兔肝纖維化模型的建立及自體骨髓干細胞移植治療
發(fā)布時間：2018-10-09 15:37
【摘要】： 目的 1.建立兔肝纖維化動物模型的制作方法和評價標準。 2.建立肝纖維化動物模型兔自體骨髓間充質(zhì)細胞轉(zhuǎn)GFP基因標記的方法。 3.觀察自體骨髓間充質(zhì)干細胞移植后肝纖維化模型兔肝內(nèi)標記細胞的數(shù)量、遷移和分布規(guī)律,并通過檢測血清肝功能指標的變化和肝組織病理分析評價臨床療效。 方法 1.兔肝纖維化模型的建立和評價:40只普通級日本大耳白兔,隨機分為實驗組和對照組,實驗組腹腔注射40%CCL_4橄欖油溶液,對照組腹腔注射等量生理鹽水。1—3周CCL_4劑量為0.2ml/kg。3—6周CCL4劑量為0.4 ml/kg。6—8周CCL_4劑量為0.5ml/kg。實驗開始后30、60、90天分別留取肝組織和血清標本,進行HE染色病理觀察和生化指標檢測。 2.自體骨髓間充質(zhì)細胞的培養(yǎng)和標記:取24只普通級模型組日本大耳白兔,隨機分為實驗組和對照組,實驗組移植自體骨髓間充質(zhì)細胞,對照組輸注生理鹽水。對實驗組模型兔進行髂骨穿刺采集骨髓,密度梯度離心法分離骨髓單個核細胞,貼壁培養(yǎng)獲得間充質(zhì)干細胞,用綠色熒光蛋白(GFP)標記移植細胞,熒光顯微鏡下觀察標記細胞數(shù)量,標記陽性率為99%。 3.自體骨髓間充質(zhì)細胞移植治療肝纖維化模型兔和療效評價:將GPP基因標記3×10~6細胞/ml的骨髓間充質(zhì)細胞經(jīng)腸系膜上靜脈植入實驗組模型動物肝內(nèi),于移植后第0、4、8、12周定期取家兔血清進行肝功能檢測,并取小部分肝組織做成石蠟切片,置于熒光顯微鏡下觀察移植細胞的數(shù)量、遷移和分布規(guī)律;另對小部分肝組織進行HE染色,觀察肝纖維化組織細胞移植后病理組織學變化。 4.統(tǒng)計分析:實驗數(shù)據(jù)以均值±標準差((?)±s)的形式表示,采用SPSS13.0軟件對血清肝功統(tǒng)計指標進行重復測量設計方差分析,多個水平組間比較用LSD法檢驗,兩個水平組間比較采用獨立樣本t檢驗,檢驗值為P≤0.05時具有統(tǒng)計學意義。 結(jié)果 1.模型兔肝組織病理學改變:對照組兔肝臟外觀呈暗紅色,病理切片示:肝小葉結(jié)構完整,肝細胞圍繞中央靜脈呈放射狀排列。實驗組8周時,肝臟呈暗紅色,表面有輕微粟粒樣改變,病理切片示:肝細胞點狀及點灶狀壞死,匯管區(qū)炎癥細胞浸潤,呈早期纖維化表現(xiàn)。12周時,肝臟呈灰褐色,有較明顯粟粒樣改變。病理切片示:肝小葉結(jié)構破壞,肝索排列紊亂,肝細胞脂肪變性,間質(zhì)纖維組織增生,有炎性細胞浸潤,為明顯肝纖維化表現(xiàn)。 2.模型兔血生化指標:隨著注射CCL4時間的延長,白球蛋白比值由原來的1.26±0.90下降到0.93±0.17,球蛋白、間接膽紅素、直接膽紅素逐漸升高,谷丙轉(zhuǎn)氨酶、谷草轉(zhuǎn)氨酶分別由36.80±7.66、23.40±14.20升高到392.00±57.93、282.00±38.54。 3.骨髓間充質(zhì)細胞的形態(tài)學觀察:原代細胞呈MSCs特征性的旋渦狀生長,排列有方向性,旋渦中心細胞呈多層分布,細胞界限不清,細胞形態(tài)多呈梭形。P1代細胞生長速度明顯增快,集落方式生長,旋渦中心細胞呈多層分布,P3代細胞長梭形更加明顯,細胞形態(tài)趨于一致。GFP標記細胞呈綠色熒光,標記陽性率為99%。 4.移植后熒光細胞觀察:肝纖維化組織中可見大量的綠色熒光細胞,密集均勻分布,與肝組織相容,熒光細胞遷移范圍廣,逐漸向病灶處遷移,在移植后第3天熒光細胞肝小葉中央靜脈周圍最多,隨著時間的推移,陽性染色逐漸增強,并逐步向肝組織內(nèi)部延伸至肝臟邊緣。 5.移植后組織病理學觀察:移植后4周,對照組肝索紊亂,間質(zhì)纖維增生,部分伸入小葉,有炎性細胞浸潤,而治療組肝組織結(jié)構恢復明顯,上述改變較輕,而且變性、壞死細胞明顯減少。移植后12周,移植組兔肝細胞變性壞死極少,中央靜脈及匯管區(qū)周圍有少量膠原纖維沉積,肝小葉結(jié)構清晰。與正常對照組比較無明顯差異。 6.移植后血清指標改變:骨髓干細胞移植后血清TP含量、ALB含量逐漸升高,與對照組比較無統(tǒng)計學意義(P＞0.05);移植組血清GLO含量逐漸降低,與對照組比較第8周、第12周有顯著差異(P＜0.05);移植組血清TBIL、DBIL含量逐漸降低,與對照組比較,第4、8、12周差異極顯著(P＜0.01);移植組血清ALT、AST活性逐漸降低,與對照組比較,第4、8、12周差異極顯著(P＜0.01)。 結(jié)論 1.腹腔注射40%四氯化碳橄欖油溶液12周,成功建立了兔肝纖維化模型:病理組織學改變和肝功能生化指標變化符合為肝纖維化標準。 2.建立了肝纖維化家兔自體骨髓間充質(zhì)細胞的轉(zhuǎn)GFP基因標記方法,標記陽性率為99%。 3自體骨髓間充質(zhì)干細胞移植能夠部分修復損傷的肝組織,抑制肝纖維化的發(fā)展,部分逆轉(zhuǎn)肝纖維化進程,使其向正常結(jié)構發(fā)展并改善肝功能。
[Abstract]:Purpose 1. A preparation method of rabbit liver fibrosis animal model is established. Evaluation criteria. 2. Establishment of an animal model of hepatic fibrosis rabbit autologous bone marrow mesenchymal transition G Methods: To observe the number, migration and distribution of labeled cells in liver of rabbit liver after autologous bone marrow mesenchymal stem cell transplantation, and to detect the change of serum liver function index. and liver Histopathological analysis and evaluation of clinical efficacy. Methods 1. Establishment and evaluation of rabbit liver fibrosis model: 40 ordinary Japanese white rabbits were randomly divided into experimental group and experimental group. In the control group, 40% CCl _ 4 olive oil solution was injected into the abdominal cavity of the experimental group, and the same amount of normal saline was injected into the control group. The dosage was 0. 5 ml/ kg at 8 weeks after the start of the experiment. The liver group was taken from 30, 60 and 90 days after the start of the experiment. The pathological observation of HE staining and the detection of biochemical indexes were carried out in the tissues and serum samples. The culture and labelling of autologous bone marrow mesenchymal cells were carried out: 24 ordinary Japanese rabbits were randomly divided into experimental groups. In the control group, autologous bone marrow mesenchymal cells were transplanted in the experimental group and normal saline was infused in the control group. The experimental group model rabbits were subjected to percutaneous puncture to acquire bone marrow and density gradient centrifugation to separate the mononuclear cells from the bone marrow, the adherent cells were cultured to obtain the mesenchymal stem cells, and the green fluorescent protein (GFP) was used to mark the cells. The number of labeled cells was observed under fluorescence microscope, and the positive rate was 99%. 3. Autobone marrow mesenchymal stem cell transplantation was used for the treatment of hepatic fibrosis model rabbits and efficacy evaluation: the GPP gene was labeled with 3 ~ 10 ~ 6 cells/ ml bone marrow mesenchymal stem cells were implanted into the experimental group via superior mesenteric vein. In animal liver, the serum of rabbit was taken for liver function test at 0, 4, 8 and 12 weeks after transplantation, and the small part of liver tissue was taken as paraffin section, and the number, migration and distribution of transplanted cells were observed under fluorescence microscope. HE staining was performed on small hepatic tissue and hepatic fibrosis was observed. The changes of pathological histology after cell transplantation were analyzed. The statistical analysis showed that the experimental data were expressed in the form of mean square deviation ((?)/ s), and the statistical indexes of liver function of serum were analyzed by SPSS 13.0 software. The results of analysis of variance and LSD method were used for comparison among multiple levels. Inspection The independent sample t test was used for the comparison between the two horizontal groups, and the test value was P 0.05. The results showed that the model rabbit liver group Histopathological changes: The appearance of the liver was dark red in the control group. The pathological section showed that the structure of the hepatic lobule was complete, and the hepatocytes were arranged radially around the central vein. In the experimental group, the liver was dark red at 8 weeks, there were slight changes in the surface of the liver, and the pathological section showed that the liver cells The focal spot and focal necrosis, infiltration of inflammatory cells in the area of remit, showed early fibrosis. The liver was gray brown at 12 weeks, and there were obvious changes in granulation samples. The pathological section showed: The hepatic lobule structure was destroyed, hepatic cable arrangement disorder, liver cell fat degeneration, interstitial fibrous tissue proliferation, inflammatory cell infiltration, and obvious hepatic fibrosis expression. 2. Model rabbit blood biochemical index: As the time of injection CCL4 was prolonged, the ratio of white globulin decreased from 1. 26 to 0. 93, 0. 17, globulin, indirect bilirubin, direct bilirubin increased gradually, glutamic pyruvic transaminase, glutamic acid and ammonia were transferred to ammonia. The enzymes were elevated to 392. 00, 57. 93, 282. 00, 38. 54. 3, respectively, from 36. 80, 7. 66, 23. 40, and 14. 20. Morphological observation: The primary cells showed the characteristic spiral growth of MSCs, the arrangement of the cells was directional, the center cells of the vortex were distributed in multiple layers, the cell boundary was not clear, and the cell morphology Multi-shuttle-shaped. The growth rate of P1-generation cells increased significantly, and the colony-forming method The results showed that the cell morphology tended to be consistent. GFP-labeled cells were green fluorescence, and the positive rate was 99%. The fluorescence cells observed after transplantation were observed in the liver fibrosis tissues. The amount of green fluorescent cells is uniformly distributed and is compatible with the liver tissue, and the fluorescence cell migration range is wide, As time progresses, positive staining gradually increases and gradually extends inside the liver tissue to the liver's edge. 5. Histopathological observation after transplantation: remove 4 weeks after implantation, the control group had hepatic cord disorder, interstitial fibrosis, partially extended into the lobules, inflammatory cell infiltration, and the recovery of liver tissue structure in the treatment group was obvious. the above changes are lighter and degeneration, necrosis, The cells were significantly reduced. After 12 weeks after transplantation, there was little degeneration and necrosis of hepatocytes in the transplantation group, and a small amount of collagen fibers were deposited around the central vein and the junction area. The structure of the hepatic lobule was clear. Compared with the control group, there was no significant difference. 6. The serum indexes after transplantation were changed: the serum TP content after bone marrow stem cell transplantation. Compared with the control group, the content of GLO decreased gradually, the content of GLO in the transplantation group decreased gradually, the content of serum TBIL and DBIL decreased gradually in the transplantation group, and the content of serum TBIL and DBIL in the transplantation group decreased gradually. photograph group Compared with the control group, the difference between the 4th, 8th and 12th week was very significant (P <0.01). (P <0.01). Conclusion 1. Intraperitoneal injection of 40% carbon tetrachloride olive oil solution 1 2 weeks, the rabbit liver fibrosis model was successfully established: the changes of pathological histology and liver function biochemical indexes were in accordance with the criteria of hepatic fibrosis.
【學位授予單位】：昆明醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R575.2;R-332
本文編號：2259859
[Abstract]:Purpose 1. A preparation method of rabbit liver fibrosis animal model is established. Evaluation criteria. 2. Establishment of an animal model of hepatic fibrosis rabbit autologous bone marrow mesenchymal transition G Methods: To observe the number, migration and distribution of labeled cells in liver of rabbit liver after autologous bone marrow mesenchymal stem cell transplantation, and to detect the change of serum liver function index. and liver Histopathological analysis and evaluation of clinical efficacy. Methods 1. Establishment and evaluation of rabbit liver fibrosis model: 40 ordinary Japanese white rabbits were randomly divided into experimental group and experimental group. In the control group, 40% CCl _ 4 olive oil solution was injected into the abdominal cavity of the experimental group, and the same amount of normal saline was injected into the control group. The dosage was 0. 5 ml/ kg at 8 weeks after the start of the experiment. The liver group was taken from 30, 60 and 90 days after the start of the experiment. The pathological observation of HE staining and the detection of biochemical indexes were carried out in the tissues and serum samples. The culture and labelling of autologous bone marrow mesenchymal cells were carried out: 24 ordinary Japanese rabbits were randomly divided into experimental groups. In the control group, autologous bone marrow mesenchymal cells were transplanted in the experimental group and normal saline was infused in the control group. The experimental group model rabbits were subjected to percutaneous puncture to acquire bone marrow and density gradient centrifugation to separate the mononuclear cells from the bone marrow, the adherent cells were cultured to obtain the mesenchymal stem cells, and the green fluorescent protein (GFP) was used to mark the cells. The number of labeled cells was observed under fluorescence microscope, and the positive rate was 99%. 3. Autobone marrow mesenchymal stem cell transplantation was used for the treatment of hepatic fibrosis model rabbits and efficacy evaluation: the GPP gene was labeled with 3 ~ 10 ~ 6 cells/ ml bone marrow mesenchymal stem cells were implanted into the experimental group via superior mesenteric vein. In animal liver, the serum of rabbit was taken for liver function test at 0, 4, 8 and 12 weeks after transplantation, and the small part of liver tissue was taken as paraffin section, and the number, migration and distribution of transplanted cells were observed under fluorescence microscope. HE staining was performed on small hepatic tissue and hepatic fibrosis was observed. The changes of pathological histology after cell transplantation were analyzed. The statistical analysis showed that the experimental data were expressed in the form of mean square deviation ((?)/ s), and the statistical indexes of liver function of serum were analyzed by SPSS 13.0 software. The results of analysis of variance and LSD method were used for comparison among multiple levels. Inspection The independent sample t test was used for the comparison between the two horizontal groups, and the test value was P 0.05. The results showed that the model rabbit liver group Histopathological changes: The appearance of the liver was dark red in the control group. The pathological section showed that the structure of the hepatic lobule was complete, and the hepatocytes were arranged radially around the central vein. In the experimental group, the liver was dark red at 8 weeks, there were slight changes in the surface of the liver, and the pathological section showed that the liver cells The focal spot and focal necrosis, infiltration of inflammatory cells in the area of remit, showed early fibrosis. The liver was gray brown at 12 weeks, and there were obvious changes in granulation samples. The pathological section showed: The hepatic lobule structure was destroyed, hepatic cable arrangement disorder, liver cell fat degeneration, interstitial fibrous tissue proliferation, inflammatory cell infiltration, and obvious hepatic fibrosis expression. 2. Model rabbit blood biochemical index: As the time of injection CCL4 was prolonged, the ratio of white globulin decreased from 1. 26 to 0. 93, 0. 17, globulin, indirect bilirubin, direct bilirubin increased gradually, glutamic pyruvic transaminase, glutamic acid and ammonia were transferred to ammonia. The enzymes were elevated to 392. 00, 57. 93, 282. 00, 38. 54. 3, respectively, from 36. 80, 7. 66, 23. 40, and 14. 20. Morphological observation: The primary cells showed the characteristic spiral growth of MSCs, the arrangement of the cells was directional, the center cells of the vortex were distributed in multiple layers, the cell boundary was not clear, and the cell morphology Multi-shuttle-shaped. The growth rate of P1-generation cells increased significantly, and the colony-forming method The results showed that the cell morphology tended to be consistent. GFP-labeled cells were green fluorescence, and the positive rate was 99%. The fluorescence cells observed after transplantation were observed in the liver fibrosis tissues. The amount of green fluorescent cells is uniformly distributed and is compatible with the liver tissue, and the fluorescence cell migration range is wide, As time progresses, positive staining gradually increases and gradually extends inside the liver tissue to the liver's edge. 5. Histopathological observation after transplantation: remove 4 weeks after implantation, the control group had hepatic cord disorder, interstitial fibrosis, partially extended into the lobules, inflammatory cell infiltration, and the recovery of liver tissue structure in the treatment group was obvious. the above changes are lighter and degeneration, necrosis, The cells were significantly reduced. After 12 weeks after transplantation, there was little degeneration and necrosis of hepatocytes in the transplantation group, and a small amount of collagen fibers were deposited around the central vein and the junction area. The structure of the hepatic lobule was clear. Compared with the control group, there was no significant difference. 6. The serum indexes after transplantation were changed: the serum TP content after bone marrow stem cell transplantation. Compared with the control group, the content of GLO decreased gradually, the content of GLO in the transplantation group decreased gradually, the content of serum TBIL and DBIL decreased gradually in the transplantation group, and the content of serum TBIL and DBIL in the transplantation group decreased gradually. photograph group Compared with the control group, the difference between the 4th, 8th and 12th week was very significant (P <0.01). (P <0.01). Conclusion 1. Intraperitoneal injection of 40% carbon tetrachloride olive oil solution 1 2 weeks, the rabbit liver fibrosis model was successfully established: the changes of pathological histology and liver function biochemical indexes were in accordance with the criteria of hepatic fibrosis.
【學位授予單位】：昆明醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R575.2;R-332
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