受体来源大鼠ADSCs分化的类肝细胞诱导大鼠肝移植免疫耐受的实验研究
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摘要
第一部分大鼠原位肝移植模型的建立
目的：建立稳定的大鼠肝移植反应模型的方法,为肝移植的相关研究提供稳定可靠的模型基础。同时对比DA→Lewis与SD→SD大鼠组合,了解高排斥反应组合模型DA-Lewis术后的生存率和生存时间。为进一步的实验摸索条件。
方法：通过Kamad“二袖套法”,建立大鼠肝移植模型。并观察大鼠术后存活率和并发症的发生情况。第一阶段：进行200次双人肝移植训练,主要是熟悉手术过程,总结经验,同时对二袖套管法进行改进；第二阶段：进行了DA→Lewis与SD→SD大鼠组合肝移植对比,对比一般状态、生存期、肝功能、病理表现。
结果：总共实施大鼠原位肝移植248例,在训练阶段主要要克服麻醉问题,血管吻合问题,如何缩短无肝期以及无肝期结束后的管理问题；在成熟期时手术,供体手术时间(35.78±5.78)min,热缺血时间(0.5±0.22)min,供肝修整时间(15.13±3.45)min,受体手术时间(57±16.5)min,受体手术开始至无肝期开始(37±7.11)min,无肝期为(21.33±3.65)min,无肝期结束至关腹(13.98±3.83)min,手术成功率为93.5%,成功地建立了稳定的大鼠肝移植模型。在正式实验中DA-Lewis表现出高排斥反应的表现,存活时间短,平均时间为8.900±0.390天,术后肝功能急剧上升,病理表现出明显的重度排斥反应。
结论：大鼠原位肝移植模型的建立,需要在一定的显微外科基础和熟悉大鼠的腹部解剖结构的前提下,经过4-6月的不间断训练,方能获得成功。正确的供肝灌注、严密的腹腔止血、熟练的显微血管缝合技术、尽量缩短的无肝期、完善的术中补液纠酸及术后复温是保证手术成功的关键。DA-Lewis组合可以成功地复制出大鼠高排斥肝移植模型。
第二部分大鼠脂肪源性干细胞诱导形成的类肝细胞的免疫调节作用的体外实验研究
目的：在脂肪源性干细胞传统的分离方法的基础上进行改进,克隆筛选纯化脂肪源性干细胞；建立大鼠脂肪源性干细胞转化为类肝细胞的程序化诱导体系。并在诱导过程中观察细胞分化的变化及对类肝细胞功能进行鉴定；并进一步研究该类肝细胞的免疫特性并探讨可能的机理。
方法：
1.消化离心法分离Lewis大鼠脂肪源性干细胞,有限密度稀释法克隆化及条件培养基筛选,进行原代培养,以克隆筛选的脂肪源性干细胞为实验组,未克隆筛选的脂肪源性干细胞为对照组,观察细胞形态,对比生长曲线、倍增时间,流式细胞仪鉴定脂肪源性干细胞表面标记CD49d、 CD34. CD44。
2.将分离纯化的Lewis大鼠脂肪源性干细胞,分三个阶段加入含有FGF-4、 FGF-a. HGF及OSM细胞因子的诱导培养体系中,使脂肪源性干细胞向肝细胞转化。用RT-PCR检测ALB、 AFP及CK18mRNA的动态变化,进一步采用免疫荧光组织化学法及对氨的代谢及尿素合成能力鉴定该细胞向肝细胞转化的性能。
3.检测诱导前后的Lewis大鼠脂肪源性干细胞对DA/lewis大鼠脾T淋巴细胞混合培养体系的免疫调节作用,并检测上清液中的IL-2、IL-10的浓度。
结果：
1.经克隆筛选的脂肪源性干细胞与未经克隆筛选的脂肪源性干细胞相比,前者形态均一,生长力旺盛,倍增时间短,多代培养后生长曲线无明显改变,通过流式细胞仪鉴定,实验组与对照组在3-6代之间均表达表面标记CD49d、CD44, CD34亻(?)表达。
2.纯化后的脂肪源性干细胞大鼠脂肪源性干细胞通过向肝细胞的分段诱导方案,在0、14、21、28d分别检测ALB、AFP及CK18mRNA的表达,表达随诱导的延长而增强,通过荧光免疫分析,类肝细胞具有合成白蛋白的功能。对氨的代谢和尿素的合成功能在9-12d出现并持续存在。
3.通过混合淋巴细胞培养以DA大鼠脾T淋巴细胞为刺激细胞,Lewis大鼠脾淋巴细胞为反应细胞的体系中淋巴细胞的增殖反应受到Lewis大鼠脂肪源性干细胞的抑制,抑制的强度与干细胞的浓度正相关,与是否诱导无关。
结论：结果证实克隆筛选的纯化方法是脂肪源性干细胞原代培养的有效、经济的纯化方法。脂肪源性干细胞在体外通过阶段诱导可成功转化为类肝细胞,该诱导分化方法可为肝细胞移植、肝组织工程等临床治疗提供一个有效可靠的种子细胞来源。经过纯化的脂肪源性干细胞在体外可以成功转化为类肝细胞,该细胞同时具有干细胞下调免疫反应及肝细胞的特征的双重特性。
第三部分脂肪源性干细胞诱导形成的类肝细胞对大鼠肝移植免疫排斥的影响
目的：在大鼠原位肝移植高排斥模型(DA→Lewis)术中经门静脉注入由脂肪源性干细胞诱导形成的类肝细胞之后,研究由脂肪源性干细胞诱导形成的类肝细胞在移植肝的定位,及其对受体肝功能、免疫排斥、生存期的影响,初步探讨脂肪源性干细胞及其分化而来的类肝细胞运用于器官移植的安全性、效能以及机制。
方法：
1.随机DA与Lewis大鼠分为4组,每组各31对,采用改良Kamada两袖套法建立DA-Lewis大鼠肝移植模型：a.A组(排斥组)行原位肝移植术；b.B组(免疫抑制组)行原位肝移植术+口服他克莫司胶囊FK506(0.15mg/kg/d,灌胃1次/d×14d,术后2d开始)；c. C组行原位肝移植术+术中门静脉输注ADSCs:d.D组行原位肝移植+术中门静脉输注诱导后的类肝细胞；
2.观察并记录每组实验大鼠的一般情况(大鼠皮毛、眼睛色泽、活动和精神状态)；每组各留6只观察生存期；
3..每组术后第3、7、10、14天随机活杀6只大鼠,进行抽血,测定肝功能并用ELISA法测定外周血的细胞因子(IL-2、INF-Y、IL-10及IL-4)水平；
4.用BrdU体外标记脂肪源性干细胞及类肝细胞,并用免疫荧光染色单标及双标记的方法了解BrdU标记的成功与否,在4个时相点将一部分肝组织制作成冰冻切片,行BrdU免疫荧光检测,了解类肝细胞在肝脏中的定位情况；
5.取每只大鼠肝脏组织做成石蜡切片,HE染色光镜下观察,并计算排斥指数了解移植肝的排斥程度；
6.于实验第30d将实验各组中尚存活的大鼠中,挑选出状态最好的大鼠进行取材,行混合淋巴培养,了解术后T淋巴细胞增殖能力。用流式细胞仪测定各组CD4+、CD8+变化,计算CD4+/CD8+。
结果：
1.4组均在相同的条件下采用相同的手术方式(改良两袖套法)建立动物模型,在术前体重及手术时间的比较4组间无明显差异(P>0.05),即具有可比性。存活率为94.66%(124/131)。
2.从术后3天以细胞核BrdU染色阳性的脂肪源性干细胞及其形成的类肝细胞开始出现,散在分布于肝组织,同时有部份阳性细胞密集于汇管区及血管旁,随着时间的延长,逐渐形成灶状分布。
3.B、C、D组中位生存时间明显高于A组(对照组),均存在统计学差异(P<0.05),D组中位生存时间最长,与C组相比有统计学意义,与B组相比无统计学差异。
4.移植术后各组大鼠血清生化检查：在移植术后3、7、10d, B、C、D组肝功能逐渐好转,A组ALT、AST、TBIL急剧上升,与存活时间表现一致。在术后第14天,A组动物已完全死亡,数据缺失；B、C、D组肝功能进一步恢复,B、D组恢复较快,C组较慢,在部分指标(ALT、AST) D组优于B组(P<0.05)。
5.A组IL-2、INF-Y明显高于其它各组(P<0.05), B、C、D组水平逐渐下降,B、D组与C组比较,下降更明显(P<0.05),B、D组之间无明显差异(P>0.05)；A组IL-10及IL-4明显低于其它各组(P<0.05), B、C、D组水平逐渐升高,B、D组与C组比较,升高更明显(P<0.05)。
6.肝脏病理示术后7d、10d,A组的急性排斥评分明显高于B、C、D组(p<0.05),B、D组随着时间延长,呈下降趋势,但两者之间无明显差异(P>0.05),C组排斥指数高于B、D组(P<0.05)。
7.混合淋巴细胞培养：以DA大鼠脾细胞为刺激细胞,移植受体的脾细胞为反应细胞的混合淋巴培养体系中,B、C及D组OD值较阳性对照组降低(p<0.05),以SD大鼠脾细胞为刺激细胞移植受体的脾细胞为反应细胞的的混合淋巴培养体系中,B组的OD值低于阳性对照组,而C、D组与SD阳性对照组之间的比较没有明显差异,C、D实验组对DA及SD两种不同的刺激细胞反映出的OD值之间的比较有统计学差异(P<0.01),说明脂肪源性干细胞处理组及类肝细胞对DA刺激细胞刺激后建立起来的免疫下调状态是特异性的。
8.A组CD4+/CD8+的比值进行性上升,显著高于B组、C和D组(P<0.05),第10天达到高峰(4.683±0.775)。术后3-10天B组、C和D组比值缓慢上升,到14天开始回落,但各时间点各组差异不大,无显著性差异。
结论
1.成功建立大鼠肝移植高排斥模型。
2.单纯脂肪源性干细胞治疗组的移植肝内有肝纤维化出现以及脂肪变性,MSCs可能参与此过程的发生,可能影响其在体内的分化,不足以完全抑制移植术后的免疫排斥反应。
3.由脂肪源性干细胞分化形成的类肝细胞治疗组受鼠对供肝产生耐受,且为供者特异性免疫耐受。
4.由脂肪源性干细胞分化形成的类肝细胞治疗组对耐受的产生发挥一定的作用,并促进了肝细胞再生从而避免使用免疫抑制剂。
Part One Establishment of Orthotopic Liver Transplantation Model in Rats
Objective:To investigate the establishing methods of orthotopic liver transplantation model in rats and provide a stable basic model for liver tansplantation research. And then we deal orthotopic liver transplantation model in rats with DA-Lewis and SD-SD, to take knowledge of the high lever of immunity rejection in DA-Lewis in survival time and pathology.
Methods:Using Kamda two-cuff-technique to establish rat orthotopic liver transplantation model. During pre-experiment, SD rats were exceuted with orthotopic liver transplantation, while in formal orthotopic liver transplantation experiment, DA and Lewis rats were used as donors and recipients respectively. After the operation, recipient survival rate and liver function and pathology were observed.
Results:Orthotopic liver transplantation models were successfully established in248rats. The main problems in the training phase were to overcome the problem of anesthesia, vascular anastomosis, to shorten the time of the anhepatic phase, to improve the management after the anhepatic phase. Analyzing50model standard operations, donor operation time was35.78±5.78min,warm ischemia time was0.5±0.22min, donor truing time was15.13±3.45min, recipient operation time was40±10.5min, the time before anhepatic was37±7.11min, anhepatic time was21.33±3.65min, the time after anhepatic is13.98±3.83min,operation success rate was97.3%. At the formal examination, the model from DA to Lewis showed high lever of immunity rejection by survival rate shortened and liver function increased rapidly and severe pathology.
Conclusion:To establish methods of orthotopic liver transplantation model in rats successfully, operators must have the process of orthotopic liver transplantation the techniques extent microsurgery、the knowledge of rat abdomen anatomy and the manipulate of aseptic surgery, and lastly hard training for half of a year was inevitable. Key factors for successful operation include right liver infusion methods, rigorous peritoneal hemostasis、skilled microvessel suture technique, shortened anhepatic time, improved fluid therapy, corrected acid and alkali balance during operation and rewarming after operation. The model of liver transplantation from DA to Lewis can successfully replicate the high rejection in rat liver transplantation model.
Part Two the research of the immunologic characteristics of the hepatic-like cells differentiationed from adipose tissue-derived stem cell in vitro
Objective:To investigate the effect of the improved method of Monoclonal screening which was used to purification adipose tissue-derived stem cells(ADSCs) in the original generation culture.To differentiated the rat ADSCs into the hepatic-like cell (iPS) in vitro by procedure induction. The rat adipose tissue-derived stem cells were differentiated into the hepatic-like cells in vitro and the immunologic characteristics of iPS were investigated.
Methods.
After isolated the Lewis rat ADSCs with the digestion centrifuge process, The ADSCs was taken with the limited density dilution method cloning and condition-culture medium screening in the original generation culture.The group with clone screening ADSCs was taken as the experimental group, without clone screening as the control group, comparied the cellular appearance, growth curve and the cell doubling generation time; the surface marks CD49d, CD34, CD44of ADSCs were determined by flow cytometry.
The rat ADSCs was differentiationed into iPS in the procedure culture system by tris-step including FGF-4、FGF-α、HGF、dexamethasone and OSM. The ALB、AFP、 CK18mRNA were determined by RT-PCR and Urine and transferring Ammonia were monitored during inducing differentiation periods. The ALB was determined by fluorescence immunohistochemisty at last. To detect the Immunoregulation function of rat ADSCs and the iPS differentiationed from ADSCs by mixed lymphocyte culture of the system of the DA/Lewis spleen cell, and the effect of immuno-regulation on IL-2and IL-10of spleen cell were determined by ELISA analyze.
Result:The appearance of ADSCs which undergone clone screening has been homogeneous. The appearance were the doubling time was shorten, the change of growth curves was not obviously after many generation, growing-power after long-term culture was vigorous and stable, and the surface marks CD49d, CD44were high expressed while the surface marks of CD34was low in both the control group and the the experimental group examined by FCM.Rat ADSCs was successfully differentiationed into iPS by the21d culture system in vitro. The expression of ALB、 AFP and CK18mRNA was determined fortified by degrees at7thd、14thd and21thd. The protein ALB was determined by immunohistochemisty. It was occurred and persistenced of Metabolism of ammonia and urea synthesis after9th-12th d. It was shown that both Lewis rat ADSCs and iPS can down-regulate the immunologic reaction of the co-culture of DA and Lewis rat spleen cell indifferently, and the immuno suppression effect was enhanced by concentration of Lewis rat ADSCs and iPS.
Conclusion:The clone screening purification method is effective、economical purification method of ADSCs. Rat ADSCs was successfully differentiationed into iPS in vitro after procedure-induction. The method of induction can provide a valid and reliable source of seed cells for hepatocytes transplantation, the clinical treatment of liver tissue engineering. Rat ADSCs was successfully differentiationed into iPS in vitro, the iPS have the characteristics both function of Liver cell regeneration and immunosuppression effect.
Part Three the influence to the immunity rejection of the orthotopic liver transplantation model in rats induced by the hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells.
Objectives:After injected the hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells by portal vein in the process of the model of the orthotopic liver transplantation in rats of the model with high-lever rejection (DA-Lewis). To investigate the homing position in the rat liver, and the influence on the liver function the degree of immunity rejection survival time. The safety、 efficiency and mechanism of the hepatic-like cells differentiation from adipose-derived mesenchymal stem cells in the orthotopic liver transplantation model in rats were discussed.
Methods:
1. The model of the orthotopic liver transplantation in rats with high-lever rejection (DA-Lewis) was established with Kamada two-cufft technique, divided the DA and Lewis rat into4group(31recipient/group) randomizing,
1) The group A (reject group):operation without any other management
2) The group B (the immunosuppressant group) operation and take FK506capsule orally (0.15mg/kg/d by Gastric lavage qdX14d begin after the second day of operation)
3) The group C:operation and injected the adipose-derived mesenchymal stem cells by portal vein in the process of operation.
4) The group D:operation and injected the hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells by portal vein in the process of operation.
2. The general condition of the luster of eyes、rat hair、activity and mental state of each experiment rat were observed and recorded;6rat were left to observe the survival time each group;
3.6rats were killed at the4th、7th、10th、14th day after operation each group. The liver function and cytokine (IL-2, INF-y and IL-10and IL-4) were detected. Cytokine were detected by the ELIS A method.
4. Brdu was used to mark the adipose-derived mesenchymal stem cell and hepatic-like cells differentiationed from adipose-derived mesenchymal stem cell in vitro. Identification of the mark of Brdu by fluorescence immunohistochemical, and detected the homing position of the cell in vivo marked by Brdu with fluorescence immunohistochemical methods.
5. The hematoxylin-eosin staining of rat liver was taken to observe the rejection activity index.
6. The mixed lymphocytes culture test was taken at30th after operation. CD4+、CD8+were measuresed by flow cytometry.
Result:
1. The animal model were built up in all4group rat under the same condition, including weight and operation time without obvious difference (P>0.05), the survival rate is94.66%.(124/131).
2. The adipose-derived mesenchymal stem cell and the hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells marked with Brdu began to appear after3days of the operation, which nucleus positive of red dye by fluorescence immunohistochemical scattered in liver tissue, and some parts of the positive cells to collect and dense around the blood vessels and abbacy. With the extension of time, gradually formed shape distributings oven.
3. Group B, C, D of median survival time is obviously higher than that of the group A (control group)(P<0.05), group D of median survival time was the longest in all group, and were longer than C group,but is no statistical difference compared with group B.
4. Liver function of ALT, AST, TBIL of group A rising sharply, but B、C、D group gradually declined in the postoperative day7th and10th, In the postoperative day14th, animals of group A has completely death, data lost; liver function of group B、C、D further recovery, group B、D recovery degree is better than group C, group D better than B group in some data(ALT,AST)(P<0.05).
5. The lever of IL-2、INF-gamma of group A higher than other group significantly (P<0.05), while level of the group B、C、D gradually declined in the postoperative day7th、10th and14th, the lever of group B and D declined more than group C (P<0.05); the lever of IL-10and IL-4of group A was significantly lower than the other group (P<0.05), but group B、C、D gradually raised, group B and D raised more than group C (P<0.05).
6. Rejection activity index of group A significantly higher than group B、C、D in the postoperative day7th and10th(p<0.05), group B、D have down trend with time prolonged, rejection index of group C was high than group B and D (p>0.05).
7. The mixed lymphocytes culture test:OD value of group B, C and D reduce compared with the positive control group (A) when the stimulate cell come from spleen cells of DA rat. OD value of group B, reduce compared with the positive control group when the stimulate cell come from spleen cells of SD rat. But the OD value of group C、D was still high as the positive control group (p<0.01). It showed that the immune tolerance of recipient was specificity.
8. The ratio of CD4+/CD8+of Group A rise progressively after operation,which significantly higher than group B, C and D (P<0.05);the ratio of group B, C and D rise slowly from3th to10th and fall down from14th after operation,but there's no statistical difference.
Conclusion:
1. Rat liver transplantation high-lever rejection model was established successfully.
2. Liver fibrosis and steatosis may appear when adipose-derived mesenchymal stem cells injected by portal vein to intro, which rent differentiation of it to liver cell, the therapy of adipose-derived mesenchymal stem cells only may not enough to protect the body from immune rejection.
3. Specificite immune tolerance was successfully established of recipient deal with the hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells by portal vein.
4. The hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells play a certain role of tolerance establish, without using immunosuppressive agents.
目的：建立稳定的大鼠肝移植反应模型的方法,为肝移植的相关研究提供稳定可靠的模型基础。同时对比DA→Lewis与SD→SD大鼠组合,了解高排斥反应组合模型DA-Lewis术后的生存率和生存时间。为进一步的实验摸索条件。
方法：通过Kamad“二袖套法”,建立大鼠肝移植模型。并观察大鼠术后存活率和并发症的发生情况。第一阶段：进行200次双人肝移植训练,主要是熟悉手术过程,总结经验,同时对二袖套管法进行改进；第二阶段：进行了DA→Lewis与SD→SD大鼠组合肝移植对比,对比一般状态、生存期、肝功能、病理表现。
结果：总共实施大鼠原位肝移植248例,在训练阶段主要要克服麻醉问题,血管吻合问题,如何缩短无肝期以及无肝期结束后的管理问题；在成熟期时手术,供体手术时间(35.78±5.78)min,热缺血时间(0.5±0.22)min,供肝修整时间(15.13±3.45)min,受体手术时间(57±16.5)min,受体手术开始至无肝期开始(37±7.11)min,无肝期为(21.33±3.65)min,无肝期结束至关腹(13.98±3.83)min,手术成功率为93.5%,成功地建立了稳定的大鼠肝移植模型。在正式实验中DA-Lewis表现出高排斥反应的表现,存活时间短,平均时间为8.900±0.390天,术后肝功能急剧上升,病理表现出明显的重度排斥反应。
结论：大鼠原位肝移植模型的建立,需要在一定的显微外科基础和熟悉大鼠的腹部解剖结构的前提下,经过4-6月的不间断训练,方能获得成功。正确的供肝灌注、严密的腹腔止血、熟练的显微血管缝合技术、尽量缩短的无肝期、完善的术中补液纠酸及术后复温是保证手术成功的关键。DA-Lewis组合可以成功地复制出大鼠高排斥肝移植模型。
第二部分大鼠脂肪源性干细胞诱导形成的类肝细胞的免疫调节作用的体外实验研究
目的：在脂肪源性干细胞传统的分离方法的基础上进行改进,克隆筛选纯化脂肪源性干细胞；建立大鼠脂肪源性干细胞转化为类肝细胞的程序化诱导体系。并在诱导过程中观察细胞分化的变化及对类肝细胞功能进行鉴定；并进一步研究该类肝细胞的免疫特性并探讨可能的机理。
方法：
1.消化离心法分离Lewis大鼠脂肪源性干细胞,有限密度稀释法克隆化及条件培养基筛选,进行原代培养,以克隆筛选的脂肪源性干细胞为实验组,未克隆筛选的脂肪源性干细胞为对照组,观察细胞形态,对比生长曲线、倍增时间,流式细胞仪鉴定脂肪源性干细胞表面标记CD49d、 CD34. CD44。
2.将分离纯化的Lewis大鼠脂肪源性干细胞,分三个阶段加入含有FGF-4、 FGF-a. HGF及OSM细胞因子的诱导培养体系中,使脂肪源性干细胞向肝细胞转化。用RT-PCR检测ALB、 AFP及CK18mRNA的动态变化,进一步采用免疫荧光组织化学法及对氨的代谢及尿素合成能力鉴定该细胞向肝细胞转化的性能。
3.检测诱导前后的Lewis大鼠脂肪源性干细胞对DA/lewis大鼠脾T淋巴细胞混合培养体系的免疫调节作用,并检测上清液中的IL-2、IL-10的浓度。
结果：
1.经克隆筛选的脂肪源性干细胞与未经克隆筛选的脂肪源性干细胞相比,前者形态均一,生长力旺盛,倍增时间短,多代培养后生长曲线无明显改变,通过流式细胞仪鉴定,实验组与对照组在3-6代之间均表达表面标记CD49d、CD44, CD34亻(?)表达。
2.纯化后的脂肪源性干细胞大鼠脂肪源性干细胞通过向肝细胞的分段诱导方案,在0、14、21、28d分别检测ALB、AFP及CK18mRNA的表达,表达随诱导的延长而增强,通过荧光免疫分析,类肝细胞具有合成白蛋白的功能。对氨的代谢和尿素的合成功能在9-12d出现并持续存在。
3.通过混合淋巴细胞培养以DA大鼠脾T淋巴细胞为刺激细胞,Lewis大鼠脾淋巴细胞为反应细胞的体系中淋巴细胞的增殖反应受到Lewis大鼠脂肪源性干细胞的抑制,抑制的强度与干细胞的浓度正相关,与是否诱导无关。
结论：结果证实克隆筛选的纯化方法是脂肪源性干细胞原代培养的有效、经济的纯化方法。脂肪源性干细胞在体外通过阶段诱导可成功转化为类肝细胞,该诱导分化方法可为肝细胞移植、肝组织工程等临床治疗提供一个有效可靠的种子细胞来源。经过纯化的脂肪源性干细胞在体外可以成功转化为类肝细胞,该细胞同时具有干细胞下调免疫反应及肝细胞的特征的双重特性。
第三部分脂肪源性干细胞诱导形成的类肝细胞对大鼠肝移植免疫排斥的影响
目的：在大鼠原位肝移植高排斥模型(DA→Lewis)术中经门静脉注入由脂肪源性干细胞诱导形成的类肝细胞之后,研究由脂肪源性干细胞诱导形成的类肝细胞在移植肝的定位,及其对受体肝功能、免疫排斥、生存期的影响,初步探讨脂肪源性干细胞及其分化而来的类肝细胞运用于器官移植的安全性、效能以及机制。
方法：
1.随机DA与Lewis大鼠分为4组,每组各31对,采用改良Kamada两袖套法建立DA-Lewis大鼠肝移植模型：a.A组(排斥组)行原位肝移植术；b.B组(免疫抑制组)行原位肝移植术+口服他克莫司胶囊FK506(0.15mg/kg/d,灌胃1次/d×14d,术后2d开始)；c. C组行原位肝移植术+术中门静脉输注ADSCs:d.D组行原位肝移植+术中门静脉输注诱导后的类肝细胞；
2.观察并记录每组实验大鼠的一般情况(大鼠皮毛、眼睛色泽、活动和精神状态)；每组各留6只观察生存期；
3..每组术后第3、7、10、14天随机活杀6只大鼠,进行抽血,测定肝功能并用ELISA法测定外周血的细胞因子(IL-2、INF-Y、IL-10及IL-4)水平；
4.用BrdU体外标记脂肪源性干细胞及类肝细胞,并用免疫荧光染色单标及双标记的方法了解BrdU标记的成功与否,在4个时相点将一部分肝组织制作成冰冻切片,行BrdU免疫荧光检测,了解类肝细胞在肝脏中的定位情况；
5.取每只大鼠肝脏组织做成石蜡切片,HE染色光镜下观察,并计算排斥指数了解移植肝的排斥程度；
6.于实验第30d将实验各组中尚存活的大鼠中,挑选出状态最好的大鼠进行取材,行混合淋巴培养,了解术后T淋巴细胞增殖能力。用流式细胞仪测定各组CD4+、CD8+变化,计算CD4+/CD8+。
结果：
1.4组均在相同的条件下采用相同的手术方式(改良两袖套法)建立动物模型,在术前体重及手术时间的比较4组间无明显差异(P>0.05),即具有可比性。存活率为94.66%(124/131)。
2.从术后3天以细胞核BrdU染色阳性的脂肪源性干细胞及其形成的类肝细胞开始出现,散在分布于肝组织,同时有部份阳性细胞密集于汇管区及血管旁,随着时间的延长,逐渐形成灶状分布。
3.B、C、D组中位生存时间明显高于A组(对照组),均存在统计学差异(P<0.05),D组中位生存时间最长,与C组相比有统计学意义,与B组相比无统计学差异。
4.移植术后各组大鼠血清生化检查：在移植术后3、7、10d, B、C、D组肝功能逐渐好转,A组ALT、AST、TBIL急剧上升,与存活时间表现一致。在术后第14天,A组动物已完全死亡,数据缺失；B、C、D组肝功能进一步恢复,B、D组恢复较快,C组较慢,在部分指标(ALT、AST) D组优于B组(P<0.05)。
5.A组IL-2、INF-Y明显高于其它各组(P<0.05), B、C、D组水平逐渐下降,B、D组与C组比较,下降更明显(P<0.05),B、D组之间无明显差异(P>0.05)；A组IL-10及IL-4明显低于其它各组(P<0.05), B、C、D组水平逐渐升高,B、D组与C组比较,升高更明显(P<0.05)。
6.肝脏病理示术后7d、10d,A组的急性排斥评分明显高于B、C、D组(p<0.05),B、D组随着时间延长,呈下降趋势,但两者之间无明显差异(P>0.05),C组排斥指数高于B、D组(P<0.05)。
7.混合淋巴细胞培养：以DA大鼠脾细胞为刺激细胞,移植受体的脾细胞为反应细胞的混合淋巴培养体系中,B、C及D组OD值较阳性对照组降低(p<0.05),以SD大鼠脾细胞为刺激细胞移植受体的脾细胞为反应细胞的的混合淋巴培养体系中,B组的OD值低于阳性对照组,而C、D组与SD阳性对照组之间的比较没有明显差异,C、D实验组对DA及SD两种不同的刺激细胞反映出的OD值之间的比较有统计学差异(P<0.01),说明脂肪源性干细胞处理组及类肝细胞对DA刺激细胞刺激后建立起来的免疫下调状态是特异性的。
8.A组CD4+/CD8+的比值进行性上升,显著高于B组、C和D组(P<0.05),第10天达到高峰(4.683±0.775)。术后3-10天B组、C和D组比值缓慢上升,到14天开始回落,但各时间点各组差异不大,无显著性差异。
结论
1.成功建立大鼠肝移植高排斥模型。
2.单纯脂肪源性干细胞治疗组的移植肝内有肝纤维化出现以及脂肪变性,MSCs可能参与此过程的发生,可能影响其在体内的分化,不足以完全抑制移植术后的免疫排斥反应。
3.由脂肪源性干细胞分化形成的类肝细胞治疗组受鼠对供肝产生耐受,且为供者特异性免疫耐受。
4.由脂肪源性干细胞分化形成的类肝细胞治疗组对耐受的产生发挥一定的作用,并促进了肝细胞再生从而避免使用免疫抑制剂。
Part One Establishment of Orthotopic Liver Transplantation Model in Rats
Objective:To investigate the establishing methods of orthotopic liver transplantation model in rats and provide a stable basic model for liver tansplantation research. And then we deal orthotopic liver transplantation model in rats with DA-Lewis and SD-SD, to take knowledge of the high lever of immunity rejection in DA-Lewis in survival time and pathology.
Methods:Using Kamda two-cuff-technique to establish rat orthotopic liver transplantation model. During pre-experiment, SD rats were exceuted with orthotopic liver transplantation, while in formal orthotopic liver transplantation experiment, DA and Lewis rats were used as donors and recipients respectively. After the operation, recipient survival rate and liver function and pathology were observed.
Results:Orthotopic liver transplantation models were successfully established in248rats. The main problems in the training phase were to overcome the problem of anesthesia, vascular anastomosis, to shorten the time of the anhepatic phase, to improve the management after the anhepatic phase. Analyzing50model standard operations, donor operation time was35.78±5.78min,warm ischemia time was0.5±0.22min, donor truing time was15.13±3.45min, recipient operation time was40±10.5min, the time before anhepatic was37±7.11min, anhepatic time was21.33±3.65min, the time after anhepatic is13.98±3.83min,operation success rate was97.3%. At the formal examination, the model from DA to Lewis showed high lever of immunity rejection by survival rate shortened and liver function increased rapidly and severe pathology.
Conclusion:To establish methods of orthotopic liver transplantation model in rats successfully, operators must have the process of orthotopic liver transplantation the techniques extent microsurgery、the knowledge of rat abdomen anatomy and the manipulate of aseptic surgery, and lastly hard training for half of a year was inevitable. Key factors for successful operation include right liver infusion methods, rigorous peritoneal hemostasis、skilled microvessel suture technique, shortened anhepatic time, improved fluid therapy, corrected acid and alkali balance during operation and rewarming after operation. The model of liver transplantation from DA to Lewis can successfully replicate the high rejection in rat liver transplantation model.
Part Two the research of the immunologic characteristics of the hepatic-like cells differentiationed from adipose tissue-derived stem cell in vitro
Objective:To investigate the effect of the improved method of Monoclonal screening which was used to purification adipose tissue-derived stem cells(ADSCs) in the original generation culture.To differentiated the rat ADSCs into the hepatic-like cell (iPS) in vitro by procedure induction. The rat adipose tissue-derived stem cells were differentiated into the hepatic-like cells in vitro and the immunologic characteristics of iPS were investigated.
Methods.
After isolated the Lewis rat ADSCs with the digestion centrifuge process, The ADSCs was taken with the limited density dilution method cloning and condition-culture medium screening in the original generation culture.The group with clone screening ADSCs was taken as the experimental group, without clone screening as the control group, comparied the cellular appearance, growth curve and the cell doubling generation time; the surface marks CD49d, CD34, CD44of ADSCs were determined by flow cytometry.
The rat ADSCs was differentiationed into iPS in the procedure culture system by tris-step including FGF-4、FGF-α、HGF、dexamethasone and OSM. The ALB、AFP、 CK18mRNA were determined by RT-PCR and Urine and transferring Ammonia were monitored during inducing differentiation periods. The ALB was determined by fluorescence immunohistochemisty at last. To detect the Immunoregulation function of rat ADSCs and the iPS differentiationed from ADSCs by mixed lymphocyte culture of the system of the DA/Lewis spleen cell, and the effect of immuno-regulation on IL-2and IL-10of spleen cell were determined by ELISA analyze.
Result:The appearance of ADSCs which undergone clone screening has been homogeneous. The appearance were the doubling time was shorten, the change of growth curves was not obviously after many generation, growing-power after long-term culture was vigorous and stable, and the surface marks CD49d, CD44were high expressed while the surface marks of CD34was low in both the control group and the the experimental group examined by FCM.Rat ADSCs was successfully differentiationed into iPS by the21d culture system in vitro. The expression of ALB、 AFP and CK18mRNA was determined fortified by degrees at7thd、14thd and21thd. The protein ALB was determined by immunohistochemisty. It was occurred and persistenced of Metabolism of ammonia and urea synthesis after9th-12th d. It was shown that both Lewis rat ADSCs and iPS can down-regulate the immunologic reaction of the co-culture of DA and Lewis rat spleen cell indifferently, and the immuno suppression effect was enhanced by concentration of Lewis rat ADSCs and iPS.
Conclusion:The clone screening purification method is effective、economical purification method of ADSCs. Rat ADSCs was successfully differentiationed into iPS in vitro after procedure-induction. The method of induction can provide a valid and reliable source of seed cells for hepatocytes transplantation, the clinical treatment of liver tissue engineering. Rat ADSCs was successfully differentiationed into iPS in vitro, the iPS have the characteristics both function of Liver cell regeneration and immunosuppression effect.
Part Three the influence to the immunity rejection of the orthotopic liver transplantation model in rats induced by the hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells.
Objectives:After injected the hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells by portal vein in the process of the model of the orthotopic liver transplantation in rats of the model with high-lever rejection (DA-Lewis). To investigate the homing position in the rat liver, and the influence on the liver function the degree of immunity rejection survival time. The safety、 efficiency and mechanism of the hepatic-like cells differentiation from adipose-derived mesenchymal stem cells in the orthotopic liver transplantation model in rats were discussed.
Methods:
1. The model of the orthotopic liver transplantation in rats with high-lever rejection (DA-Lewis) was established with Kamada two-cufft technique, divided the DA and Lewis rat into4group(31recipient/group) randomizing,
1) The group A (reject group):operation without any other management
2) The group B (the immunosuppressant group) operation and take FK506capsule orally (0.15mg/kg/d by Gastric lavage qdX14d begin after the second day of operation)
3) The group C:operation and injected the adipose-derived mesenchymal stem cells by portal vein in the process of operation.
4) The group D:operation and injected the hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells by portal vein in the process of operation.
2. The general condition of the luster of eyes、rat hair、activity and mental state of each experiment rat were observed and recorded;6rat were left to observe the survival time each group;
3.6rats were killed at the4th、7th、10th、14th day after operation each group. The liver function and cytokine (IL-2, INF-y and IL-10and IL-4) were detected. Cytokine were detected by the ELIS A method.
4. Brdu was used to mark the adipose-derived mesenchymal stem cell and hepatic-like cells differentiationed from adipose-derived mesenchymal stem cell in vitro. Identification of the mark of Brdu by fluorescence immunohistochemical, and detected the homing position of the cell in vivo marked by Brdu with fluorescence immunohistochemical methods.
5. The hematoxylin-eosin staining of rat liver was taken to observe the rejection activity index.
6. The mixed lymphocytes culture test was taken at30th after operation. CD4+、CD8+were measuresed by flow cytometry.
Result:
1. The animal model were built up in all4group rat under the same condition, including weight and operation time without obvious difference (P>0.05), the survival rate is94.66%.(124/131).
2. The adipose-derived mesenchymal stem cell and the hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells marked with Brdu began to appear after3days of the operation, which nucleus positive of red dye by fluorescence immunohistochemical scattered in liver tissue, and some parts of the positive cells to collect and dense around the blood vessels and abbacy. With the extension of time, gradually formed shape distributings oven.
3. Group B, C, D of median survival time is obviously higher than that of the group A (control group)(P<0.05), group D of median survival time was the longest in all group, and were longer than C group,but is no statistical difference compared with group B.
4. Liver function of ALT, AST, TBIL of group A rising sharply, but B、C、D group gradually declined in the postoperative day7th and10th, In the postoperative day14th, animals of group A has completely death, data lost; liver function of group B、C、D further recovery, group B、D recovery degree is better than group C, group D better than B group in some data(ALT,AST)(P<0.05).
5. The lever of IL-2、INF-gamma of group A higher than other group significantly (P<0.05), while level of the group B、C、D gradually declined in the postoperative day7th、10th and14th, the lever of group B and D declined more than group C (P<0.05); the lever of IL-10and IL-4of group A was significantly lower than the other group (P<0.05), but group B、C、D gradually raised, group B and D raised more than group C (P<0.05).
6. Rejection activity index of group A significantly higher than group B、C、D in the postoperative day7th and10th(p<0.05), group B、D have down trend with time prolonged, rejection index of group C was high than group B and D (p>0.05).
7. The mixed lymphocytes culture test:OD value of group B, C and D reduce compared with the positive control group (A) when the stimulate cell come from spleen cells of DA rat. OD value of group B, reduce compared with the positive control group when the stimulate cell come from spleen cells of SD rat. But the OD value of group C、D was still high as the positive control group (p<0.01). It showed that the immune tolerance of recipient was specificity.
8. The ratio of CD4+/CD8+of Group A rise progressively after operation,which significantly higher than group B, C and D (P<0.05);the ratio of group B, C and D rise slowly from3th to10th and fall down from14th after operation,but there's no statistical difference.
Conclusion:
1. Rat liver transplantation high-lever rejection model was established successfully.
2. Liver fibrosis and steatosis may appear when adipose-derived mesenchymal stem cells injected by portal vein to intro, which rent differentiation of it to liver cell, the therapy of adipose-derived mesenchymal stem cells only may not enough to protect the body from immune rejection.
3. Specificite immune tolerance was successfully established of recipient deal with the hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells by portal vein.
4. The hepatic-like cells differentiationed from adipose-derived mesenchymal stem cells play a certain role of tolerance establish, without using immunosuppressive agents.
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