胰岛素样生长因子-1基因转染脂肪间充质干细胞向软骨细胞分化的研究

目的 观察胰岛素样生长因子(IGF)-1基因转染的脂肪间充质干细胞(ADSCs)向软骨细胞分化的效果.方法 原代培养兔ADSCs,免疫荧光法检测细胞表面抗原CD44、CIM9;脂质体介导人IGF-1基因转染兔ADSCs联合低浓度血清培养基向软骨细胞分化诱导,RT-PCR及Western blot方法检测IGF-1的表达,MMT法绘制细胞增殖曲线、甲苯胺蓝染色软骨结节、免疫组织化学检测Ⅱ型胶原的表达.结果 脂肪间充质干细胞CD44、CD109表达阳性,基因转染后细胞IGF-1表达阳性,细胞增殖速度增快,出现软骨结节,Ⅱ型胶原表达增高.结论 从脂肪组织中能够分离出增殖旺盛的ADSCs,IGF-1在ADSCs内获得稳定表达,细胞增殖能力增强,促进其向软骨细胞分化.     

四种中药对骨愈合过程中相关基因表达的影响

目的:探索中药水蛭、海螵蛸、阿胶、骨碎补在骨折愈合过程中的干预作用,了解它们各自的调节靶点,探索建构其基因组学的途径。方法:通过在大鼠胫骨打孔的方法建立单因素干扰模型,并将300只大鼠随机分为正常组、模型组和给药组(分别用4种中药给药),每组50只,分别在实验的第4、7、14、21、28天不同时间点采用原位杂交方法对各类mRNA的变化进行动态观察,分析骨愈合过程中Ⅰ、Ⅱ、Ⅲ型前胶原mRNA、转化生长因子TGF-β1mRNA、骨形态发生蛋白BMP-2mRNA以及血管内皮生长因子VEGF-mRNA的表达情况。结果:不同中药对不同基因的作用不同,作用的时间点不同,作用强度也存在差异。其中海螵蛸在骨折早期对Ⅰ、Ⅲ型前胶原mRNA、VEGF-mRNA、BMP-2mRNA的表达升高,后期Ⅱ、Ⅲ型前胶原mRNA表达水平下降,VEGF-mRNA、TGF-β1mRNA表达量维持于较高水平;骨碎补组较模型组在BMP-2mRNA、TGF-β1mRNA、Ⅰ型前胶原mRNA的表达上差异有显著性统计意义;阿胶对骨愈合早、中期Ⅰ、Ⅱ、Ⅲ型前胶原mRNA和TGF-β1mRNA的表达与模型组比较差异存在显著性统计意义;水蛭对VEGF-mRNA的表达具有一定的促进作用。结论:海螵蛸、水蛭对血管形成有促进作用,阿胶、骨碎补和海螵蛸对骨折软骨形成早期具有促进骨诱导的作用,并对成骨细胞的增殖及合成活性有较大影响。     

Synergistic effect of low dose Cyclosporine A and human interleukin 10 overexpression on acute rejection in rat lung allotransplantation

Objective: Electroporation mediated transfer of plasmid DNA into peripheral muscle results in high transfection efficiency. The aim of this study was to investigate the effect of gene transfer of human IL-10 (hIL-10) into the tibialis anterior muscle (MTA) in combination with low dose Cyclosporine A (CsA) on acute rejection of lung allografts in the rat. Methods: Lung allotransplantation was performed from male BN donor to male Fisher F344 rats. Gene transfer was achieved by intramuscular injection into the MTA of the recipient followed by electroporation (4×20 ms impulses at 200 V/cm) 24 h prior to the transplantation. Group A (n=5) received CsA (2.5 mg/kg bw ip) for 5 days post-transplant and group B (n=5) 2.5 μg of PCIK hIL-10 (plasmid expression vector containing human CMV immediate early gene promoter and enhancer) and a low dose CsA (2.5 mg/kg bw i.p.). Graft function was assessed by blood gas at day 5 after exclusion of the native lung. Animals were sacrificed and blood was drawn to measure serum hIL-10 levels (ELISA) and tissue was sampled for histological grading of rejection. Results: Local expression of hIL-10 was confirmed at the mRNA level by in situ hybridization. All group A control animals showed severe signs of rejection. At day 5 all grafts in group B showed good gas exchange mean PaO2 233±123 mmHg, vs 44±8 mmHg in group A. Histological examination revealed moderate to severe rejection in all animals in group A (IIIB, ISHLT) in contrast to low moderate rejection in group B (II–IIIA). hIL-10 serum levels on day 5 were 14±7 pg/ml in group B vs. 0 in group A. Conclusions: Electroporation mediated hIL-10 overexpression in a peripheral muscle of the recipient in combination with low dose CsA reduces acute rejection in this model of rat lung allotransplantation.     

In Vivo Molecular Imaging Characterizes Pulmonary Gene Expression During Experimental Lung Transplantation

Experimental gene therapy is a promising strategy to prevent ischemia-reperfusion (I/R) injury and allograft rejection after lung transplantation, and methods will eventually be needed to characterize pulmonary transgene expression in vivo in humans. Therefore, we studied positron emission tomography (PET) as a means of performing in vivo molecular imaging in rodent models of lung transplantation. Rats were transfected endotracheally with adenovirus encoding a fusion gene of a mutant Herpes simplex virus-1 thymidine kinase and the green fluorescent protein gene (the former serving as an imaging reporter gene). Twenty-four hours after transfection, lungs were transplanted in groups representing normal transplantation, I/R injury and acute allograft rejection. Imaging was obtained either 24 h after transplantation to study reperfusion injury or 4 days after transplantation to study graft rejection. After imaging, lungs were excised and analyzed for thymidine kinase activity. Imaging detected transgene expression in transplanted lungs even in the presence of acute rejection or I/R injury. The PET imaging signal correlated with in vitro lung tissue assays of thymidine kinase activity (r(2) = 0.534). Thus, noninvasive molecular imaging with PET is a feasible, sensitive and quantitative method for characterizing pulmonary transgene expression in experimental lung transplantation.     

p53基因(今又生~)联合支气管动脉化疗治疗肺癌15例近期疗效观察

目的探讨瘤内注射或经支气管动脉灌注p53基因联合支气管动脉灌注化疗(BAI)治疗肺癌的安全性、疗效和给药途径。方法经病理证实的15例肺癌,首先在CT引导下经皮穿刺瘤内注射或经支气管动脉灌注p53基因(今又生~),2～5天后,行BAI灌注化疗药物;然后根据病情变化再次进行基因治疗和/或BAI。治疗后常规使用螺旋CT定期复查,评价治疗效果。结果全部15例患者均完成上述治疗,并接受随访2～11个月。有效率(CR+PR)46.7%(7/15),1例肺部肿块消失,6例肺部肿块缩小,3例纵隔淋巴结缩小,1例胸水减少,仅1例观察到肿瘤长大,14例临床症状有不同程度缓解93.3%(14/15)。p53治疗后6例出现发热(38℃～40℃),未观察到基因药物的其它严重副作用,无严重操作有关并发症出现。结论本研究结果初步显示,作为肿瘤综合治疗的一部分,p53基因治疗与BAI联合应用,是一种治疗肺癌安全、有效的方法,提高了BAI的治疗效果。经皮瘤内注射p53是否为一种较好的给药途径值得进一步探讨。     

性别差异对脓毒症大鼠肝脏Toll样受体4和髓样分化蛋白-2基因表达的影响

目的探讨性别差异对脓毒症大鼠肝脏组织Toll样受体4(TLR4)和髓样分化蛋白- 2(MD-2)基因表达的影响。方法以脂多糖(LPS)按5 mg/kg体重由大鼠腹腔注射制作脓毒症动物模型,注射后2 h留取肝脏组织检测TLR4、MD-2和肿瘤坏死因子-α(TNF-α)基因表达,同时测定各组大鼠血浆中丙氨酸氨基转移酶(ALT)及雌二醇含量。结果正常雌雄性大鼠肝脏组织均可表达少量TLR4、MD-2、TNF-α基因,其中雌性组分别为0．175±0．034、0．211±0．044、0．201±0．068; 雄性组分别为0．205±0．061、0．243±0．049、0．243±0．063,两组数据差异无统计学意义(P> 0．05),但LPS刺激后雌性大鼠肝脏组织上述指标分别为0．615±0．089、0．708±0．181、0．730± 0．118,血浆中ALT含量为(81．07±10．72)U／L;雄性组分别为0．723±0．091、1．123±0．272、 0．881±0．156,ALT含量为(106．39±14．21)U／L,雌性组各项指标均明显低于雄性大鼠(P< 0．05)。相关分析表明雌性及雄性脓毒症大鼠肝脏组织TLR4及TNF-α基因表达与相应性别大鼠血浆中雌二醇含量呈显著负相关(P<0．05)。结论 LPS刺激后大鼠肝脏组织TLR4、MD-2及 TNF-α基因表达存在性别差异,内源性雌激素的作用可能导致雌性脓毒症大鼠肝脏组织损伤较雄性轻。     

pcD2/hVEGF对缺血皮瓣存活的影响

目的 观察并探讨pcD2/hVEGF对缺血皮瓣血循环重建和皮瓣存活率的影响.方法 SD大鼠24只,分2组,每组12只.在鼠背部制作蒂位于尾侧的缺血皮瓣(7cm×1cm).实验组皮下注射pcD2/hVEGF,对照组皮下注射生理盐水,术后7天观察皮瓣存活面积比、单位面积微血管计数和及VEGF基因表达水平.结果 2组中pcD2VEGF组皮瓣存活面积比、单位面积微血管密度与对照组有显著性差异.结论 pcD2VEGF能促进缺血皮瓣存活.     

不同器官细胞因子在多细菌感染性炎症时的基因表达和临床意义

目的探讨肝、肺细胞因子基因表达与腹腔吞噬细胞上清液、循环血中细胞因子含量的关系,为临床诊治多细菌感染引发的炎症提供实验依据.方法将30只小鼠分为假手术对照组(sham组)和盲肠结扎组(CLP组).采用RT-PCR法检测肝脏和肺脏肿瘤坏死因子α(TNF-α)和白细胞介素10(IL-10)的基因表达情况,采用ELISA法检测腹腔巨噬细胞上清液和循环血液中相应细胞因子含量.结果CLP组的TNF-α、IL-10基因表达和腹腔巨噬细胞上清液、循环血液中的相应细胞因子含量均高于sham组.CLP后18h组与4h组比较,TNF-α在腹腔巨噬细胞上清液、循环血液中的活性均有显著性差异(P＜0.05),在肝、肺中基因表达有非常显著性差异(P＜0.01);IL-10在腹腔巨噬细胞上清液和循环血液中的含量无显著性差异,而在肝、肺中基因表达有显著性差异.结论发生多细菌感染性炎症时,肝、肺参与细胞因子的表达;血液中细胞因子含量不能完全代表组织器官内的基因表达情况;多细菌感染性炎症治疗应考虑靶器官细胞因子的表达状态.     

Human Tumor–infiltrating Lymphocytes Transfected with Tumor Necrosis Factor Gene Could Augment Cytotoxicity to Autologous Tumor Cells

Human tumor–infiltrating lymphocytes (TILs) derived from pleural or ascitic fluid were incubated with recombinant interleukin 2 and transfected with human tumor necrosis factor (TNF) a gene by the lipofection procedure. The resulting TILs secreted significant amounts of TNF in the culture supernatant and exhibited cytotoxicity against established cell lines, such as K562 and Daudi, and autologous tumor cells. The TNF gene–transfected TILs exhibited an augmented killing of autologous tumor cells.     

肿瘤抑制基因P53突变与肾细胞癌的关系

采用ＤＮＡ聚合酶链反应－单链构象多态性分析和直接序列测定技术，检测２５例肾细胞癌组织中Ｐ５３基因５－８外显子，２５例肾细胞癌仅有２例肿瘤组织存在Ｐ５３基因点突变，分别位于１５４和２７３密码子上，核苷酸序理分析突变形式分别为Ｇ→Ｔ，Ｃ→Ｔ。结果提示：肾细胞癌组织中Ｐ５３基因突变并不显著，表明肾细胞癌的发生可能是一个多基因变化的过程。