大黄素对大鼠原位肝移植术后急性排斥反应的干预

目的:目前国外已有少量报道证实中药大黄素具有极强的免疫抑制效应。实验拟进一步验证大黄素对同种异体大鼠肝移植术后早期急性排斥反应的干预效果。方法:实验于2004-01/2005-02在西安交通大学第一附属医院肝胆外科实验室完成。制备SD→Wistar大鼠全血供肝移植模型(n=80),按随机数字表法分为4组,每组20只。模型对照组、大黄素组、环孢素A组及环孢素A 大黄素组分别腹腔注射给予9g/L生理盐水、1.5mg/(kg·d)大黄素、3mg/(kg·d)环孢素A及3mg/(kg·d)环孢素 1.5mg/(kg·d)大黄素。术后观察大鼠一般情况,并于第7天各组分别处死10只大鼠,取肝脏标本及血清,观察移植肝组织排斥反应强度、Fractalkine(Fkn)阳性表达情况及大鼠血清中白蛋白含量及谷丙转氨酶活性,余受体继续应用药物干预直至死亡,记录其生存时间。结果:各组受体手术成功数量分别为模型对照组17只、大黄素组18只、环孢素A组18只、环孢素A 大黄素组18只。①与模型对照组相比,各用药组大鼠术后存活时间明显延长(P<0.05),以环孢素A 大黄素组存活时间最长。②与模型对照组相比,各用药组大鼠术后第7天移植肝排斥反应强度明显降低(P<0.05),血清中白蛋白含量明显升高,而谷丙转氨酶活性明显降低(P均<0.05),肝组织中Fkn表达阳性率明显降低(P<0.05),以环孢素A 大黄素组表现最为显著。结论:大黄素具有抑制同种异体大鼠肝移植急性排斥反应发生、发展的作用,与环孢素A联用具有协同作用。     

中华生存质量量表的研制

目的:在中华文化背景和中医理论指导下研制中华生存质量量表,推动国内生存质量研究的进一步发展,丰富国际生存质量研究。方法:遵照国际通用量表研制的程序化方法,在中华文化背景和中医理论指导下,结合国际生存质量概念内涵,建立中华生存质量量表的理论模型,确立了调查领域。通过设立研究组,建立条目池、编制初选量表、现场测试、文化适应和临床预调查等形成了临床初步调查表。根据研究设计,发出300份调查表,回收有效量表273份,通过对调查结果的系统统计分析进一步筛选条目。结果:应用专家重要性评分法、离散趋势法(变异系数法)、主成分分析法与因子分析法、聚类分析法、逐步回归分析法、判别分析法及克朗巴赫系数法等统计分析方法,对不同分析结果选中的条目进行综合考虑,最后形成了以3大领域、11个方面和50个条目为结构的最终中华生存质量量表。结论:基于中华文化和中医理论,按照国际量表通用的程序化方式研制的中华生存质量量表,具有国际可比性,将会得到广泛应用和同行认可。     

Inhibitory effects of low-level laser therapy on skin-flap survival in a rat model

BACKGROUND:Although several studies have demonstrated the effects of low-level laser therapy (LLLT) on skin flap viability, the role of higher doses has been poorly investigated.OBJECTIVE:To investigate the inhibitory effect of the LLLT (λ=670 nm) on the viability of random skin flaps in a rat model using an irradiation energy of 2.79 J at each point.METHODS:Sixteen Wistar rats were randomly assigned into two groups: sham laser irradiation (n=8); and active laser irradiation (n=8). Animals in the active laser irradiation group were irradiated with a 670 nm diode laser with an energy of 2.79 J/point, a power output 30 mW, a beam area of 0.028 cm², an energy density of 100 J/cm², an irradiance of 1.07 W/cm² for 93 s/point. Irradiation was performed in 12 points in the cranial skin flap portion. The total energy irradiated on the tissue was 33.48 J. The necrotic area was evaluated on postoperative day 7.RESULTS:The sham laser irradiation group presented a mean (± SD) necrotic area of 47.96±3.81%, whereas the active laser irradiation group presented 62.24±7.28%. There was a significant difference in skin-flap necrosis areas between groups (P=0.0002).CONCLUSION:LLLT (λ=670 nm) increased the necrotic area of random skin flaps in rats when irradiated with an energy of 2.79 J (100 J/cm²).     

Mixed hematologic chimerism after allogeneic marrow transplantation for severe aplastic anemia is associated with a higher risk of graft rejection and a lessened incidence of acute graft-versus-host disease

Ninety-six patients with severe aplastic anemia who received a sex- mismatched, HLA-identical allogeneic sibling marrow transplant had sequential cytogenetic analyses performed to determine the incidence and implications of posttransplant mixed hematologic chimerism. Of the 96 patients, 56 (58.3%) became mixed chimeras with coexisting host and donor cells detected in peripheral blood or marrow 14 days or later after transplant, and 40 patients (41.7%) were complete chimeras with 100% donor-type hematopoietic cells. The incidence of mixed chimerism was independent of prior blood production transfusions and infusion of donor buffy coat. The rejection rate was significantly increased in the mixed chimeric group, particularly in patients not receiving buffy coat (14 of 36 rejecting), although overall, the majority (69.7%) retained their first graft. Rejection was seen almost exclusively in patients exposed to multiple transfusions before transplantation. If patients who reject their first graft are censored, the overall incidence of grades II through IV acute graft-v-host disease (GVHD) was significantly reduced in those with mixed chimerism. Transfused patients with mixed chimerism in particular were less likely to develop grades II through IV acute GVHD. The incidence of chronic GVHD was similar in the two groups and did not significantly influence survival. In this study, mixed chimerism persisted for up to 395 days posttransplant, either the first graft being rejected or, more commonly, hematopoiesis reverting to 100% donor-type cells. Mixed lymphohematopoietic chimerism may persist in patients with aplastic anemia who have received matched allogeneic marrow transplants for significant periods before hematopoiesis reverts to donor cell type.     

骨髓源性神经样细胞体外存活、增殖的特性

目的:寻找骨髓间充质干细胞体外诱导分化为神经元样/星形胶质样细胞的有效方法,并评价其体外存活和增殖特性。方法:实验于2005-07/2006-08在华中科技大学同济医学院附属协和医院完成。采用贴壁筛选法分离培养大鼠骨髓间充质干细胞。收集第5代骨髓间充质干细胞单细胞悬液,以1×109L-1置入6孔培养板中,加入DMEM 20%胎牛血清生长培养基,24h后更换生长培养基为DMEM/F12 2?7 40μg/L碱性成纤维生长因子 20μg/L内皮生长因子诱导分化培养基,诱导10～20d,即采用多因子分阶段逐步诱导方法定向诱导骨髓间充质干细胞分化为神经前体样细胞。更换诱导分化培养基为DMEM/5%胎牛血清生长培养基,分两组进行诱导:一组向神经元样诱导分化:全反式维甲酸连续加样,首次浓度0.5μmol/L,以后每天全量加样;二组向星形胶质样细胞诱导分化:10μg/L血小板衍生生长因子BB首次加样,以后每天半量加样,均连续诱导10～14d,进而分化为神经元样/星形胶质样细胞。应用活细胞计数试剂盒CCK-8检测神经元样/星形胶质样细胞的增殖及存活情况,绘制生长曲线及细胞存活曲线。结果:①碱性成纤维生长因子 表皮生长因子连续诱导7d后骨髓间充质干细胞分化为球团样的神经前体样细胞,巢蛋白表达阳性。②全反式维甲酸、血小板衍生生长因子BB分别连续诱导10d后骨髓间充质干细胞逐步分化出神经元样、星形胶质样细胞,并分别表达纤维丝蛋白200及胶质纤维酸性阳性蛋白。③神经元样细胞早期生长较缓慢,具有一定的增殖能力,六七天后达到高峰,此后增殖能力明显下降,细胞存活率明显下降,十四五天之后细胞存活率下降到7.6%以下,难以传代培养。星形胶质样细胞初期生长慢,5d后生长加速,进入对数生长期,七八天后进入平台期,仍保持较高的细胞存活率。结论:采用多因子分阶段诱导方法可成功诱导骨髓间充质干细胞分化为神经元样/星形胶质样细胞。神经元样细胞存活、增殖能力较差,难以传代,星形胶质样细胞存活、增殖能力较强,可建立传代的二倍体细胞系。     

局部注射血管内皮生长因子基因促进大鼠坐骨神经再生：组织学与电生理指标的评估

目的:局部应用血管内皮生长因子(vascular endothelial growth factor,VEGF)蛋白可加速神经再生,促进其功能恢复。探讨局部注射pHVEGF165质粒对周围神经再生的作用。方法:实验于2004-03/06在武汉协和医院手外科实验室完成。实验动物:成年雄性Wistar大鼠45只。实验分组:随机分成3组,每组15只。即对照组、pHVEGF165质粒25μg和50μg组。实验方法:①制备pHVEGF165质粒。②制成双侧坐骨神经损伤动物模型,于神经断端间及周围肌肉内对照组局部注射生理盐水,pHVEGF165质粒25μg组和50μg组局部分别注射pHVEGF16525μg,50μg。实验评估:①术后4,6和8周行腓肠肌湿重检测与组织形态学观察。②术后6,8周行神经断面图象分析,测量有髓纤维密度、平均轴突直径、髓鞘厚度与有髓纤维总数。③术后8周通过电生理检测记录复合肌肉动作电位波幅,潜伏期,计算出运动神经传导速度。统计分析以上数据比较各组大鼠坐骨神经再生情况。结果:45只大鼠全部进入结果分析。①坐骨神经组织学检查结果:术后4周,各组大鼠神经均以大量变性的髓鞘为主;6,8周时,对照组神经断面仍可见到一些变性神经纤维,再生之神经纤维数量少,pHVEGF165质粒组的再生神经纤维数量增多也更加成熟,且pHVEGF165质粒50μg组再生神经纤维已接近正常神经组织。②腓肠肌湿重:神经损伤6周及8周,pHVEGF165质粒50μg组均明显优于对照组、pHVEGF165质粒25μg组[6周:(733.64±41.69),(491.82±40.21),(680.08±48.70)mg,8周:(906.68±35.03),(577.26±57.22),(772.74±61.14)mg,P<0.05]。③坐骨神经图象分析:6周和8周,pHVEGF165质粒25μg组、50μg组明显优于对照组(P<0.05),pHVEGF165质粒50μg组最优(P<0.05)。④电生理检测结果:术后8周,pHVEGF165质粒50μg组运动神经传导速度、复合肌肉动作电位波幅及潜伏期指标显示明显优于pHVEGF165质粒25μg组和对照组(P<0.05),pHVEGF165质粒25μg组居中。结论:局部注射phVEGF165质粒治疗周围神经损伤具有可行性,可促进神经轴突再生,加速神经功能恢复。并且这种作用可能存在剂量依赖性。