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。

重组旋毛虫53000抗原蛋白联合亚胺培南对多细菌感染脓毒症小鼠的保护作用

目的观察重组旋毛虫53000抗原蛋白（rTsP53）联合亚胺培南（IMP）对脓毒症小鼠的保护作用，初步探讨其可能机制。方法按随机数字表法将雄性BALB／e小鼠分为5组，采用盲肠结扎穿孔术（CLP）构建多细菌感染小鼠脓毒症模型（CLP组），假手术（Sham）组仅开腹、关腹，不进行结扎。CLP＋IMP组、CLP＋rTsP53组、cLP＋IMP＋rTsP53组分别于术后6h起腹腔注射IMP20mg／kg＋0．1mL白蛋白、rTsP53蛋白6mg／kg＋0．1mL生理盐水（Ns）、IMP20mg／kg＋rTsP53蛋白6mg／kg；Sham组、CLP组则给予0．1mL白蛋白＋0．1mLNS；12h重复1次，至实验结束。各组取20只小鼠观察72h存活情况；并于术后0、6、12、24、36、48、72h各取3只小鼠血标本，采用酶联免疫吸附试验（ELISA）检测血清细胞因子水平，全血培养进行活菌菌落计数。24h处死小鼠取小肠组织，透射电镜下观察小肠黏膜上皮细胞超微结构。结果①cLP＋IMP＋rTsP53组小鼠存活率明显高于CLP组、CLP＋IMP组、CLP＋rTsP53组（85％比20％、55％、25％，均P〈0．05o②Sham组各时间点全血均未培养出细菌；各实验组术后6h活菌计数均显著升高，CLP组呈先升后降趋势，于24h达峰值（×10^6cfu／L：12．74±2．33）；CLP＋rTsP53组12h起显著高于CLP组，于36h达峰值（×10^6cfu／L：22．13±4．28）后逐渐下降；CLP＋IMP组、CLP＋IMP＋rTsP53组于6h达峰值（×10^6cfu／L：5．72±0．50、5．49±0．59）后呈逐渐下降趋势，12h起即显著低于CLP组。③各实验组术后6h起血清细胞因子水平均明显高于Sham组。CLP组肿瘤坏死因子-α（TNF-α）呈先升后降趋势，于36h达峰值（ng／L：1422：67±72．19），CLP＋IMP组、CLP＋rTsP53组、cIJP＋IMP＋rTsP53组达峰值时间提前至12h（nglL：1376．29＋44．67、929．36±40．42、809．61±22．61oCLP组和CLP＋IMP组白细胞介素-6（IL-6）于24h达峰值（ng/L：215．39±16．05、191．63±8．99），CLP＋rTsP53组、cLP＋IMP＋rTsP53组达峰值时间提前至12h（ng／L：113．01±12．11、92．43±6．11℃ LP组IL-4、IL-10均逐渐升高至72h达峰值（ng／L：366．25±24．25、923．14±30．36），CLP＋IMP组IL-4、IL-10分别于12h、24h达峰值（ng／L：281．47±16．33、555．67±13．57）后逐渐下降，CLP＋rTsP53组、cLP＋IMP＋rTsP53组IL-4、IL—10均逐渐升高至72h达峰值[IL-4（ng／L）分别为453．14±18．53、410．43±15．75，IL-10（ng/L）分别为1185．61±16．74、1006．77±36．91]。cLP＋IMP＋rTsP53组12h起各炎性因子水平均显著低于CLP＋IMP组、CLP＋rTsP53组。④术后24h，cLP＋IMP＋rTsP53组小肠黏膜上皮微绒毛、细胞连接、线粒体等超微结构损伤较CLP组、CLP＋IMP组、cLP＋rTsP53组明显减轻。结论rTsP53蛋白联合IMP干预可使多细菌感染脓毒症小鼠促炎因子水平降低、抑炎因子升高、存活率提高；而rTsP53蛋白单独干预未能显现出保护作用，且血细菌计数升高。

Helicobacter pylori infection in relation to E-cadherin gene promoter polymorphism and hypermethylation in sporadic gastric carcinomas

AIM: To study Helicobacter pylori (H pylori) infection in relation to E-cadherin (E-cad) promoter polymorphism and hypermethylation in GCs.METHODS: Specimens were taken from representative cancerous lesions and adjacent non-cancerous epithelia of 67 resected GCs. Hpyloriwas detected by real-time PCR of the cagA gene from non-neoplastic epithelium.E-cad promoter polymorphism and hypermethylation were determined by restriction fragment length polymorphism analysis and methylation-specific PCR, respectively. Expression of E-cad protein was determined by immunohistochemistry.RESULTS: Hpyloriwas found in 57% of patients with GC.H pylori infection was more frequently found in tumors with the -160C/C genotype than those with the -160C/A and -160A/A genotypes (74% vs47%, P = 0.02). Hpylori infection was associated with E-cad methylation in nonneoplastic epithelium; however, no significant difference in H pylori was observed between methylated and unmethylated cancerous lesions.CONCLUSION: Patients with the -160C/C genotype might require Hpyloriinfection to promote the inactivation of CDH1, suggesting that Hpylori infection might affect GC in an initial stage because polymorphism is germ line.Mechanism of hypermethylation of CDH1 promoter in GC is complex, and Hpyloriinfection might affect it in an initial stage.

Restriction fragment length polymorphism of adhesin gene hpaA from different Helicobacter pylori strains of Chongqing, China

AIM:To assess the variability of adhesin gene hpaA between different Helicobacter pylori (H pylori) strains with PCR-restriction fragment length polymorphism (RFLP). METHODS: Twelve different H pylori strains were chosen to amplify the 710-bp segments of gene hpaA. These strains were NCTC11637, SS1; Chongqing clinical isolates CCS9801, CCS9802, CCS9803, CCS9806, CCS9809, CCS9810, CCS9813, which were gained from patients of gastritis; Mongolia gerbil adapted H pylori strains (abbreviation MG), which were gained from the following steps: gastric mucosal specimens of Mongolia gerbils infected by clinical isolate CCS9803 were cultured and detected, the positive H pylori strains were named as the first generation of Mongolia gerbil adapted H pylori strains (abbreviation MG1) and then were subcultured with healthy Mongolia gerbil to generate MG2, in turn to gain the ninth generation (abbreviation MG9). All hpaA segments, obtained from 12 different H pylori strains, were digested by Hhal and HaeIll individually and analyzed by agarose gel electrophoresis. RESULTS: In all 12 strains, the 710-bp PCR products were successfully amplified and products were cloned to pMD18-T vector respectively, then the recombinant plasmids were digested simultaneously with Ncol and Xhol to recover the small fragments. The objective fragments from 12 different H pylori strains digested with Haelll could be seen as 4 types of bands and 5 types with Hha I. According to the hpaA RFLP patterns, the 12 H pylori strains could be divided into 5 groups: group I, NCTC11637 and SS1; group II, CCS9809, which RFLP type digested with HaeIll was the same as strains of group I, but Hhal RFLP showed difference compared with the other groups; group III, CCS9810; group IV, CCS9803; group V: CCS9801, CCS9802, CCS9806, CCS9813, MGl, MG3 and MG9. The sequence data of 12 hpaA segments were analyzed by DNAsis software and it was observed that: (1) The homologies of base pair and amino acid sequence between strains NCTC11637, SS1, CCS9809 were 99.6% and 98.9%, respectively; (2) The homology of base pair and amino acid sequence between CCS9803 and CCS9810 was 97.7% and 99.1%; (3) That of the rest strains, CCS9801, CCS9802, CCS9806, CCS9813, MGl, MG3, MG9 reached 99.4% and 98.4%; (4) The base pair homologies between all hpaA fragments of different sources were higher than 94.6%, therefore the correspondence of deduced amino acid sequence was higher than 96.8% between each other. CONCLUSION: The gene hpaA from different H pylori strains revealed variation, and this might provide an effective method for molecular epidemiological survey of H pylori.