本文選題：BMSCs + GFAP　； 參考：《重慶醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的 通過構(gòu)建SD大鼠骨髓間充質(zhì)干細胞(Bone marrow-derivedmesenchymal stem cells, BMSCs)與大鼠來源C6腦膠質(zhì)瘤細胞直接共培養(yǎng)模型，探討B(tài)MSCs與C6腦膠質(zhì)瘤細胞直接共培養(yǎng)后BMSCs是否發(fā)生瘤樣轉(zhuǎn)化；通過構(gòu)建BMSCs原位注射C6腦膠質(zhì)瘤荷瘤裸鼠模型，為BMSCs在C6腦膠質(zhì)瘤活體內(nèi)瘤樣轉(zhuǎn)化的研究提供基礎(chǔ)模型，從而為BMSCs作為靶向治療載體安全用于臨床提供實驗依據(jù)。 材料與方法 1.實驗動物 SPF級SD大鼠，3-4周齡，體重約40-60g；裸鼠，雌性，5-6周齡，體重約18-20g，均購于重慶醫(yī)科大學(xué)實驗動物中心。 2.細胞來源 全骨髓貼壁篩選法分離培養(yǎng)SD大鼠BMSCs；C6腦膠質(zhì)瘤細胞系和293細胞系均由重慶醫(yī)科大學(xué)附屬兒童醫(yī)院“兒童發(fā)育與疾病研究教育部重點實驗室”腫瘤研究室惠贈； 3.實驗分組 本研究分為兩部分，第一部分分為三組：(1)實驗組：細胞膜熒光染料CM-Dil標記的BMSCs(CM-Dil+BMSCs)與C6腦膠質(zhì)瘤細胞直接共培養(yǎng)；(2)陽性對照組：C6腦膠質(zhì)瘤細胞單獨培養(yǎng)；(3)陰性對照組：BMSCs單獨培養(yǎng)。第二部分分為兩組：(1)實驗組：原位注射綠色熒光蛋白基因重組腺病毒載體(Ad-GFP)標記BMSCs (Ad-GFP+BMSCs)的C6腦膠質(zhì)瘤荷瘤裸鼠；(2)陽性對照組：未注射Ad-GFP+BMSCs的荷瘤裸鼠。 4.實驗方法 4.1全骨髓貼壁篩選法分離培養(yǎng)SD大鼠BMSCs，倒置顯微鏡觀察細胞形態(tài)。 4.2免疫熒光（Immunoflurescence, IF）法檢測CD90、CD105、CD45、GFAP蛋白表達情況。 4.3流式細胞術(shù)（Flow cytometry, FCM）檢測感染效率、分選直接共培養(yǎng)組BMSCs。 4.4RT-PCR檢測各組GFAP、PTEN、Bcl-xl、CyclinD1、CD90、CD105基因的表達水平。 4.5HE染色法觀察腫瘤組織細胞形態(tài)結(jié)構(gòu)。 結(jié)果 1. BMSCs培養(yǎng)至第三代時，細胞形態(tài)、性質(zhì)均一。IF結(jié)果顯示90%以上BMSCs表達CD90分子、CD105分子，不表達CD45分子。BMSCs與C6腦膠質(zhì)瘤細胞直接共培養(yǎng)7d后，BMSCs表達酸性膠質(zhì)纖維蛋白（Glial fibrillary acidic protein, GFAP）水平較陰性對照組明顯增高；BMSCs表面陽性分子CD90、CD105表達較陰性對照組明顯減弱。 2. FCM結(jié)果顯示CM-Dil標記BMSCs標記效率為98.3%；Ad-GFP感染BMSCs72h后感染病毒量0ul、5ul、6ul、8ul、10ul、15ul組感染效率分別為0.22%、8.58%、29.03%、36.54%、42.27%、51.43%。 3. RT-PCR結(jié)果顯示BMSCs與C6腦膠質(zhì)瘤細胞直接共培養(yǎng)7d后，BMSCs表達GFAP、PTEN、Bcl-xl、CyclinD1基因水平較陰性對照組明顯增高（P＜0.05）；BMSCs表達CD90、CD105基因水平較陰性對照組明顯降低（P＜0.05）。 4. HE染色結(jié)合激光共聚焦結(jié)果顯示BMSCs原位注射到荷瘤裸鼠瘤體7d后，，BMSCs仍存活，通過Ad-GFP綠色熒光信號對瘤體內(nèi)的BMSCs進行定位。 結(jié)論 1. BMSCs在與C6腦膠質(zhì)瘤細胞直接共培養(yǎng)后，其表面分子發(fā)生了改變且腫瘤相關(guān)基因表達增高，提示BMSCs在C6腦膠質(zhì)瘤細胞微環(huán)境中存在瘤樣轉(zhuǎn)化傾向。 2. Ad-GFP標記的BMSCs原位注射到C6腦膠質(zhì)瘤瘤體中可以存活并通過熒光標記進行定位，可以做為BMSCs在C6腦膠質(zhì)瘤活體內(nèi)瘤樣轉(zhuǎn)化研究的基礎(chǔ)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R739.41

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