组织工程化肝脏体外构建及体内移植治疗肝衰竭的实验研究
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摘要
肝脏疾病严重威胁着人类身体健康,目前可行的治疗方案主要有以下两种:肝脏移植与肝细胞移植。这两种治疗方式虽能明显改善患者症状,但也存在很多问题:如肝脏器官移植受到供体器官匮乏、高额费用和长期需要使用免疫抑制剂的限制;肝细胞移植后机体易于产生明显免疫排斥反应,从而导致移植最终失败。因此,如何寻求新的治疗技术和方法来解决上述问题,逐渐引起国内外研究人员的广泛关注,组织工程学的出现为肝病的治疗带来了希望。
组织工程作为一门以“制造和替代人体组织和器官”为主要目的的交叉学科,在过去二十余年发展过程中,先后在皮肤、软骨、神经、血管等组织再造领域取得明显进展,部分组织工程产品已进入市场。针对肝脏的组织工程研究近年来已成为组织工程的热点领域,发展尤为迅速,并取得了一系列突破性进展,研究人员利用胶原、壳聚糖、海藻酸钠等为支架,先后成功在体外再造了工程化肝脏组织。尽管如此,肝脏组织工程研究仍面临着许多问题和挑战,例如:如何提高再造肝脏组织的血管化能力以及体内移植后如何避免宿主的免疫排斥反应等。
本论文分别以胶原/Matrigel和海藻酸微囊作为细胞支架材料,在体外构建了三维立体肝脏组织片层以及微囊化肝细胞,并通过在肝细胞-材料复合体中进一步添加血管内皮细胞的方式,促进再造肝脏组织的血管化。研究结果表明,胶原/Matrigel是肝细胞良好的支架材料,肝细胞在材料内能够形成肝脏组织,并且发现在肝组织片层中有管腔样结构出现,证实了血管内皮细胞对肝细胞功能的促进作用。在微囊化肝细胞研究方面,本论文基于微囊的免疫隔离作用,用微囊共包裹肝细胞与血管内皮细胞并对肝衰竭大鼠进行移植治疗,通过对大鼠死亡率和肝功能的检测后发现,微囊共包裹肝细胞与血管内皮细胞可以明显改善肝衰竭大鼠的肝功能,血管内皮细胞的加入对于微囊化肝细胞发挥生物学功能起到了积极作用。
本论文从三维立体肝脏组织再造到微囊化肝细胞研究,主要研究内容包括以下五个部分:
第一部分:基于胶原/Matrigel的组织工程化肝脏片层体外构建研究
本部分实验首先通过改良的胶原酶消化法,原代分离培养鉴定了大鼠肝细胞,并将其培养于胶原/Matrigel基质环境中,构建了组织工程化肝脏片层。通过观察细胞形态、检测细胞间的连接以及肝细胞的白蛋白分泌,并与胶原三明治结构相比较,研究发现构建的肝脏片层中细胞聚集生长,状态良好,并且表达actin和E-cadherin连接蛋白,白蛋白的分泌也显著高于三明治结构,说明胶原/Matrigel基质的三维立体环境更有利于肝细胞的生长和功能的发挥,体外再造的三维立体肝脏组织对于肝脏疾病的治疗及相关基础性研究具有重要作用。
第二部分:基于胶原/Matrigel的肝细胞与血管内皮细胞三维立体共培养体系的建立
本部分实验将肝细胞和血管内皮细胞共培养于胶原/Matrigel基质环境中,构建了肝细胞/血管内皮细胞片层,观察片层中细胞的形态、对细胞进行H.E.染色和免疫荧光染色,并且在体外培养1、3、5、7、14d时,更换培养液,收集培养上清,检测上清培养液中的白蛋白分泌水平。结果可见肝细胞/血管内皮细胞片层中的细胞逐渐聚集生长,组织学检测发现血管内皮细胞迁移聚集,形成管腔样结构。与单纯肝细胞片层相比,肝细胞/血管内皮细胞片层组培养液中的白蛋白分泌水平明显高于单纯肝细胞组。
第三部分:微囊材料的制备及其生物学性能评价
在海藻酸钠-聚赖氨酸-海藻酸(APA)微囊的制备基础之上,用Ba2+代替Ca2+,制备出海藻酸钡-聚赖氨酸-海藻酸(BPA)微囊,并采用机械震荡法测定APA和BPA微囊的机械强度和体外模拟体液法长期培养测定APA和BPA微囊的渗透压强度,结果表明,BPA微囊比APA微囊具有较高的机械强度和长期稳定性。采用体内移植实验测定微囊在肌肉内的移植强度,结果表明,APA和BPA微囊纤维化程度不明显。但是,APA微囊移植后体积膨胀率较BPA微囊略大,且成多边形态,而BPA微囊的形态多近似球形。采用腹腔移植实验测定微囊的生物相容性,结果表明,大鼠腹腔移植BPA微囊和APA微囊后第7、14、28和56d时,回收的微囊体积均占植入微囊体积的90%以上,光镜下见回收的两种微囊绝大部分仍呈完整的球形,表面光滑,无细胞包裹,纤维化程度不明显。
第四部分:微囊化肝细胞生物学性能的体外评价
制备BPA微囊化肝细胞,通过对微囊化肝细胞进行组织学检测、Live/Dead染色以及测定白蛋白分泌水平,体外评价了肝细胞的活性和功能,结果发现,在培养过程中光镜可见微囊和肝细胞均保持完整形态;组织学染色与Live/Dead染色结果显示微囊内大多数肝细胞仍保持较好活性,肝细胞在BPA微囊内培养7d内能够维持稳定的白蛋白分泌水平,表明BPA微囊是一种适合于肝细胞生长的微囊材料。
第五部分:微囊共包裹肝细胞和血管内皮细胞移植治疗大鼠肝衰竭的实验研究
采用D-氨基半乳糖腹腔注射法建立大鼠急性肝衰竭模型,经腹腔移植空微囊、游离的肝细胞、微囊化肝细胞和共包裹肝细胞和血管内皮细胞四个实验组治疗肝衰竭大鼠,于移植后的1d、3d、5d、1w、2w、3w、4w眼眦静脉取血,测血液中谷丙转氨酶(ALT),谷草转氨酶(AST),白蛋白(ALB)水平,通过对回收微囊的形态学检测和肝衰竭大鼠肝功能检测发现,移植4w后回收的BPA微囊形状完好,纤维化程度不明显,H.E.染色结果表明移植4w后囊内的肝细胞形态依旧完整,充分说明了BPA微囊本身的免疫隔离作用保护了肝细胞在移植体内的功能发挥。移植4w内共包裹组明显改善了肝衰竭大鼠的肝功能,其中移植后2到4周,共包裹组大鼠的ALT、AST与ALB水平显著优于其他各组;此外,共包裹组肝衰大鼠死亡率降低,与单纯包裹肝细胞组相比,其第四周死亡率由50%减少到20%,表明共包裹组中血管内皮细胞维持了肝细胞的活性和功能,延长了肝细胞的存活时间,对大鼠肝衰竭的治疗起到了促进作用。
综上所述,本研究首先在体外构建了组织工程化肝细胞片层,并研究了胶原/Matrigel凝胶环境对于肝细胞的活性和功能发挥的促进作用,在此基础上,利用本支架材料体系探讨了肝细胞与血管内皮细胞的相互作用以及血管内皮细胞对肝细胞功能的促进作用。基于上述细胞生物学研究基础,并利用以往已经建立的微囊化人工细胞的研究平台,进一步研制了具有免疫隔离作用的BPA微囊材料,体外评价了BPA微囊化肝细胞的生物学功能,并共包裹了肝细胞和血管内皮细胞,移植治疗大鼠急性肝衰竭,结果发现由于微囊可以避免免疫排斥反应,共包裹的细胞对肝衰竭大鼠有明显的治疗作用,血管化微囊化肝细胞对于肝衰竭临床治疗具有潜在的应用前景。
Hepatopathy is one of the most serious global diseases that harm human health. Now,liver transplantation and hepatocyte transplantation are feasible choices to treat liver diseases. However, procedures of liver transplantation are limited by the shortage of donor organs, high cost, and lifelong dependence on immunosuppression. Hepatocytes transplantation will bring the organism a strong post-transplant immune rejection, which makes transplantation ultimately fail. Therefore, it is urgent to find new techniques and methods for the above problems. The development of tissue engineering brought new hope for the therapy of liver diseases.
Tissue engineering, as an interdisciplinary science, which purpose is constructing and replacing human tissue and organ, has made great progress in the skins, cartilage, nerve, blood vessel in the past 20 years.The development of liver tissue engineering is fast recently; meanwhile in this field great breakthroughs had been made. However, many problems and challenges are existed, for example, how to improve the vascularization of reconstructed liver tissue and to avoid host immunologic rejection after in vivo transplantation.
This study used collagen/Matrigel and alginate microcapsules as liver tissue engineering scaffolds respectively. Based on this, we promoted the vascularization of engineered liver tissue through adding the vascular endothelial cells in tissue engineered hepatocyte/vascular endothelial cell sheets. Our results demonstrated that vascular endothelial cells could promote the hepatocyte viability and function. In other hand, based on the merit of avoiding immunological rejection, we prepared microencapsules as another tissue engineering material. Then we transplanted the co-encapsulated hepatocyte and vascular endothelial cell into rats to treat hepatic failure, vascular endothelial cell has improved the biological function of microencapsulated hepatocytes.
The main study contents from the reconstruction of the 3D tissue to the evaluation of the microencapsulated hepatocytes include the following five parts:
1. Construction of 3D engineered hepatocyte sheet based on collagen/Matrigel in vitro
In this part, we firstly isolated the rat primary hepatocyte by the improved collagenase digestion, which were cultured in the collagen/Matrigel environment for the construction of engineered hepatocyte sheet. Compared with the collagen sandwich structure, hepatocytes in the engineered hepatocyte sheet grow more aggregative and express the more actin, E-cadherin and albumin by assessing the cell morphology, cell-cell conjunction and secretion of albumin. These results show the 3D collagen/Matrigel environment is good for the growth and function of hepatocytes. The in vitro reconstruction of the 3D liver tissue played important roles in the treatment of liver disease and the relative basic study.
2. Construction of 3D co-cultured the hepatocyte/vascular endothelial cell system based on collagen/Matrigel in vitro
In this part, we co-cultured the hepatocyte and vascular endothelial cell in the collagen/Matrigel environment and constructed the hepatocyte/vascular endothelial cell sheet. By assessing the cell morphology, H.E. staining, immunofluorescence and secretion of albumin, we found the vascular endothelial cells in hepatocyte/vascular endothelial cell sheet migrate together to form a vessel like structure. The albumin secretion of hepatocyte/vascular endothelial cell sheet was higher than the hepatocyte sheet, after 1, 3, 5, 7, 14 days culture in vitro.
3. Preparation of microencapsule and evaluation of their biological function
In this part , we used barium ion to substitute calcium ion to form Ba–alginate–polylysine–alginate(BPA) microencapsules based on the alginate–polylysine–alginate(APA) microencapsules. And then we tested the mechanical strength of APA microencapsules and BPA microencapsules with mechanical shaking experiments, and the osmotic pressure of two types mincroencapsules was measured by long-time culture in simulated body fluids. The results showed that BPA microencapsules have higher mechanical strength and long-time stability than APA microcapsules. We measured the transplantation strength of the microencapsules by transplanting the microencapsules into the muscle of rats. The results showed the APA and BPA microencapsules have little fibrosis, while APA microencapsules were out of shape and more swelled than BPA, BPA microencapsules were still spherical. After that, we measured the biocompatibility of the microencapsules by transplanting the microencapsules into the peritoneal cavity. After being transplanted for 7,14,28,56 days, the recovery was up to 90%, the recovered two types of microcapsules were intact and less fibrously adhesive.
4. In vitro evaluation of microencapsulated hepatocytes
We prepared the BPA microencapsulated hepatocytes and evaluated the function and viability of the hepatocytes in microencapsules by the secretion detection of Live/Dead staining and albumin secretion. The results showed the microencapsules kept the integrity morphous in the process of culture. After being cultured for 7 days, the secretion of albumin of BPA microencapsulated hepatocytes is higher than the alone cultured hepatocytes, and most hepatocytes keep viable. Therefore, the BPA microencapsules we prepared are a suitable material for hepatocyte growth.
5. Co-transplantation of encapsulated hepatocyte and vascular endothelial cell for the treatment of fulminate hepatic failure
We transplanted the empty microencapsules, isolated hepatocytes, microcapsulated hepatocytes, co-encapsulated hepatocytes and vascular endothelial cell intraperitoneally into rat models of fulminate hepatic failure induced by D-aminogalactose (D-gal). After 1d、3d、7d、2w、3w、4w transplantation, we calculated the mortality and assessed the ALT、AST、ALB in the serum of the rat model using the spectrophotometer, evaluated the integrality and recovery of microencapsules, and then carried out H.E. staining. The results showed that after 4w transplantation, the function of liver in co-encapsulated hepatocytes and vascular endothelial cell group was significantly higher than that in microencapsulated hepatocytes group, while such function in microencapsulated hepatocytes group also higher than that in isolated hepatocytes group. The recovered microencapsules were intact and less fibrously adhesive, which confirms the BPA microencapsules are suitable for the hepatocyte transplantation. Additionally, the microencapsules have good immunoprotection to protect the hepatocytes from immunological rejection and maintain the function of hepatocytes.
In conclusion, this study firstly constructed engineered collagen/Matrigel hepatocyte sheet in vitro, then evaluated the collagen/Matrigel environment made for the hepatocytes viable and function. After that, we investigate the interaction between the hepatocyte and vascular endothelial cell, as well as the promotion of the vascular endothelial cell to the function of hepatocyte. Based on this, we prepared the BPA microencapsules with immunoisolation, and evaluated the biological function of the BPA microencapsulated hepatocytes. Subsequently, we co-transplanted the encapsulated hepatocyte and vascular endothelial cell for the treatment of fulminate hepatic failure, because the microencapsule can avoid the immunological rejection, co-encapsulated cells improved the liver function of fulminate hepatic failure, it brings new approach and theoretical basis for the treatment of fulminate hepatic failure.
组织工程作为一门以“制造和替代人体组织和器官”为主要目的的交叉学科,在过去二十余年发展过程中,先后在皮肤、软骨、神经、血管等组织再造领域取得明显进展,部分组织工程产品已进入市场。针对肝脏的组织工程研究近年来已成为组织工程的热点领域,发展尤为迅速,并取得了一系列突破性进展,研究人员利用胶原、壳聚糖、海藻酸钠等为支架,先后成功在体外再造了工程化肝脏组织。尽管如此,肝脏组织工程研究仍面临着许多问题和挑战,例如:如何提高再造肝脏组织的血管化能力以及体内移植后如何避免宿主的免疫排斥反应等。
本论文分别以胶原/Matrigel和海藻酸微囊作为细胞支架材料,在体外构建了三维立体肝脏组织片层以及微囊化肝细胞,并通过在肝细胞-材料复合体中进一步添加血管内皮细胞的方式,促进再造肝脏组织的血管化。研究结果表明,胶原/Matrigel是肝细胞良好的支架材料,肝细胞在材料内能够形成肝脏组织,并且发现在肝组织片层中有管腔样结构出现,证实了血管内皮细胞对肝细胞功能的促进作用。在微囊化肝细胞研究方面,本论文基于微囊的免疫隔离作用,用微囊共包裹肝细胞与血管内皮细胞并对肝衰竭大鼠进行移植治疗,通过对大鼠死亡率和肝功能的检测后发现,微囊共包裹肝细胞与血管内皮细胞可以明显改善肝衰竭大鼠的肝功能,血管内皮细胞的加入对于微囊化肝细胞发挥生物学功能起到了积极作用。
本论文从三维立体肝脏组织再造到微囊化肝细胞研究,主要研究内容包括以下五个部分:
第一部分:基于胶原/Matrigel的组织工程化肝脏片层体外构建研究
本部分实验首先通过改良的胶原酶消化法,原代分离培养鉴定了大鼠肝细胞,并将其培养于胶原/Matrigel基质环境中,构建了组织工程化肝脏片层。通过观察细胞形态、检测细胞间的连接以及肝细胞的白蛋白分泌,并与胶原三明治结构相比较,研究发现构建的肝脏片层中细胞聚集生长,状态良好,并且表达actin和E-cadherin连接蛋白,白蛋白的分泌也显著高于三明治结构,说明胶原/Matrigel基质的三维立体环境更有利于肝细胞的生长和功能的发挥,体外再造的三维立体肝脏组织对于肝脏疾病的治疗及相关基础性研究具有重要作用。
第二部分:基于胶原/Matrigel的肝细胞与血管内皮细胞三维立体共培养体系的建立
本部分实验将肝细胞和血管内皮细胞共培养于胶原/Matrigel基质环境中,构建了肝细胞/血管内皮细胞片层,观察片层中细胞的形态、对细胞进行H.E.染色和免疫荧光染色,并且在体外培养1、3、5、7、14d时,更换培养液,收集培养上清,检测上清培养液中的白蛋白分泌水平。结果可见肝细胞/血管内皮细胞片层中的细胞逐渐聚集生长,组织学检测发现血管内皮细胞迁移聚集,形成管腔样结构。与单纯肝细胞片层相比,肝细胞/血管内皮细胞片层组培养液中的白蛋白分泌水平明显高于单纯肝细胞组。
第三部分:微囊材料的制备及其生物学性能评价
在海藻酸钠-聚赖氨酸-海藻酸(APA)微囊的制备基础之上,用Ba2+代替Ca2+,制备出海藻酸钡-聚赖氨酸-海藻酸(BPA)微囊,并采用机械震荡法测定APA和BPA微囊的机械强度和体外模拟体液法长期培养测定APA和BPA微囊的渗透压强度,结果表明,BPA微囊比APA微囊具有较高的机械强度和长期稳定性。采用体内移植实验测定微囊在肌肉内的移植强度,结果表明,APA和BPA微囊纤维化程度不明显。但是,APA微囊移植后体积膨胀率较BPA微囊略大,且成多边形态,而BPA微囊的形态多近似球形。采用腹腔移植实验测定微囊的生物相容性,结果表明,大鼠腹腔移植BPA微囊和APA微囊后第7、14、28和56d时,回收的微囊体积均占植入微囊体积的90%以上,光镜下见回收的两种微囊绝大部分仍呈完整的球形,表面光滑,无细胞包裹,纤维化程度不明显。
第四部分:微囊化肝细胞生物学性能的体外评价
制备BPA微囊化肝细胞,通过对微囊化肝细胞进行组织学检测、Live/Dead染色以及测定白蛋白分泌水平,体外评价了肝细胞的活性和功能,结果发现,在培养过程中光镜可见微囊和肝细胞均保持完整形态;组织学染色与Live/Dead染色结果显示微囊内大多数肝细胞仍保持较好活性,肝细胞在BPA微囊内培养7d内能够维持稳定的白蛋白分泌水平,表明BPA微囊是一种适合于肝细胞生长的微囊材料。
第五部分:微囊共包裹肝细胞和血管内皮细胞移植治疗大鼠肝衰竭的实验研究
采用D-氨基半乳糖腹腔注射法建立大鼠急性肝衰竭模型,经腹腔移植空微囊、游离的肝细胞、微囊化肝细胞和共包裹肝细胞和血管内皮细胞四个实验组治疗肝衰竭大鼠,于移植后的1d、3d、5d、1w、2w、3w、4w眼眦静脉取血,测血液中谷丙转氨酶(ALT),谷草转氨酶(AST),白蛋白(ALB)水平,通过对回收微囊的形态学检测和肝衰竭大鼠肝功能检测发现,移植4w后回收的BPA微囊形状完好,纤维化程度不明显,H.E.染色结果表明移植4w后囊内的肝细胞形态依旧完整,充分说明了BPA微囊本身的免疫隔离作用保护了肝细胞在移植体内的功能发挥。移植4w内共包裹组明显改善了肝衰竭大鼠的肝功能,其中移植后2到4周,共包裹组大鼠的ALT、AST与ALB水平显著优于其他各组;此外,共包裹组肝衰大鼠死亡率降低,与单纯包裹肝细胞组相比,其第四周死亡率由50%减少到20%,表明共包裹组中血管内皮细胞维持了肝细胞的活性和功能,延长了肝细胞的存活时间,对大鼠肝衰竭的治疗起到了促进作用。
综上所述,本研究首先在体外构建了组织工程化肝细胞片层,并研究了胶原/Matrigel凝胶环境对于肝细胞的活性和功能发挥的促进作用,在此基础上,利用本支架材料体系探讨了肝细胞与血管内皮细胞的相互作用以及血管内皮细胞对肝细胞功能的促进作用。基于上述细胞生物学研究基础,并利用以往已经建立的微囊化人工细胞的研究平台,进一步研制了具有免疫隔离作用的BPA微囊材料,体外评价了BPA微囊化肝细胞的生物学功能,并共包裹了肝细胞和血管内皮细胞,移植治疗大鼠急性肝衰竭,结果发现由于微囊可以避免免疫排斥反应,共包裹的细胞对肝衰竭大鼠有明显的治疗作用,血管化微囊化肝细胞对于肝衰竭临床治疗具有潜在的应用前景。
Hepatopathy is one of the most serious global diseases that harm human health. Now,liver transplantation and hepatocyte transplantation are feasible choices to treat liver diseases. However, procedures of liver transplantation are limited by the shortage of donor organs, high cost, and lifelong dependence on immunosuppression. Hepatocytes transplantation will bring the organism a strong post-transplant immune rejection, which makes transplantation ultimately fail. Therefore, it is urgent to find new techniques and methods for the above problems. The development of tissue engineering brought new hope for the therapy of liver diseases.
Tissue engineering, as an interdisciplinary science, which purpose is constructing and replacing human tissue and organ, has made great progress in the skins, cartilage, nerve, blood vessel in the past 20 years.The development of liver tissue engineering is fast recently; meanwhile in this field great breakthroughs had been made. However, many problems and challenges are existed, for example, how to improve the vascularization of reconstructed liver tissue and to avoid host immunologic rejection after in vivo transplantation.
This study used collagen/Matrigel and alginate microcapsules as liver tissue engineering scaffolds respectively. Based on this, we promoted the vascularization of engineered liver tissue through adding the vascular endothelial cells in tissue engineered hepatocyte/vascular endothelial cell sheets. Our results demonstrated that vascular endothelial cells could promote the hepatocyte viability and function. In other hand, based on the merit of avoiding immunological rejection, we prepared microencapsules as another tissue engineering material. Then we transplanted the co-encapsulated hepatocyte and vascular endothelial cell into rats to treat hepatic failure, vascular endothelial cell has improved the biological function of microencapsulated hepatocytes.
The main study contents from the reconstruction of the 3D tissue to the evaluation of the microencapsulated hepatocytes include the following five parts:
1. Construction of 3D engineered hepatocyte sheet based on collagen/Matrigel in vitro
In this part, we firstly isolated the rat primary hepatocyte by the improved collagenase digestion, which were cultured in the collagen/Matrigel environment for the construction of engineered hepatocyte sheet. Compared with the collagen sandwich structure, hepatocytes in the engineered hepatocyte sheet grow more aggregative and express the more actin, E-cadherin and albumin by assessing the cell morphology, cell-cell conjunction and secretion of albumin. These results show the 3D collagen/Matrigel environment is good for the growth and function of hepatocytes. The in vitro reconstruction of the 3D liver tissue played important roles in the treatment of liver disease and the relative basic study.
2. Construction of 3D co-cultured the hepatocyte/vascular endothelial cell system based on collagen/Matrigel in vitro
In this part, we co-cultured the hepatocyte and vascular endothelial cell in the collagen/Matrigel environment and constructed the hepatocyte/vascular endothelial cell sheet. By assessing the cell morphology, H.E. staining, immunofluorescence and secretion of albumin, we found the vascular endothelial cells in hepatocyte/vascular endothelial cell sheet migrate together to form a vessel like structure. The albumin secretion of hepatocyte/vascular endothelial cell sheet was higher than the hepatocyte sheet, after 1, 3, 5, 7, 14 days culture in vitro.
3. Preparation of microencapsule and evaluation of their biological function
In this part , we used barium ion to substitute calcium ion to form Ba–alginate–polylysine–alginate(BPA) microencapsules based on the alginate–polylysine–alginate(APA) microencapsules. And then we tested the mechanical strength of APA microencapsules and BPA microencapsules with mechanical shaking experiments, and the osmotic pressure of two types mincroencapsules was measured by long-time culture in simulated body fluids. The results showed that BPA microencapsules have higher mechanical strength and long-time stability than APA microcapsules. We measured the transplantation strength of the microencapsules by transplanting the microencapsules into the muscle of rats. The results showed the APA and BPA microencapsules have little fibrosis, while APA microencapsules were out of shape and more swelled than BPA, BPA microencapsules were still spherical. After that, we measured the biocompatibility of the microencapsules by transplanting the microencapsules into the peritoneal cavity. After being transplanted for 7,14,28,56 days, the recovery was up to 90%, the recovered two types of microcapsules were intact and less fibrously adhesive.
4. In vitro evaluation of microencapsulated hepatocytes
We prepared the BPA microencapsulated hepatocytes and evaluated the function and viability of the hepatocytes in microencapsules by the secretion detection of Live/Dead staining and albumin secretion. The results showed the microencapsules kept the integrity morphous in the process of culture. After being cultured for 7 days, the secretion of albumin of BPA microencapsulated hepatocytes is higher than the alone cultured hepatocytes, and most hepatocytes keep viable. Therefore, the BPA microencapsules we prepared are a suitable material for hepatocyte growth.
5. Co-transplantation of encapsulated hepatocyte and vascular endothelial cell for the treatment of fulminate hepatic failure
We transplanted the empty microencapsules, isolated hepatocytes, microcapsulated hepatocytes, co-encapsulated hepatocytes and vascular endothelial cell intraperitoneally into rat models of fulminate hepatic failure induced by D-aminogalactose (D-gal). After 1d、3d、7d、2w、3w、4w transplantation, we calculated the mortality and assessed the ALT、AST、ALB in the serum of the rat model using the spectrophotometer, evaluated the integrality and recovery of microencapsules, and then carried out H.E. staining. The results showed that after 4w transplantation, the function of liver in co-encapsulated hepatocytes and vascular endothelial cell group was significantly higher than that in microencapsulated hepatocytes group, while such function in microencapsulated hepatocytes group also higher than that in isolated hepatocytes group. The recovered microencapsules were intact and less fibrously adhesive, which confirms the BPA microencapsules are suitable for the hepatocyte transplantation. Additionally, the microencapsules have good immunoprotection to protect the hepatocytes from immunological rejection and maintain the function of hepatocytes.
In conclusion, this study firstly constructed engineered collagen/Matrigel hepatocyte sheet in vitro, then evaluated the collagen/Matrigel environment made for the hepatocytes viable and function. After that, we investigate the interaction between the hepatocyte and vascular endothelial cell, as well as the promotion of the vascular endothelial cell to the function of hepatocyte. Based on this, we prepared the BPA microencapsules with immunoisolation, and evaluated the biological function of the BPA microencapsulated hepatocytes. Subsequently, we co-transplanted the encapsulated hepatocyte and vascular endothelial cell for the treatment of fulminate hepatic failure, because the microencapsule can avoid the immunological rejection, co-encapsulated cells improved the liver function of fulminate hepatic failure, it brings new approach and theoretical basis for the treatment of fulminate hepatic failure.
引文
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[1]. Keeffe, E.B. Liver transplantation: Current status and novel approaches to liver replacement. Gastroenterology 120, 749, 2001.
[2]. te Velde AA, Bosman DK, Oldenburg J, et al. Three different hepatocyte transplantation techniques for enzyme deficiency disease and acute hepatic failure. Artif Organs 1992; 16: 522.
[3]. Ambrosino G, Varotto S, Basso SM, et al. Hepatocyte transplantation in the treatment of acute liver failure: microencapsulated hepatocytes versus hepatocytes attached to an autologous biomatrix. Cell Transplant 2003; 12: 43.
[4]. Strom SC, Fisher RA, Thompson MT, et al. Hepatocyte transplantation as a bride to orthotopic liver transplantation in terminal liver failure. Transplantation 1997; 63: 559.
[5]. Fox IJ, Roy-Chowdhury J. Hepatocyte transplantation. J Hepatol 2004; 40: 878.
[6]. Mito M, Kusano M, Kawaura Y. Hepatocyte transplantation in Man.Transplant Proc 1992; 24: 3052.
[7]. Arkadopoulos N, Lilja H, Sub KS, et al. Intrasplenic transplantation of allogeneic hepatocytes prolongs survival in anhepatic rats. Hepatology 1998; 28: 1365.
[8]. Demetriou AA, Felcher A, Moscioni AD. Hepatocyte transplantation: A potential treatment for liver disease. Dig Dis Sci 1991; 36: 1320.
[9]. Kawashita Y,Guha C.Yamanouchi K.et a1.Liver repopulation:a new concept of hepatoeyte transplantation. Surg Today,2005,35:71-75
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[23]. Elcin YM, Dixit V, Lewin K, et al. Xenotransplantation of fetal porcine hepatocytes in rats using a tissue engineering approach. Artif Organs,1999,23 (2) :146-152
[2]、te Velde AA, Bosman DK, Oldenburg J, et al. Three different hepatocyte transplantation techniques for enzyme deficiency disease and acute hepatic failure. Artif Organs 1992; 16: 522.
[3]、Ambrosino G, Varotto S, Basso SM, et al. Hepatocyte transplantation in the treatment of acute liver failure: microencapsulated hepatocytes versus hepatocytes attached to an autologous biomatrix. Cell Transplant 2003; 12: 43.
[4]、Strom SC, Fisher RA, Thompson MT, et al. Hepatocyte transplantation as a bride to orthotopic liver transplantation in terminal liver failure. Transplantation 1997; 63: 559.
[5]、Fox IJ, Roy-Chowdhury J. Hepatocyte transplantation. J Hepatol 2004; 40: 878.
[6]、Mito M, Kusano M, Kawaura Y. Hepatocyte transplantation in Man. Transplant Proc 1992; 24: 3052.
[7]、Arkadopoulos N, Lilja H, Sub KS, et al. Intrasplenic transplantation of allogeneic hepatocytes prolongs survival in anhepatic rats. Hepatology 1998; 28: 1365.
[8]、Demetriou AA, Felcher A, Moscioni AD. Hepatocyte transplantation: A potential treatment for liver disease. Dig Dis Sci 1991; 36: 1320.
[9]、Kawashita Y,Guha C.Yamanouchi K.et a1.Liver repopulation:a new concept of hepatoeyte transplantation. Surg Today,2005,35:71-75
[10]、Lee SW ,Wang X,Chowdhury NR,el a1.Hepatocyte tran splantation: State of the art and strategies for overcoming existing hurdles.Ann Hepatol,2004,3:48-53.
[11]、Dunn JC, Tompkins RG, Yarmush ML.Long-term in vitro function of adult hepatocytes in a collagen sandwich configuration. Biotechnol Prog. 1991 May-Jun;7(3):237-45.
[12]、Kono Y, Yang S, Roberts EA.Extended primary culture of human hepatocytes in a collagen gel sandwich system. In Vitro Cell Dev Biol Anim. 1997 Jun;33(6):467-72.
[13]、王英杰,刘鸿凌,文红伟,刘俊,郭海涛.胶原凝胶固定培养大鼠肝细胞的功能与形态观察.肝脏. 2003, Vol 8 , No. 1:15-17
[14]、Chang T M S. Semipermeable microcapsules. Science, 1964, 146:524–525.
[15]、Sun AM, Cai Z, Shi Z, Ma F, O'Shea GM. Biomater Artif Cells Artif Organs. Microencapsulated hepatocytes: an in vitro and in vivo study. 1987;15(2):483-96.
[16]、Jeng LB, Hsu BR, Fu SH, Chuang KL, Lee WC, Chen MF, Chang CH.Cotransplantation of microencapsulated hepatocytes and islets for acute hepatic failure in rats. Transplant Proc. 1996 Jun;28(3):1859-60.
[17]、Liu ZC, Chang TM. Increased viability of transplanted hepatocytes when hepatocytes are co-encapsulated with bone marrow stem cells using a novel method. Artif Cells Blood Substit Immobil Biotechnol. 2002 Mar;30(2):99-112.
[18]、Tony Manibur Rahman, Ivan Diakanov, Clare Selden and Humphrey Hodgson. Co-transplantation of encapsulated HepG2 and rat Sertoli cells improves outcome in a thioacetamide induced rat model of acute hepatic failure. Transplant International.2005(18) 1001-1009.
[19]、Gao Y, Xu J, Sun B, Jiang HC.Microencapsulated hepatocytes and islets as in vivo bioartificial liver support system. World J Gastroenterol. 2004 Jul 15;10(14):2067-71.
[20]、Liu ZC, Chang TM. Coencapsulation of hepatocytes and bone marrow stem cells: in vitro conversion of ammonia and in vivo lowering of bilirubin in hyperbilirubemia Gunn rats. Int J Artif Organs. 2003 Jun;26(6):491-7.
[21]、Ito A, Takizawa Y, Honda H, Hata K, Kagami H, Ueda M, Kobayashi T. Tissue engineering using magnetite nanoparticles and magnetic force: heterotypic layers of cocultured hepatocytes and endothelial cells, Tissue Eng., 2004, 10(5-6):833-40
[22]、Takayama G, Taniguchi A, Okano T. Identification of differentially expressed genes in hepatocyte/endothelial cell co-culture system, Tissue Eng., 2007, 13(1):159-66
[23]、Wang X, Yan Y, Xiong Z, Lin F, Wu R, Zhang R, Lu Q. Preparation and evaluation of ammonia-treated collagen/chitosan matrices for liver tissue engineering. J Biomed Mater Res B Appl Biomater. 2005 Oct;75(1):91-8.
[24]、Ohashi K, Yokoyama T, Yamato M, Kuge H, Kanehiro H, Tsutsumi M, Amanuma T,Iwata H, Yang J, Okano T, Nakajima Y. Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets. Nat Med. 2007Jul;13(7):880-5. Epub 2007 Jun 17.
[25]、Tuschl G, Mueller SO. Effects of cell culture conditions on primary rat hepatocytes-Cell morphology and differential gene expression. Toxicology. 2006 Feb 1;218(2-3):205-15.
[26]、Walkup MH, Gerber DA.Hepatic stem cells: in search of. Stem Cells. 2006 Aug;24(8):1833-40. Epub 2006 May 4.
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