免疫隔离技术在异种肝细胞移植中的应用

目的探讨微囊化猪肝细胞腹腔内移植对药物性肝衰大鼠的治疗作用，观察移植大鼠存活率、肝功能的变化。方法以海藻酸钠体外包裹经胶原酶灌注法分离的乳猪肝细胞，以SD大鼠为受体，D-氨基半乳糖腹腔内注射诱导大鼠急性肝衰竭，48h后将微囊化的猪肝细胞移植于大鼠腹腔内，观察移植大鼠存活率、绘制生存曲线，并测定肝功能的变化。结果腹腔内肝细胞移植可显著改善大鼠肝功能，转氨酶及胆红素均较对照组明显下降（P〈0．05）。与裸肝细胞移植组相比．微囊化肝细胞移植组1周存活率（78．6％ vs 66．7％）及2周存活率（42．9％ vs 25．0％）均显著提高（P〈0．01）。结论对异种肝细胞经微囊化处理后移植治疗急性肝衰大鼠，可给予肝功能代谢支持，提高移植治疗效果。     

微囊化同系、同种异体、异种肝细胞腹腔移植对急性肝衰的治疗作用

目的 探讨微囊化同系、同种异体、异种肝细胞腹腔移植对急性肝衰（ａｃｕｔｅ ｌｉｖｅｒ ｆａｉｌｕｒｅ,ＡＬＦ）的治疗作用。方法 切除大鼠９０％的肝脏,建立急性肝衰模型;分离纯化包裹同系Ｗｉｓｔａｒ鼠,同种异体ＳＤ鼠及异种豚鼠的肝细胞。移植到受体模型鼠腹腔内,观察微囊存活情况及肝衰鼠的生存率和生化改变。结果 在４８ｈ内对照组存活率仅１５．４％,而同系肝细胞组为７８．６％,ＳＤ微囊组７３．３％,ＧＰ微囊组６２．５％,７ｄ后对照组大鼠全部死亡,而其余３组存活率分别为５７．１％,、５３．３％、５０．０％。微囊化肝细胞组ＡＬＴ和ＴＢＩＬ的水平明显降低,与对照组相比差异有非常显著性（Ｐ〈０．０１）。结论 微囊包裹同种异体和异种肝细胞腹腔移植而不使用免疫抑制剂,能够阻止免疫排斥反应,给予肝功能代谢支持,阻止急性肝衰模型鼠的死     

共微囊化异种肝细胞胰岛联合移植治疗急性肝衰

目的　探讨共微囊化异种肝细胞胰岛联合移植对急性肝衰的治疗作用。方法　D 氨基半乳糖诱导小鼠急性肝衰 ;Seglen法分离大鼠肝细胞 ;Sutton法分离胰岛 ;自制双喷头成囊器制备微囊。比较生理盐水、自由肝细胞移植、微囊化肝细胞移植、共微囊化肝细胞胰岛联合移植 4组间存活率、体重、谷丙转氨酶、血清白蛋白、总胆红素、血糖等指标的变化情况。结果　共微囊化肝细胞胰岛联合移植组存活率达 10 0 % ,组间差异有显著性 (P <0 .0 5 ) ,各项生化指标也明显占优。肝细胞与胰岛 β细胞可在微囊内融合。 结论　共微囊化肝细胞胰岛联合移植对氨基半乳糖诱导的急性肝衰有最佳的治疗作用     

微囊化罗非鱼肝细胞移植治疗大鼠急性肝功能衰竭

目的 观察微囊化罗非鱼肝细胞移植对急性肝衰大鼠的治疗作用.方法 D-氨基半乳糖制备的肝衰大鼠随机分为4组:微囊化组,裸肝细胞组、空微囊组及生理盐水组,腹腔内分别植入微囊化罗非鱼肝细胞、裸肝细胞、空微囊及生理盐水.比较各组间1周死亡率.移植后动态检测各组大鼠总胆红素,比较其差异,动态观察移植物的病理变化.结果 移植后1周内微囊化组的存活率较高(57.9%),与其他各组比较差异有统计学意义(P＜0.05).移植后48 h,微囊化组总胆红素(6.21±0.86)μmol/L明显较低,与其他各组差异有统计学意义(P＜0.01).移植后1周,微囊化组可以收集到形态完整,没有粘连的微囊,其内的肝细胞尚部分保持存活.结论 微囊化罗非鱼肝细胞移植能改善肝衰大鼠的肝功能、提高存活率.     

药物性肝衰大鼠脾内移植异种肝细胞治疗前后的CD4，CD8的变化及意义

为探讨异种肝细胞脾内移植治疗大鼠药物性肝衰的意义，观察大鼠肝功能、存活率及ＣＤ４，ＣＤ８在免疫排斥反应中的作用。笔者以豚鼠肝细胞为供体，ＳＤ大鼠为受体，Ｄ-氨基半乳糖(１９.５ml/㎏)腹腔内一次性注射，制作肝衰模型。４８h后将微囊化的游离豚鼠肝细胞移植于大鼠脾脏内。以豚鼠裸肝细胞移植及生理盐水脾脏注射为对照。移植后１４d测定存活率和肝功能，检查病理切片及ＣＤ４，ＣＤ８的变化。结果示微囊化肝细胞移植组存活率８０.0％，明显高于生理盐水组（２５.0％）和裸肝细胞移植组（７０.0％）（分别为P<０.０１和P<０.０５）。微囊肝细胞移植组１４ｄ总胆红素和ＡＬＴ，明显低于生理盐水组和裸肝细胞组。移植后７２ｈ和１４ｄ分别测定受体脾脏ＣＤ４，ＣＤ８淋巴细胞，移植后７２ｈ，１４ｄ微囊化肝细胞组、裸肝细胞组CD4，CD8均呈阳性。提示：大鼠药物性肝衰微囊化肝细胞脾内移植后可维持受体存活１４ｄ，体液和细胞免疫均参与排斥反应。肝细胞微囊化处理可延迟细胞免疫的发生时间。     

微囊化大鼠肝细胞移植的组织学研究

目的 研究微囊化大鼠肝细胞腹腔移植后的存活情况以及组织学改变。方法 用二步法胶原酶门－腔静脉灌注法分离Wistar大鼠肝细胞,用Percoll梯度分离液纯化,海酸钠－氯化钡法微囊化包裹肝细胞,分别行腹腔注射移植,将纯化的肝细胞移植至SD大鼠体内（第1组）、微囊化包裹的肝细胞移植至SD大鼠体内（第2组）及Wistar大鼠体内（第3组）,观察移植后各组不同时间肝细胞存活率及其组织学变化。结果 （1）移植后第4、7d,各组间肝细胞存活率的差异均有显著性（P＜0.01）;移植后第14d,第2组与经3组间肝细胞存活率的差异无显著性;（2）移植后第4d开始,微囊周围出现纤维增生现象,第2组较第3组明显。结论 微囊化可为移植的肝细胞提供免疫屏蔽作用,从而提高肝细胞移植的存活率;微囊周围纤维增生可影响肝细胞的存活率。     

微囊化猪肝细胞移植治疗大鼠急性肝衰竭

目的探讨微囊化猪肝细胞移植(HTx)治疗大鼠急性肝衰竭(ALF)的效果。方法采用原位胶原酶循环肝灌注法分离中国实验用小型猪肝细胞,海藻酸钠-氯化钡法微囊化。应用 D-氨基半乳糖(D-gal)1．2 g／kg体重腹腔内注射制作SD大鼠ALF模型。ALF大鼠随机分为3组。注药24 h后分别将PRMI 1640培养液2 ml腹腔注射为对照(I组)、2 ml游离猪肝细胞(2×107个／ml)腹腔内移植(Ⅱ组),以2 ml微囊化猪肝细胞腹腔内移植(2×107个／ml,Ⅲ组)。观察移植后大鼠14 d存活率、谷丙转氨酶(ALT)、总胆红素(TB)、血氨(NH3)的改变和肝脏的病理变化。结果肝细胞移植后大鼠14 d存活率:I组为20．0％(5／25),Ⅱ组为66．7％(16／24),Ⅲ组为76．0％(19／ 25),3组间差异有统计学意义(P<0．05)。移植后第1天I组ALT、TB和移植前相比差异无统计学意义,Ⅱ、Ⅲ组ALT、TB下降。第4天3组ALT、TB均继续下降,Ⅱ、Ⅲ组低于I组(P<0．05), Ⅱ、Ⅲ两组间差异无统计学意义(P>0．05)。移植后第7天,ALT进一步下降,3组间差异有统计学意义(P<0．05);TBⅢ组显著低于Ⅱ组和I组(P<0．05),Ⅱ组低于I组但差异无统计学意义 (P>0．05)。移植后第14天,肝功能均明显恢复,ALT、TBⅡ、Ⅲ组均低于I组(P<0．05),Ⅱ、Ⅲ两组间差异无统计学意义(P>0．05)。各组治疗前后NH3有所改善,但各时间段及各组之间差异无统计学意义(P>0．05)。微囊组和游离肝细胞组移植后肝脏病理损害修复较快,而阴性对照组肝脏再生修复较差。结论微囊化猪肝细胞腹腔内移植可以提高药物诱导急性肝衰竭大鼠的存活率,改善急性肝衰竭大鼠的肝功能,有利于受体受损肝脏的再生修复,但对降低血氮的效果不明显。     

微囊化大鼠肝细胞腹腔移植后细胞形态结构,功能及其对急？…

目的 研究微囊化大鼠肝细胞同种异体腹腔移植后微囊细胞形态结构功能及其对急性肝衰竭的治疗作用。方法 采用微囊包被技术微囊化ＳＤ大鼠肝细胞后，移入急性肝衰型Ｗｉｓｔｒａ大鼠腹腔内，对照组为裸肝细胞和空囊。结果 三组大鼠的存活率；微囊化组７０．８％（１７／２４），裸细胞组３３．３％（８／２４）和空微囊组１６．６％（５／３０），微囊化组存活率明显高于其它两组（Ｐ〈０．０１）；移植后，微囊化组血糖升高和胆红     

微囊化异种肝细胞移植对大鼠爆发性肝衰竭治疗作用的研究

目的　探讨微囊化异种肝细胞移植对暴发性肝衰竭 (FHF)大鼠的治疗作用。方法　ACP微囊化处理原代豚鼠肝细胞 ,于诱发FHF前 2 4h移植到大鼠腹腔内大网膜附近 ,采用 90 %肝切除法诱发FHF。在FHF诱发后 2 4h取静脉血检测大鼠肝功能谷丙转氨酶 (ALT ) ,清蛋白 (ALb) ,总胆红素(TB) ,血糖 (BS)和凝血酶原时间 (PT)等指标 ;取腹腔移植入的微囊进行组织学检查观察细胞形态、功能及数量的变化 ;动态全程观察大鼠生活质量及存活时间并绘制生存曲线。结果　微囊化肝细胞移植组大鼠生活质量明显改善 ,出现共济失调等中枢神经系统症状及腹水的时间明显延长。微囊化肝细胞移植组大鼠的ALT ,Alb ,TB ,BS和PT均明显改善并且与对照组均有差异的显著性 (P <0 .0 5 )。微囊化肝细胞移植组大鼠平均生存时间为 60h ,空白对照组为 2 2h(P <0 .0 1)。组织学检查 :移植后 72h开腹 ,约有 80 %微囊呈无粘连游离状态 ,镜下见微囊周围纤维化率约为 8% ,囊内肝细胞形态正常。未微囊化肝细胞移植组则很少见到正常的肝细胞 ,且周围有大量淋巴细胞浸润。结论　对异种肝细胞进行微囊化处理后移植治疗FHF大鼠 ,可以减少移植排斥反应 ,提高治疗效果 ,是一种治疗FHF的较好方法。     

长期冻存对微囊化肝细胞形态结构功能的影响及在急性肝功能衰竭中的治疗作用

目的　研究长期液氮冻存对微囊化肝细胞结构和功能的影响。方法　将３ 组 Ｓ Ｄ 大鼠微囊化肝细胞分别用液氮冻存１５ 、３０ 、６０ 天后,检测肝细胞活率、结构、功能及其对急性肝衰的初步治疗效果。结果　冻存后３ 组的细胞活率分别为（７２ ．７５ ±３ ．９） ％ 、（７０ ．０７ ±５ ．６８） ％ 、（７２ ．３９ ±１３ ．７５） ％ ;肝细胞的形态结构正常;肝细胞葡萄糖６磷酸酶、糖原及白蛋白染色正常,培养４ 天后各组的总蛋白和尿素氮的合成分泌功能与新鲜微囊化肝细胞差异无显著性（ Ｐ＜ ０ ．０１） 。冻存组（１５ 天组） 腹腔移植能明显提高急性肝功能衰竭模型大鼠的存活率５５ ％ （１１／２０） ,与裸细胞组（３３ ％ ,８／２４） 及空囊组（１６ ．６ ％ ,５／３０） 相比差异有显著性（ Ｐ＜ ０ ．０１） 。结论　液氮可良好的保存微囊化肝细胞的形态结构及功能;冻存微囊化肝细胞腹腔移植有良好的肝功能支持作用。     

Three different hepatocyte transplantation techniques for enzyme deficiency disease and acute hepatic failure

The effects of three different techniques of hepatocyte transplantation were investigated: transplantation of free hepatocytes into the spleen and intraperitoneal transplantation of microcarrier-attached hepatocytes or of microencapsulated hepatocytes. The liver-supportive functions of these transplanted hepatocytes were analyzed using either the Gunn rat (hyperbilirubinemia) or rats with acute liver failure. In the Gunn rat intraperitoneal transplantation of microcarrier-attached hepatocytes resulted in a significant reduction of plasma bilirubin for 28 days whereas intraperitoneal transplantation of microencapsulated hepatocytes was ineffective notwithstanding immunosuppression by cyclosporin A. Intrasplenic hepatocyte transplantation was only effective in reducing plasma bilirubin for 14 days. During acute liver failure, liver support was achieved temporarily by hepatocyte transplantation in the spleen, by intraperitoneally transplanted microcarrier-attached hepatocytes, and by microencapsulated hepatocytes to equal extents, the microencapsulated hepatocytes being the least effective after 8 h of liver ischemia.     

急性肝衰竭大鼠载肝细胞生长因子纳米粒子肝细胞移植后有丝分裂指数和Ki-67表达变化及意义

目的 探讨急性肝衰竭(ALF)大鼠载肝细胞生长因子(HGF)的聚乳酸-氧-羧甲基壳聚糖(PLA-O-CMC)纳米粒子肝细胞移植后有丝分裂指数和Ki-67抗原的表达变化及意义.方法 D-氨基半乳糖腹腔内注射制作大鼠ALF模型,48 h后分别将Ⅰ型胶原(Ⅰ组)、PLA-O-CMC纳米粒子(Ⅱ、Ⅳ组)、载HGF的PLA-O-CMC纳米粒子(Ⅲ组)培养的大鼠肝细胞(均为5×107个,5 ml)移植到ALF大鼠腹腔内.以每日静脉注射HGF 10μg/kg×7 d(Ⅳ组)、5 Ml RPMI 1640腹腔内注射(Ⅴ组)作为对照.观察其14 d存活率、血清及肝组织HGF浓度、肝组织病理及有丝分裂指数(MI)、Ki-67变化.结果移植后14 d Ⅰ～Ⅴ组ALF大鼠的存活率分别为50.00％、68.75％、81.25％、75.00％、18.75％.各纳米移植组高于V组.Ⅲ组3 d时肝组织HGF浓度最高,平均为5882.91 μG／L.Ⅲ组肝组织结构恢复更快,5 d时平均MI 10.20％0、7 d时平均MI 10.20‰、7 d时Ki-67平均标记指数16.8‰,均高于Ⅴ组.结论 应用载HGF的PLA-O-CMC纳米粒子培养的大鼠肝细胞腹腔内移植治疗ALF大鼠能促进肝再生相关抗原表达.

Abstract：

Objective To evaluate the change and significance of Ki-67 antigen expression follow ing hepatocyte transplantation using hepatocyte growth factor (HGF) loaded polylactic acid-O-carboxymethylchitosan (PLA-O-CMC) nanoparticles in rats with acute liver failure (ALF). Methods ALF models of rats were established by D-galactosamine intraperitoneal injection. Rat hepatocytes type Ⅰ collagen suspension (group Ⅰ ),rat hepatocytes co-cultured with PLA-O-CMC nanoparticles ( groups Ⅱ ,Ⅳ ) and rat hepatocytes co-cultured with HGF loaded PLA-O-CMC nanoparticles ( group Ⅱ ) (5 × 107 cells each group,5 ml) were transplanted into the abdominal cavity of SD rats at 48 h after D-Gal injection. Intravenous injection of HGF ( 10 μg/kg for 7 days) ( group Ⅳ ) and RPMI 1640 injection (group Ⅴ ) were used as the negative groups. The 14th-day survival rate of rats,pathological change and mitotic index of liver,Ki-67 antigen labeling index of ALF rat livers were observed. Results The 14th-day survival rate in groups Ⅰ -Ⅴ was 50.00％,68. 75％,81.25％,75.00％,18.75％,and that in nano-transplanted groups was higher than in group Ⅴ. At the 3rd day,the concentration of HGF in liver tissue of group Ⅲ,average 5882. 91 μg/L was the highest. The hepatic lobules structure in group Ⅲ recovered faster. The average mitotic index in group Ⅱ at the 5th day was 10. 20‰ and the average Ki-67 labeling index at the 7th day was 16. 8‰,which were all higher than in group Ⅴ. Conclusion Intraperitoneal transplantation of HGF loaded PLA-O-CMC nanoparticle-attached hepatocytes for treatment of ALF can promote the liver regenerationassociated antigen expression.     

Intrasplenic transplantation of encapsulated hepatocytes decreases mortality and improves liver functions in fulminant hepatic failure from 90% partial hepatectomy in rats

BACKGROUND: Encapsulated cell therapy might be a promising approach to enable cell transplantation without immunosuppression. This study investigates the viability and hepatic function of hepatocytes encapsulated with alginate/poly-L-lysine in vitro and the effect of the intrasplenic transplantation of cultured encapsulated hepatocytes on survival in 90% hepatectomized rats as a preliminary step toward allogeneic hepatocyte transplantation without immunosuppression. MATERIALS AND METHODS: Rat hepatocytes were isolated and encapsulated using alginate/poly-L-lysine. Encapsulated hepatocytes were cultured for 28 days to measure cell viability, liver function, and morphology. Rats were treated with a 90% partial hepatectomy and then immediately underwent the intrasplenic transplantation of the cultured encapsulated hepatocytes, the capsule alone, or the allogeneic hepatocytes without the capsule. The survival rate, liver function, and cell morphology were assessed after transplantation. RESULTS: The cultured encapsulated hepatocytes maintained their viability and showed better metabolic activity than day 0 cultured encapsulated hepatocytes. The encapsulated cells strongly expressed albumin and were positive for periodic acid-Schiff staining. Electron microscopy demonstrated that the microencapsulated hepatocytes retained the structural elements of hepatic cytoplasm and nuclei. Intrasplenic transplantation of the encapsulated hepatocytes increased the survival rate and improved the hepatic function. Encapsulated hepatocytes transplanted into rat spleen survived well and retained their hepatic function. Moreover, dramatic liver regeneration was observed 48 hr after transplantation in the group that received intrasplenic transplantations of encapsulated hepatocytes. CONCLUSIONS: The intrasplenic transplantation of cultured encapsulated hepatocytes improved the survival rate of an acute liver failure rat model induced by a 90% partial hepatectomy.     

Cell-free supernatant from hepatocyte cultures improves survival of rats with chemically induced acute liver failure

Current support or replacement therapies for fulminant hepatic failure are frequently inadequate. Hepatocyte transplantation has been found to permit or facilitate recovery from chemically induced liver failure in rats. The mechanisms are unclear, but function of the transplanted cells or stimulation of host liver regeneration by factors released from the cells could improve survival; there is evidence from experiments by other investigators using cell fractions to support the latter hypothesis. We compared intact cells and the supernatant from cultured liver cells for their influence on the survival of Fischer male rats with d-galactosamine (d-Gal) induced acute liver failure (ALF). All treatments were given 20 to 24 hr after poisoning. Cell supernates were injected intrasplenically (sp), intraperitoneally (ip), or intravenously (iv). Liver cells (2 × 10⁷) suspended in Hanks' solution were injected intrasplenically. Untreated rats and rats treated with Hanks' solution or culture medium alone had a 94–100% mortality, with all deaths occurring between 38 and 74 hr after poisoning. Improved survival was seen in all experimental groups: 47% of the rats receiving intact liver cells survived; 50, 55, and 62% of the rats receiving cell-free supernate by the sp, ip, or iv routes, respectively, survived. A chronologic electron micrographic study of livers from rats serially sacrificed in parallel experiments showed that recovery from morphological changes induced by d-Gal occurred in treated rats. These studies demonstrate that intact hepatocytes are not required to improve survival in rats with drug-induced ALF. Improved survival may be achieved by factors liberated by cultured hepatocytes that enhance the regeneration of the damaged liver. Supernatant from cultured liver cells may have a therapeutic potential in acute hepatic failure.     

微囊化肝细胞移植对急性肝衰竭大鼠肝组织Smac及细胞色素cmRNA表达的影响

目的动态观察微囊化肝细胞腹腔移植对急性肝衰竭大鼠肝组织Smac／DIABLO及细胞色素c（cyt—c）mRNA表达的影响。方法D-氨基半乳糖诱导大鼠急性肝衰竭模型,并设微囊化肝细胞移植组、裸肝细胞移植组和对照组。RT—PCR法测定大鼠肝脏凋亡指标Smac／DIABLO和cyt—cmRNA表达情况。样本比较采用重复测量的多元方差分析或单因素方差分析,组间比较采用t检验。结果急性肝衰竭大鼠肝组织中Smac／DIABLO及cyhmRNA的表达较对照组明显增加（F值为4．345、14．821、47．565、42．178、62．961,P值均〈0．05）。裸肝细胞移植组和对照组肝组织中的Smac／DIABLO和cyt—cmRNA表达在48h最高,而微囊化肝细胞移植组的峰值略微前移,在24h最高。结论Smac／DIABLO及cyt—CmRNA的表达随着急性肝衰竭的进程而波动,可以较好地判断肝细胞凋亡情况。