一种新型抗体静置孵育装置及Kir2.1促胃癌干细胞侵袭转移
发布时间：2018-08-07 07:20
【摘要】：第一部分：新型免疫印迹法抗体表面静置孵育实验装置的应用效果 我们设计了一个新的免疫印迹抗体孵育湿盒可以克服传统孵育湿盒的缺点。该装置由盛水加盖的外盒和一个具疏水平面且可漂浮的内置物构成，，漂浮的内置物可保证水平，疏水平面可防止敷于偏氟乙烯薄膜(PVDF)或硝酸纤维素薄膜(NC)上的抗体弥散，盒中的水保证盒内湿度防止抗体溶液的蒸发。我们在实验中应用了该实用新型，并且该实用新型于2013年获得中国国家知识产权局的授权(专利号ZL201320144341.7)。 第二部分：Kir2.1在胃癌干细胞干性维持和侵袭转移中作用的研究 胃癌是世界范围内高发病率、高致死性的恶性消化系统肿瘤之一。尽管近年来胃癌的手术、化疗以及其他治疗方法均取得了长足进步，但其疗效仍不尽如人意，5年生存率并未得到显著提高。这一现象折射出人们对胃癌侵袭转移机制的认识还不够深入。因此，深入进行胃癌侵袭转移机制的研究是当前一项紧迫的任务。 肿瘤干细胞(cancer stem cells，CSCs)学说的提出是近几年来肿瘤研究领域的一大进展，也为我们认识胃癌侵袭和转移机制开拓了新的视角。愈来愈多的证据表明，CSCs是肿瘤侵袭转移的“种子”细胞。我们小组之前通过无血清成球培养的方法分离/富集了胃癌干细胞(gastric cancer stem cells, GCSCs)，并发现其具有高度自我更新和侵袭转移潜能，从而证明了GCSCs是胃癌侵袭转移的细胞学基础。然而GCSCs侵袭转移的机制并不清楚。 近年来，钾离子通道在肿瘤中的作用受到关注，为探讨钾离子通道是否在GCSCs干性维持及侵袭转移中发挥作用，我们进行了全细胞电压膜片钳实验，发现GCSCs中内向整流K+电流显著高于非GCSCs。然而，钾离子通道种类繁多，为确定何种钾离子通道发挥主要作用，我们进一步作了基因表达谱芯片分析，结果显示内向整流K+通道Kir2.1(potassium inwardly rectifying channel, subfamily J, member2，KCNJ-2)在GCSCs中显著高表达。我们还应用免疫组化方法检测了临床胃癌标本中Kir2.1表达，并统计分析了Kir2.1表达与临床病理参数及病人预后的关系。本研究主要方法、结果及结论如下： 1、胃癌干细胞产生高强度内向整流钾电流与高表达Kir2.1密切相关 (1)我们选择胃癌细胞系中转移能力较强的细胞系SGC7901，按本组先前建立的无血清成球培养法分离/富集GCSCs，然后以全细胞电压膜片钳检测其K+电流的产生。结果发现，相比单层生长细胞(monolayer cells，MN)，胃癌细胞球细胞(sphere cells，SC)即GCSCs在电压的刺激下可以释放更高强度的内向整流K+电流。 (2)基因表达谱芯片分析结果表明，GCSCs较MN显著高表达Kir2.1通道蛋白(基因名：KCNJ-2)的mRNA并得到qRT-PCR验证。 (3) Western blot实验从蛋白水平证明GCSCs高表达Kir2.1，免疫荧光实验显示Kir2.1通道蛋白主要表达在细胞膜但也存在于细胞质中。 (4) qPCR进一步证明只有能产生特征性内向整流K+电流的胃癌细胞才能检测到KCNJ-2基因的表达，而在没有特征性内向整流K+电流的胃癌细胞中则检测不到KCNJ-2基因的表达。这些结果表明，GCSCs产生高强内向整流K+电流，并且该电流的产生主要由于其高表达内向整流K+通道Kir2.1所致。 2、Kir2.1+胃癌细胞具有干性特性和高侵袭能力 (1)流式细胞仪分析发现SGC7901细胞中Kir2.1阳性(Kir2.1+)细胞的比率约为7.5%。 (2)与Kir2.1-细胞相比较，Kir2.1+细胞高表达干性转录因子Nanog，具有更强的细胞球(tumor-sphere)和克隆形成能力，表明Kir2.1+细胞具有干性特性。 (3)活细胞工作站观察结果显示Kir2.1+细胞较Kir2.1-细胞具有更强的运动能力，Transwell小室迁移实验显示Kir2.1+细胞较Kir2.1-细胞具有更强的迁移能力，qRT-PCR检测还表明Kir2.1+细胞较Kir2.1-细胞高表达基质金属蛋白酶2(matrixmetalloproteinase2, MMP2)。 3、siRNA沉默Kir2.1的表达抑制胃癌细胞干性和侵袭能力 应用靶向Kir2.1的siRNA敲低Kir2.1的表达后，胃癌细胞Nanog和MMP2的表达均显著下调，体外迁移和侵袭能力也受到显著抑制。 4、Kir2.1在胃癌标本中表达与病人预后密切相关 (1)应用免疫组化染色方法检测了212例胃癌标本中Kir2.1的表达，发现Kir2.1的表达与胃癌患者年龄(P=0.674)、性别(P=0.329)、组织学分型(P=0.625)无关，而与浸润深度(P=0.010)、TNM分期(P=0.002)和是否发生淋巴结转移(P=0.028)呈正相关。 (2)采用Kaplan-Meier方法计算132例有随访资料病人的生存率，结果显示高表达Kir2.1的患者预后差于低表达Kir2.1的患者，提示Kir2.1可以用作判断胃癌患者预后的指标。 该部分的主要结论如下：胃癌干细胞高表达内向整流K+通道Kir2.1，并与其干性维持和侵袭能力密切相关；Kir2.1的表达与胃癌浸润深度、TNM分期和淋巴结转移呈正相关而与患者生存时间呈负相关，可能成为判断胃癌预后的新的指标。
[Abstract]:Part I: the application effect of a new immunoblotting antibody incubating device on the surface.
We have designed a new immunoblotting antibody incubating box that can overcome the shortcomings of traditional incubating wet boxes. The device consists of an outer box filled with water and a built-in and floating built-in. The floating built-in can guarantee the level, and the hydrophobic surface can be prevented from applying to vinylidene fluoride film (PVDF) or nitrocellulose film (NC). The antibody in the box is dispersed, and the water in the box ensures the moisture in the box to prevent the evaporation of the antibody solution. We applied the utility model in the experiment, and the utility model was authorized by the China National Intellectual Property Office (Patent No. ZL201320144341.7) in 2013.
The second part: the role of Kir2.1 in the maintenance, invasion and metastasis of gastric cancer stem cells.
Gastric cancer is one of the malignant digestive system tumors with high morbidity and death in the world. Although the operation, chemotherapy and other treatment methods of gastric cancer have made great progress in recent years, the curative effect is still not satisfactory, and the 5 year survival rate has not been significantly improved. This image reflects the recognition of the mechanism of invasion and metastasis of gastric cancer. Therefore, it is an urgent task to further study the mechanism of invasion and metastasis of gastric cancer.
The theory of cancer stem cells (CSCs) is a great progress in the field of cancer research in recent years. It also opens a new perspective for our understanding of the mechanism of invasion and metastasis of gastric cancer. More and more evidence shows that CSCs is the "seed" cell of tumor invasion and metastasis. Gastric cancer stem cells (GCSCs) was isolated and enriched, and it was found to have high self renewal and invasion and metastasis potential, which proved that GCSCs is the cytological basis of invasion and metastasis of gastric cancer. However, the mechanism of GCSCs invasion and metastasis is not clear.
In recent years, the role of potassium channel in the tumor is concerned. In order to investigate whether the potassium channel plays a role in the dry maintenance and invasion of GCSCs, we have carried out a whole cell voltage patch clamp experiment. We found that the K+ current in GCSCs is significantly higher than that of non GCSCs., and there are a wide variety of potassium channels to determine what potassium ionization is. The subchannel plays a major role. We further performed gene expression chip analysis. The results showed that the inward rectifier K+ channel Kir2.1 (potassium inwardly rectifying channel, subfamily J, member2, KCNJ-2) was highly expressed in GCSCs. We also used immunohistochemical method to detect the expression of Kir2.1 in the clinical gastric cancer specimens. The relationship between Kir2.1 expression and clinicopathological parameters and prognosis was analyzed. The main methods, results and conclusions were as follows:
1, the high intensity inward rectifier potassium current produced by gastric cancer stem cells is closely related to the high expression of Kir2.1.
(1) we selected the cell line SGC7901 with strong metastatic ability in the gastric cancer cell line, and separated / enriched GCSCs according to the serum-free spheric culture method previously established in this group. Then the K+ current was detected by the whole cell voltage patch clamp. The results showed that compared with the monolayer growth cells (monolayer cells, MN), the gastric cancer cell ball cells (sphere cells, SC) is GC. SCs can release a higher intensity of inward rectifying K+ current stimulated by voltage.
(2) Gene microarray analysis showed that GCSCs significantly overexpressed Kir2.1 channel protein (gene name: KCNJ-2) mRNA than MN and was verified by qRT-PCR.
(3) the Western blot experiment showed that the GCSCs was highly expressed from the protein level, and the immunofluorescence experiment showed that the Kir2.1 channel protein was mainly expressed in the cell membrane but also in the cytoplasm.
(4) qPCR further proved that only gastric cancer cells capable of producing characteristic introversion K+ current can detect the expression of KCNJ-2 gene, and the expression of the KCNJ-2 gene is not detected in the gastric cancer cells without the characteristic inward rectifier K+ current. These results indicate that GCSCs produces high strength introverted K+ current, and the current is produced mainly. It is due to its high expression of inward rectifying K+ channel Kir2.1.
2, Kir2.1+ gastric cancer cells have dry characteristics and high invasiveness.
(1) flow cytometry analysis showed that the ratio of Kir2.1 positive (Kir2.1+) cells in SGC7901 cells was about 7.5%.
(2) compared with Kir2.1- cells, Kir2.1+ cells highly expressed the dry transcription factor Nanog, which had a stronger cell sphere (tumor-sphere) and clonogenic ability, indicating that Kir2.1+ cells have a dry character.
(3) the observation of the live cell workstation showed that the Kir2.1+ cells had stronger movement ability than the Kir2.1- cells, and the Transwell cell migration experiment showed that the Kir2.1+ cells had a stronger migration ability than the Kir2.1- cells, and the qRT-PCR detection also showed that the Kir2.1+ cells expressed the matrix metalloproteinase 2 higher than the Kir2.1- cells (matrixmetalloproteinase2, MMP2). ).
3, siRNA silenced Kir2.1 expression to inhibit gastric cancer cell dry and invasive ability.
When Kir2.1-targeting siRNA was used to knock down the expression of Kir2.1, the expression of Nanog and MMP2 in gastric cancer cells was significantly down-regulated, and the ability of migration and invasion in vitro was also significantly inhibited.
4, the expression of Kir2.1 in gastric cancer specimens is closely related to the prognosis of patients.
(1) the expression of Kir2.1 in 212 specimens of gastric cancer was detected by immunohistochemical staining. It was found that the expression of Kir2.1 was not related to the age (P=0.674), sex (P=0.329) and histological type (P=0.625) of gastric cancer patients, but was positively correlated with the depth of infiltration (P=0.010), TNM staging (P=0.002) and lymph node metastasis (P=0.028).
(2) the Kaplan-Meier method was used to calculate the survival rate of 132 patients with follow-up data. The results showed that the prognosis of patients with high expression of Kir2.1 was inferior to those with low expression of Kir2.1, suggesting that Kir2.1 could be used as an indicator of prognosis of gastric cancer patients.
The main conclusions of this part are as follows: the high expression of inward rectifying K+ channel Kir2.1 in gastric cancer stem cells is closely related to its dry maintenance and invasion ability. The expression of Kir2.1 is positively correlated with the depth of gastric cancer, TNM staging and lymph node metastasis, and is negatively related to the survival time of the patients, which may be a new index for judging the prognosis of gastric cancer.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R735.2
本文编号：2169283
[Abstract]:Part I: the application effect of a new immunoblotting antibody incubating device on the surface.
We have designed a new immunoblotting antibody incubating box that can overcome the shortcomings of traditional incubating wet boxes. The device consists of an outer box filled with water and a built-in and floating built-in. The floating built-in can guarantee the level, and the hydrophobic surface can be prevented from applying to vinylidene fluoride film (PVDF) or nitrocellulose film (NC). The antibody in the box is dispersed, and the water in the box ensures the moisture in the box to prevent the evaporation of the antibody solution. We applied the utility model in the experiment, and the utility model was authorized by the China National Intellectual Property Office (Patent No. ZL201320144341.7) in 2013.
The second part: the role of Kir2.1 in the maintenance, invasion and metastasis of gastric cancer stem cells.
Gastric cancer is one of the malignant digestive system tumors with high morbidity and death in the world. Although the operation, chemotherapy and other treatment methods of gastric cancer have made great progress in recent years, the curative effect is still not satisfactory, and the 5 year survival rate has not been significantly improved. This image reflects the recognition of the mechanism of invasion and metastasis of gastric cancer. Therefore, it is an urgent task to further study the mechanism of invasion and metastasis of gastric cancer.
The theory of cancer stem cells (CSCs) is a great progress in the field of cancer research in recent years. It also opens a new perspective for our understanding of the mechanism of invasion and metastasis of gastric cancer. More and more evidence shows that CSCs is the "seed" cell of tumor invasion and metastasis. Gastric cancer stem cells (GCSCs) was isolated and enriched, and it was found to have high self renewal and invasion and metastasis potential, which proved that GCSCs is the cytological basis of invasion and metastasis of gastric cancer. However, the mechanism of GCSCs invasion and metastasis is not clear.
In recent years, the role of potassium channel in the tumor is concerned. In order to investigate whether the potassium channel plays a role in the dry maintenance and invasion of GCSCs, we have carried out a whole cell voltage patch clamp experiment. We found that the K+ current in GCSCs is significantly higher than that of non GCSCs., and there are a wide variety of potassium channels to determine what potassium ionization is. The subchannel plays a major role. We further performed gene expression chip analysis. The results showed that the inward rectifier K+ channel Kir2.1 (potassium inwardly rectifying channel, subfamily J, member2, KCNJ-2) was highly expressed in GCSCs. We also used immunohistochemical method to detect the expression of Kir2.1 in the clinical gastric cancer specimens. The relationship between Kir2.1 expression and clinicopathological parameters and prognosis was analyzed. The main methods, results and conclusions were as follows:
1, the high intensity inward rectifier potassium current produced by gastric cancer stem cells is closely related to the high expression of Kir2.1.
(1) we selected the cell line SGC7901 with strong metastatic ability in the gastric cancer cell line, and separated / enriched GCSCs according to the serum-free spheric culture method previously established in this group. Then the K+ current was detected by the whole cell voltage patch clamp. The results showed that compared with the monolayer growth cells (monolayer cells, MN), the gastric cancer cell ball cells (sphere cells, SC) is GC. SCs can release a higher intensity of inward rectifying K+ current stimulated by voltage.
(2) Gene microarray analysis showed that GCSCs significantly overexpressed Kir2.1 channel protein (gene name: KCNJ-2) mRNA than MN and was verified by qRT-PCR.
(3) the Western blot experiment showed that the GCSCs was highly expressed from the protein level, and the immunofluorescence experiment showed that the Kir2.1 channel protein was mainly expressed in the cell membrane but also in the cytoplasm.
(4) qPCR further proved that only gastric cancer cells capable of producing characteristic introversion K+ current can detect the expression of KCNJ-2 gene, and the expression of the KCNJ-2 gene is not detected in the gastric cancer cells without the characteristic inward rectifier K+ current. These results indicate that GCSCs produces high strength introverted K+ current, and the current is produced mainly. It is due to its high expression of inward rectifying K+ channel Kir2.1.
2, Kir2.1+ gastric cancer cells have dry characteristics and high invasiveness.
(1) flow cytometry analysis showed that the ratio of Kir2.1 positive (Kir2.1+) cells in SGC7901 cells was about 7.5%.
(2) compared with Kir2.1- cells, Kir2.1+ cells highly expressed the dry transcription factor Nanog, which had a stronger cell sphere (tumor-sphere) and clonogenic ability, indicating that Kir2.1+ cells have a dry character.
(3) the observation of the live cell workstation showed that the Kir2.1+ cells had stronger movement ability than the Kir2.1- cells, and the Transwell cell migration experiment showed that the Kir2.1+ cells had a stronger migration ability than the Kir2.1- cells, and the qRT-PCR detection also showed that the Kir2.1+ cells expressed the matrix metalloproteinase 2 higher than the Kir2.1- cells (matrixmetalloproteinase2, MMP2). ).
3, siRNA silenced Kir2.1 expression to inhibit gastric cancer cell dry and invasive ability.
When Kir2.1-targeting siRNA was used to knock down the expression of Kir2.1, the expression of Nanog and MMP2 in gastric cancer cells was significantly down-regulated, and the ability of migration and invasion in vitro was also significantly inhibited.
4, the expression of Kir2.1 in gastric cancer specimens is closely related to the prognosis of patients.
(1) the expression of Kir2.1 in 212 specimens of gastric cancer was detected by immunohistochemical staining. It was found that the expression of Kir2.1 was not related to the age (P=0.674), sex (P=0.329) and histological type (P=0.625) of gastric cancer patients, but was positively correlated with the depth of infiltration (P=0.010), TNM staging (P=0.002) and lymph node metastasis (P=0.028).
(2) the Kaplan-Meier method was used to calculate the survival rate of 132 patients with follow-up data. The results showed that the prognosis of patients with high expression of Kir2.1 was inferior to those with low expression of Kir2.1, suggesting that Kir2.1 could be used as an indicator of prognosis of gastric cancer patients.
The main conclusions of this part are as follows: the high expression of inward rectifying K+ channel Kir2.1 in gastric cancer stem cells is closely related to its dry maintenance and invasion ability. The expression of Kir2.1 is positively correlated with the depth of gastric cancer, TNM staging and lymph node metastasis, and is negatively related to the survival time of the patients, which may be a new index for judging the prognosis of gastric cancer.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R735.2
【参考文献】
相关期刊论文 前1条
1 Bryan A Ong;Kenneth J Vega;Courtney W Houchen;;Intestinal stem cells and the colorectal cancer microenvironment[J];World Journal of Gastroenterology;2014年08期
本文编号：2169283
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