人脐带间充质干细胞促进肝癌细胞的增殖和转移

目的:研究人脐带来源的间充质干细胞(human umbilical cord mesenchymal stem cells,UC－MSCs)在体内?体外实验中对人肝癌细胞株HepG2的增殖和转移作用?方法:将UC－MSCs在无血清DMEM/F12培养基中培养48 h,收集上清液(MSCs－CM)?加入到HepG2的培养基中,使其在HepG2培养基的终浓度为25%?50%和75%作为实验组,DMEM/F12作为对照组,采用CCK－8法检测细胞增殖效应?其后使用Transwell法检测细胞的侵袭效应?建立裸鼠皮下肿瘤模型,健康雌性裸鼠12只随机分为A(HepG2细胞单/左侧皮下成瘤)?B(UC－MSCs+HepG2预混单/左侧皮下成瘤)?C(左侧HepG2?右侧UC－MSCs+HepG2皮下成瘤)3组(n = 4),每隔7 d监测成瘤体积变化,50 d后统一处死称量肿瘤湿重?结果:经各浓度MSCs－CM 处理后HepG2细胞增殖能力较对照组明显增高(P < 0.05);实验组侵袭能力明显高于对照组(P < 0.01)?50 d后皮下肿瘤湿重测得A组(0.054 2 ± 0.011 2)g?B组(0.292 0 ± 0.156 9)g?C组[左侧(0.089 4 ± 0.024 2)g?右侧(0.332 6 ± 0.102 9)g],并且从定期体积测量数据发现,两组体积的差异随着时间的推移逐渐增大(P < 0.05)?结论:UC－MSCs能促进HepG2的生长和转移,并且这种对肿瘤的增殖和侵袭促进作用可能与UC－MSCs分泌的一些因子有关?     

BrdU作为脂肪干细胞标记示踪法的可行性

目的:研究BrdU标记猪脂肪干细胞(adipose-derived stem cells,ADSCs)的最佳剂量及时间,探讨其作为干细胞标记示踪方法的可行性.方法:自中华实验猪背部提取脂肪组织,采用贴壁法分离培养ADSCs,取第3代细胞以终浓度分别为10、15、20、25及30 μmol/L BrdU进行标记:另一孔不含BrdU作为对照组,分别培养12、24、48、72及96 h.免疫荧光法检测各组细胞BrdU标记率,找出BrdU的最佳标记方法,通过台盼蓝排斥试验、MTT及细胞凋亡检测,观察BrdU对ADSCs生长情况的影响.对第3代的ADSCs采用最佳标记方法后更换普通培养基继续培养,适时传代,连续检测第4、5、6、7、8代ADSCs的BrdU标记率.结果:原代培养的ADSCs的形态主要为长梭形,第3代ADSCs经BrdU标记后胞核呈红色荧光,随浓度的升高及时间的延长,BrdU阳性标记率逐渐升高,以终浓度20μmol/L BrdU标记48 h后阳性率达90%以上,且连续传5代标记率仍达40%.MTT、台盼蓝排斥试验及细胞凋亡检测发现BrdU对ADSCs生长增殖基本无影响.结论:BrdU标记ADSCs的最佳剂量和时间为20 μmol/L和48 h,该方法标记率高,对细胞影响小,可用于动态研究ADSCs在移植体内生长、分化.     

HNF4α真核表达载体的构建及其在人脐带间充质干细胞中的表达

目的:构建pcDNA3.1/HNF4α重组质粒,转染人脐带间充质干细胞 (human umbilical cord mesenchymal stem cells,HUMSCs),并检测其在HUMSCs中的表达?方法:采用基因重组技术构建载体pcDNA3.1/HNF4α,经酶切和DNA测序鉴定,通过脂质体法转染HUMSCs后,进行RT-PCR和Western blot分析,免疫荧光检测转染后1周肝特异性生化指标?结果:成功构建真核表达质粒pcDNA3.1/HNF4α,转染人脐带间充质干细胞后,RT-PCR示转染后HNF4α mRNA表达,Western blot分析见HNF4α蛋白表达,免疫荧光示转染1周后HNF4α促进了HUMSCs向肝细胞方向分化?结论:成功构建真核表达载体,并在HUMSCs中正确表达,为进一步研究HNF4α在干细胞向肝细胞分化中作用提供了实验基础?     

过表达肝细胞核因子4α脐带间充质干细胞促进小鼠大部肝切后肝再生

目的：探讨人源性脐带间充质干细胞（UC-MSC）以及过表达肝细胞核因子4α（HNF4α）的UC-MSC能否促进小鼠大部肝切后肝再生。方法体外分离、培养、鉴定人UC-MSC,慢病毒转染过表达HNF4α。体外收集细胞培养上清液,将L02与上清液共培养,通过细胞增殖实验试剂盒（CCK8）检测细胞增殖活性。体内实验建立肝大部切除模型（约70%）,分别经尾静脉向肝切除小鼠移植0.9%生理盐水（NS）、MSC、MSC-HNF4α。48h后比较3组肝切后肝再生的变化,通过免疫组化来观察肝标本Ki67的表达。结果成功分离鉴定UC-MSC,并且成功建立稳定过表达HNF4α的MSC。体外实验MSC组和MSC-HNF4α组中L02的增殖能力都明显高于NS组（P＜0.01）,MSC组高于MSC-HNF4α组（P＜0.05）。同样体内实验相对于NS组,经MSC或MSC-HNF4α细胞处理的肝脏,其Ki67的表达明显高于NS组（ P＜0.01）,同样MSC组高于MSC-HNF4α组（P＜0.05）。结论 UC-MSC和过表达HNF4α的MSC都分泌一系列因子促进肝再生。     

慢加急性肝衰竭的研究进展

慢加急性肝衰竭(ACLF)是在慢性肝脏疾病的基础上发生肝脏急性衰竭和失代偿的临床病症。ACLF的短期病死率较高,主要死因为感染和器官衰竭。引起ACLF的常见急性事件包括细菌或病毒感染、酒精性肝炎以及手术,也有近40%的患者无明显诱因。系统性炎性反应和易感染是ACLF的典型病理生理学特点。ACLF治疗的关键在于识别和处理引起ACLF的急性事件,同时给患者提供多器官支持治疗以应对伴肝病的危重患者复杂的生理指标紊乱。慢性肝功能衰竭联盟(CLIF-C)评分已经用于对ACLF患者的分类和预后评估。肝移植是目前最有可能治愈患者的治疗选择,但是受者的识别、供肝的来源、移植的紧迫性和资源的高效利用都是肝移植广泛应用的壁垒。该文就ACLF的定义、损伤诱因、非手术治疗和肝移植研究进展作一综述。     

功能性磁共振成像测定耳针刺激

2000年3～5月,在瑞士巴塞尔放射学研究所,研究人员应用神经放射学技术功能性磁共振成像(fMRI),对耳针与中枢神经系统(CNS)的相关性进行了前瞻性研究。受试者为10名健康人,男女各5人,年龄18～52岁。实时记录受试者在下述5种状态时的脑兴奋运动区横断面的回波平面图像,即①安静状态时;②频率2Hz     

兔慢性肝损害急性肝衰竭动物模型的建立

Objective To establish an ideal animal model of acute-on-chronic liver failure (ACLF) in New Zealand white rabbits in order to provide a large animal model for further researches.Methods Totally 75 New Zealand rabbits were randomly divided into experimental group (n =70) and control group (n = 5 ). Rabbits in the experimental group were injected with CCl4 into the abdominal cavity twice every week and the doses of CCl4 were modified according to the index of liver function and the body weight, whereas those in the control group were treated with the same volume of saline. At the 10th week,48 New Zealand rabbits with hepatic fibrosis were randomly assigned to 4 groups and injected with CCl4 as before, D-Gal at a dose of 0. 65 g/kg body weight (BW), 0. 70 g/kg BW and 0. 75 g/kg BW, respectively. By observing and comparing the general state, survival time, biochemical indexes, and the histopathology, a method of establishing a stable animal model of acute hepatic failure was found. Results As compared with those in control group, the levels of ALT, AST, GGT, HA, LN and PC-Ⅲ in the experiment group were increased significantly, while the level of ALB was decreased at the end of 10 weeks. Typical features of hepatic fibrosis and the formation of pseudo-lobules were observed at the end of 10 weeks. After treatment with D-Gal, all rabbits in group Ⅰ survived with minimal changes in liver function tests. In group Ⅱ , there was a temporary hepatic injury, but no hepatic coma. Four of the 12 rabbits died (33. 3% ). In group Ⅲ , biochemical indexes changed obviously 12 h after the administration and hepatic injury reached its peak after 48 h. Ten of 12 rabbits were died of severe hepatic failure with a survival time of ( 53. 00 ± 25. 69) h. Histology of liver section revealed massive necrosis in nodules. In group Ⅳ , hepatic injury occurred early and severely. All the rabbits died of severe hepatic failure with a survival time of (32. 70 ± 17. 46) h. Conclusion The experimental model of ACLF could be established by injected with D-Gal in New Zealand rabbits with hepatic fibrosis, induced by CCl4 intraperitoneal injection for 10 weeks.The one induced by 0. 70 g/kg of D-galactosamine was more stable and showed similar clinical pathophysiological changes in human beings. So it can be a good experimental platform for studies of ACLF.     

兔慢性肝损害急性肝衰竭动物模型的建立

目的 建立新西兰兔的慢加急性肝衰竭(ACLF)动物模型.方法 将75只新西兰兔随机分为实验组(n=70)与对照组(n=5),实验组采用四氯化碳(CCl4)腹腔注射建立兔代偿性肝纤维化模型,每周2次,通过体质量及谷丙转氨酶(ALT)/谷草转氨酶(AST)改变调整药物剂量,共10周,获得48只肝纤维化新西兰兔,在此基础上将动物随机分为4组(n=12),Ⅰ组:继续腹腔注射CCl4,Ⅱ组:静脉注射D-氨基半乳糖(D-Gal)0.65 g/kg,Ⅲ组:静脉注射D-Gal 0.70 g/kg,Ⅳ组:静脉注射D-氨基半乳糖0.75 g/kg,观察各组动物的一般情况、生化指标及病理改变.结果 与对照组比较,肝纤维化实验组兔10周时ALT、AST、ALB、γ-谷氨酰转肽酶(GGT)、透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原(PC-Ⅲ)差异均有统计学意义(P＜0.05),可观察到肝纤维化的病理表现,出现典型的假小叶,在肝纤维化基础给予D-Gal后,Ⅰ组动物全部存活,生化指标轻度改变;Ⅱ组动物死亡率为33.3%(4/12),生化指标仅出现一过性的改变;Ⅲ组动物死亡率为83.3%(10/12),平均存活时间为(53.00±25.69)h,给药后12 h生化指标及临床表现出现改变,48 h达到高峰,病理显示肝脏大块坏死;Ⅳ组动物均死于肝衰竭,平均存活时间为(32.70±17.46)h,肝损害出现时间早,损伤剧烈.结论 对CCl4诱导的肝纤维化新西兰兔给予D-Gal急性攻击可建立ACLF模型.其中给予D-Gal 0.70 g/kg的模型稳定性好,能较大程度上的模拟临床上ACLF的病理生理过程.

Abstract：

Objective To establish an ideal animal model of acute-on-chronic liver failure (ACLF) in New Zealand white rabbits in order to provide a large animal model for further researches.Methods Totally 75 New Zealand rabbits were randomly divided into experimental group (n =70) and control group (n = 5 ). Rabbits in the experimental group were injected with CCl4 into the abdominal cavity twice every week and the doses of CCl4 were modified according to the index of liver function and the body weight, whereas those in the control group were treated with the same volume of saline. At the 10th week,48 New Zealand rabbits with hepatic fibrosis were randomly assigned to 4 groups and injected with CCl4 as before, D-Gal at a dose of 0. 65 g/kg body weight (BW), 0. 70 g/kg BW and 0. 75 g/kg BW, respectively. By observing and comparing the general state, survival time, biochemical indexes, and the histopathology, a method of establishing a stable animal model of acute hepatic failure was found. Results As compared with those in control group, the levels of ALT, AST, GGT, HA, LN and PC-Ⅲ in the experiment group were increased significantly, while the level of ALB was decreased at the end of 10 weeks. Typical features of hepatic fibrosis and the formation of pseudo-lobules were observed at the end of 10 weeks. After treatment with D-Gal, all rabbits in group Ⅰ survived with minimal changes in liver function tests. In group Ⅱ , there was a temporary hepatic injury, but no hepatic coma. Four of the 12 rabbits died (33. 3% ). In group Ⅲ , biochemical indexes changed obviously 12 h after the administration and hepatic injury reached its peak after 48 h. Ten of 12 rabbits were died of severe hepatic failure with a survival time of ( 53. 00 ± 25. 69) h. Histology of liver section revealed massive necrosis in nodules. In group Ⅳ , hepatic injury occurred early and severely. All the rabbits died of severe hepatic failure with a survival time of (32. 70 ± 17. 46) h. Conclusion The experimental model of ACLF could be established by injected with D-Gal in New Zealand rabbits with hepatic fibrosis, induced by CCl4 intraperitoneal injection for 10 weeks.The one induced by 0. 70 g/kg of D-galactosamine was more stable and showed similar clinical pathophysiological changes in human beings. So it can be a good experimental platform for studies of ACLF.     

大鼠原位肝移植胆道外引流模型的改进

目的 对原有大鼠原位肝移植胆道外引流模型进行改进,建立一个稳定、可靠的大鼠原位肝移植胆道外引流模型,为动态检测移植术后胆汁中各项指标提供可能.方法 根据Kamada和王振杰报道的术式加以多项技术改进建立大鼠原位肝移植胆道外引流模型,胆总管内置硬膜外导管外接合适口径的软管,高位空肠造瘘并置入软管,两软管引至右侧腹壁皮下对口相接.结果 共施行大鼠原位肝移植98次,24h动物存活率达94.1%,1周动物存活为88.2%.结论 此方法简便易行,成功率高,并发症少且可大大提高术后生存率和生存时间,是一种长期、动态收集胆汁的理想模型.     

永生化人肝细胞系的建立及其生物学特性

目的 建立一种由慢病毒载体表达人端粒酶反转录酶亚单位,具有原代肝细胞的特性的永生化肝细胞系.方法 采用离体两步胶原酶灌注法无菌条件下分离肝细胞,构建慢病毒载体并包装高滴度慢病毒,肝细胞被采用表达永生化基因的慢病毒转染.通过筛选功能基因阳性表达克隆建立永生化肝细胞系,倒置相差显微镜观察形态学变化,免疫组化检测细胞角蛋白18(CK18)表达,RT-PCR及Westernblot检测功能基因及蛋白表达.结果 采用功能基因阳性表达筛选建立了永生化肝细胞系,其可连续扩增传代,具有原代肝细胞的特性并未呈现致瘤性,揭示出肝脏实质细胞的形态学特征并且表达肝细胞主要的功能基因标志.结论 通过功能基因阳性表达筛选成功建立具有原代肝细胞特性的永生化肝细胞系.