWEAK-expressing adenovirus. Oval cell expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was significantly reduced in Fn14-null mice as well as in adult wild-type mice with a blocking anti-TWEAK mAb. Importantly, TWEAK stimulated the proliferation of an oval cell culture model. Finally, we show increased Fn14 expression in chronic hepatitis C and other human liver diseases relative to its expression in normal liver, which suggests a role for the TWEAK/Fn14 pathway in human liver injury. We conclude that TWEAK has a selective mitogenic effect for liver oval cells that distinguishes it from other previously described growth factors.     

脱氢表雄酮对衰老大鼠体质量和学习记忆的影响

目的:观察脱氢表雄酮对D-半乳糖致亚急性衰老模型大鼠体质量和学习记忆损害的影响。方法:实验于2003-11/2004-12在中国医学科学院完成。①分组及模型制备:24只SD大鼠随机分为D-半乳糖组和药物治疗组,每组12只。动物经皮下注射D-半乳糖100mg/kg,1次/d,共56d制备亚急性衰老模型。②给药方法:药物治疗组动物同时腹腔注射脱氢表雄酮30mg/kg、隔日一次,共28次。D-半乳糖组注射等剂量生理盐水。③检测指标:各组大鼠每周定时测量体质量1次。采用Morris水迷宫法测定各组大鼠定位航行试验逃避潜伏期、在平台象限游泳距离和时间占各象限游泳总计距离、时间的百分比,评估大鼠学习记忆功能。结果:24只大鼠均进入结果分析。①治疗后药物治疗组动物体质量增加百分比显著低于D-半乳糖组(6.23%,13.04%,P<0.001)。②药物治疗组大鼠水迷宫试验逃避潜伏期明显低于D-半乳糖组(F=557.96,P<0.001),平台象限游泳距离、时间占各象限游泳总计距离、时间百分比明显高于D-半乳糖组[(55.4±3.4)%,(28.2±2.3)%;(57.6±4.1)%,(28.5±3.0)%](P<0.001)。结论:脱氢表雄酮能够降低D-半乳糖所致的亚急性衰老模型大鼠体质量,改善衰老动物学习记忆功能。     

人脐血干细胞定向分化为神经细胞的诱导方法

目的:分析人脐血干细胞在体外和体内向神经细胞分化的不同诱导条件。资料来源:应用计算机检索Medline数据库1990-01/2005-10相关脐带血干细胞向神经细胞分化文章,检索词为“umbilicalcordblood,mesenchymalstemcells,neuraldifferentiation”,分别组合进行检索,限定文章语言种类为English,同时计算机检索中文期刊数据库2000-01/2005-10相关脐带血间充质干细胞向神经分化文章,检索词为:“脐带血,间充质干细胞,神经分化”,分别组合进行检索,限定文章语言种类为中文。资料选择:初审资料,选取包括人脐血干细胞分化为神经细胞的文章。纳入标准:①脐带血间充质干细胞。②间充质干细胞向神经细胞分化。排除标准:①脐带血造血干细胞。②文献综述,重复研究,Meta分析类文章。资料提炼:共收集到68篇符合要求的文章,查找全文,选取其中近期相关文章19篇。将所有文章按照脐血干细胞诱导分化体外实验和体内实验进行分类综合。资料综合:脐带血干细胞来源丰富、采集方便、安全,且体外扩增迅速,在体外和体内一定条件下可以分化为神经细胞,然而不同的诱导条件对其分化的影响有不同,人们运用各种手段希望能找到诱导效率较高的诱导方法,包括几种化学诱导剂联合,生长因子之间配伍,化学诱导剂与生长因子联合,或应用接近生理状态的条件培养基等。在改进诱导条件的同时人们开始关注单一表型的细胞群作为实验对象是否能有效地提高诱导效率。研究者以期找到一种高效率而具有应用价值的诱导方法,为将脐带血干细胞应用于神经科学领域提供有力的证据。结论:人脐血干细胞在体外和体内一定条件下可以分化为神经细胞,应用于中枢神经系统疾病的细胞治疗具有广阔前景,然而寻找高效实用的诱导方法仍需要进一步研究。