一種新型抗體靜置孵育裝置及Kir2.1促胃癌干細(xì)胞侵襲轉(zhuǎn)移
發(fā)布時間：2018-08-07 07:20
【摘要】：第一部分：新型免疫印跡法抗體表面靜置孵育實驗裝置的應(yīng)用效果 我們設(shè)計了一個新的免疫印跡抗體孵育濕盒可以克服傳統(tǒng)孵育濕盒的缺點。該裝置由盛水加蓋的外盒和一個具疏水平面且可漂浮的內(nèi)置物構(gòu)成，，漂浮的內(nèi)置物可保證水平，疏水平面可防止敷于偏氟乙烯薄膜(PVDF)或硝酸纖維素薄膜(NC)上的抗體彌散，盒中的水保證盒內(nèi)濕度防止抗體溶液的蒸發(fā)。我們在實驗中應(yīng)用了該實用新型，并且該實用新型于2013年獲得中國國家知識產(chǎn)權(quán)局的授權(quán)(專利號ZL201320144341.7)。 第二部分：Kir2.1在胃癌干細(xì)胞干性維持和侵襲轉(zhuǎn)移中作用的研究 胃癌是世界范圍內(nèi)高發(fā)病率、高致死性的惡性消化系統(tǒng)腫瘤之一。盡管近年來胃癌的手術(shù)、化療以及其他治療方法均取得了長足進(jìn)步，但其療效仍不盡如人意，5年生存率并未得到顯著提高。這一現(xiàn)象折射出人們對胃癌侵襲轉(zhuǎn)移機(jī)制的認(rèn)識還不夠深入。因此，深入進(jìn)行胃癌侵襲轉(zhuǎn)移機(jī)制的研究是當(dāng)前一項緊迫的任務(wù)。 腫瘤干細(xì)胞(cancer stem cells，CSCs)學(xué)說的提出是近幾年來腫瘤研究領(lǐng)域的一大進(jìn)展，也為我們認(rèn)識胃癌侵襲和轉(zhuǎn)移機(jī)制開拓了新的視角。愈來愈多的證據(jù)表明，CSCs是腫瘤侵襲轉(zhuǎn)移的“種子”細(xì)胞。我們小組之前通過無血清成球培養(yǎng)的方法分離/富集了胃癌干細(xì)胞(gastric cancer stem cells, GCSCs)，并發(fā)現(xiàn)其具有高度自我更新和侵襲轉(zhuǎn)移潛能，從而證明了GCSCs是胃癌侵襲轉(zhuǎn)移的細(xì)胞學(xué)基礎(chǔ)。然而GCSCs侵襲轉(zhuǎn)移的機(jī)制并不清楚。 近年來，鉀離子通道在腫瘤中的作用受到關(guān)注，為探討鉀離子通道是否在GCSCs干性維持及侵襲轉(zhuǎn)移中發(fā)揮作用，我們進(jìn)行了全細(xì)胞電壓膜片鉗實驗，發(fā)現(xiàn)GCSCs中內(nèi)向整流K+電流顯著高于非GCSCs。然而，鉀離子通道種類繁多，為確定何種鉀離子通道發(fā)揮主要作用，我們進(jìn)一步作了基因表達(dá)譜芯片分析，結(jié)果顯示內(nèi)向整流K+通道Kir2.1(potassium inwardly rectifying channel, subfamily J, member2，KCNJ-2)在GCSCs中顯著高表達(dá)。我們還應(yīng)用免疫組化方法檢測了臨床胃癌標(biāo)本中Kir2.1表達(dá)，并統(tǒng)計分析了Kir2.1表達(dá)與臨床病理參數(shù)及病人預(yù)后的關(guān)系。本研究主要方法、結(jié)果及結(jié)論如下： 1、胃癌干細(xì)胞產(chǎn)生高強(qiáng)度內(nèi)向整流鉀電流與高表達(dá)Kir2.1密切相關(guān) (1)我們選擇胃癌細(xì)胞系中轉(zhuǎn)移能力較強(qiáng)的細(xì)胞系SGC7901，按本組先前建立的無血清成球培養(yǎng)法分離/富集GCSCs，然后以全細(xì)胞電壓膜片鉗檢測其K+電流的產(chǎn)生。結(jié)果發(fā)現(xiàn)，相比單層生長細(xì)胞(monolayer cells，MN)，胃癌細(xì)胞球細(xì)胞(sphere cells，SC)即GCSCs在電壓的刺激下可以釋放更高強(qiáng)度的內(nèi)向整流K+電流。 (2)基因表達(dá)譜芯片分析結(jié)果表明，GCSCs較MN顯著高表達(dá)Kir2.1通道蛋白(基因名：KCNJ-2)的mRNA并得到qRT-PCR驗證。 (3) Western blot實驗從蛋白水平證明GCSCs高表達(dá)Kir2.1，免疫熒光實驗顯示Kir2.1通道蛋白主要表達(dá)在細(xì)胞膜但也存在于細(xì)胞質(zhì)中。 (4) qPCR進(jìn)一步證明只有能產(chǎn)生特征性內(nèi)向整流K+電流的胃癌細(xì)胞才能檢測到KCNJ-2基因的表達(dá)，而在沒有特征性內(nèi)向整流K+電流的胃癌細(xì)胞中則檢測不到KCNJ-2基因的表達(dá)。這些結(jié)果表明，GCSCs產(chǎn)生高強(qiáng)內(nèi)向整流K+電流，并且該電流的產(chǎn)生主要由于其高表達(dá)內(nèi)向整流K+通道Kir2.1所致。 2、Kir2.1+胃癌細(xì)胞具有干性特性和高侵襲能力 (1)流式細(xì)胞儀分析發(fā)現(xiàn)SGC7901細(xì)胞中Kir2.1陽性(Kir2.1+)細(xì)胞的比率約為7.5%。 (2)與Kir2.1-細(xì)胞相比較，Kir2.1+細(xì)胞高表達(dá)干性轉(zhuǎn)錄因子Nanog，具有更強(qiáng)的細(xì)胞球(tumor-sphere)和克隆形成能力，表明Kir2.1+細(xì)胞具有干性特性。 (3)活細(xì)胞工作站觀察結(jié)果顯示Kir2.1+細(xì)胞較Kir2.1-細(xì)胞具有更強(qiáng)的運(yùn)動能力，Transwell小室遷移實驗顯示Kir2.1+細(xì)胞較Kir2.1-細(xì)胞具有更強(qiáng)的遷移能力，qRT-PCR檢測還表明Kir2.1+細(xì)胞較Kir2.1-細(xì)胞高表達(dá)基質(zhì)金屬蛋白酶2(matrixmetalloproteinase2, MMP2)。 3、siRNA沉默Kir2.1的表達(dá)抑制胃癌細(xì)胞干性和侵襲能力 應(yīng)用靶向Kir2.1的siRNA敲低Kir2.1的表達(dá)后，胃癌細(xì)胞Nanog和MMP2的表達(dá)均顯著下調(diào)，體外遷移和侵襲能力也受到顯著抑制。 4、Kir2.1在胃癌標(biāo)本中表達(dá)與病人預(yù)后密切相關(guān) (1)應(yīng)用免疫組化染色方法檢測了212例胃癌標(biāo)本中Kir2.1的表達(dá)，發(fā)現(xiàn)Kir2.1的表達(dá)與胃癌患者年齡(P=0.674)、性別(P=0.329)、組織學(xué)分型(P=0.625)無關(guān)，而與浸潤深度(P=0.010)、TNM分期(P=0.002)和是否發(fā)生淋巴結(jié)轉(zhuǎn)移(P=0.028)呈正相關(guān)。 (2)采用Kaplan-Meier方法計算132例有隨訪資料病人的生存率，結(jié)果顯示高表達(dá)Kir2.1的患者預(yù)后差于低表達(dá)Kir2.1的患者，提示Kir2.1可以用作判斷胃癌患者預(yù)后的指標(biāo)。 該部分的主要結(jié)論如下：胃癌干細(xì)胞高表達(dá)內(nèi)向整流K+通道Kir2.1，并與其干性維持和侵襲能力密切相關(guān)；Kir2.1的表達(dá)與胃癌浸潤深度、TNM分期和淋巴結(jié)轉(zhuǎn)移呈正相關(guān)而與患者生存時間呈負(fù)相關(guān)，可能成為判斷胃癌預(yù)后的新的指標(biāo)。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R735.2
本文編號：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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R735.2
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
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
本文編號：2169283
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