多孔髓芯减压联合干细胞移植治疗股骨头坏死的早期随访结果

目的:探讨多孔髓芯减压联合自体骨髓干细胞移植治疗股骨头坏死的疗效及临床分析。方法:选择2003-02/2006-12在南京医科大学附属南京第一医院骨关节中心采用多孔髓芯减压联合干细胞移植治疗的股骨头坏死患者22例,共28髋,年龄17～48岁,根据世界骨循环研究学会(ARCO)的国际骨坏死分期标准:Ⅰ期13髋,Ⅱ期11髋,Ⅲ期4髋。长期使用激素史9例,长期酗酒史6例,外伤史5例,原因不明者2例。纳入标准:有髋关节疼痛,功能受限;经髋关节X射线片及MRI检查确诊;ARCO分期Ⅰ～Ⅲ期;患者知情同意并签署知情同意书。排除标准:其他髋关节疾病。自患者髂前上棘处行骨髓穿刺分离与培养骨髓间充质干细胞。取患肢大粗隆下大腿外侧纵向直切口约3.0cm,钝性分离至股骨,在C形臂机引导下自股骨头中心钻入3枚直径4.0mm斯氏针,选位置较好的斯氏针,将直径约8.0mm特制套管在斯氏针的引导下钻至股骨头关节软骨面下1.0～2.0mm,不穿破关节面。将一长注射器针头置入股骨头坏死中心,立即行X射线正侧位摄片,确保针头位于股骨头内,从针头向股骨头内加压注入自体骨髓间充质干细胞悬液1.5～2.0mL。术后12个月随访,每3个月1次,随访时门诊复查,拍正、侧位和蛙式位X射线片,行MRI检查,观察病情变化。使用髋关节Harris评分进行疗效评价,>90分为优,75～90分为良,60～74分为可,<60分评定为差。若Harris评分提高,X射线骨形态变化改善及MRI股骨头坏死区体积变小可认为联合治疗有效。结果:①22例患者均完成随访,进入结果分析。②随访3个月时X射线及MRI检查:2例(2髋)激素引起的Ⅲ期患者股骨头发生进一步变形及塌陷,其余患者在随访期间未出现严重并发症,不良反应及病情恶化。股骨头坏死体积由术前31.07%减小到17.46%。激素组治疗前后股骨头坏死体积差值小于外伤、酗酒组,就本随访资料而言激素组疗效不如外伤及酗酒组。③随访12个月Harris评分:由术前54.3上升到84.6,有较明显提高,其中优7髋(25.0%),良15髋(53.6%),可4髋(14.3%),差2髋(0.07%)。结论:多孔髓芯减压联合干细胞移植是治疗股骨头坏死的一种新手段,尤其适合于年青、ARCO-Ⅰ或Ⅱ期、非激素导致的股骨头坏死治疗。     

Inhibition of human lymphocyte reactivity by plasma fibronectin in vitro

The effect of purified human plasma fibronectin (FN) on the reactivity of human lymphocyte-rich mononuclear cells to mitogens and allogeneic cell interactions was studied. Concentrations of FN from 25 to 100 micrograms per 250 microL of culture consistently depressed phytohemagglutinin (PHA) responses. To exert an inhibitory effect, FN must be present within 20 hours after the addition of PHA to cells, and, therefore, it appears to interfere with early events in the transformation process. Increasing the concentration of PHA failed to reduce the inhibitory effect of FN, which suggests that the depressed response was not the result of FN-PHA complex formation, which would reduce the amount of mitogen available for stimulation. This possibility was supported by the finding that FN also inhibited the mixed lymphocyte response (MLR), in a reaction that was not dependent on the activity of soluble antigen or mitogen. In contrast, the stimulation of lymphocytes to undergo transformation that is induced by the nonlectin mitogen, sodium periodate, was unaffected by FN. Periodate-treated cells are, however, already stimulated to undergo transformation, prior to their exposure to FN. FN did not interfere with the activity of interleukin-2, nor did it indirectly regulate lymphocyte responses by modifying the production and/or effect of humoral regulatory factors released from the adherent accessory cells (macrophages). These studies show that FN is a potent immunosuppressive agent in vitro.     

人白细胞介素24mRNA在瘢痕疙瘩中的表达及意义

目的:从基因水平测量瘢痕疙瘩组织中白细胞介素24的表达水平,探讨白细胞介素24在瘢痕疙瘩发生、发展过程中的作用和意义。方法:实验于2005-10/2006-09在广东医学院整形外科研究所完成。①选取2004-06/2005-10广东医学院附属医院整形外科收治的患者,瘢痕疙瘩标本12例,正常瘢痕标本10例,行巨乳缩小、除皱术、植皮等患者正常皮肤标本12例,患者均知情同意且自愿捐献标本,实验经医学伦理委员会批准。标本取材部位为颜面、前胸、四肢等,切取后液氮保存。②低温条件下切取秤量组织,采用Trizol法提取总RNA。电泳鉴定总RNA完整性,并统一调整总RNA含量为10g/L,-70℃储存。RT-PCR二步法合成cDNA。③以正常皮肤、正常瘢痕为对照,以GAPDH作为扩增内参照基因,将正常皮肤、正常瘢痕和瘢痕疙瘩各类标本总RNA反转录的cDNA模板浓度调整相对一致进行扩增反应。以白细胞介素24mRNA与GAPDHmRNA的光密度积分值之比作为各类组织标本中白细胞介素24的相对含量,比较白细胞介素24mRNA在正常皮肤、正常瘢痕及瘢痕疙瘩组织中的表达情况。结果:①各类组织标本中总RNA抽提结果:正常皮肤、正常瘢痕和瘢痕疙瘩组织中抽提总RNA经甲醛变性凝胶电泳后显示较清晰的18s和28s条带,经紫外分光光度计测定A260/A280≈2.0。②各类组织标本中白细胞介素24mRNA的表达:白细胞介素24和GAPDH基因表达产物通过RT-PCR方法得到的特异性DNA片段长度分别为173bp和577bp。瘢痕疙瘩的白细胞介素24mRNA与GAPDHmRNA的吸光度比值明显低于正常皮肤、正常瘢痕(0.577±0.113,1.070±0.185,1.139±0.195;t=7.436×10-8～4.745×10-8,P均<0.01),正常皮肤与正常瘢痕白细胞介素24mRNA的相对表达量基本一致(t=0.405,P>0.05)。结论:瘢痕疙瘩的形成可能与白细胞介素24在组织中的表达降低有关。提示采用基因疗法提高早期瘢痕疙瘩中白细胞介素24的含量与活性,可能为瘢痕疙瘩的康复治疗提供有效途径。     

胶体磷酸铬^32P关节腔内注射改善佐剂型关节炎大鼠的相关指标

目的:观察胶体磷酸铬32P关节腔内注射治疗大鼠佐剂型关节炎的效果。方法:实验于2006-07/09在南京市第一医院动物实验室完成。选择6～8周龄清洁级SD雌性大鼠30只,按随机数字表法分为3组,正常对照组、模型组、胶体磷酸铬32P治疗组,每组10只。大鼠左足跖皮内注射完全弗氏佐剂0.1mL免疫法制备佐剂型关节炎模型。胶体磷酸铬32P治疗组于造模后10d左踝关节腔内注射37GBq/L胶体磷酸铬32P0.02mL,即0.74MBq,正常对照组和模型组分别给予等量生理盐水左踝关节腔内注射。①每2周观察1次大鼠左踝关节左右径宽度。②关节炎指数评定采用关节炎评分法(0～4分),分数越高,症状越重。③于用药后2,4,6周采用99Tcm-MDP显像感兴趣区分析法计算大鼠左踝关节区和右胫腓骨的放射性计数比。④于用药后4,6周测定血清肿瘤坏死因子和白细胞介素1β水平。⑤于用药后4,6周观察大鼠滑膜组织和软骨组织病理学改变。结果:纳入大鼠30只,均进入结果分析。①用药后2周模型组大鼠左踝关节左右径宽度大于正常对照组[分别为(7.82±0.36),(5.89±0.35)mm],差异有显著性意义(t=12.16,P<0.001),胶体磷酸铬32P治疗组大鼠左踝关节左右径宽度大于模型组,差异无显著性意义(P>0.05)。用药后6周胶体磷酸铬32P治疗组大鼠左踝关节左右径宽度小于模型组[分别为(6.87±0.27),(7.25±0.26)mm],差异有显著性意义(t=2.87,P<0.05)。②用药后2周和4周胶体磷酸铬32P治疗组大鼠关节炎指数高于模型组,用药后6周胶体磷酸铬32P治疗组大鼠关节炎指数低于模型组,两组间差异均无显著性意义(P>0.05)。③用药后2周模型组大鼠感兴趣区放射性计数比高于正常对照组(分别为2.05±0.20,1.46±0.15),差异有显著性意义(t=7.46,P<0.001)。用药后6周胶体磷酸铬32P治疗组大鼠感兴趣区放射性计数比低于模型组(分别为1.52±0.18,1.78±0.24),差异有显著性意义(t=2.45,P<0.05)。④用药后4,6周模型组大鼠血清肿瘤坏死因子和白细胞介素1β水平高于正常对照组,差异有显著性意义[用药后4周分别为(2.039±0.344),(1.115±0.192)μg/L;(0.305±0.034),(0.192±0.041)μg/L,t=7.42,6.71,P<0.001。用药后6周分别为(1.694±0.305),(1.126±0.256)μg/L;(0.259±0.027),(0.191±0.019)μg/L,t=4.03,5.83,P<0.01,0.001]。用药后4,6周胶体磷酸铬32P治疗组在血清肿瘤坏死因子和白细胞介素1β水平与模型组比较,差异无显著性意义(P>0.05)。⑤用药后4周胶体磷酸铬32P治疗组和模型组滑膜组织增生和炎症细胞浸润均较明显;用药后6周胶体磷酸铬32P治疗组与正常对照组比较仍有滑膜组织增生和炎症细胞浸润,与模型组比较滑膜组织增生程度明显减轻,而炎症细胞浸润程度稍轻。用药后6周胶体磷酸铬32P治疗组大鼠左踝关节的软骨组织未见有异常改变。结论:胶体磷酸铬32P关节腔注射可减轻完全弗氏佐剂免疫大鼠受注射关节的滑膜增生程度,改善关节肿胀症状,疗效肯定。     

A near-fatal reaction during granulocyte transfusion of a neonate

Although reactions to granulocyte transfusions in neonates are rarely reported, we observed a near-fatal pulmonary reaction, presumably due to white cell antibodies, in a neonate with Rh hemolytic disease. The hemolytic disease was being treated with exchange transfusions, and at 2 days after the infant's birth, bacterial sepsis was suspected and granulocyte transfusions were begun. The first granulocyte transfusion (Day 3) was uneventful. Five minutes after the beginning of the second granulocyte transfusion (Day 4), severe respiratory distress, hypotension, bradycardia, cyanosis, and acidosis suddenly occurred. The infant's serum obtained after the reaction contained granulocytotoxic and B-lymphocytotoxic antibodies that reacted with leukocytes from the second granulocyte donor. Antibodies could not be detected either in the initial infant serum or in maternal serum. However, an antileukocyte antibody was present in the serum of a parous woman donor. We used plasma from this woman to prepare reconstituted whole blood for the exchange transfusion that we performed immediately preceding the second granulocyte transfusion. Despite the sequence of events, an irrefutable cause-and-effect mechanism could not be established because the properties of the donor and neonatal antibodies were similar, but not identical. However, this catastrophic event emphasizes both the potential for adverse effects of granulocyte transfusions in neonates and the need for caution when transfusing blood from parous women.     

Influence of fibronectin-containing blood products on lymphocyte reactivity

Two fibronectin (FN)-containing blood products, human peripheral blood plasma and cryoprecipitate, were examined for their effect on mitogen-induced lymphocyte transformation in vitro. Responses of human peripheral blood lymphocytes to phytohemagglutinin (PHA) were depressed in the presence of a plasma concentration above that required for maximum DNA synthesis, and this concentration must be present in cultures prior to lymphocyte activation. The removal from the plasma of heparin-induced cryoprecipitate, a complex consisting of FN, heparin, and fibrinogen, resulted in a significant reduction in the inhibitory effect of the plasma on the PHA response. Plasma specifically depleted of FN by affinity chromatography on gelatin-agarose beads was 32 percent less inhibitory to the PHA-induced stimulation of cells than untreated plasma; the remaining inhibitory activity in the FN-depleted plasma samples was attributed to the presence of other normal immunosuppressive factors. The inhibitory capacity of FN in plasma was similar to that obtained with purified FN alone, which indicates that, unlike that of other known plasma inhibitors, the immunosuppressive activity of FN was not altered by the presence of other components of plasma. Cryoprecipitate used in the treatment of hemophilia contains high levels of FN, and, as anticipated, PHA-induced lymphocyte transformation was markedly depressed in the presence of solubilized cryoprecipitate. The contribution of FN to the T-cell abnormalities in patients chronically receiving cryoprecipitate and/or factor VIII concentrates derived from cryoprecipitate warrants further investigation.     

6-羟基多巴胺帕金森病模型大鼠氧化应激反应

目的:观察6-羟基多巴胺帕金森病大鼠模型的氧化应激反应及其可能的机制。方法:实验于2005-06/12在上海中医药大学科研实验中心完成。实验分组:Wistar雄性大鼠18只,随机分为正常对照组、假手术组、模型组,每组6只。实验干预:采用6-羟基多巴胺(溶于0.2g/L抗坏血酸的生理盐水中)注射于脑右侧黑质,10d后以腹腔注射阿朴吗啡0.5mg/kg诱发大鼠向一侧旋转,记录开始旋转至30min内的旋转圈数,以平均每分钟旋转圈数超过7次者为合格的帕金森病模型。假手术组6只大鼠只注射含0.2g/L抗坏血酸的等量生理盐水,其余条件与造模组手术相同。正常对照组6只大鼠只进行大鼠固定,不进行任何处理。在旋转诱发实验完成后,动物每天自由饮水与进食,共45d。实验评估:应用化学比色法测定大鼠中脑黑质纹状体部位活性氧、丙二醛、谷胱甘肽含量,谷胱甘肽过氧化物酶、超氧化物歧化酶的活性。结果:18只大鼠均进入结果分析。①活性氧含量:模型组明显高于正常对照组和假手术组[(451.13±73.42),(135.62±53.46),(161.15±61.16)U/mg,P<0.01]。②谷胱甘肽含量:模型组明显低于正常对照组和假手术组[(4.26±0.75),(7.03±1.39),(7.03±1.42)mg/g,P<0.05]。③谷胱甘肽过氧化物酶活性:模型组明显低于正常对照组和假手术组[(3.05±0.38),(3.96±0.39),(3.77±0.38)NU/g,P<0.01]。④超氧化物歧化酶活性:模型组明显低于正常对照组和假手术组[(137.74±4.65),(190.77±8.47),(199.73±8.23)NU/mg,P<0.01]。⑤丙二醛含量:模型组明显高于正常对照组和假手术组[(10.90±2.17),(4.18±2.88),(4.52±2.45)μmol/g,P<0.01]。结论:帕金森病模型大鼠的氧化反应被激活,处于氧化应激状态,氧化应激反应增强可能是帕金森病的发病机制之一。     

Hemostasis in patients with severe von Willebrand disease improves after normal platelet transfusion and normalizes with further correction of the plasma defect

BACKGROUND: A defective hemostatic effect of plasma concentrate infusion in patients with severe von Willebrand disease (vWD) has been ascribed to the absence of platelet von Willebrand factor (vWF) STUDY DESIGN AND METHODS: The role of platelet vWF in hemostasis of severe vWD was investigated. A plateletpheresis unit (4-5 × 10(11) platelets) from a normal compatible donor was transfused before any cryoprecipitate infusion to three type 3 vWD patients and to one patient with severe type 1 vWD with low levels of platelet vWF who required replacement therapy for bleeding episodes. Autologous platelets were transfused to one of the patients with type 3 vWD. RESULTS: Partial corrections of bleeding times (14-17 min vs. baseline>30 min) were observed in all patients after the transfusion of normal platelets. During cryoprecipitate infusion, bleeding times were normalized (<6 min), and bleeding episodes stopped when plasma levels of vWF activity ranged from 14 to 18 U per dL. Platelet interactions with the subendothelium increased in parallel with the correction of bleeding times. These results indicate that if approximately 20 percent of the total number of platelets have normal vWF antigen and if plasma vWF levels are at least 14 U per dL, then bleeding times will normalize and mucosal hemorrhages will stop. Transfusion of autologous platelets in one patient with type 3 vWD did not modify bleeding times or platelet adhesion on the subendothelium. CONCLUSION: The hemostatic effect of normal platelets in type 3 vWD seems to be related to the platelet vWF in the transfused platelets.