同型半胱氨酸对大鼠血管平滑肌细胞增殖及胶原生成的影响

目的:观察同型半胱氨酸的浓度对Ⅰ,Ⅲ,Ⅳ和Ⅵ型胶原的产生以及大鼠血管平滑肌细胞增殖效应的影响。方法:实验于2005-02/2006-04在瑞金医院心内科实验室完成。①采用组织块贴壁法原代培养大鼠胸主动脉平滑肌细胞,用不同浓度的同型半胱氨酸(0.025,0.05,0.1,0.2,0.5,1.0,1.5,2.0,3.0,5.0 mmol/L)刺激大鼠血管平滑肌细胞,并设对照孔(未加同型半胱氨酸)和空白孔(未加细胞)作对照。②分别作用12,24,48,72h,以四甲基偶氮唑盐法检测同型半胱氨酸对平滑肌细胞的活性细胞数量及细胞增殖情况;以5溴-2脱氧尿苷法检测同型半胱氨酸对大鼠血管平滑肌细胞DNA合成的作用;流式细胞仪观察细胞周期的变化;酶联免疫法测定胶原的含量。结果:①细胞增殖情况:在0.025,0.05 mmol/L的同型半胱氨酸浓度刺激下,12～48h血管平滑肌细胞增殖增加明显。②细胞DNA合成:与四甲基偶氮唑盐结果类似,24～48h时,在浓度为0.025,0.05 mmol/L的同型半胱氨酸刺激下细胞合成DNA增加(24h:432±33,774±52;48h:582±32,816±62),其促增殖作用增强(P<0.05);随着浓度的增加,大鼠血管平滑肌细胞合成DNA开始下降,至5 mmol/L浓度时最低。③细胞周期的变化:24h时在浓度为0.025,0.05 mmol/L的同型半胱氨酸刺激下增殖的细胞S期的百分含量较对照组和同型半胱氨酸其他浓度组明显增加。④胶原的含量:细胞的Ⅰ型和Ⅳ型胶原分泌随着同型半胱氨酸浓度的升高呈现剂量依赖性的增高模式。Ⅲ型胶原有轻度的升高,无统计学上意义。高浓度的同型半胱氨酸会减少Ⅵ型胶原的分泌,并呈剂量依赖性(r2=0.41;P<0.001)。结论:较低浓度的同型半胱氨酸刺激大鼠血管平滑肌细胞的增殖加速;并可能通过改变纤维斑块中的胶原构成类型导致了斑块的不稳定性和易损性,最终加速了粥样斑块的发生和发展。     

骨髓间质干细胞分泌胶质源性神经生长因子及对1-甲基4-苯基吡啶离子诱导PC12细胞凋亡的干预

目的:收集体外培养、纯化的骨髓间质干细胞条件培养液,检测其对多巴胺能神经元有保护作用的胶质源性神经生长因子分泌情况,并观察对1-甲基4-苯基吡啶离子诱导的PC12细胞凋亡的保护作用。方法:实验于2005-05/2006-10在中国医科大学附属一院神经内科实验室完成。①PC12细胞由协和医科大学细胞培养中心提供。1-甲基4-苯基吡啶离子(Sigma,USA,批号3707312)。②选取清洁级SD大鼠20只,麻醉后取股骨和胫骨,去净肌肉取骨髓,按1010L-1接种于含体积分数为0.2胎牛血清的低糖型DMEM培养基中,通过弃悬浮细胞及换液后可得到较纯的骨髓间质干细胞。培养第10天胰蛋白酶消化传代,当第2代细胞扩增至铺满瓶底80%时,改用含体积分数为0.05胎牛血清的低糖型DMEM条件培养液,48h后收集培养液,经超滤浓缩系统(截留分子量为10000)浓缩10倍后过滤除菌。③骨髓间充质干细胞接种于24孔板内,贴壁后多聚甲醛固定,磷酸盐缓冲液漂洗,免疫荧光法鉴定骨髓间充质干细胞表面抗原的表达。骨髓间充质干细胞消化后进行细胞计数,按1×108L-1接种于75cm培养瓶,加入含体积分数为0.2胎牛血清的DMEM培养液20mL,于培养第5,10天采用ELISA法测定细胞培养上清液中的胶质源性神经生长因子含量。④PC12细胞置于RPMI1640培养液中,内含体积分数为0.1的马血清和0.05的胎牛血清,汇集至80%时进行传代接种,液氮罐中贮存备用。实验前首先置换培养液,使血清浓度降至为仅含0.01马血清和0.01胎牛血清,24h后将培养的细胞分为4组:空白对照组,在细胞培养体系中不加入任何药物;骨髓间充质干细胞上清液组,接种后24h在细胞培养体系中分别加入骨髓间充质干细胞上清液30,60,120μL;1-甲基4-苯基吡啶离子组,接种后24h分别加入1-甲基4-苯基吡啶离子,使终浓度分别为100,200,400μmol/L;联合组,接种后24h加入200μmol/L1-甲基4-苯基吡啶离子,24h后再分别给予骨髓间充质干细胞上清液30,60,120μL。⑤各组细胞于给药后24,48h,流式细胞术检测PC12细胞的凋亡率;通过免疫细胞化学法和RT-PCR法检测PC12细胞半胱氨酸天冬氨酸蛋白酶3蛋白和mRNA的表达。结果:①骨髓间充质干细胞表面抗原鉴定:骨髓间充质干细胞可在体外分离扩增,其表面抗原CD45呈阴性,而CD44表达阳性。②骨髓间充质干细胞培养上清液中胶质源性神经生长因子水平检测:第5,10天培养上清液中的胶质源性神经生长因子浓度为(44.57±5.96)ng/L和(45.41±6.33)ng/L,差异无显著性意义(P>0.05)。③PC12细胞凋亡情况:联合组200μmol/L1-甲基4-苯基吡啶离子作用PC12细胞24h后,细胞凋亡率为(42.34±3.21)%;加入30,60,120μL骨髓间充质干细胞上清液处理24h后,细胞凋亡率分别降为(31.96±2.89)%,(17.89±1.78)%,(10.08±0.91)%,差异有显著性意义(P<0.05)。联合组药物作用48h与24h情况相似。④给药后各组半胱氨酸天冬氨酸蛋白酶3蛋白及mRNA的表达:联合组半胱氨酸天冬氨酸蛋白酶3蛋白和mRNA水平均明显低于1-甲基4-苯基吡啶离子组(t=0.05～0.32,P均<0.05)。结论:骨髓间充质干细胞能够分泌胶质源性神经生长因子,对1-甲基4-苯基吡啶离子诱导的PC12细胞凋亡产生保护作用。这种保护作用的强弱与其浓度有关,具体作用机制可能是通过抑制半胱氨酸天冬氨酸蛋白酶3蛋白和mRNA水平实现的。     

苏皖地区汉族人群不稳定型心绞痛患者与血小板反应素4基因A387P多态性的关系

目的:观察血小板反应素4基因G29926C(A387P)多态性与中国苏皖地区汉族人群不稳定型心绞痛的可能关系。方法:选择2004-11/2006-05在南京医科大学第一附属医院和江苏大学附属武进医院住院不稳定型心绞痛患者110例,病例均符合2002年AHA/ACC关于不稳定型心绞痛诊断指南的诊断标准,同期选择337例非冠心病者为对照。酚-氯仿法提取白细胞DNA,应用聚合酶链反应-限制性片段长度多态性方法分析血小板反应素4A387P多态性,取部分PCR扩增产物进一步测序鉴定,比较两组间多态性频率分布差异,探讨血小板反应素4基因多态性与不稳定型心绞痛发病的可能关系。结果:447例均进入结果分析。GC基因型在不稳定型心绞痛组和对照组的分布无统计学差异(5.5%,7.1%,P=0.54),未检测到CC纯合子。C等位基因频率在不稳定型心绞痛组和对照组分别为2.7%,3.6%(P=0.55)。GC基因型与不稳定型心绞痛无显著性关联(OR=0.75,95%CI:0.30￣1.89,P=0.54)。结论:血小板反应素4基因387A→P不是中国苏皖地区汉族人群常见的多态位点,且与不稳定型心绞痛的发病无显著相关性。     

Results of intensive therapy in childhood acute myeloid leukemia, incorporating high-dose melphalan and autologous bone marrow transplantation in first complete remission

Childhood acute myeloid leukemia (AML) has a poor prognosis with standard chemotherapy. Allogeneic bone marrow transplantation (BMT) in remission improves the outlook only for the one third of patients with sibling donors. Autologous BMT with a lower morbidity and mortality is available to all. In this study, maximum cytoreduction was achieved by intensive early chemotherapy. Final intensification, with autologous BMT was offered to all those remaining in first complete remission (CR). Patients received two induction and two consolidation courses of intensively scheduled chemotherapy. Cytoreduction was assessed on day 14 and remission was assessed after courses 2 and 4. Bone marrow was harvested after recovery from the second consolidation course or after the first maintenance course and separated on a discontinuous percoll gradient before cryopreservation. Twenty-eight of 31 consecutively enrolled patients achieved CR. Three relapsed early and, of the 25 eligible, 24 underwent autologous BMT. Twenty-three patients received high-dose melphalan and 1 received busulphan and cyclophosphamide before autologous BMT at a median of 113 days (range, 86 to 301) after initial CR. Trilineage engraftment occurred in all. Neutrophil recovery to greater than 0.5 x 10(9)/L occurred at a median of 46 days (range, 13 to 92) after autologous BMT. Platelet recovery was delayed, with a median time to achieve greater than 20 x 10(9)/L of 42 days (range, 18 to 215). With a minimum follow up of 25 months following autologous BMT only 3 children have relapsed. The 5-year event-free survival rate (EFS) from diagnosis is 68% (95% confidence interval, 46% to 90%). Five- year EFS following autologous BMT is 87% (95% confidence interval, 67% to 100%). Autologous BMT with high-dose melphalan administration after intensive chemotherapy has produced EFS equivalent to allogeneic BMT and is associated with a strikingly low relapse rate. High-dose melphalan appears to be a valuable agent for conditioning therapy in AML.     

Factor XI antigen and activity in human platelets

Washed platelets, contaminated with less than 0.20% plasma factor XI, were examined for the presence of factor XI antigen and activity. These platelets contained a factor-XI-like coagulant activity (0.67 +/- 0.11 U/10(11) platelets) that remained constant after successive washes. By means of indirect immunofluorescence, a monospecific antibody to factor XI showed specific staining of both normal platelets and platelets from patients deficient in plasma factor XI. Radiolabeled Triton extracts of washed platelets and labeled purified factor XI solutions were analyzed for factor XI antigen by Staph A immunoprecipitation analysis using antibody to purified plasma factor XI followed by SDS gel electrophoresis. On unreduced gels, the platelet material ran as a single band having an apparent molecular weight of 220,000 daltons, whereas purified plasma factor XI gave a single band at 160,000 daltons. On reduced gels, the platelet material analyzed as a single band at 52,000 daltons, whereas purified factor XI gave a single band of 80,000 daltons. Analysis of a partially purified factor XI preparation from platelets by immunoelectrophoresis revealed that the platelet preparation displayed a slightly lower cathodal electrophoretic mobility at pH 8.6 than did plasma factor XI and yet appeared to possess complete antigenic identity with plasma factor XI. These results indicate that platelets possess a form of factor XI that exists as a disulfide-linked 52,000-dalton tetramer in contrast to the plasma form that circulates as a 80,000-dalton disulfide-linked dimer.     

木通马兜铃化学成分研究

从木通马兜铃(Aristolochia manshuriensis Kom)茎皮中分得一个新化合物(5),经光谱(IR,UV,HRMS,^{1HNMR,NOEDS)鉴定为3,4-二甲氧基-10-硝基菲-1-羧酸甲酯,命名为马兜铃酸BII甲酯。另11个已知化合物是马兜铃酸Ⅰ,Ⅱ,Ⅲa,Ⅳ,Ⅳa,对羟基桂皮酸,β-谷甾醇,豆甾烷-3,6-二酮,6-羟基-豆甾-4-烯-3-酮,胡萝卜甙,二十八酸甘油单酯;其中二十八酸甘油单酯为首次从该属植物中分得。}     

p16INK4A gene alterations are not a prognostic indicator for survival in patients with hepatocellular carcinoma undergoing curative hepatectomy

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) is a common malignancy worldwide that is highly associated with chronic hepatitis B or C infection and cirrhosis. The tumor suppressor gene p16INK4A is an important component of the cell cycle and inactivation of the gene has been found in a variety of human cancers. The present study was performed to determine genetic and epigenetic alterations in the p16INK4A tumor suppressor gene and the effect of these on HCC progression. METHODS: The status of p16INK4A was evaluated in 117 HCC tumoral nodules and 110 corresponding peritumoral tissues by loss of heterozigosity (LOH) at the 9p21-22 region, homozygous deletions, single-strand conformation polymorphism-polymerase chain reaction (PCR) mutational analysis and methylation specific PCR. RESULTS: The most frequent inactivation mechanism was hypermethylation of the promoter region, which was found in 63.2% of the tumor samples and in 28.2% of the peritumoral samples. Loss of heterozygosity at the 9p21 region was detected in 27.3% and 10% of tumor and peritumoral tissues, respectively. Homozygous deletions and mutations were less common events in hepatocarcinogenesis. The authors found 5.9% of the tumor cases with exon 2 homozygous deletions and 8.6% with mutations. Two polymorphisms were detected, one at codon 148 (GCG --> ACG, Ala --> Thr) in three cases and the other in exon 3 at 540 bp (34.2% of the samples). No association was found between inactivation of p16INK4A and clinicopathological characteristics or prognosis. CONCLUSION: p16INK4A is altered frequently and early in HCC, being the predominant mechanism of inactivation promoter hypermethylation. The present results suggest that the p16INK4A gene plays an important role in the pathogenesis of HCC.