骨髓间充质干细胞肿瘤微环境直接培养模型构建及其生物学特性分析

发布时间：2018-06-25 15:31

  本文选题：BMSCs + GFAP　； 参考：《重庆医科大学》2014年硕士论文

【摘要】：目的 通过构建SD大鼠骨髓间充质干细胞(Bone marrow-derivedmesenchymal stem cells, BMSCs)与大鼠来源C6脑胶质瘤细胞直接共培养模型，探讨BMSCs与C6脑胶质瘤细胞直接共培养后BMSCs是否发生瘤样转化；通过构建BMSCs原位注射C6脑胶质瘤荷瘤裸鼠模型，为BMSCs在C6脑胶质瘤活体内瘤样转化的研究提供基础模型，从而为BMSCs作为靶向治疗载体安全用于临床提供实验依据。 材料与方法 1.实验动物 SPF级SD大鼠，3-4周龄，体重约40-60g；裸鼠，雌性，5-6周龄，体重约18-20g，均购于重庆医科大学实验动物中心。 2.细胞来源 全骨髓贴壁筛选法分离培养SD大鼠BMSCs；C6脑胶质瘤细胞系和293细胞系均由重庆医科大学附属儿童医院“儿童发育与疾病研究教育部重点实验室”肿瘤研究室惠赠； 3.实验分组 本研究分为两部分，第一部分分为三组：(1)实验组：细胞膜荧光染料CM-Dil标记的BMSCs(CM-Dil+BMSCs)与C6脑胶质瘤细胞直接共培养；(2)阳性对照组：C6脑胶质瘤细胞单独培养；(3)阴性对照组：BMSCs单独培养。第二部分分为两组：(1)实验组：原位注射绿色荧光蛋白基因重组腺病毒载体(Ad-GFP)标记BMSCs (Ad-GFP+BMSCs)的C6脑胶质瘤荷瘤裸鼠；(2)阳性对照组：未注射Ad-GFP+BMSCs的荷瘤裸鼠。 4.实验方法 4.1全骨髓贴壁筛选法分离培养SD大鼠BMSCs，倒置显微镜观察细胞形态。 4.2免疫荧光（Immunoflurescence, IF）法检测CD90、CD105、CD45、GFAP蛋白表达情况。 4.3流式细胞术（Flow cytometry, FCM）检测感染效率、分选直接共培养组BMSCs。 4.4RT-PCR检测各组GFAP、PTEN、Bcl-xl、CyclinD1、CD90、CD105基因的表达水平。 4.5HE染色法观察肿瘤组织细胞形态结构。 结果 1. BMSCs培养至第三代时，细胞形态、性质均一。IF结果显示90%以上BMSCs表达CD90分子、CD105分子，不表达CD45分子。BMSCs与C6脑胶质瘤细胞直接共培养7d后，BMSCs表达酸性胶质纤维蛋白（Glial fibrillary acidic protein, GFAP）水平较阴性对照组明显增高；BMSCs表面阳性分子CD90、CD105表达较阴性对照组明显减弱。 2. FCM结果显示CM-Dil标记BMSCs标记效率为98.3%；Ad-GFP感染BMSCs72h后感染病毒量0ul、5ul、6ul、8ul、10ul、15ul组感染效率分别为0.22%、8.58%、29.03%、36.54%、42.27%、51.43%。 3. RT-PCR结果显示BMSCs与C6脑胶质瘤细胞直接共培养7d后，BMSCs表达GFAP、PTEN、Bcl-xl、CyclinD1基因水平较阴性对照组明显增高（P＜0.05）；BMSCs表达CD90、CD105基因水平较阴性对照组明显降低（P＜0.05）。 4. HE染色结合激光共聚焦结果显示BMSCs原位注射到荷瘤裸鼠瘤体7d后，，BMSCs仍存活，通过Ad-GFP绿色荧光信号对瘤体内的BMSCs进行定位。 结论 1. BMSCs在与C6脑胶质瘤细胞直接共培养后，其表面分子发生了改变且肿瘤相关基因表达增高，提示BMSCs在C6脑胶质瘤细胞微环境中存在瘤样转化倾向。 2. Ad-GFP标记的BMSCs原位注射到C6脑胶质瘤瘤体中可以存活并通过荧光标记进行定位，可以做为BMSCs在C6脑胶质瘤活体内瘤样转化研究的基础模型。
[Abstract]:objective
By constructing SD rat bone marrow mesenchymal stem cells (Bone marrow-derivedmesenchymal stem cells, BMSCs) and the direct co culture model of C6 glioma cells from rat derived C6 glioma cells, the tumor like transformation of BMSCs was investigated after direct co culture of BMSCs and C6 brain glioma cells, and the nude mice model of glioma with C6 brain tumor was injected by BMSCs in situ. It provides a basic model for BMSCs in vivo study of C6 glioma in vivo, so as to provide experimental evidence for BMSCs as a targeting therapeutic vector and safe for clinical use.
Materials and methods
1. experimental animals
SPF SD rats were 3-4 weeks old, weighing about 40-60g. Nude mice, female, 5-6 weeks old, weighing about 18-20g, were purchased at the experimental animal center of Medical University Of Chongqing.
2. cell source
The BMSCs of SD rats was isolated and cultured by full bone marrow adherence screening method, and the C6 glioma cell line and 293 cell line were all given by the Cancer Research Laboratory of "the Key Laboratory of the Ministry of education for child development and disease research in the children's development and Disease Research Department of the children's Hospital of Medical University Of Chongqing."
3. experimental grouping
The study was divided into two parts. The first part was divided into three groups: (1) the experimental group: the BMSCs (CM-Dil+BMSCs) labeled by the cell membrane fluorescent dye CM-Dil and the C6 glioma cells were co cultured; (2) the positive control group: C6 glioma cells were cultured alone; (3) the negative control group: BMSCs alone culture. The second part was divided into two groups: (1) experimental group. In situ injection of green fluorescent protein gene recombinant adenovirus vector (Ad-GFP) to mark BMSCs (Ad-GFP+BMSCs) of C6 glioma in nude mice; (2) the positive control group: non Ad-GFP+BMSCs tumor bearing nude mice.
4. experimental method
4.1 the whole bone marrow adherence screening method was used to isolate and culture BMSCs from SD rats. The morphology of cells was observed by inverted microscope.
4.2 the expression of CD90, CD105, CD45 and GFAP protein was detected by Immunoflurescence (IF).
4.3 Flow cytometry (FCM) was used to detect the infection efficiency and direct co culture group BMSCs..
The expression levels of GFAP, PTEN, Bcl-xl, CyclinD1, CD90 and CD105 genes in each group were detected by 4.4RT-PCR.
4.5HE staining method was used to observe the morphological structure of tumor cells.
Result
When 1. BMSCs was cultured to the third generation, the cell morphology and properties of homogeneous.IF showed that more than 90% BMSCs expressed CD90, CD105 molecule, CD45 molecule.BMSCs and C6 brain glioma cells directly co cultured 7d, BMSCs expression of acidic glial fibrin (Glial fibrillary) level was significantly higher than that of negative control group. The expression of CD90 and CD105 on the surface of S was significantly lower than that in the negative control group.
The results of 2. FCM showed that the CM-Dil marker BMSCs labeling efficiency was 98.3%, Ad-GFP infection BMSCs72h infection virus 0ul, 5ul, 6ul, 8ul, 10ul, 15ul group infection efficiency were 0.22%, 8.58%, 29.03%, 36.54%, 42.27%, 51.43%..
3. RT-PCR results showed that after direct co culture of BMSCs and C6 glioma cells, BMSCs expressed GFAP, PTEN, Bcl-xl, and CyclinD1 significantly increased (P < 0.05) in the negative control group (P < 0.05), and the BMSCs expression CD90 was significantly lower than that of the negative control group (0.05).
4. HE staining combined with laser confocal results showed that BMSCs was still alive after the injection of BMSCs in situ to tumor bearing nude mice 7d, and the BMSCs in the tumor was located by Ad-GFP green fluorescence signal.
conclusion
1. BMSCs was co cultured with C6 glioma cells, and the surface molecules changed and the expression of tumor related genes increased, suggesting that BMSCs had a tumor like transformation in the microenvironment of C6 glioma cells.
The 2. Ad-GFP labeled BMSCs in situ injected into the C6 glioma tumor can survive and locate by fluorescence labeling, which can be used as the basic model for the study of BMSCs in the tumor like transformation of C6 brain glioma.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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