胎盤羊膜間充質(zhì)干細胞的生物學特性及細胞移植對膠質(zhì)瘤生長抑制作用的實驗研究
發(fā)布時間：2018-08-25 11:36
【摘要】：間充質(zhì)干細胞(mesenchymal stem cells, MSCs)是一種具有自我更新和向中胚層來源細胞分化能力的多能干細胞。MSCs具有低免疫原性，細胞移植無免疫排斥反應，對腫瘤有靶向性和抑瘤性等，使其在腫瘤治療和損傷修復等研究中成為熱點。MSCs的來源取自成人骨髓最為常見，但取材時需要骨髓穿刺，且人骨髓中的MSCs含量極低，細胞數(shù)量和擴增、分化能力隨年齡增長均顯著下降，使其臨床應用受到限制。近年來，從胎盤羊膜間質(zhì)中獲取羊膜間充質(zhì)干細胞（amniotic mesenchymal stem cells,AMSCs）成為MSCs的新來源，具有材料供應豐富、取材方便、操作安全、污染機率少，細胞增殖能力強、擴增速度快等優(yōu)勢，為干細胞移植治療開辟新的途徑。 人腦膠質(zhì)瘤是中樞神經(jīng)系統(tǒng)中最常見的原發(fā)性惡性腫瘤，侵襲性強，發(fā)病早期即向周圍正常腦組織內(nèi)呈指狀浸潤性生長，術后易復發(fā)，患者預后差，死亡率高。近年來研究表明，MSCs具有向人腦膠質(zhì)瘤的特異性定向遷移能力，除了部分遷移到膠質(zhì)瘤內(nèi)，主要是呈“膠囊樣”分布于腦膠質(zhì)瘤體與正常腦實質(zhì)的邊界，并有部分細胞能“追蹤”瘤體外散在的膠質(zhì)瘤細胞，可成為膠質(zhì)瘤基因治療的理想載體。MSCs本身對于腦膠質(zhì)瘤具有抑瘤效應。體內(nèi)外研究表明MSCs可抑制膠質(zhì)瘤細胞增殖，可能與誘導細胞凋亡、阻礙細胞周期等有關。但胎盤來源的AMSCs對人腦膠質(zhì)瘤是否具有趨瘤和抑瘤效應，機制如何，有待進一步研究。 本實驗旨在建立有效的AMSCs體外培養(yǎng)擴增體系，經(jīng)誘導分化探討AMSCs的細胞生物學特性；通過體外實驗，觀察AMSCs定向遷移至膠質(zhì)瘤病變區(qū)域的能力及對腫瘤細胞生長的作用；建立裸鼠人腦膠質(zhì)瘤模型，瘤內(nèi)注射AMSCs，觀察移植后AMSCs的存活、遷移及對腫瘤生長的作用，初步探討AMSCs在體內(nèi)外趨瘤抑瘤效應的可能機制。 第一部分AMSCs的分離培養(yǎng)和細胞生物學特性鑒定 目的：建立有效的AMSCs體外培養(yǎng)擴增體系，通過誘導分化探討AMSCs的細胞生物學特性。 方法：取健康產(chǎn)婦正常剖宮產(chǎn)足月胎兒的新鮮胎盤組織，鈍性分離羊膜層，取羊膜組織消化培養(yǎng)，倒置顯微鏡觀察AMSCs的細胞形態(tài)；流式細胞儀檢測細胞表型；采用特定誘導條件將AMSCs分別向軟骨細胞、骨細胞、脂肪細胞和神經(jīng)組織方向誘導分化，采用特異性染色對誘導后細胞進行鑒定。RT-PCR法分析軟骨細胞、骨細胞、脂肪細胞及神經(jīng)組織特異性基因在誘導前后AMSCs中的表達。 結(jié)果：（1）倒置顯微鏡觀察AMSCs均呈典型的成纖維細胞樣貼壁生長，流式細胞儀分析顯示，AMSCs高表達CD73、CD90和CD105，不表達CD14、CD34、CD45和HLA-DR。 （2）AMSCs經(jīng)成骨誘導后，細胞均由長梭形向立方形轉(zhuǎn)變，von Kossa染色可見細胞呈集落生長并出現(xiàn)鈣結(jié)節(jié)；經(jīng)成軟骨誘導2w后，AMSCs形態(tài)逐漸變得扁平，甲苯胺藍染色可見細胞被染成藍色；經(jīng)成脂肪誘導2w后，細胞內(nèi)有明顯的脂滴出現(xiàn)，油紅O染色陽性；經(jīng)成神經(jīng)誘導24h后，AMSCs呈神經(jīng)膠質(zhì)細胞樣或/和神經(jīng)元樣改變，多數(shù)細胞呈現(xiàn)GFAP免疫熒光標記陽性。 （3）RT-PCR結(jié)果顯示：AMSCs向軟骨細胞、骨細胞、脂肪細胞誘導后，表達PLIN（脂肪細胞）、ACAN（軟骨細胞）和RUNX2（骨細胞）特異性基因；向神經(jīng)組織誘導前AMSCs即表達nestin mRNA、GFAP mRNA、mushashi-1mRNA以及β-tubulin III mRNA，誘導2d，除了表達以上基因外，還有NF mRNA表達，誘導5d，僅Nestin mRNA的表達有所下降。 結(jié)論：從人胎盤羊膜組織中經(jīng)過特定的分離消化培養(yǎng)易于獲得AMSCs，且增殖能力強和傳代穩(wěn)定。研究結(jié)果表明AMSCs具有MSCs干細胞標記及細胞生物學特性，具有多向分化能力。 第二部分AMSCs對膠質(zhì)瘤細胞生長抑制作用的體外研究 目的：觀察AMSCs向人腦膠質(zhì)瘤的定向遷移能力及對腫瘤細胞生長的作用，初步探討AMSCs體外趨瘤抑瘤效應的可能作用機制。 方法：在Transwell培養(yǎng)下室分別接種不同密度的人腦膠質(zhì)瘤細胞系U251細胞，觀察接種于上室的AMSCs的定向遷移能力。將生長良好的P3代AMSCs培養(yǎng)上清轉(zhuǎn)移到微型濃縮器中，離心制備上清濃縮蛋白，加入U251細胞培養(yǎng)基作用后，采用Transwell侵襲實驗檢測U251細胞侵襲力的變化，，透射電鏡觀察U251細胞形態(tài)學變化，AnnexinV-FITC-PI雙染法檢測細胞早期凋亡情況，RT-PCR法分析腫瘤細胞Casepase-3、Bax和Bcl-2的mRNA表達情況。 結(jié)果：（1）Transwell遷移實驗結(jié)果表明U251細胞可在體外共培養(yǎng)時增強AMSCs的定向遷移能力，且其效應與腫瘤細胞接種密度呈依賴性。 （2）經(jīng)AMSCs上清濃縮蛋白作用后，Transwell侵襲實驗結(jié)果表明U251細胞侵襲力降低。 （3）透射電鏡下可見細胞核固縮，核內(nèi)染色質(zhì)密集、趨邊凝聚明顯，質(zhì)膜脫落及凋亡小體等典型的凋亡形態(tài)特征。 （4）Annexin V-FITC-PI雙染結(jié)果顯示：U251細胞在AMSCs上清濃縮蛋白作用24h時后的凋亡率為9.34±4.27％，而在48h后凋亡率為42.93±11.54％，二者之間有顯著性差別(P0.05)，提示細胞凋亡率隨著作用時間的增加而升高。 （5）RT-PCR檢測結(jié)果表明：AMSCs上清濃縮蛋白作用24h后，U251細胞Casepase-3、Bax的mRNA表達水平明顯高于對照組，在48h時進一步升高，實驗組與對照組比較有顯著性差異(P0.05)；Bcl-2mRNA表達水平在24h和48h均低于對照組，實驗組與對照組比較有顯著性差異(P0.05)。 結(jié)論：膠質(zhì)瘤微環(huán)境中分泌的各種趨化因子可增強AMSCs定向遷移能力，與接種的膠質(zhì)瘤細胞密度呈依賴性。AMSCs可抑制膠質(zhì)瘤細胞的侵襲能力。AMSCs可在體外抑制膠質(zhì)瘤細胞的增殖，并誘導其凋亡，其機理可能是Bcl-2/Bax比值下降，激活caspase-3，最終導致膠質(zhì)瘤細胞凋亡。 第三部分AMSCs對膠質(zhì)瘤生長抑制作用的體內(nèi)研究 目的：裸鼠瘤內(nèi)注射AMSCs，觀察AMSCs的存活、遷移以及移植對實體腫瘤的作用，初步探討AMSCs趨瘤抑瘤的可能機制。 方法：采用雄性裸鼠腋窩皮下注射U251細胞制作人腦膠質(zhì)瘤模型，隨機分為3組。對照組正常飼養(yǎng)，不予任何處理；PBS組瘤內(nèi)注射0.2ml PBS緩沖液；AMSCs移植組瘤內(nèi)注射BrdU標記的0.2ml AMSCs懸液，每天測量腫瘤大小。AMSCs移植14d后處死，取出腫瘤組織，HE染色觀察腫瘤病理特征改變；免疫熒光染色觀察AMSCs存活、遷移情況；透射電鏡觀察腫瘤細胞超微結(jié)構改變；RT-PCR法分析腫瘤組織Casepase-3、Bcl-2、Bax的mRNA表達情況。 結(jié)果：（1）裸鼠皮下人腦膠質(zhì)瘤造模成功率高，腫瘤接種3d后在腋窩皮下可見腫塊，7d后皮下腫瘤可達5mm左右，腫瘤大小隨時間的延長而增大。對照組和PBS組腫瘤的生長基本一致，AMSCs移植組腫瘤生長出現(xiàn)明顯抑制，與對照組和PBS組相比有顯著性差異（P0.05），實驗表明瘤內(nèi)注射AMSCs可顯著抑制裸鼠膠質(zhì)瘤的生長。 （2）光鏡觀察可見腫瘤細胞排列致密，毛細血管增生明顯，部分可見壞死，周邊可見腫瘤呈浸潤性生長，侵入肌組織之間。病理性核分裂相多見，呈異型性。 （3）免疫熒光染色顯示瘤內(nèi)有較多的BrdU陽性細胞。 （4）透射電鏡下可觀察到腫瘤細胞核漿比高，核固縮、染色質(zhì)趨邊凝聚及凋亡小體等典型的凋亡形態(tài)特征。 （5）RT-PCR檢測結(jié)果表明AMSCs移植組腫瘤組織Casepase-3、Bax的mRNA表達水平明顯高于對照組(P0.05)，Bcl-2mRNA水平顯著低于對照組(P0.05)。 結(jié)論：裸鼠膠質(zhì)瘤模型瘤內(nèi)移植AMSCs可在瘤內(nèi)存活、遷移，通過誘導細胞凋亡等過程抑制膠質(zhì)瘤的生長，對腦膠質(zhì)瘤的臨床治療，可能成為更為有效的途徑。
[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.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R739.41
本文編號：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.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R739.41
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