目的 建立经SD大鼠颈动脉途径胃左动脉插管移植肝细胞的动物模型.方法 将20只SD大鼠经颈动脉插管进入腹腔干,临时阻断脾动脉及肝总动脉,将亚甲蓝注入胃左动脉,观察亚甲美蓝的分布;将CFDA SE标记的肝细胞注入胃左动脉,通过荧光显微镜观察肝细胞在胃的分布情况.结果 亚甲美蓝染胃成蓝色;荧光显微镜发现C FD A SE标记的肝细胞在胃壁显示.结论 经胃左动脉移植CFDA SE标记的肝细胞成功分布于胃壁的肌层及黏膜下层,成功建立经SD大鼠颈动脉途径插管经胃左动脉的动物模型,为研究肝细胞移植提供一种新的动物模型.

BACKGROUND: Hepatocyte transplantation has achieved some success in animal experiments for the treatment of metabolic diseases and acute liver failure. However, the clinical efficacy of hepatocyte transplantation is unsatisfactory. The difference between the experimental results and the clinical efficacy may be related to the hepatocyte transplantation approach. OBJECTIVE: To evaluate the therapeutic effects of hepatocyte transplantation by the intubation via the intubation of the splenic artery and intrasplenic injection in rats with acute hepatic failure, providing more optimal transplantation approaches and methods. METHODS: Hepatocytes were isolated and cultured by the modified Seglen's method (two-step). Acute hepatic failure was induced by D-gal in Sprague-Dawley rats (provided by the Experimental Animal Center of Chongqing Medical University in China). After 24 hours, an intubation tube was inserted into the splenic artery in 65 rats with acute hepatic failure, which was successful in 60 rats. Then these 60 rat models were randomly divided into three groups (n=20 per group). Intrasplenic injection group received about 2×107 hepatocytes through intrasplenic injection and 0.4 mL of Hank's solution through the splenic artery. Splenic artery group received 0.4 mL of Hank's solution through intrasplenic injection and 2×107 hepatocytes through the splenic artery. Model group received 0.4 mL of Hank's solution through intrasplenic injection and 0.4 mL of Hank's solution through the splenic artery. Survival rate and liver function of the rats was observed within 14 days after transplantation. The distribution of CFDA-SE-labeled hepatocytes transplanted via the splenic artery was observed under fluorescence microscope at 7 days after transplantation, and meanwhile, the distribution and proliferation of transplanted hepatocytes in the spleen were observed using hematoxylin-eosin staining. Synthesis of albumin in the spleen was observed by immunofluorescence staining. RESULTS AND CONCLUSION: (1) 80％-90％ hepatocytes survived after isolation. (2) At 14 days after transplantation, the survival rates of rats in the three groups were significantly different: intrasplenic injection group> splenic artery group> model group. (3) The liver function of rats was significantly improved in the intrasplenic injection group and the splenic artery group, especially in the former group. (4) CFDA-SE-labeled hepatocytes (green fluorescence) were scattered in the rat spleen and liver at 7 days after transplantation via the splenic artery. (5) At 14 days after transplantation, immunofluorescent staining of albumin demonstrated some positive cells in the rat spleen in the intrasplenic injection group and splenic artery group. (6) At 7 days after transplantation, transplanted hepatocytes were concentrated and colonized in the red pulp of the spleen. In conclusion, hepatocyte transplantation through catheterization of the splenic artery via carotid route can improve the survival of rats with acute hepatic failure and ameliorate the hepatic function, but intrasplenic injection is significantly superior to the injection via the splenic artery.

目的 脾内同种异体肝细胞移植治疗急性肝功能衰竭(acute liver failure ALF)的SD大鼠,观察ALF大鼠肝功能变化情况,探讨移植肝细胞在脾内的功能和增殖情况.方法 采用D-氨基半乳糖(D-gal)诱导大鼠ALF,24h后实验分为两组,A组(n=20):经脾脏注射Hank's 0.4 mL;B组(n=20):经脾脏移植2×10 7个原代肝细胞.观察不同时间点大鼠的血清中转氨酶、总胆红素变化情况,免疫荧光检测脾内白蛋白分泌作用,HE染色观察脾内肝细胞增殖情况.结果 B组大鼠肝功能10 d恢复,A组大鼠肝功能14 d恢复正常,B组的转氨酶、总胆红素水平恢复明显好于A组(P＜0.05).羧基荧光素琥珀酰亚胺酯(CFDA SE)标记的肝细胞脾内移植7d后,受体大鼠脾脏可以看到散在绿色荧光分布.B组移植60 d后,共焦白蛋白免疫荧光结果表明,脾内有白蛋白绿色荧光信号,HE染色可以看到肝细胞在脾脏红髓中簇集在一起并定植下来.结论 同种异体肝细胞脾内移植能短暂改善ALF大鼠的肝功能.ALF恢复正常之后,脾内移植的肝细胞60d后仍具有一定的增殖能力和分泌白蛋白的功能.

背景:研究表明肌成纤维细胞具有促进肝祖细胞分化为胆管细胞作用,但对于肌成纤维细胞在体内胆管发育中作用仍不清楚.目的:观察小鼠肝内胆管发育的特点,以及肌成纤维细胞在胆管发育过程中的作用.方法:SPF级C57小鼠购自重庆医科大学实验动物中心,实验方案经重庆医科大学动物实验伦理委员会批准,批准号为2017-182.采用14.5 d、16.5 d孕鼠及刚出生(0 d)和出生后10 d的小鼠,麻醉后取其胎鼠肝脏(E14.5、E16.5)和出生后小鼠肝脏(P0、P10),采用免疫组织化学技术观察 α 平滑肌肌动蛋白、Sox9及细胞角蛋白19在E14.5、E16.5小鼠胎肝和P0、P10小鼠肝脏中的表达.结果与结论:①E14.5小鼠胎肝中,α平滑肌肌动蛋白阳性肌成纤维细胞环绕门静脉周围,Sox9阳性胆管细胞紧靠α平滑肌肌动蛋白阳性肌成纤维细胞,并形成单层细胞排列的胆管板;②E16.5小鼠胎肝中,紧靠α平滑肌肌动蛋白阳性肌成纤维细胞的细胞角蛋白19阳性胆管细胞形成双层胆管细胞结构;③P0小鼠肝脏中,细胞角蛋白19阳性胆管细胞发育形成具有管腔的胆管结构,并且α平滑肌肌动蛋白阳性肌成纤维细胞开始包绕胆管;④P10小鼠肝脏中,胆管进一步发育扩张,门静脉周围未形成胆管的胆管细胞开始消失.并且α平滑肌肌动蛋白阳性肌成纤维细胞环绕胆管,胆管旁发育形成肝动脉;⑤结果表明了小鼠肝内胆管发育的特点,发现α平滑肌肌动蛋白阳性肌成纤维细胞与肝内胆管发育的关系密切,且在肝内胆管发育中发挥了重要作用.

Objective To analyze the homology, biofilm-forming ability, and risk factors of prevalent A, B, C clones(carrying blaOXA-23 and blaOXA-51 genes mostly) of carbapenem-resistant Acinetobacter baumannii (CRAB) relative to carbapenemsusceptible Acinetobacter baumannii (CSAB). Methods A total of 87 prevalent A, B, C clones of CRAB and CSAB strains were collected from the First Affiliated Hospital of Chongqing Medical University. Multilocus sequence typing (MLST) was used for homologyanalysis of clone A, B, C strains. Biofilm-forming ability of CRAB and CSAB was measured quantitatively via crystal violet staining. Results Clone A was measured to be homologous type of ST238, while clones B and C were ST238 type. In general, CRAB prevalent clones showed weaker biofilm-forming ability than CSAB strains (0.470±0.301 versus 0.913±0.626, P＜0.05). CRAB clones A, B, and C varied in ability of biofilm formation. Clone A had comparative biofilm-forming ability to clone C (P=0.432). Both clone A and C were weaker than clone B in biofilm-forming ability (both P＜0.001). Biofilm-forming ability was not associated with blaOXA-23 or blaOXA-51 genes in CRAB strains (both P＞0.05). Conclusions Prevalent CRAB clone A, B, C are derived from the same origin. We are the first to report the prevalence of ST238 and the homologous types in a hospital. Biofilm-forming ability is negatively correlated with carbapenem resistance of Acinetobacter baumannii, which suggests that clone prevalence is mainly related to antibiotic resistance acquisition and antibiotic selective pressure. Biofilm-forming ability varies with the prevalent CRAB clone. The wide spread of clone B is of concern.

Objective To study the virulence and environmental adaptability of extensively drug-resistant Acinetobacter baumannii (XDRAB) ST238 clone, and explore the potential mechanisms of the persistent prevalence of this clone. Methods MTT method was employed to examine the in vitro growth of these strains. The competition index (CI) were determined by in vitro competition experiment to compare with A. baumannii ATCC 19606. The gelatinase activity, D-mannose related O/AB hemagglutination activity, twitching motility, and biofilm formation were studied to analyze its virulence. The median lethal dose (LD50) was determined for mice in sepsis peritoneal infection model. Results The in vitro growth (P>0.05) or competition experiment (CI=0.29, P>0.05) did not show significant difference between ST238 clone and the reference strain ATCC 19606. The gelatinase (P>0.05) and hemagglutination activity (P>0.05) did not show significant difference between ST238 clone and the susceptible strain. However, the susceptible reference strain ATCC 19606 did show stronger motility and biofilm formation ability than ST238 clone (P<0.05). The LD50 of ST238 clone and reference strain ATCC 19606 was 6.9 and 6.7 log CFU/mL, respectively. There was no significant difference in the mean time to death of mice after challenging with 9.3 log CFU/mL (P>0.05). Conclusions A. baumannii ST238 clone acquired extensively drug-resistant ability but without loss of environmental adaptability and virulence. This biological nature may contribute to its persistent prevalence in our hospital.

Objective To investigate the antibiotic resistance of nosocomial infection by Acinetobacter baumannii in our hospital in 2009,and analysis the outbreak of carbapenem-resistant Acinetobacter baumannii and the genotype of OXA-carbapenemases.Methods Antimicrobial susceptibility testing was performed using broth micro dilution on 272 strains of A.baumannii isolated from our hospital in 2009 according to the NCCLS guidelines for MIC testing.The homology of 102 strains of carbapenem-resistant A.baumannii(CRAB) in 2009 was studied by PFGE,the genotype of OXA-carbapenemases(bla_(OXA-51like),bla_(OXA-23like),bla_(OXA-24like) and bla_(OXA-58like)) was detected by a novel multiplex polymerase chain reaction(PCR).Results The drug resistance rate of A.baumannii to common antimicrobial agents increased year by year,the resistance rate tominocycline,doxycycline,polymyxin B was lowest, resistance to carbapenems raised faster.PFGE results showed that there was outbreak of A/B/C cloning strains in ICU and pneumology department etc,and scattered between monthes in 2009.In 102 strains of carbapenem-resistant A.baumannii,71.6%of strains produced carbape nemases which were confirmed as OXA-23 by multiplex PCR, and 84.3%of strains produced OXA-51 carbape nemases,only 2 strains produced OXA-24 carbape nemases,3 strains produced OXA-58 carbape nemases.Conclusion There was outbreak of A/B/C cloning strains in ICU and pneumology department etc in our hospital,OXA-23 and OXA-51 carbape nemases were the most popular genotypes. Because of high resistance rate of drugs especially carbapenems and prevalence of cloning strains,it is necessary to strengthen the clinic monitoring to prevent and contral the clinical infection of A.baumannii.

目的建立SD大鼠药物性肝硬化模型,探讨原代肝细胞移植治疗大鼠肝硬化的疗效。方法经四氯化碳（Carbon tetrachloride,CCL4）诱导大鼠肝硬化模型,将40只模型大鼠分为2组,每组20只,Ⅰ组经脾尾注射约2×107个肝细胞,Ⅱ组经脾尾注射0.4 ml Hank’s液。观察每组大鼠的存活情况及肝功能变化;HE染色观察脾脏病理变化;免疫荧光观察植入的肝细胞在大鼠脾内合成白蛋白（Serum albumin level,ALB）的功能。结果经CCL4诱导10周后,大鼠肝脏经HE染色发现假小叶形成,表明成功建立了肝硬化模型;移植术后8周,Ⅰ组大鼠存活率（75%）显著高于Ⅱ组（35%）,差异有统计学意义（P＜0.01）;Ⅰ组大鼠的血清ALB水平较Ⅱ组明显升高（P＜0.05）,血氨（Serum ammonia level,AMM）水平较Ⅱ组明显降低（P＜0.05）;Ⅰ组大鼠移植术后1、8周,大量肝细胞在脾脏红髓中定植下来;Ⅰ组大鼠移植术后5周,植入的肝细胞在大鼠脾内具有合成白蛋白的功能。结论 CCL4能稳定诱导肝硬化动物模型;同种异体肝细胞移植能提高肝硬化大鼠的生存率,并改善肝性脑病和低蛋白血症。

目的 调查分析我院2006～2010年临床分离的革兰阴性菌对碳青霉烯类抗生素的耐药性.方法 采用纸片扩散法进行抗菌药物敏感实验,采用WHONET5.4软件及SPSS13.0软件进行数据分析.结果 大肠埃希菌和肺炎克雷伯菌对亚胺培南和美罗培南的耐药率呈上升趋势,但最高不超过4.5％.铜绿假单胞菌对亚胺培南的耐药率2006年和2007年监测显示为21.3％和41％,2008年达到44.8％,在2009年下降为12.4％,但2010年又迅速增加到30.1％.鲍曼不动杆菌对亚胺培南耐药率的逐年上升更为显著,从2006年9 1％上升到2010年的68％,差异具有统计学意义(P＜0.01).嗜麦芽窄食单胞菌对碳青霉烯类抗生素天然耐药,2006年对亚胺培南耐药率为30％,但自2007年起就增加到80.8％,最高达100％.结论 肠杆菌科细菌对碳青霉烯类敏感性高,近年耐药率有上升趋势.铜绿假单胞菌和鲍曼不动杆菌对碳青霉烯类耐药率高,尤其是鲍曼不动杆菌的分离率逐年增加,耐药形势严峻.临床应合理使用抗生素,减少多重耐药株的产生.

Objective To investigate the epidemiological characteristics of nosocomial infection by Acinetobacter baumannii(A.baumannii) and its antibiotic resistance change in our hospital,and to analyze the reason for A.baumannii outbreak and prevalence.Methods Five-hundred and ninety-one strains of A.baumannii isolated from our hospital from 2006 to 2009 were selected for a systematic study on A.baumannii infection distribution and change of its antibiotic resistance spectrum.The homology of 102 strains of Pandrug-resistant A.baumannii(PDR-Ab) including A.baumannii resistant to carbapenems in 2009 were studied using PFGE,and antimicrobial susceptibility test was performed using agar dilution method according to the NCCLS guidelines for MIC testing.Results The infection of A.baumannii in our hospital mainly occurred in pneumology department,intensive care units(ICU),neurology department,cardiac surgery and neurosurgery department.The drug resistance rate of A.baumannii to common antibiotics increased year by year.The resistance rates to minocycline,doxycycline and polymyxin B were the lowest(43.7%,32.9%,2.5%,respectively),while the resistance rate to carbapenems raised fast,accompanied by a dramatically increased dosage of carbapenems.PFGE results showed that an outbreak of A/B clone strains of A.baumannii in ICU and neurology department.Conclusion Unreasonable use of Carbapenems and prevalence of A.baumannii clone strains result in an increasing infection rate of hospital-acquired PDR-Ab.It is necessary to strengthen the control of nosocomial infection and reasonable antibiotics administration to improve the curative effect and reduce A.baumannii infection.