供体脂肪干细胞对大鼠小体积肝移植免疫调节及促移植肝再生的研究
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摘要
第一部分
30%小体积肝移植大鼠模型手术技巧及改良
背景：
活体肝移植显著缓解了供肝短缺矛盾,如何使供体献出的肝量最小,同时受体得到最大的临床受益成为了目前的研究热点,而制作稳定的小体积肝移植模型是上述研究的基础。
目的：
探索一种简便、稳定的小体积肝移植大鼠模型的建立方法,为进一步对脂肪源性间充质干细胞对大鼠小体积肝移植术后免疫及肝再生影响的研究提供最佳的模型。
方法：
供受体均为SD大鼠,雌雄各半。以Kamada“二袖套法”非动脉化原位肝移植大鼠模型为基础并参考国内外文献,制作30%小体积肝移植模型,其中原位减除肝叶后再进行灌注的方法获取供肝40对为I组；采用供肝冷灌注后全肝切取体外减除肝叶的方法获取供肝20对为II组,I、II组共60对为改良前模型组。采用供肝冷灌注后原位减除肝叶获取供肝,并对供肝切取、血管／胆管的套入方法以及手术诸多细节进行优化和改良制作模型80对为改良组(III组)。其中改良前组以肝中叶为供肝,供受体体重相近；改良组以肝中叶+右叶为供肝,供体体重<受体100-120g。Ⅰ、Ⅱ、Ⅲ分组主要观察供体手术各阶段时间,比较术后1天、7天血浆谷氨酰丙氨酸转移酶(ALT)、总胆红素(TB)、血氨(AMON)值,术后7天肝脏病理学HE染色观察组织损伤及排斥反,用于比较不同供肝灌注方法和肝叶减除方法在供肝获取上优劣。改良前、后分组主要观察手术成功率,术后并发症和术后第1天、7天、14天的生存率及生存情况分析,用于证实改良模型的优势。
结果：
改良后模型(Ⅲ组)较改良前不论是Ⅰ组(体内肝叶减除)还是Ⅱ组(体外肝叶减除组)的供肝获取时间和肝叶减除时间均显著缩短(P<0.05)；Ⅱ组的供肝冷缺血时间显著长于Ⅰ组和Ⅲ组(P<0.05)；而改良前后的无肝期、受体手术时间无显著差别(P>0.05)。改良后的小体积肝移植模型手术成功率较改良前显著提高(92.5%vs80.0%,P=0.029),而且术后1天、7天及14天存活率均高于改良前模型,其中1天存活率两者差异无统计学意义(85.0%vs66.7%,P=0.071),7天和14天两者的差异有统计学意义(62.5%vs37.6%,P=0.032；50.0%vs20.0%,P=0.01)。生存分析发现改良模型较改良前的术后中位生存时间延长了9天(14d vs5d,P=0.002)。改良模型的手术总并发症例次较改良前组显著较少(43/60vs9/80,P<0.001),其中改良前模型发生2种或以上并发症的有17例,而改良模型无1例发生2种或以上并发症。分层分析发现,改良组供肝断面出血、供肝灌注不良、肝后下腔静脉狭窄、气体栓塞的发生例次显著少于改良前组(P<0.05),而其他并发症的发生两组差异无统计学差异。
结论：
采用肝中叶+肝右叶供肝同时供体体重<受体100-120g的方法以及对操作技术的改良,能建立稳定的30%小体积肝移植大鼠模型,而且在操作简便、减少并发症、提高手术成功率和术后存活率等方面有显著优势。
第二部分
大鼠脂肪间充质干细胞的分离、培养、鉴定及体外诱导向肝细胞分化的研究
背景：
2001年英国的Zuk等从抽脂术抽取的脂肪组织悬液中分离、培养得到一群贴壁生长的多能干细胞,此后其他研究小组也证实了这些细胞是一群具有向多胚层分化潜能的成体干细胞,即脂肪间充质干细胞(Adipos-derived Mesenchymal Stem Cells, AMSCs).由于AMSCs有与BMSCs相似的分化潜能,而且分离培养要求较低,相同体积组织获取的干细胞数量更多,加之脂肪储备量巨大,取材方便容易,对供体创伤小,使AMSCs必将成为最佳的种子细胞来源。目前,对于AMSCs的分离培养由于种属来源、取材方式和部位的不同,方法各异,差异和相互矛盾的地方较多。
目的：
本实验探讨从大鼠腹股沟皮下脂肪组织中分离培养AMSCs简易、高效的方法,以及其鉴定和体外向肝样细胞分化潜能等方面的问题,为后续AMSCs对小体积肝移植供肝保护的研究做准备。
方法：
取健康雄性SD大鼠,体重200-220g,乙醚吸入麻醉后,超净工作台内取腹股沟皮下脂肪,仔细剔除血管及淋巴组织(机械性纯化)后,采用胶原酶消化直接贴壁法分离AMSCs。1：3传代培养,绘制第Ⅲ,Ⅴ,Ⅸ代细胞生长曲线,计算细胞倍增时间。流式细胞仪检测第Ⅴ代AMSCs表面抗原及细胞周期,电镜观察细胞超微结构,台盼兰染色检测细胞活力。取本实验第一部分建立成功的30%小体积肝移植存活24h后的模型SD大鼠肝脏,均浆离心后的上清液作为分化诱导培养液,对第V代AMSCs进行体外诱导培养,观察细胞形态变化并于诱导后12d和24d免疫组化检测A1b、CK18。
结果：
1.原代细胞4-6h后开始沉降贴壁,5d左右细胞大量增殖,约第10天细胞长满培养瓶底,细胞为长梭形,呈成纤维细胞样或漩涡样生长,随着传代次数增多,细胞形态逐渐单一。体外培养条件下各代AMSCs经历生长滞缓期(接种后第1-3d),对数增值期(接种后第3-6d)和生长平台期(接种第7d后),生长曲线呈“S”形,细胞倍增时间58.4±6.2h,传至第1X代细胞增殖能力无明显减弱。透射及扫描电镜观察第V代AMSCs具有幼稚细胞的结构特征。
2.流式细胞检测Go/G1细胞占78.3%,S期占9.2%,G2/M期12.5%；CD44表达率约98.5%,CD90表达率约92.6%,CD49d表达率约90.5%,CD29表达率约73.1%,CD45表达率约0.3%,CD34表达率约0.6%。证实所得到的细胞为AMSCs,且纯度较高。
3.诱导第5天开始梭形的AMSCs细胞两极缩短,开始发生形态变化,12d后多角形细胞明显增多,胞质丰富,胞质内颗粒丰富,胞核明显,出现双核细胞。免疫组化检测到诱导后12d和24d的细胞爬片Alb和CK18表达呈阳性。
4.第V代AMSCs台盼兰染色后,计算细胞活力为96.6±1.7%。
结论：
采用胶原酶消化直接贴壁法,加上对切取的脂肪在培养前进行仔细的机械纯化,大鼠腹股沟皮下脂肪能够获取大量质量较高的AMSCs。
采用大鼠小体积肝移植术后24h的成活供肝制备的诱导培养液,能在体外成功诱导AMSCs向肝样细胞分化,为后续的体内移植研究提供了理论基础。
第三部分
联合供体脂肪间充质干细胞移植诱导30%小体积肝移植大鼠免疫低反应及促肝再生机制的初探
背景：
小体积肝移植由于供肝体积小,移植后的肝细胞在经受缺血再灌注和急性排斥反应损伤的同时还必须迅速的再生,才能使移植获得成功。间充质干细胞(MSCs)免疫源性低,不论其来源供体或受体以及第三方,均不易引起排斥或移植物抗宿主反应,还能减轻受体对移植物的排斥反应甚至产生特异性的免疫耐受,同时还具有多向分化潜能(如向肝细胞分化)。其中脂肪间充质干细胞(AMSCs)不但具有MSCs的分化潜能和免疫调节能力,而且储备量巨大,取材方便容易,对供体创伤小,因而使AMSCs的应用有望成为提高小体积供肝移植成功率的最佳方法。本部分实验将第二部分所获得的AMSCs输入第一部分成功建立的30%小体积肝移植大鼠模型,希望能够诱导免疫低反应的同时促进供肝的再生,并通过血清学和病理学检测来初步探讨一些相关的机制。
目的：
观察AMSCs诱导30%小体积肝移植大鼠模型免疫低反应的同时促进移植肝再生的效果,并对相关机制进行初步探讨,为进一步广泛和深入的研究,以及临床应用提供一些基础性依据。
方法：
应用第二部分的方法所获得雄性Lewis大鼠的AMSCs,将其移植入采用第二部分改良方法成功建立的Lewis-Wistar大鼠30%小体积肝移植模型。分为A组(对照组)；B组(FK506组)；C组(AMSCs单次输入组)；D组(AMSCs术前预输注和术后多次输入组)。对比各组术后的生存情况,各监测时间点的肝脏酶谱,血清IL-2、INF-γ、IL-4、IL-10、TGFβ1值,移植肝病理学表现,免疫组化检测PCNA、Ki-67、TGF-β1、ICAM-1、IDO以及细胞凋亡在移植肝的表达。C组和D组行SRY原位杂交+Alb/CK18免疫组化双染色检测AMSCs的定植和分化。D组大鼠制作脾细胞悬液进行单向混合淋巴细胞反应(MLR)和IL-2逆转试验,体内过继转移试验用以了解是否诱导特异性免疫耐受。
结果：
1.中位生存时间：A组8±0.55d；B组144±1.1d；C组10±1.1d；D组21±2.2d,D组生存时间显著长于A-C组(P=0.001)。
2.肝脏酶谱：ALT、AST和TB在术后各组均明显升高,A组和C组术后随时间呈持续上升,B组在使用FK506期间各指标虽有回落,但撤药后再度迅速回升,在术后第7天时A-C组间各指标无显著差别,均显著高于D组。
3.血清细胞因子：术后各组别各细胞因子均明显升高(显著高于正常对照),其中IL-2和INF-γ在A组和C组术后随时间迅速上升,术后第5天B组(FK506停药第1天)与D组的上升幅度均较A组和C组低,但之后B组的上升明显加快,在第7天时D组显示出下降趋势,但包括B组在内的其余三组则显著持续升高,此三组间比较无差异,而且均显著高于D组。而IL-4和IL-10在各组别的变化与上述两细胞因子刚好相反。TGF-β1在各组别术后都显著升高,其A组和C组随时间持续升高,B组在术后第5天时血清TGF-β1的升高被抑制,曾一度显著低于A组和C组,但此后又再次显著升高,第7天时升高的程度和A、C组已无差别,D组则随时间逐渐下降,术后1-5天下降较快,而后5-7天则下降缓慢,术后第7天虽显著低于A-C组,但仍显著高于用于正常对照的本底值(263.1±62.8vs19.8±9.3)。
4.移植肝免疫组化：IDO(定位于胞浆)在各组模型大鼠术后均有明显的表达,而且随时间的推移各组的表达强度变化不大,组间无统计学差异。ICAM-1(定位于胞膜)和TGF-β1(定位于胞浆)在各组移植肝均有明显表达,A-C组术后持续高水平表达,D组在术后第5天时表达逐渐下降,第7天时显著低于A-C组。PCNA(定位于胞核)和Ki-67(定位于胞核)在术后第1天各组别移植肝内均明显表达,但A-C组随后则显著下降,术后第7天每高倍镜视野仅可.见数个或十余个阳性细胞,而D组术后两者的表达始终维持较高的水平,在各时间点的阳性细胞数均显著高于其他三组,且在术后第5和第7天部分阳性细胞可见双核和多核的增值细胞。凋亡小体在术后各组移植肝均可见,散在分布于肝实质,D组每高倍镜视野仅可见数个,显著少于A-C组,而且炎症反应较重的汇管区则见不到凋亡小体。
5.SRY原位杂交+CK18/A1b免疫组化双染色：术后第5天,C组仅在汇管区见数个褐色胞核细胞滞留(SRY阳性细胞),而D组汇管区均可见大片状褐色胞核细胞,同时在肝实质区域也可见散在胞浆紫兰色(CK18阳性细胞)而胞核呈褐色的SRY+CK18套染阳性细胞,占9.7±1.7%。术后第7天,C组仅在个别汇管区见到数枚胞核呈褐色的细胞停留,而肝实质内无阳性细胞,D组汇管区褐色胞核细胞减少,而肝实质内双阳性细胞则明显增多,占31.4±10.5%,部分成群集聚并可见双核或多核细胞。SRY+A1b套染双阳性细胞在两组移植肝也呈现相似的变化。
6.移植肝免疫排斥分级：术后第1天,各组移植物肝小叶结构基本正常,汇管区可见以少量淋巴细胞为主的混合炎性细胞浸润,血管、胆管基本完好,小叶间静脉内膜轻度水肿增厚,肝实质结构紧密,无明显坏死或炎性侵润,Banff评分0-1级。
术后第5天,A组和C组移植肝小叶结构存在,但大量淋巴细胞为主的混合炎性细胞浸润,胆管周围被炎性细胞包裹,血管内皮下炎性浸润水肿增厚明显,肝实质少量炎性浸润。Banff评分II级；B组表现与A组和C组相似,但汇管区炎性浸润较轻,血管壁水肿轻微,Banff评分I级；D组肝小叶结构正常,少量淋巴细胞为主的炎性浸润,与第1天比较变化不明显,Banff评分0-1级。
术后第7天,A组、B组和C组汇管区和肝实质均见大量以淋巴细胞为主的混合炎性浸润,并伴静脉周围肝细胞坏死,Banff评分III级；D组汇管区淋巴细胞浸润较前明显增多,血管壁水肿增加,但结构完好,肝实质炎性浸润较少,Banff评分0-1级。
7.单相混合淋巴细胞反应(MLR)及IL-2逆转试验：D组对供体品系Lewis大鼠和无关品系SD大鼠的淋巴细胞刺激百分率(SI%)分别为32.7±3.7%和39.7±12.3%,无统计学差别(P=0.257),反应体系中加入IL-2后能够使D组对两种刺激因素的免疫低反应均得到逆转,SI%分别升高为89.9±7.9%和91.44±7.6%,前后对比有显著统计学差异(P<0.001,P=0.002)。
8.体内过继试验：过继大鼠对供体品系的Lewis大鼠和无关品系SD大鼠所产生的迟发型超敏反应FI值为0.55±0.18mm和0.59±0.11mm,两组比较无统计学差异(P=0.644),但与正常大鼠对以上两品系大鼠所产生的FI值(0.86±0.21mml,0.91±0.11mm)相比较均有统计学差异(P=0.036,P=0.002)。
结论：
1.术前预输注和术后多次输注AMSCs能够通过多种机制抑制排斥反应的同时促进了移植肝的再生,加速了肝脏酶谱的恢复,显著延长了小体积移植肝和受体的存活时间,而且AMSCs能定植于移植物,并向肝样细胞分化。
2.术前预输注和术后连续多次输注AMSCs,可能通过影响IL-2, INF-γ和IL-4, IL-10的分泌,使Th0细胞向Th2表型方向偏移；通过表达和/或促移植物表达IDO等途径,来抑制受体对供肝的免疫排斥反应。并且AMSCs对降低受体的免疫排斥反应可能存在剂量依赖性。
3. AMSCs能减少TGF-β1在血液中的含量,有可能对全身和局部的炎症反应均有可能产生很强的抑制作用,但同时AMSCs在移植肝内定植后,可能表达或促周围细胞表达TGF-β1来抑制T细胞的活性和增值,表达ICAM-1来趋化T细胞,通过细胞间接触或其他途径抑制T细胞的活化和增值,从而诱导受体对移植物的低免疫反应效应。
5. AMSCs可能通过释放一些细胞因子或其他途径对凋亡相关基因或调控基因产生影响,而减少移植肝实质细胞的凋亡,有益于减轻移植肝的再灌注损伤。
6.使用AMSCs能够对受体免疫产生抑制,但并未表现出特异性耐受,未形成特异性免疫过继。
7.多次输注AMSCs可能上调移植肝内PCNA和Ki-67的表达,使小体积供肝术后维持较高增值状态。
Introduction:
At present, allogeneic orthotopic liver transplantation was the only effective means to cure the end-stage liver disease, but its development was limited by the shortage of organs。In order to expand the supply of organs and partially overcome the grafts shortage, split liver transplantation and living-related liver transplantation have played a leading role。But the safety matching of donor and receptor was still the toughest problem on the current。For instance, the liver was supposed to cut as small as possible in order to keep the safety and the less complication incidence of donor in living-related liver transplantation. However, the benefits of them were limited in adult recipients when the volume of the grafts was small. The small-for-size grafts were hard to tolerant the damage of ischemia reperfusion and the immunologic rejection simultaneously. Once the hyperplasia of the hepatic cells couldn't stand against the damage given by the factors mentioned above, the Small-for-size grafts would be lost。This was the antinomy which the clinician at work on liver transplantation had to face。To find out the method which not only could make small-for-size grafts regenerate quickly, but also could lower immune rejection or lead to immunological tolerance was the problem waiting to solve in living donor liver transplantation at present。
The research about liver regeneration mainly concentrates in the factors of regeneration. But the half-life of outside source liver regeneration factors was very short and the factors needed to be input again and again in order to have its possibility effect. However, various hepatic growth factors have not been commercialized production. The decrease of hepatic cells was absolute in small-for-size liver transplantation. The hope that liver cell could regenerate rapidly and effectively relying on the various hepatic growth factors was extremely difficult.
Part Ⅰ
Liver transplantation in rats using30%small-for-size grafts and the surgical techniques improvement
Background:
The techniques of partial liver transplantation, using a living donor graft, expanded the supply of organs and partially overcame the grafts shortage. But these benefits were limited in adult recipients when the volume of the grafts was small. To establish a model of small-for-size liver transplantation, using a simple and effective way, was the basis for the study mentioned above.
Objective:
To explore a simple and effective way of establishing a30%small-for-size liver transplantation in rats.
Methods:
280Spraque-Dawley rats were selected as the donors and recipients to establish30%Small-for-size orthotropic live transplantation using two-cuff techniques. Animals were divided into two groups depending on the techniques modified or not.60pairs of rats were divided into before modification group, using the way according to reference10to15and the median lobe of the liver as graft. These60pairs of rats were divided into two groups again depending on hepatectomy in vivo or in vitro. Group Ⅰ, performed hepatectomy in vivo before liver irrigation; group Ⅱ, performed hepatectomy in vitro after liver irrigation.80pairs of rats transplanted using the way of improvement by us were divided into modification group (group Ⅲ), in which hepatectomy was performed in suit after liver irrigation, the median and right lobes of liver were used as graft, body weights of donor were100-120g less than those of recipients, two-cuff technique and bile duct stent techniques were improved. Time of operation, survival and technical complications were compared among these groups.
Results:
The time (min) of hepatectomy and graft harvest were significantly shorter in group Ⅲ than those in group Ⅱ and Ⅰ (9.0±0.7vs.15.0±1.5vs.15.6±1.4, P=0.005;56.6±3.4vs.73.6±2.3vs.74.6±3.0, P=0.002). The cold ischemia time in group II was significantly longer than those in group Ⅰ and Ⅲ (81.0±2.2vs.62.6±3.1vs.59.8±3.2, P=0.001). The time of anhepatic and recipient operation were not significantly difference among them. The incidence of bleeding, bile leakage IVC stricture, graft less perfusion and gas embolism were significantly less in modification group than those in before modification group. The rats in modification group had a higher transplanting successful rate (92.5%), more7-d and14-d survivors post operation (62.5%&50.0%) and longer median survival time(14d) than those in before modification group(P<0.05).
Conclusion:
The way of modification by us was a more effective and simple for establishing a30%small-for-size liver transplantation in rats with higher transplanting successful rate and survival rate but fewer complications after operation.
Part II
Isolation, proliferation, Identification and differentiation into hepatocyte-like cells of rat adipos-derived mesenchymal stem cells
Objective:
To explore the rat AMSCs acquisition method in vitro、biological characteristics、 the capacity of differentiation into hepatocyte-like cells in vitro.
Methods:
We fetch inguinal fat of male SD rats to primary culture, observe cell growth and cell morphology, depict cell growth curve, take the fifth generation cell and flow cytometry identified this cells and determine cell purity. Preparation of conditioned induced medium in vitro, use the fifth generation cell to prepared cell climbing film, add induced medium culture cell, after12,24d immunohistochemistry detected cell Alb and CK18.
Results:
①The cell covered the bottom about10days after primary culture, the cell were fibroblast-like or vortex-like, with the more passage times, the cell morphology more single, cell growth curve like "S". The third to ninth generation cells had the strongest proliferation.
②By flow cytometry to detect cell surface markers, CD44expression rate about98.5%, CD90expression rate about92.6%, CD49d expression rate about90.5%, CD29expression rate about73.1%, CD45expression rate about0.3%, CD34expression rate about0.6%. Through the combination between them surface markers confirm this cell is AMSCs, and has high purity.
③Cell climbing film began morphological changes from long fusiform into edge shape or oval through5days induce. Some cell appeared dual-core, this is unique to liver cells12days after induced. Alb and CK18expression positive on12,24days.
Conclusion:
①AMSCs can be got by a special cultured method from the adipose tissue, and cultured in vitro can be passaged for many times, a lot of amplification, while maintaining its biological characteristics. They had a very strong proliferation, and the third to ninth generations had strongest proliferation.
②Confirm these cell is AMSCs by flow cytometry detected a variety of cell surface antigens and the combination of many different surface antigens, and these cell has high purity.
③MSCs can differentiate into hepatocyte-like cells in a suitable culture medium, and express the liver cell antigen; it has the part function of liver cells.
Part III
Mechanism of immune hyporesponsiveness and liver regeneration induced by the Adipose-derived Mesenchymal Stem Cells from donor in a rat30%Small-for-Size Liver Transplantation model
Objective:
To observe the effect that AMSCs induced immune response in rat model of30%small-for-size liver transplantation, while promoting transplant liver regeneration, and Preliminary investigate the mechanisms. Provide some basis for the further extensive and in-depth study, and the application of clinical.
Methods:
We transplanted the AMSCs of the male Lewis rats, obtained as the method in part one, into the rats underwent the30%small-for-size liver transplantation as the method in part two. The rats were divided into group A (comparison group), group B (FK506group), group C (AMSCs single input group) and group D (AMSCs preoperative and postoperative multiple input group). The survival and the rejection in different groups were observed. The serum level of liver enzymes, IL-2, INF-γ, IL-4, IL-10and TGF-β1was examined by ELISA. The variation of PCNA、Ki-67、TGF-、β1、 NF-κB、ICAM-1、IDO and apoptosis in the grafts was detected by immunochemical methods. The Y chromosome (SRY) in the grafts of group C and D was detected by hybridization in situ. The one-way mixed lymphocyte reaction (MLR), recombined IL-2reverse assay of one-way MLR and adoptive transfer assay were performed in group D.
Results:
①The median survival time of rats was8±0.55d in group A,14±1.1d in group B,10±1.1d in group C and21±2.2d in group D respectively. The survival time of rats in group D was longer than that in group A to C significantly.
②The serum level of liver enzymes increased after the operation in all groups significantly. It rose continuously in group A to C and decreased in group D as time goes by. The serum level of liver enzymes was significantly lower than that in group A to C on the7th day after operation.
③The serum level of the cytokines increased after operation in all groups significantly. The level of IL-2and INF-γ raised continuously and rapidly in group A and C as time goes by. But it rose slowly in group B and D. Furthermore, it decreased in group D on the7th day after the operation. The level of IL-4and IL-10changed oppositely in all groups against that of IL-2and INF-γ. The level of TGF-β1increased after operation in all groups significantly. It rose continuously in group A and C as time goes by. It was tend to rise slowly in group B on the5th day, but rose quickly again on the7th day after the operation. In group D it was decreased gradually as time goes by. Its level was lower in group D than it in group A to C on the7th day after the operation.
④The immunochemical examination of grafts:The IDO conveyed in the cytoplasm and its level was not changed significantly in all groups as time goes by after the operation. The ICAM-1conveyed in the membrane and the TGF-β1in the cytoplasm. Their level raised continuously in group A to C as time goes by. They decreased gradually on the5th day after the operation and were lower on the7th day in group D than theirs in group A to C. PCNA and Ki-67conveyed in the nucleus. Their level decreased rapidly after the operation in group A to C as time goes by. Otherwise, their convey always maintained a high level after the operation. The number of apoptotic bodies in the grafts in group D was less than that in group A to C.
⑤The double positive cells of the SRY hybridization in situ(brown nuclei) and Alb/Ck18immunohistochemistry (purple/violet blue cytoplasm) would be observed in the grafts in group C and D on the5th and7th day after the operation. They increased gradually in the number in group D as time goes by and decreased quickly in group C. The number of the cells in group D was higher than that in group C significantly on the5th and7th day after the operation.
⑥The score of Banff was grade0to1in all groups on the1st day after the operation. It was grade2in the group A and C, grade1in the group B and grade0to1in group D on the5th day after the operation. It was grade3in the group A to C but still grade0to1in group D on the7th day after the operation.
⑦The SI%of one-way MLR answered to donor strain(Lewis) and unrelated strain(SD) was32.7±3.7%and39.7±12.3%respectively. There was not significantly difference between them. The SI could be reversed by IL-2similarly between them. In vivo adoptive transfer assay was not observed in group D.
Conclusion:
The way of AMSCs infusion repeatedly on the Pre and post liver transplantation using small-for-size graft in rats could inhibit the acute rejection while promote the graft regeneration through a variety of mechanisms. It would have the liver function recovered faster and prolong the survival time of the rats. The AMSCs infused could colonized in the graft and differentiated to the hepatocyte-like cells but would not induce the immune tolerance.
30%小体积肝移植大鼠模型手术技巧及改良
背景：
活体肝移植显著缓解了供肝短缺矛盾,如何使供体献出的肝量最小,同时受体得到最大的临床受益成为了目前的研究热点,而制作稳定的小体积肝移植模型是上述研究的基础。
目的：
探索一种简便、稳定的小体积肝移植大鼠模型的建立方法,为进一步对脂肪源性间充质干细胞对大鼠小体积肝移植术后免疫及肝再生影响的研究提供最佳的模型。
方法：
供受体均为SD大鼠,雌雄各半。以Kamada“二袖套法”非动脉化原位肝移植大鼠模型为基础并参考国内外文献,制作30%小体积肝移植模型,其中原位减除肝叶后再进行灌注的方法获取供肝40对为I组；采用供肝冷灌注后全肝切取体外减除肝叶的方法获取供肝20对为II组,I、II组共60对为改良前模型组。采用供肝冷灌注后原位减除肝叶获取供肝,并对供肝切取、血管／胆管的套入方法以及手术诸多细节进行优化和改良制作模型80对为改良组(III组)。其中改良前组以肝中叶为供肝,供受体体重相近；改良组以肝中叶+右叶为供肝,供体体重<受体100-120g。Ⅰ、Ⅱ、Ⅲ分组主要观察供体手术各阶段时间,比较术后1天、7天血浆谷氨酰丙氨酸转移酶(ALT)、总胆红素(TB)、血氨(AMON)值,术后7天肝脏病理学HE染色观察组织损伤及排斥反,用于比较不同供肝灌注方法和肝叶减除方法在供肝获取上优劣。改良前、后分组主要观察手术成功率,术后并发症和术后第1天、7天、14天的生存率及生存情况分析,用于证实改良模型的优势。
结果：
改良后模型(Ⅲ组)较改良前不论是Ⅰ组(体内肝叶减除)还是Ⅱ组(体外肝叶减除组)的供肝获取时间和肝叶减除时间均显著缩短(P<0.05)；Ⅱ组的供肝冷缺血时间显著长于Ⅰ组和Ⅲ组(P<0.05)；而改良前后的无肝期、受体手术时间无显著差别(P>0.05)。改良后的小体积肝移植模型手术成功率较改良前显著提高(92.5%vs80.0%,P=0.029),而且术后1天、7天及14天存活率均高于改良前模型,其中1天存活率两者差异无统计学意义(85.0%vs66.7%,P=0.071),7天和14天两者的差异有统计学意义(62.5%vs37.6%,P=0.032；50.0%vs20.0%,P=0.01)。生存分析发现改良模型较改良前的术后中位生存时间延长了9天(14d vs5d,P=0.002)。改良模型的手术总并发症例次较改良前组显著较少(43/60vs9/80,P<0.001),其中改良前模型发生2种或以上并发症的有17例,而改良模型无1例发生2种或以上并发症。分层分析发现,改良组供肝断面出血、供肝灌注不良、肝后下腔静脉狭窄、气体栓塞的发生例次显著少于改良前组(P<0.05),而其他并发症的发生两组差异无统计学差异。
结论：
采用肝中叶+肝右叶供肝同时供体体重<受体100-120g的方法以及对操作技术的改良,能建立稳定的30%小体积肝移植大鼠模型,而且在操作简便、减少并发症、提高手术成功率和术后存活率等方面有显著优势。
第二部分
大鼠脂肪间充质干细胞的分离、培养、鉴定及体外诱导向肝细胞分化的研究
背景：
2001年英国的Zuk等从抽脂术抽取的脂肪组织悬液中分离、培养得到一群贴壁生长的多能干细胞,此后其他研究小组也证实了这些细胞是一群具有向多胚层分化潜能的成体干细胞,即脂肪间充质干细胞(Adipos-derived Mesenchymal Stem Cells, AMSCs).由于AMSCs有与BMSCs相似的分化潜能,而且分离培养要求较低,相同体积组织获取的干细胞数量更多,加之脂肪储备量巨大,取材方便容易,对供体创伤小,使AMSCs必将成为最佳的种子细胞来源。目前,对于AMSCs的分离培养由于种属来源、取材方式和部位的不同,方法各异,差异和相互矛盾的地方较多。
目的：
本实验探讨从大鼠腹股沟皮下脂肪组织中分离培养AMSCs简易、高效的方法,以及其鉴定和体外向肝样细胞分化潜能等方面的问题,为后续AMSCs对小体积肝移植供肝保护的研究做准备。
方法：
取健康雄性SD大鼠,体重200-220g,乙醚吸入麻醉后,超净工作台内取腹股沟皮下脂肪,仔细剔除血管及淋巴组织(机械性纯化)后,采用胶原酶消化直接贴壁法分离AMSCs。1：3传代培养,绘制第Ⅲ,Ⅴ,Ⅸ代细胞生长曲线,计算细胞倍增时间。流式细胞仪检测第Ⅴ代AMSCs表面抗原及细胞周期,电镜观察细胞超微结构,台盼兰染色检测细胞活力。取本实验第一部分建立成功的30%小体积肝移植存活24h后的模型SD大鼠肝脏,均浆离心后的上清液作为分化诱导培养液,对第V代AMSCs进行体外诱导培养,观察细胞形态变化并于诱导后12d和24d免疫组化检测A1b、CK18。
结果：
1.原代细胞4-6h后开始沉降贴壁,5d左右细胞大量增殖,约第10天细胞长满培养瓶底,细胞为长梭形,呈成纤维细胞样或漩涡样生长,随着传代次数增多,细胞形态逐渐单一。体外培养条件下各代AMSCs经历生长滞缓期(接种后第1-3d),对数增值期(接种后第3-6d)和生长平台期(接种第7d后),生长曲线呈“S”形,细胞倍增时间58.4±6.2h,传至第1X代细胞增殖能力无明显减弱。透射及扫描电镜观察第V代AMSCs具有幼稚细胞的结构特征。
2.流式细胞检测Go/G1细胞占78.3%,S期占9.2%,G2/M期12.5%；CD44表达率约98.5%,CD90表达率约92.6%,CD49d表达率约90.5%,CD29表达率约73.1%,CD45表达率约0.3%,CD34表达率约0.6%。证实所得到的细胞为AMSCs,且纯度较高。
3.诱导第5天开始梭形的AMSCs细胞两极缩短,开始发生形态变化,12d后多角形细胞明显增多,胞质丰富,胞质内颗粒丰富,胞核明显,出现双核细胞。免疫组化检测到诱导后12d和24d的细胞爬片Alb和CK18表达呈阳性。
4.第V代AMSCs台盼兰染色后,计算细胞活力为96.6±1.7%。
结论：
采用胶原酶消化直接贴壁法,加上对切取的脂肪在培养前进行仔细的机械纯化,大鼠腹股沟皮下脂肪能够获取大量质量较高的AMSCs。
采用大鼠小体积肝移植术后24h的成活供肝制备的诱导培养液,能在体外成功诱导AMSCs向肝样细胞分化,为后续的体内移植研究提供了理论基础。
第三部分
联合供体脂肪间充质干细胞移植诱导30%小体积肝移植大鼠免疫低反应及促肝再生机制的初探
背景：
小体积肝移植由于供肝体积小,移植后的肝细胞在经受缺血再灌注和急性排斥反应损伤的同时还必须迅速的再生,才能使移植获得成功。间充质干细胞(MSCs)免疫源性低,不论其来源供体或受体以及第三方,均不易引起排斥或移植物抗宿主反应,还能减轻受体对移植物的排斥反应甚至产生特异性的免疫耐受,同时还具有多向分化潜能(如向肝细胞分化)。其中脂肪间充质干细胞(AMSCs)不但具有MSCs的分化潜能和免疫调节能力,而且储备量巨大,取材方便容易,对供体创伤小,因而使AMSCs的应用有望成为提高小体积供肝移植成功率的最佳方法。本部分实验将第二部分所获得的AMSCs输入第一部分成功建立的30%小体积肝移植大鼠模型,希望能够诱导免疫低反应的同时促进供肝的再生,并通过血清学和病理学检测来初步探讨一些相关的机制。
目的：
观察AMSCs诱导30%小体积肝移植大鼠模型免疫低反应的同时促进移植肝再生的效果,并对相关机制进行初步探讨,为进一步广泛和深入的研究,以及临床应用提供一些基础性依据。
方法：
应用第二部分的方法所获得雄性Lewis大鼠的AMSCs,将其移植入采用第二部分改良方法成功建立的Lewis-Wistar大鼠30%小体积肝移植模型。分为A组(对照组)；B组(FK506组)；C组(AMSCs单次输入组)；D组(AMSCs术前预输注和术后多次输入组)。对比各组术后的生存情况,各监测时间点的肝脏酶谱,血清IL-2、INF-γ、IL-4、IL-10、TGFβ1值,移植肝病理学表现,免疫组化检测PCNA、Ki-67、TGF-β1、ICAM-1、IDO以及细胞凋亡在移植肝的表达。C组和D组行SRY原位杂交+Alb/CK18免疫组化双染色检测AMSCs的定植和分化。D组大鼠制作脾细胞悬液进行单向混合淋巴细胞反应(MLR)和IL-2逆转试验,体内过继转移试验用以了解是否诱导特异性免疫耐受。
结果：
1.中位生存时间：A组8±0.55d；B组144±1.1d；C组10±1.1d；D组21±2.2d,D组生存时间显著长于A-C组(P=0.001)。
2.肝脏酶谱：ALT、AST和TB在术后各组均明显升高,A组和C组术后随时间呈持续上升,B组在使用FK506期间各指标虽有回落,但撤药后再度迅速回升,在术后第7天时A-C组间各指标无显著差别,均显著高于D组。
3.血清细胞因子：术后各组别各细胞因子均明显升高(显著高于正常对照),其中IL-2和INF-γ在A组和C组术后随时间迅速上升,术后第5天B组(FK506停药第1天)与D组的上升幅度均较A组和C组低,但之后B组的上升明显加快,在第7天时D组显示出下降趋势,但包括B组在内的其余三组则显著持续升高,此三组间比较无差异,而且均显著高于D组。而IL-4和IL-10在各组别的变化与上述两细胞因子刚好相反。TGF-β1在各组别术后都显著升高,其A组和C组随时间持续升高,B组在术后第5天时血清TGF-β1的升高被抑制,曾一度显著低于A组和C组,但此后又再次显著升高,第7天时升高的程度和A、C组已无差别,D组则随时间逐渐下降,术后1-5天下降较快,而后5-7天则下降缓慢,术后第7天虽显著低于A-C组,但仍显著高于用于正常对照的本底值(263.1±62.8vs19.8±9.3)。
4.移植肝免疫组化：IDO(定位于胞浆)在各组模型大鼠术后均有明显的表达,而且随时间的推移各组的表达强度变化不大,组间无统计学差异。ICAM-1(定位于胞膜)和TGF-β1(定位于胞浆)在各组移植肝均有明显表达,A-C组术后持续高水平表达,D组在术后第5天时表达逐渐下降,第7天时显著低于A-C组。PCNA(定位于胞核)和Ki-67(定位于胞核)在术后第1天各组别移植肝内均明显表达,但A-C组随后则显著下降,术后第7天每高倍镜视野仅可.见数个或十余个阳性细胞,而D组术后两者的表达始终维持较高的水平,在各时间点的阳性细胞数均显著高于其他三组,且在术后第5和第7天部分阳性细胞可见双核和多核的增值细胞。凋亡小体在术后各组移植肝均可见,散在分布于肝实质,D组每高倍镜视野仅可见数个,显著少于A-C组,而且炎症反应较重的汇管区则见不到凋亡小体。
5.SRY原位杂交+CK18/A1b免疫组化双染色：术后第5天,C组仅在汇管区见数个褐色胞核细胞滞留(SRY阳性细胞),而D组汇管区均可见大片状褐色胞核细胞,同时在肝实质区域也可见散在胞浆紫兰色(CK18阳性细胞)而胞核呈褐色的SRY+CK18套染阳性细胞,占9.7±1.7%。术后第7天,C组仅在个别汇管区见到数枚胞核呈褐色的细胞停留,而肝实质内无阳性细胞,D组汇管区褐色胞核细胞减少,而肝实质内双阳性细胞则明显增多,占31.4±10.5%,部分成群集聚并可见双核或多核细胞。SRY+A1b套染双阳性细胞在两组移植肝也呈现相似的变化。
6.移植肝免疫排斥分级：术后第1天,各组移植物肝小叶结构基本正常,汇管区可见以少量淋巴细胞为主的混合炎性细胞浸润,血管、胆管基本完好,小叶间静脉内膜轻度水肿增厚,肝实质结构紧密,无明显坏死或炎性侵润,Banff评分0-1级。
术后第5天,A组和C组移植肝小叶结构存在,但大量淋巴细胞为主的混合炎性细胞浸润,胆管周围被炎性细胞包裹,血管内皮下炎性浸润水肿增厚明显,肝实质少量炎性浸润。Banff评分II级；B组表现与A组和C组相似,但汇管区炎性浸润较轻,血管壁水肿轻微,Banff评分I级；D组肝小叶结构正常,少量淋巴细胞为主的炎性浸润,与第1天比较变化不明显,Banff评分0-1级。
术后第7天,A组、B组和C组汇管区和肝实质均见大量以淋巴细胞为主的混合炎性浸润,并伴静脉周围肝细胞坏死,Banff评分III级；D组汇管区淋巴细胞浸润较前明显增多,血管壁水肿增加,但结构完好,肝实质炎性浸润较少,Banff评分0-1级。
7.单相混合淋巴细胞反应(MLR)及IL-2逆转试验：D组对供体品系Lewis大鼠和无关品系SD大鼠的淋巴细胞刺激百分率(SI%)分别为32.7±3.7%和39.7±12.3%,无统计学差别(P=0.257),反应体系中加入IL-2后能够使D组对两种刺激因素的免疫低反应均得到逆转,SI%分别升高为89.9±7.9%和91.44±7.6%,前后对比有显著统计学差异(P<0.001,P=0.002)。
8.体内过继试验：过继大鼠对供体品系的Lewis大鼠和无关品系SD大鼠所产生的迟发型超敏反应FI值为0.55±0.18mm和0.59±0.11mm,两组比较无统计学差异(P=0.644),但与正常大鼠对以上两品系大鼠所产生的FI值(0.86±0.21mml,0.91±0.11mm)相比较均有统计学差异(P=0.036,P=0.002)。
结论：
1.术前预输注和术后多次输注AMSCs能够通过多种机制抑制排斥反应的同时促进了移植肝的再生,加速了肝脏酶谱的恢复,显著延长了小体积移植肝和受体的存活时间,而且AMSCs能定植于移植物,并向肝样细胞分化。
2.术前预输注和术后连续多次输注AMSCs,可能通过影响IL-2, INF-γ和IL-4, IL-10的分泌,使Th0细胞向Th2表型方向偏移；通过表达和/或促移植物表达IDO等途径,来抑制受体对供肝的免疫排斥反应。并且AMSCs对降低受体的免疫排斥反应可能存在剂量依赖性。
3. AMSCs能减少TGF-β1在血液中的含量,有可能对全身和局部的炎症反应均有可能产生很强的抑制作用,但同时AMSCs在移植肝内定植后,可能表达或促周围细胞表达TGF-β1来抑制T细胞的活性和增值,表达ICAM-1来趋化T细胞,通过细胞间接触或其他途径抑制T细胞的活化和增值,从而诱导受体对移植物的低免疫反应效应。
5. AMSCs可能通过释放一些细胞因子或其他途径对凋亡相关基因或调控基因产生影响,而减少移植肝实质细胞的凋亡,有益于减轻移植肝的再灌注损伤。
6.使用AMSCs能够对受体免疫产生抑制,但并未表现出特异性耐受,未形成特异性免疫过继。
7.多次输注AMSCs可能上调移植肝内PCNA和Ki-67的表达,使小体积供肝术后维持较高增值状态。
Introduction:
At present, allogeneic orthotopic liver transplantation was the only effective means to cure the end-stage liver disease, but its development was limited by the shortage of organs。In order to expand the supply of organs and partially overcome the grafts shortage, split liver transplantation and living-related liver transplantation have played a leading role。But the safety matching of donor and receptor was still the toughest problem on the current。For instance, the liver was supposed to cut as small as possible in order to keep the safety and the less complication incidence of donor in living-related liver transplantation. However, the benefits of them were limited in adult recipients when the volume of the grafts was small. The small-for-size grafts were hard to tolerant the damage of ischemia reperfusion and the immunologic rejection simultaneously. Once the hyperplasia of the hepatic cells couldn't stand against the damage given by the factors mentioned above, the Small-for-size grafts would be lost。This was the antinomy which the clinician at work on liver transplantation had to face。To find out the method which not only could make small-for-size grafts regenerate quickly, but also could lower immune rejection or lead to immunological tolerance was the problem waiting to solve in living donor liver transplantation at present。
The research about liver regeneration mainly concentrates in the factors of regeneration. But the half-life of outside source liver regeneration factors was very short and the factors needed to be input again and again in order to have its possibility effect. However, various hepatic growth factors have not been commercialized production. The decrease of hepatic cells was absolute in small-for-size liver transplantation. The hope that liver cell could regenerate rapidly and effectively relying on the various hepatic growth factors was extremely difficult.
Part Ⅰ
Liver transplantation in rats using30%small-for-size grafts and the surgical techniques improvement
Background:
The techniques of partial liver transplantation, using a living donor graft, expanded the supply of organs and partially overcame the grafts shortage. But these benefits were limited in adult recipients when the volume of the grafts was small. To establish a model of small-for-size liver transplantation, using a simple and effective way, was the basis for the study mentioned above.
Objective:
To explore a simple and effective way of establishing a30%small-for-size liver transplantation in rats.
Methods:
280Spraque-Dawley rats were selected as the donors and recipients to establish30%Small-for-size orthotropic live transplantation using two-cuff techniques. Animals were divided into two groups depending on the techniques modified or not.60pairs of rats were divided into before modification group, using the way according to reference10to15and the median lobe of the liver as graft. These60pairs of rats were divided into two groups again depending on hepatectomy in vivo or in vitro. Group Ⅰ, performed hepatectomy in vivo before liver irrigation; group Ⅱ, performed hepatectomy in vitro after liver irrigation.80pairs of rats transplanted using the way of improvement by us were divided into modification group (group Ⅲ), in which hepatectomy was performed in suit after liver irrigation, the median and right lobes of liver were used as graft, body weights of donor were100-120g less than those of recipients, two-cuff technique and bile duct stent techniques were improved. Time of operation, survival and technical complications were compared among these groups.
Results:
The time (min) of hepatectomy and graft harvest were significantly shorter in group Ⅲ than those in group Ⅱ and Ⅰ (9.0±0.7vs.15.0±1.5vs.15.6±1.4, P=0.005;56.6±3.4vs.73.6±2.3vs.74.6±3.0, P=0.002). The cold ischemia time in group II was significantly longer than those in group Ⅰ and Ⅲ (81.0±2.2vs.62.6±3.1vs.59.8±3.2, P=0.001). The time of anhepatic and recipient operation were not significantly difference among them. The incidence of bleeding, bile leakage IVC stricture, graft less perfusion and gas embolism were significantly less in modification group than those in before modification group. The rats in modification group had a higher transplanting successful rate (92.5%), more7-d and14-d survivors post operation (62.5%&50.0%) and longer median survival time(14d) than those in before modification group(P<0.05).
Conclusion:
The way of modification by us was a more effective and simple for establishing a30%small-for-size liver transplantation in rats with higher transplanting successful rate and survival rate but fewer complications after operation.
Part II
Isolation, proliferation, Identification and differentiation into hepatocyte-like cells of rat adipos-derived mesenchymal stem cells
Objective:
To explore the rat AMSCs acquisition method in vitro、biological characteristics、 the capacity of differentiation into hepatocyte-like cells in vitro.
Methods:
We fetch inguinal fat of male SD rats to primary culture, observe cell growth and cell morphology, depict cell growth curve, take the fifth generation cell and flow cytometry identified this cells and determine cell purity. Preparation of conditioned induced medium in vitro, use the fifth generation cell to prepared cell climbing film, add induced medium culture cell, after12,24d immunohistochemistry detected cell Alb and CK18.
Results:
①The cell covered the bottom about10days after primary culture, the cell were fibroblast-like or vortex-like, with the more passage times, the cell morphology more single, cell growth curve like "S". The third to ninth generation cells had the strongest proliferation.
②By flow cytometry to detect cell surface markers, CD44expression rate about98.5%, CD90expression rate about92.6%, CD49d expression rate about90.5%, CD29expression rate about73.1%, CD45expression rate about0.3%, CD34expression rate about0.6%. Through the combination between them surface markers confirm this cell is AMSCs, and has high purity.
③Cell climbing film began morphological changes from long fusiform into edge shape or oval through5days induce. Some cell appeared dual-core, this is unique to liver cells12days after induced. Alb and CK18expression positive on12,24days.
Conclusion:
①AMSCs can be got by a special cultured method from the adipose tissue, and cultured in vitro can be passaged for many times, a lot of amplification, while maintaining its biological characteristics. They had a very strong proliferation, and the third to ninth generations had strongest proliferation.
②Confirm these cell is AMSCs by flow cytometry detected a variety of cell surface antigens and the combination of many different surface antigens, and these cell has high purity.
③MSCs can differentiate into hepatocyte-like cells in a suitable culture medium, and express the liver cell antigen; it has the part function of liver cells.
Part III
Mechanism of immune hyporesponsiveness and liver regeneration induced by the Adipose-derived Mesenchymal Stem Cells from donor in a rat30%Small-for-Size Liver Transplantation model
Objective:
To observe the effect that AMSCs induced immune response in rat model of30%small-for-size liver transplantation, while promoting transplant liver regeneration, and Preliminary investigate the mechanisms. Provide some basis for the further extensive and in-depth study, and the application of clinical.
Methods:
We transplanted the AMSCs of the male Lewis rats, obtained as the method in part one, into the rats underwent the30%small-for-size liver transplantation as the method in part two. The rats were divided into group A (comparison group), group B (FK506group), group C (AMSCs single input group) and group D (AMSCs preoperative and postoperative multiple input group). The survival and the rejection in different groups were observed. The serum level of liver enzymes, IL-2, INF-γ, IL-4, IL-10and TGF-β1was examined by ELISA. The variation of PCNA、Ki-67、TGF-、β1、 NF-κB、ICAM-1、IDO and apoptosis in the grafts was detected by immunochemical methods. The Y chromosome (SRY) in the grafts of group C and D was detected by hybridization in situ. The one-way mixed lymphocyte reaction (MLR), recombined IL-2reverse assay of one-way MLR and adoptive transfer assay were performed in group D.
Results:
①The median survival time of rats was8±0.55d in group A,14±1.1d in group B,10±1.1d in group C and21±2.2d in group D respectively. The survival time of rats in group D was longer than that in group A to C significantly.
②The serum level of liver enzymes increased after the operation in all groups significantly. It rose continuously in group A to C and decreased in group D as time goes by. The serum level of liver enzymes was significantly lower than that in group A to C on the7th day after operation.
③The serum level of the cytokines increased after operation in all groups significantly. The level of IL-2and INF-γ raised continuously and rapidly in group A and C as time goes by. But it rose slowly in group B and D. Furthermore, it decreased in group D on the7th day after the operation. The level of IL-4and IL-10changed oppositely in all groups against that of IL-2and INF-γ. The level of TGF-β1increased after operation in all groups significantly. It rose continuously in group A and C as time goes by. It was tend to rise slowly in group B on the5th day, but rose quickly again on the7th day after the operation. In group D it was decreased gradually as time goes by. Its level was lower in group D than it in group A to C on the7th day after the operation.
④The immunochemical examination of grafts:The IDO conveyed in the cytoplasm and its level was not changed significantly in all groups as time goes by after the operation. The ICAM-1conveyed in the membrane and the TGF-β1in the cytoplasm. Their level raised continuously in group A to C as time goes by. They decreased gradually on the5th day after the operation and were lower on the7th day in group D than theirs in group A to C. PCNA and Ki-67conveyed in the nucleus. Their level decreased rapidly after the operation in group A to C as time goes by. Otherwise, their convey always maintained a high level after the operation. The number of apoptotic bodies in the grafts in group D was less than that in group A to C.
⑤The double positive cells of the SRY hybridization in situ(brown nuclei) and Alb/Ck18immunohistochemistry (purple/violet blue cytoplasm) would be observed in the grafts in group C and D on the5th and7th day after the operation. They increased gradually in the number in group D as time goes by and decreased quickly in group C. The number of the cells in group D was higher than that in group C significantly on the5th and7th day after the operation.
⑥The score of Banff was grade0to1in all groups on the1st day after the operation. It was grade2in the group A and C, grade1in the group B and grade0to1in group D on the5th day after the operation. It was grade3in the group A to C but still grade0to1in group D on the7th day after the operation.
⑦The SI%of one-way MLR answered to donor strain(Lewis) and unrelated strain(SD) was32.7±3.7%and39.7±12.3%respectively. There was not significantly difference between them. The SI could be reversed by IL-2similarly between them. In vivo adoptive transfer assay was not observed in group D.
Conclusion:
The way of AMSCs infusion repeatedly on the Pre and post liver transplantation using small-for-size graft in rats could inhibit the acute rejection while promote the graft regeneration through a variety of mechanisms. It would have the liver function recovered faster and prolong the survival time of the rats. The AMSCs infused could colonized in the graft and differentiated to the hepatocyte-like cells but would not induce the immune tolerance.
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