大鼠肝细胞生长因子与肝再生增强因子融合基因真核表达质粒的构建及鉴定

Objective To construct and identify an eukaryotic expression plasmid containing rat hepatocyte growth factor(rHGF)gene and rat augmenter of liver regeneration(rALR)gene,so that to provide experimental basis for developing new treatments of hepatic fibrosis.Methods The gene fragments of rHGF and rALR were amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR by polymerase chain reaction(PCR),respectively,then were spliced by overlap extension PCR with a linker,and the fusion gene rHGF-linker-rALR was constructed.The fusion gene was directionally inserted into the eukaryotic expression plasmid pcDNA3.1 between restriction sites of Kpn Ⅰ and Xba Ⅰ to construct the recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR,and the new constructed recombinant plasmid was identified by double restriction digestion and DNA sequencing.Results DNA fragments of 2200 bp and 400 bp were observed after the electrophoresis of products amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR,respectively,which was consistent with the theoretical value.The electrophoresis of fusion gene rHGF-linker-rALR obtained by overlap extension PCR technique showed only a 2 600 bp DNA fragment,which was in accordance with the expected value.Electrophoresis of products of pcDNA3.1-rHGF-linker-rALR digested with Kpn Ⅰ and Xba Ⅰ showed two DNA fragments with 2600 bp and 5400 bp,which were both consistent with the expected value.The sequences were confirmed correctly by DNA sequencing.Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR is successfully constructed,which provides experimental basis for developing gene therapy of hepatic fibrosis.     

大鼠肝细胞生长因子与肝再生增强因子融合基因真核表达质粒的构建及鉴定

Objective To construct and identify an eukaryotic expression plasmid containing rat hepatocyte growth factor(rHGF)gene and rat augmenter of liver regeneration(rALR)gene,so that to provide experimental basis for developing new treatments of hepatic fibrosis.Methods The gene fragments of rHGF and rALR were amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR by polymerase chain reaction(PCR),respectively,then were spliced by overlap extension PCR with a linker,and the fusion gene rHGF-linker-rALR was constructed.The fusion gene was directionally inserted into the eukaryotic expression plasmid pcDNA3.1 between restriction sites of Kpn Ⅰ and Xba Ⅰ to construct the recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR,and the new constructed recombinant plasmid was identified by double restriction digestion and DNA sequencing.Results DNA fragments of 2200 bp and 400 bp were observed after the electrophoresis of products amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR,respectively,which was consistent with the theoretical value.The electrophoresis of fusion gene rHGF-linker-rALR obtained by overlap extension PCR technique showed only a 2 600 bp DNA fragment,which was in accordance with the expected value.Electrophoresis of products of pcDNA3.1-rHGF-linker-rALR digested with Kpn Ⅰ and Xba Ⅰ showed two DNA fragments with 2600 bp and 5400 bp,which were both consistent with the expected value.The sequences were confirmed correctly by DNA sequencing.Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR is successfully constructed,which provides experimental basis for developing gene therapy of hepatic fibrosis.     

高危组肾移植患者血浆置换后进行HLA配型1例

患者,男性,43岁,既往诊断为原发性肾小球肾炎所致尿毒症.2005年1月肾移植术后,因为发生超急性排斥而导致移植肾丧失功能,患者血液透析维持治疗并欲进行第二次肾移植.     

肝细胞生长因子与肝再生增强因子重组表达质粒对大鼠肝纤维化的影响

Objective To evaluate the therapeutic effects of recombinant expression plasmid containing hepatocyte growth factor (HGF) and augmenter of liver regeneration (ALR) on rats with hepatic fibrosis. Methods Ninety Sprague-Dawley rats, which had been established into hepatic fibrosis models, were equally divided into 6 groups: blank group, pcDNA3.1 therapy group,pcDNA3.1-HGF therapy group, pcDNA3. 1-ALR therapy group, pcDNA3.1-HGF and pcDNA3. 1-ALR combined therapy group, and pcDNA3. 1-HGF-ALR therapy group. Zero point one μmol of blank or plasmid was injected into model rats in each group by tail vein once a day for 3 days. Model rats in blank group didn't receive any treatment. Additional 10 rats were chosen as control group, which were not given any interference during the experiment. All rats were sacrificed 4 days after end of treatment. Liver tissues were reserved for observing pathologic changes after HE staining and detecting proliferating cell nuclear antigen (PCNA) and c-jun by immunohistochemistry. Measurement data were compared by single-factor analysis of variance. Comparison between groups was done by SNK test. Enumeration data were analyzed by Fisher's exact test. Results In blank group and pcDNA3.1 therapy group, hyperplasia of fibrous connective tissue was very obvious, false lobules were formed. There was no significant difference between these two groups (x2 =0. 317,P= 1. 000).In the 4 remaining groups, hepatic fibrosis all achieved different degree of amelioration, and the therapeutic effect of pcDNA3.1-HGF-ALR was optimal. In control group, the expressions of PCNA and c-jun in liver tissues were low, with absorbance value of 8.6±1.9 and 3.2 ± 1.2, respectively. In blank group and pcDNA3. 1 therapy group, the expressions of PCNA and c-jun were obviously increased, with absorbance value of 24. 1±3.0, 24.5±4.3 and 23.8±3.1, 24.9±4.2, respectively,which were significant different from control group (all P＜0.01). In the 4 remaining groups, the expressions of PCNA were all obviously increased, and expressions of c-jun were all obviously decreased. The maximum change scope was observed in pcDNA3. 1-HGF-ALR therapy group.Conclusions The recombinant expression plasmid pcDNA3. 1-HGF-ALR can effectively ameliorate experimental hepatic fibrosis of rats. The anti-fibrosis effects are achieved probably by up-regulating PCNA expression and down-regulating c-jun expression.     

下颌骨骨折颌间牵引对牙体牙周组织的影响

目的：评价带钩牙弓夹板颌间弹力牵引治疗下颌骨骨折对牙体牙周组织的影响。方法：牙弓夹板固定前、拆除牙弓夹板后第二天及牙弓夹板拆除后3个月，分3次调查41例患者（固定组）与41例对照健康人（对照组）的软垢指数，牙周治疗需要指数、龋失补牙数。结果：3次检查固定组一的龋均无显著差异（P＞0．05），第二次检查两组的软垢指数，牙周治疗需要指数差异显著（分别为P＜0．01，P＜0．05），第三次检查固定组软 指数稍下降，但仍明显高于对照组（P＜0．01），两组牙周治疗需要指数差异更为明显（P＜0．001）。固定组第二、三次检查软垢指数、牙周治疗需要指数均明显高于第一次检查，结论：牙弓夹板颌间弹力牵引治疗颌骨骨折，对牙体组织影响不明显，但对牙周组织有较大不利影响。     

根中1/3折恒前牙拔出后再植的临床研究

目的评估恒前牙根中1/3折拔出采用体外根充根管钉固定后再植术的临床疗效。方法23例共31颗患牙采用拔出体外根充根管钉固定后再植。结果23例31颗患牙治愈21颗,治愈率为67.74%,其中20岁以内的治愈率为75%,20岁以上的为40%,两者差异呈显著性（P〈0.02）。结论年轻恒牙拔出采用体外根充根管钉固定后再植术较成年恒牙效果显著,但对于根尖孔未成形、根尖孔敞开的年轻恒牙,在治疗过程中应注意勿伤及其牙乳头。     

盲袢瘘的防治体会

任何一种新改进的术式都应在长期大量的实践中考证,才能得出正确的评价。作者的实事求是的态度对盲袢式胆管空肠吻合术并发盲袢瘘的问题作了较详细的总结,很有意义。本文提示:①任何手术均应严格掌握其适应证,取利避害。②本术式是胆肠内引流术的一种,它也只能在有效地解除肝内梗阻,去除肝内病灶的前提下应用,不能把解决肝内病变的努力,留待术后经盲袢的有限处理上。③一旦发生盲袢并发症,病人的负担和处理的困难都是不小的。