骨髓间充质干细胞移植治疗大鼠急性肝功能衰竭

目的探讨骨髓间充质干细胞（BMSCs）移植对急性肝功能衰竭的治疗作用。方法大鼠90%肝脏切除制备急性肝功能衰竭模型,实验组大鼠肝内移植2×106个BMSCs,对照组仅注射生理盐水。观察大鼠的生存情况,RT-PCR检测BMSCs在肝脏的植入,血清检测肝功能确认BMSCs移植对肝脏修复的作用。结果移植BM-SCs的实验组大鼠存活80%,对照组大鼠仅存活20%。RT-PCR显示,BMSCs移植后定植于大鼠肝脏内。肝功能检测显示,实验组大鼠的血清丙氨酸氨基转移酶水平、天冬氨酸氨基转移酶水平显著降低,血清白蛋白水平显著升高。结论 BMSCs移植可以显著提高急性功能衰竭大鼠的生存率,促进其肝功能的恢复,对急性肝功能衰竭的治疗具有积极作用。     

鼠成骨细胞诱导兔BMSCs成骨的实验观察

目的观察乳鼠成骨细胞诱导乳兔骨髓间充质干细胞（BMSCs）的成骨情况。方法取SD大鼠乳鼠颅盖骨,采用组织块培养法培养成骨细胞;取乳兔长骨,采用全骨髓培养法培养BMSCs;用Transwell双层细胞培养板共育,实验组于培养上室接种成骨细胞,对照组培养上室不接种细胞,两组培养下室均接种BMSCs。观察BMSCs形态变化,用酶标仪检测诱导分化后BMSCs的ALP活性,RT—PCR检测BMSCs中的骨钙素、骨形态发生蛋白-2（BMP-2）、Ⅰ型胶原基因。结果共培养后实验组BMSCs形态逐渐向成骨样细胞转化,对照组无明显变化;实验组BMSCs中ALP活性高于对照组,实验组的BMSCs的骨钙素、BMP-2、Ⅰ型胶原基因有所表达,对照组无表达。结论乳鼠成骨能够诱导乳兔BMSCs向成骨细胞分化。     

白细胞介素-10与减体积大鼠肝移植后肝再生的关系

目的 探讨白细胞介素-10(IL-10)与减体积大鼠肝移植术后移植肝再生的关系。方法 建立减体积大鼠肝移植模型,实验分为:肝切除组、全肝移植组和减体积肝移植组,分别于术后1、2、4、7d取肝组织,免疫组织化学检测各组IL-10的表达,流式细胞仪检测移植肝的增殖活性。结果 肝切除组、全肝移植组和减体积肝移植组肝细胞增生活跃,术后4d增殖高峰分别为26.3±0.9、35.8±2.2、32.4±1.8。IL-10与移植后肝再生呈负相关(r=-0.58,P<0.01)。结论 减体积肝移植和全肝移植术后肝脏具有同样的增殖活性,但增殖峰值较肝切除延迟。IL-10对移植肝肝再生具有明显的调控作用,同时受免疫系统产生的其它细胞因子和激素的影响。     

5-羟色胺2A受体阻断剂对大鼠肝脏再生的影响

目的观察5-羟色胺2A受体阻断剂酮色林对大鼠肝部分切除后肝再生的影响,了解5-羟色胺及其受体在肝脏再生中的作用。方法 80只雄性Wistar大鼠随机分为实验组和对照组。采用肝大部分切除术建立肝再生模型,术后16 h分别给予腹腔内注射酮色林（实验组）和生理盐水（对照组）,采用免疫组化及流式细胞技术动态观察并比较两组大鼠术后24、36、48、72 h肝脏Ki67、增殖细胞核抗原的表达情况。结果大鼠肝大部切除术后24、36 h肝脏表达Ki67、增殖细胞核抗原最为活跃,而后表达逐渐下降。实验组大鼠肝脏表达Ki67、增殖细胞核抗原较对照组显著下降（P〈0.05）。结论 5-羟色胺2A受体阻断剂酮色林显著抑制大鼠肝大部切除术后的肝脏再生,说明5-羟色胺具有一定的促进肝再生的作用,2A受体是其重要的信号传导受体之一。     

5-HT及其受体在大鼠肝再生过程中的作用

目的分析5-羟色胺（5-HT）及其受体在大鼠肝脏再生过程中的作用。方法将50只雄性Wistar大鼠随机分成实验组和对照组。实验组大鼠于肝大部分切除术后24、36、48和72h处死,对照组行假手术。采用流式细胞技术检测大鼠肝脏增殖细胞核抗原（PCNA）表达、免疫组化法检测Ki67及5-HT表达、实时荧光定量PCR检测5-HT2A、2B受体亚型的表达。结果肝大部切除术后大鼠肝脏重量逐渐增加,PCNA和Ki67表达于术后24和36h达高峰;在肝切除术后各个时间点5-HT2A、2B受体在肝脏中的表达均显著升高,以术后36h最高;术后36h空肠嗜铬细胞5-HT含量高于24h,且都高于正常大鼠。结论肝脏再生过程中5-HT合成及其受体表达均显著上调,可能与肝脏的再生有关。     

骨髓间充质干细胞移植对大鼠脊神经急性损伤的疗效

目的探讨骨髓间充质干细胞(BMSCs)移植对大鼠脊神经急性损伤的疗效。方法取Wistar健康大鼠1只,采集骨髓,贴壁法分离BMSCs并进行培养和标记,培植细胞数约为5×104/μl的BMSCs培养液,供移植用。建立Wistar脊神经急性损伤大鼠模型40只,随机分为移植组和对照组,每组20只。损伤1 w后,将BMSCs缓慢注入移植组大鼠脊髓损伤中心,对照组注入生理盐水,观察和检测移植后两组大鼠的后肢功能康复效果以及神经生长因子(NGF)和脑源性神经营养因子(BDNF)的蛋白表达情况。结果损伤3⁸ w后移植组后肢功能康复效果明显优于对照组(P<0.05),8 w后移植组NGF和BDNF蛋白表达明显高于对照组(P<0.05)。结论 BMSCs同种异体移植可以显著提高脊神经急性损伤大鼠下肢运动功能的康复,可能与BMSCs移植对促进大鼠脊神经的再生与修复有关。     

骨髓基质细胞移植冶疗大鼠脑缺血的实验研究

目的研究骨髓基质细胞（BMSCs）对脑缺血治疗作用和机制。方法采用线栓法建立大鼠大脑中动脉缺血模型。对照组和实验组于建模成功后24 h分别经尾静脉注入等体积的PBS液和已制备好的同种异体BMSCs悬液。脑缺血治疗后1、3、7、14、28 d时行神经功能缺损评分;TTC染色、HE染色判断病变的范围及病理改变;采用免疫组化染色检测各时间点脑组织Bc l-2、Bax蛋白的表达。结果与对照组比较,在移植后3、7、14、28 d,实验组大鼠神经功能缺损评分、梗死体积及Bax蛋白表达明显降低（P〈0.05）,Bc l-2蛋白表达明显升高（P〈0.05）。结论经静脉途径移植BMSCs可显著促进脑缺血梗死大鼠的神经功能恢复,缩小梗死体积;通过上调Bc l-2蛋白表达、下调Bax蛋白表达,减少神经细胞凋亡可能是其主要机制之一。     

骨髓干细胞移植可影响心肌梗死后心肌细胞的凋亡

目的观察骨髓单个核细胞（bone marrowmononuclear cells,BM-MNCs）移植对大鼠急性心肌梗死（AMI）后心肌细胞凋亡的影响。方法健康雄性Wistar大鼠30只,随机均分为实验组和对照组。用结扎冠状动脉左前降支的方法建立大鼠AMI模型,将制备的BM-MNCs悬液和培养液分别经心外膜下植入实验组和对照组梗死心肌周围。移植术后4周末,检测心肌细胞凋亡情况及Bcl-2、Fas、FasL蛋白在心肌细胞中表达水平,并观察梗死区心肌内移植BM-MNCs及其周边区组织形态学特点。结果移植后4周末,实验组心肌细胞凋亡指数显著低于对照组（P<0.05）;与对照组相比,实验组Bcl-2蛋白吸光值显著升高（P<0.05）,Fas、FasL蛋白吸光值显著降低（P<0.05）;实验组心肌梗死区内有BrdU标记阳性的BM-MNCs存活。结论同种异体大鼠BM-MNCs移植可调节Bcl-2、Fas、FasL蛋白表达,抑制AMI后心肌细胞凋亡发生。     

缺血预处理抑制减体积肝移植大鼠早期肝细胞凋亡的研究

目的探讨缺血预处理（IPC）对减体积肝移植大鼠再灌注损伤早期细胞凋亡的影响。方法建立50%减体积大鼠肝移植模型,将72只成年雄性SD大鼠随机分为两组：对照组和IPC组,检测术后2、6、24 h血清丙氨酸转氨酶（ALT）水平变化及组织病理学变化;TUNEL法检测术后24 h肝细胞凋亡指数;免疫组织化学检测bcl-2和caspase-3蛋白表达。结果与对照组比较,IPC组术后6、24 h ALT水平下降,术后24 h肝损伤减轻,肝细胞凋亡指数、caspase-3表达均下降,而抗凋亡蛋白bcl-2表达增加（P均〈0.01）。结论 IPC明显减轻减体积肝移植术后再灌注损伤,其机制与通过上调bcl-2、下调caspase-3蛋白表达,从而抑制肝脏细胞凋亡相关。     

氢质子波谱成像对热缺血再灌注损伤大鼠移植肝细胞再生能力的评价作用

目的 探讨磁共振氢质子波谱(1H-MRS)评价大鼠原位肝移植(OLT)热缺血模型肝细胞再生能力的意义,以及热缺血对于移植肝再生能力的影响. 方法 以实验组热缺血10min的大鼠肝移植模型和对照组无热缺血大鼠肝移植模型各30只为研究对象,移植术后分6个时间点(6 h、1 d、3 d、7 d、14 d、30 d)对每只大鼠行肝脏常规T1WI、T2WI成像及氢质子波谱扫描.扫描后取肝脏,测定肝细胞的细胞增殖核抗原(PCNA)表达,同时检测肝酶代谢水平. 结果 术后5个时间点实验组的肝细胞PCNA阳性率、胆碱峰与水峰的峰高比值均高于对照组,实验组和对照组胆碱峰/水峰峰高比值均与相应PCNA的阳性率呈显著正相关(r实验=0.819,P＜0.01;r对照=0.543,P＜0.01).实验组和对照组的血清ALT、AST术后明显升高,尤以术后6 h～3 d最为显著,实验组的ALT、AST明显高于对照组. 结论 热缺血再灌注损伤对于移植后肝细胞的再生能力有明显的影响,1H-MRS的胆碱峰可以无创伤地评价移植肝的肝细胞再生能力.     

基质细胞衍生因子1对骨髓单个核细胞向肝脏迁移分化的影响

目的 探索基质细胞衍生因子1对骨髓单个核细胞向肝脏迁移、分化的影响。方法建立小鼠CCl4-AAF肝损伤模型，从小鼠骨髓中分离出骨髓单个核细胞，以荧光染料PKH26标记后经尾静脉输入肝损伤模型的小鼠体内，实验组立即给予肝内注射基质细胞衍生因子1，对照组给予肝内注射盐水，12d后取肝组织，在荧光显微镜下观察两组骨髓单个核细胞向肝脏迁移的差异，并用免疫组化法测定移植细胞的白蛋白表达。结果 PKH26标记阳性的细胞20倍镜下实验组中每张切片平均迁移数为（195．40±9．095）个，对照组平均迁移数为（169．80±7.983）个（P〈0．05）。免疫组化显示移植细胞可以表达白蛋白。结论 基质细胞衍生因子1可以促进骨髓单个核细胞向肝脏迁移，并且迁移至肝脏的骨髓单个核细胞可以向肝细胞分化。     

脐带间充质干细胞移植对急性肝功能衰竭的修复及移植途径的优化

目的：探讨脐带间充质干细胞（hUCMSC）移植对急性肝功能衰竭大鼠的治疗作用,并优化移植治疗途径。方法采用组织块贴壁法收集 hUCMSC,流式细胞术检测细胞表面标志物。将 SD 大鼠随机分为经尾静脉注射移植治疗组,经肝叶注射移植治疗组,模型对照组,空白对照组。急性肝衰竭动物模型采用一次性腹腔注射2．5 mL／kg CCl4橄榄油（1∶1）混合溶液建立,24 h 后移植治疗组分别经尾静脉和肝叶注射 hUCMSC 细胞悬液;治疗后0 h、24 h、48 h、72 h、96 h和1周收集血清分析肝功能恢复情况;治疗后3 d、1周、2周留取肝脏标本,观察病理学恢复情况,用 Real-time PCR 检测鼠肝中人 CK8、CK18和 AFP 基因 mRNA 转录水平,免疫组化法检测人 CK18的表达。结果移植治疗组 TBil 和 ALT 含量较模型对照组有明显恢复（P ＜0．05）,肝细胞再生程度提高,炎细胞减少,肝脏病理学修复作用增强。在造模后1周、2周时,经尾静脉移植治疗组和经肝叶注射移植治疗组的肝组织中,CK8、CK18和 AFP 基因 mRNA 的相对转录量与模型对照组相比差异有统计学意义（P ＜0．05）。经尾静脉注射移植治疗组和经肝叶移植治疗组中,移植后的 hUCMSC 在鼠肝中诱导分化后表达人肝细胞相关蛋白 CK18。经尾静脉和经肝叶注射移植治疗组的肝功能、移植细胞分化程度比较差异无统计学意义（P ＞0．05）。结论采用 hUCMSC 移植治疗急性肝衰竭大鼠模型后,能促进肝功能和肝脏病理学修复。移植后的 hUCMSC 自身能分化为具有肝细胞功能的类肝样细胞。经尾静脉与经肝叶注射移植疗效相似。     

Effect of cyclosporine on liver regeneration after orthotopic reduced-size hepatic transplantation in the rat

These experiments were undertaken to study the effects of cyclosporine A (CsA) on liver regeneration after an isogeneic orthotopic reduced-size hepatic transplantation (RSHT) in rats. Male Wistar rats were treated with or without a daily injection of CsA beginning 24 hr before surgery and were subjected to a 68% partial hepatectomy. A isogeneic orthotopic reduced-size hepatic transplantation was performed in recipient rats pretreated with or without CsA. A daily injection of CsA was continued until the recipient rats were sacrificed. Animals were sacrificed at various time points (12, 24, 36, 48, and 72 hr) postoperatively. The incorporation of bromodeoxyuridine (BrdU) into the DNA of the remnant hepatocytes was evaluated by immunohistochemical staining with a monoclonal antibody against BrdU. CsA (10 mg/kg/day) significantly augmented BrdU incorporation into hepatocytes after hepatectomy. The maximum labeling index (LI) was observed at 24 hr after hepatectomy. In contrast, the maximum LI in the recipient rats not receiving CsA was seen at 36 hr after RSHT, and 10 mg/kg/day of CsA decreased the LI at 36 hr after RSHT. A lower dose of CsA (3 mg/kg/day), however, significantly increased the LI in the recipient rats (P<0.01), and it reached a peak at 24 hr after RSHT when compared to the transplant recipients not receiving CsA. The time course of the increase in the LI in the transplant recipient rats receiving 3 mg/kg/day of CsA was similar to that observed in the rats after hepatectomy. This dosage improved the delay in the reduced-size hepatic transplant LI reaching its peak. These findings suggest that after RSHT the liver graft is more sensitive to both hepatotrophic and hepatotoxic effects of CsA.This work was supported in part by Mika Fund.     

Establishment of a new pig model for auxiliary partial orthotopic liver transplantation

AIM: To establish a new pig model for auxiliary partial orthotopic liver transplantation (APOLT). METHODS: The liver of the donor was removed from its body. The left lobe of the liver was resected in vivo and the right lobe was used as a graft. After the left lateral lobe of the recipient was resected, end-to-side anastomoses of suprahepatic inferior vena cava and portal vein were performed between the donor and recipient livers, respectively. End-to-end anastomoses were made between hepatic artery of graft and splenic artery of the host. Outside drainage was placed in donor common bile duct. RESULTS: Models of APOLT were established in 5 pigs with a success rate of 80%. Color ultrasound examination showed an increase of blood flow of graft on 5th d compared to the first day after operation. When animals were killed on the 5th d after operation, thrombosis of hepatic vein (HV) and portal vein (PV) were not found. Histopathological examination of liver samples revealed evidence of damage with mild steatosis and sporadic necrotic hepatocytes and focal hepatic lobules structure disorganized in graft. Infiltration of inflammatory cells was mild in portal or central vein area. Hematologic laboratory values and blood chemical findings revealed that compared with group A (before transplantation), mean arterial pressure (MAP), central venous pressure (CVP), buffer base (BB), standard bicarbonate (SB) and K+ in group B (after portal vein was clamped) decreased (P<0.01). After reperfusion of the graft, MAP, CVP and K+ restored gradually. CONCLUSION: Significant decrease of congestion in portal vein and shortened blocking time were obtained because of the application of in vitro veno-venous bypass during complete vascular clamping. This new procedure, with such advantages as simple vessel processing, quality anastomosis, less postoperative hemorrhage and higher success rate, effectively prevents ischemia reperfusion injury of the host liver and deserves to be spread.     

Outcomes of split versus reduced-size grafts in pediatric liver transplantation

BACKGROUND: Split-liver transplantation, where two grafts are created from a single donor organ, is a means of overcoming donor organ scarcity. There are few data comparing outcomes of split with reduced-size liver grafts, which is the most common type of cadaveric graft in pediatric liver transplantation. The aims of the present paper were to compare survival and complication rates between split and reduced-size cadaveric grafts in pediatric patients receiving a liver transplant in Brisbane. METHODS: Review of the Queensland Liver Transplant Service database was undertaken. All pediatric patients who received either a cadaveric split or reduced-size graft between 1985 and 2000 were examined. The incidence of patient and graft survival, vascular complications and biliary complications were identified. RESULTS: A total of 251 liver transplants were performed of which 138 were reduced-size grafts and 30 were split grafts. There were no differences in etiology of liver disease, mean age, weight, and urgency of transplant between the two groups. One-year patient and graft survivals were comparable at 73% and 67%, respectively, in both groups. There was no difference in the incidence of vascular complications between groups. Biliary complications were significantly more common after split grafts when compared with reduced-size grafts (21%vs 4%, P < 0.0001) but did not affect patient or graft survival. CONCLUSIONS: Survival and vascular complications after split-liver grafts were comparable to outcomes after reduced-size grafts. Biliary complications occur more commonly with split-liver grafts but did not affect patient or graft survival. It is recommended that every pediatric recipient be considered for a split-liver graft.     

自体骨髓间充质干细胞对肝硬化患者门静脉高压血流动力学的影响

目的观察白体骨髓间充质干细胞对乙型肝炎肝硬化失代偿患者门静脉血流动力学的影响。方法将2011年02月-2012年01月收治的乙型肝炎肝硬化失代偿住院患者46例配对分为两组。治疗组23例,对照组23例。两组患者在性别、年龄、诊断、生化及影像学指标方面差异无统计学意义。所有患者在签定知情同意书后进行治疗。对照组仅给予抗病毒及护肝利尿支持等治疗。治疗组抽取自体骨髓200ml,体外分离纯化骨髓间充质干细胞在体外诱导培养后制成10ml细胞悬液,经肝动脉注入肝脏,分别在治疗后第8、12周观察患者的门静脉血流动力学指标的变化。组内比较采用配对t检验,组间比较采用成组t检验。结果经骨髓间充质干细胞治疗8周和12周,治疗组门静脉内径（DPV）和脾静脉内径（DSV）下降,DPV在8周和12周分别为（13．26±1．3l）和（12．83±1．38）mm,与治疗前比较,t值为2．290和3．421,P〈0．05和P〈0．01;DSV在8周和12周分别为（8．39±1．38）和（8．02±1．24）mm,与治疗前比较,t值为2．079和2．787,P〈0．05和P〈0．01。与同期对照组比较,DPV在8周和12周t值为2．382和2．602,P值均〈0．05;DSV在8周和12周t值为3．236和4．185,P值均〈0．01,差异均有统计学意义。门静脉最大血流速度（PVX）增快,8周和12周分别为（20．72±4．63）和（20．58±3．46）cm／s,与治疗前比较,t值为2．833和3．198,P值均〈0．01,差异有统计学意义。与同期对照组（17．12±4．78）和（17．20±3．87）cm／s比较,差异有统计学意义（t值为2．530和3．123,P〈0．05和P〈0．01）。结论自体骨髓间充质干细胞可以明显改善乙型肝炎肝硬化失代偿患者的门静脉血流动力学指标。     

First case of cadaveric liver transplantation in Japan

The first case of liver transplantation from a brain-dead donor in Japan is described. The recipient was a 43-year-old man with familial amyloid polyneuropathy who manifested various neuropathic symptoms and autonomic dysfunction at the time of transplantation. The graft had three arteries, for which a single trunk was created at the back table. A side-to-side cavacaval anastomosis was performed as an outflow reconstruction. To avoid portal congestion, a temporary shunt between the right posterior branch of the portal vein and the vena cava was constructed, instead of a venovenous bypass. The graft preservation time was 7.2 h and the operation time was 12.2 h. Although sufficient blood flow in the hepatic artery, portal vein, and hepatic vein was confirmed intra- and postoperatively, using Doppler ultrasound, transient graft dysfunction was observed immediately after surgery, but there was spontaneous improvement. The patient was discharged 100 days after transplantation.     

丹黄方对部分肝叶切除大鼠肝组织中肝细胞生长因子的影响

[目的]探讨丹黄方对部分肝叶切除大鼠肝组织中肝细胞生长因子（hepatocyte growth factor，HGF）mRNA表达的影响。[方法]采用部分肝叶切除肝再生模型。24只大鼠随机分为假手术组、手术组、丹黄方组和促肝细胞生长素（pHGF）组。除假手术组外，其余3组接受部分肝叶切除手术。从手术前3d至手术后48h，丹黄方组给予丹黄方（10g／kg）灌胃及0．85％氯化钠（4．0ml／kg）腹腔注射，每天1次；pHGF组给予0．85％氯化钠（4．0ml／kg）灌胃和pHGF（1ml／100g）腹腔注射，每天1次；手术组及假手术组仅给予0．85％氯化钠（4．0ml／kg）灌胃和腹腔注射。采用RT-PCR方法检测HGFmRNA的表达。[结果]丹黄方组肝组织中HGFmRNA的表达显著高于假手术组和手术组（P〈0．01），与pHGF组比较差异无统计学意义（P〉0．05）。[结论]丹黄方具有促进部分肝叶切除大鼠肝组织中HGFmRNA的表达作用。     

The liver protective effect of methylprednisolone on a new experimental acute-on-chronic liver failure model in rats

Background

Acute-on-chronic liver failure is a severe, life-threatening entity and the comprehension of this disease is incomplete. Currently, a reasonable surgical model of acute-on-chronic liver failure is still lacking. The aim of this study was to establish a new model of acute-on-chronic liver failure in rats and to investigate the protective effects of methylprednisolone on this model.

Methods

An obstructive jaundice model in rats was established. Two weeks later, the animals were subjected to a choledochoduodenostomy and a reduced-size hepatic ischaemia/reperfusion injury. Animals were randomly divided into a control group, a methylprednisolone injected via the tail vein group and a methylprednisolone injected via the portal vein group. The survival rates and serum levels of alanine transaminase, aspartate aminotransferase, total bilirubin, tumour necrosis factor alpha, and interferon gamma of the rats were measured and the pathological changes in liver tissues were observed.

Results

The survival rate was significantly improved in the methylprednisolone treatment groups. Serum levels of the biochemical indexes were the lowest in the portal vein injection group. Liver tissues under microscopy presented severe pathological injury in the control group.

Conclusion

This model could be useful for further research into acute-on-chronic liver failure and methylprednisolone may be a potential therapeutic agent for this disease.