人脐静脉内皮细胞永生化细胞系的建立、鉴定及应用
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摘要
【背景】
肿瘤的治疗是目前全世界面临的难题,传统的放疗、化疗及手术治疗疗效欠佳,探索新的肿瘤治疗方法成为医学研究领域的当务之急。1971年Folkman教授[1]提出了肿瘤血管依赖性理论。自此之后,肿瘤血管抑制治疗成为肿瘤领域的研究热点,并且取得了一些成果。但是,由于缺少肿瘤血管的靶向性分子,临床治疗效果不理想。而肿瘤血管与正常血管相比存在“异质性”[2]的研究使得肿瘤血管的靶向治疗成为可能。
在前期的工作中,我们建立了人低分化胃腺癌的肾包膜下异种移植瘤模型,利用噬菌体随机展示肽库进行体内筛选,在荷瘤鼠体内成功筛选出了能与胃癌血管特异性结合的环七肽GX1(CGNSNPKSC)[3]。实验证实,GX1对胃癌的血管具有良好的靶向性并且能够在一定程度上抑制血管生成[4],具有良好的应用前景。目前肿瘤血管抑制治疗多采用内皮细胞与肿瘤细胞体外共培养模型,内皮细胞多采用来源广泛、易于获得的人脐静脉内皮细胞(HUVECs)。但是由于原代人脐静脉内皮细胞分离工作量大,体外培养传代有限等限制了这一领域的研究。因此本课题就围绕人脐静脉内皮细胞永生化细胞系的建立、鉴定及应用展开,以期成功建立HUVECs的永生化细胞系,为GX1后续研究及肿瘤的血管抑制治疗提供一个很好的细胞模型。
【目的】
获得HUVECs的永生化细胞系,从而为GX1后续研究及肿瘤的血管抑制治疗提供一个很好的细胞模型。
【方法】
1.通过胶原酶消化法分离原代的脐静脉内皮细胞(HUVECs)。
2.通过慢病毒介导SV40LT感染原代HUVECs。
3.通过电镜观察、免疫细胞荧光化学、RT-PCR及管状成形试验等方法从形态学、特征性标志物的表达等方面对原代及转染后的HUVECs进行鉴定。
4.利用免疫荧光化学及IP的方法验证GX1的受体在永生化HUVECs上的表达,从而证实永生化HUVECs可作为一种新的细胞模型用于肿瘤血管抑制治疗的研究。
【结果】
1.成功分离培养了原代的HUVECs。在相差倒置显微镜下,分离的细胞呈单层生长,呈多角形或短梭状;扫描电镜及透射电镜下,细胞表面多绒毛及凸起,且发现内皮细胞的特征性结构W-P小体;八因子及KDR染色阳性;管状形成试验阳性。
2.成功包装SV40LT的慢病毒载体,并转染入HUVECs,转染后的HUVECs可以持续传代,形态学、特征性标志物及管状形成试验与原代HUVECs无明显差异; RT-PCR证实SV40LT成功转入,且原代与转染后的HUVECs八因子及KDR的表达在mRNA水平无明显差异。
3.免疫荧光化学及IP结果均证实永生化HUVECs上有GX1受体的表达,其可以替代原代HUVECs用于GX1的相关研究。
【结论】
1.慢病毒介导的SV40LT可以成功诱导HUVECs永生化。
2.永生化HUVECs可以作为一种新的细胞模型用于肿瘤血管抑制治疗。
【Background】
Cancer therapy is a worldwide problem. Traditional therapies are not satisfying, such as radiotherapy, chemotherapy and operation. There is an urgent need to study a new therapy to cancer. Folkman introduced tumor vascular theory in 1971, and the anti-angiogenesis has become a promising approach for cancer therapy. The heterogeneity between normal and tumorous vasculature makes the specific vascular targets suitable for therapies targeting tumors. However, one of reasons to low efficiency is lacking specific molecules targeting vascular in the therapy.
Using phage display technology, we have identified the peptide GX1 (CGNSNPKSC) as having the ability to target the gastric cancer vasculature in xenograft study. We found that GX1 targeted specifically to the gastric cancer vasculature and inhibited the angiogenesis. The current model of target treatment usually adopted endothelial cells and tumor cells co-culture model. Due to abundant source and easy expansion, human umbilical vein endothelial cells (HUVECs) become the most commonly cells in study of tumor angiogenesis. However, the complex isolation and limited passage of HUVECs in vitro hampered our study. Therefore, this study mainly focuses on the establishment, identification and application of immortalized HUVECs. The immortalized HUVECs provide an ideal cell model for examining GX1 receptor and tumor anti-angiogenesis.
【Objectives】
We aim to obtain the immortalized HUVECs and establish an ideal cell model for the study of anti-angiogenesis.
【Methods】
1. Isolation of Human Umbilical Vein Endothelial Cells using collagenase digestive method.
2. Transfection of the primary HUVECs by lentvirus vetor contained SV40LT.
3. The primary HUVECs and the transfected cells were identified by light microscope and scanning electron microscope. Tube formation assay and specific markers were studied by Immunofluorescence and real-time PCR.
4. Immunofluorescence staining and IP were performed to detect the expression of receptor of GX1 in immortalized HUVEC.
【Results】
1. The primary HUVECs was successfully isolated. Cells showed confluent monolayer with the cobblestone morphology under light microscope and much villi on cell surfaces under scanning electron microscope. The characteristic W-P body was found in the primary HUVECs. The primary HUVECs exhibited FactorⅧand KDR expression and the tube formation in Matrigel.
2. The HUVECs were transformed by the lentvirus vector containing with SV40LT into the HUVECs. The immortalized HUVECs shared the similar characteristics with the primary HUVECs, such as the cobblestone morphology, W-P body, FactorⅧ, KDR and the ability of tube formation.
3. The receptor of GX1 was detected in the immortalized HUVECs by using the methods of Immunofluorescence staining and immunoprecipitation.
【Conclusions】
1. The immortalized HUVECs were successfully transformed by SV40LT.
2. The immortalized HUVECs can be used in the study of anti-angiogenesis as a cell model.
肿瘤的治疗是目前全世界面临的难题,传统的放疗、化疗及手术治疗疗效欠佳,探索新的肿瘤治疗方法成为医学研究领域的当务之急。1971年Folkman教授[1]提出了肿瘤血管依赖性理论。自此之后,肿瘤血管抑制治疗成为肿瘤领域的研究热点,并且取得了一些成果。但是,由于缺少肿瘤血管的靶向性分子,临床治疗效果不理想。而肿瘤血管与正常血管相比存在“异质性”[2]的研究使得肿瘤血管的靶向治疗成为可能。
在前期的工作中,我们建立了人低分化胃腺癌的肾包膜下异种移植瘤模型,利用噬菌体随机展示肽库进行体内筛选,在荷瘤鼠体内成功筛选出了能与胃癌血管特异性结合的环七肽GX1(CGNSNPKSC)[3]。实验证实,GX1对胃癌的血管具有良好的靶向性并且能够在一定程度上抑制血管生成[4],具有良好的应用前景。目前肿瘤血管抑制治疗多采用内皮细胞与肿瘤细胞体外共培养模型,内皮细胞多采用来源广泛、易于获得的人脐静脉内皮细胞(HUVECs)。但是由于原代人脐静脉内皮细胞分离工作量大,体外培养传代有限等限制了这一领域的研究。因此本课题就围绕人脐静脉内皮细胞永生化细胞系的建立、鉴定及应用展开,以期成功建立HUVECs的永生化细胞系,为GX1后续研究及肿瘤的血管抑制治疗提供一个很好的细胞模型。
【目的】
获得HUVECs的永生化细胞系,从而为GX1后续研究及肿瘤的血管抑制治疗提供一个很好的细胞模型。
【方法】
1.通过胶原酶消化法分离原代的脐静脉内皮细胞(HUVECs)。
2.通过慢病毒介导SV40LT感染原代HUVECs。
3.通过电镜观察、免疫细胞荧光化学、RT-PCR及管状成形试验等方法从形态学、特征性标志物的表达等方面对原代及转染后的HUVECs进行鉴定。
4.利用免疫荧光化学及IP的方法验证GX1的受体在永生化HUVECs上的表达,从而证实永生化HUVECs可作为一种新的细胞模型用于肿瘤血管抑制治疗的研究。
【结果】
1.成功分离培养了原代的HUVECs。在相差倒置显微镜下,分离的细胞呈单层生长,呈多角形或短梭状;扫描电镜及透射电镜下,细胞表面多绒毛及凸起,且发现内皮细胞的特征性结构W-P小体;八因子及KDR染色阳性;管状形成试验阳性。
2.成功包装SV40LT的慢病毒载体,并转染入HUVECs,转染后的HUVECs可以持续传代,形态学、特征性标志物及管状形成试验与原代HUVECs无明显差异; RT-PCR证实SV40LT成功转入,且原代与转染后的HUVECs八因子及KDR的表达在mRNA水平无明显差异。
3.免疫荧光化学及IP结果均证实永生化HUVECs上有GX1受体的表达,其可以替代原代HUVECs用于GX1的相关研究。
【结论】
1.慢病毒介导的SV40LT可以成功诱导HUVECs永生化。
2.永生化HUVECs可以作为一种新的细胞模型用于肿瘤血管抑制治疗。
【Background】
Cancer therapy is a worldwide problem. Traditional therapies are not satisfying, such as radiotherapy, chemotherapy and operation. There is an urgent need to study a new therapy to cancer. Folkman introduced tumor vascular theory in 1971, and the anti-angiogenesis has become a promising approach for cancer therapy. The heterogeneity between normal and tumorous vasculature makes the specific vascular targets suitable for therapies targeting tumors. However, one of reasons to low efficiency is lacking specific molecules targeting vascular in the therapy.
Using phage display technology, we have identified the peptide GX1 (CGNSNPKSC) as having the ability to target the gastric cancer vasculature in xenograft study. We found that GX1 targeted specifically to the gastric cancer vasculature and inhibited the angiogenesis. The current model of target treatment usually adopted endothelial cells and tumor cells co-culture model. Due to abundant source and easy expansion, human umbilical vein endothelial cells (HUVECs) become the most commonly cells in study of tumor angiogenesis. However, the complex isolation and limited passage of HUVECs in vitro hampered our study. Therefore, this study mainly focuses on the establishment, identification and application of immortalized HUVECs. The immortalized HUVECs provide an ideal cell model for examining GX1 receptor and tumor anti-angiogenesis.
【Objectives】
We aim to obtain the immortalized HUVECs and establish an ideal cell model for the study of anti-angiogenesis.
【Methods】
1. Isolation of Human Umbilical Vein Endothelial Cells using collagenase digestive method.
2. Transfection of the primary HUVECs by lentvirus vetor contained SV40LT.
3. The primary HUVECs and the transfected cells were identified by light microscope and scanning electron microscope. Tube formation assay and specific markers were studied by Immunofluorescence and real-time PCR.
4. Immunofluorescence staining and IP were performed to detect the expression of receptor of GX1 in immortalized HUVEC.
【Results】
1. The primary HUVECs was successfully isolated. Cells showed confluent monolayer with the cobblestone morphology under light microscope and much villi on cell surfaces under scanning electron microscope. The characteristic W-P body was found in the primary HUVECs. The primary HUVECs exhibited FactorⅧand KDR expression and the tube formation in Matrigel.
2. The HUVECs were transformed by the lentvirus vector containing with SV40LT into the HUVECs. The immortalized HUVECs shared the similar characteristics with the primary HUVECs, such as the cobblestone morphology, W-P body, FactorⅧ, KDR and the ability of tube formation.
3. The receptor of GX1 was detected in the immortalized HUVECs by using the methods of Immunofluorescence staining and immunoprecipitation.
【Conclusions】
1. The immortalized HUVECs were successfully transformed by SV40LT.
2. The immortalized HUVECs can be used in the study of anti-angiogenesis as a cell model.
引文
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