片状工程化肝组织体外构建及大鼠原位移植的实验研究

发布时间：2018-07-01 15:57

本文选题：肝组织工程 + 新生大鼠肝细胞　；参考：《中国人民解放军军医进修学院》2012年硕士论文

【摘要】：背景：肝移植是终末期肝病的有效治疗手段，但供肝的严重缺乏一直制约其发展，因此解决这一难题必须寻求新的治疗技术。肝组织工程是利用肝脏细胞与载体支架复合构建可植入的类肝组织，具有缓解器官供体短缺的治疗前景。从研究角度，由于肝脏血供丰富，较大尺度类肝组织在大鼠肝脏原位移植手术操作困难，至今缺乏安全有效的方法。本课题拟在体外构建较大尺度工程化肝组织并探索在大鼠体内原位移植的方法，为开辟一种新的治疗途径奠定实验基础。目的：为解决较大尺度类肝组织体内移植后难以存活的问题，本项目拟在体外构建预血管化的片状工程化肝组织，并在大鼠肝脏表面制造轻度渗血创面后将植入物贴覆其上，使构建物与肝脏尽快建立血液联通，以探索将大块工程化肝组织与原位肝脏整合的新方法。方法：1.种子细胞的制备：胰酶冷消化法获得新生大鼠肝细胞，肺组织块贴壁法分离大鼠肺微血管内皮细胞，常规培养。四唑盐(MTT)比色法评价细胞的增殖活性，免疫荧光法检测肝细胞白蛋白表达及内皮细胞CD31的表达。2.五种片状支架材料细胞相容性的体外评价：仿血管束支架材料、海绵状胶原材料、薄膜状胶原材料、聚乳酸、壳聚糖表观形貌及扫描电镜观察。新生大鼠肝细胞、内皮细胞分别与五种材料共培养。运用扫描电镜观察细胞/材料黏附情况，MTT比色法检测细胞增殖情况，白蛋白、尿素氮检测试剂盒检测肝细胞功能。3.片状工程化肝组织体外构建两步法：首先将内皮细胞/血管内皮生长因子与体积1cm3的片状支架材料复合并共培养两小时；再将肝细胞与预血管化处理的支架材料复合1小时，构建成片状工程化肝组织。4.大鼠的肝表面贴覆移植（n=15）：先将肝脏包膜造成轻微渗血创面，再将构建的片状类肝组织以肝表面贴覆的方法植入，医用纤维蛋白胶喷洒固定，4周后取材进行大体观察及组织切片HE染色、白蛋白及CD31免疫组化染色。结果：1.分离得到的新生大鼠肝细胞约为1.5×106/鼠肝，活率95%以上，呈团簇生长。MTT值在培养7d升至峰值，随后逐渐下降。白蛋白染色阳性；分离得到的大鼠肺微血管内皮细胞贴壁率约为80%，原代细胞存活率达98%以上。贴壁后呈单层镶嵌排列铺路石状。MTT值在2-4天成倍数生长。CD31染色阳性。2.五种材料表观形貌各有不同，种子细胞与支架复合后，电镜下观察细胞分布以海绵状胶原材料最适合细胞黏附。细胞的增殖情况经MTT检测显示：肝细胞的增殖依次为海绵状胶原材料仿血管束支架材料薄膜状胶原材料壳聚糖聚乳酸；内皮细胞的增殖依次为海绵状胶原材料仿血管束支架材料薄膜状胶原材料壳聚糖聚乳酸。肝细胞培养上清中白蛋白和尿素的含量测定表明海绵状胶原材料结果最佳；经上述实验筛选后，选择海绵状胶原材料作为后续构建工程化肝组织的载体支架。3.体外构建的片状工程化肝组织大小约（1.5×1.5×0.6）cm3，镜下观察可见：海绵状胶原材料上，预培养2小时的血管内皮细胞已围绕胶原细丝贴壁聚集成团，达到了预血管化的目的。大量肝细胞被胶原细丝包绕密集成团，几乎布满所有孔隙。4.片状工程化肝组织通过肝表面贴覆法能够简便安全地植入大鼠肝脏原位，并且与受体肝脏完全整合，形成体积1cm3的新生组织，H.E.染色显示新生组织内肝细胞形态良好，相互连接成片，，内皮细胞可在组织内部形成大小不等的管腔,内有红细胞分布。免疫组化染色显示新生组织表达白蛋白，内部形成的管腔表达CD31。结论：1.新生大鼠肝细胞及肺微血管内皮细胞分别可以通过胰酶冷消化法及组织块贴壁法简便、高效的获得，在体外培养条件下具有增殖活性且保持原有的分泌功能，可以作为构建工程化肝组织的种子细胞应用于肝组织工程研究。2.海绵状胶原材料与肝脏细胞相容性良好，适合作为构建片状工程化肝组织的载体支架。3.种子细胞分别于不同时间与支架材料复合的类肝组织构建法能够成功在体外构建出预血管化的片状工程化肝组织。4.肝表面原位贴覆法可以简便安全地将较大尺度的工程化肝组织移植到大鼠肝脏原位，并能在肝表面形成体积1cm3且具有一定功能的类肝组织，这种方法具有潜在的应用前景，为后续原位修复受损肝脏提供可行方案。
[Abstract]:Background: liver transplantation is an effective treatment for end-stage liver disease, but the serious lack of donor liver has been restricting its development. Therefore, a new treatment technology must be sought to solve this problem. Liver tissue engineering is to build an implanted liver like tissue with the combination of liver cells and carrier scaffolds, which has the prospect of alleviating the shortage of organ donors. Due to the rich blood supply of the liver, the large scale liver tissue in the rat liver in situ transplantation is difficult to operate, and it is lacking a safe and effective method. This project intends to construct a large scale engineering liver tissue in vitro and explore the method of orthotopic transplantation in the rat, which lays the foundation for opening a new treatment way.
Objective: in order to solve the problem that the large scale liver tissue is difficult to survive in vivo after transplantation in vivo, this project intends to construct a prevascularized slice engineering liver tissue in vitro, and overlay the implant on the surface of the rat liver after producing a slight bleeding wound to make the construction and liver establish blood Unicom as soon as possible in order to explore the large engineering liver. A new method of tissue and orthotopic liver integration.
Methods: 1. seed cells were prepared: neonatal rat hepatocytes were obtained by cold digestion of pancreatin, the lung microvascular endothelial cells were isolated from the lung tissue block, and the proliferation activity of the cells was evaluated by the four Zolo salt (MTT) colorimetric method, and the immunofluorescence method was used to detect the expression of.2. and the expression of CD31 in the hepatocytes and the expression of five flake stents. In vitro evaluation of material cell compatibility: vascular bundle scaffold material, cavernous collagen material, thin film collagen material, polylactic acid, chitosan surface morphology and scanning electron microscope observation. The neonatal rat hepatocytes and endothelial cells were co cultured with five kinds of materials respectively. The adhesion of cells / materials was observed by scanning electron microscope, and the MTT colorimetric assay was used to detect the cell adhesion. Cell proliferation, albumin, urea nitrogen detection kit for the detection of liver cell function.3. slice engineered liver tissue in vitro construction of the two step method: first, the endothelial cell / vascular endothelial growth factor and volume 1cm3 flake scaffold materials were combined and co cultured for two hours, and then the hepatocytes and the pretreated scaffold materials were combined for 1 hours. The liver surface clad transplantation (n=15) of.4. rats was constructed into a flaky engineering liver tissue (n=15): the liver capsule was first made to cause a slight bleeding wound, and then the constructed slices of liver tissue were implanted with the liver surface, and the fibrin glue was sprinkled with medical fibrin glue. After 4 weeks, the material was observed and the tissue section was stained with HE, albumin and CD31 immune group. Chemical dyeing.
Results: 1. the isolated neonatal rat hepatocytes were about 1.5 x 106/ liver, the survival rate was more than 95%. The.MTT value of the cluster growth was increased to the peak value at the culture 7d, and then decreased gradually. The albumin staining was positive. The adherent rate of the isolated rat lung microvascular endothelial cells was about 80% and the primary Bao Cunhuo rate was above 98%. The apparent morphology of the.MTT value of the column paving stone like.MTT in the growth of.CD31 staining positive.2. was different. After the seed cells were combined with the scaffold, the distribution of the cells with the sponge like collagen was the most suitable for the cell adhesion. The proliferation of the cells by MTT showed that the proliferation of the hepatocytes was in order of the sponge like collagen material. The membrane like collagen material of the vascular bundle scaffold materials chitosan polylactic acid. The proliferation of endothelial cells is the chitosan polylactic acid collagen material. The determination of the content of albumin and urea in the supernatant of hepatocyte culture supernatant shows that the result of the sponge like collagen material is the best. After selecting the cavernous collagen material as the carrier scaffold for the subsequent construction of the engineered liver tissue, the size of the liver tissue was about (1.5 * 1.5 * 0.6) cm3. Under the microscope, it was observed that on the sponge like collagen material, the precultured vascular endothelial cells were gathered around the collagen filaments and gathered together to achieve the prevascularization. Objective. A large number of hepatocytes are surrounded by collagenous filaments, and almost all of the pore.4. - like engineered liver tissues can be easily and safely implanted in the rat liver in situ through the liver surface cladding method. The liver cells are completely integrated with the recipient liver to form a new tissue with volume 1cm3, and H.E. staining shows that the liver cells in the newborn tissues are in good shape. The endothelial cells can form a lumen with different sizes within the tissue, and the red cells are distributed within the tissue. The immunohistochemical staining shows that the newborn tissues express albumin and the internal formation of the lumen is CD31..
Conclusion: 1. the hepatocytes and pulmonary microvascular endothelial cells in neonatal rats can be easily obtained by the cold digestion method of pancreatic enzyme and the method of tissue block adherence, which can be used as the seed cells for the construction of the liver tissue engineering to study the.2. sea. The cavernous collagen material has good compatibility with the liver cells. It is suitable to be used as a carrier scaffold for the construction of flaky engineering liver tissue. The construction of.3. seed cells at different time and scaffold material, respectively, can be successfully constructed in vitro to construct a prevascularized.4. liver surface in situ cladding method. The large scale of the liver tissue is transplanted into the rat liver in situ, and the liver tissue can be formed on the surface of the liver with a volume of 1cm3 and has a certain function. This method has potential application prospects and provides a feasible scheme for the subsequent orthotopic repair of damaged liver.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.1

【参考文献】