胎盘羊膜间充质干细胞的生物学特性及细胞移植对胶质瘤生长抑制作用的实验研究
发布时间：2018-08-25 11:36
【摘要】：间充质干细胞(mesenchymal stem cells, MSCs)是一种具有自我更新和向中胚层来源细胞分化能力的多能干细胞。MSCs具有低免疫原性，细胞移植无免疫排斥反应，对肿瘤有靶向性和抑瘤性等，使其在肿瘤治疗和损伤修复等研究中成为热点。MSCs的来源取自成人骨髓最为常见，但取材时需要骨髓穿刺，且人骨髓中的MSCs含量极低，细胞数量和扩增、分化能力随年龄增长均显著下降，使其临床应用受到限制。近年来，从胎盘羊膜间质中获取羊膜间充质干细胞（amniotic mesenchymal stem cells,AMSCs）成为MSCs的新来源，具有材料供应丰富、取材方便、操作安全、污染机率少，细胞增殖能力强、扩增速度快等优势，为干细胞移植治疗开辟新的途径。 人脑胶质瘤是中枢神经系统中最常见的原发性恶性肿瘤，侵袭性强，发病早期即向周围正常脑组织内呈指状浸润性生长，术后易复发，患者预后差，死亡率高。近年来研究表明，MSCs具有向人脑胶质瘤的特异性定向迁移能力，除了部分迁移到胶质瘤内，主要是呈“胶囊样”分布于脑胶质瘤体与正常脑实质的边界，并有部分细胞能“追踪”瘤体外散在的胶质瘤细胞，可成为胶质瘤基因治疗的理想载体。MSCs本身对于脑胶质瘤具有抑瘤效应。体内外研究表明MSCs可抑制胶质瘤细胞增殖，可能与诱导细胞凋亡、阻碍细胞周期等有关。但胎盘来源的AMSCs对人脑胶质瘤是否具有趋瘤和抑瘤效应，机制如何，有待进一步研究。 本实验旨在建立有效的AMSCs体外培养扩增体系，经诱导分化探讨AMSCs的细胞生物学特性；通过体外实验，观察AMSCs定向迁移至胶质瘤病变区域的能力及对肿瘤细胞生长的作用；建立裸鼠人脑胶质瘤模型，瘤内注射AMSCs，观察移植后AMSCs的存活、迁移及对肿瘤生长的作用，初步探讨AMSCs在体内外趋瘤抑瘤效应的可能机制。 第一部分AMSCs的分离培养和细胞生物学特性鉴定 目的：建立有效的AMSCs体外培养扩增体系，通过诱导分化探讨AMSCs的细胞生物学特性。 方法：取健康产妇正常剖宫产足月胎儿的新鲜胎盘组织，钝性分离羊膜层，取羊膜组织消化培养，倒置显微镜观察AMSCs的细胞形态；流式细胞仪检测细胞表型；采用特定诱导条件将AMSCs分别向软骨细胞、骨细胞、脂肪细胞和神经组织方向诱导分化，采用特异性染色对诱导后细胞进行鉴定。RT-PCR法分析软骨细胞、骨细胞、脂肪细胞及神经组织特异性基因在诱导前后AMSCs中的表达。 结果：（1）倒置显微镜观察AMSCs均呈典型的成纤维细胞样贴壁生长，流式细胞仪分析显示，AMSCs高表达CD73、CD90和CD105，不表达CD14、CD34、CD45和HLA-DR。 （2）AMSCs经成骨诱导后，细胞均由长梭形向立方形转变，von Kossa染色可见细胞呈集落生长并出现钙结节；经成软骨诱导2w后，AMSCs形态逐渐变得扁平，甲苯胺蓝染色可见细胞被染成蓝色；经成脂肪诱导2w后，细胞内有明显的脂滴出现，油红O染色阳性；经成神经诱导24h后，AMSCs呈神经胶质细胞样或/和神经元样改变，多数细胞呈现GFAP免疫荧光标记阳性。 （3）RT-PCR结果显示：AMSCs向软骨细胞、骨细胞、脂肪细胞诱导后，表达PLIN（脂肪细胞）、ACAN（软骨细胞）和RUNX2（骨细胞）特异性基因；向神经组织诱导前AMSCs即表达nestin mRNA、GFAP mRNA、mushashi-1mRNA以及β-tubulin III mRNA，诱导2d，除了表达以上基因外，还有NF mRNA表达，诱导5d，仅Nestin mRNA的表达有所下降。 结论：从人胎盘羊膜组织中经过特定的分离消化培养易于获得AMSCs，且增殖能力强和传代稳定。研究结果表明AMSCs具有MSCs干细胞标记及细胞生物学特性，具有多向分化能力。 第二部分AMSCs对胶质瘤细胞生长抑制作用的体外研究 目的：观察AMSCs向人脑胶质瘤的定向迁移能力及对肿瘤细胞生长的作用，初步探讨AMSCs体外趋瘤抑瘤效应的可能作用机制。 方法：在Transwell培养下室分别接种不同密度的人脑胶质瘤细胞系U251细胞，观察接种于上室的AMSCs的定向迁移能力。将生长良好的P3代AMSCs培养上清转移到微型浓缩器中，离心制备上清浓缩蛋白，加入U251细胞培养基作用后，采用Transwell侵袭实验检测U251细胞侵袭力的变化，，透射电镜观察U251细胞形态学变化，AnnexinV-FITC-PI双染法检测细胞早期凋亡情况，RT-PCR法分析肿瘤细胞Casepase-3、Bax和Bcl-2的mRNA表达情况。 结果：（1）Transwell迁移实验结果表明U251细胞可在体外共培养时增强AMSCs的定向迁移能力，且其效应与肿瘤细胞接种密度呈依赖性。 （2）经AMSCs上清浓缩蛋白作用后，Transwell侵袭实验结果表明U251细胞侵袭力降低。 （3）透射电镜下可见细胞核固缩，核内染色质密集、趋边凝聚明显，质膜脱落及凋亡小体等典型的凋亡形态特征。 （4）Annexin V-FITC-PI双染结果显示：U251细胞在AMSCs上清浓缩蛋白作用24h时后的凋亡率为9.34±4.27％，而在48h后凋亡率为42.93±11.54％，二者之间有显著性差别(P0.05)，提示细胞凋亡率随着作用时间的增加而升高。 （5）RT-PCR检测结果表明：AMSCs上清浓缩蛋白作用24h后，U251细胞Casepase-3、Bax的mRNA表达水平明显高于对照组，在48h时进一步升高，实验组与对照组比较有显著性差异(P0.05)；Bcl-2mRNA表达水平在24h和48h均低于对照组，实验组与对照组比较有显著性差异(P0.05)。 结论：胶质瘤微环境中分泌的各种趋化因子可增强AMSCs定向迁移能力，与接种的胶质瘤细胞密度呈依赖性。AMSCs可抑制胶质瘤细胞的侵袭能力。AMSCs可在体外抑制胶质瘤细胞的增殖，并诱导其凋亡，其机理可能是Bcl-2/Bax比值下降，激活caspase-3，最终导致胶质瘤细胞凋亡。 第三部分AMSCs对胶质瘤生长抑制作用的体内研究 目的：裸鼠瘤内注射AMSCs，观察AMSCs的存活、迁移以及移植对实体肿瘤的作用，初步探讨AMSCs趋瘤抑瘤的可能机制。 方法：采用雄性裸鼠腋窝皮下注射U251细胞制作人脑胶质瘤模型，随机分为3组。对照组正常饲养，不予任何处理；PBS组瘤内注射0.2ml PBS缓冲液；AMSCs移植组瘤内注射BrdU标记的0.2ml AMSCs悬液，每天测量肿瘤大小。AMSCs移植14d后处死，取出肿瘤组织，HE染色观察肿瘤病理特征改变；免疫荧光染色观察AMSCs存活、迁移情况；透射电镜观察肿瘤细胞超微结构改变；RT-PCR法分析肿瘤组织Casepase-3、Bcl-2、Bax的mRNA表达情况。 结果：（1）裸鼠皮下人脑胶质瘤造模成功率高，肿瘤接种3d后在腋窝皮下可见肿块，7d后皮下肿瘤可达5mm左右，肿瘤大小随时间的延长而增大。对照组和PBS组肿瘤的生长基本一致，AMSCs移植组肿瘤生长出现明显抑制，与对照组和PBS组相比有显著性差异（P0.05），实验表明瘤内注射AMSCs可显著抑制裸鼠胶质瘤的生长。 （2）光镜观察可见肿瘤细胞排列致密，毛细血管增生明显，部分可见坏死，周边可见肿瘤呈浸润性生长，侵入肌组织之间。病理性核分裂相多见，呈异型性。 （3）免疫荧光染色显示瘤内有较多的BrdU阳性细胞。 （4）透射电镜下可观察到肿瘤细胞核浆比高，核固缩、染色质趋边凝聚及凋亡小体等典型的凋亡形态特征。 （5）RT-PCR检测结果表明AMSCs移植组肿瘤组织Casepase-3、Bax的mRNA表达水平明显高于对照组(P0.05)，Bcl-2mRNA水平显著低于对照组(P0.05)。 结论：裸鼠胶质瘤模型瘤内移植AMSCs可在瘤内存活、迁移，通过诱导细胞凋亡等过程抑制胶质瘤的生长，对脑胶质瘤的临床治疗，可能成为更为有效的途径。
[Abstract]:Mesenchymal stem cells (MSCs) are a kind of pluripotent stem cells with the ability of self-renewal and differentiation into mesodermal-derived cells. MSCs have low immunogenicity, no immunorejection, targeted and tumor-suppressing properties, which make them a hot spot in the research of tumor treatment and damage repair. The clinical application of amniotic mesenchymal stem cells (AMS) derived from amniotic mesenchymal cells (AMS) has been limited in recent years. As a new source of MSCs, it has many advantages, such as abundant material supply, convenient material selection, safe operation, less contamination probability, strong cell proliferation ability, rapid expansion speed, etc. It opens up a new way for stem cell transplantation treatment.
Human gliomas are the most common primary malignant tumors in the central nervous system. They are highly invasive and tend to recur after surgery. Recent studies have shown that MSCs have specific directional migration ability to human gliomas except for partial migration. In glioma, it is mainly distributed in the boundary between glioma and normal brain parenchyma in a "capsule-like" manner, and some cells can "track" glioma cells scattered in vitro. MSCs can be an ideal carrier for gene therapy of glioma. Cell proliferation may be related to inducing apoptosis and blocking cell cycle. However, whether placental-derived AMSCs have tumor-chemotactic and tumor-suppressive effects on human glioma and the mechanism need to be further studied.
The aim of this study was to establish an effective culture and amplification system of AMSCs in vitro, to investigate the cellular biological characteristics of AMSCs by inducing differentiation, to observe the ability of directional migration of AMSCs to glioma lesion area and its effect on tumor cell growth in vitro, to establish a human glioma model in nude mice, and to inject AMSCs into tumors to observe the AMSCs after transplantation. Survival, migration and tumor growth of AMSCs in vivo and in vitro, to explore the possible mechanism of tumor chemotaxis and inhibition of AMSCs.
Part 1 Isolation and culture of AMSCs and identification of cell biological characteristics
AIM: To establish an effective culture and amplification system of AMSCs in vitro and to investigate the cellular biological characteristics of AMSCs by inducing differentiation.
Methods: Fresh placenta tissues of full-term fetuses of healthy parturients were obtained from normal cesarean section. Amniotic membrane was obtusely isolated and digested. The morphology of AMSCs was observed by inverted microscope. The phenotype of AMSCs was detected by flow cytometry. To induce differentiation, specific staining was used to identify the cells after induction. RT-PCR was used to analyze the expression of chondrocytes, osteocytes, adipocytes and neural tissue-specific genes in AMSCs before and after induction.
Results: (1) AMSCs showed typical fibroblast-like adherent growth under inverted microscope. Flow cytometry analysis showed that AMSCs overexpressed CD73, CD90 and CD105, but did not express CD14, CD34, CD45 and HLA-DR.
(2) After induction of osteogenesis, AMSCs changed from long spindle to cubic shape. Von Kossa staining showed that AMSCs grew in colony and appeared calcium nodules. After induction of cartilage for 2 weeks, AMSCs gradually became flat, toluidine blue staining showed that AMSCs cells were stained blue. After induction of adipogenesis for 2 weeks, there were obvious lipid droplets and oil red O in cells. Immunofluorescence staining showed that AMSCs showed glial-like or/or neuronal-like changes 24 hours after induction of neurogenesis, and GFAP immunofluorescence staining was positive in most cells.
(3) The results of RT-PCR showed that AMSCs expressed PLIN (adipocyte), ACAN (chondrocyte) and RUNX2 (osteocyte) specific genes in chondrocytes, osteocytes and adipocytes after induction; AMSCs expressed nestin mRNA, GFAP mRNA, mushashi-1 mRNA and beta-tubulin III mRNA before induction into neural tissues, and induced for 2 days, in addition to the above genes. With the expression of NF mRNA, the expression of Nestin mRNA decreased only after induction of 5D.
CONCLUSION: AMSCs can be easily obtained from human placental amniotic membrane by specific isolation, digestion and culture. AMSCs have strong proliferative ability and stable passage.
The second part is the inhibitory effect of AMSCs on the growth of glioma cells in vitro.
AIM: To observe the directional migration of AMSCs to human glioma cells and its effect on tumor cell growth, and to explore the possible mechanism of tumor chemotaxis and inhibition of AMSCs in vitro.
Methods: Human glioma U251 cells with different densities were inoculated in Transwell culture chamber to observe the directional migration ability of AMSCs inoculated in the superior chamber. Invasive ability of U251 cells was detected by invasive assay, morphological changes of U251 cells were observed by transmission electron microscopy, early apoptosis was detected by Annexin V-FITC-PI double staining, and Casepase-3, Bax and Bcl-2 mRNA expression was analyzed by RT-PCR.
Results: (1) Transwell migration test showed that U251 cells could enhance the directional migration of AMSCs in vitro, and the effect was dependent on the density of tumor cells.
(2) after AMSCs supernatant protein concentration, Transwell invasion test showed that U251 cell invasion decreased.
(3) The typical morphological features of apoptosis were observed under transmission electron microscopy, such as nuclear pyknosis, dense chromatin, obvious edge coagulation, plasmalemma exfoliation and apoptotic bodies.
(4) The results of Annexin V-FITC-PI double staining showed that the apoptotic rate of U251 cells treated with AMSCs supernatant concentrate protein for 24 hours was 9.34 [4.27], while that of U251 cells treated with AMSCs supernatant concentrate protein for 48 hours was 42.93 [11.54]. There was a significant difference between them (P 0.05), suggesting that the apoptotic rate increased with the time.
(5) The results of RT-PCR showed that the expression of Casepase-3 and Bax mRNA in U251 cells was significantly higher than that in the control group after 24 hours of AMSCs supernatant concentrate treatment, and further increased at 48 hours. The expression of Bcl-2 mRNA in the experimental group was significantly lower than that in the control group at 24 hours and 48 hours (P 0.05). Sex differences (P0.05).
CONCLUSION: Chemokines secreted in the microenvironment of glioma can enhance the directional migration of AMSCs, which is dependent on the density of inoculated glioma cells. AMSCs can inhibit the invasion of glioma cells. AMSCs can inhibit the proliferation of glioma cells in vitro and induce their apoptosis. The mechanism may be the decrease of Bcl-2/Bax ratio and the activation of caspa. Se-3 eventually leads to apoptosis of glioma cells.
The third part is the in vivo study of the inhibitory effect of AMSCs on glioma growth.
Objective: To observe the survival, migration and transplantation of AMSCs in nude mice by intratumoral injection of AMSCs, and to explore the possible mechanism of tumor chemotaxis and inhibition of AMSCs.
Methods: Human glioma models were made by subcutaneous injection of U251 cells into the axilla of male nude mice and randomly divided into three groups.The control group was fed normally without any treatment.The PBS group was injected with 0.2 ml PBS buffer. HE staining was used to observe the pathological changes of tumor tissue, immunofluorescence staining was used to observe the survival and migration of AMSCs, transmission electron microscopy was used to observe the ultrastructural changes of tumor cells, and RT-PCR was used to analyze the mRNA expression of Casepase-3, Bcl-2 and Bax in tumor tissue.
Results: (1) The success rate of subcutaneous human glioma modeling in nude mice was high. The tumors could be seen subcutaneously in axillary region 3 days after inoculation. The size of the tumors increased with time. The growth of tumors in control group and PBS group was basically the same. The growth of tumors in AMSCs transplantation group was significantly inhibited compared with that in control group and PBS group. Significant difference (P0.05) showed that intratumoral injection of AMSCs could significantly inhibit the growth of glioma in nude mice.
(2) Light microscopic observation showed that the tumor cells arranged densely, capillary hyperplasia was evident, and some necrosis was seen. Peripheral tumor showed infiltrative growth, invading between muscle tissues. Pathological mitosis was common and atypical.
(3) immunofluorescence staining showed that there were more BrdU positive cells in the tumor.
(4) Typical morphological features of apoptosis were observed under transmission electron microscopy, such as high nuclear-cytoplasmic ratio, nuclear condensation, chromatin edgewise condensation and apoptotic bodies.
(5) The results of RT-PCR showed that the expression of Casepase-3 and Bax mRNA in tumor tissues of AMSCs transplantation group was significantly higher than that of control group (P 0.05), and the level of Bcl-2 mRNA was significantly lower than that of control group (P 0.05).
Conclusion: AMSCs transplantation in nude mice glioma model can inhibit the growth of glioma by inducing apoptosis, which may be a more effective way to treat glioma.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41
本文编号：2202758
[Abstract]:Mesenchymal stem cells (MSCs) are a kind of pluripotent stem cells with the ability of self-renewal and differentiation into mesodermal-derived cells. MSCs have low immunogenicity, no immunorejection, targeted and tumor-suppressing properties, which make them a hot spot in the research of tumor treatment and damage repair. The clinical application of amniotic mesenchymal stem cells (AMS) derived from amniotic mesenchymal cells (AMS) has been limited in recent years. As a new source of MSCs, it has many advantages, such as abundant material supply, convenient material selection, safe operation, less contamination probability, strong cell proliferation ability, rapid expansion speed, etc. It opens up a new way for stem cell transplantation treatment.
Human gliomas are the most common primary malignant tumors in the central nervous system. They are highly invasive and tend to recur after surgery. Recent studies have shown that MSCs have specific directional migration ability to human gliomas except for partial migration. In glioma, it is mainly distributed in the boundary between glioma and normal brain parenchyma in a "capsule-like" manner, and some cells can "track" glioma cells scattered in vitro. MSCs can be an ideal carrier for gene therapy of glioma. Cell proliferation may be related to inducing apoptosis and blocking cell cycle. However, whether placental-derived AMSCs have tumor-chemotactic and tumor-suppressive effects on human glioma and the mechanism need to be further studied.
The aim of this study was to establish an effective culture and amplification system of AMSCs in vitro, to investigate the cellular biological characteristics of AMSCs by inducing differentiation, to observe the ability of directional migration of AMSCs to glioma lesion area and its effect on tumor cell growth in vitro, to establish a human glioma model in nude mice, and to inject AMSCs into tumors to observe the AMSCs after transplantation. Survival, migration and tumor growth of AMSCs in vivo and in vitro, to explore the possible mechanism of tumor chemotaxis and inhibition of AMSCs.
Part 1 Isolation and culture of AMSCs and identification of cell biological characteristics
AIM: To establish an effective culture and amplification system of AMSCs in vitro and to investigate the cellular biological characteristics of AMSCs by inducing differentiation.
Methods: Fresh placenta tissues of full-term fetuses of healthy parturients were obtained from normal cesarean section. Amniotic membrane was obtusely isolated and digested. The morphology of AMSCs was observed by inverted microscope. The phenotype of AMSCs was detected by flow cytometry. To induce differentiation, specific staining was used to identify the cells after induction. RT-PCR was used to analyze the expression of chondrocytes, osteocytes, adipocytes and neural tissue-specific genes in AMSCs before and after induction.
Results: (1) AMSCs showed typical fibroblast-like adherent growth under inverted microscope. Flow cytometry analysis showed that AMSCs overexpressed CD73, CD90 and CD105, but did not express CD14, CD34, CD45 and HLA-DR.
(2) After induction of osteogenesis, AMSCs changed from long spindle to cubic shape. Von Kossa staining showed that AMSCs grew in colony and appeared calcium nodules. After induction of cartilage for 2 weeks, AMSCs gradually became flat, toluidine blue staining showed that AMSCs cells were stained blue. After induction of adipogenesis for 2 weeks, there were obvious lipid droplets and oil red O in cells. Immunofluorescence staining showed that AMSCs showed glial-like or/or neuronal-like changes 24 hours after induction of neurogenesis, and GFAP immunofluorescence staining was positive in most cells.
(3) The results of RT-PCR showed that AMSCs expressed PLIN (adipocyte), ACAN (chondrocyte) and RUNX2 (osteocyte) specific genes in chondrocytes, osteocytes and adipocytes after induction; AMSCs expressed nestin mRNA, GFAP mRNA, mushashi-1 mRNA and beta-tubulin III mRNA before induction into neural tissues, and induced for 2 days, in addition to the above genes. With the expression of NF mRNA, the expression of Nestin mRNA decreased only after induction of 5D.
CONCLUSION: AMSCs can be easily obtained from human placental amniotic membrane by specific isolation, digestion and culture. AMSCs have strong proliferative ability and stable passage.
The second part is the inhibitory effect of AMSCs on the growth of glioma cells in vitro.
AIM: To observe the directional migration of AMSCs to human glioma cells and its effect on tumor cell growth, and to explore the possible mechanism of tumor chemotaxis and inhibition of AMSCs in vitro.
Methods: Human glioma U251 cells with different densities were inoculated in Transwell culture chamber to observe the directional migration ability of AMSCs inoculated in the superior chamber. Invasive ability of U251 cells was detected by invasive assay, morphological changes of U251 cells were observed by transmission electron microscopy, early apoptosis was detected by Annexin V-FITC-PI double staining, and Casepase-3, Bax and Bcl-2 mRNA expression was analyzed by RT-PCR.
Results: (1) Transwell migration test showed that U251 cells could enhance the directional migration of AMSCs in vitro, and the effect was dependent on the density of tumor cells.
(2) after AMSCs supernatant protein concentration, Transwell invasion test showed that U251 cell invasion decreased.
(3) The typical morphological features of apoptosis were observed under transmission electron microscopy, such as nuclear pyknosis, dense chromatin, obvious edge coagulation, plasmalemma exfoliation and apoptotic bodies.
(4) The results of Annexin V-FITC-PI double staining showed that the apoptotic rate of U251 cells treated with AMSCs supernatant concentrate protein for 24 hours was 9.34 [4.27], while that of U251 cells treated with AMSCs supernatant concentrate protein for 48 hours was 42.93 [11.54]. There was a significant difference between them (P 0.05), suggesting that the apoptotic rate increased with the time.
(5) The results of RT-PCR showed that the expression of Casepase-3 and Bax mRNA in U251 cells was significantly higher than that in the control group after 24 hours of AMSCs supernatant concentrate treatment, and further increased at 48 hours. The expression of Bcl-2 mRNA in the experimental group was significantly lower than that in the control group at 24 hours and 48 hours (P 0.05). Sex differences (P0.05).
CONCLUSION: Chemokines secreted in the microenvironment of glioma can enhance the directional migration of AMSCs, which is dependent on the density of inoculated glioma cells. AMSCs can inhibit the invasion of glioma cells. AMSCs can inhibit the proliferation of glioma cells in vitro and induce their apoptosis. The mechanism may be the decrease of Bcl-2/Bax ratio and the activation of caspa. Se-3 eventually leads to apoptosis of glioma cells.
The third part is the in vivo study of the inhibitory effect of AMSCs on glioma growth.
Objective: To observe the survival, migration and transplantation of AMSCs in nude mice by intratumoral injection of AMSCs, and to explore the possible mechanism of tumor chemotaxis and inhibition of AMSCs.
Methods: Human glioma models were made by subcutaneous injection of U251 cells into the axilla of male nude mice and randomly divided into three groups.The control group was fed normally without any treatment.The PBS group was injected with 0.2 ml PBS buffer. HE staining was used to observe the pathological changes of tumor tissue, immunofluorescence staining was used to observe the survival and migration of AMSCs, transmission electron microscopy was used to observe the ultrastructural changes of tumor cells, and RT-PCR was used to analyze the mRNA expression of Casepase-3, Bcl-2 and Bax in tumor tissue.
Results: (1) The success rate of subcutaneous human glioma modeling in nude mice was high. The tumors could be seen subcutaneously in axillary region 3 days after inoculation. The size of the tumors increased with time. The growth of tumors in control group and PBS group was basically the same. The growth of tumors in AMSCs transplantation group was significantly inhibited compared with that in control group and PBS group. Significant difference (P0.05) showed that intratumoral injection of AMSCs could significantly inhibit the growth of glioma in nude mice.
(2) Light microscopic observation showed that the tumor cells arranged densely, capillary hyperplasia was evident, and some necrosis was seen. Peripheral tumor showed infiltrative growth, invading between muscle tissues. Pathological mitosis was common and atypical.
(3) immunofluorescence staining showed that there were more BrdU positive cells in the tumor.
(4) Typical morphological features of apoptosis were observed under transmission electron microscopy, such as high nuclear-cytoplasmic ratio, nuclear condensation, chromatin edgewise condensation and apoptotic bodies.
(5) The results of RT-PCR showed that the expression of Casepase-3 and Bax mRNA in tumor tissues of AMSCs transplantation group was significantly higher than that of control group (P 0.05), and the level of Bcl-2 mRNA was significantly lower than that of control group (P 0.05).
Conclusion: AMSCs transplantation in nude mice glioma model can inhibit the growth of glioma by inducing apoptosis, which may be a more effective way to treat glioma.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41
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