本文選題：肝組織工程 + 新生大鼠肝細(xì)胞　； 參考：《中國人民解放軍軍醫(yī)進(jìn)修學(xué)院》2012年碩士論文

【摘要】：背景：肝移植是終末期肝病的有效治療手段，但供肝的嚴(yán)重缺乏一直制約其發(fā)展，因此解決這一難題必須尋求新的治療技術(shù)。肝組織工程是利用肝臟細(xì)胞與載體支架復(fù)合構(gòu)建可植入的類肝組織，具有緩解器官供體短缺的治療前景。從研究角度，由于肝臟血供豐富，較大尺度類肝組織在大鼠肝臟原位移植手術(shù)操作困難，至今缺乏安全有效的方法。本課題擬在體外構(gòu)建較大尺度工程化肝組織并探索在大鼠體內(nèi)原位移植的方法，為開辟一種新的治療途徑奠定實驗基礎(chǔ)。 目的：為解決較大尺度類肝組織體內(nèi)移植后難以存活的問題，本項目擬在體外構(gòu)建預(yù)血管化的片狀工程化肝組織，并在大鼠肝臟表面制造輕度滲血創(chuàng)面后將植入物貼覆其上，使構(gòu)建物與肝臟盡快建立血液聯(lián)通，以探索將大塊工程化肝組織與原位肝臟整合的新方法。 方法：1.種子細(xì)胞的制備：胰酶冷消化法獲得新生大鼠肝細(xì)胞，肺組織塊貼壁法分離大鼠肺微血管內(nèi)皮細(xì)胞，常規(guī)培養(yǎng)。四唑鹽(MTT)比色法評價細(xì)胞的增殖活性，免疫熒光法檢測肝細(xì)胞白蛋白表達(dá)及內(nèi)皮細(xì)胞CD31的表達(dá)。2.五種片狀支架材料細(xì)胞相容性的體外評價：仿血管束支架材料、海綿狀膠原材料、薄膜狀膠原材料、聚乳酸、殼聚糖表觀形貌及掃描電鏡觀察。新生大鼠肝細(xì)胞、內(nèi)皮細(xì)胞分別與五種材料共培養(yǎng)。運用掃描電鏡觀察細(xì)胞/材料黏附情況，MTT比色法檢測細(xì)胞增殖情況，白蛋白、尿素氮檢測試劑盒檢測肝細(xì)胞功能。3.片狀工程化肝組織體外構(gòu)建兩步法：首先將內(nèi)皮細(xì)胞/血管內(nèi)皮生長因子與體積1cm3的片狀支架材料復(fù)合并共培養(yǎng)兩小時；再將肝細(xì)胞與預(yù)血管化處理的支架材料復(fù)合1小時，構(gòu)建成片狀工程化肝組織。4.大鼠的肝表面貼覆移植（n=15）：先將肝臟包膜造成輕微滲血創(chuàng)面，再將構(gòu)建的片狀類肝組織以肝表面貼覆的方法植入，醫(yī)用纖維蛋白膠噴灑固定，4周后取材進(jìn)行大體觀察及組織切片HE染色、白蛋白及CD31免疫組化染色。 結(jié)果：1.分離得到的新生大鼠肝細(xì)胞約為1.5×106/鼠肝，活率95%以上，呈團(tuán)簇生長。MTT值在培養(yǎng)7d升至峰值，隨后逐漸下降。白蛋白染色陽性；分離得到的大鼠肺微血管內(nèi)皮細(xì)胞貼壁率約為80%，原代細(xì)胞存活率達(dá)98%以上。貼壁后呈單層鑲嵌排列鋪路石狀。MTT值在2-4天成倍數(shù)生長。CD31染色陽性。2.五種材料表觀形貌各有不同，種子細(xì)胞與支架復(fù)合后，電鏡下觀察細(xì)胞分布以海綿狀膠原材料最適合細(xì)胞黏附。細(xì)胞的增殖情況經(jīng)MTT檢測顯示：肝細(xì)胞的增殖依次為海綿狀膠原材料仿血管束支架材料薄膜狀膠原材料殼聚糖聚乳酸；內(nèi)皮細(xì)胞的增殖依次為海綿狀膠原材料仿血管束支架材料薄膜狀膠原材料殼聚糖聚乳酸。肝細(xì)胞培養(yǎng)上清中白蛋白和尿素的含量測定表明海綿狀膠原材料結(jié)果最佳；經(jīng)上述實驗篩選后，選擇海綿狀膠原材料作為后續(xù)構(gòu)建工程化肝組織的載體支架。3.體外構(gòu)建的片狀工程化肝組織大小約（1.5×1.5×0.6）cm3，鏡下觀察可見：海綿狀膠原材料上，預(yù)培養(yǎng)2小時的血管內(nèi)皮細(xì)胞已圍繞膠原細(xì)絲貼壁聚集成團(tuán)，達(dá)到了預(yù)血管化的目的。大量肝細(xì)胞被膠原細(xì)絲包繞密集成團(tuán)，幾乎布滿所有孔隙。4.片狀工程化肝組織通過肝表面貼覆法能夠簡便安全地植入大鼠肝臟原位，并且與受體肝臟完全整合，形成體積1cm3的新生組織，H.E.染色顯示新生組織內(nèi)肝細(xì)胞形態(tài)良好，相互連接成片，，內(nèi)皮細(xì)胞可在組織內(nèi)部形成大小不等的管腔,內(nèi)有紅細(xì)胞分布。免疫組化染色顯示新生組織表達(dá)白蛋白，內(nèi)部形成的管腔表達(dá)CD31。 結(jié)論：1.新生大鼠肝細(xì)胞及肺微血管內(nèi)皮細(xì)胞分別可以通過胰酶冷消化法及組織塊貼壁法簡便、高效的獲得，在體外培養(yǎng)條件下具有增殖活性且保持原有的分泌功能，可以作為構(gòu)建工程化肝組織的種子細(xì)胞應(yīng)用于肝組織工程研究。2.海綿狀膠原材料與肝臟細(xì)胞相容性良好，適合作為構(gòu)建片狀工程化肝組織的載體支架。3.種子細(xì)胞分別于不同時間與支架材料復(fù)合的類肝組織構(gòu)建法能夠成功在體外構(gòu)建出預(yù)血管化的片狀工程化肝組織。4.肝表面原位貼覆法可以簡便安全地將較大尺度的工程化肝組織移植到大鼠肝臟原位，并能在肝表面形成體積1cm3且具有一定功能的類肝組織，這種方法具有潛在的應(yīng)用前景，為后續(xù)原位修復(fù)受損肝臟提供可行方案。
[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.
【學(xué)位授予單位】：中國人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R318.1

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