骨髓间充质干细胞分离培养及其向肿瘤局部归巢机制的初步探索

发布时间：2018-01-14 02:29

本文关键词：骨髓间充质干细胞分离培养及其向肿瘤局部归巢机制的初步探索　出处：《华中科技大学》2010年硕士论文　论文类型：学位论文

【摘要】：【研究背景及目的】骨髓间充质干细胞是一类具有多能分化潜能的细胞,可以向骨组织,心肌,软骨组织,肌肉等分化,并且可以特异迁移到损伤部位及肿瘤局部,被认为是具有应用前景的基因治疗细胞载体。然而,间充质干细胞靶向归巢肿瘤局部的机制却不太清楚。本研究在于分离人类骨髓间充质干细胞(Mesenchymal stem cells,MSCs),并探讨其体内靶向归巢肿瘤局部的机制。【方法】(1)密度梯度离心法获得骨髓间充质干细胞,并体外培养扩增。(2)流式细胞仪及激光共聚焦显微镜检测骨髓间充质干细胞表面标志分子CD44、CD105、CD90、CD34、CD45、CD19;不同条件培养基诱导骨髓间充质干细胞的成骨分化、软骨分化及脂肪分化,倒置显微镜观察。(3)建立小鼠原位乳腺肿瘤模型,尾静脉注射菲力磁(SPIO)体外标记的骨髓间充质干细胞,利用冰冻切片对骨髓间充质干细胞在小鼠肿瘤局部的分布情况进行检测。(4)取新鲜乳腺癌组织,剪碎,胶原酶消化成单细胞悬液,细胞标记抗CD31单克隆抗体,免疫磁柱分离抗CD31单克隆抗体阳性细胞,即肿瘤血管内皮细胞,并且体外原代培养。(5)流式细胞仪及激光共聚焦显微镜检测肿瘤血管内皮细胞标志性分子CD31、CD34、vWF。(6)Transwell模型验证肿瘤细胞对间充质干细胞的体外趋化能力。(7)Transwell模型血管内皮细胞对骨髓间充质干细胞体外趋化能力,结晶紫染色,显微镜照相,每个滤膜随机选取5个200倍视野计数穿膜细胞数,穿膜细胞数表示骨髓间充质干细胞的体外迁移能力。【结果】(1)原代贴壁细胞呈长梭形。体外培养一周,细胞形态均一,扩增速度快,克隆形成能力强。(2)流式细胞仪检测发现骨髓间充质干细胞表面分子标志物CD44、CD105、CD90阳性率分别为99.79±3.35% , 99.62±3.78%,98.93±3.62%,阴性标志物CD34、CD45、CD19为4.32±1.01%,4.04±1.05%,6.04±0.95%;激光共聚焦显微镜下,可见骨髓间充质干细胞阳性标志物在细胞内存在较高表达。不同的条件培养液成功诱导骨髓间充质干细胞向骨组织、脂肪组织及软骨组织分化,验证了骨髓间充质干细胞多向分化潜能。(3)体外SPIO标记的骨髓间充质干细胞通过尾静脉注射到荷瘤小鼠体内,细胞主要聚集在肿瘤局部。(4)免疫磁珠方法分离的肿瘤血管内皮细胞,6-12小时贴壁生长,早期细胞呈小多角、球形,逐渐生长成梭形,于3～4d后融合,为单层呈铺路石状排列。(5)流式细胞仪检测发现,CD31、CD34、vWF阳性率分别为92.8±2.32%,99.87±1.95%,86.26±2.57%(6)Boyden小室结果显示:在相同时间点,下室接种MDA -MB-231及MDA -MB-435s细胞组与对照组相比,穿膜细胞数无明显改变。(7)Boyden小室结果显示:肿瘤血管内皮细胞组,骨髓间充质干细胞穿膜细胞数明显多于对照组(p㩳0.05) 【结论】体外可获得较高纯度的骨髓间充质干细胞,且易于培养扩增。骨髓间充质干细胞体内靶向归巢肿瘤局部的能力与肿瘤局部血管内皮细胞有关。目的:研究Snail与宫颈癌侵袭转移间的关系,并探讨其分子机制。方法:构建正义全长Snail真核表达载体并转染宫颈癌细胞系Hela、Siha。采用Western blot方法分析相关基因表达,细胞伤痕愈合实验,Transwell模型穿膜细胞计数检测细胞侵袭转移能力。结果:(1)在转染PEGFPC1/Snail的宫颈癌细胞株Hela、SiHa中E-cadherin表达明显下调,Snail和Vimentin则表达上调(p0.05)。(2)宫颈癌细胞系Hela、Siha转染正义全长Snail真核表达载体,12小时细胞伤痕愈合能力显著提高(p0.05);转染PEGFPC1/Snail后,Hela、SiHa细胞12小时穿膜细胞数明显增多(p0.05)。Snail过表达可显著促进宫颈癌细胞株Hela、SiHa的侵袭转移潜能。结论:转录因子Snail促进宫颈癌上皮细胞间质化转变(EMT)并提高其侵袭转移能力。
[Abstract]:[background and objective] bone marrow mesenchymal stem cell is a kind of multipotent differentiation potential of cells, to bone tissue, muscle, cartilage tissue, muscle specific differentiation, and can migrate to the injury site and local tumor, gene therapy is considered to be a promising cell carrier. However, MSCs mesenchymal stem cells targeting tumor homing mechanism was not clear. This study is the isolation of human bone marrow mesenchymal stem cells (Mesenchymal stem cells, MSCs), and investigate its in vivo targeting homing mechanism of local tumor.
[Methods] (1) bone marrow mesenchymal stem cells obtained by density gradient centrifugation and cultured in vitro. (2) by flow cytometry and confocal microscopy for detection of bone marrow mesenchymal stem cell surface marker CD44, CD105, CD90, CD34, CD45, CD19; group of bone marrow mesenchymal stem osteogenic differentiation of different cell culture conditions, cartilage differentiation and adipocyte differentiation, inverted microscope. (3) establish a mouse model of breast cancer in situ, intravenous injection of Feridex (SPIO) labeled bone marrow mesenchymal stem cells, bone marrow mesenchymal stem cells were detected in the distribution of tumor by frozen section. (4) from the fresh breast cancer tissue, cut into pieces, collagenase digested into single cell suspension cells labeled with anti CD31 monoclonal antibody, anti CD31 monoclonal antibody positive cells separation immunomagnetic column, namely tumor vascular endothelial cell, and cultured in vitro. (5) Flow cytometry and confocal microscopy for detection of tumor vascular endothelial cell markers CD31, CD34, vWF. (6) Transwell model verification of tumor cells in vitro on mesenchymal stem cell chemotaxis ability. (7) the Transwell model of vascular endothelial cells on bone marrow mesenchymal stem cells in vitro chemotaxis ability, crystal violet staining, microscopy, each membrane 5 randomly selected 200 times magnification counting transmembrane cell number, the number of penetrating cells expressed mesenchymal stem cells migration ability in vitro.
[results] (1) adherent cells were fusiform. In vitro cultured for one week, the cells were all, amplification speed, clone formation ability. (2) flow cytometry demonstrated that bone marrow mesenchymal stem cell markers CD44, surface molecules CD105, CD90 positive rate was 99.79. 3.35%, 99.62 + 3.78%, 98.93 + 3.62%, negative markers CD34, CD45, CD19 was 4.32 + 1.01%, 4.04 + 1.05%, 6.04 + 0.95%; laser confocal microscopy showed that bone marrow mesenchymal stem cell markers in positive cell memory in high expression. Different conditioned medium induced bone marrow mesenchymal mesenchymal stem cells into bone, adipose tissue and cartilage tissue differentiation, verified the bone marrow mesenchymal stem cell differentiation in vitro. (3) SPIO labeled bone marrow mesenchymal stem cells by intravenous injection into tumor bearing mice, tumor cells mainly gathered in the local. (4) by immunomagnetic beads method Tumor vascular endothelial cells from 6-12 hours, the adherent growth of cells in the early stage of a small multi angle, spherical, gradually grow into the spindle, in 3 ~ 4D after fusion, monolayer arranged like cobblestone. (5) flow cytometry showed that CD31, CD34, vWF positive rate was 92.8. 2.32%, 99.87 + 1.95%, 86.26 + 2.57% (6) Boyden assay results showed that: at the same time, MDA -MB-231 and MDA under room inoculation of -MB-435s cells compared with the control group, the number of penetrating cells had no obvious change. (7) Boyden assay results showed that the tumor vascular endothelial cells group, bone marrow mesenchymal stem cell penetrating cell number was higher than that of control group (P? 0.05)
[Conclusion] bone marrow mesenchymal stem cells with high purity can be obtained in vitro, and are easy to culture and amplify. The ability of bone marrow mesenchymal stem cells to target tumor locally is related to local vascular endothelial cells.
Objective: To study the relationship between the invasion and metastasis of Snail and cervical cancer, and to explore its molecular mechanism. Methods: to construct the sense of full-length Snail eukaryotic expression vector and transfected into cervical cancer cell lines Hela and Siha. by Western blot method for analysis of gene expression, cell wound healing assay, Transwell cell count model of transmembrane cell invasion and metastasis. Results: (1) in PEGFPC1/Snail transfected cervical cancer cell line Hela, down regulated the expression of E-cadherin in SiHa, Snail and Vimentin expression (P0.05). (2) cervical cancer cell lines Hela, Siha transfected with full-length Snail eukaryotic expression vector, 12 hours cell wound healing significantly increased (P0.05); transfection PEGFPC1/Snail, Hela, SiHa cells 12 hours transmembrane cell number increased significantly (P0.05) over expression of.Snail can significantly promote cervical cancer cell lines Hela, SiHa invasion and metastasis potential. Conclusion: the transcription factor Snail promotes The interstitial transformation (EMT) of cervical cancer epithelial cells and the ability to invasion and metastasis are improved.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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